| Objective:TGF-β1 was used to activate hepatic stellate cells to explore the inhibitory effect and mechanism of Gansu granules on the main effective substances,so as to initially reveal the possible mechanism of Gansu granules against liver fibrosis and provide a basis for its further development and application.Methods:1.Literature research Consult the relevant literature reports of Gansu Granules in recent years,including the source,clinical application and experimental research reports of Gansu Granules for literature data analysis.2.Experimental Study Establish the LX-2 activation model of human hepatic stellate cells induced by TGF-β1,screen the effect dose of the TFPCP by MTT method,set three doses of high、medium and low,and use enzyme-linked immunity The adsorption method was used to detect the content of type I collagen(Col I)and fibronectin(FN)in the cell culture fluid at different doses,the scratch test was used to detect the cell migration rate,the collagen contraction test was used to detect the collagen contraction,and the flow cytometry was used to detect the apoptosis Rate;RT-PCR method was used to detect the expression of TGF-β1/ Smads signaling pathway-related protein m RNA,and Western Blot method was used to detect the expression of apoptosis-related proteins and TGF-β1/Smads signaling pathway-related protein;and observe the administration of TGF-β1/Smads signaling pathway inhibitor LY2109761,the effect of TFPCP on collagen contraction of activated LX-2 cells and the expression of TGF-β1 / Smads signaling pathway-related proteins.Results:1.Literature research characteristics of gansu granule After analysing and researching the literature on Gansu Granules,it is found that Gansu Granules is one of the commonly used Chinese medicine preparations for the treatment of liver diseases.Its main functions are enzyme lowering,liver protection,and yellowing.Its chemical components are mainly flavonoids And phenolic compounds.Experimental research and clinical application literature have shown that Gansu granules have a therapeutic effect on liver fibrosis,pharmacological and pharmacological studies can prove that the total flavonoids of Ganhuangcao are currently recognized as the main medicinal substances for liver protection and anti-fatty liver,It is an important effective substance for the treatment of liver fibrosis.2.Establishment of the activation model of hepatic stellate cells and determination of the pharmacodynamic concentration of TFPCP TGF-β1 concentration of 2,4,6,8,10,12,14,16μg/L can promote the proliferation of LX-2 cells(P<0.01),the proliferation activity at 6 μg/L and α in cell culture medium-The content of SMA is the highest,and the effect is weakened when the dose is lower or higher,suggesting that 6 μg/L is the optimal concentration of TGF-β1 to activate LX-2,so it is the activation concentration of LX-2 cells.The screening results of the TFPCP showed that the cell survival rate was greater than 80%when the TFPCP concentration of Chrysanthemum indicum was 13mg/L,and the drug showed obvious cytotoxicity when the concentration was greater than 17mg/L,so 5,9,13mg/L was chosen for subsequent experiments.3.Effect of TFPCP on activation of general state and apoptosis of hepatic stellate cells The results of the MTT method showed that the TFPCP can inhibit the proliferation of LX-2 cells activated by TGF-β1,and the higher the concentration of the drug,the more obvious the inhibitory effect was,and there was a significant inhibitory effect at 13 mg/L(P <0.01).The scratch experiment showed that the TFPCP can inhibit the migration of LX-2 cells after TGF-β1 activation,and the inhibitory effect was significant at 9 mg/L(P<0.01).Collagen contraction experiments showed that collagen contraction in the TGF-β1 group increased significantly,and the collagen contraction degree of each group after the TFPCP decreased compared with the simple TGF-β1 group,and the effect was dose-dependent.The detection of the cytoplasmic matrix showed that the TFPCP can significantly inhibit the increase of FN and ColⅠ in the LX-2 cell sap after activation(P <0.01).Flow cytometry showed that TGF-β1 had no significant effect on apoptosis,the TFPCP could promote the apoptosis of activated LX-2 cells,and the effect was dose-dependent,9mg/L was statistically significant(P<0.01);Western blot results showed that the expression of Bcl-2increased and the expression of Bax decreased in LX-2 cells activated by TGF-β1,but there was no significant difference compared with the unactivated group The expression of Bcl-2 in LX-2 cells with the TFPCP significantly decreased while the expression of Bax increased significantly(P <0.05).4.Effects of TFPCP on activation of TGF-β1/Smads signaling pathway related proteins in hepatic stellate cells The RT-PCR experiment showed that the TFPCP can significantly inhibit the expression of α-SMA m RNA in LX-2 cells(P<0.01).In addition,the expression of TGF-β1/Smads signaling pathway-related proteins Smad2 and Smad3 m RNA were significantly reduced after the TFPCP(P<0.01);while the expression of Smad7 was slightly increased,when the drug concentration reached 13mg/L,The difference was statistically significant(P<0.05).Similarly,western blot results showed that the expression of Smad3,p-Smad2,and p-Smad3 all decreased to varying degrees after the total flavonoids of Rhizoma chinensis,while the expression of Smad7 increased slightly,and was statistically significant at 9 mg/L(P<0.01).5.Effect of TFPCP on inhibition of TGF-β1/Smads signaling pathway in activated hepatic stellate cells After using the specific TGF-β1 / Smads signaling pathway inhibitor LY21097611 to act on LX-2,the collagen contraction results showed that the TFPCP and LY2109761 alone can inhibit collagen contraction(P <0.05),while After pretreatment with LY2109761,the TFPCP were given,and the inhibitory effect was greater than that of simple administration(P <0.01).The expression of p-Smad3(P <0.05),and LY210976 pretreatment and then given the total flavonoids of Chrysanthemum,the inhibitory effect is greater than that of simple administration(P <0.01).Conclusions:1.Gansu granules have anti-hepatic fibrosis effect,and the TFPCP are its important effective substances.2.The TFPCP can inhibit the activation of TGF-β1 activated hepatic stellate cells LX-2,which can inhibit the proliferation and secretion matrix function.The mechanism is related to the induction of apoptosis and the inhibition of TGF-β1/Smads signaling pathway. |
