Hepatic fibrosis (HF) is a pathological process in which extracellular matrix accumulation and damage repair persist. Advanced HF leads to liver cirrhosis and hepatocellular carcinoma, and is a key link for treating liver diseases [1]. Alcoholic liver disease is one of the major causes of nonviral liver cirrhosis worldwide [2]. Research over the past few decades has revealed several key mechanisms of alcohol-mediated hepatocyte damage, including CYP2E1-mediated oxidative stress, mitochondrial glutathione stress, endoplasmic reticulum (ER) stress, malondialdehyde-acetaldehyde protein adducts, centrilobular hypoxia, autophagy suppression, and lysosomal dysfunction [3]. These mechanisms may likely co-exist and synergistically promote cellular damage through sensitization of hepatocytes and initiation of hepatocytes and other cell types, ultimately leading to hepatocellular death characterized by necrosis, necroptosis, or apoptosis [4].

It is widely recognized that hepatocyte damage is the initiating factor that triggers a series of complex changes in the liver to stimulate the occurrence and development of HF [5]. Hepatocytes possess a large number of ER, and many liver diseases are correlated with ER stress and ER stress-mediated apoptosis, such as HF, alcoholic liver disease, nonalcoholic fatty liver disease, viral hepatitis, acute liver disease failure, liver cancer, and drug-induced liver disease. A variety of toxic, metabolic, and inflammatory insults can directly promote the activation of hepatic stellate cells (HSCs) and cause liver damage and disease. A common feature of these injuries is the activation of apoptosis and/or necrosis. Enhanced apoptosis of hepatocytes amplifies inflammatory damage and promotes the development of fibrosis and ultimately liver cancer [6]. Therefore, inhibition of hepatocellular apoptosis has been suggested to be a plausible treatment for liver injury, especially HF.

Recent studies have shown that peroxisome proliferator-activated receptor gamma (PPAR-γ) plays an important role in HF by affecting hepatocytes [7]. PPAR-γ has several biological roles, including regulation of adipogenesis, lipid metabolism, cell proliferation and apoptosis, cell differentiation, and the inflammatory response [8]. PPAR-γ is derived from the nuclear receptor gene superfamily and affects downstream gene transcription through a ligand-dependent pathway. PPAR-γ is a negative regulator of the Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway, and TLR4 activates inflammation in CCl4-induced liver injury by stimulating NF-κB, followed by increased secretion of proinflammatory cytokines and stimulation of an inflammatory response leading to apoptosis [9]. Therefore, inhibiting PPAR-γ-dependent hepatocyte apoptosis may be an effective therapeutic strategy for the treatment and prevention of HF.

Though much progress has been made in understanding the mechanism of PPAR-γ-regulated HF, it remains largely unclear. Accumulating evidence has demonstrated that DNA methylation promotes the progression of liver fibrogenesis [10]. DNA methylation is one of the most studied epigenetic markers and is altered in HF tissues. PPAR-γ is downregulated in HSCs and hepatocytes of patients with alcoholic liver disease, and a recent study suggested liver inflammation and fibrosis were associated with hypermethylation of the PPAR-γ promoter [11,12]. Recently, Zhang et al. showed that demethylation of CpGs by PPAR-γ could inhibit pulmonary fibrosis in mice [13]. Therefore, we speculate that the DNA methylation changes of the PPAR-γ gene are transmitted to its downstream and affect the TLR4/NF-κB signaling pathway, thereby participating in the promotion of liver fibrosis.

Glycyrrhizic acid (GA), a natural product derived from the traditional Chinese medicinal plant Glycyrrhiza glabra, shows anti-inflammatory, antifibrosis, and hepatoprotective effects in vivo [14]. Liang B et al. reported that GA ameliorates CCl4-induced HF by inhibiting hepatocyte apoptosis, which may provide potential therapeutic strategies for HF [15]. However, the mechanism by which GA inhibits hepatocyte apoptosis in HF remains unknown.

