Purslane (Portulaca oleracea L.) Extract Attenuates Obesity-Induced Inflammation and Enhances Mitochondrial Biogenesis via the miR-221/222 and AMPK/SIRT1 Axis in Rats

Abstract

Purslane (Portulaca oleracea L.) exhibits anti-inflammatory and antioxidant properties, but its role in regulating adipose tissue inflammatory microRNAs and mitochondrial biogenesis in skeletal muscle under high-fat diet (HFD) conditions remains unclear. This study aimed to evaluate the effects of purslane extract (PE) on HFD-induced inflammation and mitochondrial biogenesis, focusing on regulation of the miR-221/222 cluster in white adipose tissue (WAT) and the AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) axis in skeletal muscle. Male Sprague-Dawley rats were divided into four groups: normal chow diet, HFD, HFD+0.2% PE, and HFD+0.4% PE for 12 weeks. PE supplementation significantly reduced body weight gain, improved serum and hepatic lipid profiles, and decreased circulating inflammatory cytokines (P<0.05). In WAT, PE reduced expression of pro-inflammatory cytokines and M1 macrophage markers while increasing expression of a M2 marker. Additionally, PE inhibited phospho-nuclear factor-kappa B levels and suppressed miR-221/222 expression in WAT. In skeletal muscle, PE enhanced the expression of genes related to mitochondrial biogenesis and restored mitochondrial content. Furthermore, PE increased AMPK, total SIRT, and SIRT1 activities, supporting its role in improving mitochondrial function in skeletal muscle. These findings suggest that the beneficial effects of PE against HFD-induced obesity are mediated, at least in part, by suppression of the WAT miR-221/222 cluster and activation of the AMPK/SIRT1 axis in skeletal muscle, thereby alleviating inflammation and enhancing mitochondrial biogenesis.

INTRODUCTION

Obesity is characterized by excessive fat accumulation in white adipose tissue (WAT), resulting from an energy imbalance, and can trigger chronic, low-grade inflammation and metabolic dysregulation, contributing to increased risks of type 2 diabetes, cardiovascular disease, and metabolic dysfunction-associated steatotic liver disease (Saltiel and Olefsky, 2017). Hypertrophic adipocytes release various pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and monocyte chemoattractant protein 1 (MCP-1), which act to disrupt endocrine function and recruit macrophages into WAT (Kang et al., 2016; Kawai et al., 2021). The transcription factor nuclear factor-kappa B (NF-κB), a central regulator of inflammation, plays a critical role in obesity-induced inflammatory signaling pathways (Baker et al., 2011). Activated NF-κB regulates production of pro-inflammatory cytokines, with upregulated expression of the M1 macrophage markers cluster of differentiation (CD) 68, inducible nitric oxide synthase 2 (NOS2), and CD11c (Cinti et al., 2005; Zeyda et al., 2010), but downregulated expression of the anti-inflammatory cytokine arginase 1 (Arg1), which collectively promote macrophage polarization from the anti-inflammatory M2 phenotype to the pro-inflammatory M1 phenotype (Baker et al., 2011; Cha et al., 2018). MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level and have emerged as key modulators of inflammatory responses and metabolic regulation (Landrier et al., 2019; Yamaguchi et al., 2022; Gentile et al., 2023). In obesity, miR-221 and miR-222 are upregulated and contribute to inflammation of adipose tissue and insulin resistance (Yamaguchi et al., 2022; Gentile et al., 2023).

Mitochondria play a central role in energy metabolism, and impaired mitochondrial function leads to reduced mitochondrial biogenesis and mitochondrial DNA (mtDNA) content (de Mello et al., 2018). Bonnard et al. (2008) reported that the number of mitochondria and content of mtDNA were significantly reduced in the skeletal muscle of obese mice. AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) are key regulators of mitochondrial homeostasis, where AMPK increases the intracellular NAD⁺/NADH ratio, thereby enhancing the activity of SIRT1, which subsequently deacetylates and activates peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), a master regulator of mitochondrial biogenesis (Cantó and Auwerx, 2009). Activated PGC-1α induces nuclear respiratory factor 1 (NRF1), which in turn upregulates mitochondrial transcription factor A (TFAM), leading to increased mtDNA content and enhanced mitochondrial biogenesis (Chen et al., 2022). Additionally, peroxisome proliferator-activated receptor delta (PPARδ), a ligand-activated transcription factor, promotes AMPK signaling and upregulates carnitine palmitoyl transferase-1B (CPT-1B), a key enzyme involved in mitochondrial fatty acid oxidation (Aguilar-Recarte et al., 2021). Therefore, targeting the AMPK/SIRT1 pathway to regulate mitochondrial function may serve as a promising strategy to improve metabolic health.

