| Background:The liver is the largest organ in the human body and the largest gland in the digestive system.It participates in important functions such as protein synthesis,glycogen storage and detoxification.The incidence of acute liver injury is increasing day by day,seriously jeopardizing the health of patients.Clinically,these conditions often cause liver damage,such as viruses,trauma,and chemicals such as carbon tetrachloride(CCL4).Numerous studies have shown that mitochondrial dysfunction and its mediated excessive oxidative stress and inflammation are involved in the development of liver injury.Rotenone,known as the mitochondrial complex-1 inhibitor,partially inhibits the mitochondrial respiratory chain and reduces oxidative stress levels and mitochondrial dysfunction.However,under disease conditions,abnormal mitochondria can release cytotoxic products,reactive oxygen species(ROS)and pro-apoptotic factors,leading to organ damage.Therefore,we speculate that rotenone treatment may improve organ damage by inhibiting the activity of damaged mitochondria.Consistent with this view,studies have shown that rotenone has antioxidant activity under pathological conditions,rotenone can alleviate inflammation and oxidative stress in chronic kidney disease,and can also reduce the inflammatory response of osteoarthritis.However,the specific protective effects and mechanisms of acute liver injury have not been clarified.Objective:To study the protective effects and mechanisms of rotenone in acute liver injury induced by carbon tetrachloride,and to provide basis and reference for the treatment of acute liver injury.Methods:Twenty four C57BL/6 mice,aged 8-10 weeks,weight 20-24g,male,were selected as experimental animals.Eight mice were randomly selected as control group.The remaining 16 C57BL/6 mice were used to construct animal models of acute liver injury induced by CCL4.After successful modeling,they were randomly divided into the CCL4 model group(n=8)and rotenone treated group(n=8).The control group and the CCL4 model group were all given the normal jelly diet intervention,and the mice in rotenone treated group were given 250ppm rotenone(about 40mg/kg/day)on the basis of the normal diet for continuous 3d intervention.Plasma enzyme levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in three groups were determined by serum biochemical automatic analyzer.The levels of IL-6 and 4-hydroxynonenal(HNE)in liver were determined by enzyme-linked immunosorbent assay(ELISA).Mice were sacrificed after 3 days of intervention,liver tissues were taken,and HE staining and immunohistochemical staining were completed.The levels of oxidative stress:superoxide dismutase 2(SOD2),malondialdehyde(MDA)and mitochondrial damage:mitochondrial transcription factor A(TFAM),mitochondrial NADH dehydrogenase subunit 1(ND1),mitochondrial cytochrome b(cytb)in the three groups were measured by western blot.The mRNA levels of inflammatory factors in three groups were determined by real-time fluorescence PCR.All data in this study were processed by SPSS 18.0 software.Results:(1)ALT and AST levels in the CCL4 model group were higher than those in the control group(P<0.01).ALT and AST levels in rotenone treated group were lower than those in CCL4 model group(P<0.05).(2)Gross morphology of liver tissue and HE staining results showed that CCL4 treatment showed obvious changes in gross morphology of liver,showing scattered white granules,gray,brittle property and increased liver volume,and rotenone significantly alleviated these changes.Extensive cell necrosis was found in the liver of rotenone treated group,most of which were normal after rotenone treated.The results of liver histopathological score showed that the liver pathology score of carbon tetrachloride group was higher than that of the control group(P<0.001).The liver tissue score of rotenone treated group was lower than that of carbon tetrachloride group(P<0.01).(3)CCL4 could reduce the gene expression levels of TFAM,mtNDl and Cytb.In the rotenone treated group,TFAM,mtND1 and Cytb levels were restored after rotenone intervention.The change in TFAM was consistent at the protein level by Western blotting;Meanwhile,the activity of mitochondrial complex 1 was detected in the experiment,and the results showed that compared with the CCL4 group;(4)The expression of SOD2 was measured by Western blot,and the significant decrease of this antioxidant enzyme was observed.In rotenone treated animals,this reduction in SOD2 in the liver was completely restored.Consistent with the recovery of SOD2,the increase of MDA and 4-HNE was significantly blocked in the liver tissues of rotenone-treated mice.The antioxidant effect of rotenone in the experimental environment was further confirmed by DHE probe.(5)Inflammatory genes were up-regulated by CCL4 treatment,which was inhibited by rotenone therapy.In addition,IL-6 protein levels in the liver were measured by ELISA and immunohistochemistry.Furthermore,we found that the enhanced IL-6 protein levels in the liver measured by ELISA and immunohistochemistry was largely normalized;(6)Rotenone alone had no effect on body weight and had little effect on liver weight,ALT and AST levels;Moreover,250ppm rotenone had little effect on liver morpHology after intervention.The expression levels of TFAM and mtND1 genes in the liver of mice treated with rotenone alone were detected.The results showed that rotenone did not affect the protein levels of TFAM and mRNA levels of mtND1.The expression level of CYP2E1(metabolic enzyme of CCL4)was analyzed by qRT-PCR and Western blot,and the results showed that rotunone alone did not significantly affect the expression of CYP2E1.Conclusion:This study demonstrated that inhibition of mitochondrial complex-1 by rotenone remarkably protected liver against CCL4-induced acute injury possibly by inhibiting mitochondrial oxidative stress and inflammation The findings from current research also suggested a potential strategy for the treatment of acute liver injury by modulating mitochondrial complex-1 activity. |
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