MiR-21 mediates the protection of kaempferol against hypoxia/reoxygenation-induced cardiomyocyte injury via promoting Notch1/PTEN/AKT signaling pathway

Jinxi Huang; Zhenhui Qi

doi:10.1371/journal.pone.0241007

. 2020 Nov 5;15(11):e0241007. doi: 10.1371/journal.pone.0241007

MiR-21 mediates the protection of kaempferol against hypoxia/reoxygenation-induced cardiomyocyte injury via promoting Notch1/PTEN/AKT signaling pathway

Jinxi Huang ^1,^*, Zhenhui Qi ¹

Editor: Meijing Wang²

PMCID: PMC7644004 PMID: 33151961

Abstract

Kaempferol, a natural flavonoid compound, possesses potent myocardial protective property in ischemia/reperfusion (I/R), but the underlying mechanism is not well understood. The present study was aimed to explore whether miR-21 contributes to the cardioprotective effect of kaempferol on hypoxia/reoxygenation (H/R)-induced H9c2 cell injury via regulating Notch/phosphatase and tensin homologue (PTEN)/Akt signaling pathway. Results revealed that kaempferol obviously attenuates H/R-induced the damages of H9c2 cells as evidence by the up-regulation of cell viability, the down-regulation of lactate dehydrogenase (LDH) activity, the reduction of apoptosis rate and pro-apoptotic protein (Bax) expression, and the increases of anti-apoptotic protein (Bcl-2) expression. In addition, kaempferol enhanced miR-21 level in H9c2 cells exposed to H/R, and inhibition of miR-21 induced by transfection with miR-21 inhibitor significantly blocked the protection of kaempferol against H/R-induced H9c2 cell injury. Furthermore, kaempferol eliminated H/R-induced oxidative stress and inflammatory response as illustrated by the decreases in reactive oxygen species generation and malondialdehyde content, the increases in antioxidant enzyme superoxide dismutase and glutathione peroxidase activities, the decreases in pro-inflammatory cytokines interleukin (IL)-1β, IL-8 and tumor necrosis factor-alpha levels, and an increase in anti-inflammatory cytokine IL-10 level, while these effects of kaempferol were all reversed by miR-21 inhibitor. Moreover, results elicited that kaempferol remarkably blocks H/R-induced the down-regulation of Notch1 expression, the up-regulation of PTEN expression, and the reduction of P-Akt/Akt, indicating that kaempferol promotes Notch1/PTEN/AKT signaling pathway, and knockdown of Notch1/PTEN/AKT signaling pathway induced by Notch1 siRNA also abolished the protection of kaempferol against H/R-induced the damage of H9c2 cells. Notably, miR-21 inhibitor alleviated the promotion of kaempferol on Notch/PTEN/Akt signaling pathways in H9c2 cells exposed to H/R. Taken together, these above findings suggested thatmiR-21 mediates the protection of kaempferol against H/R-induced H9c2 cell injuryvia promoting Notch/PTEN/Akt signaling pathway.

Introduction

Myocardial ischemia/reperfusion (I/R) injury is a major complication of reperfusion therapy for myocardial infarction, representing a great threat to human health [1, 2]. The underlying molecular mechanisms of myocardial I/R injury are complex, including intracellular calcium overload, acute oxygen stress injury, reduction of nitric oxide bioavailability, mitochondrial damage, and inflammation [3–5]. Although several therapeutic strategies, such as remote ischemic preconditioning and ischemic post-conditioning, have been revealed to attenuate myocardial I/R injury, no effective efficacious therapy for protecting the heart against myocardial I/R exists in clinical practice until now [6, 7]. Hence, it is urgent to discover or develop novel pharmacological agents and therapeutic strategies for I/R-stimulated myocardial damage and the underlying molecular mechanisms.

Kaempferol (3,5,7-trihydroxy-4’-methoxyflavone), a naturally occurring flavonoid, is isolated from the roots of Alpinia officinarum which used as a classical tonic agent [8]. Previous studies have revealed that kaempferol has a series of biological activities including anti-inflammatory, anti-oxidant, anti-bacterial, anti-tumor, neuroprotective, and myocardial protective properties [9, 10]. Recent studies find that kaempferol is capable of suppressing myocardial I/R injury [11–13]. The study from Dong Wang et al. reveals that kaempferol improves I/R-induced cardiac dysfunction and myocardial injury involved in reducing myocardial infarct size, cardiomyocyte apoptosis, oxidative stress, and inflammatory responses [11]. Another study also shows that kaempferol mitigates myocardial ischemic injury in isolated rat depending on it’s antioxidant activity and inhibition of GSK-3β activity [13]. However, little is known regarding the underlying molecular mechanism of action of kaempferol as a therapeutic agent for treating myocardial I/R injury.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs subgroup containing 18–23 nucleotides [14], that functionally modulate specific messenger RNA targets involved in extensive physiological and pathological processes [15, 16]. Increasing evidence reveals that various miRNAs engage in the modulation of cardiovascular formation and function [17, 18], and are the key molecular players in the diagnosis and prevention of myocardial I/R injury [19, 20]. Particularly, miR-21 is highly expressed in cardiomyocytes and has also been shown to regulate important processes such as cardiac fibroblasts, apoptosis, and inflammation in myocardial I/R injury [21–24]. Research confirms that miR-21 expression is significantly reduced after I/R, and miR-21 overexpression reduces myocardial infarct size and cardiomyocytes apoptosis during chronic I/R injury [25, 26]. Recently, miR-21 is also available to involve in a variety of myocardial protective drugs against I/R-induced heart damages in vivo and in vitro experiments [27–29]. However, the role of miR-21 in the protection of kaempferol on myocardial I/R injury has not been previously reported.

Notch1 signaling pathway is an evolutionarily conserved pathway that is broadly involved in regulating apoptosis, oxidative stress, and inflammatory responses in response to harmful stimuli [30–32]. Previous studies have reported that the Notch1 signaling pathway is also involved in the attenuation of myocardial I/R injury [33, 34]. Research bore out that Nothch1 participates in the inhibitory effect of myocardial protective drugs such as polydatin [35] and Berberine [36] on myocardial I/R injury under diabetic condition by ameliorating oxidative stress damage via activating PTEN/Akt signaling pathway. However, the role of Notch1/PTEN/Akt signaling pathway in the cardioprotection of kaempferol has not been studied. Notably, research from Cao J et al. demonstrates that miR-146a and miR-21 cooperate to accelerate vascular smooth muscle cell proliferation via modulating the Notch signaling pathway in atherosclerosis [37]. Hence, the present study further demonstrated whether Notch1/PTEN/Akt signaling pathway participates in the contribution of miR-21 to kaempferol-induced cardioprotection against H/R injury.

