巫山神茶对H2O2诱导293T细胞氧化损伤的改善作用

以巫山神茶为研究对象,通过DPPH、ABTS、OH及O₂^-自由基清除实验考察其体外抗氧化活性。采用MTT法测定巫山神茶对293T细胞氧化损伤的细胞存活率,并使用试剂盒比色法和实时荧光定量PCR法测定MDA、SOD、CAT、GSH、GSH-Px含量及相关基因的表达。另外,以HPLC法分析了巫山神茶的主要活性成分。结果表明,巫山神茶对DPPH、ABTS、羟基及超氧阴离子自由基有显著的清除效果,且呈现浓度-效应依赖性。不同浓度巫山神茶(40、100、160 μg/mL)处理后的293T细胞存活率均超过93%,说明无明显的毒性作用。对比正常组,过氧化氢(0.3 mmol/L)可显著(P<0.05)导致293T细胞氧化应激损伤,经巫山神茶处理后受损细胞的存活率提高,其中高浓度(160 μg/mL)处理下的细胞的存活率达75.6%。同时,巫山神茶处理还能显著(P<0.05)降低MDA含量,提高CAT、SOD、GSH及GSH-Px含量。实时荧光定量PCR检测相关抗氧化基因也提示巫山神茶能上调CAT、SOD、GSH及GSH-Px的mRNA表达量。此外,通过HPLC分析,从巫山神茶中检测到以下9种生物活性成分:绿原酸、表儿茶素没食子酸酯、对香豆酸、异槲皮苷、二氢槲皮素、槲皮苷、黄芩苷、槲皮素、根皮素。总之,巫山神茶具有较好的体外抗氧化能力,能够改善由H₂O₂诱导的293T细胞氧化损伤且效果显著,其药理价值可考虑进一步深入研究。

Protective Effect of Wushanshencha on Oxidative Damage of 293T Cells Induced by Hydrogen Peroxide

Taking the Wushanshencha( WST) as the research object,the antioxidant activity of WST in vitro was studied by1,1-diphenyl-2-picrylhydrazyl radical( DPPH·),2,2’-azino-bis-( 3-ethylbenz-thiazoline-6-sulfonic acid radical ion)( ABTS+·),hydroxyl radical(·OH) and superoxide anion(·O2-) scavenging tests.The 3-( 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide( MTT) assay was used to determine the cell viability of 293 T cells after oxidative damage.The contents of malondialdehyde( MDA),superoxide dismutase( SOD),catalase( CAT),glutathione( GSH),glutathione peroxidase( GSH-Px) and the expression of related antioxidant genes were determined by kit colorimetry and quantitative realtime polymerase chain reaction( qRT-PCR). In addition,the active compounds of WST were analyzed by high performance liquid chromatography( HPLC).The results showed that WST had obvious scavenging effects on DPPH·,ABTS+·,hydroxyl and superoxide anion radicals,and it had concentration-effect dependent. The survival rates of 293 T cells treated with different concentrations of WST( 40,100,160 μg/m L) exceeded 93%,indicating no significant toxic effects. Compared with the normal group,hydrogen peroxide( 0.3 mmol/L) treatment could significantly cause oxidative stress damage in 293 T cells( P < 0.05).After treatment with WST,the survival rate of damaged cells was improved,and the survival rate of cells treated with high concentration( 160 μg/m L) reached 75.6%. At the same time,WST treatment could significantly reduce MDA contents( P < 0.05),and increased CAT,SOD,GSH and GSH-Px content.Real-time fluorescent quantitative PCR also indicated that the mRNA expression levels of related antioxidant genes CAT,SOD,GSH and GSH-Px could be up-regulated by WST. In addition,the following 9 bioactive components were detected from the extracts of WST by HPLC analysis( Chlorogenic acid,(-)-epicatechin gallate,p-coumaric acid,isoquercitrin,dihydroquercetin,quercetin,baicalin,quercetin,phloretin). In summary,WST had excellent antioxidant activity in vitro and would improve the oxidative damage of 293 T cells induced by hydrogen peroxide,and its pharmacological value could be considered for further study.