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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 observe the protective effect of Di Lianxin Granule on the injury of mouse fibroblasts induced by H_2O_2 and to explore the possible mechanism. Methods: L929 cells were induced by H_2O_2 to establish oxidative damage model. MTT method was used to detect the effect of Lianxinxin on proliferation of L929 cells. Fluorescence microplate reader and Hoechst33258 staining were used to detect the effect of Lianxinxin Granule on oxidative stress-induced apoptosis and intracellular reactive oxygen species (ROS) The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) in the supernatant were measured by colorimetry. Griess The levels of NO, TNF-α, IL-1β, IL-6 and IL-6 in supernatants were determined by ELISA and ELISA. -4, IL-10, Caspase-3 and Caspase-9 in L929 cells were determined by colorimetric assay. The expressions of Bcl-2, Bcl-x L and Bax, expression. Results: When Lian-xian particles concentration was less than 100μg / ml, the proliferation of L929 cells could be promoted both with Erzhong particles and H_2O_2 alone, but when the concentration was more than 400μg / ml or with H_2O_2 more than 200μg / ml, all inhibited the cell growth; even with Erxin particles (25,50,100μg / ml) could inhibit the apoptosis of L929 cells, reduce the intracellular ROS content; increase the supernatant SOD, CAT and GSH -Px, decrease the content of MDA, decrease the contents of proinflammatory cytokines (iNOS, NO, TNF-α, IL-1β and IL-6) in the supernatant and up- regulate the expressions of Bcl-2 and Bcl- Down-regulate the expression of pro-apoptotic proteins Bax and Bad, and decrease the activity of Caspase-3 and Caspase-9. CONCLUSION: Di Lianxin Granule has a good protective effect on oxidative damage of L929 cells induced by H_2O_2. It mainly overexpresses ROS by inhibiting L929 cells and enhances the activities of GSH-Px, SOD and CAT. Reduce the content of MDA, inhibit the secretion of iNOS, NO, TNF-α, IL-1β and IL-6 and other proinflammatory cytokines, promote the secretion of IL-4 and IL-10 anti-inflammatory cytokines and up-regulate the anti-apoptotic protein Bcl- , Bcl-x L, downregulate the expression of pro-apoptotic proteins Bax, Bad and Caspase-3 and Caspase-9 activity, thereby increasing the survival rate of L929 cells, to exert the protective effect on damaged L929 cells.