Enhancing chronic wound healing with Thai indigenous rice variety, Kaab Dum: Exploring ER stress and senescence inhibition in HaCaT keratinocyte cell line

Witchuda Payuhakrit; Pimchanok Panpinyaporn; Wilunplus Khumsri; Gorrawit Yusakul; Ratsada Praphasawat; Nitra Nuengchamnong; Sarawoot Palipoch

doi:10.1371/journal.pone.0302662

. 2024 May 15;19(5):e0302662. doi: 10.1371/journal.pone.0302662

Enhancing chronic wound healing with Thai indigenous rice variety, Kaab Dum: Exploring ER stress and senescence inhibition in HaCaT keratinocyte cell line

Witchuda Payuhakrit ^1,², Pimchanok Panpinyaporn ¹, Wilunplus Khumsri ³, Gorrawit Yusakul ⁴, Ratsada Praphasawat ⁵, Nitra Nuengchamnong ⁶, Sarawoot Palipoch ^7,^*

Editor: Gopinath Ponnusamy Manogaran⁸

PMCID: PMC11095683 PMID: 38748716

Abstract

Kaab Dum, a prominent indigenous rice variety cultivated in the Pak Phanang Basin of Nakhon Si Thammarat, Thailand, is the focus of our study. We investigate the therapeutic potential of indigenous Kaab Dum rice extract in the context of chronic wounds. Our research encompasses an examination of the nutritional compositions and chemical profiles of Kaab Dum rice extract. Additionally, we assess how the extract affects chronic wounds in TGF-β-induced HaCaT cells. Our evaluation methods include the detection of cellular oxidative stress, the examination of endoplasmic reticulum (ER) stress, wound healing assays, analysis of cell cycle arrest and the study of cellular senescence through senescence-associated β-galactosidase (SA-β-gal) staining. Our research findings demonstrate that TGF-β induces oxidative stress in HaCaT cells, which subsequently triggers ER stress, confirmed by the expression of the PERK protein. This ER stress results in cell cycle arrest in HaCaT cells, characterized by an increase in p21 protein, a cyclin-dependent kinase inhibitor (CDKI). Ultimately, this leads to cellular senescence, as confirmed by SA-β-gal staining. Importantly, our study reveals the effectiveness of Kaab Dum rice extract in promoting wound healing in the chronic wound model. The extract reduces ER stress and senescent cells. These beneficial effects are potentially linked to the antioxidant and anti-inflammatory properties of the rice extract. The findings of our study have the potential to make significant contributions to the development of enhanced products for both the prevention and treatment of chronic wounds.

Introduction

Rice (Oryza sativa L.) is an essential staple food for a significant portion of the global population. It is rich in carbohydrates, low in fat, and packed with essential nutrients like vitamins, protein, minerals and dietary fiber [1]. In southern Thailand, specifically the Pak Phanang Basin of Nakhon Si Thammarat province, numerous indigenous rice varieties flourish through extensive cultivation. These indigenous rice varieties can exhibit diverse nutritional profiles, with some being rich in specific nutrients like antioxidants, dietary fiber, vitamins or minerals, making them valuable for nutrition and health [2]. Based on data collected in the field, Kaab Dum is among the most prominent indigenous rice variety cultivated in the Pak Phanang Basin. As reported by Thailand’s Rice Department, Kaab Dum rice is an indigenous rice variety with a history of cultivation spanning more than 50 years. In terms of physical appearance, the grain husks are straw-colored, while the polished rice is white. It is categorized as non-glutinous rice. Kaab Dum rice is characterized by its upright growth, with dark purple stems, leaves that are a mix of purple and green, and purple husks. The grains are short, and each rice stalk produces approximately 300–400 grains.

Chronic wounds pose a significant medical challenge, marked by inadequate healing processes and extended recovery periods, encompassing conditions such as diabetic ulcers, pressure wounds and vascular lesions [3]. Global epidemiological data emphasize the prevalence of chronic wounds, which range from 1.51 to 2.21 cases per 1,000 individuals, with a notable incidence among the elderly [4, 5]. The etiology of chronic wounds is multifaceted, involving factors like overproduction of proinflammatory cytokines, oxidative stress, cellular senescence, infections and impaired stem cell function [6, 7]. Persistent inflammation, a pivotal aspect of wound healing, impedes proper healing, resulting in chronic wounds [8]. Transforming growth factor-β (TGF-β), a crucial cytokine in wound healing, has a dual role, initially facilitating healing and potentially contributing to chronic wound pathogenesis when its activity is prolonged [9–11]. Apart from the TGF-β, senescence was also previously reported as another main factor in chronic wound association [12]. Cellular senescence, characterized by halted cell functionality and cell cycle arrest, can arise from various factors, including genotoxic stress, metabolic alterations, oxidative stress, mitochondrial dysfunction or activation of oncogenes. Emerging research indicates that cellular senescence plays a key role in chronic wound development, affecting cell reduction through cytokine production from senescent cells [12, 13].

Numerous lines of evidence support a strong correlation between cellular senescence, inflammation and chronic wound progression. Cellular senescence is a regulatory response triggered by various forms of cellular stress, including DNA damage, telomere erosion, oxidative damage and protein misfolding [14]. In chronic wounds, senescent cells accumulate, leading to a persistent inflammatory environment that impairs stem cells, affecting processes like angiogenesis, matrix remodeling, cell plasticity and growth [15]. These senescent cells maintain viability but exhibit changes in metabolic activity and develop a complex senescence-associated secretory phenotype (SASP), which can compromise tissue repair and regeneration [16]. SASP components, including cytokines, matrix remodeling proteins and growth factors can change the microenvironment to become an inflammation which is the main factor contributing to chronic wound development. Therefore, cellular senescence plays a significant role in developing chronic wounds, and strategies aimed at preventing cell senescence are emerging as promising therapeutic approaches.

This study aims to investigate the therapeutic potential of indigenous Kaab Dum rice extract in chronic wound. Specifically, it examines how Kaab Dum rice extract modulates TGF-β-induced responses in human keratinocytes (HaCaT cell line) and its potential in mitigating cellular senescence-associated chronic wound development. Additionally, the research explores the effectiveness of Kaab Dum rice extract in stimulating wound healing through profound mechanisms, involving the attenuation of oxidative stress and endoplasmic reticulum (ER) stress.

Materials and methods

Rice extraction

The polished (white) and unpolished (brown) Kaab Dum rice obtained from the Rice Research Center in Nakhon Si Thammarat Province, Thailand were ground into fine powder, with each sample weighing 100 g (n = 3). Ethanol: water 18% v/v was added. The mixture was stirred, and extraction was performed using the ultrasonicate-assisted extraction technique for 90 sec at room temperature. The extract was then filtered to collect the extracted substances and the residual rice grains were extracted two more times using the same process. The substances obtained from all three extractions were combined and solvent evaporation was carried out using an air-evaporation instrument and freeze-drying.

Analysis of nutritional compositions of Kaab Dum rice extract

The Kaab Dum rice extract underwent analysis at the Central Laboratory in Thailand. The analysis covered a range of nutrients and components, including vitamins (B1, B2 and B3), ash content, carbohydrates, fat content, moisture content, protein content, as well as levels of magnesium, phosphorus, potassium, sodium and zinc.

Analysis of chemical profiles of Kaab Dum rice extract

Chemical component separation was performed using an Agilent 1260 Infinity Series HPLC-DAD System (Agilent Technologies, Waldbronn, Germany) equipped with a Luna C-18 (2) column (4.6 × 150 mm, 5 µm, Phenomenex Inc., Torrance, CA, USA). The column temperature was maintained at 35°C. The mobile phase consisted of water (A) and acetonitrile (B), both containing 0.1% (v/v) formic acid. The gradient elution profile started with 5% acetonitrile (B) for 30 sec, followed by a gradual increase in acetonitrile concentration to 95% over a period of 30 sec and then held at 95% acetonitrile for 10 sec. The flow rate was set at 0.5 mL/min, and the injection volume was 10 µL. The molecular weight analysis of substances was carried out using a 6540 Ultra-High-Definition Accurate Q-TOF-mass spectrometer (Agilent Technologies, Singapore) with ionization through electrospray ionization (ESI). The molecular weight analysis range was m/z 100–1000 Da. The instrument conditions for molecular weight analysis included a nitrogen gas (N2) flow rate of 7 L/min, a temperature of 350°C, a nebulizer gas pressure of 30 psi, a capillary voltage of 3.5 kV, fragmentation at 100 V, skimmer voltage at 65 V, Vcap at 3500 V, octopole RFP at 750 V, and collision energy at 10, 20, and 40 V using ultra-pure nitrogen gas (99.9995%). The compound types were analyzed using Agilent MassHunter Data Acquisition Software, Version B.05.01, and Agilent MassHunter Qualitative Analysis Software B.06.00 (Agilent Technologies, Santa Clara, CA, USA), in combination with databases such as METLIN PCD/PCDL database (Agilent Technologies, Santa Clara, CA, USA) and the public database Human Metabolome Database (http://www.hmdb.ca, accessed on 19 December 2022).

Cell culture experiments

Preparation of the extract

The Kaab Dum rice extract was dissolved in dimethyl sulfoxide (DMSO) to 0.1 g/ml as a stock concentration and mixed thoroughly using a vortex mixer. Subsequently, it was filtered through a 0.45 µm filter, stored at -20°C and protected from light.

HaCaT keratinocyte culture

HaCaT cells obtained from the American Type Culture Collection (ATCC) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose concentration, supplemented with 10% fetal bovine serum and 1% w/v penicillin/streptomycin. The cells were then incubated in a controlled humidity incubator at 37°C under a 5% CO₂ atmosphere.

