Proanthocyanidin A2 attenuates the activation of hepatic stellate cells by activating the PPAR-γ signalling pathway

Abstract Liver fibrosis is the pathological process of chronic liver diseases induced by hepatic stellate cells. Proanthocyanidin A2 (PA2) has multiple pharmacological activities. In this study, we aimed to explore the effects of PA2 on hepatic stellate cell (HSC) activation in liver fibrosis. LX-2 cells were treated with TGF-β1 to establish a fibrosis cell model. Cell viability was evaluated using cell counting kit-8. The levels of fibrosis-related factors (collagen I, fibronectin, and α-SMA) were examined using quantitative real-time polymerase chain reaction, western blot, and immunofluorescence assay. The molecular mechanisms of PA2 were evaluated by RNA-seq, bioinformatic analysis, and western blot. The results showed that PA2 suppressed cell viability, and downregulated fibrosis-related factors induced by TGF-β1, suggesting PA2 suppressed the activation of HSCs. PA2 treatment-induced differentially expressed mRNAs are predicted to be associated with the PPAR-γ pathway. PA2 reversed the downregulation of PPAR-γ and the upregulation of phosphorylated (p)-Smad2 and Smad3. A rescue experiment illustrated that the inactivation of the PPAR-γ pathway reversed the effects of PA2 on cell viability and HSC activation. In conclusion, PA2 inhibited TGF-β1-induced activation of HSCs by activating the PPAR-γ/Smad pathway. The findings suggested that PA2 may be an effective treatment for liver fibrosis.


Introduction
Liver fibrosis is a pathological process of almost all chronic liver diseases [1].Early hepatic fibrosis is reversible, while late hepatic fibrosis is the main risk factor to cause liver cancer that threat to human health seriously [2].Hepatitis B and C virus infections are common causes of fibrosis, which can also be induced by alcohol, fatty liver disease, hepatitis, and cholestasis [3].Changing diet, strengthening exercise, and controlling metabolic syndrome can help slow the progression of fibrosis [4].However, there is no standard cure for advanced liver fibrosis.The most typical characteristic of liver fibrosis is the excessive accumulation of extracellular matrix (ECM).When the liver is damaged, hepatic stellate cells (HSCs) are activated to promote the secretion of ECM by liver myoblasts, leading to fibrous scarring [5][6][7].Thus, inhibiting the activation of HSCs and promoting ECM degradation may play an anti-fibrosis effect.However, the pathological role and mechanism of HSCs in liver fibrosis have not been fully elucidated.
Proanthocyanidin A2 (PA2) belongs to the tea polyphenol family and has a variety of biological activities, such as anti-virus, anti-inflammation, anti-oxidation, and anti-cancer [8,9].Previous studies showed that PA2 plays a central role in several diseases, including liver disease, vascular proliferative diseases, and virus-infected diseases [10][11][12].PA2 can be isolated from flowers, seeds, and pericarp of litchi [9,13,14].However, the involvement of PA2 influences in the progression of liver fibrosis is unknown, Thus, we sought to evaluate the impacts of PA2 on the activation of HSCs in liver fibrosis.
In addition, the transforming growth factor (TGF)-β1/ Smad pathway is involved in liver fibrosis.When the liver is damaged, inflammatory factors including TGF-β1 are released to further activate HSCs in vitro and in vivo [21].Moreover, PPAR-γ has been reported to suppress the activation of the TGF-β1/Smad signalling pathway in HSCs, thereby attenuating fibrosis [22].Thus, the effects of PA2 on the regulation of the PPAR-γ/Smad pathway in HSCs needed to be explored.
In this study, based on the biological activities of PA2, we speculated that PA2 attenuated liver fibrosis via inactivating the HSCs by regulating the PPAR-γ pathway.The findings suggested that PA2 might be a novel drug for liver fibrosis treatment.

Determination of cell survival rate
LX-2 cells were seeded into 96-well plates at 2000 cells/well concentration.Subsequently, the cells were treated with various concentrations of PA2 (0, 25, 50, 100, and 200 μM) with or without 5 ng/mL TGF-β1 for 24 h [23].To inhibit the PPAR-γ pathway, the LX-2 cells were treated with 5 μM T0070907 for 24 h.Then, cell viability was examined.CCK-8 solution (10 μL) was added to each well and incubated with the cells for 4 h.The absorbance value per well was detected at 450 nm.
For detecting protein levels, LX-2 cells were lysed in RIPA buffer.After measuring protein concentration using the BCA kit, cell lysates were run on the 10% SDS-PAGE and transferred to PVDF membranes.The membranes were incubated with specific primary antibodies at 4 °C overnight.Then, the membranes were incubated with a secondary antibody for 1 h at room temperature.The band signals were visualised using the ECL reagent.

