Research Of Prohibition Protein Gene On Oxidative Damage And Molecular Mechanisms In Renal Tubular Epithelial Cells Injury

OBJECTIVE This study were performed to investigate on the antiproliferative protein (prohibitin,PHB), transfection of exogenous genes in the role and mechanism of injury of hypoxic renal tubular epithelial cells (renal tubular epithelial cell, RTEC), for the prevention and treatment of renal interstitial fibrosis (renal tubulointerstitial fibrosis, RIF) to provide new ideas.METHODS Construction of pcDNA3.1(+)-PHB1plasmid, pcDNA3.1(+)-PHB2plasmid and of pcDNA3.1(+)-eGFP was plasmid, the use of liposomes Lipofectamine2000transfection in vitro renal tubular epithelial cell line in NRK-52E. Using expression of green fluorescence in negative control pcDNA3.1(+)-eGFP was detected under a fluorescence microscope transfection efficiency.48h after transfection to establish hypoxia models, and the establishment of the normal control group, each group was divided into five subgroups:blank control group, negative plasmid control group, liposome control group, transfection the PHB1group and transfected with PHB2group. Modeling12h re-oxygenation after1h, real-time fluorescence quantitative polymerase chain reaction (real-time fluorescent quantitative polymerase chain reaction, RT-PCR), was used to detect PHB1, PHB2, and α-smooth muscle actin (alpha-smooth muscel actin wasof a-SMA) mRNA expression in NRK-52E cells; western blot (western blot) was also used to detect PHB1, PHB2and a-SMA protein expression in NRK-52E cells; flow cytometry was used to detect the cell mitochondrial membrane potential in NRK-52E; colorimetric detection of cell culture supernatant (of malondialdehyde, MDA) content was also performed.RESULTS1. Successful recovery in NRK-52E cells were inoculated into6well plates by the number of3×105. Transfected cells could reach80-90%confluence after24h. In accordance with the liposome instructions recommended proportion of transfected NRK-52E cells, the transfection efficiency was about80%.2.(1)Normal group and model group transfected with PHB1group, transfected with PHB2group in NRK-52E cells, mRNA and protein expressions of PHB1and PHB2were significantly higher than the corresponding group blank control group, negative control group, liposome control group (P<0.05). There was no significant difference among blank control group, negative control group and the liposome control group (P>0.05);(2) α-SMA mRNA and protein expressions of normal group, model group transfected with PHB1group, and transfected with PHB2group were significantly lower than that of the blank control group, negative control group, liposome control group (P<0.05). There was no significant difference among blank control group, negative control group and the liposome control group (P>0.05).(3)Compared with normal group, the mRNA and protein expressions of PHB1, PHB2in model group were significantly lower (P<0.05), and mRNA and protein expressions of a-SMA in model group were significantly increased (P<0.05).3.(1) Mitochondrial membrane potential in normal and model groups in the transfected with PHB1group, and transfection with the PHB2groups, was significantly lower than the blank control group, negative control group, and the liposome control group (P<0.05). There was no significant difference among blank control group, negative control group and the liposome control group (P>0.05).(2) Compared with normal group, the mitochondrial membrane potential in model group was significantly decreased (P<0.05).4.(1) MDA content in normal group, model group transfected with PHB1group, or transfection with PHB2group was significantly lower than the control group, negative control group, and the liposome control group (P<0.05). Between the blank control group, negative control group, liposome control group, no significant difference (P>0.05) was found.(2) Compared with normal group, MDA content in model group was significantly increased (P <0.05).CONCLUSION After transfection of exogenous PHB1and PHB2gene expression, the stability of the RTEC mitochondrial membrane potential was increased, and MDA formation and the amount of a-SMA expression were reduced. It seems that PHB can play a protective role against hypoxic-induced injury of RTEC...