Assessment of cell viability using 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay

HaCaT cells were cultured in 96-well plates for 24 h. Then, they were exposed to different concentrations of the polished and unpolished Kaab Dum rice extract. The cells were then further incubated for 24 h at 37°C in a 5% CO₂ atmosphere. Following incubation, a media containing MTT solution with a concentration of 0.5 mg/ml was added to each well, with a volume of 100 µL per well. The cells were incubated for an additional 2 h to allow the formation of purple formazan crystals in viable cells. Subsequently, the cells were dissolved using DMSO at a volume of 100 µL per well to measure the absorbance of the formazan solution, which directly correlates with the number of viable cells. The absorbance was measured at a wavelength of 570 nm using an automatic microplate reader (EMax® Plus Microplate Reader, Molecular devices) [17].

Detection of cellular oxidative stress using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining

To assess cellular oxidative stress status, the DCFH-DA method was employed. HaCaT cells were cultured in 96-well plates at a density of 1.5x10⁴ cells per well in completed media with a volume of 200 µL. The cells were then incubated in a CO₂ incubator at 37°C for 24 h to allow them to adhere to the well plate. Subsequently, the culture medium was removed, and chronic wound conditions were simulated by adding transforming growth factor-β (TGF-β: Abbkine, PRP100190) at a concentration of 10 ng/ml for 48 h. Then, the cells were treated with polished Kaab Dum rice extract at concentrations of 0.02 and 0.1 mg/ml, as well as with unpolished Kaab Dum rice extract at concentrations of 0.2 and 1 mg/ml, and further incubated for 24 h in a volume of 200 µL. Afterward, the culture medium was removed and replaced with fresh media containing DCFH-DA at a concentration of 10 µmol, and the cells were incubated for another hour. Following incubation, the level of fluorescence representing oxidative stress was measured at an excitation/emission wavelength of 485/535 nm using a Fluorescence microplate reader (The Spark® multimode microplate reader, TECAN, Switzerland) [18].

Detection of ER stress using fluorescent technique

To investigate the expression of PERK protein in HaCaT cells, the cells were cultured in 6-well plates containing coverslips for 24 h. The cells were then incubated in a CO₂ incubator at 37°C for an additional 24 h to allow them to adhere to the cover slips. Subsequently, chronic wound conditions were simulated by adding TGF-β at a concentration of 10 ng/ml for 48 h. Then, the cells were treated with polished Kaab Dum rice extract at concentrations of 0.02 and 0.1 mg/ml, as well as with unpolished Kaab Dum rice extract at concentrations of 0.2 and 1 mg/ml, and further incubated for 24 h. Following incubation, the cells were fixed and then subjected to immunostaining. The primary antibody targeting PERK protein (Abcam: ab65142) was applied, followed by a secondary antibody. The cell nuclei were stained with DAPI to visualize protein expression under a fluorescence microscope (Nikon ECLIPSE Ni, Japan). Five fields were randomly selected and photographed with a digital camera (Nikon Digital Sight 10, Japan) using NIS-Elements software and measured for the mean intensity of PERK using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).

Wound healing assay

HaCaT cells were cultured for 24 h in 6-well plates until they reached approximately 70% confluence. Subsequently, a pipette with a size of 200 µL was used to create a wound between the cells to simulate the formation of chronic wounds. This was followed by the addition of TGF-β at a concentration of 10 ng/ml for 48 h to induce chronic wound conditions. Then, the cells were treated with polished Kaab Dum rice extract at concentrations of 0.02 and 0.1 mg/ml, as well as with unpolished Kaab Dum rice extract at concentrations of 0.2 and 1 mg/ml. The cells were then incubated for an additional 24 and 48 h. Subsequently, images were captured using an inverted phase contrast microscope (CKX3, Olympus, Japan) at 24 and 48 h, and the extent of cell migration was compared between the group treated with Kaab Dum rice extract and the chronic wound control group [19].

Detection of cell-cycle arrest using flow cytometry

Cultured HaCaT cells in 6-well plates and incubated them in a CO₂ incubator at 37°C with 5% CO₂ for 24 h. Simulated chronic wound conditions by adding TGF-β at a concentration of 10 ng/ml for 48 h. Then, the cells were treated with polished Kaab Dum rice extract at concentrations of 0.02 and 0.1 mg/ml, as well as with unpolished Kaab Dum rice extract at concentrations of 0.2 and 1 mg/ml. Subsequently, transferred the cells back to the same incubator conditions for 24 h. Then, washed the cells with Phosphate buffer saline (PBS) and detached them from the 6-well plates using trypsinization. Collected the cells in microtubes and centrifuged them at 1,200 rpm for 5 min. Then, re-suspended the cells in binding buffer with a volume of 200 µL and stained them with anti-p21 antibody (Invitrogen: 33–7000) at a volume of 2 µL for 15 min and stained with secondary antibody conjugated with Alexa Fluor® 488 (Abcam, ab150077). Incubated the mixture at room temperature in the dark for 10 min. Finally, quantified the expression levels of p21 protein using FACScan (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Detection of cellular senescence using senescence-associated β-galactosidase (SA-β-gal) staining

HaCaT cells were cultured for 24 h in 6-well plates. Chronic wound conditions were simulated by adding TGF-β at a concentration of 10 ng/ml for 48 h. Subsequently, the cells were treated with polished Kaab Dum rice extract at concentrations of 0.02 and 0.1 mg/ml, as well as with unpolished Kaab Dum rice extract at concentrations of 0.2 and 1 mg/ml, and incubated for an additional 24 h. Afterward, the cells were fixed using 0.2% glutaraldehyde as a fixative reagent and washed with PBS. They were then stained with SA-β-gal enzyme using SA-β-gal staining (Sigma, CAS 7240-90-6) for 16 h at 37°C, in the absence of CO₂ and light. The cells were examined under a microscope, and a comparison was made between the group treated with rice extract and the chronic wound control group.

Statistical analysis

All data are presented as mean ± standard deviation (Mean ± SD). Differences between the means of all test groups were analyzed using one-way analysis of variance (ANOVA) and pairwise comparisons of means were performed using Tukey’s multiple comparison test with a confidence level of 95% (p-value < 0.05) using Prism version 9 software.

Results

The nutritional compositions of Kaab Dum rice extracts

The analysis encompassed various nutritional components, including vitamin B1, B2, B3, ash, carbohydrates, fat, moisture, protein, magnesium, phosphorus, potassium, sodium and zinc. The results revealed that unpolished Kaab Dum rice extract exhibits higher levels of vitamin B1, B2, B3, fat, protein, iron, magnesium, phosphorus and potassium compared to polished rice extract, as detailed in Table 1.

Table 1. The nutritional compositions of extracts from polished and unpolished Kaab Dum rice.

Compositions	Unit	Polished Kaab Dum rice	Unpolished Kaab Dum rice
Vitamin B1 (Thiamine)	mg/100g	0.078	0.124
Vitamin B2 (Riboflavin)	mg/100g	0.011	0.019
Vitamin B3 (Nicotinic acid)	mg/100g	0.265	0.429
Ash	g/100g	1.1	1.56
Carbohydrate	g/100g	76.6	74.9
Fat	g/100g	1.28	2.03
Moisture	g/100g	12.69	12.77
Protein	g/100g	8.33	8.74
Copper	mg/kg	0.397	0.266
Iron	mg/kg	5.349	8.046
Magesium	mg/kg	579.73	1,070.26
Phosphorus	mg/kg	1,581.99	2,623.27
Potassium	mg/kg	1,273.87	2,162.94
Sodium	mg/kg	32.51	28.67
Zinc	mg/kg	27.395	24.553

Open in a new tab

Chemical profiles of Kaab Dum rice extract

Chemical composition analysis of polished Kaab Dum rice extract revealed the presence of various compounds, including sugars (3-beta-Cellobiosylglucose and D-fructose, 6-O-alpha-D-glucopyranosyl-), lipids (glycerophosphocholine, C16 sphinganine, dehydrophytosphingosine, 1-myristoyl-glycero-3-phosphocholine, 1-(9Z,12Z,15Z-octadecatrienoyl)-sn-glycero-3-phosphocholine, (9Z,12Z-octadecadienoyl)-lysophosphatidylethanolamine, 2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine, 1-eicosapentaenoyl-glycero-3-phosphocholine, glycerophospho-N-palmitoyl ethanolamine, 1-palmitoyl phosphatidylcholine, and linoleoyl ethanolamide), an amino alcohol (2-amino-3-methyl-1-butanol), an essential amino acid (tryptophan) and choline (palmitoylcholine), as depicted in Fig 1A and detailed in Table 2.

Fig 1 — LC-ESI-QTOF-MS/MS chromatograms of polished (A) and unpolished (B) Kaab Dum rice extracts.

Table 2. Chemical compositions of polished Kaab Dum rice extract.

