Experimental Study Of Anti-Hepatic Fibrosis And Molecular Mechanisms Of Compound Liuyueqing

The hepatic fibrosis is one of the most important pathological characteristicic in chronic liver diseases.Hepatic fibrosis is a reversible pathological process that the synthesis of extracellular matrix(ECM)is much more than its degradation,resulting in excessive ECM deposition in the liver. Inhibiting and reversing process of hepatic fibrosis has become a very important therapeutic strategy nowadays.Because the hepatic stellate cell(HSC)activation is the final common way to the form of hepatic fibrosis,and is the key link to the generation and development of hepatic fibrosis,so that HSC is chosen as a treatment target in more and more anti-hepatic fibrosis medicinal research.In recent years,anti-hepatic fibrosis research with traditional Chinese medicine obtains progresses greatly.Our researches are focused on Compound Liuyueqing(CLYQ).The confirmed functions of CLYQ are protective effect on acute chemical liver injuries in mice,inhibitory effect on duck hepatitis B virus DNA in ducks and on HBV DNA in the HepG2.2.15 cells.This research is designed to investigate anti-hepatic fibrosis effects and molecular action mechanisms of CLYQ in carbon tetrachloride(CCl₄)-induced rat hepatic fibrosis animal model and HSC-T6 cell model,so as to provide theoretical and experimental basis for the exploration of Guangxi ethnic features herbs for anti-hepatic fibrosis treatment.Part 1 Experiment in vivo The Effect of CLYQ on the Anti-hepatic Fibrosis and its Mechanism in RatsObjective:To study the effect of CLYQ against CCl₄-induced hepatic fibrosis in rats.Methods:100 male Sprague-Dawley(SD)rats were divided into 2 groups randomly:normal control group and hepatic fibrosis model group.Hepatic fibrosis model of SD rats were induced by subcutaneous injection(s.c.)of CCl₄ on the dorsum at dose of 0.3 ml of 40%CCl₄/100 g(40%CCl₄=4ml CCl₄:6ml peanut oil),two times a week.For rats in the normal control group,normal saline(NS)were given,s.c.,0.3 ml of NS/100g,two times a week.Two rats in hepatic fibrosis model group and one in normal control group were chosen to examine whether or not the pathological change of the hepatic fibrosis had in liver on 4th week,5th week and the 6th week respectively.60 SD hepatic fibrosis rats confirmed by the pathological inspection were divided into 5 groups randomly:â‘ normal control group:isometric NS;â‘¡model control group:isometric NS;â‘¢positive control group:colchicine,0.2 mg/kg/d;â‘£high dose of CLYQ group(CLYQh):30 g/kg/d;⑤middle dose of CLYQgroup(CLYQm):15 g/kg/d;â‘¥low dose of CLYQ group(CLYQ1):7.5 g/kg/d.All rats were treated with drugs for four consecutive weeks by intragastric administration(i.g.).24 hours after the last administration of drug,all rats were anesthetized with the sodium pentobarbitol,and the abdominal cavity under aseptic conditions was opened,then the blood of the rats was taken from abdominal aortic,and separated blood serum was finally frozen at -20℃for use. About 50-100 mg hepatic tissue was treated with the liquid nitrogen rapidly,then being frozen in -80℃freezer for extracting total RNA.Two pieces of hepatic tissue in the identical spot of liver,size 1×1×1 cm³,were taken,one was soaked in 10%neutral formalin solution for the light microscope observation and immunohistochemistry,the other was soaked in 2.5%glutaric dialdehydes for the transmission electron microscope observation.The serological indexes,e.g.superoxide dismutase(SOD), malondialdehyde(MDA),hykroxyproline(Hyp),hyaluronic acid(HA),tissue inhibitor of metalloproteinase-1(TIMP-1),were detected by enzyme linked immunoabsorbent assay(ELISA).The degree of fibrosis in hepatic tissue was observed under light microscope by HE staining.Ultra-structural changes were observed under transmission electron microscopy.Collagen typeâ… (Colâ… )protein expression in hepatic tissues was detected by immunohistochemistry.The relative quantification of Colâ… mRNA expression in hepatic tissues was detected by real time fluorescence quantitative PCR.Results:(1)The influence of CLYQ on serological indexes in hepatic fibrosis rats.Compared with the normal control group,the activity of SOD in the serum of rats decreased significantly in the model group(P＜0.01),while the contents of MDA,Hyp,HA and TIMP-1 in the serum increased significantly(P＜0.01), suggesting that hepatic fibrosis model has been successfully established.Compared with the model control group,the activity of SOD in the serum was increased significantly in all CLYQ groups(P＜0.01),while the contents of MDA,Hyp,HA and TIMP-1 in the serum decreased significantly(P＜0.01).As the drug concentration increased,and the effects also gradually increased, showing dose-effect relationship.