Mechanism Research Of Atorvastatin Protecting Endothelial Cells Injury Induced By High Glucose

Objective: The aim is to investigate the effect of Atorvastatin on the inflammation via TLR4/NF-?B pathway activation induced by high glucose in human umbilical vein endothelial cells(HUVECs).Methods: 1)The expression of TLR4 and NF-?B mRNA in HUVECs which cultured by different time(6h?12h?24h?48h)in different concentration of glucose(5.5?10?20?30?40 D-GS mmol/L D-GS)were detected by qRT-PCR to choose the suitable time and glucose concentration;2)Detecting the expression of TLR4?MyD88 and NF-?B in HUVECs which cultured in high glucose(30mM)for 12 h by qRT-PCR and Western blot,the expression of relative inflammatory cytokines IL-6 ? IL-8 and TNF?were detected by qRT-PCR and enzyme linked immunosorbent assay,and the expression of NO in the supernatant was detected by nitrate reductase method;3)HUVECs were stimulated by high glucose(30 mmol/L D-GS)for 12 h,then,detecting the cell inhibition rate of HUVECs which treated with different concentration of Atorvastatin(0?1?2.5?5?10?20?40?80umol/L Atorvastatin)for 24 h by MTT assay,and calculating the half maximal inhibitory concentration(IC50);Then observation the cell morphology of optimum concentration groups by microscope;Finally,stimulating the HUVECs by high glucose(30 mmol/L D-GS)for 12 h and treating with different concentration of Atorvastatin(0?1?2.5?5umol/L Atorvastatin)for 24 h,andthe TLR4 andNF-?B expressionwere detected by qRT-PCR and Western blot to choose the best Atorvastatinconcentration.4)HUVECs were divided into four groups,control(5.5mmol/L D-GS),Atorvastatin(5.5mmol/L D-GS +5umol/L Atorvastatin),high glucose(30 mmol/L D-GS),high glucose with Atorvastatin(30 mmol/L D-GS +5umol/L Atorvastatin).The expression of TLR4?MyD88 and NF-?B were detected by qRT-PCR and Western blot;subcellular localization of NF-?B was assessed by immunocytochemistry;the levels of IL-6?IL-8 and TNF? in the supernatant were detected by enzyme linked immunosorbent assay;and the expression of NO in the supernatant was detected by nitrate reductase method.Results:1)TLR4 and NF-?B mRNA expressionwas higher in high glucose(30mmol/L D-GS)for 12 h than in other groups(P <0.05);2)Compared to control group(5.5mmol/L D-GS),the mRNA and protein expression of TLR4/NF-?B pathway(TLR4?NF-?B)and relative inflammatory cytokines(IL-6?IL-8?TNF-??NO)were significantly increased in high glucose(30mmol/L D-GS);3)The IC50 of Atorvastatin in HUVECs tested by MTT was 10.3?mol/L,so,it has the minimal effect on HUVECs morphology when the concentration of Atorvastatin lower than 10.3?mol/L;compared to high glucose(30mmol/L D-GS),the TLR4 and NF-?B expression were inhibited with the elevated Atorvastatin concentration,and is was the most obvious at 5?mol/L(P <0.01).4)Compared to control group(5.5mmol/L D-GS),the expression of TLR4/NF-?B pathway(TLR4 ? NF-?B)and relative inflammatory cytokines(IL-6?IL-8?TNF-??NO)were significantly increased in high glucose(30mmol/L D-GS)(P <0.05);however,this was abolished in high glucose with Atorvastatin(30 mmol/L D-GS +5umol/L Atorvastatin)(P <0.05),including MyD88.Conclusions:High glucose can induce the release of inflammation cytokines(IL-6?IL-8?TNF-? and NO)via TLR4/NF-?B pathway activation in endothelial cells,which promote the inflammation response of diabetic vascular,Atorvastatin can significantly inhibit the inflammation induced by high glucose by regulating signaling pathway of TLR4/NF-?B,which may provide scientific basis for the treatment of diabetic vasculopathy.