Purslane (Portulaca oleracea L.), a member of the Portulacaceae family, is widely distributed in tropical and subtropical regions (Rahimi et al., 2019), and has long been used as a medicinal herb owing to the contents of various bioactive compounds, including polyphenols, omega-3 fatty acids, minerals, and vitamins (Fernández-Poyatos et al., 2021). Moreover, purslane has been reported to exhibit anti-inflammatory, antioxidant, antidiabetic, and anti-obesity effects (Rahimi et al., 2019; Jung et al., 2021a). However, it remains unclear whether purslane extract (PE) influences the inflammatory miR-221/222 cluster and mitochondrial biogenesis under high-fat diet (HFD)-induced conditions. Therefore, this study aimed to investigate the effects of PE on obesity-associated inflammation and mitochondrial biogenesis in HFD-fed rats, focusing on the regulation of miR-221/222 in WAT and the AMPK/SIRT1 axis in skeletal muscle.

MATERIALS AND METHODS

Preparation of PE

PE was kindly provided by EastHill and prepared as previously described (Jang et al., 2022). In brief, dried purslane was pulverized, extruded with a twin-screw device, and extracted with water. The resulting supernatant was concentrated by vacuum evaporation and subsequently spray-dried into powder. The total phenolic content of the PE was 5.43 mg/g, with chlorogenic acid (2.52 mg/g), rutin (1.37 mg/g), gallic acid (1.13 mg/g), and ferulic acid (0.41 mg/g) identified as the predominant phenolic compounds (Jang et al., 2022).

Animals and diets

The study protocol was approved by the Institutional Animal Care and Use Committee of Ewha Womans University (approval no. 21-002-t) and conducted in accordance with the “Guide for the Care and Use of Laboratory Animals.” Male Sprague-Dawley rats (n=36; age, 4 weeks; body weight, 60±5 g) were purchased from Doo-Yeol Biotech and housed in individual cages under a 12-h light/dark cycle at a constant temperature of 22±2°C and relative humidity of 55±5%. After 1 week of acclimatization to a normal chow diet (NCD; 2018S Teklad rodent diet; Envigo RMS, LLC), the rats were randomly divided into four groups (n=9 per group): control (NCD group), 45% HFD (HFD group), HFD supplemented with 0.2% PE (PEL group), and HFD supplemented with 0.4% PE (PEH group). The experimental diets were modified based on the American Institute of Nutrition-93G formulation (Reeves et al., 1993). The compositions of the diets are detailed in Supplementary Table 1. The rats were fed the experimental diets ad libitum for 12 weeks. Body weight and food intake were recorded twice per week. At the end of the feeding period, the rats were anesthetized with a 5:2 (v/v) mixture of Zoletil 50 (Virbac Laboratories) and Rompun (Bayer Korea) before sacrifice. Serum samples were collected and stored at −40°C, while liver, WAT, and skeletal muscle were rapidly excised and stored at −70°C until analysis.

Serum metabolite analysis

Serum triglycerides (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were measured using commercial kits (Asan Pharmaceutical Co., Ltd.). Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formula (Friedewald et al., 1972). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using kits from Embiel Co., Ltd. Nitric oxide (NO) levels were measured with the Griess Reagent Kit (Invitrogen Corp.) using a microplate reader (Varioskan Flash; Thermo Fisher Scientific) at 548 nm.

Hepatic lipid analysis

Hepatic lipids were extracted using the Bligh and Dyer method (Bligh and Dyer, 1959) with some modifications. Liver samples (0.1 g) were homogenized with a mixture of chloroform and methanol, then vortexed and filtered through Whatman no. 6 filter paper (Whatman International Ltd.). Filtrates were evaporated and reconstituted in a mixture of n-hexane and isopropanol (3:2, v/v), then stored at −40°C. Hepatic TG and TC levels were quantified using commercial kits (Asan Pharmaceutical Co., Ltd.).

Histological examination

Epididymal WAT (eWAT) was fixed in 10% formalin overnight, embedded in paraffin, and cut into slices, which were incubated at 40°C, stained with hematoxylin and eosin (H&E) (Jung et al., 2021b), and imaged under a light microscope (Olympus Corp.) at 200× magnification. Adipocyte size was measured using ImageJ software (https://imagej.net/ij/).

