Effects Of Chymase Inhibitor On Hepatic Fibrosis Of Experimental Rats

Background and Aims:Hepatic fibrosis is the indispensable way of many liver diseases to liver cirrhosis and also the key for aggravation of chronic hepatitis, hepatic cirrhosis etc.Hepatic fibrosis is characterized by excess deposition of extracellular matrix(ECM)due to the increase of synthesis and relative decrease of degradation.Hepatic fibrosis is a dynamic process which is a reversible pathological change.However,it becomes irreversible once hepatic fibrosis develops into the structural change of hepatic lobule and formation of pseudo-lobule(hepatic cirrhosis stage)occurs.Up to now,anti-hepatic fibrosis drug with widely approved effect has not been available for clinical treatment.Therefore,researches on prevention and therapeutic methods for hepatic fibrosis remain one of the hot but difficult topics.Chymase,a kind of chymotase belonging to serine proteinase,mainly resides in the secreted granules of the mast cells and in the intercellular substance.Studies in recent years show that once chymase is secreted into the intercellular substances by activated mast cell,it acts as follows:acting as pro-inflammatory factor in coordination with histamine;acting collagenase;adjusting ECM;converting angiotensinⅠ(AngⅠ)to angiotensinⅡ(AngⅡ)in situ.It has been proved that chymase plays an important role in fibrosis of heart,kidneys and lungs.Chymase inhibitor is able to improve heart fibrosis.Liver of human being also has mast cells where chymase exists.Earlier studies showed that chymase concentrations differed significantly in liver tissue of chronic hepatitis patients with different liver fibrosis stages.Thus presume chymase takes part in the formation of hepatic fibrosis.Suppose that was true,if we intervene the hepatic fibrosis with chymase inhibitor,the formation of hepatic fibrosis will be relieved. Therefore the present study was performed to prove such a scientific hypothesis mentioned above,to verify chymase roles on hepatic fibrosis and find new target and potential drug for anti-hepatic fibrosis.Methods:Thirty male Wistar rats were randomly divided into three groups,i.e. normal group,model group and interventional group.The interventional and model groups were all intraperitoneally injected with 1%DMN(10mg/kg,3 times per week)for 4 weeks to establish hepatic fibrosis models,and the normal group was given equal volume of normal sodium as control.The interventional group was simultaneously intragastriced with SBTI(10mg/kg,once a day,for 4 weeks as well), while the other two groups were given equal volume of normal sodium.Four weeks later,all rats were anaesthetized and weighed,then blood was collected from the heart and the liver and spleen were weighed;liver histological observation was performed after hematoxylin and eosin(HE)staining and Masson staining;ultramicrostructure of hepatic cell was observed with a transmission electron microscope(TEM);serum hyaluronic acid(HA),laminin(LN),typeⅢprocollagen(PCⅢ)and typeⅣcollagen (CⅣ)were detected with enzyme linked immunosorbent assays(ELISA); chymase-like activity in liver tissue was examined with radio immuno assay(RIA).Results:①The body weights(mean±s)of rats in normal group,model group and interventional group were 326.2±15.1g,279.2±15.8g and 321.6±19.3g respectively; the humid weights of liver were 10.60±0.95g,12.53±1.09g and 10.91±0.58g respectively;the humid weights of spleen were 0.90±0.06g,1.20±0.17g and 1.00±0.12g respectively;liver and spleen index were(3.25±0.22%,0.28±0.03%), (4.48±0.22%,0.43±0.05%)and(3.63±0.11%,0.31±0.03%)respectively.Compared with rats in normal group,those in model group had less body weights,higher humid weights of liver and spleen,higher liver and spleen indices(P＜0.05).Compared with normal group,the body weight,humid weight of liver and spleen and spleen indices in interventional group changed little(P＞0.05),but liver index increased remarkably (P＜0.05).In contrast to rats in model group,those in interventional group had higher weights and less liver and spleen,so the liver and spleen indices decreased significantly(P＜0.05).②Histological examination:HE and Masson staining showed that the hepatic lobules of rats in normal groups exhibited normal structure,all the hepatic cells were radially arranged with the central vein as center,no degeneration and necrosis was observed in hepatic cells.The hepatic lobules of rats in model group was destroyed significantly,fibrous connective tissue hyperplasyed obviously extending into the hepatic lobules and pseudo-foliole emerged.Lymphocyte infiltrated obviously in portal tract.In interventional group,slight disorder were observed in hepatic lobule structure,no typical pseudo-lobule was found,little lymphocyte infiltration was noticed in portal tract.The grade of liver inflammation and stage of liver fibrosis in interventional group is lighter than those in model group(P＜0.05).③Electron microscope demonstrated that hepatic cell damage in interventional group lessened significantly than that in model group.Its cell had clear and intact cellular organs as in normal group.There was less collagen fibers in interventional group than in model group,but more than in normal group.④The levels of serum HA,LN,PCⅢand CⅣof rats in interventional group(165.6±14.8ng/mL,102.4±11.4ng/mL, 172.8±13.8μg/L and 69.6±7.3μg/L respectively)were slightly higher than those in normal group(being 102.5±14.1ng/mL,97.2±13.5ng/mL,90.6±16.3μg/L and 38.4±5.7μg/L,and P value being＜0.05,＞0.05,＜0.05 and＜0.05 respectively),but significantly lower than those in model group(198.6±16.3ng/mL,116.9±13.4ng/mL, 201.6±12.7μg/L and 84.3±6.9μg/L respectively,P＜0.05).⑤Chymase-like activity in liver of interventional group(0.216±0.018 U/mg protein)was a little higher than that of normal group(0.165±0.051 U/mg protein,P＜0.05),but significantly lower than that of model group(0.288±0.089 U/mg protein,P＜0.05).Conclusion:①Chymase inhibitor can significantly depress the activity of chymase in liver of experimental hepatic fibrosis rats,resulting in a decreased formation of AngⅡin situ;②chymase inhibitor is able to decrease the levels of serum HA,LN,PCⅢand CⅣ,lessen the grade of liver inflammation and improve stage of hepatic fibrosis;③chymase inhibitor might play some protection to hepatic cell.Therefore,it can be deduced that chymase participates in the synthesis of liver fiber and the process of liver inflammation.Chymase inhibitor may develop a potential anti-hepatic fibrosis medicine in the future.