GA is a potential agonist of PPAR-γ [16]. In a pulmonary fibrosis model, PPAR-γ acted as a key mediator of epigenetic pulmonary fibrosis, and GA attenuated PPARγ-mediated inhibition of fibrosis in a DNA methyltransferase (DNMT)-dependent manner [13]. Thus, we hypothesized that GA elevated the expression of PPAR-γ through epigenetic regulation of PPAR-γ, thereby downregulating the NF-κB signaling pathway to inhibit rat hepatic BRL-3A cell injury.

In the present study, we used an alcohol-induced model of hepatocyte injury (rat BRL-3A hepatocytes), a cellular model of HF, to study the effects of GA on demethylation by PPAR-γ.

Antibodies and Reagents

GA was purchased from Chengdu Biotech Co., Ltd. (Sichuan, China). Both RSG and DAC were obtained from Beijing Top Science Technology & Biological Co., Ltd. (Beijing, China). Anti-caspase-3 (cs-9662S), anti-Bcl-2 (cs-3498S), and anti-Bax (cs-14796S) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-DNMT1 (ab-GR3259795-4) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-NF-κB PP65 (af-2006), anti-PPAR-γ (af-6284), and anti-β-actin (af-7018-100) antibodies were purchased from Affinity Biosciences (Jiangsu, China). Anti-NF-κB P65 (YM-3111) antibodies were purchased from ImmunoWay Biotechnology Company (Texas, USA). Anti-TLR4 (AP-52189) antibodies were purchased from Abgent Biotechnology Co., Ltd. (San Diego, USA). Anti-IL-1β (A16288) antibodies were purchased from ABclonal Technology Co., Ltd. (Boston, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit antibodies were purchased from MultiSciences (Lianke) Biotech Co., Ltd. All cell culture reagents were obtained from Gibco/Invitrogen (Grand Island, NY, USA).

Cell Culture

Rat hepatocyte BRL-3A cell line (gifted by Professor Wang Zheng of Hunan Agricultural University) was preserved in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose with 2% (static culture) or 10% (activated culture) fetal bovine serum (FBS) and antibiotics at 37°C and 5% CO₂.

CCK-8 Assay for Cell Viabilities

The cells used in this study were in the logarithmic growth phase. A total of 1×105 cells/mL were inoculated in a 96-well plate and then subjected to treatment with 600 mm ethanol. After 8 h of culture, the ethanol-containing medium was discarded, and a different drug-containing medium was added for another 8 h of incubation. CCK-8 assay was performed to detect cell viabilities in BRL-3A hepatocytes following ethanol and GA treatment.

Measurement of ALT and AST Levels

Cells treated with different factors were collected after washing with PBS and then scraped off with a cell spatula. The cells were added to a homogeneous medium and subjected to ultrasonic crushing in an ice-water bath. ALT and AST kits were used to detect enzyme activities in the cell homogenate.

Oil Red Staining

Six milliliters of oil-red working solution were mixed with 4 mL of double-distilled water and placed at room temperature for 10 minutes after filtering. After the intervention, the cells were rinsed with PBS. Next, 10% formaldehyde was added to fix the cells for 30 min. Subsequently, 1 mL of oil red solution was added to each well for 10 minutes, and the residual dye was removed with 75% alcohol. The cells were observed under a microscope and photographed.

WB Assay

Cell protein was extracted in RIPA buffer with a protease inhibitor cocktail, and protein concentrations were measured using a BCA protein kit. Next, SDS-PAGE was performed to resolve proteins, followed by the transfer of proteins to polyvinylidene difluoride membranes for the WB assay. The membranes with proteins were blocked with 5% skimmed milk in TBS-T. The blots were incubated with primary antibodies overnight at 4°C, followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactivity was detected by ECL, and the band intensities were quantified by ImageJ software.

