南昆山毛叶茶对tBHP诱导损伤NIH3T3细胞的保护效果

Protective Effect of Camellia ptilophylla Chang on the tBHP-Induced NIH3T3 Cells

本研究以南昆山毛叶茶为材料,探讨其水提物对叔丁基过氧化氢(tert-butyl hydroperoxide,tBHP)诱导小鼠成纤维细胞NIH3T3氧化损伤的保护作用。以tBHP诱导NIH3T3细胞建立氧化损伤模型,采用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)法测定细胞存活率,乳酸脱氢酶(lactate dehydrogenase,LDH)检测试剂盒测定细胞膜完整性。同时,检测细胞内活性氧(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)含量,以及过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、超氧化物歧化酶(superoxide dismutase,SOD)抗氧化酶活性,评价其抗氧化能力,荧光探针测定线粒体膜电位(mitochondrial membrane potential,MMP)变化,分光光度法和Western blot检测caspase-3、caspase-9、细胞色素c的表达以探究其损伤保护机制。结果表明,与tBHP组相比,南昆山毛叶茶水提物在25~100μg/mL浓度范围内,细胞存活率由49.68%显著提高至81.69%;LDH活性由520.70 U/L降低至346.42 U/L。同时,可显著抑制ROS、MDA产生,均为tBHP组的0.02倍,增加GSH含量至tBHP组的3.07倍,提高CAT、GSH-Px、SOD等抗氧化酶活性,分别为tBHP组的1.42倍、2.48倍和1.77倍,提高MMP,抑制caspase-3、caspase-9、细胞色素c的表达。说明南昆山毛叶茶水提物能修复抗氧化系统,抑制线粒体细胞凋亡途径,有效减缓tBHP诱导的NIH3T3细胞氧化损伤。本研究初步揭示了南昆山毛叶茶的抗氧化机理,为这一特殊资源的更广泛开发利用提供了抗氧化功能特性的理论研究基础。

In this study,the protective effect of water extract from Camellia ptilophylla Chang on the tert-butyl hydroperoxide(tBHP)-induced oxidative damage in mouse fibroblast NIH3T3 cells was investigated.Cell viability was analyzed by methyl thiazolyl tetrazolium(MTT)method and the cell membrane integrity was determined by a lactate dehydrogenase(LDH)kit.Besides,the levels of intracellular reactive oxygen species(ROS),malondialdehyde(MDA),and glutathione(GSH),as well as the activities of catalase(CAT),glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)were measured to evaluate the antioxidant capacity of Camellia ptilophylla Chang.To further explore the underlying mechanism,the mitochondrial membrane potential(MMP)was detected by a fluorescent probe,and the expressions of caspase-3,caspase-9 and cytochrome c were measured by spectrophotometry and western blot.The results showed that,in comparison with the tBHP group,the water extract of Camellia ptilophylla Chang significantly increased the cell viability of tBHP-induced NIH3T3 from 49.68%to 81.69%.Meanwhile,the LDH activity decreased from 520.70 U/L to 346.42 U/L when the cells were incubated with 25~100μg/mL of water extract from Camellia ptilophylla Chang.Moreover,the GSH content increased to 3.07 folds while the production of ROS and MDA decreased to 0.02 folds.Furthermore,the increase in the activities of major antioxidase(CAT,GSH-Px and SOD)to 1.42、2.48、1.77 folds respectively,and MMP as well as the inhibition in the expressions of caspase-3,caspase-9 and cytochrome c were observed.These results suggested that the water extract of Camellia ptilophylla Chang could effectively protect NIH3T3 cells from the tBHP-induced oxidative damage by repairing the antioxidant system and inhibiting the mitochondrial apoptotic pathway.The present study preliminarily revealed the antioxidant mechanism of Camellia ptilophylla Chang.It advanced our knowledge of the antioxidant properties of Camellia ptilophylla Chang and provided us with useful information for its further utilization.