(2)The influence of CLYQ on histomorphology in hepatic fibrosis rats.The pathological results of HE staining showed that the hepatic structural integrity in normal control group is not abnormal,but the hepatic lobule structure of the model control group was destroyed in rats,hepatic lobule was substituted by pseudo-lobule constituted by hepatic cell tubercle(size different, circular or ellipse),There is hyperplasia around fibrous nodules with a few infiltration of inflammatory cells.The hepatic lobule structure was complete and portal area had a few fiber structure in high and middle doses of CLYQ group.In rats from low dose of CLYQ group,hepatic cell arrangement was in disorder,the hepatic lobule structure was destructed,but still complete,around tubercle fiber structure proliferated,a few phlogocyte infiltrated.The results of transmission electron microscopy showed that the hepatic cell shape of the normal control group is clear in rats.The collagenous fiber of the model group obviously proliferated aroud hepatic cells.But after treament with CLYQ,most hepatic fibrosis tissues obviously become normal,the structures were good,a few apoptotic bodies were seen.(3)The influence of CLYQ on Colâ… protein expression in hepatic tissues of hepatic fibrosis rats.The results of immunohistochemistry showed that Colâ… protein expression in hepatic tissues was a low level in normal rats and no Colâ… protein expression in cytoplasm.Colâ… protein was expressed extensively in hepatic tissues in model rats and it could be seen in cytoplasm with strongly positive in immunohistochemical staining.After treament with CLYQ,the area and the intensity of Colâ… protein expression in hepatic tissues reduced and weaken significantly(P＜0.05,P＜0.01).Colâ… protein expression in the cytoplasma obviously reduced with dosage decreased.(4)The influence of CLYQ on Colâ… mRNA expression in hepatic fibrosis rats.Compared with the model control group,various dosage group of CLYQ could down regulate significantly Colâ… mRNA expression(P＜0.01)and showed a dose-dependent relationship according to the results of real time fluorescence quantitative PCR.Conclusions:1.CLYQ can enhance the activity of SOD and reduce the content of MDA in the serum in hepatic fibrosis rats,suggesting that it has the function of suppressing lipid peroxidation.Its mechanism may be related to attenuating free radical and inhibiting the lipid peroxidation.2.CLYQ can suppress fibroblast cells and collagenous fibre proliferation in hepatic tissues of hepatic fibrosis rats,suppress the secretions of Hyp,HA, TIMP-1 in hepatic tissues of hepatic fibrosis rats,down-regulate the expression of Colâ… mRNA and its protein in hepatic tissues of hepatic fibrosis rats.The results suggest that CLYQ can antagonize liver injury induced by CCl₄ in SD rats,and reduce the collagen synthesis and the deposition,thus suppressing the deposition of ECM.Hence CLYQ has the marked anti-hepatic fibrosis.Part 2 Experiment in vitro The Effect of CLYQ on HSC in Rats.Objective:To investigate the effects and mechanism of CLYQ on the proliferation,apoptosis and inhibition of fiber synthesis of HSC.Methods:CLYQ was diluted to 5 different concentrations and 6 replicates in each concentrations.Then as a model in vitro,the HSC-T6 cells were cultured with 5 different concentrations of CLYQ in incubator(37℃,5%CO₂). Cytotoxicity caused by CLYQ was detected by MTT assay,then median toxic concententration(TC₅₀)and largest no-toxic concententration(TC₀)of CLYQ were calculated.Finally,the inhibitory action of CLYQ on HSC-T6 cells proliferation was appraised.CLYQ concentrations of above 90%HSC-T6 cell survival were chosen in the experiment,they were 3.6 mg/ml,1.8 mg/ml,0.9 mg/ml respectively.The blank control group and positive control group were cultured With the