Molecular Docking Studies

The structural information of the human PPAP-γ protein was obtained from the PDB database (http://www.pdb.org/pdb/home/home.do). The PDB structural information of the protein was downloaded, and the Discovery Studio software was used to calculate the binding energies of GA with PPAP-γ and to visualize the simulation results.

PPAR-Γ Gene Pyrosequencing

Bisulfite conversion was performed using either an EpiTect Bisulfite Kit (Qiagen) or an EZ DNA Methylation-Gold Kit (Cambridge Biosciences). A total of 250–500 µg of DNA with an A260/A280 absorbance ratio > 1.7 was used for bisulfite conversion.

Statistical Analysis

GraphPad Prism 8.0.2 was used for data analysis. The measurement data were expressed as the mean ± standard (`x ± SD). Comparisons between the groups were performed by one-way ANOVA, and pairwise comparisons were performed by the least significant difference method. Differences were considered statistically significant at p < 0.05.

Alcohol induces BRL-3A hepatocyte injury through the PPAR-γ/TLR4/NF-κB signaling pathway

To confirm that alcohol-induced hepatocyte injury is related to the PPAR-γ/NF-κB signaling pathway, we used PPAR-γ agonists for intervention in the signaling pathway. The results showed that the PPAR-γ agonist rosiglitazone (RSG) decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (Fig. 1A, B). Western blot (WB) assay results revealed that the expression level of PPAR-γ was increased and the expression level of NF-κB pp65/p65 was significantly decreased (Fig. 1C, D, and E). These findings indicated that RSG inhibits alcohol-induced hepatocyte injury by activating the PPAR-γ/NF-κB pathway in BRL-3A hepatocytes.

Alcohol-induced hepatocyte injury through the PPAR-γ/TLR4/NF-κB signaling pathway is regulated by DNA methylation

To confirm that alcoholic hepatocyte damage is associated with DNA methylation levels, we observed that 5-aza-2?-deoxycytidine (DAC, a methylase inhibitor) had a protective effect on BRL3A hepatocytes. As shown in Fig. 2A and B, compared to the control group, AST and ALT levels in the alcohol-stimulating group were significantly increased, and ALT and AST activities were significantly decreased in the DAC treatment group at 500 and 1000 ng/mL concentrations. In addition, oil red staining experiments showed that the number of hepatocytes decreased and the number of lipid droplets was significantly increased after alcohol stimulation, and DAC significantly alleviated this damage (Fig. 2C-H).

We further assessed the effect of DAC on the PPAR-γ/TLR4/NF-κB signaling pathway by WB assay. In ethanol-induced BRL-3A hepatocytes, the expression level of PPAR-γ was significantly reduced, and that of NF-κB pp65/p65 and IL-1β was significantly elevated (Fig. 3A, C, D, E, and F). Moreover, DAC effectively suppressed hepatocyte apoptosis by inhibiting the expression of caspase-3 and Bax/Bcl-2 (Fig.3A, G, and H). Besides, the expression of DNMT1 protein was also increased in BRL3A hepatocytes after alcohol stimulation, and this effect was significantly inhibited by DAC (Fig. 3A and B). Taken together, these results suggest that alcohol-induced hepatocyte injury through the PPAR-γ/NF-κB signaling pathway is regulated by DNA methylation.

GA protects against alcohol-induced hepatocyte injury by activating the PPAR-γ/NF-κB pathway in BRL-3A hepatocytes                                                                     

Effect of GA on rat hepatocyte BRL-3A cell viability

The effect of GA was studied in an in vitro model of the rat hepatocyte BRL-3A cell line. The cytotoxic effect after 24 h of incubation of BRL-3A hepatocytes with 0 to 1000 µM GA was measured by the Cell Counting Kit-8 assay. As shown in Fig. 4, GA concentrations up to 500 nM had no apparent cytotoxic effect on the viability of BRL-3A hepatocytes. On the basis of this trial result, GA concentrations ranging from 100 to 500 nM were used in the subsequent experiments.

