Based On TGF-β₁/Smad And NF-κB Dual Signaling Pathways To Explore The Mechanism Of Anti-hepatic Fibrosis Of Panax Stipuleanatus

Panax stipuleanatus is the root and rhizome of Panax stipuleanatus H.T.Tsai et K.M.Feng,a plant of the genus Panax ginseng,which has the relieving effect of inflammation,pain and liver protection even resistance to cancer".It contains oleanane type pentacyclic three The terpenoid saponin component having a good effect on the treatment of hepatitis.At present,Panax stipuleanatus is a kind of medicinal plant resources with high economic value to be developed.Previous studies have found that Panax stipuleanatus saponins have hepatoprotective effect,but its mechanism of function is still unclear.Therefore,this article examines the mechanism that how Pingbian notoginsenoside protect the liver.On the one hand,using the classic inflammatory cell model lipopolysaccharide（LPS）to induce mouse macrophages Raw264.7 cells to explore the effect of Panax stipuleanatus saponins on NF-κB pathway especially,the influence of related proteins and cytokines on this pathway.On the other hand,using activated rat hepatic stellate cells（HSC-T6）and carbon tetrachloride（CCl₄）-induced rat liver fibrosis model was investigated in vitro and in vivo.Saponin inhibits liver fibrosis mediated by TGF-β₁/Smad signaling pathway.The main research contents and results of this study are as follows:1.Effects of Panax stipuleanatus saponins（PS）,Panax stipuleanatus saponins R₁（PS-R₁）and Panax stipuleanatus saponins R₂（PS-R₂）on LPS-induced NF-κB signaling pathway in Raw246.7 cells.1.1 The effect of Panax stipuleanatus saponins on the survival rate of Raw264.7cells.PS at a concentration of 0.02～1 mg/mL has no inhibitory effect on cell survival rate.PS-R₁ and PS-R₂at a concentration of 1～30μmol/L has no inhibitory effect on cell survival rate.When the concentrations of PS-R₁ and PS-R₂ reach 50μmol/L,the cell activity is inhibited to a certain extent.1.2 The inhibitory effect of Panax stipuleanatus saponins on LPS-induced production of nitric oxide（NO）in Raw264.7 cells.PS at a concentration of 0.02～0.5mg/mL can significantly reduce the NO content secreted by Raw264.7 cells（p<0.05,p<0.01,p<0.001）,and there is a certain dose dependence.PS-R₁ can significantly reduce the NO content secreted by Raw264.7 cells at a concentration of 1-30μmol/L（p<0.05,p<0.01）,while PS-R₂ has no inhibitory effect.1.3 The effect of Panax stipuleanatus saponins on LPS-induced Raw264.7 cells to produce tumor necrosis factor（TNF）-αcontent changes.PS at a concentration of0.1～0.5 mg/mL,PS-R₁ and PS-R₂at a concentration of 1～30μmol/L all have a down-regulating effect on the content of TNF-αin the supernatant of Raw264.7 cells induced by LPS（p<0.05,p<0.01）.1.4 The effect of Panax stipuleanatus saponins on the transcription factor（NF-κB）and interleukin IL-1βmRNA levels in Raw264.7 cells induced by LPS.PS（0.5mg/mL）inhibited the mRNA expression of IL-1β（p<0.001）,but had no significant inhibitory effect on NF-κB;PS-R₁（30μmol/L）and PS-R₂（30μmol/L）Significantly inhibited the mRNA expression of NF-κB and IL-1βin cells（p<0.01）.1.5 Effect of Panax stipuleanatus saponins on NF-κB protein expression in Raw264.7 cells induced by LPS.PS inhibited the expression of nuclear NF-κB protein in Raw264.7 macrophages and was concentration-dependent.When the concentration reaches 0.5 mg/mL,its inhibitory effect is most significant.PS-R₁and PS-R₂ had no significant inhibitory effect on the expression of nuclear NF-κB protein.2.Effect of Panax stipuleanatus saponins on TGF-β₁/Smad signaling pathway in rat hepatic stellate cells2.1 The effect of Panax stipuleanatus saponins on the survival rate of rat hepatic stellate cells（HSC-T6）.PS at a concentration of 0.02～1 mg/mL had no significant inhibitory effect on the growth and growth of HSC-T6 cells;PS-R₁ had no significant inhibitory effect on HSC-T6 cells at a concentration of 0.05～30μmol/L.When the concentration reached 50μmol/L,it had a certain inhibitory effect on the growth and growth of HSC-T6 cells;PS-R₂ had no significant inhibitory effect on HSC-T6 cells when the concentration was 1～100μmol/L.2.2 The effect of Panax stipuleanatus saponins on transforming growth factor-β₁（TGF-β₁）,smooth muscle protein（α-SMA）,Smad3 and Smad4 mRNA levels in HSC-T6 cells.When the concentration of PS is 0.1～0.5 mg/mL,the mRNA levels ofα-SMA,Smad3 and Smad4 in HSC-T6 cells are significantly reduced,and it has a certain concentration dependence（p<0.05,p<0.01,P<0.001）.PS at a concentration of 0.5mg/mL had a significant inhibitory effect on TGF-β₁mRNA expression（p<0.001）,but had no effect at a low concentration.PS-R₁ significantly reducedα-SMA mRNA levels in HSC-T6 cells at a concentration of 1～30μmol/L（p<0.01,p<0.001）.At a high concentration of 30μmol/L,PS-R₁ significantly reduced the levels of TGF-β₁,Smad3,and Smad4（p<0.05,p<0.001）.PS-R₂ had a significant