isometric complete culture medium and the colchicine 6.25μg/ml respectively.After each group was incubated for 24h,cell supernate was aspirated and stored at -20℃.The contents of Hyp,HA and TIMP-1 in HSC-T6 cell supernate were detected by ELISA.HSC-T6 cell apoptosis were detected by flow cytometry (FCM)after digested by 0.25%pancreas enzyme,stained by the acridine orange (AO)staining,then Annex V-FITC and PI staining combined with FCM.The ultrastructure of HSC-T6 cell were observed with the transmission electron microscope.The expression of Colâ… protein in HSC-T6 cell was detected by immunocytochemistry.The relative quantification of Colâ… mRNA expression in HSC-T6 cell was detected by real-time fluorescence quantitative PCR.Results:(1)The influence of CLYQ on hepatic stellate cells proliferation and extracellular matrix.After 24 h incubation with CLYQ,the various concentration group of CLYQ displayed strong inhibitory action to the proliferation of HSC-T6 cell (P＜0.05,P＜0.01),and this inhibitory action enhanced with increase in drug concentration.All these evidences suggested that this inhibitory effect showed a dosage-effect relationship.Experimental results in vitro showed that TC₅₀is 6.191 mg/ml,TC₀ is 3.504 mg/ml,which suggested that the CLYQ cytotoxicity to the HSC-T6 cell is very low,and the anti-proliferation of CLYQ on HSC-T6 cell is not caused by nonspecific cytotoxicity.Comparing with the blank control group,the various concentration group of CLYQ could degrade significantly the contents of Hyp,HA and TIMP-1 in cell supernate(P＜0.05,P＜0.01).At the same time the action strengthen gradually with increase in CLYQ concentration,presenting the dose-effect relationship.The results suggested that CLYQ had a significant anti-proliferation on HSC-T6 cell in vitro.(2)The effect of CLYQ on apoptosis of HSC-T6 cell.The results of AO staining showed that vast majority of HSC-T6 cells in the control group had a good condition.The cytoplasm was red,and the nucleus was green.In the various concentration group of CLYQ,majority of cell status is good,apoptosis of HSC-T6 cell increased with the increase in dosage,at the same time the nucleus assumes deep yellow and disruptive,and the nuclear matter inflow cytoplasm.Compared with the blank control group,the effect of CLYQ on the HSC-T6 apoptosis proportion had a significant difference(P＜0.01) and the rate of apoptosis cells had dose-dependent relationship.The results of flow cytometry showed that the apoptosis rate of HSC-T6 cell in blank control group was 4.82,10.1 in the CLYQ 3.6 mg/ml group,6.67 in 1.8 mg/ml group,5.18 in 0.9 mg/ml group respectively.The results suggested that CLYQ could promote the apoptosis of HSC-T6 cells to some extent.The results of transmission electron microscope showed that the morphology in blank control group cell was clear,cell membrane and karyotheca are complete,cell surface has massive microvilli.A few of necrosis cell and many varying degrees of apoptosis of HSC-T6 cell were seen after cultured with CLYQ.(3)The influence of CLYQ on Colâ… protein expression in HSC-T6 cell.The results of immunocytochemistry showed that staining of HSC-T6 cells was strongly positive in the control group before CLYQ was applied,showing a tawny brown or deep brown distributed in the cytoplasm.Colâ… protein expression in the HSC-6 cells was significantly weakened in all CLYQ concentration group,the positive parts become light yellow color,and the gray value has significant difference comparing with that before applying CLYQ (P＜0.01).(4)The influence of CLYQ on Colâ… mRNA expression in HSC-T6 cell.The results of real time fluorescence quantitative PCR showed that the Colâ… mRNA expression in HSC-T6 cell is down-regulated significantly in CLYQ 3.6 mg/ml,1.8 mg/ml group when compared with the blank control group (P＜0.01).It was possible that the part of anti-hepatic fibrosis of CLYQ resulted from suppressing the Colâ… gene expression level.Conclusions:1.CLYQ can inhibit the proliferation of HSC and induce apoptosis of HSC in vitro,so that reducing the synthesis of ECM and hepatic fibrosis.2.CLYQ can down-regulate the expression of Colâ… mRNA and its protein in HSC,finally reducing the synthesis of ECM,which may be one of the possible molecular mechanism againist hepatic fibrosis.