Immunohistochemical (IHC) staining

CD68 protein expression in eWAT sections was detected using an antibody against CD68 (cat. no. GTX101895; Santa Cruz Biotechnology) and the Polink-2 HRP Anti-rat DAB Detection Kit (OriGene Technologies, Inc.). The eWAT sections were stained with diaminobenzidine, counterstained with Mayer’s hematoxylin (ScyTek Laboratories, Inc.), and imaged at 200× magnification under a light microscope.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA from eWAT and muscle was extracted using TRIzol reagent (GeneAll Biotechnology Co., Ltd.) and reverse-transcribed into complementary DNA (cDNA) using Moloney Murine Leukemia Virus Reverse Transcriptase (Bioneer Corp.) for mRNA or the miR cDNA Synthesis Kit (Applied Biological Materials Inc.) for miRNA. The qRT-PCR assay was performed using GreenStar qPCR Master Mix (Bioneer Corp.) with a Rotor-Gene Q thermal cycler (Qiagen). The primer sequences for detection of the target genes are presented in Supplementary Table 2. Specific primers for miR-221, miR-222, and RNU6 were purchased from Applied Biological Materials Inc. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and RNU6 were used as internal controls for mRNA and miRNA normalization, respectively. Relative gene expression levels were calculated using the 2^−ΔΔCt method (Livak and Schmittgen, 2001).

Enzyme-linked immunosorbent assay (ELISA)

Serum concentrations of TNF-α and IL-6 were measured using ELISA kits (BioLegend), and MCP-1 using a kit from LABISKOMA. NF-κB p65 phosphorylation was measured using the phospho-NF-κB p65 ELISA Kit (RayBiotech) after nuclear extraction with the Nuclear Extraction Kit (Abcam). All values were normalized to total protein concentrations measured using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific).

Transmission electron microscopy (TEM)

Muscle tissues were fixed in 2% glutaraldehyde and 1% osmium tetroxide in phosphate buffer, dehydrated, embedded, and cut into sections (thickness, 60－70 nm), which were examined under a transmission electron microscope (H-7650; Hitachi, Ltd.) at 80 kV and 20,000× magnification.

mtDNA contents

Total DNA was isolated from muscle using the Puregene DNA Isolation Kit (Qiagen). The relative mtDNA content was determined by qRT-PCR, by assessing the mitochondrial gene encoding Cox1 (cytochrome c oxidase subunit 1) and using Gapdh as a nuclear reference gene (Supplementary Table 2).

AMPK and SIRT activities assay

AMPK activity was measured using the AMPK Kinase Assay Kit (MBL Life Science). SIRT1 activity was analyzed using a fluorometric assay kit (Abcam). Total SIRT activity was also measured using a kit from Abcam. Fluorescence for SIRT1 activity was detected at 340/460 nm, and absorbance for AMPK and total SIRT activities was measured at 450 nm using a microplate reader (Thermo Fisher Scientific). All enzyme activities were normalized to protein concentrations determined by the BCA assay and expressed as fold change relative to the HFD group.

Statistical analysis

All data are expressed as the mean±standard error of the mean of nine rats. Analyses were performed with IBM SPSS Statistics for Windows (version 25.0; IBM Corp.). Differences between the NCD and HFD groups were determined with the one-tailed Student’s t-test. Differences among the HFD-based groups (HFD, PEL, and PEH) were assessed using one-way analysis of variance, followed by Tukey’s post hoc test. A P-value<0.05 was considered statistically significant.

RESULTS

PE reduced body weight gain and adiposity

After the 12-week feeding period, the final body weight and total weight gain were both significantly increased in the HFD group as compared to the NCD group (Table 1). At the end of the study, the final body weight and total weight gain were reduced by 6.9% and 7.7% in the PEH group as compared to the HFD group (P<0.05; Table 1). There were no significant differences in food or energy intake among the HFD, PEL, and PEH groups (Table 1), indicating that the observed weight reduction was not attributable to decreased caloric intake. Histological analysis of eWAT using H&E staining revealed that adipocyte size was markedly increased in the HFD group relative to the NCD group. This hypertrophy was significantly attenuated in both PE-treated groups, with adipocyte size in the PEL and PEH groups reduced by 42.0% and 46.5%, respectively, as compared to the HFD group (P<0.05; Fig. 1A and 1B). Furthermore, the total WAT mass, which includes eWAT, mesenteric WAT, and retroperitoneal WAT, was also significantly reduced in the PEL and PEH groups as compared to the HFD group (P<0.05; Fig. 1C).