GA reduced the production of AST, ALT, and lipid droplets in BRL-3A hepatocytes.

The microplate method showed that AST and ALT levels in the model group were significantly higher than those in the control group (p < 0.05). Compared to the model group, GA significantly reduced AST and ALT levels in BRL-3A hepatocytes induced by alcohol (p < 0.05 or p < 0.01) (Fig. 5A and B). Oil red staining analysis revealed that GA reduced the increase in lipid droplet secretion in hepatocytes induced by alcohol (Fig. 5C-H).

GA inhibits hepatocyte apoptosis through the PPAR-γ/TRL4/NF-κB signaling pathway in BRL-3A hepatocytes

To confirm the protective effect of GA on alcohol-induced hepatocyte apoptosis, we analyzed the expression levels of several oxidative stress-related proteins, such as Bax/Bcl-2 and caspase-3. The results of the WB assay showed that the expression of Bax/Bcl-2 and caspase-3 proteins in the model group was significantly higher than that in the normal control group (p < 0.05). Compared to the model group, GA significantly reduced the increase in Bax/Bcl-2 and caspase-3 proteins in BRL-3A hepatocytes induced by alcohol in a dose-dependent manner (Fig. 6A, F, and G).

The PPAR-γ/TRL4/NF-κB signaling pathway is related to the progression of various liver diseases, including HF; therefore, the regulation of the PPAR-γ/TRL4/NF-κB signaling pathway is an important approach to treating HF. The results of the WB assay showed that the expression levels of the TLR4, NF-κB pp65/p65, and IL-1β proteins in BRL-3A hepatocytes of the normal control group were relatively low. Compared to the normal control group, the expression levels of TLR4, NF-κB pp65/p65, and IL-1β proteins in the model group were significantly increased (p < 0.05). Compared to the model group, the GA treatment groups showed a significant reduction in the increase in TLR4, NF-κB pp65/p65, and IL-1β protein expression levels in BRL-3A hepatocytes induced by alcohol (p < 0.05 or p < 0.01). In the GA treatment group, with the increase in the concentration of GA in a certain dose range, its inhibitory effect on the levels of TLR4, NF-κB pp65/p65, and IL-1β proteins in BRL-3A hepatocytes induced by alcohol was gradually enhanced, and the difference was statistically significant. The results are shown in Figures 6A, C, D, and E.

These results suggest that GA can inhibit the expression of Bax/Bcl-2 and caspase-3 proteins, thus inhibiting the apoptosis of hepatocyte BRL-3A cells through the PPAR-γ/NF-κB signaling pathway.

GA exerts anti-hepatocyte injury effect by inhibiting PPAR-γ methylation

Inhibitory effect of GA on DNMT1

The results of WB assays showed that the expression of the DNMT1 protein in the model group was significantly higher than that in the normal control group (p < 0.05). Compared to the model group, GA significantly reduced the increase in the DNMT1 protein in BRL-3A cells induced by alcohol in a dose-dependent manner (p < 0.05 or p < 0.01) (Fig.7).

Effect of GA on the methylation of the PPAR-γ gene as detected by pyrosequencing

To confirm that GA attenuates ethanol-induced damage by inhibiting the methylation of PPAR-γ, we subsequently verified this conjecture by DNA methylation sequencing. As expected, the specific analysis showed that the methylation level of the 500 μM GA treatment group and the blank control group was significantly lower than that of the 600 mM ethanol group. Statistical analysis of the sequencing experiment revealed that the amplified fragment included six CpG sites (Table 1). The CpG 1, CpG 3, and CpG 4 blank groups showed significant differences in the methylation level when compared with the 600 mM ethanol group, while the CpG 3 and Cp 4 blank groups showed significant differences in the methylation level when compared with the 600 mM ethanol group following treatment with 500 μM GA. Specifically, the methylation levels of the CpG 3 site in the promoter region of the PPAR-γ gene were 2.44±0.14, 3.83±0.83, and 2.47±0.61 in the normal rat liver BRL-3A hepatocyte group, the 600 mM ethanol group, and the 500 μM GA treatment group, respectively, and the difference was statistically significant (p < 0.05). The methylation levels of the CpG 4 site were 3.53±0.36, 4.46±0.27, and 3.8±0.21 in the normal rat hepatic BRL-3A hepatocyte group, the 600 mM ethanol group, and the 500 μM GA treatment group, respectively, and the difference was statistically significant (p < 0.05). The differences in the methylation levels of the other CpG sites were not statistically significant (p > 0.05), as shown in Fig. 8.

