Study On The Protective Effect Of The Major Individual Phenolic Compounds From Lycopus Lucidus Turcz.on Hepatocyte Injury In Vitro

Objective Basing on the in vitro experiment of BRL hepatocyte injured by CC1₄to clarify the protective effects and mechanisms of the major individual phenolic compounds including caffeic acid（CA）and rosmarinic acid（RA）from Lycopus lucidus Turcz.And to further reveal the biological activities of the major individual phenolic compounds from Lycopus lucidus Turcz..Meanwhile,to provide theoretical basis for the development and utilization of Lycopus lucidus Turcz.resources.Methods The experiment was divided control group,CCl₄ model group and CA/RA-treated groups.A model of hepatocyte injury in vitro was built by treating the BRL hepatocyte with CCl₄ at 13.79 mmol/L.The CA/RA-treated groups were pretreated the BRL hepatocyte with CA and RA before exposure to CCl₄.The cell survival rate was determined by the MTT method,and the aspartate aminotransferase（AST）,alanine aminotransferase（ALT）,and lactate dehydrogenase（LDH）levels were measured by a fully automatic biochemical analyzer.ELISA kits were used to detect the levels of reactive oxygen species（ROS）,cytochrome c（Cyt c）and 8-hydroxydeoxyguanosine（8-OHDG）in the cells.The m RNA expression levels of nuclear factor E2 related factor 2（Nrf2）,heme oxygenase 1（HO-1）,quinone oxidoreductase 1（NQO1）,glutathione reductase（GSR）and superoxide dismutase（SOD）in cells were determined by real-time fluorescent quantitative PCR.The expression levels of COX-2,inducible nitric oxide synthase（i NOS）,interleukin-6（IL-6）and Caspase-3 protein were detected by immunohistochemistry detection analysis of cell climbing pieces.Results CA and RA had cytotoxicity at a concentration of 1.6 mg/m L,and CA and RA could effectively increase the cell viability of BRL hepatocyte injured by CC1₄in the range of 0.2 to0.8 mg/m L（P<0.05）.The increases in levels of AST,ALT,LDH,ROS,Cyt c and 8-OHDG were significantly decreased in CC1₄-induced BRL hepatocyte pretreated with CA and RA（P<0.05）.The q RT-PCR test results showed that the m RNA expression of Nrf2,HO-1,NQO1,GSR and SOD in normal group was no significant change compared with the model group（P>0.05）.However,the m RNA expression of the tested genes in the CC1₄-induced BRL hepatocyte pretreated with CA and RA markedly increased in comparison with the model group（P<0.05）.Immunohistochemical results showed that the expression levels of COX-2,i NOS,IL-6 and Caspase-3 in the model group were significantly higher than those in the control group,while the levels of protein expression in the CA and RA treated groups were decreased in various degrees compared with model group.Conclusion CCl₄ exposure can cause oxidative stress and inflammatory response in BRL hepatocyte leading to liver cell damage and apoptosis.Pretreated with CA and RA can effectively inhibit the damage of CCl₄ induced liver injury with a dose-dependent effect.Its mechanism of action may be to directly enhance the free radical scavenging ability or up-regulate the expression level of Nrf2,then activate the Nrf2-ARE antioxidant pathway and improve the cell’s antioxidant defense ability at the transcription level,thereby reducing oxidative damage,the occurrence of inflammatory response and apoptosis.