The Effect Of Atorvastatin On The Liver Of ApoE-/- Mice Under Inflmmatory Stress And The Investigation Of Underlying Mechanisms

Objective: Hypercholesteremia is a primary risk factor for cardiovascular diseases(CVD). Statins not only effectively reduce serum low-density lipoprotein cholesterol(LDL-C) levels, but also remarkably decrease morbidities and mortalities of CVD. Therefore, statins are widely used for the primary and secondary prevention of CVD, and atorvastatin is the most used statin. Liver injury is one of the common adverse effects induced by statins. However, the risk factors and mechanism of statin-induced liver injury are not fully understood. Inflammatory stress is a susceptibility factor for drug-induced liver injury, but whether it sensitizes the liver to statin-induced injury is unclear. The development of drug-induced liver injury is determined on the battle between the hazardous stress and adaptive responses in the hepatocytes. It is reported that many drugs including statins can induce overproduction of reactive oxygen species(ROS), thus lead to oxidative stress associated injury. On the other hand, the activation of nuclear factor erythroid 2-related factor 2(Nrf2), a master regulator of many antioxidant genes, can protect against drug-indcued liver injury. It is yet unknown whether statin-induced liver injury can be explained by the imbalance between ROS overproduction and Nrf2 mediated antioxidant capacities. The aim of this study was to investigate the effect of atorvastatin on the liver in Apo E-/- mice under inflammatory stress, and explore the underlying mechanisms.Methods: Male Apo E-/- mice were fed a western diet to establish the hypercholesteremia model, and were injected with casein subcutaneously every other day to establish chronic systemic inflammation. Apo E-/- mice were randomly divided into four groups: 1)control group: mice were subcutaneously injected with 0.5ml saline only; 2)atorvastatin group: mice were subcutaneously injected with 0.5ml saline and treated with atorvastatin(10mg/kg/d); 3)casein group: mice were subcutaneously injected with 0.5ml 10% casein only; 4)casein+atorvastatin group: mice were subcutaneously injected with 0.5ml 10% casein and treated with atorvastatin(10mg/kg/d). The levels of serum IL6, TNFα and FFA and hepatic FFA in mice were determined by ELISA. The levels of serum TC, LDL-C, TG, ALT and AST were also examined. Hepatic TG contents were determined using the colorimetric method. The degree of liver steatosis was visulised by HE and oil red O staining. F4/80 immunohistochemistry and sirus red staining were used to evaluate the degree of hepatic inflammation and fibrosis, respectively. The m RNA and protein expressions of lipogenic genes(FAS, ACC and SREBP1), cytokines/chemokine(IL1β, TNFα and MCP1) and fibrosis related genes(αSMA, Col IV and TGFβ) were detected by RT-PCR and western blot, respectively. Hepatic O2- levels were measured with the oxidative fluorescent dye dihydroethidium(DHE). Hepatic H2O2 and MDA contents were determined using the colorimetric method. The distribution and protein expressions of Nrf2 in nuclear and cytoplasm of the liver were evaluated by immunohistochemistry staining and western blot, respectively. The m RNA expressions of Nrf2 targets genes(HO-1, NQO1 and SOD2) were detected by RT-PCR. The activities of CAT and SOD in livers of mice were examined using colorimetric methods.Results: The levels of serum IL6 and TNFα were significantly elevated in mice injected with casein(P<0.05). Atorvastatin reduced serum TC and LDL-C levels significantly(P<0.05), but had no effect on serum TG and FFA levels. In inflamed condition, atorvastatin elevated serum ALT and AST levels significantly(P<0.05), exacerbated liver steatosis, characterized by increments of hepatic TG and FFA(P<0.05), and increased the m RNA and protein expressions of lipogenic genes(FAS, ACC and SREBP1), cytokines/chemokine(IL1β, TNFα and MCP1) and fibrosis related genes(αSMA, Col IV and TGFβ)(P<0.05). In contrast, in non-inflamed condition, atorvastatin had no obvious effect on liver enzymes as well as hepatic steatosis, inflammation and fibrosis. Moreover, the contents of hepatic O2-, H2O2 and MDA were not obviously changed in mice treated with atorvastatin in non-inflamed condition, but the protein expression of Nrf2 and m RNA expressions of Nrf2 targets genes as well as activities of CAT and SOD were significantly increased(P<0.05). In contrast, hepatic O2-, H2O2 and MDA contents were significantly increased in mice of casein+atorvastatin group(P<0.05), but Nrf2-mediated adaptive antioxidant response was impaired.Conclusions: Atorvastatin had no adverse effect on the liver of mice in non-inflamed condition, which may be associated with the activation of Nrf2-mediated antioxidant response. However, under inflammatory stress, atorvastatin induced hepatic oxidative stress injury, and exacerbated hepatic steatosis, inflammation and fibrosis, which may be partly due to the lost of Nrf2-mediated adaptive response.