Fig 1: The Specific PPAR Activator Rosiglitazone (RSG) Reverses Alcohol-Induced BRL-3A Hepatocyte Injury By Inhibiting NF-Κb Activation.

Fig. 1. The specific PPAR activator rosiglitazone (RSG) reverses alcohol-induced BRL-3A hepatocyte injury by inhibiting NF-κB activation. (A and B) ALT and AST in cells secreted into the culture supernatant were measured by ELISA after treatment of hepatocytes with 600 mM ethanol and 5,10,20 µM RSG for 24 hours. (C) The expression of p65, p-P65, and PPAR-γ proteins was examined by western blotting. (D and E) The relative protein expressions were normalized to β-actin. Values are mean SD (n = 3). *P < 0.05 vs. control group (#P < 0.05 vs. ethanol group); **P < 0.01 vs. control group (##P < 0.01 vs. ethanol group) indicate a significant difference compared with the appropriate control group.

Fig. 2. 5-aza-2?-deoxycytidine (DAC, a methylase inhibitor) reverses alcohol-induced BRL-3A hepatocyte injury. (A and B)ALT and AST in cells secreted into the culture supernatant were measured by ELISA after treatment of hepatocytes with 600 mM Ethanol and 100,500,1000 µM DAC for 24 hours. (C-H) Assessment of lipid droplet deposition in hepatocytes by Oil Red O staining Values are mean SD (n = 3). *P < 0.05 vs. control group (#P < 0.05 vs. ethanol group); **P < 0.01 vs. control group (##P < 0.01 vs. ethanol group) indicate a significant difference compared with the appropriate control group.

Fig 2 Aza-2?-Deoxycytidine (DAC, A Methylase Inhibitor) Reverses Alcohol-Induced BRL-3A Hepatocyte Injury.

Fig 3: DAC Enhances PPAR-Γ Activation and Inhibits Activation of the NF-KB Pathway in BRL-3A Hepatocytes.

Fig. 3. DAC enhances PPAR-γ activation and inhibits activation of the NF-KB pathway in BRL-3A hepatocytes. Protein levels of DNMT1, PPAR-γ, TLR4, P65,p-P65,caspase-3, Bcl-2, Bax and IL-1β were determined by Western blotting and normalized to β-actin.Values are mean SD (n = 3). *P < 0.05 vs. control group (#P < 0.05 vs. Ethanol group); **P < 0.01 vs. control group (##P < 0.01 vs. Ethanol group) indicate significant difference compared with the appropriate control group.

Fig 4: The Effect of GA on the Viability of Rat Hepatocytes BRL-3A Cells.

Fig 5: GA Reverses Alcohol-Induced BRL-3A Hepatocyte Injury.

Fig. 5. GA reverses alcohol-induced BRL-3A hepatocyte injury. (A and B)ALT and AST in cells secreted into the culture supernatant were measured by ELISA after treatment of hepatocytes with 600 mM Ethanol and 100,200,500 µM GA for 24 hours. (C-H) Assessment of lipid droplet deposition in hepatocytes by Oil Red O staining.Values are mean SD (n = 3). *P < 0.05 vs. control group (#P < 0.05 vs. Ethanol group); **P < 0.01 vs. control group (##P < 0.01 vs. Ethanol group) indicate significant difference compared with the appropriate control group.