Table 1.

Effects of PE on physiological variables of rats fed a HFD for 12 weeks

Variable	NCD	HFD	PEL	PEH
Initial body weight (g)	75.08±1.13	74.42±1.20	73.79±0.85	73.20±0.87
Final body weight (g)	461.94±11.59***	537.22±9.27a	521.24±10.35ab	500.27±7.56b
Body weight gain (g)	386.86±11.33***	462.80±8.59a	447.45±10.26ab	427.08±7.81b
Food intake (g/d)	24.75±0.58***	19.95±0.51	20.35±0.63	20.51±0.63
Energy intake (kcal/d)	76.73±1.81***	91.77±2.37	93.59±2.90	94.36±2.91

Values are presented as the mean±standard error of the mean (n=9).

***P<0.001 indicate significant differences between the NCD and HFD groups. Different letters (a,b) within a row indicate significant differences (P<0.05) among the three groups (HFD, PEL, and PEH).

PE, purslane extract; HFD, high-fat diet; NCD, normal chow diet; PEL, HFD supplemented with 0.2% PE; PEH, HFD supplemented with 0.4% PE.

Fig. 1.

Effects of purslane extract (PE) on high-fat diet (HFD)-induced adiposity. (A) Representative images of H&E-stained sections of eWAT (200× magnification; scale bar=100 μm). (B) Average adipocyte size. (C) Relative weight of adipose tissue. Values are presented as the mean±standard error of the mean (n=9). *P<0.05, **P<0.01, and ***P<0.001 indicate significant differences between the NCD and HFD groups. Different letters (a,b) indicate significant differences (P<0.05) among the three groups (HFD, PEL, and PEH). NCD, normal chow diet; PEL, HFD supplemented with 0.2% PE; PEH, HFD supplemented with 0.4% PE; H&E, hematoxylin and eosin; eWAT, epididymal white adipose tissue; mWAT, mesenteric white adipose tissue; rWAT, retroperitoneal white adipose tissue; tWAT, total white adipose tissue.

PE improved hepatic and serum lipid profiles and reduced serum inflammatory cytokine levels

The liver and serum metabolic parameters are presented in Table 2. Hepatic lipid accumulation was markedly improved following PE supplementation. In the PEH group, total lipid, TG, and TC levels in the liver were reduced by 9.7%, 45.5%, and 59.9%, respectively, relative to the HFD group. Serum TG levels were 28.1% lower in the PEH group than the HFD group, whereas there were no significant differences observed in serum TC levels among the experimental groups. Serum LDL-C levels were reduced by 45.6% and 36.5% in the PEL and PEH groups, respectively, as compared to the HFD group. Conversely, HDL-C levels were increased by 1.36-fold and 1.23-fold in the PEL and PEH groups, respectively. Meanwhile, there were no significant differences in serum AST and ALT levels among the groups, suggesting that PE supplementation did not induce hepatocellular toxicity. PE supplementation also attenuated systemic inflammation. Serum TNF-α levels were reduced by 13.0% and 15.7% in the PEL and PEH groups, respectively, relative to the HFD group. Serum IL-6 and MCP-1 levels in the PEH group were decreased by 44.5% and 30.0%, respectively, as compared to the HFD group. Additionally, serum NO levels were 23.6% lower in the PEH group than the HFD group.

Table 2.

Effects of PE on serum and hepatic metabolic parameters of rats fed a HFD for 12 weeks

