Roles Of Rho A/ROCK Signal Pathway And The Protective Effects And Mechanisms Of Fasudil In Hepatic Fibrosis Rats

Hepatic fibrosis is a repair response to chronic hepatic injury caused by many factors.And it is a necessary stage and common pathological basis of chronic hepatitis which develop cirrhosis and hepatic failure.Therefore,the prevention,treatment and even reversion the process of hepatic fibrosis is the key step of blocking hepatic cirrhosis.Many studies confirmed that hepatic fibrosis is due to the destroyed dynamic balance of collagen synthesis,deposition,degradation and absorption.The synthesis and deposition of collagen is greater than the degradation and absorption,extracellular matrix(extracellularmatrix,ECM)excessive deposition.All of these comprise the typical characteristics of hepatic fibrosis.Physiological and pathological processes in the process of inflammation,oxidative stress,immune response and cell apoptosis plays an important role.China is of a high incidence of hepatic disease.Up till now,there is no effective anti drugs of hepatic disease.How to prevent or reverse the excessive deposition of ECM has become the key to the treatment of chronic hepatic disease,but also the focus of domestic and foreign scholars.Rho A,which act as “molecular switches”,is a member of the Ras superfamily of proteins.ROCK is one of the most important effector molecules downstream of Rho A and is a member of the serine/threonine(Ser/Thr)protein kinase family.In recent years,the study found that activation of the Rho A/ROCK signaling pathway can mediate cell adhesion and migration,actin cytoskeleton formation and cell proliferation and apoptosis and other biological functions,these effects play an important role in a variety of organs and tissues of chronic inflammatory fibrosis.Studies have shown that selective blockade of this signaling pathway can improve the progression and prognosis of fibrotic organs.As the only one kind of Rho-kinase selective inhibitors in clinic,fasudil was mainly applied for the prevention of cerebral vasospasm after subarachnoid hemorrhage,and to improve symptoms of cerebral ischemia.It is quickly transfered into tissue,including the hepatic,kidney,spleen and intestine in high dose after administration.Our previous studies found that fasudil could inhibit lipid peroxidation and antioxidant,and exert a protective effect on myocardial fibrosis.But the effect in the process of hepatic fibrosis is not clear at present.This study was designed to establish three rat models of hepatic fibrosis induced by different factors,investigate the role of Rho A/ROCK signal transduction pathway in hepatic fibrosis by pharmacological,morphology,molecular biology and other experimental methods,and explored the possible mechanisms of fasudil in hepatic fibrosis.Part one Roles of Rho A/ROCK signal pathway and effects of fasudil on inflammatory response of hepatic fibrosis in diabetic ratsObjective: To establish the hepatic fibrosis model of type 1 diabetic rats induced by streptozotocin,and investigate the roles of Rho A/ROCK signal pathway and the effect of fasudil on inflammatory response of hepatic fibrosis.Methods:Fifty SD rats were randomly divided into normal control and diabetes groups.Ten SD rats fed with normal rat chow comprised the normal control(NC)group.Other male rats received an intraperitoneal injection of 60 mg/kg streptozotocin dissolved in 0.01 M sodium citrate buffer(pH 4.5).Rats should fast for 6 hours prior to STZ induction.STZ-injected rats were included in the study after confirmation of hyperglycemia(≥ 15 mM glucose,5 days later)by a portable glucometer.Subsequently,forty diabetic rats were randomly divided into four groups: untreated diabetic(DM),low-dose fasudil-treated(L-Fas),high-dose fasudil-treated(H-Fas)and captopril-treated(DC)groups(n = 10 per group).The rats in the two fasudil groups were treated with fasudil(2 mg/kg b.i.d.,L-Fas group and 10 mg/kg b.i.d.,H-Fas group)separately by intraperitoneal injection.Cap group were gavaged with captopril(30 mg/kg)once a day,whereas DM and NC rats were injected intraperitoneally with equivalent volumes of 0.01 M sodium citrate buffer.Rats in each group were sacrificed after 8 weeks.The body weight and hepatic weight of each animal were recorded,and calculate hepatic weight index.Detect AST,ALT and HbA1 c in serum.The concentrations of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in the hepatic were estimated by ELISA.The expressions of MMP-9,TIMP-1,TGF-β1,NF-κBp65 and IκBα in hepatic tissue were respectively detected by Western-blotting.The expression of Coll α1 mRNA,Rho A mRNA and ROCK-1 mRNA were respectively detected by semiquantitative PCR.Results:1 The general condition of normal control group rats is good,and the rats in model group appeared typical diabetes.The average body weight of DM group was significantly lower than that of group NC,which decreased 27.14%(P < 0.05),while LW and LWI were significantly increased(P < 0.01).High dose fasudil(H-Fas)significantly reduced the decline of body weight,and the hepatic weight was