Ginsenoside Rg1 Rsgulatesns The Progression Of Hepatic Fibrosis Via TGF-B/samd Signaling Pathway

Hepatic fibrosis?HF?is a transitional deposition of extracellular matrix?ECM?caused by various factors such as viruses,alcohol,drugs,and immune damage.It is an inevitable development of liver cirrhosis and liver cancer.Process[1].However,there is currently no effective drug for treating liver fibrosis in the clinic,which has attracted widespread attention from researchers at home and abroad,and has become a research hotspot and a difficult point in recent years.Panax ginseng is a traditional Chinese medicine with anti-inflammatory[2],anti-cancer[3] and anti-aging[4].The study found that ginsenoside Rg1?G-Rg1?has anti-inflammatory,anti-apoptotic and anti-oxidation effects,which proves that it has liver and anti-fibrosis effects[5].A large number of studies suggest that Oxidative stress?OS?directly promotes the activation of HSC and promotes the secretion of ECM in the progression of fibrosis;under the stimulation of pathological factors,excessive reactive oxygen species?ROS?accumulate,Lead to excessive deposition of ECM,progressing to liver fibrosis.Parola et al [6]believe that ROS play a key role in liver fibrosis.At the same time,it is considered that G-Rg1 inhibits HSC proliferation and activation and inhibits fibrosis by inhibiting ROS[7].The study found that ginsenoside Rg1 significantly increased the expression of antioxidant enzymes and mediates Nrf2 expression and nuclear translocation against rat liver fibrosis in a rat model of alcohol and CCl₄-induced liver fibrosis[8].The study found that EMT plays an important role in liver fibrosis,and preventing the development of EMT can reverse liver fibrosis [9].It is reported in the literature that ginsenoside Rg1 has anti-rat renal interstitial fibrosis by inhibiting EMT [10],which is shown to reduce alpha smooth muscle actin??-SMA?and increase Expression of E-cadherin.TGF-?-induced EMT is mainly dependent on the Smad pathway [11-12].We speculate whether G-Rg1 can exert liver fibrosis through TGF-?/Smad.We constructed a CCl₄-induced liver fibrosis animal model and a TGF-?-induced HSC-T6 liver fibrosis cell model to explore and preliminarily explain the epithelial-interval of G-Rg1 through the TGF-?/Smad signaling pathway in hepatic stellate cells.Metabolism and anti-fibrosis,while also regulating the intracellular oxidative stress and the transport of Nrf2 in the cytoplasm and nucleus,while fibrosis.In this study,we explored the possibility of G-Rg1 becoming a first-line drug for liver fibrosis treatment through experimental research,and provided a new treatment strategy and new treatment ideas for the treatment of liverfibrosis.Chapter 1 Intervention of ginsenoside Rg1 on CCl₄-inducedHepatic fibrosis in a mice modelObjective: Although the effects of ginsenoside Rg1 on hepatic fibrosis have been reported,there is no definitive conclusion.To clarify this process and its possible mechanism,we explored the G-Rg1 in the animal model of liver fibrosis induced by CCl₄.The prevention and treatment of liver fibrosis.Methods: Twenty male Kunming mice aged 4-6 weeks were randomly divided into 4 groups: normal control group?5 rats?,randomly divided into blank control group G-Rg1 low dose group?30 mg/kg?,G-Rg1 high dose group?60mg/kg?and CCl₄ model group,G-Rg1 low dose group?30mg/kg?after 4 weeks of adaptive feeding,,G-Rg1 high dose group?60mg/kg?and CCl₄ model group were injected intraperitoneally at 3ml/kg 10% CCl₄?diluted with olive oil?while high dose group and low dose group were injected with the specified amount of G-Rg1 The body weight of the mice was recorded at the time of Rg1 administration.At the 9th week after the administration,the mice were fasted for 12 hours,then sacrificed by cervical dislocation,and serum and liver tissue samples were collected.Hepatic tissue samples were observedby HE and Masson staining.Alanine aminotransferase?ALT?,aspartate aminotransferase,type IV collagen,hyaluronic acid and laminin were detected by ELISA.The content of LN was detected by immunohistochemistry in the expression of TGF-?,Collagen I and E-cadherin in liver tissue,and the expression of ?-SMA in liver tissue was detected by Western Blot.Results: After 9 weeks of intraperitoneal injection of CC14,the mice in the hepatic fibrosis model group lost weight,while the G-Rg1 treatment group showed significant improvement,and there was a significant dose-effect relationship.HE and Masson staining showed that the model group and the G-Rg1 treatment group showed obvious hepatic tissue disorder and obvious fiber deposition in the portal area.MRI results also support this conclusion.ELISA showed that G-Rg1 could significantly reverse the serum levels of ALT,AST,HA,LN and IV-C in liver fibrosis mice.Immunohistochemistry showed that G-Rg1 could regulate the expression of EMT pathway-related proteins,and Western Blot results showed that G-Rg1 can reduce the expression of ?-SMA in hepatic fibrosis mice.Conclusion: CCl₄ can induce acute Hepatic fibrosis in mice,and G-Rg1 can significantly inhibit CCl₄-induced fibrosis.G-Rg1 can significantly increase the expression of E-cadherin,Collagen I/III and TGF-?protein in liver of mice with hepatic fibrosis.G-Rg1 regulatesCCl₄-induced hepatic fibrosis in mice and may be associated with the EMT pathway.Chapter 2 explores the effect of ginsenoside Rg1 onTGF-?