The Effect Of High Glucose On The MTOR Signal Pathway In Human Mesangial Cells And The Interventive Effect Of Resveratrol

Objective To investigate the effect of high glucose on the human mesangial cells, and to investigate the intervention effect of rapamycin and resveratrol and to explore the mechanism.Methods HMC were cultured under DMEM with normal glucose (NG) to 80% confluence, washed once with serum-free DMEM, and growth-arrest in serum-free DMEM in NG for 24 hours to synchronize the cell growth.After this time the medium was changed to the following groups.(1) To investigate the effects of rapamycin on human mesangial cell cultured in high glucose.human mesangial cells (HMC) were cultured in vitro.The cultured cells were then divided into normal glucose(NG group,5.6mmol/L),high glucose (HG group, 30mmol/L),normal glucose plus Rapamycin 40nmol/L(Rap group),high glucose plus Rapamycin at 10,20,40nmol/L respectively definition is HG+Rapl group, HG+Rap2 group and HG+Rap3 group.HMC proliferation ability within 12,24,48h was detected by MTT colorimetry. Collecting HMC and the corresponding supernatants contents of medium after cultured 48h, the expression of mTOR and P70S6K were observed using RT-PCR. The type IV collagen in supernatant contents of medium was detected by radioimmunoassay method.The secretion of TGF-β1 in supernatant contents of medium was determined by ELISA.(2) To investigate the effects of resveratrol on human mesangial cell cultured in high glucose.The cultured cell were divided into normal glucose(NG group,5.6mmol/L), high glucose (HG group,30mmol/L),normal glucose plus Resveratrol 10-4mol/L(Res group),high glucose plus Resveratrol at 10-6,10-5,10-4mol/L respectively definition is HG+Resl group, HG+Res2 group and HG+Res3 group.HMC proliferation ability within 12,24,48h was detected by MTT colorimetry.Collecting HMC and the corresponding supernatants contents of medium after cultured 48h, the lever of TGF-β1, Col-IV, SOD, TAC and MDA in supernatants were measured in every group.The expression of mTOR and P70S6K were determineded using RT-PCR in NG group, HG group, Res group and HG+Res3 group. Results 1. The cell proliferation of HG group was significantly promoted at 12, 24 and 48 hours compared with NG group (p< 0.01), but no changes can be found among the groups of Rap, Res and NG group; the various concentration of Rap and Res had inhibit the value of MTT compared to HG group(P<0.05, P＜0.01), moreover, the ability of suppression increased along with the increment concentration of drugs at the given scope, but still had higher than NG group.2. The lever of TGF-(31 and Col-Ⅳin HG group had higher than that in NG group (p< 0.01). HG+rap and HG+Res had lower than HG group, but still higher than NG group (p< 0.01). 3. Compared with NG Group, HG group showed a significant decrease of SOD activity and TAC lever (p<0.05, p< 0.01), with an increase of MDA(p<0.05, p< 0.01), when HG+Rap and HG+Res, SOD activity and TAC improved(p<0.05, p< 0.01), and SOD, TAC lower than NG group, while MDA was higher than NG group.4. Compared with NG group, both mTOR and P70S6K mRNAs expression increased in HG group.However, Rap and Res could decrease the expression of these two mRNAs(p<0.05, p< 0.01).Conclusion 1. High glucose promoted the proliferation of human mesangial cells, and incremented the secretion of TGF-β1 and typeⅣcollagen, moreover, induced oxidative stress, and elicit up-regulation the mRNA's expression of mTOR and P70S6K.2. Rapamycin improved proliferation and deposion of extracellular of mesangial cells cultured in high glucose by decreasing the expression of mTOR and P70S6k.3. Resveratrol treatmentment not only significantly resist the proliferation, accumulation of extracellular matrix and oxidative stress caused by high glucose, but also decrease the mRNA's expression of mTOR and P70S6K.