significantly decreased(P < 0.01).2 ALT、AST、TNF-α、IL-6 and the glycosylated hemoglobin(HbA1C)in DM group were significantly higher than those in control group(P < 0.01),which indicated that hyperglycemia induced a series of inflammatory reaction resulting in abnormal hepatic function in rats.Fasudil treatment resulted in markedly decreased levels of ALT,AST,IL-6 and TNF-α in serum compared with levels in the DM group(P < 0.01),with the high dose more effective than the low dose.These indices were lower in the captopril-treated group than in the DM group,except for Hb A1 C.3 The results of H&E staining showed that the structure of hepatic lobules was destroyed in the model group.The hepatocytes in the DM rats were generally abnormal.The structure of the hepatic lobule was destroyed,many vacuoles can be found in hepatocytes,and hepatocyte ballooning with inflammatory cell infiltration was present.Fiber hyperplasia was observed around the central vein.These alterations were obviously ameliorated in fasudil-treated diabetic rats.Masson trichrome staining showed that the collagen fibers in the model group were proliferated.After treatment,the extracellular matrix was decreased(P < 0.05),and the H-Fas group was more effective(P < 0.01).4 Diabetes dramatically increased the expression levels of TGF-β1,TIMP-1,NF-κBp65,and decreased the expression of MMP-9 and IκB(P < 0.01),indicating an increase of fibrosis of the hepatic.Treatment with fasudil significantly reduced TGF-β1,TIMP-1,NF-κBp65 levels in the hepatics of rats,and the expression of MMP-9 and IκB was enhanced correspondingly(P < 0.01)The study found significantly increased compared with normal control group NF-kappa Bp65 expression in hepatic of rats in the DM group,the application of fasudil treatment after this increase trend slowed.The expression of I kappa B showed the opposite trend.5 Diabetes increased the levels of Coll α1 mRNA in a statistically significant manner,indicating an increase of fibrosis in the hepatic(P < 0.01).Furthermore,gene expression of Coll α1 was significantly decreased in the fasudil-treated groups,suggesting a compensatory mechanism to prevent fibrosis(P < 0.01).Meanwhile,Coll α1,Rho A and ROCK-1 mRNA expression was significantly higher in the DM group than in the NC group,suggesting that the Rho A/ROCK pathway was activated in the diabetic hepatic.Part two Roles of Rho A/ROCK signal pathway and effects of fasudil on apoptosis in hepatic fibrosis of rats induced by CCl4Objective: To study the roles of Rho A/ROCK signal pathway and the effect of fasudil on apoptosis in hepatic fibrosis of rats induced by carbon tetrachloride(CCl4)and explore the possible mechanism.Methods:SD rats were randomly divided into normal control and model groups.The model group was injected with 50% CCl4 oil mixture,1 ml/kg,2 times a week,lasting for 8 weeks.CCl4 rats were randomly divided into four groups: CCl4 group,Fas low dose group,Fas high dose group and captopril group,10 rats in each group.The same volume of buffer,Fas 2 mg/kg/d,Fas 10 mg/kg/d,intraperitoneal injection and captopril 30 mg/kg/d gavage respectively,whereas DM and NC rats were injected intraperitoneally with equivalent volumes of 0.01 M sodium citrate buffer.After the Rats sacrificed,the body weight and hepatic weight were recorded.AST and ALT in serum were detected as routine.The morphological changes of the hepatic were observe by hematoxylin eosin staining(H&E)and Masson’s trichrome staining.The expression of Bcl-2 and Bax were detected by Western-blotting.RT-PCR detected the expression of caspase-3 mRNA,Rho A mRNA and Rock-1 mRNA in hepatic tissue.Results:1 Compared with the normal group,the rats were of drooping spirit,slow reaction,dry body weight and decreased body weight.The mean body weight of rats was decrease about 29.34% than that of group NC(P < 0.05),while LW were increased about 83.55%(P < 0.01).2 The serum ALT and AST in model group were significantly higher than those in control group(P < 0.01),which showed that the hepatic function was severely damaged.After the treatment of fasudil,the content of ALT,AST decreased significantly.The content of ALT and AST decreased 25.47% and 9.87% respectively in the L-Fas group,and in the high dose group the figures were 45.34% and 21.84%(P < 0.01).Compared with model group,the serum ALT and AST of captopil group were significantly lower(P < 0.01).3 In the NC group,the hepatic lobule structure was complete,and there was no steatosis and inflammatory cell.In the model groups,the hepatic lobules were destroyed,and the hepatic cells arranged in disorder.The fat vacuoles of different sizes were formed,and hepatocyte ballooning presented.The apoptotic body could be found.A large number of lymphocytes infiltrated,with central vein offset or absent.The hepatic lobules were obviously necrosis,collagen hyperplasia was significant,and the inner part of the hepatic lobules was extended.The thick fibers wrapped around the hepatic cells and the false lobules formated.In the treatment group,there was a small amount of fibrous hyperplasia,which only confined