Immunofluorescence assay
Cell slides were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and transparent with 0.2% Triton X 100 for 10 min.After blocking using normal goat serum, the sliders were incubated with anti-fibronectin (ab268020, 1:50, Abcam) at 4 °C overnight and incubated with goat anti-rabbit IgG (ab150077, 1:500, Abcam) at 37 °C for 1.5 h.DAPI is used to analyse nuclear localisation.After the sliders were sealed, the results were observed under a confocal laser microscope (KEYENCE, Osaka, Japan).

RNA sequencing
LX-2 cells were treated with PA2 and distilled water (negative control).Total TNA was isolated from LX-2 cells.The purity of total RNA was measured by ultraviolet spectrophotometer (Thermo Fisher Scientific, Waltham, USA), and the integrity was evaluated by agarose gel electrophoresis.RNA was purified using the Dynabeads mRNA purify kit.Then, the cDNA library was constructed by reverse transcribing cDNA first and second chains.Finally, the sequencing was performed on an Illumina HiSeq 4000 according to the manufacturer's protocol.

Bioinformatic analysis
The KEGG enrichment analysis of differentially expressed mRNAs and the related genes of the PPAR-γ pathway was predicted using the INPUT platform (http://cbcb.cdutcm.edu.cn/INPUT/Home/).

Statistical analysis
The data in this study are presented as the mean ± SD by analysis using the GraphPad Prism 7.0 software.Student's t-test and one-way ANOVA were used for compared the differences between groups.p < 0.05 was the statistical significance.

PA2 inhibits cell survival
To screen the concentrations of PA2, LX-2 cells were treated with 25, 50, 100, and 200 μM PA2, and cell survival was evaluated by CCK-8 assay.The results indicated that compared with the control group, PA2 did not affect cell survival at the concentrations of 25 and 50 μM, and significantly suppressed cell survival at 100 and 200 μM in a dosedependent manner (Figure 1(B)).Thus, LX-2 cells treated with 50 μM PA2 were used in the following study.

PA2 inhibits TGF-β1-induced activation of HSCs
Subsequently, the effects of PA2 on HSC activation were investigated.Cell viability was markedly increased by TGF-β1 treatment, and PA2 inhibited cell viability (Figure 2(A)).The levels of fibrosis markers including collagen I, fibronectin, and α-SMA were elevated by TGF-β1, which were abrogated by PA2 (Figure 2(B-E)).In addition, the levels of fibronectin were also evaluated by IF staining assay and the results showed that TGF-β1 increased fibronectin levels, while PA2 reversed the increase (Figure 2(F)).

PA2 promotes the activation of the PPAR-γ signalling pathway
To explore the underlying mechanism, RNA-seq was performed in LX-2 cells treated with PA2 and distilled water.The results showed that there are multiple mRNAs are differentially expressed.The top ten upregulated mRNAs and downregulated mRNAs were shown in the heatmap (Figure 3(A)).The data illustrated that these differentially expressed mRNAs are related to the PPAR-γ signalling pathway, insulin resistance, fatty acid metabolism, and primary bile acid biosynthesis (Figure 3(B)).Moreover, the results of KEGG analysis indicated that the PPAR-γ signalling pathway is related to several mRNAs, including HMGCS1, FADS2, ACADM, SLC27A5, RXRG, APOA5, CPT1A, and FABP1 (Figure 3(C)).The protein levels of PPAR-γ were downregulated by TGF-β1 treatment and were reversed by PA2 (Figure 3(D)).The levels of PPAR-γ-regulated Smad pathway-related factors were also examined.TGF-β1 treatment increased the levels of p-Smad2, and p-Smad3, while PA2 abrogated the increase in their levels (Figure 3(D)).The levels of Smad2 and Smad3 were not regulated by TGF-β1 and PA2 (Figure 3(D)).

PA2 inhibits TGF-β1-induced HSC activation via the PPAR-γ pathway
Finally, a rescue experiment was performed.T0070907 was added to treat LX-2 cells to inactivate the PPAR-γ pathway.PA2 inhibited cell viability, which was promoted by T0070907 (Figure 4(A)).The mRNA and protein levels of collagen I, fibronectin, and α-SMA downregulated by PA2 were abrogated by T0070907 (Figure 4(B-E)).The fluorescence signal of fibronectin was decreased by PA2 and was elevated by T0070907 (Figure 4(F)).

