The Protective Effect Of L-carnitine On The CCl₄-induced Cytotoxicity In HL7702Cells

OBJECTIVE: To establish CCl₄-induced apoptotic （oxidative damage） model of HL7702cells,screen and determine the effective concentration of L-carnitine （LC）; and investigate the mechanism of LCprotecting HL7702cells.METHODS: CCl₄-induced oxidative apoptotic model of HL7702cells was established by L49（3）orthogonal design. Two factors and three levels were CCl₄concentrations （4,8,16mmol·L-1）, injury time（3,6,9h）. MTT assay was used to determine the effective concentration of LC（10-5000μmol·L-1）on cellviability. The cells were divided into four groups: control group,5.68mmol/L vitaminC group, CCl₄modelgroup, flavonoids from L-carnitine groups （1000,3000,5000μmol·L-1flavonoids from L-carnitine）. Theviability of HL7702cells were detected by MTT assay Alanine aminotransferase （ALT）,aspartateaminotransferase（AST）,Superoxide dismutase （SOD）, lipid peroxidation malondialdehyde （MDA） levelswere measured by enzyme biochemical methods; The production of ROS in HL7702cells was tested usingflow cytometry（FCM）. Hoechst33258was used to determine the morphological characteristic of apoptoticcells;Protein expression levels of NF-κB/p65, p-NF-κB/p65, Bcl-2and Bax were determined by Westernblot analysis.RESULTS: Results of orthogonal experiment suggest that8mmol·L-1CCl₄for6h make up thebest oxidative damage model; CCl₄downgrated the viability of HL7702cells and upgrated the levels ofalanine aminotransferase （ALT）, aspartate aminotransferase（AST）, malondialdehyde （MDA）, decreased thelevel of SOD; strongly enhanced the intracellular ROS level; cell shrinkage and chromatin aggregation.CCl₄could increased transcriptional activation and translocation of NF-κB/p65to nucleus and decreasedBcl-2/Bax ratio. Flavonoids from L-carnitine （LC） could strongly reduced ROS level, decreased the levelsof ALT,AST,MDA, increased the level of SOD, morphological changes of apoptosis were also inhibited byLC; transcriptional activation and translocation of NF-κB/p65to nucleus were decreased and Bcl-2/Baxratio was increased by LC dose-dependently.CONCLUSION: The moderate oxidative damage and apoptotic model of HL7702cell wassuccessfully established (8mmol·L^-1CCl₄,6h);3000-5000μmol·L-1LC had no cytotoxic effect on HL7702cells; The protective effect of LC on HL7702cells may attributes to its antioxidant effect and anti-apoptotic effects which related to increased protein expression of NF-κB/p65and Bcl-2/Bax ratio.