地连二心颗粒对H_2O_2致小鼠成纤维细胞损伤的保护作用研究

Protective effects of Dilian Erxin granule against oxidative stress damage induced by H_2O_2^- in mouse fibroblast cells

目的:观察地连二心颗粒对H_2O_2致小鼠成纤维细胞损伤的保护作用并探讨其可能机制。方法:用H_2O_2诱导L929细胞建立氧化损伤模型。MTT法检测不同浓度地连二心颗粒对L929细胞增殖的影响,荧光酶标仪和Hoechst33258染色检测地连二心颗粒对氧化应激诱导细胞凋亡及胞内活性氧(ROS)含量的影响,比色法测定上清液中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)的活性及丙二醛(MDA)含量,用Griess法和ELISA法检测上清液中一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、白介素-6(IL-6)和白介素-4(IL-4)、白介素-10(IL-10)含量,比色法测定L929细胞中Caspase-3、Caspase-9的活性,Western blot检测L929细胞中Bcl-2、Bcl-x L和Bax、Bad蛋白表达。结果:当地连二心颗粒浓度低于100μg/ml时,无论单用地连二心颗粒还是与H_2O_2联用均对L929细胞增殖具有促进作用,但是当其浓度大于400μg/ml或与H_2O_2联用大于200μg/ml时,均对其细胞生长具有抑制作用;地连二心颗粒(25、50、100μg/ml)可抑制L929细胞凋亡,降低细胞内ROS含量;升高上清液中SOD、CAT和GSH-Px活性,降低MDA含量,降低上清液中促炎因子i NOS、NO、TNF-α、IL-1β、IL-6含量,上调抗凋亡蛋白Bcl-2、Bcl-x L蛋白表达,下调促凋亡蛋白Bax、Bad蛋白表达,降低Caspase-3和Caspase-9的活性。结论:地连二心颗粒对H_2O_2致L929细胞氧化损伤具有较好的保护作用,它主要通过抑制损伤的L929细胞过量产生ROS,增强内源性抗氧化酶GSH-Px、SOD、CAT的活性,降低MDA含量,抑制其分泌i NOS、NO、TNF-α、IL-1β和IL-6等促炎因子,促进其分泌IL-4、IL-10抗炎因子,上调抗凋亡蛋白Bcl-2、Bcl-x L表达,下调促凋亡蛋白Bax、Bad蛋白表达及降低Caspase-3和Caspase-9活性,进而提高L929细胞存活率,来发挥对受损L929细胞的保护作用。

Objective： To study the protective effects of Dilian Erxin granule against oxidative damage induced by H2O2-in mouse fibroblast cells and its mechanism.Methods：-Oxidative stress injury in mouse fibroblast cell was established with H2O2.The cell proliferation was examined by MTT assay,ROS content was detected by fluorescence enzyme label,the mophology of apoptotic cells observed by hoechst33258 through fluorescence microscope; The levels of superoxide dismutase（ SOD）,glutathione peroxidase（ GSH-Px）,hydrogen peroxidase（ CAT）,malondialdehyde（ MDA） were also measured by colorimetry,and the supernatant liquid inducible nitric oxide synthase（ iNOS）,nitric oxide（ NO）,tumor necrosis factor-α（ TNF-α）,interleukin-1β（ IL-1β）,interleukin-6（ IL-6）,interleukin-4（ IL-4） and interleukin-10（ IL-10）were measured by Griess and ELISA.The activities of Caspase-3 and Caspase-9 were also determimed by Caspase colorimetric assay kit in each group.The protein expressions of Bcl-2,Bcl-x L,Bax and Bad were detected by western blot.Results： Compared with the control group,Dilian Erxin granule could promote the proliferation of L929 cells no matter with H2O2 or not when its concentration was lower than100μg/m L; But when its concentration was higher than 400μg/m L,Dilian Erxin granule significantly inhibited the growth of the cells whether H2O2 existed or not.Dilian Erxin granule（ 25,50,100μg/ml） inhibited L929 cell apoptosis,decreased the content of intra-cellular ROS,improved activities of SOD,GSH-PX,CAT in the supernatant liquid,decreased MDA level,decreased contents of iNOS,NO,TNF-α,IL-1β,IL-6 in the supernatant liquid,increased contents of IL-4,IL-10 in the supernatant liquid from L929 cells,compared with the model group,upregulated the antiapoptotic protein Bcl-2,Bcl-x L expressions,downregulated the proapoptotic protein Bax,Bad expressions,reduced the activities of Caspase-3 and Caspase-9（ P 〈 0.05 or P 〈 0.01,respectively）.Conclusion： The results suggested that Dilian Erxin granule exerted beneficially protective effects on oxidative stress injuries of L929 cells induced by H2O2-.The influence may be related to some actions,such as inhibiting ROS excess production,reinforcing endogenous antioxidase activities,inhibiting iNOS,NO,TNF-α,IL-1β,IL-6 promote inflammatory factor secretion,promoting IL-4,IL-10 antiinflammatory cytokine secretion,upregluting the antiapoptotic protein Bcl-2 Bcl-x L expressions,downregulating the proapoptotic protein Bax,Bad expressions,decreasing activities of the Caspase-3 and Caspase-9,and improving the survival rate of L929 cell.