RT (min)	m/z	Adduct	MS/MS	Tentative Identification	Formula	Error (ppm)
2.753	104.1072	[M+H]+	60.0807,58.0651	2-Amino-3-methyl-1-butanol	C₅H₁₃NO	-2.01
3.057	258.1105	[M+H]+	184.0733,104.1067,60.0807	Glycerophospho choline	C₈H₂₀NO₆P	-1.55
3.115	527.1592	[M+Na]+	365.1050,277.0834,203.0522,104.1069	3-beta-Cellobiosylglucose	C₁₈H₃₂O₁₆	-1.79
3.258	365.1062	[M+Na]+	289.0492,271.0408,247.0402,229.0287,203.0523,185.0419	D-Fructose, 6-O-alpha-D-glucopyranosyl-	C₁₂H₂₂O₁₁	-2.1
8.629	205.0975	[M+H]+	188.0702,146.0596	(±)-Tryptophan	C₁₁H₁₂N₂O₂	-1.69
14.541	325.2283	[M+H]+	233.1649,148.1119,86.0965	Diampromide	C₂₁H₂₈N_2O	-2.64
17.486	274.2748	[M+H]+	198.9290,88.0756	C16 Sphinganine	C₁₆ H₃₅ N O₂	-2.71
18.608	316.2852	[M+H]+	143.0010,60.0444	Dehydrophyto sphingosine	C₁₈H₃₇ NO₃	-1.83
23.409	342.3374	[M+H]+	283.2633,57.0701	Palmitoylcholine	C₂₁H₄₄NO₂	-0.57
24.205	468.3084	[M+H]+	184.0732,85.0968	1-Myristoyl-glycero-3-phosphocholine	C₂₂H₄₆NO₇P	0.14
25.118	518.3246	[M+H]+	459.2494,184.0731,104.1067	1-(9Z,12Z,15Z-Octadecatrienoyl)-sn-glycero-3-phosphocholine	C₂₆H₄₈NO₇P	-0.93
26.343	478.2939	[M+H]+	337.2736,95.0849	(9Z,12Z-Octadecadienoyl)-lysophosphatidylethanolamine	C₂₃H₄₄NO₇P	-2.27
26.714	520.3413	[M+H]+	461.2046,337.2734,258.1097,184.0733,86.0963	2-(9Z,12Z-Octadecadienoyl)-sn-glycero-3-phosphocholine	C₂₆H₅₀NO₇P	-2.95
27.37	542.3231	[M+H]+	483.2480,337.2740,199.1695,104.1067	1-Eicosapentaenoyl-glycero-3-phosphocholine	C₂₈H₄₈NO₇P	1.87
27.991	454.2939	[M+H]+	313.2736,216.0634,155.0103,62.0599	Glycerophospho-N-palmitoyl ethanolamine	C₂₁H₄₄NO₇P	-2.39
28.696	496.3415	[M+H]+	313.2745,258.1102,184.0734,86.0964	1-Palmitoylphosphati dylcholine	C₂₄H₅₀NO₇P	-3.49
29.035	480.3094	[M+H]+	339.2891,216.0638,155.0105,62.0603	Glycerophospho-N-oleoyl ethanolamine	C₂₃H₄₆NO₇P	-1.95
32.228	324.2901	[M+H]+	306.2796,62.0599	Linoleoyl ethanolamide	C₂₀H₃₇NO₂	-1.22

Open in a new tab

Similarly, the chemical composition analysis of unpolished Kaab Dum rice extract detected the presence of lipids (dehydrophytosphingosine, 4E,14Z-Sphingadiene, 1-myristoyl-glycero-3-phosphocholine, 1-(9Z,12Z,15Z-octadecatrienoyl)-sn-glycero-3-phosphocholine, (9Z,12Z-octadecadienoyl)-lysophosphatidylethanolamine, 2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine, 1-eicosapentaenoyl-glycero-3-phosphocholine, glycerophospho-N-palmitoyl ethanolamine, and 1-palmitoyl phosphatidylcholine), an amino alcohol (2-amino-3-methyl-1-butanol), an essential amino acid (tryptophan), a benzylamino group (10-benzylamino-1-decanol) and (S)-Nerolidol 3-O-[a-L-rhamnopyranosyl-(1->4)-a-L-rhamnopyranosyl-(1->2)-b-D-glucopyranoside]. These components are depicted in Fig 1B and detailed in Table 3.

Table 3. Chemical compositions of unpolished Kaab Dum rice extract.

RT (min)	m/z	Adduct	MS/MS	Tentative Identification	Formula	Error (ppm)
2.713	104.1072	[M+H]+	60.0807,58.0651	2-Amino-3-methyl-1-butanol	C₅H₁₃NO	-2.01
8.687	205.0973	[M+H]+	188.0704,146.0597,74.0241	(±)-Tryptophan	C₁₁H₁₂N₂O₂	-0.71
14.349	325.2285	[M+H]+	233.1648,148.1116,91.0544,86.0965	Diampromide	C₂₁H₂₈N₂O	-3.26
15.313	264.2327	[M+H]+	219.1741,203.1429	10- Benzylamino-1-decanol	C₁₇H₂₉NO	-1.55
18.391	316.2853	[M+H]+	194.9154,60.0444	Dehydrophytosphingosine	C₁₈H₃₇NO₃	-2.15
19.028	298.2748	[M+H]+	280.2633,67.0543	4E,14Z-Sphingadiene	C₁₈H₃₅NO₂	-2.49
24.847	490.2915	[M+Na]+	431.2163,285.2424,104.1067	1-Myristoyl-glycero-3-phosphocholine	C₂₂H₄₆NO₇P	-2.22
25.096	518.3249	[M+H]+	459.2471,335.2589,258.1095,184.0730,104.1066	1-(9Z,12Z,15Z-Octadecatrienoyl)-sn-glycero-3-phosphocholine	C₂₆H₄₈NO₇P	-1.51
26.325	478.2942	[M+H]+	417.2388,337.2735,263.2365,155.0101,62.0599	(9Z,12Z-Octadecadienoyl)-lysophosphatidylethanolamine	C₂₃H₄₄NO₇P	-2.89
26.704	520.3414	[M+H]+	337.2731,184.0734,86.0961	2-(9Z,12Z-Octadecadienoyl)-sn-glycero-3-phosphocholine	C₂₆H₅₀NO₇P	-3.14
26.704	542.3227	[M+H]+	483.2478,337.2734,146.9815,86.0962	1-Eicosapentaenoyl-glycero-3-phosphocholine	C₂₈H₄₈NO₇P	2.61
27	677.3735	[M+H]+	515.3195,347.0896	(S)-Nerolidol 3-O-[a-L-Rhamnopyranosyl-(1->4)-a-L-rhamnopyranosyl-(1->2)-b-D-glucopyranoside]	C₃₃H₅₆O₁₄	1.16
27.361	542.3231	[M+H]+	483.2480,337.2739,104.1067	1-Eicosapentaenoyl-glycero-3-phosphocholine	C₂₈H₄₈NO₇P	1.87
27.992	454.2942	[M+H]+	393.2399,313.2737,155.0104,62.0599	Glycerophospho-N-palmitoyl ethanolamine	C₂₁H₄₄NO₇P	-3.05
28.708	496.3415	[M+H]+	313.2733,258.1099,184.0735,86.0962	1-Palmitoylphosphatidylcholine	C₂₄H₅₀NO₇P	-3.49
29.04	480.3094	[M+H]+	339.2887,155.0082,62.0597	Glycerophospho-N-oleoyl ethanolamine	C₂₃H₄₆NO₇P	-1.95

Open in a new tab

Cytotoxicity of Kaab Dum rice extract on HaCaT cells

The results demonstrated that polished Kaab Dum rice extract, tested at concentrations of 0.001, 0.01, 0.1, 1 and 10 mg on HaCaT cells, resulted in % cell viabilities of 117.59 ± 13.80, 92.85 ± 15.12, 81.00 ± 6.31, 67.12 ± 5.68 and 3.99 ± 0.77, respectively. Notably, a concentration of 0.1 mg was identified as a safe range for investigating effects of polished Kaab Dum rice extract on the cells (Fig 2A). Toxicity assessments of polished Kaab Dum rice extract at concentrations ranging from 0.02 to 0.1 mg revealed % cell viabilities of 95.26 ± 18.66, 97.44 ± 7.46, 97.89 ± 2.69, 92.34 ± 6.40 and 88.11 ± 8.58, respectively. These findings indicate that the highest safe concentration of polished Kaab Dum rice extract did not induce toxicity in HaCaT cells, as illustrated in the Fig 2B.

Fig 2 — A and C: Safety dose for polished and unpolished extract, respectively. B and D: The highest safe concentration dose for polished and unpolished extract, respectively. Significance level: **** (p < 0.0001).

Similarly, unpolished Kaab Dum rice extract, tested at concentrations of 0.001, 0.01, 0.1, 1 and 10 mg on HaCaT skin cells, resulted in % cell viabilities of 107.78 ± 7.74, 112.37 ± 11.14, 111.05 ± 2.17, 80.76 ± 8.97 and 4.43 ± 0.48, respectively. A concentration of 1 mg was identified as a safe range for studying the effects of unpolished Kaab Dum rice extract on the cells (Fig 2C). Toxicity assessments of brown Kaab Dum rice extract at concentrations ranging from 0.2 to 1 mg showed % cell viabilities of 100.86 ± 13.45, 100.26 ± 26.44, 89.68 ± 15.06, 88.09 ± 22.42 and 86.12 ± 15.77, respectively. These results indicate that the highest safe concentration of unpolished Kaab Dum rice extract did not induce toxicity in HaCaT skin cells, as depicted in the Fig 2D.

