Role Of Cathepsin B In Liver Damage Induced By Olaquindox

Objective The current study is to explore the effect of Olaquindox (OLA) on rat liverfunction, inflammation, oxidative damage, apoptosis and protein expression of cathepsinB by establishing olaquindox poisoning rat model. Meanwhile, the apoptosis model ofHepG-2cells was treated with cathepsin B inhibitors (CBI) in order to explore the role ofcathepsin B on the cell apoptosis induced by olaquindox. Our finding will provid cluesand evidences for further exploring liver damage mechanism of olaquindox andinformation for prevention targets.Methods1Thirty two adult male SPF rats, weighing180～220g,were randomlydivided into control group,20mg/kg olaquindox group,40mg/kg olaquindox group and60mg/kg olaquindox group after the adaptive feeding a week. The rats of oladuindoxgroup were administered with20,40,60mg/kg olaquindox solution by oral for4weeks.Rats in the control group were administered with equal volume of distilled water.2ELISA method was applied to measure the contents of Cathepsin B, tumor necrosis factor(TNF-) and interleukin6(IL-6) of liver.The activities of alanine aminotransferase(ALT), aspertate aminotransferase (AST), alkaline phosphatase (ALP) and lactatedehydrogenase (LDH) in serum were determined. The total antioxidant capacity (T-AOC), Superoxide dismutase (SOD), Glutathione peroxidase (GSH-Px) and Catalase(CAT) were measured by using the kits.3HE staining was used to observe rat liver tissuepathological changes.4Flow cytometry and transmission electron microscopy wereapplied to detect apoptosis in rat liver tissue or cells.5The protein expression levels ofcathepsin B in liver or cells were detected by using immunohistochemical method.6Establish HepG-2apoptosis model induced olaquindox: HepG-2was cultured inincubator with37℃and5%CO2, cells was divided into five groups: control group,100ug/ml olaquindox group, the200ug/ml olaquindox group,400ug/ml olaquindox group,400ug/ml olaquindox+inhibitors CBI group. After24hours treatment,0.25%trypsinwas usd to digest.7Cell proliferation rate of HepG-2induced olaquindox was detectedby MTT method.Results1Effect of olaquindox exposure on rat liver function:With olaquindoxdose increased, ALT, AST, ALP and LDH activity in liver tissue of rats gradually increase compared with control group (P<0.05).The activity of ALT，AST andolaquindox dose was positively correlated(r=0.967P=0.033，r=0.980P=0.020)in theserum of rats.2Effect of olaquindox exposure on rat liver oxidative damage: the activityof T-AOC and SOD, CAT in20mg/kg,40mg/kg,60mg/kg groups decrease comparedwith control group(P<0.05). the activity of GSH-Px in rats of40mg/kg and60mg/kggroups decrease compared with control group (P<0.05).3Effect of olaquindox exposureon rat liver inflammation: Hepatic portal area of rats in the olaquindox group had adifferent degree of inflammatory cell infiltration. Compared with the control group, thecontents of TNF-α and IL-6were gradually increased with olaquindox dose increased(P<0.05).4Effect of olaquindox exposure on rat liver cell apoptosis: The rat liver cellapoptosis was induced by olaquindox, and the early apoptosis rate is gradually increasewith olaquindox dose increased (P<0.05). The early apoptosis rate of rats in40mg/kg and60mg/kg olaquindox groups increased by231.25%and478.12%respectively than that ofcontrol group(P<0.05).5The changes of the content and protein expression of cathepsinB in olaquindox exposure rat liver: The protein expression of cathepsin B mainly locatedin the cytoplasm of rat liver portal area and around the necrotic lesions.The contentcathepsin B in liver of40mg/kg and60mg/kg olaquindox group was increasedrespectively by34.04%and72.34%(P<0.05).6Effect of olaquindox on HepG-2cellproliferation：Olaquindox exposure inhibited HepG-2cell proliferation. With theincrease of olaquindox dose, cell survival rate significantly decreased (P<0.05).7Effect of CBI on cell proliferation in HepG-2induced by olaquindox：The cell relativesurvival rate of olaquindox group and CBI group were lower than that of the controlgroup(P<0.05)，The cell relative survival rate of CBI group was higher than that of theolaquindox group(P<0.05).8Effect of CBI on cell apoptosis and the content of ROS inHepG-2induced by olaquindox：The cell apoptosis and the content of ROS in HepG-2were gradually increased with the increase of olaquindox dose (P<0.05), and theapoptosis rate and the content of ROS in CBI group is lower than that of the olaquindoxgroup (P<0.05).9The cathepsin B and caspase-3protein expression in HepG-2cells:The count of cathepsin B was gradually increased with the increase of olaquindoxdose.The protein expression of cathepsin B and caspase-3were gradually increase withthe dose of olaquindox. The protein expression of cathepsin B and caspase-3in CBIgroup was higher than the control group, but lower than the OLA group(P<0.05). Conclusions1OLA can cause oxidative damage and inflammation in lever, which leadto liver cell apoptosis of rat liver tissue. The protein expression level of cathepsin Bincreases, indicating that cathepsin B may be involved in rats liver injury induced byOLA.2OLA can cause HepG-2cell apoptosis, and apoptosis related proteins cathepsinB and caspase3expression levels increase, suggesting cathepsin B protein is involved inapoptosis induced by OLA.