The Effect Of High Glucose On The Expression Of Circadian Clock Gene In Glomerular Mesanginal Cell

Objective: Circadian rhythm is one of the most popular phenomenon of the nature,circadian clock is the system which set and regulate this kind of day and night cycle of the body,and is a kind of the circadian oscillator for a period which is approximately 24 hours.Circadian clock is synchronize with sunlight cycle,metabolism and energy intake.A number of studies have found that the circadian clock gene expression is closely related to blood glucose level,circadian rhythm disorder is one of the risk factors of diabetes mellitus(DM),but there is no research to study the effect of high glucose on the circadian clock gene expression before,so this research observes the expression of circadian clock genes stimulated by different concentrations of high glucose and reactive oxygen species(ROS)inhibitor which is named N-acctyl-L-cystcinc(NAC)in mouse glomerular mesanginal cells,we detect the ROS level and expression of inflammation factors at the same time,aiming to explore the control effects and corresponding mechanism of the high glucose in circadian clock expression and inflammatory reaction.Methods: 1.Mouse glomerular mesangial cells cultured withdifferent concentrations of high glucose as stimulating factors,NAC as the intervention factor,were divided the cells into 4 groups:(1)normal control group(group NC): 5.6 mmol/L glucose;(2)different concentrations of high glucose group(group HG): 10 mmol/L glucose(HG1),20 mmol/L glucose(HG2)and 30mmol/L glucose(HG3);(3)Osmotic pressure group(group OP): 5.6 mmol/L glucose +24.4mmol/L mannitol;(4)The NAC intervention group(group NAC+HG): 10?mol/L NAC added in the best effect concentration of high glucose medium.2.Each group was induced24 h,then detected the expression of CLOCK,BMAL1,REV-ERB?,Per1,Per2,Per3,Cry1,Cry2 mRNA by RT-PCR,ROS was measured by2',7'-Dichlorofluorescin Diacetate(DCFH-DA),while the expression of Interleukin-6(IL-6)and C-C chemokine ligand 2(CCL-2,which is also called monocyte chemotactic protein 1,MCP-1)were measured by ELISA.3.Statistical analysis: we used statistical software SPSS 20.0 for data analysis,data is represented by the mean and standard deviation(x?±s),One-way analysis of variance and LSD-t were used to analyze all experimental data,homogeneity test of variance was compared in every group,P<0.05 was considered to be statistically significant.Results:1.Compared with normal control group,the expression of CLOCK,BMAL1,REV-ERB?,Per1,Per2,Per3,Cry1,Cry2 mRNA were up-regulated in high glucose group of mouse glomerular mesangial cells,CLOCK,BMAL1,REV-ERB?,Per1,Per2,Per3,Cry2 mRNA increasedby dose-dependent manner(P<0.05),the levels of ROS,IL-6 and CCL-2expression were also up-regulated in high glucose group by dose-dependent manner of mouse glomerular mesangial cells(P<0.05).2.Compared with high glucose group,the expression of CLOCK,BMAL1,REV-ERB?,Per1,Per2,Per3,Cry1,Cry2 mRNA,the level of ROS,the expression of IL-6 and CCL-2 were decreased by NAC intervention in mouse glomerular mesangial cells(P<0.05).Conclusion:Our data support that high glucose can increase the expression of CLOCK,BMAL1,REV-ERB?,Per1,Per2,Per3,Cry1,Cry2 mRNA by oxidative stress mechanism in mouse glomerular mesangial cells,and may play a role in inflammation of diabetes nephropathy(DN).