Effect of Kaab Dum rice extract on oxidative stress in HaCaT cells

The assessment of oxidative stress induced by polished Kaab Dum rice extract was conducted using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) method. The test groups were categorized as follows: the control group, the TGF-β wound group (10 ng/ml TGF-β), the wound group treated with low concentration of polished Kaab Dum rice extract (10 ng/ml TGF-β + 0.02 mg/ml PKD), and the wound group treated with high concentration of polished Kaab Dum rice extract (10 ng/ml TGF-β + 0.1 mg/ml PKD). After 24 h, the assessment of cellular oxidant levels in HaCaT cells showed values of 128.21 ± 4.44, 90.78 ± 1.44 and 76.78 ± 10.48, respectively, compared to the control (100%). In all test groups, cellular oxidant levels in the skin cells were significantly reduced, with statistical significance at p < 0.01, p < 0.001 and p < 0.0001, respectively, when compared to the wound group. For unpolished Kaab Dum rice extract, the test groups were categorized as follows: the control group, the TGF-β wound group (10 ng/ml TGF-β), the wound group treated with low concentration of unpolished Kaab Dum rice extract (10 ng/ml TGF-β + 0.2 mg/ml UKD), and the wound group treated with high concentration of unpolished Kaab Dum rice extract (10 ng/ml TGF-β + 1 mg/ml UKD). After 24 h, the assessment of cellular oxidative stress in HaCaT cells showed values of 128.21 ± 4.44, 90.90 ± 4.01 and 85.61 ± 6.03, respectively, compared to the control (100%). In all test groups, cellular oxidative stress in the skin cells was significantly reduced, with statistical significance at p < 0.001 and p < 0.0001, respectively, when compared to the wound group (Fig 3).

Fig 3 — Oxidative stress represents 100% control. Statistical significance: *** (p < 0.001), **** (p < 0.0001).

Inhibition of ER stress induction by Kaab Dum rice extract

The results of the inhibition of ER stress induction by Kaab Dum rice extract, as assessed by monitoring the expression of PERK protein in HaCaT cells using the immunofluorescence technique. For polished Kaab Dum rice extract, the test groups were categorized as the control group, the TGF-ꞵ wound group (10 ng/ml TGF-ꞵ), the wound group treated with low concentration polished Kaab Dum rice extract (10 ng/ml TGF-ꞵ + 0.02 mg/ml PKD), and the wound group treated with high concentration polished Kaab Dum rice extract (10 ng/ml TGF-ꞵ + 0.1 mg/ml PKD). After 24 h, the assessment of PERK protein expression in HaCaT cells revealed ER stress values of 3.59 ± 0.02, 12.51 ± 1.17, 7.32 ± 0.34 and 3.65 ± 0.21, respectively. In all test groups, the induction of ER stress in skin cells was significantly reduced, with statistical significance at p < 0.0001 when compared to the wound group. For unpolished Kaab Dum rice extract, the test groups were categorized as the control group, the TGF-ꞵ wound group (10 ng/ml TGF-ꞵ), the wound group treated with low concentration unpolished Kaab Dum rice extract (10 ng/ml TGF-ꞵ + 0.2 mg/ml UKD), and the wound group treated with high concentration unpolished Kaab Dum rice extract (10 ng/ml TGF-ꞵ + 1 mg/ml UKD). After 24 h, the assessment of PERK protein expression in HaCaT cells revealed ER stress values of 3.59 ± 0.02, 12.51 ± 1.17, 9.22 ± 0.20 and 6.67±0.78, respectively. In all test groups, the induction of ER stress in skin cells was significantly reduced, with statistical significance at p < 0.01 and p < 0.0001 when compared to the wound group (Fig 4A and 4B).

Fig 4 — A: Expression of PERK protein after 24 h. B: Intensity of PERK protein after 24 h. Statistical significance: ** (p < 0.01), **** (p < 0.0001).

Effect of Kaab Dum rice extract on the migration (wound closure) of HaCaT cells induced as chronic wounds by TGF-β

For polished Kaab Dum rice extract, the test groups were divided into the following groups: the control group, the TGF-β-induced wound group (10 ng/ml TGF-β), the low-concentration polished Kaab Dum rice extract-treated wound group (10 ng/ml TGF-β + 0.02 mg/ml PKD), and the high-concentration polished Kaab Dum rice extract-treated wound group (10 ng/ml TGF-β + 0.1 mg/ml PKD). After 24 h, cell migration (wound closure) was measured, and the percentages of cell migration for the test groups were found to be 25.89 ± 7.73, 76.30 ± 4.28, and 102.16 ± 15.31, respectively, compared to the control group (100%). All test groups exhibited a statistically significant increase in cell migration compared to the wound group (Fig 5A and 5B). Furthermore, after 48 h, cell migration (wound closure) was measured again, and the percentages of cell migration for the test groups were found to be 51.14 ± 8.15, 87.79 ± 2.63 and 119.64 ± 8.62, respectively, compared to the control group. Once again, all test groups showed a statistically significant increase in cell migration compared to the wound group (Fig 5A and 5C).

Fig 5 — A: Wound closure at 24 and 48 h. B: Percentage of wound healing at 24 h. C: Percentage of wound healing at 48 h. Statistical significance: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001).

For unpolished Kaab Dum rice extract, the test groups were divided into the following groups: the control group, the TGF-β-induced wound group (10 ng/ml TGF-β), the low-concentration unpolished Kaab Dum rice extract-treated wound group (10 ng/ml TGF-β + 0.2 mg/ml UKD), and the high-concentration unpolished Kaab Dum rice extract-treated wound group (10 ng/ml TGF-β + 1 mg/ml UKD). After 24 h, the percentages of cell migration for the test groups were found to be 41.54 ± 8.70, 56.79 ± 15.15, and 68.48 ± 8.90, respectively, compared to the control group (100%). All test groups exhibited a statistically significant increase in cell migration compared to the wound group (Fig 5A and 5B). Furthermore, after 48 h, cell migration (wound closure) was measured again, and the percentages of cell migration for the test groups were found to be 46.29 ± 1.46, 52.36 ± 4.34 and 70.58 ± 8.89, respectively, compared to the control group. Once again, all test groups showed a statistically significant increase in cell migration compared to the wound group (Fig 5A and 5C).

Effect of Kaab Dum rice extract on the expression of p21 protein

For polished Kaab Dum rice extract, the inhibition of p21 protein expression, which is related to cell cycle arrest was assessed using the flow cytometry. The test groups were categorized as follows: the control group, the TGF-β wound group (10 ng/ml TGF-β), the wound group treated with low-concentration polished rice extract (10 ng/ml TGF-β + 0.02 mg/ml PKD), and the wound group treated with high-concentration polished rice extract (10 ng/ml TGF-β + 0.1 mg/ml PKD). After 24 h, the measurement of p21 protein expression in HaCaT cells revealed the percentage of positive cells as follows: 3.99 ± 0.46, 34.29 ± 1.10, 10.24 ± 1.11 and 3.45 ± 0.53, respectively. All test groups exhibited a statistically significant reduction in p21 protein expression in skin cells compared to the wound group.For unpolished Kaab Dum rice extract, the test groups were divided into: the control group, the TGF-β wound group (10 ng/ml TGF-β), the wound group treated with low-concentration unpolished rice extract (10 ng/ml TGF-β + 0.2 mg/ml UKD), and the wound group treated with high-concentration unpolished rice extract (10 ng/ml TGF-β + 1 mg/ml UKD). After 24 h, the measurement of p21 protein expression in HaCaT cells revealed the percentage of positive cells as follows: 3.99 ± 0.46, 34.29 ± 1.10, 11.71 ± 1.87 and 8.20 ± 1.51, respectively. All test groups showed a statistically significant reduction in p21 protein expression in skin cells compared to the wound group (Fig 6A and 6B).

Fig 6 — A: p21 protein expression. B: Percentage of p21 protein expression in positive cells. Significance level: **** (p < 0.0001).

Effects of Kaab Dum rice extract on senescence of HaCaT cells induced as chronic wounds by TGF-ꞵ

For polished Kaab Dum rice extract, the test groups were categorized as follows: the control group, the TGF-β-induced wound group (10 ng/ml TGF-β), the low-concentration polished Kaab Dum rice extract-treated wound group (10 ng/ml TGF-β + 0.02 mg/ml PKD), and the high-concentration polished Kaab Dum rice extract-treated wound group (10 ng/ml TGF-β + 0.1 mg/ml PKD). After 24 h, the percentage of positively stained blue cells, indicative of senescence due to the SA-β-gal enzyme reaction in HaCaT skin cells, was determined to be 4.67 ± 1.15, 64.33 ± 8.50, 30.67 ± 5.69 and 14.00 ± 6.24, respectively. All test groups exhibited a statistically significant reduction in the presence of blue-stained senescent cells compared to the wound group. For unpolished Kaab Dum rice extract, following a similar approach, the test groups were divided into: the control group, the TGF-β-induced wound group (10 ng/ml TGF-β), the low-concentration unpolished Kaab Dum rice extract-treated wound group (10 ng/ml TGF-β + 0.2 mg/ml UKD), and the high-concentration unpolished Kaab Dum rice extract-treated wound group (10 ng/ml TGF-β + 1 mg/ml UKD). After 24 hours, the percentage of positively stained blue cells, reflecting senescence due to the SA-β-gal enzyme reaction in HaCaT skin cells, was measured and found to be 6.33 ± 3.06, 62.00 ± 18.33, 34.67 ± 11.02 and 20.33 ± 4.73, respectively. All test groups exhibited a statistically significant reduction in blue-stained senescent cells compared to the wound group (Fig 7A and 7B).

Fig 7 — A: Senescent cells stained blue with SA-β-gal enzyme reaction. B: Percentage of positive cells. Significance: * (p < 0.05), ** (p < 0.01), *** (p < 0.001).

