摘要
目的:观察WNK_3激酶高表达对白介素-1β(IL-1β)诱导人胚肾细胞(HEK293细胞)凋亡的作用。方法:HEK293细胞分为3组:Vector+NS组、Vector+IL-1β组和WNK_3+IL-1β组。Vector+NS组、Vector+IL-1β组转染对照Vector,WNK_3+IL-1β组转染WNK_3。药物干预时,Vector+NS组加入等量0.9%氯化钠溶液,Vector+IL-1β组、WNK_3+IL-1β组加入10 ng/m L IL-1β。分别于培养0、12、24、36和48 h时,采用CCK-8试剂盒检测细胞活性。孵育0、18、36 h时应用Western blot法检测Caspase-3、cleaved Caspase-3、Caspase-9、cleaved Caspase-9等凋亡蛋白。在IL-1β干预0、30、60 min时采用Western blot法检测JNK通路蛋白。结果:Vector+IL-1β组在给药后细胞活性逐渐下降,在48 h达到最低值,WNK_3+IL-1β组下降幅度较缓和,与同时间点Vector+IL-1β组比较细胞活性回升,在48 h时差异有统计学意义(P<0.01)。IL-1β孵育后cleaved Caspase-3和cleaved Caspase-9表达量增加。与36 h的Vector+IL-1β组比较,WNK_3+IL-1β组的cleaved Caspase-3显著减少,2组的cleaved Caspase-9在18 h及36 h时差异亦存在统计学意义(P<0.05)。WNK_3转染后p-JNK的表达水平较未转染WNK_3组的上升幅度降低(P<0.05)。结论:IL-1β诱导HEK293细胞活性下降,而转染WNK_3质粒可以有效逆转部分细胞活性。WNK_3激酶通过减少JNK的磷酸化抑制Caspase途径的活化,减少细胞凋亡,起到在不利条件下保护肾脏细胞的作用。
Objective： To observe the effect of high expression of WNK3 on apoptosis in HEK293cells induced by interleukin-1β. Methods： The HEK293 cells were divided into 3 groups （Vector＋NS group, Vector＋IL-1β group and WNK3＋IL-1β group）, then tansfection was performed. Liposome 2000 was added to the 1st and 2na group with PCMV-HA-Vector as the blank and negative control respectively; WNK3 was added to the 3ra group. After treated with 10 ng/mL IL-1β （the 2na and 3rd group） or the same amount of normal saline （the 1st group）, the CCK8 analysis was performed for the detection of cell activity after incubation for 0 h, 12 h, 24 h, 36 h and 48 h at 37 ℃ respectively. Cell proteins were collected after 0 h, 18 h and 36 h incubation respectively for detection of Caspase-3, Cleaved Caspase-3, Caspase-9, Cleaved Caspase-9. In addition, cell protein were collected 0 min, 30 min and 60 min after the treatment oflL-1β for JNK detection. Results： No significant difference of cell activity was observed in the Vector＋NS group within 48 h. In the Vector＋IL-1β group, cell activity began to decline soon after administration. Significant difference could be found after 24 h and the cell activity reached the lowest at 48 h （P〈0.01）. Cell activity of WNK3＋IL-1β group declined in a moderate manner and the significant difference appeared at 36 h and 48 h when comparing with that at 0 h in the same group （both P〈0.05 ）. However, in comparison with the Vector＋IL-1β group at the same time point, cell activity of WNK3＋IL- 1β group was still a little higher and the significant difference could be observed at 48 h （P〈0.01）. The expression of Caspase-9 and Caspase-3 began to decline 18 h after the administration with an increasing peak of cleaved Caspase-9 which decreased slightly at 36 h. An obvious cleaved Caspase-3 could also be observed 36 h after theadministration. In the WNK3＋IL-1β group, the decline of Caspase-3 was more moderate when comparing with that in the Vector＋IL-1β group. A small quantity of cleaved Caspase-3 could be detected and showed an statistical significance in comparison with that of the same group at 0 h （P〈0.05） with the WNK3; the expression of p-JNK declined but t-JNK did not change. Conclusion： IL-1β may decrease activity of HEK293 cells while transfection of WNK3 may partly reverse this affection of IL-113. The over expression of WNK3 may inhibit the activation of JNK pathways induced by IL-1β, decrease the activation of Caspase pathway to reduce apoptosis, and finally pro- tect the kidney cells under adverse conditions.
出处
《温州医科大学学报》
CAS
2016年第1期1-6,共6页
Journal of Wenzhou Medical University
基金
国家自然科学基金青年基金资助项目(81200513)
钱江人才项目(2011R10049)
温州市科技局科研基金资助项目(H20110014)