Parameter	NCD	HFD	PEL	PEH
Liver
Total lipids (mg/g)	32.4±0.76***	37.6±0.73a	37.3±0.93a	33.9±0.34b
TG (μmol/g)	1.12±0.27**	2.51±0.34a	2.45±0.19a	1.37±0.10b
TC (μmol/g)	0.93±0.22***	1.80±0.22a	1.38±0.20ab	0.72±0.15b
Serum
TG (mmol/L)	0.53±0.06	0.54±0.05a	0.42±0.02ab	0.38±0.03b
TC (mmol/L)	2.29±0.07**	2.06±0.05	1.96±0.08	1.98±0.04
HDL-C (mmol/L)	1.14±0.07***	0.94±0.03b	1.28±0.07a	1.16±0.06a
LDL-C (mmol/L)	0.87±0.04	0.90±0.04a	0.49±0.07b	0.57±0.04b
AST (IU/L)	50.1±6.86	51.7±6.75	47.3±5.38	48.2±5.67
ALT (IU/L)	1.75±0.55	2.47±0.39	2.25±0.30	2.70±0.51
TNF-α (pg/mL)	5.25±0.22*	6.23±0.21a	5.42±0.22b	5.25±0.34b
IL-6 (pg/mL)	32.3±2.60	34.2±4.45a	25.4±4.70ab	19.0±1.00b
MCP-1 (ng/mL)	7.85±0.52	8.05±0.66a	7.39±0.32ab	5.63±0.54b
NO (μmol/L)	31.5±2.29	34.8±3.12a	27.3±1.91ab	26.6±0.85b

Values are presented as the mean±standard error of the mean (n=9).

*P<0.05, **P<0.01, and ***P<0.001 indicate significant differences between the NCD and HFD groups. Different letters (a,b) within a row indicate significant differences (P<0.05) among the three groups (HFD, PEL, and PEH).

PE, purslane extract; HFD, high-fat diet; NCD, normal chow diet; PEL, HFD supplemented with 0.2% PE; PEH, HFD supplemented with 0.4% PE; TG, triglyceride; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; AST, aspartate aminotransferase; ALT, alanine aminotransferase; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; NO, nitric oxide.

PE attenuated HFD-induced inflammation and downregulated miR-221/222 in visceral WAT

To assess the anti-inflammatory effects of PE, we evaluated inflammatory cytokines and miR-221/222 expression in eWAT following HFD exposure. The mRNA and protein levels of the pro-inflammatory cytokines TNF-α, IL-6, and MCP-1 were decreased in the PE-fed groups as compared to the HFD group (Fig. 2A and 2B). IHC analysis of CD68 revealed that the number of crown-like structures (CLS) in eWAT was 2.80-fold higher in the HFD group than the NCD group, while PE supplementation markedly decreased CLS formation by 63.5% (PEL) and 72.1% (PEH), respectively (Fig. 2C and 2D). In line with these findings, expression levels of the M1 macrophage markers Cd68, Nos2, and CD11c were downregulated, while expression of the anti-inflammatory M2 marker Arg1 was upregulated in the PE-fed groups (Fig. 2E). Moreover, expression of phospho-NF-κB was elevated by 1.46-fold in the HFD group relative to the NCD group but was suppressed by 36.0% in the PEH group (Fig. 2F). The expression levels of miR-221 and miR-222 were upregulated by 2.70- and 3.17-fold, respectively, in the HFD group as compared to the NCD group, while PE intake downregulated miR-221 levels by 63.8% and 60.8% in the PEL and PEH groups, respectively, and miR-222 levels by 50.2% and 39.7% (Fig. 2G and 2H).

Fig. 2.

Effects of purslane extract (PE) on high-fat diet (HFD)-induced inflammation in visceral WAT. mRNA (A) and protein (B) levels of TNF-α, IL-6, and MCP-1. (C) Representative images of IHC staining for CD68 (arrows indicate CLS; 200× magnification; scale bars=100 μm). (D) The abundance of dying adipocytes marked by CLS in eWAT sections is expressed as the percentage of all adipocytes. (E) mRNA levels of M1/M2 macrophage markers. (F) Level of phosphorylated NF-κB. (G and H) miR-221/222 expression. Values are presented as the mean±standard error of the mean (n=9). *P<0.05, **P<0.01, and ***P<0.001 indicate significant differences between the NCD and HFD groups. Different letters (a,b) indicate significant differences (P<0.05) among the three groups (HFD, PEL, and PEH). NCD, normal chow diet; PEL, HFD supplemented with 0.2% PE; PEH, HFD supplemented with 0.4% PE; WAT, white adipose tissue; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin 6; MCP-1, monocyte chemoattractant protein 1; IHC, immunohistochemistry; CLS, crown-like structures; Cd68, cluster of differentiation 68; Nos2, nitric oxide synthase 2; Cd11c, cluster of differentiation 11c; Arg1, arginase 1; miR, microRNA; NF-κB, nuclear factor-kappa B.