Discussion

Kaab Dum rice is a traditional local rice variety that has been cultivated for more than 50 years in the Pak Phanang Basin, Nakhon Si Thammarat province. It is cooked for consumption, and due to its firm texture when cooked, it is also used as a raw material for processing various products, especially as a key ingredient in making traditional local delicacies in the region. An analysis of rice compositions revealed that unpolished rice contains higher magnesium levels than polished rice, with Kaab Dum rice displaying particularly elevated magnesium content. Magnesium plays a pivotal role as a cofactor for enzymes involved in vital processes, including protein synthesis, muscle and nerve transmission, active transport of calcium and potassium ions across cell membranes, structural bone development and immunological functions like macrophage activation and lymphocyte proliferation [20]. In addition, Kaab Dum rice contains lower sodium levels. Sodium is crucial for regulating water and fluid balance, as well as controlling blood pressure. There is a significant connection between sodium intake and various health events, including strokes and coronary heart disease [21]. Furthermore, the analysis reveals that Kaab Dum rice is rich in zinc. Zinc deficiency can lead to impaired physical growth and development, impacting the epidermal, gastrointestinal, central nervous, immune, skeletal and reproductive systems [22].

Chemical analysis of Kaab Dum rice extract has identified a range of compounds, with sugars, lipids, an essential amino acid and choline as the major constituents. Tryptophan, an essential amino acid, plays a crucial role in protein synthesis, which is vital for various biological functions such as tissue growth, repair and maintenance. It also serves as a precursor for serotonin, a neurotransmitter that regulates mood, sleep and appetite [23]. C16 Sphinganine, an essential component of sphingolipids, is involved in critical processes like cell growth, differentiation and apoptosis [24]. Glycerophospho-N-oleoyl ethanolamine, a naturally occurring lipid molecule, plays a role in various physiological processes, particularly in the regulation of appetite and energy balance [25, 26]. Another lipid molecule, linoleoyl ethanolamide, is also involved in appetite regulation, the endocannabinoid system and may have potential roles in obesity management [27].

Chronic wounds, characterized by their prolonged inability to heal within the expected time frame, pose a significant medical challenge [28]. TGF-β coordinates critical processes such as re-epithelialization, angiogenesis, fibroblast proliferation and extracellular matrix (ECM) production, leading to granulation tissue formation and wound contraction. In the remodeling phase, TGF-β influences the balance between collagen degradation by matrix metalloproteinases (MMPs) and the presence of endogenous tissue inhibitors of MMPs (TIMPs), whose expression it also regulates [11, 29, 30]. TGF-β is focusing in this study because the previous evidence showed TGF-β as the main factor producing chronic wounds, including diabetic ulcers [10] and pressure wounds [31]. Our study uses TGF-β to induce chronic wound conditions, where TGF-β also induces oxidative stress by stimulating the production of reactive oxygen species (ROS). Our research demonstrates that TGF-β induces oxidative stress in HaCaT cells, while the application of Kaab Dum rice extract effectively reduces cellular oxidant levels. This oxidative stress can subsequently lead to ER stress, resulting in the accumulation of misfolded or unfolded proteins, in accordance with previous studies [32, 33]. Protein kinase R-like endoplasmic reticulum kinase (PERK) is a key mediator of ER stress, playing a crucial role in the regulation of essential cellular functions [34]. However, when ER stress becomes irreversible, it can lead to the deterioration of cellular functions, often resulting in cell death [35]. Our study found that TGF-β-induced chronic wounds exhibited signs of ER stress, indicated by the presence of PERK. Furthermore, ER stress hindered the migration of HaCaT keratinocytes. However, the administration of Kaab Dum rice extract significantly improved ER stress and the migration of keratinocytes. We propose that the high magnesium and zinc content in the extract may contribute to the reduction of oxidative stress. Furthermore, the presence of tryptophan in Kaab Dum rice extract has the potential to enhance antioxidant status, alleviate inflammation and ER stress in TGF-β-induced HaCaT keratinocytes. These tryptophan-related effects are consistent with findings in a previous study [36].

The cyclin-dependent kinase inhibitors (CDKIs) play a crucial role in regulating cell cycle processes. P21, a CDKI, exerts anti-proliferative effects through a P53-dependent mechanism. It is activated by p16INK4A, leading to the inhibition of CDK2-cyclin E complex. This inhibition results in the hypo-phosphorylation of pRB, leading to cell cycle arrest [37]. Senescence is often identified by the presence of SA-β-gal activity, which is a reliable marker in cultured cells and mammalian tissues [12]. Our study shows that TGF-β induces senescence in HaCaT cells by upregulating p21 protein and SA-β-gal expression, while treatment with Kaab Dum rice extract reduces p21 protein and SA-β-gal expression. We attribute these effects to the antioxidant and/or anti-inflammatory properties of the nutritional and chemical components found in Kaab Dum rice extract, including linoleoyl ethanolamide, phosphatidylcholine, magnesium, zinc and tryptophan. Previous studies have indicated that both linoleoyl ethanolamide and phosphatidylcholine exert anti-inflammatory effects [38, 39].

Conclusions

This study induced chronic wounds in HaCaT cells using TGF-β, which led to ER stress, as confirmed by the expression of the PERK protein. Subsequently, this ER stress caused cell cycle arrest in HaCaT cells, evidenced by an increase in a CDKI, p21 protein. This process resulted in the cells entering a state of senescence, as confirmed by staining with the SA-β-gal. Of particular interest, the study revealed the effectiveness of extracts from Kaab Dum rice in promoting wound healing in the chronic wound model by reducing ER stress and senescent cells. Nevertheless, further research is necessary to gain a deeper understanding of the underlying mechanisms involved in this process. Moreover, this research holds the potential to pave the way for the development of natural products for the treatment of chronic wounds, addressing a significant medical concern.

Supporting information

S1 File

(XLSX)

pone.0302662.s001.xlsx^{(22.4KB, xlsx)}

Acknowledgments

We are thankful to the President and all the staff of Walailak University and Mahidol University for their kind support.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

the Office of the Royal Development Projects Board, Thailand.