down-regulation effect onα-SMA mRNA levels in HSC-T6 cells at a concentration of 0.5～30μmol/L（p<0.01,p<0.001）,but had no significant effect on TGF-β₁mRNA levels.At a high concentration of 30μmol/L,PS-R₂ significantly inhibited the levels of Smad3 and Smad4 mRNA in HSC-T6 cells（p<0.05,p<0.01）.2.3 The effect of Panax stipuleanatus saponins on the expression ofα-SMA,Smad3,Smad4 and NF-κB protein in HSC-T6 cells.Western Blot analysis showed that PS at a concentration of 0.1～0.5 mg/mL significantly reduced the expression of Smad3,α-SMA,and NF-κB protein in HSC-T6 cells in a dose-dependent manner.There is no obvious inhibitory effect on Smad4 protein expression.PS-R₁and PS-R₂at the concentration of 1～30μmol/L,significantly reduced the expression of Smad3 protein in HSC-T6 cells,and has a certain concentration dependence.PS-R₁ and PS-R₂ have a certain inhibitory effect on the protein expression ofα-SMA and Smad4 in HSC-T6cells.2.4 The molecular docking of Panax stipuleanatus saponins with proteins Smad3and Smad4.The results showed that PS-R₂ had the best binding effect with Smad3protein and the highest affinity.PS-R₂ binds to the active site of Smad3 protein and has a good match with the active site protein.Gln386,Val 385,Asn400,Thr 246,Ser235 and Tyr 237 that fit into the active site form hydrogen bond interactions.These interactions promote The small molecule stably binds to the active site of the protein to exert biological activity.3.The effect of Panax stipuleanatus saponins on TGF-β₁/Smad signaling pathway in CCl₄-induced liver fibrosis rats.3.1 The effect of Pingbian Notoginsenoside on ALT and AST in serum of rats with hepatic fibrosis induced by CCl₄.Compared with the blank group,the serum levels of ALT and AST in the model group increased significantly（p<0.001）.Compared with the model group,the PS（100,150,200 mg/kg）of each dose administration group significantly reduced the content of ALT and AST（p<0.05,p<0.01,p<0.001）.PS-R₂medium-dose group（10 mg/kg）and high-dose group（30 mg/kg）significantly reduced the content of ALT and AST（p<0.01,p<0.001）,and there was no significant difference in the low-dose group.3.2 The effect of Panax stipuleanatus saponins on CCl₄ induced liver fibrosis in rat liver pathological sections.The pathological section analysis of rat liver histopathology showed that in the blank group,hepatocytes had normal morphology,no fibrous deposition and no pathological changes;howver,in the model group,hepatocytes were disordered,hepatocyte necrosis was obvious,and fibrous tissue hyperplasia in the confluent area was significant as well.There were obvious pathological changes.Compared with the model group,the proliferation of fibrous connective tissue in the manifold of rats in the low-dose group of PS and PS-R₂（100,1 mg/kg）was slightly reduced;the medium-and high-dose group of PS（150,200mg/kg）and PS-R₂ medium and high dose groups（10,30 mg/kg）,there was only a small amount of fibrous tissue hyperplasia in the rat liver confluent area,hepatocyte degeneration and necrosis was significantly reduced,and a small amount of lymphocyte infiltration;among them,the high dose liver injury was less severe than the positive group.3.3 The effect of Panax stipuleanatus saponins on the expression of NF-κB and p-NF-κB protein in the NF-κB signaling pathway in liver tissue of CCl₄-induced liver fibrosis rats.Western Blot analysis showed that compared with the model group,PS at a concentration of 100-200 mg/kg significantly reduced the expression of NF-κB and p-NF-κB protein in liver tissue.At a concentration of 1-30 mg/kg,PS-R₂significantly reduced the protein expression of NF-κB and p-NF-κB in liver tissue,and showed a concentration-dependent manner.3.4 The effect of Panax stipuleanatus saponins on the expression of Smad3 and Smad4 protein in TGF-β₁/Smad signaling pathway in liver tissue of CCl₄-induced liver fibrosis rats.Compared with the model group,at the concentrations of 100-200 mg/kg and 1-30 mg/kg of PS and PS-R₂ respectively,significantly reduced the protein expression of Smad3 and Smad4 in liver tissue,and had a certain effect as concentration dependence.3.5 Panax stipuleanatus saponins immunohistochemical results of TGF-β₁,Smad3and Smad4 protein in the TGF-β₁/Smad signaling pathway in liver tissue of CCl₄-induced liver fibrosis rats.Immunohistochemical test results showed that compared with the blank group,the TGF-β₁ and Smad3/4 protein levels in the rat liver of the model group were significantly increased compared with the model group,in the PS high-dose group（200 mg/kg）TGF-β₁,Smad3/4 protein levels were administered significantly down-regulate,while the inhibition of PS-R₂ to these protein was not significant.In summary,the total saponins of Panax stipuleanatus and its monomeric saponins have an inhibitory effect on liver fibrosis,which may be related to the TGF-β₁/Smad and NF-κB dual signaling pathways.