Here, the results showed that miR-21 mediates kaempferol-induced the inhibition on H/R-induced the damage of cardiomyocytes through reducing oxidative stress and inflammatory response. In addition, miR-21 contributes to kaempferol-led to the enhancement of Notch1/PTEN/Akt signaling pathway, and inhibition of Notch1/PTEN/Akt signaling pathway blocks the cardioprotection of kaempferol under H/R condition, indicating that miR-21 mediates the protective effect of kaempferol on myocardial I/R injury via promoting Notch1/PTEN/AKT signaling pathways in vitro. These results potentially provide a new perspective on understanding cardioprotective effect of kaempferol.

Materials and methods

Cell culture and H/R treatment

The rat heart-derived cardiac myoblast H9c2 cardiomyocytes were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St Louis, USA) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (both from GIBCO, Grand Island, NY, USA) in a humidified incubator with 95% air and 5% CO₂ at 37°C. In order to establish an in vitro model of myocardial I/R injury, H9c2 cells were maintained in a hypoxic chamber (95% N₂ and 5% CO₂) with serum- and glucose-free DMEM at 37°C for 6 h, to induce hypoxia. After hypoxia, the cells were reoxygenated under normoxic conditions (95% air/5% CO₂) with DMEM containing 10% FBS for 12 h at 37°C [38]. Cells cultured under normal conditions in a humidified incubator with 95% air and 5% CO₂ were served as negative controls.

Experimental group

To determine the protection of kaempferol on H/R injury, cells were randomly allocated to four groups: 1) Control group, no treatment; 2) H/R group, hypoxia (6 h)/reoxygenation (12 h); 3) kaempferol (Kae) + H/R group, pretreatment with different concentrations of kaempferol (5, 10, 20, or 30 μM) for 2 h prior to H (6 h)/R (12 h); and 4) kae alone group, pretreatment with kaempferol (5, 10, 20, or 30 μM) for 20 h. To further explore the role of miR-21 in the cardioprotection of kaempferol, cells were randomly allocated to six groups: 1) Control group, no treatment; 2) H/R group, H(6 h)/R(12 h); 3) kae + H/R group, pretreatment with kaempferol (20 μM) for 2 h prior to H (6 h)/R (12 h); 4) Kae + H/R + NC, transfection with negative control (NC) followed by treatment with kaempferol (20 μM) for 2 h and then co-treatment with H (6 h)/R (12 h); 5) Kae + H/R + miR-21 I, transfection with miR-21 inhibitor (miR-21 I) followed by treatment with kaempferol (20 μM) for 2 h and then co-treatment with H (6 h)/R (12 h); and 6) miR-21 I alone group. To demonstrate the role of Notch1/PTEN/Akt signaling pathway in the cardioprotection of kaempferol, cells were randomly allocated to five groups:1) Control group, no treatment; 2) H/R group, H (6 h)/R(12 h); 3) Kae + H/R group, pretreatment with kaempferol (20μM) for 2 h prior to H (6 h)/R (12 h); 4) Kae + H/R + Control siRNA, transfection with control siRNA followed by treatment with kaempferol (20 μM) for 2 h and then co-treatment with H (6 h)/R (12 h); and 5) Kae + H/R + Notch1 siRNA, transfection with Notch1 siRNA followed by treatment with kaempferol (20 μM) for 2 h and then co-treatment with H (6 h)/R (12 h).

Cell Counting Kit (CCK)-8 assay

The cell viability was analyzed by a CCK-8 assay kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to a standardized method. Briefly, after the corresponding administration, cells were incubated with 10 μL of CCK-8 solution at 37°C for 3 h. The absorbance at 450 nm was measured using a microplate reader (Bio-Rad Benchmark, Hercules, CA, USA).

Lactate dehydrogenase (LDH) assay

Cell damage was also reflected by the measurement of LDH activity in the culture supernatant using an LDH detection kit (Shanghai Genmed Pharmaceutical Technology Co Ltd., Shanghai, China) according to the manufacturer's protocol. In brief, the culture medium was collected and the adherent H9c2 cells were lysed according to the instructions. LDH activity was assessed using a microplate reader (Biotek Corporation, VT, USA) at 490 nm.

Cell transfection

MiR-21 inhibitor and negative control were synthesized by Biomics Biotechnologies (Nantong, China). Control small interfering RNA (siRNA) and Notch1 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). The sequences were as following: miR-21 inhibitor sense 5′-UAGCUUAUCAGACUGAUGUUGA-3′ and anti-sense 5′-UCAACAUCAGUCUGAUAAGCUA-3′, negative control sense 5’-UUCUCCGAACGUGUCACGUTT-3’ and anti-sense 5’-ACGUGACACGUUCGGAGAATT-3’, Control siRNA forward 5’-CCUACGCCACCAAUUUCGU-3’ and anti-sense 5’-ACGAAAUUGGUGGCGUAGG-3’, and Notch1-siRNA sense 5’-GCACGCGGAUUAAUUUGCA-3’ and anti-sense 5’-UGCAAAUUAAUCCGCGUGC-3’. H9c2 cells were plated to reach 70–90% confluency and then were transfected with miR-21 inhibitor, negative control, Control siRNA or Notch1 siRNA at a final concentration of 100 nM using Lipofectamine 3000 (Life technologies corporation, Gaithersburg, MD, USA) in Opti-MEM medium (Gibco, Carlsbad, CA, USA) according to the manufacturer’s instructions. Transfected cells were subjected to H/R after 48 h of transfection. The efficiency of transfection was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

RT-qPCR assay

Total RNA was extracted from the cells with the TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. For mRNA detection, RNA (1 μg) was reversed transcribed to cDNA using the RevertAid™ cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Real-time PCR amplification was performed on a CFX96 RT-PCR system (Biorad, Hemel Hempstead, UK) using iTaq™ Universal SYBR^® Green Supermix (Bio-Rad, USA) according to manufacturer’s specification. Reaction conditions were as follows: pre-denaturation at 96°C for 5 min, followed by 45 cycles of denaturation at 95°C for 30 s and annealing at 56°C for 45 s, and a final extension at 72°C for 60 s. β-actin served as an internal control. For miRNA detection, One-step real-time qPCR was performed, and the expression of miR-21 was detected using the EzOmics miRNA qPCR Detection Primer Set (Biomics, USA) and EzOmics One-Step qPCR Kit (Biomics, USA). PCR was carried out at 42°C for 45 min; 95°C for 10 min, followed by 45 cycles of amplification. The primers used synthesized by Invitrogen (Shanghai, China) and as follows: Notch-1, forward: 5′-ATGACTGCCCAGGAAACAAC-3′ and reverse: 5’-GTCCAGCCATTGACACACAC-3’; β-actin, forward: 5′-AGGGAAATCGTGCGTGAC-3′ and reverse, 5′-CGCTCATTGCCGATAGTG-3′; miR-21, forward: 5’-GCACCGTCAAGGCTGAGAAC-3’ and reverse: 5’-CAGCCCATCGACTGGTG-3’; and U6, forward: 5’-CTCGCTTCGGCAGCACA-3’ and reverse: 5’-AACGCTTCACGAATTTGCGT-3’. U6 was used as an internal control. The relative expression was calculated using the 2^–ΔΔCt method [39].