References

1.Sen S, Chakraborty R, Kalita P. Rice—not just a staple food: A comprehensive review on its phytochemicals and therapeutic potential. Trends in Food Science & Technology. 2020;97:265–85. [Google Scholar]
2.Summpunn P, Panpipat W, Manurakchinakorn S, Bhoopong P, Cheong LZ, Chaijan M. Comparative analysis of antioxidant compounds and antioxidative properties of Thai indigenous rice: Effects of rice variety and processing condition. Molecules. 2022;27(16):5180. doi: 10.3390/molecules27165180 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Frykberg RG, Banks J. Challenges in the treatment of chronic wounds. Adv Wound Care (New Rochelle). 2015;4(9):560–82. doi: 10.1089/wound.2015.0635 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Zhu X, Olsson MM, Bajpai R, Järbrink K, Tang WE, Car J. Health-related quality of life and chronic wound characteristics among patients with chronic wounds treated in primary care: A cross-sectional study in Singapore. Int Wound J. 2022;19(5):1121–32. doi: 10.1111/iwj.13708 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Goh OQ, Ganesan G, Graves N, Ng YZ, Harding K, Tan KB. Incidence of chronic wounds in Singapore, a multiethnic Asian country, between 2000 and 2017: a retrospective cohort study using a nationwide claims database. BMJ Open. 2020;10(9):e039411. doi: 10.1136/bmjopen-2020-039411 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Gould L, Abadir P, Brem H, Carter M, Conner-Kerr T, Davidson J, et al. Chronic wound repair and healing in older adults: current status and future research. J Am Geriatr Soc. 2015;63(3):427–38. doi: 10.1111/jgs.13332 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Wilkinson HN, Hardman MJ. Senescence in wound repair: Emerging strategies to target chronic healing wounds. Frontiers in Cell and Developmental Biology. 2020;8:773. doi: 10.3389/fcell.2020.00773 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.MacLeod AS, Kwock JT. Inflammation in wound repair: Role and function of inflammation in wound repair. Wound Healing 2018:177–94. [Google Scholar]
9.O’Kane S, Ferguson MW. Transforming growth factor beta s and wound healing. Int J Biochem Cell Biol. 1997;29(1):63–78. doi: 10.1016/s1357-2725(96)00120-3 [DOI] [PubMed] [Google Scholar]
10.Jude EB, Blakytny R, Bulmer J, Boulton AJ, Ferguson MW. Transforming growth factor-beta 1, 2, 3 and receptor type I and II in diabetic foot ulcers. Diabet Med. 2002;19(6):440–7. doi: 10.1046/j.1464-5491.2002.00692.x [DOI] [PubMed] [Google Scholar]
11.Wang XJ, Han G, Owens P, Siddiqui Y, Li AG. Role of TGF beta-mediated inflammation in cutaneous wound healing. J Investig Dermatol Symp Proc. 2006;11(1):112–7. doi: 10.1038/sj.jidsymp.5650004 [DOI] [PubMed] [Google Scholar]
12.Wei X, Li M, Zheng Z, Ma J, Gao Y, Chen L, et al. Senescence in chronic wounds and potential targeted therapies. Burns Trauma. 2022;10:tkab045. doi: 10.1093/burnst/tkab045 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Schultz GS, Sibbald RG, Falanga V, Ayello EA, Dowsett C, Harding K, et al. Wound bed preparation: a systematic approach to wound management. Wound Repair Regen. 2003;11 Suppl 1:S1–28. doi: 10.1046/j.1524-475x.11.s2.1.x [DOI] [PubMed] [Google Scholar]
14.He S, Sharpless NE. Senescence in health and disease. Cell. 2017;169(6):1000–11. doi: 10.1016/j.cell.2017.05.015 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Wang Z, Shi C. Cellular senescence is a promising target for chronic wounds: A comprehensive review. Burns Trauma. 2020;8:tkaa021. doi: 10.1093/burnst/tkaa021 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Kumari R, Jat P. Mechanisms of cellular senescence: Cell cycle arrest and senescence associated secretory phenotype. Frontiers in Cell and Developmental Biology. 2021;9: 645593. doi: 10.3389/fcell.2021.645593 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Naktubtim C, Payuhakrit W, Uttarawichien T, Hassametto A, Suwannalert P. YAP, a novel target regulates F-actin rearrangement-associated CAFs transformation and promotes colorectal cancer cell progression. Biomed Pharmacother. 2022;155:113757. doi: 10.1016/j.biopha.2022.113757 [DOI] [PubMed] [Google Scholar]
18.Aimvijarn P, Payuhakrit W, Charoenchon N, Okada S, Suwannalert P. Riceberry rice germination and UVB radiation enhance protocatechuic acid and vanillic acid to reduce cellular oxidative stress and suppress B16F10 melanogenesis relating to F-actin rearrangement. Plants. 2023;12(3):484. doi: 10.3390/plants12030484 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Uttarawichien T, Khumsri W, Suwannalert P, Sibmooh N, Payuhakrit W. Onion peel extract inhibits cancer cell growth and progression through the roles of L1CAM, NF-κB, and angiogenesis in HT-29 colorectal cancer cells. Prev Nutr Food Sci. 2021;26(3):330–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Al Alawi AM, Majoni SW, Falhammar H. Magnesium and human health: Perspectives and research directions. Int J Endocrinol. 2018;2018:9041694. doi: 10.1155/2018/9041694 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Aburto NJ, Ziolkovska A, Hooper L, Elliott P, Cappuccio FP, Meerpohl JJ. Effect of lower sodium intake on health: systematic review and meta-analyses. BMJ: British Medical Journal. 2013;346:f1326. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Roohani N, Hurrell R, Kelishadi R, Schulin R. Zinc and its importance for human health: An integrative review. J Res Med Sci. 2013;18(2):144–57. [PMC free article] [PubMed] [Google Scholar]
23.Kanova M, Kohout P. Tryptophan: A unique role in the critically Ill. Int J Mol Sci. 2021;22(21):11714. doi: 10.3390/ijms222111714 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Kanno T, Gotoh A, Nishizaki T. Sphingosine arrests the cell cycle and induces apoptosis by targeting sphingosine-dependent protein kinase and protein kinase Cδ in vitro. Personalized Medicine Universe. 2014;3:22–7. [Google Scholar]
25.Tovar R, Gavito AL, Vargas A, Soverchia L, Hernandez-Folgado L, Jagerovic N, et al. Palmitoleoylethanolamide is an efficient anti-obesity endogenous compound: Comparison with oleylethanolamide in diet-induced obesity. Nutrients. 2021;13(8):2589. doi: 10.3390/nu13082589 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Brown J, Karimian E, Ayala J. Oleoylethanolamide: A fat ally in the fight against obesity. Physiology & Behavior. 2017;176:50–8. doi: 10.1016/j.physbeh.2017.02.034 [DOI] [PubMed] [Google Scholar]
27.Ho M, Anderson GH, Lin L, Bazinet RP, Kubant R. Ethanolamides of essential α-linolenic and linoleic fatty acids suppress short-term food intake in rats. Food Funct. 2020;11(4):3066–72. [DOI] [PubMed] [Google Scholar]
28.Falanga V, Isseroff RR, Soulika AM, Romanelli M, Margolis D, Kapp S, et al. Chronic wounds. Nature Reviews Disease Primers. 2022;8(1):50. doi: 10.1038/s41572-022-00377-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Liarte S, Bernabé-García Á, Nicolás FJ. Role of TGF-β in skin chronic wounds: A keratinocyte perspective. Cells. 2020;9(2):306. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Finnson KW, McLean S, Di Guglielmo GM, Philip A. Dynamics of transforming growth factor beta signaling in wound healing and scarring. Adv Wound Care (New Rochelle). 2013;2(5):195–214. doi: 10.1089/wound.2013.0429 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Strong AL, Bowles AC, MacCrimmon CP, Lee SJ, Frazier TP, Katz AJ, et al. Characterization of a murine pressure ulcer model to assess efficacy of adipose-derived stromal cells. Plast Reconstr Surg Glob Open. 2015;3(3):e334. doi: 10.1097/GOX.0000000000000260 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Chong WC, Shastri MD, Eri R. Endoplasmic reticulum stress and oxidative stress: A vicious nexus implicated in bowel disease pathophysiology. Int J Mol Sci. 2017;18(4):771. doi: 10.3390/ijms18040771 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Richter K, Kietzmann T. Reactive oxygen species and fibrosis: further evidence of a significant liaison. Cell and Tissue Research. 2016;365(3):591–605. doi: 10.1007/s00441-016-2445-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Liu Z, Lv Y, Zhao N, Guan G, Wang J. Protein kinase R-like ER kinase and its role in endoplasmic reticulum stress-decided cell fate. Cell Death & Disease. 2015;6(7):e1822. doi: 10.1038/cddis.2015.183 [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Sano R, Reed JC. ER stress-induced cell death mechanisms. Biochimica et Biophysica Acta (BBA)—Molecular Cell Research. 2013;1833(12):3460–70. doi: 10.1016/j.bbamcr.2013.06.028 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Liu G, Tao J, Lu J, Jia G, Zhao H, Chen X, et al. Dietary tryptophan supplementation improves antioxidant status and alleviates inflammation, endoplasmic reticulum stress, apoptosis, and pyroptosis in the intestine of piglets after lipopolysaccharide challenge. Antioxidants (Basel). 2022;11(5):872. doi: 10.3390/antiox11050872 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Wagner KD, Wagner N. The senescence markers p16INK4A, p14ARF/p19ARF, and p21 in organ development and homeostasis. Cells. 2022;11(12):1966. doi: 10.3390/cells11121966 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Ishida T, Nishiumi S, Tanahashi T, Yamasaki A, Yamazaki A, Akashi T, et al. Linoleoyl ethanolamide reduces lipopolysaccharide-induced inflammation in macrophages and ameliorates 2,4-dinitrofluorobenzene-induced contact dermatitis in mice. Eur J Pharmacol. 2013;699(1–3):6–13. doi: 10.1016/j.ejphar.2012.11.030 [DOI] [PubMed] [Google Scholar]
39.Treede I, Braun A, Sparla R, Kühnel M, Giese T, Turner JR, et al. Anti-inflammatory effects of phosphatidylcholine. J Biol Chem. 2007;282(37):27155–64. doi: 10.1074/jbc.M704408200 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0302662.r001

Decision Letter 0

Gopinath Ponnusamy Manogaran

9 Jan 2024

PONE-D-23-34554Enhancing chronic wound healing with Thai indigenous rice variety, Kaab Dum: exploring ER stress and senescence inhibition in HaCaT keratinocyte cell linePLOS ONE

Dear Dr. Palipoch,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Feb 23 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Gopinath Ponnusamy Manogaran, PhD

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Note from Emily Chenette, Editor in Chief of PLOS ONE, and Iain Hrynaszkiewicz, Director of Open Research Solutions at PLOS: Did you know that depositing data in a repository is associated with up to a 25% citation advantage (https://doi.org/10.1371/journal.pone.0230416)? If you’ve not already done so, consider depositing your raw data in a repository to ensure your work is read, appreciated and cited by the largest possible audience. You’ll also earn an Accessible Data icon on your published paper if you deposit your data in any participating repository (https://plos.org/open-science/open-data/#accessible-data).

3. Thank you for stating the following financial disclosure:

“the Office of the Royal Development Projects Board, Thailand.”

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

4. Please provide a complete Data Availability Statement in the submission form, ensuring you include all necessary access information or a reason for why you are unable to make your data freely accessible. If your research concerns only data provided within your submission, please write "All data are in the manuscript and/or supporting information files" as your Data Availability Statement.

5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Prof Dr/Editor,

Greetings!

Thank you for your invitation to review the manuscript entitled ‘’ Enhancing chronic wound healing with Thai indigenous rice variety, Kaab Dum: exploring ER stress and senescence inhibition in HaCaT keratinocyte cell line’’.

Please, see below my comments:

Introduction:

1. Please, explain the relationship between senescence, inflammation, and delayed wound healing.

Materials and methods

2. Please, mention the ethanol: water (V/V) used for your extract.

3. Please, mention the final concentration of your stock in DMSO.

4. Please, mention the solute for MTT solution. Is it medium or PBS?

5. The concentration of MTT is 0.5 mg per L or ml???

6. What is the concentration of DMSO after addition of your extract to the medium???

7. What is the type of fixative you used in SA-β-gal staining assay?

8. Correct the title of Table 1 ………polished and unpolished……..

Results

9. Please, revise the abbreviation of TGF-β in all result section.

10. In the wound healing assay, the image 3 and 4 of 0 h is not similar in size as others.

Discussion

11. The discussion section containing too long paragraphs.

Reviewer #2: This article presents a study on the therapeutic potential of Kaab Dum rice extract in the context of chronic wound healing. The investigation focused on the effects of the indigenous rice variety on TGF-β-induced HaCaT cells, specifically examining oxidative stress, endoplasmic reticulum (ER) stress, cell cycle arrest, and cellular senescence. The findings suggest a reduction of oxidative stress, inhibition of ER stress, and promotion of wound healing. However, there are areas where the study could be strengthened:

1. A more detailed investigation into the mechanisms by which Kaab Dum rice extract affects wound healing is necessary to fully understand its therapeutic potential.

