circ_0001478靶向miR-335-3p/ADAM10通路加重缺血再灌注急性肾损伤肾小管上皮细胞损伤与凋亡的机制研究OA

Objective To investigate the regulatory role of circ_0001478 on apoptosis of renal tubular epithelial cells in ischemia-reperfusion acute kidney injury(IR-AKI)by targeting the miR-335-3p/ADAM metallopeptidase domain 10(ADAM10)pathway.Methods A total of 24 C57BL/6J mice aged 8-10 weeks were divided into 4 groups(n=6 each):the NC group,the NC+IR group,the circ_0001478 siRNA group,and the circ_0001478 siRNA+IR group.At 24 h before model establishment,the NC and NC+IR groups received negative control siRNA(NC siRNA,7.5 mg/kg)via tail vein injection,while the circ_0001478 siRNA and circ_0001478 siRNA+IR groups received circ_0001478 siRNA(7.5 mg/kg)via tail vein injection,once every 12 h for two consecutive injections.The NC+IR and circ_0001478 siRNA+IR groups were subjected to IR-AKI modeling.Blood samples and kidney tissues were collected 48 h after successful modeling.Fluorescence in situ hybridization(FISH)was used to observe the cellular localization of circ_0001478 and miR-335-3p in renal tu-bular cells.Quantitative real-time PCR(RT-qPCR)was used to detect the gene expression of circ_0001478,miR-335-3p,and ADAM10 in kidney tissues.ELISA was used to detect blood urea nitrogen(BUN),serum creatinine(Scr),and IL-6.Colorimetric method was used to detect myeloperoxidase(MPO)activity,and thio-barbituric acid(TBA)method was used to detect malondialdehyde(MDA)level in kidney tissues.Western blot was used to detect the protein expression levels of caspase 3,cleaved caspase 3,and ADAM10 in kidney tissues.Hematoxylin-eosin(HE)staining was used to observe pathological changes in kidney tissues under light microscopy.Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay was used to observe apoptosis of renal tubular epithelial cells.Results FISH results showed that both circ_0001478 and miR-335-3p were expressed in the cytoplasm of renal tubular epithelial cells.Compared with the NC group,the NC+IR group showed significantly increased fluorescence intensity of circ_0001478(P<0.05),while the flu-orescence intensity of miR-335-3p was significantly decreased(P<0.05).Compared with the NC group,the NC+IR and circ_0001478 siRNA+IR groups showed significantly increased levels or activities of circ_0001478 gene,BUN,Scr,IL-6,MDA,MPO,cleaved caspase 3,and ADAM10 gene and protein in tissues or ser-um(P<0.05),while miR-335-3p gene expression in kidney tissues was significantly decreased(P<0.05).HE staining and TUNEL assay showed aggravated renal tubular injury and apoptosis of renal tubular epitheli-al cells,respectively(P<0.05).Compared with the NC+IR group,the circ_0001478 siRNA+IR group showed significantly decreased levels or activities of circ_0001478 gene,BUN,Scr,IL-6,MDA,MPO,cleaved caspase 3,and ADAM10 gene and protein in tissues or serum(P<0.05),while miR-335-3p gene expression in kidney tissues was significantly increased(P<0.05).HE staining and TUNEL assay showed alleviated renal tubular injury and apoptosis of renal tubular epithelial cells,respectively(P<0.05).Conclusion In the mouse IR-AKI model,circ_0001478 inhibits miR-335-3p expression and promotes ADAM10 production,there-by promoting the expression of inflammatory factors and cleaved caspase 3,aggravating renal tubular epitheli-al cell injury and apoptosis,and ultimately leading to AKI.