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-κB pathway activation induced by high glucose in human umbilical vein endothelial cells(HUVECs).Methods: 1)The expression of TLR4 and NF-κB 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-κB 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 TNFαwere 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-κB 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-κB were detected by qRT-PCR and Western blot;subcellular localization of NF-κB was assessed by immunocytochemistry;the levels of IL-6、IL-8 and TNFα 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-κB 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-κB pathway(TLR4、NF-κB)and relative inflammatory cytokines(IL-6、IL-8、TNF-α、NO)were significantly increased in high glucose(30mmol/L D-GS);3)The IC50 of Atorvastatin in HUVECs tested by MTT was 10.3μmol/L,so,it has the minimal effect on HUVECs morphology when the concentration of Atorvastatin lower than 10.3μmol/L;compared to high glucose(30mmol/L D-GS),the TLR4 and NF-κB expression were inhibited with the elevated Atorvastatin concentration,and is was the most obvious at 5μmol/L(P <0.01).4)Compared to control group(5.5mmol/L D-GS),the expression of TLR4/NF-κB pathway(TLR4 、 NF-κB)and relative inflammatory cytokines(IL-6、IL-8、TNF-α、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-α and NO)via TLR4/NF-κB 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-κB,which may provide scientific basis for the treatment of diabetic vasculopathy.