Fig 6: GA Enhances PPAR-Γ Activation and Inhibits Activation of the NF-KB Pathway in BRL-3A Hepatocytes.

Fig. 6. GA enhances PPAR-γ activation and inhibits activation of the NF-KB pathway in BRL-3A hepatocytes. Protein levels of PPAR-γ, TLR4, P65,p-P65,caspase-3, Bcl-2, Bax and IL-1β were determined by Western blotting and normalized to β-actin.Values are mean SD (n = 3). *P < 0.05 vs. control group (#P < 0.05 vs. Ethanol group); **P < 0.01 vs. control group (##P < 0.01, ###P < 0.001 vs. Ethanol group) indicate significant difference compared with the appropriate control group.

Fig 7: GA Inhibits DNMT1 Activation In BRL-3A Hepatocytes.

Fig. 7. GA inhibits DNMT1 activation in BRL-3A hepatocytes. Protein levels of DNMT1 were assessed by western blot. Data were shown as means SD (n = 3). *P < 0.05 vs. control group (#P < 0.05 vs. Ethanol group);**P < 0.01 vs. control group (###P < 0.001 vs. Ethanol group) indicate significant difference compared with the appropriate control cells.

Fig 8: Pyrosequencing Analysis of the Effect of GA on the Methylation of the PPAR-Γ Gene.

Fig. 8. Pyrosequencing analysis of the effect of GA on the methylation of the PPAR-γ gene. Data were shown as means SD (n = 3). *P < 0.05 vs. control group (#P < 0.05 vs. Ethanol group);**P < 0.01 vs. control group indicate significant difference compared with the appropriate control cells.

Table 1: Methylation Levels of Cpg1-9 Sites in the Promoter Region of PPAR-Γ Gene after Glycyrrhizic Acid Administration.

The typical process of HF begins with hepatocyte damage, leading to chronic inflammation and activation of HSCs [17]. The main contributing factors include viral infections, alcohol, or metabolic syndrome inducing nonalcoholic steatohepatitis (NASH). Evasion of hepatocyte injury and apoptosis has become an important therapeutic strategy to block and reverse the process of HF. Yinchenhao decoction regulates the PERK-CHOP-GADD34 pathway and reduces the Bax/Bcl-2 ratio, thereby reducing hepatocyte apoptosis and inhibiting liver injury [18]. Silymarin effectively blocks the organic ion uptake transporter on the surface of hepatocytes and reduces the uptake of xenobiotics (including mushroom poisons) by cells, thereby effectively protecting intact hepatocytes or cells that have not been irreversibly damaged [19]. Bone marrow mesenchymal stem cells inhibited hepatocyte apoptosis by activating the TGF-β1/Bax signaling pathway to treat HF and liver injury [20].

In the present study, a hepatic BRL-3A cell injury model was effectively developed following exposure to 600 µM ethanol for 8 h and incubation in a culture medium for 4 h, as measured by the CCK-8 assay. The expression levels of AST, ALT, Bax/Bcl-2, and caspase-3 were significantly increased, which indicated hepatic BRL-3A cell damage; AST and ALT levels were determined by ELISA, while Bax, Bcl-2, and caspase-3 levels were determined by WB. Changes in cell morphology and lipid droplets were used to assess the activation of HSCs. This model and the pharmacological evaluation index have been widely used to study the pathogenesis of HF [21].

Previous studies have shown that the TLR4/NF-κB signaling pathway plays an important role in the pathogenesis of HF [22]. The transduction of the TLR4/NF-κB signaling pathway can effectively inhibit the occurrence of cellular inflammatory response [23]. The expression of TLR4 is upregulated during HF, thereby activating the downstream signaling pathways, which in turn triggers a series of chain reactions. NF-κB is one of the signaling pathways related to cellular inflammation regulated by TLR4 [24]. As a ligand-dependent activating transcription factor, PPAR-γ is considered to be a key component in regulating inflammatory responses [25]. PPAR-γ can directly or indirectly inhibit the TLR4/NF-κB signaling pathway and inflammatory responses [26]. In the present study, the expression of PPAR-γ was downregulated and that of TLR4/NF-κB p65 was upregulated in the alcohol-induced model.