2. The absence of in vivo studies or animal models is a significant limitation. Such studies are crucial for assessing the physiological relevance of the rice extract in a more complex biological system.

3. Comparing the effectiveness of Kaab Dum rice extract with other treatments for chronic wounds would provide context and further insight.

4. Detailed information about the reagents used in the study should be provided, including the source, stock number, tradename, company, city, and country. This information is critical for the reproducibility of the research.

5. An inconsistency in Figure 5 regarding the cell scratching assay should be addressed to ensure the validity of the results. The scratch area at 0 hr for the groups treated with TGF-β+0.02 mg/ml PKD and TGF-β+0.1 mg/ml PKD does not match the initial scratch area of the other groups.

6. What is the authors' rationale for choosing TGF-β induced HaCaT cells as a model of chronic wound? What type of chronic wound is this? Further justification and discussion are needed.

Reviewer #3: Dear Authors,

Your manuscript is well-structured and informative. Addressing the following comments will enhance your study's clarity and scientific rigor.

1. Antibiotic-Antimycotic Solution: For clarity, kindly mention the composition of the antibiotic-antimycotic solution used in your experiments.

2. MTT Solution Concentration: Clarify whether the MTT solution concentration of 0.5 mg/L should instead be reported as 0.5 mg/mL for accuracy.

3. Transforming Growth Factor-β (TGF-β): Provide the source and product number of the TGF-β used in your study for better reproducibility.

4. Rice Extract Concentrations: Explain the rationale behind using different concentrations for polished and unpolished Kaab Dum rice extracts (0.02 and 0.1 mg/mL for polished; 0.2 and 1 mg/mL for unpolished). What factors influenced this selection?

5. Scratch Wound as Chronic Wound: Justify why the scratch wound model was chosen to represent a chronic wound in your study.

6. % of Control and % of Inhibition: Clarify the meaning of "% of control." Does it refer to "% of inhibition"? Please provide clarity on these percentages for better understanding.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Xuqiang Nie

Reviewer #3: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: Comments.docx

pone.0302662.s002.docx^{(13.8KB, docx)}

PLoS One. 2024 May 15;19(5):e0302662. doi: 10.1371/journal.pone.0302662.r002

Author response to Decision Letter 0

25 Jan 2024

Dear Editor

We edited and added information according to the editor's suggestions as follows:

1. Our manuscript meets PLOS ONE's style requirements.

2. We state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." in our cover letter.

3. We provide a complete Data Availability Statement in the submission form, we write "All data are in the manuscript".

4. I have an ORCID iD and that it is validated in Editorial Manager.

We made changes based on reviewers' comments. Details are in the attached file “response to reviewers”.

Yours sincerely,

Assoc.Prof.Dr. Sarawoot Palipoch

Corresponding Author

Attachment

Submitted filename: Response to reviewers.docx

pone.0302662.s003.docx^{(22.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0302662.r003

Decision Letter 1

Gopinath Ponnusamy Manogaran

6 Mar 2024

PONE-D-23-34554R1Enhancing chronic wound healing with Thai indigenous rice variety, Kaab Dum: exploring ER stress and senescence inhibition in HaCaT keratinocyte cell linePLOS ONE

Dear Dr. Palipoch,

Please submit your revised manuscript by Apr 19 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Gopinath Ponnusamy Manogaran, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #2: I appreciate the authors' responses and I have only a couple of residual questions.

1. The LC-ESI-QTOF-MS/MS chromatogram should be marked with the main peak information.

2. A few typographical errors also remain (i.e. the word " TGF-β" on pg 20-21, Table 2, line 4, [M+Na}? ).

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Xuqiang Nie

Reviewer #3: No

**********

PLoS One. 2024 May 15;19(5):e0302662. doi: 10.1371/journal.pone.0302662.r004

Author response to Decision Letter 1

7 Mar 2024

Dear Editor and Reviewers

We made changes based on reviewers' comments. Details are in the attached file “response to reviewers”.

Yours sincerely,

Assoc.Prof.Dr. Sarawoot Palipoch

Corresponding Author

Attachment

Submitted filename: Response to reviewers.docx

pone.0302662.s004.docx^{(16.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0302662.r005

Decision Letter 2

Gopinath Ponnusamy Manogaran

10 Apr 2024

Enhancing chronic wound healing with Thai indigenous rice variety, Kaab Dum: exploring ER stress and senescence inhibition in HaCaT keratinocyte cell line

PONE-D-23-34554R2

Dear Dr. Palipoch,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Gopinath Ponnusamy Manogaran, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File

(XLSX)

pone.0302662.s001.xlsx^{(22.4KB, xlsx)}

Attachment

Submitted filename: Comments.docx

pone.0302662.s002.docx^{(13.8KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

pone.0302662.s003.docx^{(22.9KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

pone.0302662.s004.docx^{(16.6KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

[pone.0302662.ref001] 1.Sen S, Chakraborty R, Kalita P. Rice—not just a staple food: A comprehensive review on its phytochemicals and therapeutic potential. Trends in Food Science & Technology. 2020;97:265–85. [Google Scholar]

[pone.0302662.ref002] 2.Summpunn P, Panpipat W, Manurakchinakorn S, Bhoopong P, Cheong LZ, Chaijan M. Comparative analysis of antioxidant compounds and antioxidative properties of Thai indigenous rice: Effects of rice variety and processing condition. Molecules. 2022;27(16):5180. doi: 10.3390/molecules27165180 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref003] 3.Frykberg RG, Banks J. Challenges in the treatment of chronic wounds. Adv Wound Care (New Rochelle). 2015;4(9):560–82. doi: 10.1089/wound.2015.0635 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref004] 4.Zhu X, Olsson MM, Bajpai R, Järbrink K, Tang WE, Car J. Health-related quality of life and chronic wound characteristics among patients with chronic wounds treated in primary care: A cross-sectional study in Singapore. Int Wound J. 2022;19(5):1121–32. doi: 10.1111/iwj.13708 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref005] 5.Goh OQ, Ganesan G, Graves N, Ng YZ, Harding K, Tan KB. Incidence of chronic wounds in Singapore, a multiethnic Asian country, between 2000 and 2017: a retrospective cohort study using a nationwide claims database. BMJ Open. 2020;10(9):e039411. doi: 10.1136/bmjopen-2020-039411 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref006] 6.Gould L, Abadir P, Brem H, Carter M, Conner-Kerr T, Davidson J, et al. Chronic wound repair and healing in older adults: current status and future research. J Am Geriatr Soc. 2015;63(3):427–38. doi: 10.1111/jgs.13332 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref007] 7.Wilkinson HN, Hardman MJ. Senescence in wound repair: Emerging strategies to target chronic healing wounds. Frontiers in Cell and Developmental Biology. 2020;8:773. doi: 10.3389/fcell.2020.00773 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref008] 8.MacLeod AS, Kwock JT. Inflammation in wound repair: Role and function of inflammation in wound repair. Wound Healing 2018:177–94. [Google Scholar]

[pone.0302662.ref009] 9.O’Kane S, Ferguson MW. Transforming growth factor beta s and wound healing. Int J Biochem Cell Biol. 1997;29(1):63–78. doi: 10.1016/s1357-2725(96)00120-3 [DOI] [PubMed] [Google Scholar]

[pone.0302662.ref010] 10.Jude EB, Blakytny R, Bulmer J, Boulton AJ, Ferguson MW. Transforming growth factor-beta 1, 2, 3 and receptor type I and II in diabetic foot ulcers. Diabet Med. 2002;19(6):440–7. doi: 10.1046/j.1464-5491.2002.00692.x [DOI] [PubMed] [Google Scholar]

[pone.0302662.ref011] 11.Wang XJ, Han G, Owens P, Siddiqui Y, Li AG. Role of TGF beta-mediated inflammation in cutaneous wound healing. J Investig Dermatol Symp Proc. 2006;11(1):112–7. doi: 10.1038/sj.jidsymp.5650004 [DOI] [PubMed] [Google Scholar]

[pone.0302662.ref012] 12.Wei X, Li M, Zheng Z, Ma J, Gao Y, Chen L, et al. Senescence in chronic wounds and potential targeted therapies. Burns Trauma. 2022;10:tkab045. doi: 10.1093/burnst/tkab045 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref013] 13.Schultz GS, Sibbald RG, Falanga V, Ayello EA, Dowsett C, Harding K, et al. Wound bed preparation: a systematic approach to wound management. Wound Repair Regen. 2003;11 Suppl 1:S1–28. doi: 10.1046/j.1524-475x.11.s2.1.x [DOI] [PubMed] [Google Scholar]

[pone.0302662.ref014] 14.He S, Sharpless NE. Senescence in health and disease. Cell. 2017;169(6):1000–11. doi: 10.1016/j.cell.2017.05.015 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref015] 15.Wang Z, Shi C. Cellular senescence is a promising target for chronic wounds: A comprehensive review. Burns Trauma. 2020;8:tkaa021. doi: 10.1093/burnst/tkaa021 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref016] 16.Kumari R, Jat P. Mechanisms of cellular senescence: Cell cycle arrest and senescence associated secretory phenotype. Frontiers in Cell and Developmental Biology. 2021;9: 645593. doi: 10.3389/fcell.2021.645593 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref017] 17.Naktubtim C, Payuhakrit W, Uttarawichien T, Hassametto A, Suwannalert P. YAP, a novel target regulates F-actin rearrangement-associated CAFs transformation and promotes colorectal cancer cell progression. Biomed Pharmacother. 2022;155:113757. doi: 10.1016/j.biopha.2022.113757 [DOI] [PubMed] [Google Scholar]