Caspase-3 activity measurement

Caspase-3 activity was measured in lysates of cells using a FluorAce Apopain Assay kit (BioRad, Hercules, CA, USA) following the manufacturer's instructions. In brief, the lysates of cells were incubated with DEVD-pNA substrate (200 μM, a substrate of caspase-3) at 37°C for 4 h. The absorbance at 405 nm was measured by a microplate reader (Biotek Corporation, VT, USA).

Cell apoptosis assay

Cell apoptosis was determined using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, San Diego, CA, USA) followed by flow cytometry. Briefly, H9c2 cells were collected by 0.25% trypsin, washed twice with PBS, and centrifuged at 1, 000 × g for 5 min. The cells were then incubated with 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI) for 20 min at room temperature in the dark. Apoptotic rate was analyzed by flow cytometry (FACSCalibur; BD Biosciences) with Cell Quest 3.3 software (BD Biosciences, Franklin Lakes, NJ, USA).

Endogenous reactive oxygen species (ROS) production measurement

Endogenous ROS production was detected using 2’,7’-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich, St Louis, USA) according to the manufacturer's protocol. After treatment with corresponding administration, H9c2 cells were collected and incubated with DCFH-DA (25 μM) at 37°C for 20 min in the dark and were washed twice times with PBS. ROS generation was determined using a FACSCalibur flow cytometer with Cell Quest 3.3 software (BD Biosciences, Franklin Lakes, NJ, USA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Determination of NO level in the culture supernatant

NO production in culture medium was measured using the Nitrate/Nitrite Assay Kit (Beyotime Institute of Biotechnology, Nanjing, China; cat. number S0021M) according to the instructions. Briefly, cell culture medium (50 μL) were collected, centrifuged at 1, 000 × g for 10 min and mixed with Griess Reagent I (50 μL) and Griess Reagent II (50 μL), both included in the kit, in a 96-well microtiter plate for 10 min at 37°C. The absorbance at 540 nm was read using a microplate reader (Biotek Corporation, VT, USA).

Detection of MDA content, SOD and GSH-Px activities

The content of MDA (cat. number A003-1), the activities of SOD (cat. number A001-3) and GSH-Px (cat. number A007-1-1) in treated H9c2 cells were measured by using corresponding commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Enzyme-linked immunosorbent assay

The culture supernatant was collected and centrifuged at 10000 × g at 4°C for 10 min. The concentrations of cytokines including IL-1β (cat. number H002), IL-6 (cat. number H007), TNF-α (cat. number H052), IL-8 (cat. number H008), and IL-10 (cat. number H009), in the culture supernatant, were detected by a commercial enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions, respectively. Optical density was measured at 450 nm.

Western blot analysis

After treatment, H9c2 cells were collected and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology; cat. number P0013B) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Beyotime Institute of Biotechnology; cat. number ST506). Protein concentration was measured by BCA protein assay (Thermo Fisher Scientific, Grand Island, NY, USA; cat. number 23227) and equal amounts of protein (30 μg) were fractionated by 12% sodium dodecyl sulfate-polyacrylamide gel and then transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA; cat. number C2035). After blocked with 5% nonfat milk powder in Tris buffer containing 0.1% Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated with the appropriate following primary antibodies: anti-Bax (dilution of 1: 2000; cat. number #2772, Cell Signaling Technology, MA, USA), anti-Bcl-2 (dilution of 1: 1000; cat. number ab32124, Abcam, Britain), anti-Notch1(dilution of 1: 2000; cat. number #3608, Cell Signaling Technology), anti-PTEN (dilution of 1: 1000; cat. number #9188, Cell Signaling Technology), anti-P-AKT (dilution of 1: 2000; cat. number #4060, Cell Signaling Technology), anti-AKT (dilution of 1: 2000; cat. number #4691, Cell Signaling Technology), and anti-GAPDH (dilution of 1: 2000; cat. number #5174, Cell Signaling Technology) overnight at 4°C. GAPDH was used as an internal control. After washing three times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated second antibodies (dilution of 1: 2000, cat. number #7074, Cell Signaling Technology) for 2 h at room temperature. Images were visualized with an enhanced BeyoECL Plus reagents (Beyotime Institute of Biotechnology; cat. number P0018M) and analyzed by ImageJ 1.49 (National Institute of Health, Bethesda, MD).

Statistical analysis

Results are presented as the means ± standard deviation (SD) of at least three independent replicates. Data comparisons between the two groups were analyzed using one-way ANOVA followed by the Tukey multiple comparisons test. When the sample does not meet the normality check, a nonparametric test was performed followed by the Dunnett's T3 test. Data was analyzed by GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P< 0.05 was considered to indicate a statistically significant difference.

Results

Kaempferol attenuates hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and promotes miR-21 expression in H9c2 cells

To determine the protective effect of kaempferol on H/R injury, the functions of kaempferol under H/R condition were conducted by detecting cell viability and LDH activity. As shown in Fig 1, pretreatment with kaempferol (10, 20, and 30 μM) obviously increased cell viability (Fig 1A) and decreased LDH activity compared to H/R group (Fig 1B). Kaempferol (20 μM) significantly led to an increase in cell viability and a decrease in LDH release (P < 0.01), and the inhibitory efficiency of kaempferol (30 μM) was not significantly higher than that of kaempferol (20 μM) (P> 0.05). Therefore, kaempferol at a concentration of 20 μM were performed in subsequent experiments. To further investigate the protection of kaempferol against H/R injury, the impact of kaempferol on apoptosis were detected by Annexin V-FITC/PI staining (Fig 1C). The results revealed that H/R results in a significant increase in apoptosis rate compared with the control group, while kaempferol pretreatment led to the decrease in apoptosis rate compared with H/R group (Fig 1D). Furthermore, western blot results (Fig 1E) revealed that pretreatment with kaempferol also reverses H/R-induced the increase in the expression of pro-apoptotic protein (Bax) and the decrease in the expression of anti-apoptotic protein (Bcl-2) in H9c2 cells (Fig 1F).

Fig 1 — H9c2 cells were pretreated with kaempferol (5, 10, 20, and 30 μM) for 2 h and then co-treated with H (6 h)/R (12 h). (A) CCK-8 assay for cell viability. (B) LDH detection kit for LDH activity in cellular supernatant. Data are presented as the means ± SD, n = 3. Compared with the control group, ^##P < 0.001; compared with the H/R group, *P < 0.05, **P < 0.01. H9c2 cells were pretreated with kae (20 μM) for 2 h and then co-treated with H (6 h)/R (12 h). (C) Annexin V-FITC staining assay for apoptosis rates. (D) A statistical representation of apoptosis rates. (E) Western blot analysis for Bax and Bcl-2 expression. (F) Quantitative analysis of Bax and Bcl-2 expressions normal to GAPDH. (G) RT-PCR for miR-21 level. Data are presented as the means ± SD, n = 3. Compared with the control group, ^#P < 0.05, ^##P < 0.001; compared with the H/R group, *P < 0.05, **P < 0.01. Kae, kaempferol; H/R, hypoxia/reoxygenation.

