Effect Of Active Vitamin D3 On PARP1/SIRT1/mTOR Signaling Pathway In Human Renal Tubular Epithelial Cells Induced By High Glucose

Objectives:To explore the effects of high and low glucose culture and different concentrations of active vitamin D3（1,25dihydroxyvitamin D3）,on the proliferation of human renal tubular epithelial cells（HK2）in high glucose culture;2.To explore the effects of high and low glucose and active vitamin D3 on HK2 morphology and cell migration;3.To explore the effect of active vitamin D3 on PARP1/SIRT1/m TOR signaling pathway in human renal tubular epithelial cells induced by high glucose.Methods:1.Cultured HK2 cells in vitro and grouped them:Low glucose group（LG group 5.5mmo/l）,high glucose group（HG group 25mmol/l）,high glucose+10^-7mol/l active vitamin D 3group(HG+10^-7VD group),high glucose+10^-8mol/l active vitamin D3group(HG+10^-8VD),high glucose+10^-9mol/l active vitamin D3 group(HG+10^-9VD)The effects of high and low glucose and different concentrations of 1,25（OH）2D3(10^-7mol/l,10^-8mol/l,10^-9mol/l)on proliferation of HK2 under high glucose culture were determined by cck8 method.2.In order to observe the effects of high sugar and active vitamin D3 on the morphology and migration of HK2 cells,the cells were divided into:low sugar group（LG group5.5mmo/l）,high sugar group（HG group 25mmol/l）,high sugar+active vitamin D group（HG+VD group）,high sugar+INO-1001 group（HG+INO-1001 group）to observe cell morphological changes and scratch experiments to observe cell migration;The cells were then divided into:low glucose group（LG group 5.5mmo/l）,high sugar group（HG group25mmol/l）,low sugar+active vitamin D3 group（LG+VD group）,high sugar+active vitamin D3 group（HG+VD group）,low sugar+INO-1001 group（LG+INO-1001 group）,high sugar+INO-1001 group（HG+INO-1001 group）,and RT-q PCR technology was used to detect m RNA expression of PARP1/SIRT1/m TOR signaling pathway correlated factors;Western blot was used to detect the expression of PARP1/SIRT1/m TOR signaling pathway-related protein molecules in the above groups.Results:1.After 24h of culture,the proliferation of HG group was significantly faster than that of LG group（P<0.05）,and there was no difference between the two groups after 48h and 72h of culture（P>0.05）.In high glucose environment,compared with HG control group,the proliferation of high and medium dose groups of active vitamin D3 was significantly inhibited after 24h and 48h intervention（P<0.05）.At 24h,48h and 72h,there was no dose relationship between different concentrations of active vitamin D3groups（P>0.05）.2.In the LG group,HK2 cells were oval in shape and grew like cobblestone pavement,with close arrangement among cells.After 48h of high glucose culture,some cells showed fusiform or irregular morphological changes.Most of the cells in HG+VD group and HG+INO-1001 group were still oval after 48h culture,and the cell morphology did not change significantly.3.Scratch assay results showed that,compared with LG group,HG group migrated faster at 12h（P<0.05）,The mobility of HG+VD group and high glucose+INO-1001 group was lower than that of HG group（P<0.05）.After 24h,the mobility of HG group was still higher than that of LG group（P<0.05）,but the mobility of HG+VD group and HG+INO-1001 group was decreased compared with HG group（P<0.05）.4.RT-q PCR test detected the m RNA expression levels of each target factor after the intervention of high and low glucose culture,active vitamin D 3and PARP-1 inhibitor INO-1001.The results showed that compared with HG group,the m RNA expressions of VDR and SIRT-1 in HG+VD group and high glucose+INO-1001 group were significantly increased（P<0.05）,while the m RNA expressions of PARP-1 and m TOR were significantly decreased（P<0.05）.5.Western blot assay was used to detect the expression levels of each target protein after the intervention of high and low glucose culture,active vitamin D3 and PARP-1 inhibitor INO-1001.The results showed that compared with the control group,the relative expression levels of VDR and SIRT-1 protein in VD group and INO-1001 group were significantly increased（P<0.05）.The relative expression levels of PARP-1 and m TOR were significantly decreased（P<0.05）.Conclusion:1.Active vitamin D3 may alleviate the damage of HK2 induced by hyperglycemia by inhibiting its proliferation and migration;2.Active vitamin D3 may delay the damage of HK2 induced by hyperglycemia by regulating PARP1/SIRT1/m TOR signaling pathway.