-mediated proliferation and apoptosis of hepaticstellate cells.Objective: TGF-? plays an important role in hepatic fibrosis.Many literatures have shown that TGF-? can affect the proliferation of HSC cells,while the effect of G-Rg1 on TGF-?-induced proliferation is not clear.A TGF-?-induced proliferation model was used to examine the effect of G-Rg1 on TGF-?-mediated HSC cell proliferation and apoptosis.Methods:HSC-T6 cells were treated with different concentrations of TGF-?.Cell viability was determined by CCK-8 method after 24 h.The effects of different concentrations of G-Rg1 on TGF-?-mediated HSC-T6 and blank control group were compared.After HSC-T648 h,the cell viability was determined by CCK-8 method,and the half-inhibitory concentration of G-Rg1 was calculated.After treating HSC-T6 cells with G-Rg1,the cells were collected and stained for cell apoptosis by flow cytometry.At the same time,the expression of apoptosis-related proteins Bax and Bcl-2 were determined by Western Blot.Finally,the cells were divided into Control group,G-Rg1 group,TGF-? group and G-Rg1 TGF-?group.The cells were collected and stained for flow.Apoptosis rate was determined by cytometry.Results:TGF-? significantly increased the proliferation of HSC-T6 cells in a dose-dependent manner at a concentration of 0-4 ng/ml.G-Rg1 significantly reversed TGF-?-induced HSC-T6 cell proliferation and passed through the cells.Morphology can clearly observe the transformation of TGF-?-treated cells into spindle-shaped epithelial cells;flow-through results show that G-Rg1 can significantly promote the apoptosis of HSC-T6 cells,and G-Rg1 can promote the expression of apoptosis protein Bax.Inhibition of anti-apoptotic protein Bcl-2 expression;and by comparing the changes in apoptotic rate of differently treated HSC-T6 cells,it was found that G-Rg1 can significantly induce apoptosis in the presence of TGF-?.Conclusion:TGF-? can significantly promote the growth of HSC-T6cells;G-Rg1 can protect the liver by inducing Bax expression and inhibiting the expression of Bcl-2 to induce apoptosis of HSC-T6 cells.Chapter 3 Ginsenoside Rg1 inhibits Hepatic fibrosis viaTGF-?/Smad signaling pathway in vitroObjective:We have demonstrated that G-Rg1 in vivo can attenuate CCl₄-induced fibrosis and inhibit TGF-?-induced proliferation in vivo,butthe specific mechanism is unclear.This section detects G-Rg1 in TGF-?-stimulated HSC-T6 cells.The role of the EMT signaling pathway,while verifying that G-Rg1 can affect the intracellular redox reaction,and finally affect the progression of Hepatic fibrosis.Methods:HSC-T6 cells were treated with 1 ng/ml TGF-?,and then the cell lysates were collected.The expression of Collagen I,Collagen III and TGF-? was determined by Western Blot after the concentration was determined.The expression of smad2/3,total smad2/3,E-cadherin and Vimentin was detected by phosphorylation of related proteins,and the cellular localization and expression changes of Collagen I and E-cadhein were observed by cell immunofluorescence;40?g/ml G-Rg1 was used to treat HSC-T6 cells,and then cell lysates were collected.Western blot was used to determine the expression changes of fibrosis-related maker proteins Collagen I,Collagen III and TGF-?.Epithelial-mesenchymal transition-related protein maker phosphorylated smad2/3,total The expression of smad2/3,E-cadherin and Vimentin was changed,and the cellular localization and expression changes of Collagen I and E-cadhein were observed by cellular immunofluorescence.The ROS levels of different treated HSC-T6 cells were determined by CM-H2 DCFDA probe method.Changes;cytoplasmic proteins and nuclear proteins were extracted by cytoplasmic nuclear protein extraction kit,and the changes of Nrf2 were determined by Western Blot.Results : After treatment with TGF-?,the protein levels of the fibrosis-related proteins Collagen I,Collagen III and TGF-? were significantly up-regulated,and the phosphorylated smad2/3 was significantly increased,the E-cadherin protein level was significantly down-regulated,and the Vimentin protein level was significantly decreased.Significantly up-regulated;cellular immunofluorescence results confirmed this result;after treatment of HSC-T6 cells with G-Rg1,the protein levels of the fibrosis-related proteins Collagen I,Collagen III and TGF-? were significantly down-regulated,and phosphorylated smad2/3 was evident.Decreased,E-cadherin protein levels were significantly up-regulated,while Vimentin protein levels were significantly down-regulated;cellular immunofluorescence results confirmed this result;and CM-H2 DCFDA probe method results showed that TGF-? significantly up-regulated oxidation in HSC-T6 cells Level,while G-Rg1 can make the intracellular redox level balance;at the same time,G-Rg1 can promote the transport of Nrf2 protein from the cytoplasm to the nuclear,and regulate the level of cellular redox.Conclusion:TGF-? can promote the EMT process of HSC-T6 cells and induce cell fibrosis.G-Rg1 can inhibit the decrease of E-cadhein protein level induced by TGF-?and increase the Vimterin protein,indicating that G-Rg1 can significantly inhibit TGF-?-induced EMT and Fibrosis;G-Rg1 can reduce the level of ROS in HSC-T6 cells,and alsopromote the transport of Nrf2 to the nucleus.G-Rg1 regulates TGF-?-induced fibrosis of HSC-T6 may be related to the regulation of intracellular redox.