Several studies have proved the possible involvement of miR-21 in ischemia-reperfusion (I/R) injury by regulating key signaling elements in cell survival and apoptosis [40]. The present article further found that following kaempferol pretreatment, the expression of miR-21 was obviously increased in comparison with the H/R group (Fig 1G). Taken together, these data indicated that kaempferol prevents H9c2 cells against H/R injury and enhances miR-21 level in H/R-exposed H9c2 cells.

MiR-21 inhibitor reverses the protective effect of kaempferol on H/R injury in H9c2 cells

To postulate whether miR-21 mediates the cardioprotection of kaempferol against H/R injury, miR-21 inhibitor was utilized in kaempferol functional assays. RT-PCR results showed that miR-21 inhibitor transfection has succeeded in reducing miR-21 expression compared with negative control (NC) transfection (Fig 2A). Beside, miR-21 inhibitor transfection reversed kaempferol-induced the upregulation of cell viability (Fig 2B) and the downregulation of LDH activity (Fig 2C) compared with NC transfection in H/R-treated H9c2 cellsmiR-21 inhibitor Fig. Additionally, miR-21 inhibitor exhibited an increased apoptosis rate when compared with controls NC transfection (Fig 2D) in cotreated with kaempferol and H/R group. Caspase-3 plays a vital function in the intrinsic and extrinsic process of apoptosis [41]. The results further presented that kaempferol inhibits H/R-induced the increase in caspase-3 activity, while this effect was reversed by miR-21 inhibitor transfection (Fig 2E). Furthermore, western blot results (Fig 2F) showed that kaempferol-resulted in a decrease in Bax expression and an increase in Bcl-2 expression in H/R-exposed H9c2 cells are also blocked by miR-21 inhibitor transfection (Fig 2G). These data indicated that miR-21 mediates the protective effect of kaempferol on H/R injury partly by inhibiting mitochondrial-dependent apoptosis in H9c2 cells.

Fig 2 — H9c2 cells were transfected with miR-21 inhibitor (miR-21 I) or negative control (NC). (A) RT-PCR for miR-21 level. Data are presented as the means ± SD, n = 3. Compared with the control group, ^NSP > 0.05; compared with the NC transfection group, **P < 0.01. H9c2 cells were transfected with miR-21 I or NC followed by treatment with kaempferol (20 μM) for 2 h and then co-treatment with H (6 h)/R (12 h). (B) CCK-8 assay for cell viability. (C) LDH detection kit for LDH activity in cellular supernatant. (D) Annexin V-FITC staining assay for apoptosis. (E) Determination of caspase-3 activity by commercial kit. (F) Western blot analysis for Bax and Bcl-2 expression. (G) Quantitative analysis of Bax/Bcl-2 normal to GAPDH. Data are presented as the means ± SD, n = 3. Compared with the control group, ^#P < 0.05, ^##P < 0.001; compared with the H/R group, *P < 0.05, **P < 0.01; compared with the Kae + H/R group, ^NSP > 0.05; compared with the Kae + H/R + NC group, ^$P < 0.05, ^$$P < 0.01. NS: No significance. Kae, kaempferol; H/R, hypoxia/reoxygenation.

MiR-21 inhibitor eliminates kaempferol-induced the inhibition on oxidative stress in H/R-treated H9c2 cells

Oxidative stress, defined as an imbalance between production and destruction of free radicals, participates in apoptosis, mitochondrial dysfunction, and the pathogenesis and progression of myocardial I/R injury [4]. Hence, the present further studied whether kaempferol regulates reactive oxygen species (ROS) production and antioxidant defense under H/R condition and whether miR-21 participates in this process. As illustrated in Fig 3, kaempferol mostly reduced the endogenous ROS production (Fig 3A) and malondialdehyde (MDA) content (Fig 3B), the latter of which is a measure of ROS-related lipid peroxidation. Consistently, these effects were abolished by miR-21 inhibitor transfection. In addition, kaempferol markedly enhanced the activities of antioxidant enzymes including superoxide dismutase (SOD) (Fig 3C) and glutathione peroxidase (GSH-Px) (Fig 3D) compared with H/R group in H9c2 cells, which were both reversed bymiR-21 inhibitor transfection. MiR-21 inhibitor alone transfection had no effects on oxidative stress. These results revealed that miR-21 mediates the protection of kaempferol against oxidative injury under the H/R condition in H9c2 cells.

Fig 3 — H9c2 cells were transfected with miR-21 inhibitor (miR-21 I) or negative control (NC) followed by treatment with kaempferol (20 μM) for 2 h and then co-treatment with H (6 h)/R (12 h). (A) ROS production was detected by 2’,7’-dichlorofluorescein diacetate (DCFH-DA). (B) Malondialdehyde (MDA) content, (C) superoxide dismutase (SOD), and (D) glutathione peroxidase (GSH-Px) were measured by corresponding commercial kits. Data are presented as the means ± SD, n = 3. Compared with the control group, ^#P < 0.05, ^##P < 0.001; compared with the H/R group, *P < 0.05, **P < 0.01; compared with the Kae + H/R group, ^NSP > 0.05; compared with the Kae + H/R + NC group, ^$P < 0.05, ^$$P < 0.01.Kae, kaempferol; H/R, hypoxia/reoxygenation.

MiR-21 inhibitor mitigates the protective effect of kaempferol on inflammatory response under H/R injury in H9c2 cells

Considerable evidence has revealed a causative function of inflammatory response in I/R-induced cardiac injury [42]. The present study further demonstrated that kaempferol significantly reduces the concentrations of pro-inflammatory cytokines IL-1β (Fig 4A), IL-8 (Fig 4B) and TNF-α (Fig 4C) and remarkably increases the concentration of anti-inflammatory cytokine IL-10 (Fig 4D) as relative to H/R group. However, the protective effect of kaempferol on inflammatory response was obviously attenuated by miR-21 inhibitor transfection. Nitric oxide synthase (iNOS)/subsequent nitric oxide (NO) pathway is known to be involved in the regulation of many physiological processes, such as inflammation and cardiomyocyte survival [43, 44]. The results further confirmed that H/R-induced the excessive increases in iNOS activity (Fig 4E) and NO level (Fig 4F) in H9c2 cells were attenuated by kaempferol. However, miR-21 inhibitor transfection reversed the inhibition of kaempferol on the iNOS/NO pathway (Fig 4E and 4F). Overall, these results implied that miR-21 mediates kaempferol-alleviated H/R-induced inflammatory response partially by inhibiting iNOS/NO pathway.