to the portal area.4 The ratio of Bcl-2 / Bax determines the survival of the cell or apoptosis.In the model groups,the expression of Bax was significantly increased,while the expression of Bcl-2 was decreased,which suggested the apoptosis increased(P < 0.01).After the intervention by fasudil and captopril,the expression of Bax was decreased,while Bcl-2 was significantly increased,which showed the drugs involved were effective in the apoptotic process.5 In the model groups,the expression of caspase-3 mRNA in hepatic were significantly higher than the control group(P < 0.01),and the expression of Rho A mRNA and ROCK-1 mRNA were also increased.The expression of caspase-3 mRNA was significantly decreased in fasudil-treated groups(P < 0.05),with the high dose group more effective.The expression of Rho A mRNA and ROCK-1 mRNA were inhibited too,suggesting the pathway were actived in apoptosis process.Captopril group had similar results.Part three Roles of Rho A/ROCK signal pathway and effects of fasudil on oxidative stress response in hepatic fibrosis of rats induced byporcine serumObjective: To investigate the roles of Rho A/ROCK signal pathway and the effects of fasudil on oxidative stress response in rat hepatic fibrosis model induced by porcine serum.Methods:SD rats were randomly divided into normal control and model groups.Each rat in model groups was injected with 0.5 ml porcine serum intraperitoneally,twice a week for 8 weeks.The model rats were divided into four groups as last part mentioned.Normal fed after 8 weeks,the rats were sacrificed.The content of malondialdehyde(MDA)and the activities of superoxide dismutase(SOD)in hepatic tissue were respectively measured.The morphological changes and the degree of hepatic fibrosis were observed by hematoxylin eosin staining(H&E)and Masson trichrome staining.The expression of TGF-β1 mRNA、Rho A m RNA and Rock-1 mRNA in hepatic tissue were measureed by semi-quantitative PCR.Results:1 Compared with the normal group,the model group was similar to the normal group,and the number of individual hair color was slightly yellow than that of the normal group,and there was no significant change in body weight(P > 0.05).The weight and hepatic weight of rats in model group were not obvious,and the hepatic was similar to that of normal group(P > 0.05).2 The serum ALT and AST in model group were significantly higher than those in control group(222.85% and 273.34% respectively)(P < 0.01),which showed that the hepatic function was severely damaged.Fasudil treatment significantly improved hepatic function in rats(P < 0.01),in a dose-dependent manner.Compared with model group,the serum ALT and AST of Captopil group were significantly lower(P < 0.05).3 H&E staining observation shows that,given the low or high dose fasudil treatment,the histological changes of the level of performance is significantly improved,with hepatic cell arrangeing rules and fiber tissue decreasing.Masson staining showed that the hepatic fibrosis area decreased with the increase dosage of fasudil(P < 0.05).4 Compared with the normal control group,the content of MDA in the hepatic tissue of the model group was significantly higher than that of the control group(77.78%),and the SOD activity decreased about 50.01%(P < 0.01).Compared with the model group,fasudil reduced the MDA content in the fasudil low dose and high dose group,while incresed SOD activity significantly(P < 0.01).MDA was decreased in the Captopil treated group and SOD activity increased significantly(P < 0.05).5 Compared with the normal control group,the expression of TGF-β1 mRNA was significantly increased in the model group(P < 0.01).The TGF-β1 mRNA significantly reduced in fasudil treatment groups,which showed that the compensatory mechanism of oxidative stress was started.Rho A mRNA and ROCK-1 mRNA decreased in Fasudil-treated groups,which indicated that the Rho A/ROCK pathway was involved in the process of hepatic fibrosis.Conclusions:1 In the model of hepatic fibrosis in type 1 diabetes rats induced by streptozotocin,Rho kinase signal pathway is actived in hepatic fibrosis of diabetic rat.It can reduce the production of inflammatory factors,which in turn affect the activation of the NF-κB pathway,and give play to the role of anti-inflammatory,so as to delay the progression of hepatic fibrosis.2 Inhibiting the activation of Rho A/ROCK pathway can improve the expression of Bcl-2/Bax and down-regulating the expression of caspase-3,thus inhibit apoptosis and delay the progression of hepatic fibrosis in the model of CCl4-induced hepatic fibrosis.3 The activation of the Rho A/ROCK pathway suggested that fasudi scavenged oxygen free radicals to repress oxidative stress in order to improved hepatic fibrosis.In conclusion,the Rho A/ROCK pathway is active in the process of hepatic fibrosis.The ROCK inhibitor can reduce inflammatory response,lesson apoptosis and repress oxidative stress in order to reduce liver damage,reduce collagen expression and delay the progress of hepatic fibrosis.So it may be a new way of the prevention and treatment of hepatic fibrosis.