The Effect Of Novel Turtle Shell Decoction On The Cycle,Apoptosis And The Expression Of Colla1 By Down-Regulating CTGF/Itgα5 Signal Pathway In Hepatic Stellate Cells

Background:Hepatic fibrosis(HF)is a necessary process for chronic liver disease to end stage liver disease,Early HF can be reversed through etiology removal and drug treatment,If HF continues,it will cause permanent liver damage,develop into cirrhosis and even liver cancer.After nearly 30 years of research,the pathogenesis and etiology of HF have been better understood,but no anti-fibrosis drugs have been approved yet.Therefore,it is of great significance to further explore its pathogenesis and find effective antagonists for the prevention and treatment of chronic liver disease.The pathogenesis of HF is still unclear,Many studies have displayed that the activation of hepatic stellate cells(HSCs)is an important link in the formation of HF.HSCs can synthesize and secrete connective tissue growth factor(CTGF),As a matrix protein,CTGF can promote cell proliferation,differentiation and tissue repair,and is highly expressed in activated HSCs.The stimulation of CTGF can promote the deposition of extracellular matrix(ECM)of HSCs in rats,which can aggravate fibrosis,while silencing CTGF or neutralizing CTGF protein can down-regulate the ECM expression of HSCs.There are three kinds of receptors on the surface of HSCs,such as growth factor,angiotensin and integrin.As an important member of integrin receptor,integrin α5(Itgα5)binding with CTGF participates in the differentiation,proliferation,migration and adhesion of hepatic progenitor cells(HPCs)and HSCs,promotes the activation of HSCs as myofibroblast like cells(MFC),and promotes ECM,especially type Ⅰ collagen a1(Col1a1)synthesis and secretion lead to excessive deposition of ECM and aggravate fibrosis reaction,indicating that CTGF and Itgα5 can promote the phenotype transformation of HSCs and participate in the formation of HF.Therefore,The regulation of CTGF/Itgα5 signal pathway plays an important role in the development of HF.The existing methods of HF treatment include chemical drugs,gene therapy and biological agent application,but the curative effect is not obvious.In recent years,traditional Chinese medicine has some advantages in clinical application of chronic liver diseases including HF.Among them,Turtle shell pills(TSP),derived from Chapter 4 of "The Golden Chamber should be used to treat the pulse syndrome of malaria",has the effects of dredging blood and activating collaterals,dispersing the liver and dispersing nodules,and regulating immunity.It has obtained positive curative effect in the treatment of chronic hepatitis,HF,liver cirrhosis,liver cancer and other diseases,but it is not suitable for long-term use because of its large toxic and side effects.The treatment of chronic liver disease needs long-term medication.After years of exploration,our research group has made a novel turtle shell decoction(NTSD)by removing the ingredients with large side effects in TSP and adding many kinds of drugs to strengthen the body and replenish qi.NTSD has a more mild effect.Previous experiments found that NTSD improves the histology of HF better than TSP.In this study,hepatic stellate cell T6(HSC＿T6)(a kind of HSCs)was used as experimental model cells to explore the effect of NTSD on the cycle,apoptosis and the expression of ECM by regulating CTGF/Itgα5 signal pathway in HSCs,which can further clarify the development mechanism of HF,provide new strategies for its prevention and treatment,and lay an experimental foundation for NTSD as an anti-fibrosis drug for clinical use.Objectives:1.Eexplore the regulatory effect of NTSD on CTGF/Itgα5 signal pathway.2.Study the effect of NTSD on the cycle,apoptosis and the expression of ECM by regulating CTGF/Itgα5 signal pathway.Methods:1.Preparation of medicament: 11 kinds of traditional Chinese medicines(turtle shell,bupleurum,ground beetle beetle,etc.)were made into NTSD decoction,containing 0.71g/ml crude drug,and packed separately for frozen storage.The positive control drug TSP is prepared with 0.71g/ml of crude drug solution.2.Experimental grouping and cell growth and proliferation detection: HSC＿T6 was cultured on 96-well plate for 24 hours,then the culture medium was replaced with medium containing NTSD as the experimental group.HSC＿T6 treated with TSP as positive control group,untreated HSC＿T6 was used as normal control group.The 96-well plates were taken out at 24,48,72 and 96 hours respectively,and the growth and proliferation of cells in each group were observed under microscope.Then the culture medium was changed,and cell count kit-8(CCK-8)reagent was added.The culture was continued for about 3 hours,and the plates were taken out and mixed evenly.The