miR-182-5p调控FABP4表达对草酸钙结晶性肾损伤的影响

Effect of miR-182-5p regulating FABP4 expression on calcium oxalate crystal kidney injury

目的探讨抑制miR-182-5p对草酸钙结晶性肾损伤的影响及其可能的作用机制。方法将72只C57BL/6小鼠随机分为对照组、模型组、阴性对照(NC)抑制剂组、miR-182-5p抑制组、miR-182-5p抑制+NC沉默组、miR-182-5p抑制+脂肪酸结合蛋白4(FABP4)沉默组,每组12只。除对照组外,其他组均构建肾草酸钙结晶小鼠模型,建模成功后,NC抑制剂组、miR-182-5p抑制组、miR-182-5p抑制+NC沉默组、miR-182-5p抑制+FABP4沉默组分别通过尾静脉注射NC antagomir、miR-182-5p antagomir、miR-182-5p antagomir和NC shRNA、miR-182-5p antagomir和FABP4shRNA,对照组、模型组通过尾静脉注射等量的生理盐水。检测尿离子钙、血清肌酐(Scr)及血尿素氮(BUN)浓度及尿草酸浓度;HE染色、pizzolato染色分别检测小鼠肾组织病理变化、草酸钙结晶沉积;分析小鼠肾组织中超氧化物歧化酶(SOD)、丙二醛(MDA)及肾小管上皮细胞凋亡情况;q RT-PCR、Western blot分别检测肾组织中miR-182-5p、FABP4蛋白表达;双荧光素酶报告基因实验验证miR-182-5p与FABP4的关系。结果与对照组比较,模型组和NC抑制剂组小鼠病理评分,尿离子钙、尿草酸、Scr、BUN、MDA水平、草酸钙结晶沉积、肾小管上皮细胞凋亡率及miR-182-5p表达显著升高,SOD水平及FABP4蛋白表达显著降低,差异均有统计学意义(P<0.05);与模型组、NC抑制剂组比较,miR-182-5p抑制组、miR-182-5p抑制+NC沉默组小鼠病理评分、尿离子钙、尿草酸、Scr、BUN、MDA水平、草酸钙结晶沉积、肾小管上皮细胞凋亡率及miR-182-5p表达显著降低,SOD水平及FABP4蛋白表达显著升高,差异均有统计学意义(P<0.05);与miR-182-5p抑制组、miR-182-5p抑制+NC沉默组比较,miR-182-5p抑制+FABP4沉默组小鼠肾组织病理评分、尿离子钙、尿草酸、Scr、BUN、MDA水平、草酸钙结晶沉积、肾小管上皮细胞凋亡率显著升高,SOD水平及FABP4蛋白表达显著降低,差异均有统计学意义(P<0.05)。双荧光素酶报告基因实验结果显示,与mimic NC和WT-FABP4共转染组比较,miR-182-5p mimic和WT-FABP4共转染组HEK293T细胞的荧光素酶活性显著降低,差异有统计学意义(P<0.05)。结论抑制miR-182-5p通过上调FABP4抑制氧化应激,降低肾小管细胞凋亡,进而发挥对小鼠草酸钙结晶性肾损伤的保护作用。

Objective To investigate the effect of inhibiting miR-182-5p on calcium oxalate crystal kidney injury and its possible mechanism.Methods A total of 72 C57BL/6 mice were randomly divided into control group,model group,negative control(NC)inhibition group,miR-182-5p inhibition group,miR-182-5p inhibition+NC silence group and miR-182-5p inhibition+fatty acid-binding protein 4(FABP4)silence groups,each group with 12 animals.Except for the control group,the other groups were all constructed renal calcium oxalate crystal mouse models.After the model was constructed,NC inhibitor group,miR-182-5p inhibitor group,miR-182-5p inhibitor+NC silencing group,miR-182-5p inhibitor+FABP4silencing group were injected with NC antagomir,miR-182-5p antagomir,miR-182-5p antagomir and NC shRNA,miR-182-5p antagomir and FABP4 shRNA,control group and model group were injected with the same volume of normal saline through tail vein.Urinary ionized calcium,serum creatinine(Scr),blood urea nitrogen(BUN)concentration and urinary oxalic acid concentration were detected.HE staining and pizzolato staining were used to detect pathological changes and calcium oxalate crystal deposits in mouse kidney tissues,respectively.Superoxide dismutase(SOD),malondialdehyde(MDA)and renal tubular epithelial cell apoptosis in mouse kidney tissue were analyzed.qRT-PCR and Western blot were used to detect miR-182-5p and FABP4 protein expression in kidney tissue,respectively.Dual luciferase reporter gene experiment was used to verify the relationship between miR-182-5p and FABP4.Results Compared with the control group,the pathological scores of mice,urinary ionized calcium,urinary oxalic acid,Scr,BUN,MDA levels,calcium oxalate crystal deposition,renal tubular epithelial cell apoptosis rate and miR-182-5p expression were significantly increased,the SOD level and FABP4 protein expression were significantly decreased in the model group and NC inhibitor group,and the differences were statistically significant(P<0.05);compared with the model group and NC inhibitor group,the pathological scores of mice,urinary ionized calcium,urinary oxalic acid,Scr,BUN,MDA levels,calcium oxalate crystal deposition,renal tubular epithelial cell apoptosis rate and miR-182-5p expression were significantly decreased,the level of SOD and the expression of FABP4 protein were significantly increased in the miR-182-5p inhibition group and the miR-182-5p inhibition+NC silencing group,and the difference was statistically significant(P<0.05);compared with the miR-182-5p inhibition group and the miR-182-5p inhibition+NC silencing group,the pathological scores of mice,urinary ionized calcium,urinary oxalic acid,Scr,BUN,MDA levels,calcium oxalate crystals deposition and the renal tubular epithelial cells apoptosis rate were significantly increased,the SOD level and FABP4 protein expression were significantly decreased in the miR-182-5p inhibition+FABP4 silencing group,and the differences were statistically significant(P<0.05).The results of the dual-luciferase reporter gene assay showed that compared with the mimic NC and WT-FABP4 co-transfection groups,the luciferase activity of HEK293T cells was significantly decreased in the miR-182-5p mimic and WT-FABP4 co-transfection groups,the difference was statistically significant(P<0.05).Conclusion Inhibition of miR-182-5p could inhibit oxidative stress by up-regulating FABP4,reduce renal tubular cell apoptosis,and play a protective effect on calcium oxalate crystal kidney injury in mice.