Profilin1激活自噬参与草酸介导的肾小管上皮细胞凋亡

Profilin1 activates autophagy to participate in apoptosis of renal tubular epithelial cells induced by oxalate

目的探讨Profilin1(PFN1)激活自噬参与草酸介导的肾小管上皮细胞凋亡的影响机制。方法选取2022年4月至2022年7月在本院确诊为肾结石的30例患者作为结石组,同期选取30例无肾结石患者作为对照组,收集两组研究对象的尿液标本,采用ELISA法检测两组患者尿液中的PFN1浓度。使用HK-2细胞作为体外研究对象,分为细胞对照组(基础培养基)、草酸组(2 mmol/L草酸)、阴性对照组(2 mmol/L草酸+siRNA阴性对照)、si-PFN1组(2 mmol/L草酸+si-PFN1)和si-PFN1+雷帕霉素组(2 mmol/L草酸+si-PFN1+雷帕霉素)。通过PCR和蛋白质免疫印迹实验检测细胞内的PFN1表达,然后使用基因工程技术对HK-2细胞进行PFN1基因敲减,检测各组的Caspase3酶活性含量、线粒体膜电位水平以及自噬标志分子Beclin1的表达。通过药物干预对PFN1的作用进行挽救实验,再次检测各组的Caspase3酶活性含量和线粒体膜电位水平。结果结石组患者尿液中的PFN1浓度显著高于对照组(501.83±81.40 vs.236.67±77.09,P<0.05)。在对PFN1进行siRNA敲减后,与细胞对照组比较,草酸组的PFN1 mRNA(1.07±0.21 vs.0.45±0.91)和蛋白表达量(0.27±0.05 vs.0.16±0.03)增加,差异均有统计学意义(均P<0.01);与细胞对照组比较,草酸组的Caspase3酶活性增加,而线粒体膜电位水平下降;与阴性对照组比较,si-PFN1组的Caspase3酶活性降低,而线粒体膜电位水平增加;与细胞对照组比较,草酸组的Beclin1蛋白和mRNA表达增加(1.06±0.16 vs.0.28±0.07,P<0.05);与阴性对照组比较,si-PFN1组的Beclin1蛋白和mRNA表达降低(0.69±0.14 vs.1.13±0.11,P<0.05)。使用自噬激活剂后,与si-PFN1组比较,si-PFN1+雷帕霉素组的Caspase3酶活性上升,而线粒体膜电位降低。结论PFN1参与了草酸介导的肾小管上皮细胞凋亡,其作用机制可能是激活自噬而导致的,而PFN1有望作为肾结石防治的靶点。

ObjectiveTo investigate the effect of Profilin1(PFN1)activation of autophagy involved in oxalic acid-mediated apoptosis of renal tubular epithelial cells.MethodsThirty patients diagnosed with kidney stones in our hospital from April 2022 to July 2022 were selected as the stone group,and 30 patients without kidney stones were selected as the control group during the same period.Urine specimens were collected from the study subjects of the two groups,and the concentration of PFN1 in the urine of the two groups was detected by ELISA.HK-2 cells were used as in vitro study subjects,which were divided into cell control group(basal medium),oxalic acid group(2 mmol/L oxalic acid),negative control group(2 mmol/L oxalic acid+siRNA negative control),si-PFN1 group(2 mmol/L oxalic acid+si-PFN1),and si-PFN1+rapamycin group(2 mmol/L oxalic acid+si-PFN1+rapamycin).Intracellular PFN1 expression was detected by qPCR and protein immunoblotting assay,and then PFN1 knockdown was performed in HK-2 cells using genetic engineering technology,and the Caspase3 enzyme activity content,mitochondrial membrane potential level,and the expression of the autophagy marker molecule Beclin1 were further detected in each group.Rescue experiments on the effect of PFN1 by drug intervention were performed,and the content of Caspase3 enzyme activity and the level of mitochondrial membrane potential were again detected in each group.ResultsThe concentration of PFN1 in the urine of patients in the stone group was significantly higher than that in the control group(501.83±81.40 vs.236.67±77.09,P<0.05).After siRNA knockdown of PFN1,PFN1 mRNA(1.07±0.21 vs.0.45±0.91)and protein expression(0.27±0.05 vs.0.16±0.03)were increased in the oxalic acid group compared with the cellular control group(all P<0.05).Caspase3 enzyme activity was increased and mitochondrial membrane potential levels were decreased in the oxalic acid group compared to the cellular control group.Caspase3 enzyme activity was decreased in the si-PFN1 group and mitochondrial membrane potential level was increased compared to the negative control group.Beclin1 protein and mRNA expression was increased in the oxalic acid group compared with the cellular control group(1.06±0.16 vs.0.28±0.07,P<0.05).Beclin1 protein and mRNA expression was decreased in the si-PFN1 group compared with the negative control group(0.69±0.14 vs.1.13±0.11,P<0.05).After the use of autophagy activator,Caspase3 enzyme activity was increased and mitochondrial membrane potential was decreased in the si-PFN1+rapamycin group compared with the si-PFN1 group.ConclusionsPFN1 is involved in oxalic acid-mediated apoptosis of renal tubular epithelial cells,which may result from the activation of autophagy,and PFN1 is expected to serve as a target for the prevention and treatment of renal stones.