The Roles Of RhoA/Rho Kinase And TGF-β₁/CTGF Pathway In Diabetic Hepatic Fibrosis In Rats With Type2Diabetes

Objective: Diabetes can cause a variety of chronic complications.Diabetes-induced chronic liver injury, specifically for increasing fibrosis, isattracted to the attention of researchers. While,the molecluar mechanisms ofdiabetes-induced hepatic fibrosis are not fully understood. Currently there isno effective treatment for hepatic fibrosis. The purpose of this study is todetermine whether the RhoA/ROCK is involved in the pathogenesis ofdiabetes-induced hepatic fibrosis in rats with type2diabetes, as well as therelationship of RhoA/ROCK with TGF-β₁/CTGF pathway, and clarify themechanisms of liver fibrosis induced by diabetes, with the objective ofproviding a novel therapeutic target for the treatment of diabetic hepaticfibrosis.Methods: An animal model of type2diabetes was developed by high fatdiet combined with one-time intraperitoneal injection of low dosestreptozotocin （STZ,30mg/kg）. The control group was established. Bloodglucose （BG） and body weight（BW） were measured monthly. At week10,Glucose infusion rate （GIR） was confirmed by hyperinsulinemic–euglycemicclamp. Fasting blood glucose （FBG）, fasting insulin （FINS）, triglycerides （TG）and cholesterol （TC） was measured. Then, diabetic rats were randomlydivided into untreated diabetic group and treated diabetic rats that receivedfasudil （10mg/kg/day i.p.）. At week24, FBG, FINS, TG, TC, aspartateaminotransferase （AST）, alanine aminotransferase （ALT） and glycosylatedhemoglobin （HbA1c） were measured. The morphology of liver tissue wasobserved by HE staining. The deposition and contents of collagen wereevaluated by Masson staining and the hydroxyproline （HYP） determination.The mRNA expressions of transforming growth factor （TGF-β₁） andconnective tissue growth factor （CTGF） in liver tissue were assessed by reverse transcription polymerase chainreaction （RT-PCR）.Immunohistochemistry staining for TGF-β₁was performed. Thephosphorylation of myosin phosphatase target subunit1（p-MYPT1） wasmeasured by Western blot analysis, representing ROCK activity.Results:1General stateEating, drinking, movement of the rats in control group were normal. Thefur is smooth and shiny gloss. The diabetic rats appeared polyuria, polydipsiaand polyphagia. The movement of the diabetic rats was significantly reduced,and the fur was dark and messy.2Changes in body weight （BW）At week10, compared with control group, the BW was markedlyincreased in untreated diabetic rats （P＜0.05）. At week24，compared withcontrol group, the BW of untreated diabetic rats and fasudil-treated diabeticrats were significant lower （P＜0.05）. There were no markedly differences inBW between untreated diabetic rats and fasudil-treated diabetic rats （P＞0.05）.3Insulin sensitivityAt week10, GIR in untreated diabetic rats was significantly lower thancontrol group （5.32±0.83mg/kg/min vs11.35±0.79mg/kg/min，P＜0.01）. Atweek24, insulin resistance index （HOMA-IR=FBG×FINS÷22.5） wascalculated. Compared with control group, the HOMA-IR was significantlyhigher in untreated diabetic rats and fasudil-treated diabetic rats（117.90±21.20and107.36±21.20vs15.51±3.65, respectively, P＜0.01）.There were no significant differences in HOMA-IR between untreated diabeticrats and fasudil-treated diabetic rats （117.90±21.20vs107.36±21.20, P＞0.05）.4Blood biochemical parametersAt week10, compared with control group, FBG, FINS, TG and TC weremarkedly increased in untreated diabetic rats （P＜0.01, P＜0.05）. At week24,compared with control group, FBG, FINS, TG, TC, HbA1c, ALT and AST were significantly elevated in untreated diabetic rats （P＜0.01）; but ALT andAST in fasudil-treated diabetic rats were significantly decreased comparedwith untreated diabetic rats （P＜0.01）. There were no significant differencesin FBG, FINS, TG, TC, HbA1c between untreated diabetic rats andfasudil-treated diabetic rats （P＞0.05）.5HE stainingIn control group, the structure of liver lobules was clear, hepatocyteswere arranged in order. The size and shape of hepatocytes were normal. While,hepatocytes were generally abnormal in the untreated diabetic rats comparedwith control group. The structure of liver lobule was destroyed, hepatocytescontained many vacuoles and hepatocyte ballooning with inflammatory cellinfiltration was present. Fibre hyperplasia was observed around the centralvein. These alterations were obviously ameliorated in fasudil-treated diabeticrats.6Masson stainingCompared with control group, the deposition of collagen around hepaticcentral vein in the untreated diabetic rats was significantly increased （densityof collagen:0.083±0.109vs0.051±0.008，P＜0.01）. However, the depositionof collagen in fasudil-treated diabetic rats was significantly reduced comparedwith the untreated diabetic rats （density of collagen:0.059±0.009vs0.083±0.109, P＜0.01）.7The contents of collagenThe contents of collagen in liver tissues were significantly increased inthe untreated diabetic rats compared with that in control group （268.79±10.85μg/g vs164.89±10.15μg/g，P＜0.01）. The contents of collagen in livertissues were markedly reduced in fasudil-treated diabetic rats compared withthat in the untreated diabetic rats （168.47±9.63μg/g vs268.79±10.85μg/g，P＜0.01）. There were no significant differences in the contents of collagen inliver tissues between fasudil-treated diabetic rats and control group （168.47±9.63μg/g vs164.89±10.15μg/g, P＞0.05）.8Immunohistochemistry staining There was faint positive expression of TGF-β₁in liver tissues in controlgroup. Very strong positive staining for TGF-β₁was found in hepatocytecytoplasmic and the protein expression of TGF-β₁in liver tissues from theuntreated diabetic rats was significantly greater than that in control group（positive area:0.231±0.104vs0.169±0.100, P＜0.01）. The protein expressionof TGF-β₁in liver tissues from fasudil-treated diabetic rats was markedlyreduced compared with that in the untreated diabetic rats （positive area:0.178±0.08vs0.231±0.104, P＜0.01）.9Gene expressionThe mRNA expression of TGF-β₁and CTGF in liver tissues weresignificantly up-regulated in the untreated diabetic rats compared with that incontrol group （P＜0.01）. Compared with the untreated diabetic rats, themRNA expression of TGF-β₁and CTGF in liver tissues were markedlyreduced in fasudil-treated diabetic rats （P＜0.01）. There were no significantdifferences between fasudil-treated diabetic rats and control group （P＞0.05）.10Protein expressionCompared with control group, the phosphorylation of MYPT1wassignificantly enhanced in liver tissues from the untreated diabetic rats （P＜0.01）. The phosphorylation of MYPT1was significantly reduced in livertissues from fasudil-treated diabetic rats compared with that from theuntreated diabetic rats （P＜0.01）. There were no significant differences in thephosphorylation of MYPT1in liver tissues between fasudil-treated diabeticrats and control group （P＞0.05）.Conclusions：The animal model of type2diabetes was established byhigh fat diet combined with one-time intraperitoneal injection of low-dosestreptozotocin. Hyperglycemia can activate the RhoA/ROCK in liver tissues.This pathway participates in regulating TGF-β₁/CTGF pathway in liver tissuesand plays a critical role in the development of diabetic liver fibrosis. ROCKinhibitor, fasudil, can effectively ameliorate diabetic liver fibrosis, which isindependent of blood fat and glycemic control. This study suggests ROCKmay be a new therapeutic target for diabetic liver fibrosis.