PE enhanced mitochondrial biogenesis and activated the AMPK/SIRT1 pathway in skeletal muscle

To further elucidate the metabolic effects of PE, impacts on mitochondrial biogenesis and activation of the AMPK/SIRT1 pathway in skeletal muscle were examined. The mRNA expression levels of Nrf1, Tfam, and Pgc-1α, key regulators of mitochondrial biogenesis, were significantly upregulated by 1.94-, 1.69-, and 2.14-fold, respectively, in the PEH group as compared to the HFD group (P<0.05; Fig. 3A). Similarly, the expression levels of Pparδ, Cpt-1B, and Sirt1, which are associated with mitochondrial fatty acid oxidation and function, were elevated by 2.31-, 1.69-, and 1.92-fold, respectively (Fig. 3A). The mtDNA content, which was reduced by 41.6% in the HFD group as compared to the NCD group, was significantly increased by 1.31- and 1.45-fold in the PEL and PEH groups, respectively, as compared to the HFD group (P<0.05; Fig. 3B). Consistently, TEM analysis revealed that the number and size of mitochondria, which were reduced in the HFD group, were notably restored in the PE-fed groups (Fig. 3C). AMPK, total SIRT, and SIRT1 activities were markedly suppressed in the HFD group by 50.9%, 11.2%, and 52.8%, respectively, but were increased in the PE-fed groups. Specifically, AMPK activity was elevated by 1.58- and 1.98-fold in the PEL and PEH groups, while total SIRT and SIRT1 activities were increased by 2.09- and 1.18-fold, respectively, in the PEH group relative to the HFD group (P<0.05; Fig. 3D－3F).

Fig. 3.

Effects of purslane extract (PE) on mitochondria biogenesis and AMP-activated protein kinase/sirtuin 1 (AMPK/SIRT1) activities in skeletal muscle. (A) mRNA expression of genes involved in mitochondrial function. (B) mtDNA contents AMPK. (C) Representative TEM images [red arrows indicate the position of mitochondria (M); 20,000× magnification; scale bars=2 μm]. (D), total SIRT (E), and SIRT1 (F) activities. Values are presented as the mean±standard error of the mean (n=9). *P<0.05, **P<0.01, and ***P<0.001 indicate significant differences between the NCD and HFD groups. Different letters (a,b) indicate significant differences (P<0.05) among the three groups (HFD, PEL, and PEH). NCD, normal chow diet; HFD, high-fat diet; PEL, HFD supplemented with 0.2% PE; PEH, HFD supplemented with 0.4% PE; Pparδ, peroxisome proliferator-activated receptor delta; Nrf1, nuclear respiratory factor 1; Tfam, mitochondrial transcription factor A; Pgc-1α, peroxisome proliferator-activated receptor gamma coactivator-1 alpha; Sirt1, sirtuin 1; Cpt-1B, carnitine palmitoyl transferase-1B; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; TEM, transmission electron microscopy; SIRT, sirtuin.

DISCUSSION

Obesity is accompanied by an inflammatory response and causes mitochondrial biogenesis and dysfunction, which are characterized by a reduction in the number and function of mitochondria in patients and animals with obesity (Jheng et al., 2012; Nammi et al., 2004). In this study, we investigated the effects of PE on obesity-induced inflammation and muscle mitochondrial biogenesis in HFD-fed rats. Furthermore, we explored whether PE regulates WAT miR-221/222 and the AMPK/SIRT1 axis in muscle. The total phenolic compound content of PE, including chlorogenic acid, rutin, gallic acid, and ferulic acid, was 5.43 mg/g. Based on previous reports, the dietary levels of PE used in this study (0.2% and 0.4%) correspond to effective oral doses (150 and 300 mg/kg BW/d) shown to reduce body weight and improve lipid profiles without hepatotoxicity in rats (Abdalla, 2010; Won and Kim, 2011; Chen et al., 2012). In addition, high dietary supplementation of P. oleracea powder (5%－10%) has been reported to improve obesity-related metabolic parameters without liver toxicity in mice fed a HFD (Jung et al., 2021a). These findings support that the 0.2% and 0.4% PE levels used in this study are within a safe and biologically active range capable of exerting beneficial metabolic effects. PE supplementation for 12 weeks reduced body weight gain and WAT weight without altering food and energy intake among the experimental groups. PE improved serum and hepatic lipid levels of HFD-fed rats. A previous study reported that P. oleracea L. powder reduced weight gain and WAT mass in HFD-induced obese mice (Jung et al., 2021a). Jang et al. (2022) showed that PE improved serum and hepatic lipid levels in rats fed a high-cholesterol diet. In addition, chlorogenic acid, one of the constituents of PE, reduced weight gain and WAT mass, while improving plasma lipid profiles, in HFD-fed mice (Wang et al., 2019). Our results indicate that PE inhibits weight gain and improves serum and liver lipid profiles, suggesting potential beneficial effects against obesity.