[pone.0302662.ref018] 18.Aimvijarn P, Payuhakrit W, Charoenchon N, Okada S, Suwannalert P. Riceberry rice germination and UVB radiation enhance protocatechuic acid and vanillic acid to reduce cellular oxidative stress and suppress B16F10 melanogenesis relating to F-actin rearrangement. Plants. 2023;12(3):484. doi: 10.3390/plants12030484 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref019] 19.Uttarawichien T, Khumsri W, Suwannalert P, Sibmooh N, Payuhakrit W. Onion peel extract inhibits cancer cell growth and progression through the roles of L1CAM, NF-κB, and angiogenesis in HT-29 colorectal cancer cells. Prev Nutr Food Sci. 2021;26(3):330–7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref020] 20.Al Alawi AM, Majoni SW, Falhammar H. Magnesium and human health: Perspectives and research directions. Int J Endocrinol. 2018;2018:9041694. doi: 10.1155/2018/9041694 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref021] 21.Aburto NJ, Ziolkovska A, Hooper L, Elliott P, Cappuccio FP, Meerpohl JJ. Effect of lower sodium intake on health: systematic review and meta-analyses. BMJ: British Medical Journal. 2013;346:f1326. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref022] 22.Roohani N, Hurrell R, Kelishadi R, Schulin R. Zinc and its importance for human health: An integrative review. J Res Med Sci. 2013;18(2):144–57. [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref023] 23.Kanova M, Kohout P. Tryptophan: A unique role in the critically Ill. Int J Mol Sci. 2021;22(21):11714. doi: 10.3390/ijms222111714 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref024] 24.Kanno T, Gotoh A, Nishizaki T. Sphingosine arrests the cell cycle and induces apoptosis by targeting sphingosine-dependent protein kinase and protein kinase Cδ in vitro. Personalized Medicine Universe. 2014;3:22–7. [Google Scholar]

[pone.0302662.ref025] 25.Tovar R, Gavito AL, Vargas A, Soverchia L, Hernandez-Folgado L, Jagerovic N, et al. Palmitoleoylethanolamide is an efficient anti-obesity endogenous compound: Comparison with oleylethanolamide in diet-induced obesity. Nutrients. 2021;13(8):2589. doi: 10.3390/nu13082589 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref026] 26.Brown J, Karimian E, Ayala J. Oleoylethanolamide: A fat ally in the fight against obesity. Physiology & Behavior. 2017;176:50–8. doi: 10.1016/j.physbeh.2017.02.034 [DOI] [PubMed] [Google Scholar]

[pone.0302662.ref027] 27.Ho M, Anderson GH, Lin L, Bazinet RP, Kubant R. Ethanolamides of essential α-linolenic and linoleic fatty acids suppress short-term food intake in rats. Food Funct. 2020;11(4):3066–72. [DOI] [PubMed] [Google Scholar]

[pone.0302662.ref028] 28.Falanga V, Isseroff RR, Soulika AM, Romanelli M, Margolis D, Kapp S, et al. Chronic wounds. Nature Reviews Disease Primers. 2022;8(1):50. doi: 10.1038/s41572-022-00377-3 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref029] 29.Liarte S, Bernabé-García Á, Nicolás FJ. Role of TGF-β in skin chronic wounds: A keratinocyte perspective. Cells. 2020;9(2):306. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref030] 30.Finnson KW, McLean S, Di Guglielmo GM, Philip A. Dynamics of transforming growth factor beta signaling in wound healing and scarring. Adv Wound Care (New Rochelle). 2013;2(5):195–214. doi: 10.1089/wound.2013.0429 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref031] 31.Strong AL, Bowles AC, MacCrimmon CP, Lee SJ, Frazier TP, Katz AJ, et al. Characterization of a murine pressure ulcer model to assess efficacy of adipose-derived stromal cells. Plast Reconstr Surg Glob Open. 2015;3(3):e334. doi: 10.1097/GOX.0000000000000260 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref032] 32.Chong WC, Shastri MD, Eri R. Endoplasmic reticulum stress and oxidative stress: A vicious nexus implicated in bowel disease pathophysiology. Int J Mol Sci. 2017;18(4):771. doi: 10.3390/ijms18040771 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref033] 33.Richter K, Kietzmann T. Reactive oxygen species and fibrosis: further evidence of a significant liaison. Cell and Tissue Research. 2016;365(3):591–605. doi: 10.1007/s00441-016-2445-3 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref034] 34.Liu Z, Lv Y, Zhao N, Guan G, Wang J. Protein kinase R-like ER kinase and its role in endoplasmic reticulum stress-decided cell fate. Cell Death & Disease. 2015;6(7):e1822. doi: 10.1038/cddis.2015.183 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref035] 35.Sano R, Reed JC. ER stress-induced cell death mechanisms. Biochimica et Biophysica Acta (BBA)—Molecular Cell Research. 2013;1833(12):3460–70. doi: 10.1016/j.bbamcr.2013.06.028 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref036] 36.Liu G, Tao J, Lu J, Jia G, Zhao H, Chen X, et al. Dietary tryptophan supplementation improves antioxidant status and alleviates inflammation, endoplasmic reticulum stress, apoptosis, and pyroptosis in the intestine of piglets after lipopolysaccharide challenge. Antioxidants (Basel). 2022;11(5):872. doi: 10.3390/antiox11050872 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref037] 37.Wagner KD, Wagner N. The senescence markers p16INK4A, p14ARF/p19ARF, and p21 in organ development and homeostasis. Cells. 2022;11(12):1966. doi: 10.3390/cells11121966 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0302662.ref038] 38.Ishida T, Nishiumi S, Tanahashi T, Yamasaki A, Yamazaki A, Akashi T, et al. Linoleoyl ethanolamide reduces lipopolysaccharide-induced inflammation in macrophages and ameliorates 2,4-dinitrofluorobenzene-induced contact dermatitis in mice. Eur J Pharmacol. 2013;699(1–3):6–13. doi: 10.1016/j.ejphar.2012.11.030 [DOI] [PubMed] [Google Scholar]

[pone.0302662.ref039] 39.Treede I, Braun A, Sparla R, Kühnel M, Giese T, Turner JR, et al. Anti-inflammatory effects of phosphatidylcholine. J Biol Chem. 2007;282(37):27155–64. doi: 10.1074/jbc.M704408200 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Enhancing chronic wound healing with Thai indigenous rice variety, Kaab Dum: Exploring ER stress and senescence inhibition in HaCaT keratinocyte cell line

Witchuda Payuhakrit

Pimchanok Panpinyaporn

Wilunplus Khumsri

Gorrawit Yusakul

Ratsada Praphasawat

Nitra Nuengchamnong

Sarawoot Palipoch

Roles

Abstract

Introduction

Materials and methods

Rice extraction

Analysis of nutritional compositions of Kaab Dum rice extract

Analysis of chemical profiles of Kaab Dum rice extract

Cell culture experiments

Preparation of the extract

HaCaT keratinocyte culture

Assessment of cell viability using 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay

Detection of cellular oxidative stress using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining

Detection of ER stress using fluorescent technique

Wound healing assay

Detection of cell-cycle arrest using flow cytometry

Detection of cellular senescence using senescence-associated β-galactosidase (SA-β-gal) staining

Statistical analysis

Results

The nutritional compositions of Kaab Dum rice extracts

Table 1. The nutritional compositions of extracts from polished and unpolished Kaab Dum rice.

Chemical profiles of Kaab Dum rice extract

Fig 1.

Table 2. Chemical compositions of polished Kaab Dum rice extract.

Table 3. Chemical compositions of unpolished Kaab Dum rice extract.

Cytotoxicity of Kaab Dum rice extract on HaCaT cells

Fig 2. Cytotoxicity of Kaab Dum rice extracts on HaCaT cells using the MTT assay for 24 h.

Effect of Kaab Dum rice extract on oxidative stress in HaCaT cells

Fig 3. Effect of polished (PKD) and unpolished (UKD) Kaab Dum rice extracts on oxidative stress in TGF-ꞵ-induced HaCaT cells using the DCFH-DA method.

Inhibition of ER stress induction by Kaab Dum rice extract

Fig 4. Effect of polished (PKD) and unpolished (UKD) Kaab Dum rice extracts on ER stress inhibition in HaCaT cells induced as chronic wounds by TGF-ꞵ, observed under a fluorescence microscope at 40X magnification.

Effect of Kaab Dum rice extract on the migration (wound closure) of HaCaT cells induced as chronic wounds by TGF-β

Fig 5. Effect of polished (PKD) and unpolished (UKD) Kaab Dum rice extracts on the migration (wound closure) of TGF-β-induced HaCaT cells using a wound healing assay at 24 and 48 h.

Effect of Kaab Dum rice extract on the expression of p21 protein

Fig 6. Effect of polished (PKD) and unpolished (UKD) Kaab Dum rice extracts on p21 protein expression in TGF-ꞵ-induced HaCaT cells using flow cytometry at 24 h.

Effects of Kaab Dum rice extract on senescence of HaCaT cells induced as chronic wounds by TGF-ꞵ

Fig 7. Effects of polished (PKD) and unpolished UKD) Kaab Dum rice extracts on senescence of TGF-ꞵ-induced HaCaT cells using senescence-associated β-galactosidase (SA-β-gal) staining at 24.

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Gopinath Ponnusamy Manogaran

Roles

Author response to Decision Letter 0

Decision Letter 1

Gopinath Ponnusamy Manogaran

Roles

Author response to Decision Letter 1

Decision Letter 2

Gopinath Ponnusamy Manogaran

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases