Dendrobium Nobile Lindl. Alkaloids Protect Against CCl₄-induced Liver Mitochondrial Oxidative Damage Via The Activation Of Nrf2 Signaling Pathway

Objective:To investigate the protective effects of Dendrobium nobile Lindl.alkaloids（DNLA）on carbon tetrachloride（CCl₄）-induced hepatic mitochondrial oxidative damage and the underlying mechanism.Methods:Wild-type（WT）and Nrf2 knockout(Nrf2^-/-)mice were randomly divided into normal control group,CCl₄ model group,DNLA low dose group（10 mg/kg）and DNLA high dose group（20 mg/kg）,10 mice per group.The mice were given the DNLA for 7 days,and the normal control group and the CCl₄ model group were given equal volume ofsolvent.After 2 hours of the last administration,acute liver injury was induced by intraperitoneal injection of 0.2%CCl₄（20μL/kg,ip）solution.The mice were sacrificed 24 hours posttreatment and liver and serum samples were collected.The levels of serum ALT and AST were determined by assay kit.The hepatic histopathological changes were observed by hematoxylin-eosin staining（HE）and the ultrastructural changes of liver were observed by transmission electron microscopy.The cellular ROS content was measured by flow cytometry,and the contents of mitochondrial MDA and H₂O₂ were detected by assay kit.In addition,8-OHdG was detected by enzyme linked immunosorbent assay（ELISA）.The measurement of mitochondrial Mn-SOD activity and the content of GSH were conducted by a commercial kit.The ATP content in liver tissue was determined using an Enhanced ATP Assay Kit,and mitochondrial function was assessed by measuring oxygen consumption of isolated mice liver mitochondria.At last,the leakage of cytochrome c in mitochondria was detected by Western blotting,and TUNEL staining was used to detect apoptosis.Results:The results showed that in WT mice the pre-administration of DNLA significantly reduced the levels of ALT and AST in the serum of mice,however,the serum levels of ALT and AST in the Nrf2^-/-mice CCl₄ model group were significantly higher than those in the WT mice.And pathological morphology and electron microscopy examination showed that DNLA can significantly improve hepatic cell degeneration,necrosis and inflammatory cell infiltration.At the same time,it also improved mitochondrial swelling,nuclear membrane shrinkage,and chromatin edge set induced by CCl₄.Compared with the CCl₄ model group,the content of H₂O₂,MDA,and 8-OHdG in the DNLA-administered group were significantly decreased,but in Nrf2^-/-mice challenged with CCl₄,DNLA pretreatment did failed to prevent CCl₄-induced oxidative damage by detecting mitochondrial oxidative damage-related indicators.In WT mice,DNLA significantly increased the antioxidant capacity in mitochondria,however,Mn-SOD activity and GSH content were still at low levels in spite of pretreatment with DNLA in Nrf2^-/-mice.Furthermore,pretreatment with DNLA remarkably reversed the inhibition of mitochondrial respiration,indicating that DNLA protected mitochondria from dysfunction caused by CCl₄.However,in Nrf2^-/-mice,pretreatment with DNLA had a weaker influence on the respiratory function of liver mitochondria than in WT mice.In addition,the administration of DNLA improved elevated ATP production,and decreased CCl₄-induced apoptosis in WT mice,whereas the DNLA-mediated protection on mitochondrial function were diminished in the Nrf2 null mice.Conclusion:In conclusion,our findings demonstrate that DNLA protects against mitochondrial oxidative injury in liver through improving mitochondrial antioxidant capacity and the activation of Nrf2 signaling pathway.