| BackgroundHepatic fibrosis is a necessary stage in many chronic liver diseases developing to liver cirrhosis and this process is reversible proved by research. But if Hepatic fibrosis is left untreated, it may be transformed into liver cirrhosis. The survival rate of end-stage liver cirrhosis is only16%in3years, directly death in liver cirrhosis is on the sixth place in human total death of the whole world.The mainly formation mechanism of liver fibrosis is that:after the liver damage by inflammation, there are excessive proliferation and abnormal deposition of Extracellular matrix (ECM), leading an abnormal changes of liver structure and function. Transforming growth factor-β1(TGF-β1) is the main reason of Hepatic fibrosis by activate hepatic stellate cells, regulate synthesis and degradation of extracellular matrix, and it is the important target for liver fibrosis. The effect of TGF-β1is mainly realized by TGF-β1/Smad signal transduction pathway. The treatment for Hepatic fibrosis on Modern Medicine is blocking the inducement and there is no specific medicine for Hepatic fibrosis on clinic. In contrast, Traditional Chinese Medicine has various advantages and achieved effect on the treatment of Hepatic fibrosis such as multiple components, multiple links, and multiple targets, has broad prospects for development and application. Compound Qizhu Tablet has many functions such as clearing heat and removing toxicity, benefiting qi for activating blood circulation and it has proved its effect in the treatment of chronic hepatitis B as protection of liver, dropping enzyme and anti-liver fibrosis. Our previous studies have shown that Compound Qizhu Tablet has a protective effect on acute and chronic liver injury and hepatic fibrosis rat model.The experiment aims to observe the systematic effect of Compound Qizhu Tablet on Hepatic fibrosis, and make further research on the mechanism. The conclusion of this experiment, providing exploratory research to develop the treatment of Hepatic fibrosis drugs from Traditional Chinese Medicine.Objective: This study aims to research the effect of TGF-β1/Smad signaling pathway and cell factor as TIMPs/MMPs and ILs influenced by Compound Qizhu Tablet on CC14rats hepatic fibrosis model and verify the mechanism partly on molecular biology and cell biology; To find out the effective components of Compound Qizhu Tablet by HPLC; To observe the influence of effective components of Compound Qizhu Tablet on HSC-T6cell effected by exogenous TGF-β1, by testing cell apoptosis, cell cycle and TGF-β1/Smad signal transduction pathway effects, to provide further experimental evidence for the treatment of liver fibrosis in Traditional Chinese Medicine.Methods:Experiment1:Treatment of liver fibrosis of Compound Qizhu Tablet in vivo experimentsSeventy SD rats were randomly divided into normal group, model group, Qizhu high-dose group (water preparation), Qizhu low dose group (water preparation), Qizhu high-dose group (ethanol preparation), Qizhu low dose group (ethanol preparation), and colchicine group. All the rats except normal group received40%CCl4subcutaneous injection twice a week for8weeks, rats was firstly given40%CCl4(5mL per1OOg body weight) and3mL per100g body weight secondly. Rats in both Qizhu high-dose group and small dose group were given16.2,9.72g per100g body weight by ig. Qizhu decoction respectively while rats in colchicine group were fed with0.5mg per100g body weight colchicine. Liver histology and liver and spleen index were observed after the model is done. The expression of TNF-a, IL-4,IL-8,IL-10,TIMP-1,MMP-1,MMP-2,MMP-13in hepatic tissue was detected by enzyme linked immune sorbent assay (ELISA),and as same as the expression of TIMP-1, MMP-1, MMP-2,MMP-13in the blood serum. To detect the protein expression levels of Smad2, Smad3, Smad4, Smad7, and TGF-β1by Western-blot, the mRNA expression levels of Smad2, Smad3, Smad4, Smad7, and TGF-β1by real time quantitative RT-PCR.2. Experiment2:HPLC for Compound Qizhu TabletHPLC Method was applied to establish the Compound Qizhu Tablet. The separation was performed on a Agilent C18column (250mm×4.6mm,5μm) with a gradient elution composed of acetonitrile(A) and water (B). The column temperature was set at30℃and the flow rate was0.8Ml/min.The detective wave length was at280nm. HPLC fingerprint was performed on the Similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine (A)Experiment3:Treatment of liver fibrosis of the effective components of Compound Qizhu Tablet in vitro experimentsHSC-T6cells were cultured in DMEM medium supplemented with10%fetal bovine serum,50U penicillin/mL and50μg streptomycin/mL. For sub culturing purposes, cells were detached by treatment with0.25%trypsin/0.02%EDTA at37℃. Cultures were used at75%confluence. Cultures were divided into control group, Polydatin group, Hypophyllanthin group,Astragaloside group, Peoniflorin group, and model group is add exogenous TGF-β1. ALL groups are measured for proliferation and cell cycle change at24h by flow cytometry; The mRNA and protein expression levels of Bcl-2、Bax、CyclinD1、Smad2、Smad3、Smad4、Smad7、TGF-β1were detected by real time quantitative RT-PCR and Western-blot respectively at24h.Data are expressed as Mean±SD. The Student’s t-test was used to determine the statistical significance of the experimental data.ResultsExperiment1:Treatment of liver fibrosis of Compound Qizhu Tablet in vivo experiments1.Compared with normal group, body weight of rats in model group decreased (P<0.01), liver index, spleen index increased (P<0.01); Compared with the model group, body weight of rats in treatment group increased, the high dose group were improved significantly (P<0.01); spleen index decreased (P<0.05); On the rat gross anatomy, the treatment group appearance similar to the normal group; According to the pathological condition, the rats in model group were observed that