高糖培养下系膜细胞JAK_2/STAT_3信号转导通路活性变化及与活性氧簇作用的研究

Effects of High Glucose on the JAK_2/STAT_3 Signal Transduction Pathway and Reactive Oxygen Species in Mesangial Cells

目的:通过观察高糖培养条件下大鼠肾小球系膜细胞P-STAT3的表达变化,探讨糖尿病状态下JAK2/STAT3途径活性的变化及该通路与活性氧簇(ROS)之间的相互作用。方法:用传代培养的大鼠肾小球系膜细胞同步化后分组:(1)正常糖浓度组(含5.5mmol/L),高糖浓度组(25mmol/L),甘露醇组(5.5mmol/L糖+19.5mmol甘露醇),正常糖+AG-490(浓度10μmol/L)组,高糖+AG-490(浓度10μmol/L)组。继续观察培养后用WesternBlot及细胞免疫化学方法检测系膜细胞STAT3、P-STAT3表达的变化。(2)正常糖浓度组(N),高糖浓度组(H),甘露醇组(M),正常糖+Apocynin组(N+A,Apocynin浓度为100μmol/L),高糖+Apocynin组(H+A,Apocynin浓度为100μmol/L),收集上清液,用比色法检测系膜细胞ROS水平。(3)NADPH氧化酶抑制剂Apocynin预处理,分组同(2),Apocynin提前1h加入,与正常糖或高糖同时培养后,用WesternBlot方法检测系膜细胞P-STAT3表达。结果:(1)高糖培养大鼠肾小球系膜细胞P-STAT3的表达较正常糖浓度组明显升高,甘露醇组与正常糖浓度组相比差异无统计学意义;各组之间STAT3表达差异无统计学意义。(2)高糖条件下,ROS产生明显升高,NADPH氧化酶抑制剂Apocynin可明显降低ROS的产生。(3)高糖条件下,Apocynin经预处理,在正常糖浓度和高糖浓度同时培养48h后,正常糖浓度组和正常糖+Apocynin组对比P-STAT3的表达差异无明显区别;高糖+Apocynin组较正常糖浓度组有明显区别,但与高糖组相比明显降低。结论:高糖通过磷酸化方式激活大鼠肾小球系膜细胞JAK2/STAT3信号转导通路;高糖作用下,肾小球系膜细胞ROS产生增加,并具有时间依赖性;高糖状态下ROS可激活肾小球系膜细胞的JAK2/STAT3信号传导通路,证明ROS可能参与糖尿病肾病的发生和发展过程。

Objective：To study the changes of JAK2/STAT3 pathway in the diabetic state by observing the changes of P-STAT3 of the rat glomerular mesangial cells （GMC） cultured in high glucose state, and study the effects of JAK2/STAT3 pathway on the generation of ROS and the secretion of ECM. Methods： （ 1 ）Synchronized GMCs were divided into following groups： the normal group,High glucose group, Mannitol group, the normal ＋ AG-490 group, the high glucose ＋ ACT- 490 group. After 24～48 hours＇ incubation, the GMCs were analyzed by Western Blot and Immunocytochemistry to observe the changes of STAT3 and P-STAT3 expressions in mesangial cells. （2） Collect the cells at 0h, 12 h and 36 h respectively,and then determine the O2^-levels with colorimetric method. （3） After treatment with Apocynin for 1 h, incubate the GMC of each group for 48 hours. The change of P - STAT3 expression in GMC was detected by Western Blot. Results： （ 1 ）The P - STAT3 expression in the GMC that cultured with high glu- cose is significantly higher than that in the normal group. And there is no statistical difference between the mannitol and the normal group;and also there are no significant differences among the groups on the STAT3 exprexsions. （2） At on 02- level in each group is almost same and the 02- level in the High glucose group is obviously increased and higher than that in the normal （P（0.05）. There is no significant difference between the Mannitol and the normal group in the 02 level, and the 02 level in the high glucose ＋ Apoc- ynin is lower than that in the high glucose group （P 〈 0.05 ）. （3） The P- STAT3 expression in GMC of the H group is obviously higher than that in the N group at 48 h;There is no difference between the NA and the N groups;The P-STAT3 expression in the H＋ A group is significantly decreased but still higher than that in the N group. Conclusion： Under high glucose conditions,JAK2/ STAT3 signal transduction pathway can be activated in the phosphorylation manner. ROS in GMC can be increased in a time - dependent manner under high glucose condition and NAI）PH oxidase is the stimulator for the generation of ROS, so the Apocynin of NADPH oxidase inhibitors can inhibit ROS level effectively. Under high glucose condition with the activated JAK2/STAT3 pathway, blockage of ROS can only decrease the P - STAT3 expression in the GMC partly, which means that the pathway may be involved inthe regulation of the secretion of ECM.