Mechanism Of Tibetan Medicine Lang Qing A Ta Regulating TGF-β1/Smads Signaling Pathway To Inhibit Proliferation And Activation Of Hepatic Stellate Cells

Objective:To investigate the mechanism of Tibetan medicine Lang Qing A Ta by regulating TGF-β1/SMADS signaling pathway to intervene the proliferation and activation of hepatic stellate cells and play an anti-hepatic fibrosis role.Methods:1.Animal experimental studies:Forty female C57BL/6 mice aged 6-8 weeks were selected to establish an animal model of liver fibrosis by intraperitoneal injection of poly I:C twice a week.The effect of the model was evaluated from the general state,blood biochemical indexes,serum mitochondrial antibody,liver tissue HE staining and immunohistochemical detection of CD4+and CD8+T lymphocyte expression in the liver tissues of mice in control group and model group.2.Study on the mechanism of Lang Qing A Ta anti-liver fibrosis in vivo:After HF mice were constructed,they were randomly divided into 7 groups with 10 mice in each group.They were given distilled water,positive drug group(ursodeoxycholic acid 91.67mg/kg/d),Traditional Chinese medicine group(Lang Qing A Ta medium dose 7.36g/kg/d,Lang Qing A Ta low dose 3.68g/kg/d),Lang Qing A Ta combined ursodeoxycholic acid group and other drugs by intragastric administration for 8 weeks.Biochemical indices and pathological changes of HE staining were detected,and the expressions of TGF-β1,Tβ RⅠ,TβR Ⅱ and Smad2/3 in liver tissues were detected by immunohistochemistry and qPCR,so as to explore the mechanism of this formula in treating HF.3.Preparation and identification of drug-containing serum:Forty clean SD rats were randomly divided into Lang Qing A Ta high-dose group(73.6g/kg/d),medium-dose group(36.8g/kg/d),low-dose group(18.4g/kg/d)and blank group(0.9%NaCl).After centrifugal sampling,the composition of the serum containing Lang Qing A Ta drug was analyzed by mass spectrometry and compared with the standard substance to identify the main drug components.4.Effects of Lang Qing A Ta on proliferation of rat activated hepatic stucid cells(HSC-T6):MTT method was used to detect the proliferation of HSC-T6 cells.After the hSC-T6 cells were incubated with Lang Qing A Ta drug-containing serum of high,medium and low dose groups for 24h,the changes of OD values among each group were measured to observe the changes of cell proliferation.5.Lang Qing A Ta regulates TGF-βl/Smads pathway and affects the proliferation and activation mechanism of hepatic stellate cells:Lang Qing A Ta high,medium and low dose concentration groups and blank control group were set.Under the same culture conditions,the drug serum of each group was used to intervene the activated hepatic stellate cells for 24h.After protein extraction,the protein expressions of TGF-β1,Smad2/3 and Smad7 were detected by Western blot.To determine the effect on TGF-β1/Smads signaling pathway.Results:(1)C57BL/6 mice induced intraperitoneally by intraperitoneal injection of polycycyrine sodium showed that blood biochemical indexes,immunohistochemical EXPRESSION of CD4+and CD8+T lymphocytes,and liver pathological changes were consistent with hepatic fibrosis.(2)UDCA group and Lang Qing A Ta group(medium and low dose)could significantly reduce the levels of ALT and AST in HF mice,the difference was statistically significant(P<0.05),alleviate the inflammatory lesions in hepatic portal area of liver pathology(P<0.05),and improve the accumulation content of α-I collagen in liver tissue of model group mice.The difference was statistically significant(P<0.05).The combination of Lang Qing A Ta and positive drug administration group also showed good treatment effect,but the improvement degree of each indicator was not as good as that of Lang Qing A Ta group or UDCA group alone.Immunohistochemistry and PCR were used to detect the expression of TGF-β1,TβRⅠ,TβRⅡ,Smad2 and Smad3.The results showed that the administration group had a certain degree of improvement compared with the model group,especially the Lang Qing A Ta high-dose group.There were statistically significant differences in TGF-β1,TβRⅠ,TβRⅡ and Smad2 compared with model group(P<0.05).(3)After the preparation of serum containing Lang Qing A Ta drugs,the mass spectrometry analysis showed that it was consistent with Lang Qing A Ta active components.(4)OD value of HSC-T6 cells decreased significantly after Lang Qing A Ta serum concentration in all groups,and the inhibitory effect on HSC-T6 proliferation was obvious,but there was no statistical difference in low Lang Qing A Ta serum concentration group.(5)The results of western blotting showed that the protein contents of TGF-β1 and Smad2/3 in the low-dose group containing Lang Qing A Ta serum were not significantly different from those in the blank control group(P>0.05),while the protein contents of TGF-β1 and Smad2/3 in the medium-high dose group containing Lang Qing A Ta serum were significantly decreased,the difference was statistically significant(P<0.05).The expression of Smad7 protein in Lang Qing A Ta containing serum was high in high dose group,and the difference was statistically significant(P<0.05).Conclusion:(1)The animal model of liver fibrosis was successfully established by intraperitoneal injection of Poly I:C in C57BL/6 mice;(2)Lang Qing A Ta has a significant improvement effect on biochemical indexes and liver pathology in the mouse model of liver fibrosis,and has an anti-liver fibrosis effect.(3)The mechanism of Lang Qing A Ta anti-hepatic fibrosis was related to TGF-β1/Smads.(4)The mass spectrometry analysis of Lang Qing A Ta drug-containing serum after preparation showed that the target drug component was contained,which met the requirements of subsequent experiments.(5)Lang Qing A Ta drug-containing serum had a significant inhibitory effect on hSC-T6 cells induced by proliferation and activation.(6)Lang Qing A Ta affects the proliferation and activation of hepatic stellate cells by regulating TGF-β1/Smads signaling pathway,and plays an anti-hepatic fibrosis role.