Fig 4 — H9c2 cells were transfected with miR-21 inhibitor (miR-21 I) or negative control (NC) followed by treatment with kaempferol (20 μM) for 2 h and then co-treatment with H (6 h)/R (12 h). The levels of IL-1β (A), IL-8 (B), TNF-α (C) and IL-10 (D), and the activity of iNOS were determined by ELISA, respectively. (F) The content of NO was analyzed by the Nitrate/Nitrite Assay Kit. Data are presented as the means ± SD, n = 3. Compared with the control group, ^#P < 0.05, ^##P < 0.001; compared with the H/R group, *P < 0.05, **P < 0.01; compared with the Kae + H/R group, ^NSP > 0.05; compared with the Kae + H/R + NC group, ^$P < 0.05, ^$$P < 0.01. Kae, kaempferol; H/R, hypoxia/reoxygenation.

Kaempferol attenuates H/R injury via promoting Notch1/PTEN/Akt signaling pathway in H9c2 cells

Recently, the Notch1 signaling pathway has attracted growing concerns for its myocardial protective actions [33, 34]. To explore whether the Notch1 signaling pathway plays a role in the present circumstance, the effect of kaempferol on Notch 1 expression was investigated. Western blot analysis results (Fig 5A) showed that H/R treatment decreases Notch1 protein expression, which was reversed by kaempferol (Fig 5B). It is reported that phosphatase and tensin homolog deleted on chromosome ten (PTEN) can be negatively regulated by Notch 1 signaling, and is the upstream and negative regulator of AKT pathway [36]. The effects of kaempferol on PTEN/Akt pathway were further investigated. Western blot results showed that kaempferol obviously decreases PTEN expression (Fig 5C) and increases P-Akt/Akt ratio (Fig 5D) compared with H/R group. These results indicated the promotion of kaempferol on the Notch1/PTEN/Akt signaling pathway under the H/R condition.

Fig 5 — H9c2 cells were pretreated with kaempferol (20 μM) for 2 h and then co-treated with H/R. (A) Protein expressions were measured by Western blot analysis. Quantitative analysis of Notch1 (B), PTEN (C), and P-Akt/Akt (D) normal to GAPDH. Data are presented as the means ± SD, n = 3. Compared with the control group, ^##P < 0.01; compared with the H/R group, *P < 0.05, **P < 0.01. H9c2 cells were transfected with control siRNA and Notch1 siRNA, respectively. (E) Western blot analysis for Notch1 normal to GAPDH. Data are presented as the means ± SD, n = 3. Compared with the control group, ^NSP > 0.05; compared with the control siRNA group, **P < 0.01. H9c2 cells were transfected with control siRNA or Notch1 siRNA followed by treatment with kaempferol (20 μM) for 2 h and then co-treated with H/R. (F) RT-PCR for mRNA level and Quantitative analysis. (G) CCK-8 assay for cell viability. (H) LDH detection kit for LDH activity in cellular supernatant. Data are presented as the means ± SD, n = 3. Compared with the control group, ^##P < 0.001; compared with the H/R group, **P < 0.01; compared with the Kae + H/R + control siRNA group, ^$P < 0.05, ^$$P < 0.01. Notch1, neurogenic locus notch homolog protein‑1; PTEN, phosphatase and tensin homolog deleted on chromosome ten.

To confirm the function of the Notch1/PTEN/Akt signaling pathway, the loss of function assay was carried out. Notch1 siRNA transfection significantly reduced the Notch1 protein expression compared with the control siRNA transfection (Fig 5E). In addition, RT-PCR results showed thatNotch1 siRNA transfection also blocked kaempferol-induced an increase in Notch1 miRNA level and a decreases in PTEN miRNA level, and an increase in Akt miRNA level in H/R-treated H9c2 cells (Fig 5F). These data indicated that Notch1 siRNA lead to the blockage of kaempferol-induced the activation of Notch1/PTEN/Akt signaling pathway under H/R condition. On these foundations, results further revealed that Notch1 siRNA attenuates kaempferol-induced the up-regulation of cell viability and the down-regulation of LDH activity in H9c2 cells exposed H/R. All in all, these data implied that Notch1/PTEN/Akt signaling pathway mediates the cardioprotection of kaempferol against H/R injury in H9c2 cells.

MiR-21 contributes to kaempferol-led to the activation of Notch1/PTEN/Akt signaling pathway in H9c2 cells exposed to H/R

To clarify the further molecular mechanism of miR-21-mediated the beneficial effects of against H/R injury, the effects of miR-21 on the Notch1/PTEN/Akt signaling pathway were investigated. Western blot results (Fig 6A) showed that compared with NC transfection, miR-21 inhibitor transfection reversed kaempferol-induced an increase in Notch1 expression (Fig 6B), a decrease in PTEN (Fig 6C), and an increase in P-Akt/Akt (Fig 6D) in H/R-treated H9c2 cells. MiR-21 inhibitor alone transfection did not affect Notch1/PTEN/Akt signaling pathway. These results suggested that miR-21 mediates kaempferol-induced the enhancement of the Notch1/PTEN/Akt signaling pathway along with myocardial protection.

Fig 6 — H9c2 cells were transfected with miR-21 inhibitor (miR-21 I)or negative control (NC) followed by treatment with kaempferol (20 μM) for 2 h and then co-treatment with H (6 h)/R (12 h). (A) Western blot analysis for protein expression. (B) Quantitative analysis of Notch1 expression (C), PTEN expression, and (D) P-Akt/Akt. Data are presented as the means ± SD, n = 3. Compared with the control group, ^#P < 0.05, ^##P < 0.001; compared with the H/R group, *P < 0.05, **P < 0.01; compared with the Kae + H/R group, ^NSP > 0.05; compared with the Kae + H/R + NC group, ^$P < 0.05, ^$$P < 0.01.

Discussion

In the present study, the underlying protective mechanisms of kaempferol on cardiac cell injury in response to H/R challenge were investigated. We found that miR-21 mediates the profound myocardial protective effect of kaempferol against H/R injury by reducing oxidative stress and informatory response. Additionally, the Notch1/PTEN/Akt signaling pathway contributed to the therapeutic effect of kaempferol on H/R injury. Furthermore,miR-21 was required for kaempferol-induced the activation of Notch1/PTEN/Akt signaling pathway H9c2 cells exposed to H/R. Taken together, these results identified a specific mechanism by which kaempferol attenuates H/R injury in H9c2 cells via promoting Notch1/PTEN/Akt signaling pathway in miR-21-dependent manner.