optical density(OD)value was detected by the Microplate Reader,and the cell proliferation curve of each group was drawn.3.Cell cycle and apoptosis experiment: HSC＿T6 was routinely cultured for 24 hours,then the culture medium was replaced with medium containing NTSD,and the cells treated with TSP and not treated were used as control.The cells were collected and counted at 72 hours,fixed with 80% alcohol,stained with propidium iodide(PI),and detected by flow cytometry(FCM)for cell cycle.Take cell suspension of each group,add buffer to resuspend cells,add Annexin V-FITC apoptosis detection reagent,incubate and centrifuge at room temperature,resuspend cells in buffer,and detect the apoptosis of cells in each group by FCM.4.Quantitative PCR detection the expression of Ctgf,Itgα5 and Col1a1 of cells in each group: The total RNA of each group HSC＿T6 cells was used as a template,and quantitative PCR was used to detect the expression of Ctgf,Itgα5 and Col1a1 of cells in each group at gene level.GAPDH is used as internal reference.5.Detection the expression of Ctgf,Itgα5 and Col1a1 in each group by Western-Blot at protein level: transfer the total protein of cells in each group onto PVDF membrane,and immerse them in the sealing solution of secondary antibody of Ctgf,Itgα5 and Col1a1,incubate,add the corresponding secondary antibody,and then incubate again,and combine with the luminescent substrate.GAPDH is used as internal reference.6.Detection the expression of Ctgf,Itgα5 and Col1a1 in each group by Immunofluoresce nce cytochemistry at protein level: HSC＿T6 cultured by climbing were cultured in the medium containing NTSD for 48-72 h,fixed and repaired with antigen,and were incubated with prima ry antibody of of Ctgf,Itgα5 and Col1a1 together respectively,and then incubated with the co rresponding secondary antibody again,closed cell climbing.After that,the expression of each protein was detected by fluorescence microscope.Results:1.NTSD can inhibit HSC＿T6 cell growth and proliferationIt can be seen under the inverted fluorescence microscope that HSC＿T6 treated with NTSD for 48-72 h become slightly round and smaller,as compared with the control cells.CCK-8 test identified that NTSD could inhibit HSC＿T6 growth,and the growth of the HSC＿T6 treated with MTSD were lower than that of controls and the differences were statistically significant after the cells were treated for 48 h(P<0.01).2.NTSD can inhibit the growth and proliferation of HSC＿T6 at S phase of cell cycleFCM test showed that compared with the control group,the S phase of HSC＿T6 treated by NTSD was significantly increased(P<0.01),suggesting that NTSD could inhibit the growth and proliferation of HSC＿T6 at S phase of cell cycle.3.NTSD can induce HSC＿T6 cell apoptosisThe results of FCM revealed that compared with the control group,the apoptosis rate of HSC＿T6 treated by NTSD was significantly increased(P<0.01),suggesting that NTSD could induce HSC＿T6 cell apoptosis.4.One of the mechanisms of NTSD inhibiting the growth of HSC＿T6 may be due to NTSD down-regulates the expression of Ctgf,Itgα5 and Col1a1 at gene levelThe results of quantitative PCR showed that one of the mechanisms of NTSD inhibiting the growth of HSC＿T6 may be due to NTSD down-regulates the expression of Ctgf,Itgα5 and Col1a1 because the down-regulation of Ctgf,Itgα5 and Col1a1 in HSC＿T6 treated with NTSD at gene level is most obvious,as compared with the control cells(P<0.01).5.One of the mechanisms of NTSD inhibiting the growth of HSC＿T6 may be due to NTSD down-regulates the expression of Ctgf,Itgα5 and Col1a1 at protein level.The results of immunofluorescence cytochemistry and immunoblotting test confirmed that one of the mechanisms of NTSD inhibiting the growth of HSC＿T6 may be due to NTSD downregulates the expression of Ctgf,Itgα5 and Col1a1 because the down-regulation of Ctgf,Itgα5and Col1a1 in HSC＿T6 treated with NTSD at protein level is most obvious,as compared with the control cells(P<0.01).Conclusions:the mechanism of NTSD inhibiting HSC＿T6 cells in S cell cycle,promoting their apoptosis,and then inhibit ing their growth and proliferation may be through regulating CTGF/Itg α 5 signal path.Significance:In this experiment,NTSD was used to regulate the molecular expression of Ctgf,Itgα5and Col1a1,and explore its effects on the growth,proliferation,cell cycle,apoptosis and ECM secretion of HSCs.It was found that NTSD was used to regulate CTGF/Itgα5 signal pathway may be a new strategy to inhibit the growth and proliferation of HSCs and reduce ECM secretion.Compared with TSP,NTSD has a more obvious effect on inhibiting the growth and proliferation of HSCs and reducing their ECM secretion.The research results laid a new experimental foundation for the effective prevention and treatment of HF.