NF-κB is a key transcriptional factor that upregulates genes encoding pro-inflammatory cytokines and promotes M1 macrophage polarization (Baker et al., 2011). TNF-α and IL-6 are representative pro-inflammatory cytokines, while MCP-1 acts as a chemotactic factor that triggers macrophage recruitment to adipose tissue (Samarghandian et al., 2017; Miao et al., 2019). In the present study, PE administration effectively reduced the circulating levels of these inflammatory cytokines, particularly in the PEH group, suggesting alleviation of systemic inflammation. CLS, defined as clusters of macrophages surrounding dead or dying adipocytes, serve as markers of chronic inflammation and can be identified by CD68 expression (Cinti et al., 2005; Chang et al., 2021a). In obesity, increased M1 macrophage polarization in adipose tissue enhances local inflammatory responses (Lumeng et al., 2007). M1 macrophages express the inflammatory markers CD68, CD11c, and NOS2 (Martinez and Gordon, 2014), the latter being responsible for NO production (Förstermann and Sessa, 2012). In contrast, Arg1, secreted by M2 macrophages, exerts anti-inflammatory effects by counteracting NOS2 activity (Brigo et al., 2021). Our findings demonstrate that PE administration reduced WAT mass and adipocyte hypertrophy, accompanied by diminished macrophage infiltration and CLS formation. These histological changes were supported by molecular evidence showing downregulation of Tnf-α, Il-6, and Mcp-1 at both the mRNA and protein levels in WAT. Moreover, PE suppressed expression of the M1 macrophage markers Cd68, CD11c, and Nos2, while upregulating expression of Arg1, indicating a shift toward the anti-inflammatory M2 phenotype. Serum NO levels were also reduced, and phospho-NF-κB levels in adipose tissue were inhibited, further supporting the anti-inflammatory action of PE. These findings are consistent with those of previous studies (Chang et al., 2020; Jung et al., 2021a; Wang et al., 2023). For example, Jung et al. (2021a) reported that administration of P. oleracea L. powder alleviated WAT weight in HFD-fed mice, while Wang et al. (2023) found that polysaccharides from P. oleracea L. suppressed Tnf-α and Il-6 mRNA expression in diabetic mice, and Chang et al. (2020) demonstrated that P. oleracea L. oligopeptides inhibited NO and pro-inflammatory cytokine production as well as NF-κB activity in RAW264.7 cells. Collectively, our results suggest that PE alleviates obesity-induced inflammation by reducing pro-inflammatory cytokines and mediators, suppressing macrophage infiltration, and promoting a shift from M1 to M2 macrophage polarization in adipose tissue.

miRs are small non-coding RNAs that regulate gene expression at the post-transcriptional level and play pivotal roles in both metabolic and inflammatory processes (Ambros, 2004). Among them, the miR-221/222 cluster has been implicated in cancer progression and inflammatory disease development (Song et al., 2017). Previous studies have shown that overexpression of miR-221/222 in human aortic endothelial cells promotes excessive production of reactive oxygen species, whereas inhibition has an opposite effect via modulation of PGC-1α (Xue et al., 2015). Additionally, suppression of miR-221 in fibroblast-like synoviocytes is reported to attenuate expression of pro-inflammatory cytokines (Yang and Yang, 2015). Notably, NF-κB has been shown to induce expression of miR-221 and miR-222, while inhibition of miR-221 suppresses NO synthesis and activates NF-κB signaling (Galardi et al., 2011; Chen et al., 2015). More recently, Yamaguchi et al. (2022) demonstrated that adipocyte-specific inhibition of miR-221/222 ameliorated diet-induced obesity, highlighting potential roles in adipose tissue inflammation and metabolic dysfunction. However, the regulatory effect of PE on miR-221/222 expression in WAT under HFD conditions remains poorly understood. In the present study, PE administration downregulated expression of miR-221 and miR-222 in WAT of HFD-fed rats, suggesting that the anti-inflammatory effects of PE may, at least in part, be mediated through suppression of the miR-221/222 cluster, providing new insights into the molecular mechanisms underlying the therapeutic potential PE against metabolic disorders.