Studies have proved that many flavonoids possessed unique antioxidants and protection against myocardial I/R injury [45–47]. Kaempferol, a naturally occurring flavonoid, also has a very good antioxidant, anti-inflammatory, and cardioprotective properties in myocardial I/R injury [11, 12]. Kaempferol mitigates myocardial ischemic injury by attenuating inflammation and apoptosis in rats [12]. The study from Zhen Guo et al. also reveals that kaempferol protects cardiomyocytes against anoxia/reoxygenation-induced injury through inhibiting mitochondria-mediated apoptosis pathway [48]. Analogously, the present study confirmed that kaempferol pretreatment attenuates H/R-induced cytotoxicity and apoptosis in H9c2, indicating the cardioprotection of kaempferol against H/R injury. However, the underlying mechanism of kaempferol under H/R injury remains unclear.

MicroRNA-21 (miR-21) is a highly specific miRNA in heart, which can be potential therapeutic targets for the treatment of cardiovascular-related diseases, such as heart failure [49], atherosclerosis [50], and myocardial infarction [51]. More recently, the functions of miR-21 inhibitor in myocardial I/R injury have received significant attention. Y-Q Pan et al. report that myocardial I/R injury leads to a significant decreased miR-21 expression, and miR-21 overexpression effectively inhibited myocardial apoptosis and inflammatory factors release in myocardial I/R-administrated rats [26]. Other researches also reveal that miR-21 mediates a variety of myocardial protective drugs against myocardial I/R injury [29, 52]. Interestingly, a study confirms that kaempferol inhibits vascular smooth muscle cell migration by modulating miR-21 expression [53]. However, the role of miR-21 in the protective effects of kaempferol on myocardial I/R injury have not been reported. In the present study, the results firstly revealed that kaempferol pretreatment remarkably increases miR-21 expression during H/R in H9c2 cells. And, loss-of-function experiments confirmed that down-regulation of miR-21 attenuates the protective effects of kaempferol against H/R-induced cytotoxicity and apoptosis. These data indicated that miR-21 mediates the cardioprotection of kaempferol against H/R injury. However, the molecular mechanisms involved in the contribution of miR-21 to the cardioprotection of kaempferol remain elusive.

More and more studies confirm that kaempferol protects against I/R injury by attenuating oxidative stress, inflammation, and apoptosis [12, 13]. Consistent with these studies, our findings showed that kaempferol decreases ROS generation and MDA content and increases SOD and GSH-Px activities in H9c2 cells exposed to H/R. Additionally, kaempferol reduced pro-inflammatory cytokinesIL-1β, IL-8, and TNF-α levels and promoted anti-inflammatory cytokine IL-10 level as well as attenuating iNOS activity and NO level under H/R condition. These results indicated that kaempferol elicited cardioprotection against I/R injury depending on it’s anti-oxidant and anti-inflammatory activities. Hai-Xiang Xu et al. prove that miR-21 inhibitor exacerbates oxidative stress and attenuates the antioxidative defense system induced by H/R [27]. Another study reveals that miR-21 mediates the protective effects of hydrogen sulfide against ischemia injury through attenuating inflammatory injury in cardiomyocytes [54]. Consistent with these studies, the present showed that miR-21 inhibitor eliminates kaempferol-induced anti-oxidant and anti-inflammatory activities in H/R-treated H9c2 cells. These results suggested that miR-21 contributes to the anti-oxidant and anti-inflammatory activities of kaempferol during H/R injury.

The Notch1 signaling pathway regulates a wide range of physiological processes, including cell proliferation, apoptosis, transcriptional regulation, and ROS generation, playing important regulatory functions during I/R process [33–35]. Previous studies reveal that pharmacological activation of the Notch1 signaling pathway attenuated myocardial oxidative stress and inflammation and preserved heart function during I/R injury [55, 56], and knockdown of Notch1 exacerbated cardiac damage following I/R damage by promoting oxidative stress [57]. On the other hand, Liming Yu et al. prove that Notch1/Hairy and enhancer of split (Hes)-mediated activation of phosphatase and tensin homolog (PTEN)/Akt signaling plays a crucial role in polydatin-induced protection against I/R injury by ameliorating oxidative/nitrative stress damage [35]. However, there are few studies on the role of the Notch1 signaling pathway in the biological function of kaempferol. Our results showed that kaempferol promotes Notch1 expression, inhibits PTEN expression, and enhances Akt phosphorylation in H9c2 cells exposed to H/R, indicating the activation of Notch1/PTEN/Akt signaling pathway induced by kaempferol during H/R. Besides, knockdown of Notch1 induced by Notch1 siRNA reversed the protection of kaempferol against H/R injury. These results indicated that promoting Notch1/PTEN/Akt signaling pathway contributes to the cardioprotection of kaempferol against H/R injury.

The research draws out the crosstalk between miR-21 and the Notch1/PTEN pathway in Human Colorectal Cancer [58]. Jian Cao et al. demonstrates that miR-21 cooperate to modulate the proliferation of vascular smooth muscle cell via regulating Notch2signaling pathway [37]. Notably, during I/R and H/R, forced miR-21 expression promotes Akt signaling activity via suppressing PTEN expression and further inhibited apoptosis and cardiocyte injury [59]. However, the effects of miR-21 on the Notch1/PTEN/Akt pathway under the cardioprotection of kaempferol have not been explored. In the present study, our results further found that miR-21 inhibitor reverses kaempferol-induced the activation of Notch1/PTEN/Akt signaling pathway under H/R condition in H9c2 cells, indicating that kaempferol protects H9c2 cells against H/R injury by promoting Notch1/PTEN/Akt signaling pathway in a miR-21-dependent manner.

However, there is still more to explore behind these experimental results. Firstly, our study is limited to in vitro cell experiment, it is of great necessity to perform the animal investigation. Secondly, other study demonstrates that the Notch pathway controls miR-21 expression. During myocardial I/R injury, the possible interplay between miR-21 and the Notch1 signaling pathway has not been further investigated. Thirdly, how miR-21 participates in the regulation of kaempferol on Notch1/PTEN/Akt signaling pathway during myocardial I/R injury remains to be further studied.

Taken together, the results showed that miR-21 mediates the protection of kaempferol against H/R injury by inhibiting oxidative stress and inflammation via promoting Notch1/PTEN/Akt signaling pathway in cardiomyocytes.

