S100A9促进体外SD大鼠星形胶质细胞凋亡及其机制的实验研究

Experimental Study on the Effect of S100A9 on the Apoptosis of Astrocytes in SD Rats in vitro and its Mechanism

本实验以S100A9蛋白和星形胶质细胞为原材料,采用CCK-8法检测不同浓度S100A9对星形胶质细胞的增殖抑制效应以及检测FPS-ZM1、TAK-242对S100A9作用于星形胶质细胞后的保护作用;应用流式细胞术检测细胞凋亡变化;采用ELISA法检测S100A9作用于细胞后上清液中IL-1、IL-6和TNF-α的水平;使用Western blotting检测细胞表达JNK、P38-MAPK、Bcl-2蛋白浓度水平的变化。研究发现,与对照组相比,0.5mg/m LS100A9作用24h后,星形胶质细胞出现显著的增殖抑制效应,且凋亡率显著增高（p〈0.01）。S100A9＋TAK-242组、S100A9＋FPS-ZM1＋TAK-242组作用24 h后细胞增殖抑制显著降低且凋亡率显著降低（p〈0.01）,S100A9＋FPS-ZM1组作用后凋亡率与S100A9组无显著差异（p〈0.05）。与对照组相比,S100A9单独作用24h后星形胶质细胞分泌IL-1、IL-6和TNF-α显著增加（p〈0.01）,S100A9＋FPS-ZM1组、S100A9＋TAK-242组、S100A9＋FPS-ZM1＋TAK-242组,上述因子无明显变化。与对照组相比,S100A9组JNK、P38-MAPK蛋白浓度升高,而Bcl-2蛋白浓度降低;S100A9＋TAK-242组、S100A9＋FPS-ZM1＋TAK-242组JNK、P38-MAPK蛋白浓度均降低,Bcl-2蛋白浓度升高（p〈0.05）;S100A9＋FPS-ZM1组3种蛋白浓度变化与S100A9组无明显差异。综上表明,S100A9可能通过上调JNK、P38-MAPK及下调Bcl-2蛋白表达导致细胞凋亡从而抑制其增殖。TLR-4受体的特异性抑制剂TAK-242减轻了S100A9促凋亡作用,表明S100A9可能通过结合星形胶质细胞膜上TLR-4受体促进凋亡作用。S100A9通过结合细胞膜上TLR-4受体和RAGE受体刺激细胞炎性因子产生增多。

In this study,S100 A9 protein and astrocytes were used as raw materials,the inhibitory effect of S100 A9 ontheproliferation ofastrocytes and theprotective effectof FPS-ZM1 and TAK-242 on astrocytesinsulted by S100 A9 were investigated by CCK-8 method;the apoptosis occurred to each group were detected by flow cytometry;the levels of IL-1,IL-6 and TNF-α in supernatant were measured by ELISA;the expressions of JNK,P38-MAPK and Bcl-2 proteins were detected by Western blotting.The results suggested thatcompared with the control group,the inhibitory effect of astrocytes and the apoptotic rate were significantly increased after 0.5 mg/m L S100 A9 insulted thecells for 24 hours（P〈0.01）.Both S100 A9＋TAK-242 and S100 A9＋TAK-242＋FPS-ZM1 groups acted for 24 hours,the inhibition of cell proliferation and the rate of apoptosis were significantly decreased（P〈0.01）,the apoptoticrate of S100 A9＋FPS-ZM1 group was not significantly different from that of S100 A9 group（P〈0.05）.Compared with the control group,astrocytes secreted IL-1,IL-6 and TNF-α significantly increased after S100 A9 alone（P〈0.01）,but there have no significant changes were observed insulted the cells among the S100 A9＋FPS-ZM1、S100 A9＋TAK-242 and S100 A9＋FPS-ZM1＋TAK-242 groups.The levels of JNK,P38-MAPK protein were increased significantly in the S100 A9 group,and the Bcl-2 protein level was decreased dramaticly;the levels of JNK,P38-MAPK protein in S100 A9＋TAK-242 group were decreased,and the level of Bcl-2 protein was increased（P〈0.05）;there was no significant difference of these three proteins between S100 A9＋FPS-ZM1 group and S100 A9 group.In summary,S100 A9 might cause apoptosis result in cell proliferation inhibition by up regulation of JNK,P38-MAPK and down-regulate the expression of Bcl-2 protein.TAK-242,a specific inhibitor of TLR-4 receptor,attenuates the pro-apoptotic effect of S100 A9,while the effect of RAGE receptor inhibitor FPS-ZM1 was not significant,indicating that S100 A9 might promote apoptosis bybinding TLR-4 receptors on the astrocyte membrane.S100 A9 stimulates the production of inflammatorycytokines bybinding to TLR-4 receptors and RAGE receptor on the cell membrane.