The Expression And Function Of Prohibitin In Hypoxic Damage Of Mesangial Cells

OBJECTIVE:To Investigate the function of Prohibitin1 and Prohibitin2 in hypoxic damage of glomerular mesangial cells,by detecting the expression levels of Prohibitin1and Prohibitin2 after hypoxic damage.METHODS:The glomerular mesangial cells were randomly divided into two groups:the normal group and the model group.Normal group was cultured in normal medium and without any treatment.Model group was cultured in hypoxia incubator chamber for 24h and 48h,which filled with the gas that composed of 950 mL·L^-11 nitrogen and 50 mL·L^-11 carbon dioxide.After 24h and48h of hypoxia,cells were collected for detection of relevant indicators.Morphological changes were observed under light microscopy;Reactive oxygen species and mitochondrial membrane potential were detected by Kit;Cell viability were detected by Cell Counting Kit-8;Apoptosis rate were detected by flow cytometry;PHB_1,PHB₂,TGF-?1 and?-SMA mRNA expressions were detected by Real-time-PCR;PHB1,PHB2,TGF-?1,?-SMA,Col-IV and FN protein expressions were detected by Western blot.RESULTS:1.Under light microscopy,the mesangial cells in the normal group exhibited a long shuttle type,the cell body was stretched and full,arranged in an orderly manner,and the space between the cells was moderate.The cells in the model group exhibited a elongated type,the cell body was slender with the morphology was atrophic,and the space between the cells was widened and deepened,also the fragments of cells were increased,and the change in hypoxia 48h group was more obvious than that in hypoxia 24h group.2.Compared with the normal group,the mRNA and protein expressions of PHB1 and PHB2 in model group were obviously decreased?All P<0.05?;compared with 24h group of hypoxia,the mRNA and protein expressions of PHB1 and PHB2 in hypoxia 48h group were obviously decreased?All P<0.05?.3.Compared with the normal group,contents of ROS in model group were obviously increased?P<0.05?,mitochondrial membrane potential in model group were obviously decreased?P<0.05?;compared with hypoxia 24h group,contents of ROS in hypoxia 48h group were obviously increased?P<0.05?,and mitochondrial membrane potential in hypoxia 48h group were obviously decreased?P<0.05?.4.Compared with the normal group,the mRNA and protein expressions of TGF-?1,?-SMA and the protein expression of Col-?and FN in model group were obviously increased?All P<0.05?;compared with hypoxia 24h group,the mRNA and protein expressions of TGF-?1,?-SMA and the protein expression of Col-?and FN in hypoxia 48h group were obviously increased?All P<0.05?.5.Compared with the normal group,apoptosis rate in model group were obviously increased?P<0.05?,activity of cells in model group were obviously decreased?P<0.05?;compared with hypoxia 24h group,apoptosis rate in hypoxia 48h group were obviously increased?P<0.05?,and the activity of cells in hypoxia 48h group were obviously decreased?P<0.05?.6.Correlation analysis:In model group,the protein expression of PHB1 and PHB2 was negatively correlated with the protein of TGF-?1,?-SMA,Col-?and FN,also negatively correlated with contents of ROS and apoptosis rate?All P<0.05?;The other,the protein expression of PHB1 or PHB2 was positively correlated with mitochondrial membrane potential and activity of cells?All P<0.05?.CONCLUSIONS:In the hypoxic model of mesangial cells,the expression of PHB1 and PHB2 significantly decreased was involved in the process of hypoxia damage,with the hypoxia time increased,the expression of PHB1 and PHB2 were decreased more obvious,and the cell damage was more serious.The possible mechanism is that the expression of PHB1 and PHB2 decreased resulted in contents of ROS increased,mitochondrial membrane potential decreased,and the expression of TGF-?1 and?-SMA increased then leading to ECM accumulation.