Supporting information

S1 File

(DOC)

Click here for additional data file.^{(54.5MB, doc)}

Data Availability

All relevant data are within the manuscript.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0241007.r001

Decision Letter 0

Meijing Wang

5 Aug 2020

PONE-D-20-20790

MiR-21 mediates the protection of kaempferol against hypoxia/reoxygenation injury via modulating Notch1/PTEN/AKT signaling pathways in H9c2 cells

PLOS ONE

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Kaempferide is a nature flavanol. Previously, other reports demonstrated that kaempferol improves I/R-induced cardiac dysfunction and myocardial injury via reducing myocardial infarct size, cardiomyocyte apoptosis, oxidative stress. In this study, Huang and Qi reported that kaempferol mediated protective effects in H9c2 cells against hypoxia/reoxygenation injury could be mediated by modulating of miR-21 expression to regulate Notch1/PTEN/AKT signaling pathways. The overall of the study is well designed and executed. Some major and minor issued need to be addressed are as below:

1. I/R injury induced cardiomyocyte drop-out is mostly associated with necrosis rather than in vitro H/R cell model associated cell apoptosis. Thus, using H9c2 cells in H/R model in this study may not fully reflect the true mechanism of kaempferide mediated in vivo cardiac-protective effects again I/R injury.

2. In Figure1 and Figure 2 D, the Flow cytometry analysis results of apoptosis (Fitc-Annexin positive) , late stage apoptosis (FITC-A and PI double positive) and necrosis (PI positive) is not consistent.

3. This study only examined overall Notch-1 transcription and protein level, but did not check the Notch intracellular domain (NICD). As NICD is a more direct reflection of Notch signaling activation, so it should be included in the WB analysis.

4. In methods section at Page 10, some of the words are written in Chinses which need to be replaced by English.

Reviewer #2: Ref: PONE-D-20-20790

In the present article entitled “MiR-21 mediates the protection of kaempferol against hypoxia/ reoxygenation injury via modulating Notch1/PTEN/AKT signaling pathways in H9c2 cells” Huang et al. have explored the involvement of miR-21 in the cardioprotective effect of kaempferol on hypoxia/reoxygenation (H/R)-induced H9c2 cell injury. Further they have tested the involvement of Notch/PTEN/Akt signaling pathway in such cardioprotective effect of kaempferol. The study design is simple and the results are well supported by the data. However, some points need to be addressed to make the study robust for the publication.

1. There are many grammatical errors in the manuscript. Especially the use of “the” needs to be checked throughout the manuscript.

2. Title is not clear. “protection of kaempferol against hypoxia/reoxygenation injury…” needs to be rephrased.

3. Authors have used “Kaempferide” or “kaempferol” or “kae” interchangeably. Please stick with one nomenclature.

4. Page 11, line 11 “Particularly, miR-21 is highly expressed in cardiomyocytes and has also been shown to regulate important processes of myocardial I/R injury”. Please provide some mechanistic information of how miR-21 is involved in myocardial I/R injury by citing other papers like Roy et al., PMID: 19147652; Gu et al., PMID: 25809568; Xu et al., PMID: 25159851.

5. Page 16, line 7, there is some text in different language. Please fix.

6. Statistical analysis. The samples size used in this paper is majorly 3. In this smaller number of samples, the normality check is not possible. Have the authors used non-parametric tests? Please mention.

7. Figure 1, Authors have selected 20 μM concentration of kae for all experiments. But in figure 1A and 1B, the concentration of kea used was 30 μM (without H/R, last bar). Authors should present data from 20 μM (without H/R) in some experiments.

8. Figure 1D: The bar graph (H/R group) presented does not match with the scatter plot data shown in Figure 1C.

9. Page 20, line 10, Figures 1F and 1G are not cited.

10. Page 20, line 13, “and apoptosis [37]. The present further found that following kae pretreatment”. It should be “The present article further…...”

11. Figure 2D: The bar graph (H/R and Kae+H/R+miR-21 I groups) presented does not match with the scatter plot data.

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PLoS One. 2020 Nov 5;15(11):e0241007. doi: 10.1371/journal.pone.0241007.r002

Author response to Decision Letter 0

21 Sep 2020

Dear Editors and Reviewers:

Thank you for your letter and for the comments concerning our manuscript entitled “MiR-21 mediates the protection of kaempferol against hypoxia/reoxygenation injury via modulating Notch1/PTEN/AKT signaling pathways in H9c2 cells (ID: PONE-D-20-20790)”. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the reviewer’s comments are as following:

Journal Requirements:

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

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Response: The manuscript has been corrected according to the PLOS ONE's style requirements.

2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

Response: The original uncropped and unadjusted images underlying all blot or gel results are all reported in a Supporting Information files “Full-length blots are presented in Supplementary Figures”.

Response: The ORCID iD has been added.

Review Comments to the Author

Response: Thanks for your constructive suggestion. We don't know if the reviewers have misunderstanding. A wide array of studies confirms that I/R injury induced cardiomyocyte drop-out is mostly associated with in vitro H/R cell model associated cell apoptosis (SEEN: 1. Dang X, Qin Y, Gu C, Sun J, Zhang R, Peng Z. Knockdown of Tripartite Motif 8 Protects H9C2 Cells Against Hypoxia/Reoxygenation-Induced Injury Through the Activation of PI3K/Akt Signaling Pathway. Cell Transplant. 2020;29:963689720949247; 2. Li Y, Liu X. Novel insights into the role of mitochondrial fusion and fission in cardiomyocyte apoptosis induced by ischemia/reperfusion. J Cell Physiol. 2018;233(8):5589-5597; 3. Badalzadeh R, Mokhtari B, Yavari R. Contribution of apoptosis in myocardial reperfusion injury and loss of cardioprotection in diabetes mellitus. J Physiol Sci. 2015 May;65(3):201-1.). In the present study, we only used H9c2 cell to explore the protective mechanism of kaempferide on myocardial I/R injury. Of course, in vitro experiments will make our article more convincing, which is our next experimental plan. This part has been explained in the penultimate paragraph of our discussion.

Response: These are two separate sets of experiments. The apoptotic rate of Figure 1D and Figure 2D was not consistent, which may be related to cell state, experimental operation process and drug storage time and so on.

Response: Thanks for your meaningful advice. We are sorry we didn't pay attention to this part. Due to the limitation of time and antibody as well as the impact of the COVID-19 pandemic, we can’t make up the experiment for the time being. We have looked at a lot of other relevant literature, which also only detected the expression of Notch1 (SEEN: 1. Solanki A, Yánez DC, Lau CI, Rowell J, Barbarulo A, Ross S, Sahni H, Crompton T. The transcriptional repressor Bcl6 promotes pre-TCR induced differentiation to CD4+CD8+ thymocyte and attenuates Notch1 activation. Development. 2020:dev.192203; 2. Thabassum Akhtar Iqbal S, Tirupathi Pichiah PB, Raja S, Arunachalam S. Paeonol Reverses Adriamycin Induced Cardiac Pathological Remodeling through Notch1 Signaling Reactivation in H9c2 Cells and Adult Zebrafish Heart. Chem Res Toxicol. 2020; 33(2):312-323; 3. Zheng J, Li J, Kou B, Yi Q, Shi T. MicroRNA-30e protects the heart against ischemia and reperfusion injury through autophagy and the Notch1/Hes1/Akt signaling pathway. Int J Mol Med. 2018; 41(6):3221-3230.). In present study, we detected the changes in downstream targets PTEN/AKT signaling of Notch1 pathway, which can indirectly reflect the activation of Noctch1 pathway. On this basis, the loss of NICD expression does not affect our conclusion. Of course, in future studies, we will supplement and further explore the role of Notch1/PTEN/AKT signaling pathway in the protective effects of kaempferol on myocardial ischemia/reperfusion injury.