Alcohol abuse is associated with epigenetic dysregulation of human DNA methylation changes and experimental liver injury, and epigenetic factors are considered to play a role in alcoholic hepatitis [27]. Hypermethylation of PTEN activates the P13K/AKT and ERK pathways, which are involved in the activation of HSCs and the regulation of HF [28]. Zhao Q et al. reported that the hypermethylation of the PPAR gamma promoter is associated with liver inflammation and fibrosis. [12] In the present study, we showed that the methylase inhibitor and RSG inhibited the overexpression of TLR4 and NF-κB p65 by increasing PPAR-γ activation. Hence, the activation of the PPAR-γ-dependent NF-κB signaling pathway and the secretion of inflammatory cytokines were directly associated with increased DNA methylation in alcohol-stimulated hepatocytes. This evidence strongly supports that PPAR-γ likely acts as a “fibrosis suppressor” and that the PPAR-γ level is maintained by DNA demethylation, thus achieving anti-HF effects through various profibrotic signaling pathways and cellular processes. Further studies showed that DNMT1 plays an important role in regulating PPAR-γ methylation.

Phytochemical components derived from many medicinal plants are known to have epigenetic regulatory capacity and have emerged as relatively safe alternatives for treating epigenetic disorders [29]. GA shows anti-fibrosis effects in animal models; however, the precise pharmacological modes of action of GA are not well understood [15, 30]. GA is a triterpenoid saponin, and some of its metabolites have a hypomethylation effect [31]. GA is known to inhibit PPAR-γ methylation and show beneficial effects on pulmonary fibrosis [13]. In the present study, we demonstrate that GA recovery of fibrotic PPAR-γ loss is largely abrogated in the presence of alcohol or during DNMT1 overexpression in lung epithelial fibroblasts, and GA normalizes the aberrant expression of antioxidant enzymes and inflammatory cytokines in a PPAR-γ-dependent manner. This finding suggests that GA-induced PPAR-γ promoter hypomethylation prevents fibrotic PPAR-γ suppression, which contributes significantly to the anti-HF functions of GA.

To further confirm the proposed hypothesis, a pyrosequencing assay was performed to verify the effect of GA on PPAR-γ methylation. We observed that treatment with GA significantly attenuated methylation at CpG 3 and Cp 4 sites of PPAR-γ as compared to that in BRL-3A hepatocytes treated with alcohol alone.

In conclusion, the novel findings of the present study demonstrated that GA treatment effectively alleviated alcohol-induced hepatocyte injury by inhibiting the TLR4/NF-κB pathway and reducing DNMT1-mediated PPAR-γ methylation. On the basis of these results, GA could be considered a promising anti-HF agent. In future studies, we plan to assess whether GA has beneficial effects on alcohol-induced HF in mice, a widely accepted and validated in vivo model for experimental studies of HF.

Funding

This research was supported by the National Natural Science Foundation of China, No. 82174271.

Acknowledgments

The authors thank the Head of the Science and Technology Innovation Center/State Key Laboratory Breeding Base of Chinese Medicine Powder and Innovative Medicine, Yuhong Wang, for the possibility to use the equipment for the crucial experiments for the paper (Science and Technology Innovation Center/State Key Laboratory Breeding Base of Chinese Medicine Powder and Innovative Medicine, Hunan University of Chinese Medicine, Changsha 410125, China) and Wang Zheng (College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410208, China) for the donated rat hepatocyte BRL-3A cells.

Competing interests

The authors declare no conflict of interest.

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