肾损伤因子-1在脂多糖诱导的HK-2细胞炎症反应中的作用

The biological effects of kidney injury molecule-1 in lipopolysaccharide mediated HK-2 cell inflammatory response

目的:分析肾损伤因子-1(KIM-1)在脂多糖(LPS)诱导HK-2细胞炎症模型中的表达并探讨其可能参与的生物学进程。方法:LPS刺激人肾小管上皮HK-2细胞诱导细胞炎症模型,CCK-8比色法分析LPS对HK-2细胞株体外生长的抑制作用;RT-PCR和Western blot实验分析KIM-1在正常组和LPS诱导组中的表达差异;设计siRNA靶向干扰KIM-1基因的表达,分析其对LPS诱导下HK-2细胞生长抑制作用的影响;Hoechst33342/PI双染法分析LPS诱导下对照组和siRNA干扰组细胞凋亡的影响。结果:50、100 μg/mL终浓度的LPS处理24 h 和48 h后能抑制HK-2细胞增殖,与对照组比差异有统计学意义(P<0.05);KIM-1和IL-6基因和蛋白表达水平在LPS处理组中较对照组明显上调,且具有浓度依赖性,差异有统计学意义(P<0.05);LPS处理组中Hoechst33342染色阳性细胞比例明显高于对照组;siRNA转染组中KIM-1和IL-6基因和蛋白表达水平均明显低于siRNA-NC组,在LPS作用下siRNA转染组HK-2细胞增殖率明显高于siRNA-NC组,差异有统计学意义(P<0.05);在siRNA转染组中,Hoechst33342染色强度均低于siRNA-NC组,提示靶向KIM-1基因siRNA转染可以抑制LPS诱导的细胞凋亡作用。结论:LPS可以诱导HK-2细胞增殖受抑和凋亡,并且上调KIM-1和IL-6基因表达;KIM-1可能通过调节细胞凋亡和生长抑制过程参与细胞炎症反应。

Objective: To investigate the expression of kidney injury molecule-1 (KIM-1) in lipopolysaccharide (LPS)-induced HK-2 cells and explore the biological process it maybe involved. Methods: Human renal tubular epithelial HK-2 cells were treated with LPS to establish the sepsis-induced renal cell inflammatory model. CCK-8 assay was used to detect the growth inhibition effect of LPS on HK-2 cells. The mRNA and protein expression levels of KIM-1 were detected by RT-PCR and Western blot respectively. The siRNA sequences targeted on KIM-1 gene were designed and tranfected to HK-2 cells,and then the effects of LPS on the growth inhibition of transfected HK-2 cells were detected by CCK-8 assays. Hoechst33342/PI staining was performed to detect the apoptosis in siRNA and control groups. Results: The proliferation of HK-2 cells was inhibited after treatment with LPS at final concentration of 50 μg/mL and 100 μg/mL for 24 h and 48 h,and the differences were statistically significant compared with the control group (P<0.05). The mRNA and protein levels of KIM- 1 and IL-6 gene were greatly up-regulated after treatment with LPS in a dose-dependent manner,the differences being statistically significant (P<0.05). The proportion of Hoechst3342 positive cells in LPS treatment group was significantly higher than that in control group. The expression levels of KIM-1 and IL-6 genes in siRNA groups were significantly lower than that in siRNA-NC group,with significant differences (P<0.05); The proliferation rate was significantly higher in siRNA transfection group,compared with the siRNA-NC group,and the differences were statistically significant (P<0.05). The fluorescence intensity of Hoechst3342 staining in siRNA transfection groups was lower than that in siRNA-NC group,suggesting that siRNA transfection targeting on KIM- 1 gene could inhibit the apoptosis induced by LPS. Conclusion: LPS can induce the proliferation inhibition and apoptosis of HK-2 cells,and up-regulate the expression of KIM-1 and IL-6 genes. KIM-1 may participate in the inflammatory reaction by regulating the process of apoptosis and cell growth inhibition.