The Inhibitory Effect And Mechanism Of Notoginsenoside R1 On Hepatic Stellate Cells Activation In Liver Fibrosis

Hepatic fibrosis is a dynamic process.Chronic liver damage caused by various etiology,such as hepatitis virus,alcohol,and non-alcoholic steatohepatitis,can lead to liver fibrosis.Liver fibrosis is essentially an excess of extracellular matrix synthesis and reduced degradation.Hepatic stellate cell（HSC）activation has been recognized as the main driver of liver fibrosis.HSC are liver fat-storage cells,which store a large number of lipid droplets in quiescent state.When stimulated by various factors,they are activated into myofibroblasts,and their proliferation,migration,contraction,and pro-fibrotic phenotypes are enhanced.With the continuous discovery of new pathways and new mediators such as autophagy,endoplasmic reticulum stress,oxidative stress,retinol and cholesterol metabolism,epigenetics,etc.,the complexity of HSC activation has been revealed.Among these intercellular or intracellular mediators and signaling that can activate HSC,many are potential targets for anti-fibrotic therapy.Notoginsenoside R1（NR1）,extracted from traditional Chinese medicine Sanqi,has the effect of anti-inflammatory and oxidative stress.It is speculated that NR1 may have a significant inhibitory effect on liver fibrosis.Objective:（1）To verify whether NR1 has anti-hepatic fibrosis effect;（2）To clarify whether NR1 can inhibit the activation of HSC,and whether inflammatory response,oxidative stress response,fatty acid metabolism,etc.play roles in activating HSC.（3）To find out the direct binding pattern of NR1 to inhibit the activation of HSC.Methods and Results:（1）Liver fibrosis murine models were successfully established by intraperitoneal injection of Carbon tetrachloride（CCl₄）,and then NR1 was conducted to treat liver fibrosis.At the end of the experiment,the weight of the mice in NR1 groups increased significantly compared with liver fibrosis group;ALT and AST of the mice in NR1 groups were significantly lower than those in liver fibrosis group;H&E staining showed that the livers of the mice in NR1 groups were not significantly damaged,and the inflammatory infiltration was light;Sirius Red and Masson staining both showed that the livers of the mice in NR1 groups were basically intact with less collagen deposition.WB and q PCR experiments verified HSC activation-related indicators in liver tissues.The results showed thatα-SMA and COL1A1 in NR1-treated mice were significantly decreased at both m RNA and protein levels.This part of the experiment verified that NR1 can improve the nutritional level of mice,reduce inflammation,inhibit the activation of HSC in liver tissues,reduce the degree of liver fibrosis,and protect the liver in vivo.（2）In vitro model was constructed by stimulating JS-1,LX-2 and primary mouse hepatic stellate cell with TGF-β1.The results showed that NR1 could effectively inhibit the expression ofα-SMA and COL1A1 via q PCR,WB,and immunocytofluorescence（ICF）.In addition,NR1 could inhibit HSC migration and proliferation by scratch assay,CCK-8proliferation assay,etc.（3）Differentially expressed genes were obtained through transcriptome sequencing analysis.Differentially expressed genes were found to be mainly enriched in inflammatory response（|NES|=1.9,p<0.001,FDR<0.001）,oxidative stress（|NES|=|-1.6|,p=0.001,FDR=0.011）and lipid regulation（|NES|=|-1.78|,p=0.000,FDR=0.016）and other biological functions or signaling pathways via Gene Set Enrichment Analysis（GSEA）.q PCR,WB and ICF experiments were involved to verify the genes and signaling pathways.1)NR1 can inhibit the nuclear translocation of NF-κb,reduce the phosphorylation of NF-κB,IκBα,and P38,and inhibit the activation of HSC;2)The expression of antioxidant genes including Sirt1,HO-1,etc.were up-regulated after NR1 intervention;3)The expression of PPAR-γwas down-regulated after TGF-β1 stimulation,was up-regulated after NR1intervention,which was negatively correlated with the expression ofα-SMA and COL1A1.It revealed that the TGF-β1/Smads signaling pathway and the fibrotic phenotype also changed after down-regulating and up-regulating PPAR-γexpression by using PPAR-γselective inhibitors and agonists,respectively.This indicated that PPAR-γmediates NR1 to inhibit the activation of HSC by inhibiting the TGF-β1signaling pathway,and plays an anti-fibrotic effect.（4）Docking between NR1 and PPAR-γmolecules by predicting the binding partern and predicting the power of affinity.Conclusion:NR1 can effectively inhibit the activation of HSC and play a role in improving liver fibrosis in vivo and in vitro.NR1 exerts anti-fibrotic effects by inhibiting hepatic inflammatory response,oxidative stress and regulating lipid metabolism.As an important lipid-regulating gene,PPAR-γmediates the regulation of TGF-β1/Smads signaling pathway by NR1,and further plays an anti-fibrotic role.We achieved molecular docking of NR1 with PPAR-γ.