the liver cells swelling, widely balloon degeneration and fibrosis, the rats in treatment group showed Compound Qizhu Tablet can improve liver inflammatory lesions, reduce liver cell degeneration or necrosis, stop or reverse the hyperplasia of fibrous tissue. It showed that liver fibrosis model replication is established successfully, and Compound Qizhu Tablet has a preventive effect on liver fibrosis.2. Compared with normal group, the expression of IL4,IL8,TNF-a,TIMP-1,MMP-2in liver fibrosis rats increased, the expression of IL-10,MMP-1,MMP-13decreased (P<0.01);Compound Qizhu Tablet can reduce the expression of IL4, IL-8, TNF-a in liver fibrosis rats, increase the expression of IL-10(P<0.01); At the same time, Compound Qizhu Tablet can reduce the expression of reduce the expression of TIMP-1,MMP-2in liver fibrosis rats, increase the expression of MMP-1,MMP-13(P<0.01), it showed the same TIMPs/MMPs expression in the serum of liver fibrosis rats (P<0.01).3.Compared with normal group, protein and gene expression of TGF-β1, Smad2, Smad3, Smad4in model group increased, protein and gene expression of Smad7decreased (P<0.05); Compared with model group, protein and gene expression of TGF-β1, Smad2, Smad3,Smad4in treatment group decreased,and expression of Smad7increased (P<0.05) Experiment2:HPLC for Compound Qizhu TabletResult of HPLC in Compound Qizhu Tablet showed that Polydatin, Hypophyllanthin, and Peoniflorin is the effective components of Compound Qizhu Table; And ethanol preparation is more effective than water preparation.Experiment3:Treatment of liver fibrosis of the effective components of Compound Qizhu Tablet in vitro experiments1. Hypophyllanthin,Polydatin in each dosage group and dose group of Paeoniflorin, Astragaloside showed effect of proliferation inhibition on HSC-T6cells in12h,24h,48h,and the most obvious performance in on24h.2. Hypophyllanthin, Polydatin, Astragaloside can increase protein and gene expression of Bax in normal group and model group HSC-T6cells, decrease the expression of Bcl-2; Paeoniflorin can increase protein and gene expression of Bax in normal group and model group HSC-T6cells, decrease the protein expression of Bcl-2.3. Hypophyllanthin can hold the HSC-T6cells of model group in G1phase by decrease the protein and gene expression of cell cycle related factors CyclinD1.4. Compared with normal group, HSC-T6in model group protein and gene expression of TGF-β1, Smad2,Smad3increased, protein and gene expression of Smad7decreased; Hypophyllanthin can decrease protein expression of Smad2, gene expression of Smad3, increase protein and gene expression of Smad7and decrease protein and gene expression of endogenous TGF-β1; Polydatin can decrease protein and gene expression of Smad2, decrease protein and gene expression of endogenous TGF-β1; Paeoniflorin can decrease protein and gene expression of Smad3, protein expression of Smad2, increase protein and gene expression of Smad7, and decrease protein and gene expression of endogenous TGF-β1; Astragaloside can increase protein and gene expression of Smad7and decrease protein expression of endogenous TGF-β1.Conclusions1. Compound Qizhu Tablet can improve the weight of the rats of liver fibrosis model, reduce the enlargement of the liver and spleen wet weigh, and restore the damage of fibrosis in liver tissue.It shows Compound Qizhu Tablet can protect liver fibrosis rats and treat liver fibrosis.2.Compound Qizhu Tablet can reduce the expression of IL4, IL-8, TIMP-1, MMP-2,TNF-a in liver fibrosis rats, increase the expression of IL-10, MMP-1, MMP-13.It shows Compound Qizhu Tablet treat hepatic fibrosis though regulate TTMPs/MMPs, ILs and TNF-a in the liver, reduce the expression of extracellular matrix in liver and balance liver collogen.3.Compound Qizhu Tablet can decrease protein and gene expression of Smad2, Smad3, Smad4,and increase expression of Smad7, regulat the TGF-β1/Smad Signal Transduction Pathway to decrease the expression of TGF-β1and Inhibiting activation of hepatic stellate cells, reduce the secretion of extracellular matrix, reduce the degree of liver fibrosis.4. According to HPLC on Compound Qizhu Tablet, Hypophyllanthin, Phyllanthus, polydatin are the effective component of Compound Qizhu Tablet, ethanol preparation is better than water preparation in the extraction of the monomer, it showed ethanol preparation regulate TIMPs/MMPs, TGF-β1/Smad signal transduction pathway more effective than water preparation With the relevant of in the extraction of the effective component.5.Hypophyllanthin,Polydatin in each dosage group and dose group of Paeoniflorin, Astragaloside showed effect of proliferation inhibition on HSC-T6cells in12h,24h,48h,and the most obvious performance in on24h.It showed that treatment of Compound Qizhu Tablet on Hepatic fibrosis is relative to its effective component’s effect of proliferation inhibition on HSC-T6cell.6. Hypophyllanthin, Polydatin, Astragaloside and Paeoniflorin increase protein and gene expression of Bax in normal group and model group HSC-T6cells; decrease the expression of Bcl-2. Compound Qizhu Tablet’s effect on Hepatic fibrosis is relative to proliferation inhibition on HSC-T6cells, as well as the effective component’s effect on Promoting HSC-T6apoptosis.7. Hypophyllanthin can hold the HSC-T6cells of model group in G1phase by decrease the protein and gene expression of cell cycle related factors CyclinD1and the proliferation inhibition on HSC-T6cells is relative to the control of cell cycle.8. Effect on hepatic fibrosis of Compound Qizhu Tablet is relative to its effective component such as Hypophyllanthin, Polydatin, Astragaloside and Paeoniflorin, which can respectively control the TGF-β1/Smad signal transduction pathway, inhibition TGF-β1expression and activation of hepatic stellate cells, reduced ECM deposition in liver tissue. |
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