4. In methods section at Page 10, some of the words are written in Chinses which need to be replaced by English.

Response: Thanks for the editor’s careful suggestion. “少了反向序列” has been replaced by “and reverse: 5’-GTCCAGCCATTGACACACAC-3’” in the reversed manuscript.

Reviewer #2: Ref: PONE-D-20-20790

1. There are many grammatical errors in the manuscript. Especially the use of “the” needs to be checked throughout the manuscript.

Response: Thanks for the editor’s good evaluation and kind suggestion. We are really sorry for the typography, format, and layout that are a bit non-standard, and the spelling and syntax errors in our manuscript. We have carefully examined the format and grammar, and corrected errors and deficiencies in articles. Meanwhile, we also invited an English professional and experienced colleagues to improve the scientific writing in our manuscript. The use of “the” has been checked throughout the manuscript. The “unnecessary the” has been removed in the reversed manuscript.

2. Title is not clear. “protection of kaempferol against hypoxia/reoxygenation injury…” needs to be rephrased.

Response: “protection of kaempferol against hypoxia/reoxygenation injury” has been changed to “protective effect of kaempferol on hypoxia/reoxygenation injury”.

3. Authors have used “Kaempferide” or “kaempferol” or “kae” interchangeably. Please stick with one nomenclature.

Response: “Kaempferide” and “kae” have all been changed to “kaempferol”.

Response: These three references are inserted into the reversed manuscript as references 22, 23 and 24.

5. Page 16, line 7, there is some text in different language. Please fix.

Response: Thanks for the editor’s careful suggestion. “少了反向序列” has been replaced by “and reverse: 5’-GTCCAGCCATTGACACACAC-3’” in the reversed manuscript.

Response: The related part “When the sample does not meet the normality check, a nonparametric test was performed followed by the Dunnett's T3 test.” has been added to the “Statistical analysis”.

Response: The effects of kea (5, 10, 20, and 30 μM) alone on cell viability and LDH activity have all been detected. Considering the beauty of the drawing, we only added the kea (30 μM) alone group. Taking into account the reviewers' comments, the effects of kea (20) alone on cell viability and LDH activity have been added to Figure 1A and Figure 1B.

8. Figure 1D: The bar graph (H/R group) presented does not match with the scatter plot data shown in Figure 1C.

Response: The mistake in control group and Kea alone group in Figure 1D have been corrected.

9. Page 20, line 10, Figures 1F and 1G are not cited.

Response: The Figures 1F and 1G have been added to Result (Kaempferol (Kae) attenuates the damage of cardiomyocytes and promotes miR-21 expression in hypoxia/reoxygenation (H/R)-treated H9c2 cell).

10. Page 20, line 13, “and apoptosis [37]. The present further found that following kae pretreatment”. It should be “The present article further…...”

Response: “The present further found” has been corrected to “The present article further found”.

11. Figure 2D: The bar graph (H/R and Kae+H/R+miR-21 I groups) presented does not match with the scatter plot data.

Response: Our statistical results are based on three independent experiments. Hence, sometimes statistical images may be slightly different from typical images. In Figure 2D, there is nothing wrong between the bar graph and the scatter plot data.

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PLoS One. doi: 10.1371/journal.pone.0241007.r003

Decision Letter 1

Meijing Wang

7 Oct 2020

MiR-21 mediates the protection of kaempferol against hypoxia/reoxygenation-induced cardiomyocyte injury via promoting Notch1/PTEN/AKT signaling pathway

PONE-D-20-20790R1

Dear Dr. Huang,

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.

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Kind regards,

Meijing Wang, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: (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 #1: No

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0241007.r004

Acceptance letter

Meijing Wang

27 Oct 2020

PONE-D-20-20790R1

MiR-21 mediates the protection of kaempferol against hypoxia/reoxygenation-induced cardiomyocyte injury via promoting Notch1/PTEN/AKT signaling pathway

Dear Dr. Huang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Dr. Meijing Wang

Academic Editor

PLOS ONE

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Data Availability Statement

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PERMALINK

MiR-21 mediates the protection of kaempferol against hypoxia/reoxygenation-induced cardiomyocyte injury via promoting Notch1/PTEN/AKT signaling pathway

Jinxi Huang

Zhenhui Qi

Roles

Abstract

Introduction

Materials and methods

Cell culture and H/R treatment

Experimental group

Cell Counting Kit (CCK)-8 assay

Lactate dehydrogenase (LDH) assay

Cell transfection

RT-qPCR assay

Caspase-3 activity measurement

Cell apoptosis assay

Endogenous reactive oxygen species (ROS) production measurement

Determination of NO level in the culture supernatant

Detection of MDA content, SOD and GSH-Px activities

Enzyme-linked immunosorbent assay

Western blot analysis

Statistical analysis

Results

Kaempferol attenuates hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and promotes miR-21 expression in H9c2 cells

Fig 1. Kaempferol protects cardiomyocyte injury and increases miR-21 expression in H9c2 cell-subjected with H/R.

MiR-21 inhibitor reverses the protective effect of kaempferol on H/R injury in H9c2 cells

Fig 2. MiR-21 inhibitor attenuates kaempferol-induced the inhibitory effects on H/R-induced cytotoxicity and apoptosis in H9c2 cells.

MiR-21 inhibitor eliminates kaempferol-induced the inhibition on oxidative stress in H/R-treated H9c2 cells

Fig 3. MiR-21 inhibitor reverses kaempferol-induced inhibition on oxidative stress in H9c2 cells exposed to H/R.

MiR-21 inhibitor mitigates the protective effect of kaempferol on inflammatory response under H/R injury in H9c2 cells

Fig 4. MiR-21 inhibitor blocks kaempferol-induced the inhibition on inflammatory response in H9c2 cells exposed to H/R.

Kaempferol attenuates H/R injury via promoting Notch1/PTEN/Akt signaling pathway in H9c2 cells

Fig 5. Kaempferol blocks H/R-induced the inhibition on Notch1/PTEN/Akt signaling pathway in H9c2 cells exposed to H/R.

MiR-21 contributes to kaempferol-led to the activation of Notch1/PTEN/Akt signaling pathway in H9c2 cells exposed to H/R

Fig 6. MiR-21 inhibitor abolishes kaempferol-induced the activation of Notch1/PTEN/Akt signaling pathway in H9c2 cells exposed to H/R.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Meijing Wang

Roles

Author response to Decision Letter 0

Decision Letter 1

Meijing Wang

Roles

Acceptance letter

Meijing Wang

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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