Mitochondria play a central role in maintaining energy homeostasis and are essential for lipid metabolism (Bournat and Brown, 2010). In obesity, mitochondrial dysfunction is commonly observed, often accompanied by a reduction in mtDNA content and impaired mitochondrial biogenesis (Todosenko et al., 2023). These alterations contribute to diminished activity of key metabolic regulators and enzymes in skeletal muscle (Bournat and Brown, 2010). AMPK serves as a crucial energy sensor that becomes activated in response to an increase in the AMP/ATP ratio, which subsequently promotes fatty acid oxidation and activates PGC-1α (Garcia and Shaw, 2017). SIRT1 further enhances mitochondrial metabolism by deacetylating and activating PGC-1α (Nemoto et al., 2005). Activated PGC-1α, in turn, stimulates expression of NRF1 and Tfam, which are both essential for mitochondrial biogenesis and function (Austin and St-Pierre, 2012; Chen et al., 2022). Additionally, PGC-1α plays a role in regulating the production of reactive oxygen species to minimize oxidative stress. CPT-1B, located on the outer mitochondrial membrane, catalyzes the rate-limiting step of fatty acid oxidation (van Weeghel et al., 2018), and its activity is enhanced by AMPK-mediated activation of PPARδ in skeletal muscle (Aguilar-Recarte et al., 2021). Previous studies have shown that bioactive compounds in PE, such as chlorogenic acid, rutin, and gallic acid, can regulate genes involved in mitochondrial metabolism (Seo et al., 2015; Zhou et al., 2016; Chang et al., 2021b). Chlorogenic acid is reported to stimulate AMPK phosphorylation and upregulate Pgc-1α, Nrf1, and Tfam expression in the liver (Zhou et al., 2016). Similarly, rutin was demonstrated to increase mtDNA content, AMPK activity, and mRNA expression of Pgc-1α, Nrf1, Tfam, and Sirt1 in skeletal muscle of HFD-fed rats (Seo et al., 2015), while gallic acid and rutin were shown to enhance mitochondrial gene expression in C2C12 myotubes (Chang et al., 2021b). In the present study, PE administration upregulated expression of Pgc-1α, Nrf1, Tfam, Sirt1, Pparδ, and Cpt-1B, increased AMPK and SIRT1 activities, and elevated mtDNA content in the skeletal muscle of HFD-fed rats. Here, we report for the first time that PE activated the AMPK/SIRT1 axis, thereby enhancing mitochondrial biogenesis and fatty acid oxidation in skeletal muscle, ultimately contributing to improved mitochondrial function in diet-induced obesity.

In conclusion, this study demonstrates that PE mitigated HFD-induced obesity and inflammation, partly through inhibition of the miR-221/222 cluster and NF-κB pathway in WAT. Furthermore, PE promoted mitochondrial biogenesis in skeletal muscle via activation of the AMPK/SIRT1 axis, indicating potential as a functional ingredient for metabolic health (Fig. 4). However, to confirm translational applicability, further studies are warranted to identify the bioactive components of PE and elucidate the underlying mechanisms in greater detail. Although beneficial effects were observed in a rodent model, the human equivalent dose, long-term safety, and clinical relevance of these findings remain to be established. While serum liver enzyme levels were not elevated in this study, a comprehensive toxicological evaluation is necessary to verify the safety profile of PE at the tested doses.

Fig. 4.

A schematic diagram illustrating the potential regulatory mechanisms of purslane extract (PE) on adipose tissue inflammation and muscle mitochondrial biogenesis in high-fat diet (HFD)-fed rats. WAT, white adipose tissue; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin 6; MCP-1, monocyte chemoattractant protein 1; NF-κB, nuclear factor-kappa B; Cd, cluster of differentiation; Nos2, nitric oxide synthase 2; Arg1, arginase 1; miR, microRNA; AMPK, AMP-activated protein kinase; SIRT1, sirtuin 1; Pgc-1α, peroxisome proliferator-activated receptor gamma coactivator-1 alpha; Pparδ, peroxisome proliferator-activated receptor delta; Nrf1, nuclear respiratory factor 1; Cpt-1B, carnitine palmitoyl transferase-1B; Tfam, mitochondrial transcription factor A; mtDNA, mitochondrial DNA.

SUPPLEMENTARY MATERIALS

Supplementary materials can be found via https://doi.org/10.3746/pnf.2025.231