Discussion
Liver fibrosis is the main cause of death from chronic liver diseases.Effective and specific anti-fibrosis drugs have significant benefits for the treatment of many liver diseases [24].PA2 has a variety of biological activities, indicating its potential value in various diseases.For example, because of its anti-adhesion effect, PA2 isolated from cranberry inhibits the adhesion of Escherichia coli and Proteus mirabilis to urinary  epithelial cells, thereby alleviating urinary tract infection [25].Additionally, suppresses IL-4-induced CCL26 production, leading to difficulty maintaining and progressing allergic asthma disease [26].Moreover, PA2 decreases proinflammatory cytokine levels and suppresses oxidative stress of LPS-treated Raw264.7 cells, protecting cells from inflammatory damage by regulating the NF-κB, MAPK, and Nrf2 signalling pathways [27].However, most research has focused on how to efficiently extract PA2 from a variety of plants [28,29], and there is still little research on PA2 in human diseases.At present, there is no relevant study on whether it can play a role in the progression of liver fibrosis.Litchi has a good effect on relieving symptoms of various diseases, such as chest tightness, flatulence, cough, neuralgia, etc. [30].
Researchers are concerned about the presence of polyphenols in the peel, such as epicatechin and procyanidin [30,31].PA2 is enriched in Litchi.Thus, we focused on the role of PA2 in liver fibrosis.In view of the important role of HSCs in the progression of hepatic fibrosis, we studied the effect of PA2 on HSC activation.Collagen I, fibronectin, and α-SMA are critical markers of activated HSCs, which levels are upregulated in activated HSCs in response to liver injury [32].In the present study, the results illustrated that PA2 inhibited cell viability and downregulated the levels of fibrosis markers induced by TGF-β1.The results suggest that PA2 inactivated the HSC activation and has an anti-liver fibrotic effect.
To explore the molecular mechanism by which PA2 acted.Through RNA-seq analysis, we found that PA2 induced differential expression of several genes.Among these genes, the top 10 up-regulated and the top 10 down-regulated genes can be significantly enriched into the PPAR-γ pathway.Thus, we sought to identify the role of the PPAR-γ signalling pathway in hepatic fibrosis.PPAR-γ pathway is associated with liver diseases, including fatty liver, hepatitis, and cholestatic liver diseases [33,34].PPAR-γ pathway is involved in inflammation, lipid metabolism, and senescence in liver fibrosis [19].Multiple studies have shown that PPAR-γ is involved in the activation of HSCs.For instance, promoting the activation of the PPAR-γ pathway inhibits the activation and autophagy of HSCs, then attenuates CCl4-induced liver fibrosis [31].Methoxyeugenol activates PPAR-γ to inhibit HSCs activation, leading to the inhibition of cell proliferation, contractility loss, and downregulation of fibroblast markers [20].Additionally, PPAR-γ agonist inhibits the activation of HSCs, thereby attenuating liver fibrosis and inflammation [35].PPAR-γ plays a crucial role in fibrosis diseases of multiple organs by modulating the downstream pathways, such as MAPK, TGF-β, and NF-κB signalling pathways [36].The TGF-β/Smad pathway is involved in renal, hepatic, pulmonary and cardiac fibrosis.TGF-β1 directly activates downstream Smad signalling, including Smad2 and Smad3, inducing pro-fibrotic gene overexpression [37].Several studies have revealed that the TGF-β/Smad pathway is blocked when HSC activation is inhibited [23,38].In this study, TGF-β1 decreased PPARγ levels and increased p-Smad2 and p-Smad3 levels, whereas PA2 reversed the effects of TGF-β1, suggesting that PA2 inactivating the PPAR-γ/Smad pathway.Moreover, inactivation of the PPAR-γ pathway abrogated the suppression of viability and fibrosis, suggesting that PA2 attenuated liver fibrosis by activating the pathway.
In conclusion, the results of this study first showed that PA2 attenuated TGF-β1-induced activation of HSCs by activating the PPAR-γ/Smad signalling pathway.The findings provide solid theoretical support for the application of PA2 in the treatment of hepatic fibrosis.However, we only studied the PPAR-γ/Smad pathway as the underlying mechanism of PA2.Whether other members of PPARs are involved in HSC activation remains further studied.

Figure 3 .
Figure 3. PA2 promotes the activation of the PPAR-γ signalling pathway.(A) the heatmap showed that the top ten upregulated and top ten downregulated mRnAs were analysed using RnA-seq.(B) the enrichment analysis of the Dysregulated mRnAs was analysed by the bioinformatic analysis.(C) the PPAR-γ signalling pathway-related factors were predicted using the bioinformatic analysis.(D) the protein levels of PPAR-γ, Smad2, p-Smad2, Smad3, and p-Smad3 were measured.n = 3.