The Association Of Prohibitin With Oxidative Damage And Its Molecular Mechanisms In The Renal Interstitial Fibrosis Rats And In Rtec Injury Induced By Hypoxia

Part Ⅰ The association of prohibitin with oxidative damage and its molecular mechanisms in the renal interstitial fibrosis ratsOBJECTIVE To explore the association of prohibitin (PHB) with oxidative damage and its molecular mechanisms in the renal interstitial fibrosis (RIF) rats by detecting the change of PHB, reactive oxygen species (ROS), malonaldehyde (MDA), etc. in renal tissue of renal interstitial fibrosis (RIF) rats.METHODS Eighty Wistar male rats were assigned into two groups randomly:sham operation group (SHO) and model group subjected to unilateral ureteral obstruction (GU); n=40, respectively. Rats were anaesthetized by intraperitoneal injection of chloral hydrate. The left ureter of rats in GU group was dissociated to be obstructed. The rats in SHO were dissociated left ureter only, and not to be obstructed. Twenty rats of the two groups were killed on the end of2-week and4-week after surgery respectively. Renal pathological examination using Masson staining was performed and the RIF index was calculated. Real-time fluorescent quantitative polymerase chain reaction (RT-PCR), immunohistochemistry method and Western-blot were performed to detect the mRNA and protein expressions of PHB1, PHB2, transforming growth factor-β1(TGF-β1). Immunohistochemistry method and Western-blot were conducted to detect the protein expressions of collagen-IV (Col-IV) and fibronectin (FN). Terminal-deoxynucleotidyl transferse-mediated nick end labeling (TUNEL) was perfromed to determinated the cell apoptosis in renal interstitium. The contents of ROS, MDA, superoxide dismutase (SOD) and glutathione (GSH) were also detected.RESULTS1.①When compared with those in SHO group, the mRNA and protein expressions of PHB1or PHB2in GU group were reduced (P＜0.01). The mRNA and protein expressions of PHB1or PHB2in GU group in4-week were markedly increased than those in GU group in2-week (P＜0.01).②The mRNA and protein expressions of TGF-β1, the protein expressions of Col-IV and FN, and the RIF index in GU group were up-regulated when compared with those in SHO group (P＜0.01). The mRNA and protein expressions of TGF-β1, the protein expressions of Col-IV and FN, and the RIF index in GU group in4-week were up-regulated when compared with those in GU group in2-week (P＜0.01).③When compared with those of SHO group, the contents of ROS and MDA in GU group were increased (P＜0.01), the concentrations of SOD and GSH in GU group were reduced (P＜0.01), and the cell apoptosis index in GU group was up-regulated (P＜0.01). The concentrations of ROS and MDA, and the cell apoptosis index in GU group in4-week were increased than those in GU group in2-week (P＜0.01). The concentrations of SOD and GSH in GU group in4-week were reduced when compared with those in GU group in2-week (P＜0.01). 2. Correlation analysis:The PHB1or PHB2protein expression was inverse correlated with the RIF index and cell apoptosisin index, the protein expression of TGF-β1, Col-IV, FN, and ROS or MDA content (P＜0.05). The PHB1or PHB2protein was positively correlated with the content of SOD or GSH (P＜0.05). The protein expression of PHB1was positively correlated with the protein expression of PHB2(P＜0.05). There was a positive correlation between ROS content and TGF-β1, Col-IV, FN, RIF index or cell apoptosis index (P＜0.05).CONCLUSION There was an association between PHB1/PHB2and oxidative damage in renal tissue of RIF rats. The molecular mechanisms might be as follows:PHB might be involved in the progression of RIF by regulating the ROS content, and the expressions of TGF-β1, Col-Ⅳ and FN. PartⅡ The association of prohibitin with oxidative damage and its molecular mechanisms in RTEC injury induced by hypoxiaOBJECTIVE To explore the association of prohibitin with oxidative damage and its molecular mechanisms in RTEC injury induced by hypoxia in vitro.METHODS The viral vectors of pLP1-PHB1+, pLP1-PHB2+, pLP1-PHB1-, pLPl-PHB2-, negative control vector of pLPl-eGFP were synthesized. The cells were divided into seven groups:normal control group (control group), hypoxic-injury group (model group), PHB1+group, PHB2+group, PHB1-group, PHB2-group, and negative control group. The hypoxic treatment was performed in PHB1+group, PHB2+group, PHB1-group, PHB2-group, and negative control group for48hours after adding the gene interference agents. The methods of hypoxic treatment were as follows:The culture plates with RTEC were placed in a vacuum drying cans. Suction device was used to draw the residual gas in the vacuum tank. The hypoxic gas with the mixed gas volume fraction of95%N2and5%CO2was filled in the vacuum tank. The vacuum tank was sealed to induce the hypoxic injury. All the samples were harvested for detection after48hour using hypoxic treatment (including the model group). RT-PCR was performed to detect the mRNA expression of PHB1, PHB2or TGF-β1. Western-blot was conducted to detect the protein expressions of PHB1, PHB2, TGF-β1, Col-Ⅳ and FN. Cell apoptosis and mitochondrial membrane potential (△Ψm) were detected using flow cytometry. The contents of reactive oxygen species (ROS), malonaldehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) were also detected. Scanning electron microscopy and transmission electron microscopy were used to detect the changes in cell morphology and mitochondrial morphological.RESULTS1. When compared with those in control group, the mRNA and protein expressions of PHB1or PHB2in model group were reduced (P＜0.01). When compared with those in model group, the mRNA and protein expressions of PHB1or PHB2in PHB1-or PHB2-group were markedly reduced(P＜0.01), and the mRNA and protein expressions of PHB1or PHB2in PHB1+or PHB2+group were markedly up-regulated (P＜0.01).2. When compared with those in control group, the mRNA and protein expressions of TGF-β1, and the protein expressions of Col-IV and FN were increased (P＜0.01). When compared with those in model group, the mRNA and protein expressions of TGF-β1, and the protein expressions of Col-Ⅳ and FN in PHB1-or PHB2-group were markedly increased (P＜0.01), and the mRNA and protein expressions of TGF-β1, and the protein expressions of Col-Ⅳ and FN in PHB1+or PHB2+group were markedly down-regulated (P＜0.01).3. When compared with those in control group, the contents of ROS and MDA in model group were increased (P＜0.01), the concentrations of SOD and GSH in model group were reduced (P＜0.01). When compared with those in model group, the contents of ROS and MDA in PHB1-or PHB2-group were increased (P＜0.01), the concentrations of SOD and GSH in PHB1-or PHB2-group were reduced (P＜0.01); and the contents of ROS and MDA in PHB1+or PHB2+group were reduced (P＜0.01), the concentrations of SOD and GSH in PHB1+or PHB2+group were increased (P＜0.01). 4. When compared with that in control group, the△Ψm in model group was reduced (P＜0.01), the cell apoptosis rate in model group was increased (P＜0.01), cell morphology shrinking was observed, vacuolization change was observed in the mitochondrial morphology. When compared with that in model group, the△Ψm in PHB1-or PHB2-group was reduced (P＜0.01), the cell apoptosis rate in PHB1-or PHB2-group was increased (P＜0.01), cell morphology shrinking was aggravated, vacuolization change was aggravated in the mitochondrial morphology; the△Ψm in PHB1+or PHB2+group was increased (P＜0.01), the cell apoptosis rate in PHB1+or PHB2+group was reduced (P＜0.01), cell morphology shrinking was alleviated, vacuolization change was alleviated in the mitochondrial morphology.5. Correlation analysis:the PHB1or PHB2protein was inverse correlated with the expression of TGF-β1, Col-IV, FN, ROS, or MDA, and cell apoptosis rate (P＜0.05). The PHB1or PHB2protein was positively correlated with the expression of SOD, GSH or△Ψm (P＜0.05). The protein expression of PHB1was positively correlated with the protein expression of PHB2(P＜0.05). There was a positive correlation between ROS content and TGF-β1, Col-IV, FN, cell apoptosis rate (P＜0.05).CONCLUSION In RTEC culture in vitro, the low expression of PHB1or PHB2was associated with the oxidative damage. The increased PHB1and PHB2played a protective role against oxidative damage. Its molecular mechanisms might be as follows:PHB1and PHB2reduced the contents of ROS, ROS, etc., and reduced the TGF-β1expression and ECM accumulation.