Effects Of Yiqi Huoxue Tongluo Prescription On Inflammation And Apoptosis In High Glucose Stimulated Huvecs Viaactivation Of The JNK Signaling Pathway

Objective:To explore the effects and mechanism of Yiqi Huoxue Tongluo prescription(YHTP)on apoptosis and inflammation in high-glucose-stimulated endothelial cells,and investigate the relationship between them and JNK signaling pathway.To explain the molecular mechanism of Yiqi Huoxue Tongluo prescription on diabetic peripheral neuropathy based on JNK signaling pathway.Methods:1.The human umbilical vein endothelial cells(HUVECs)incubated in vitro for12 h,24h or 48 h in different culture medium(glucose concentration 5.55mmol/L,11.1mmol/L,22.2mmol/L,33.3mmol/L,44.4mmol/L respectively).MTT assay,Hoechst 33258 fluorescent staining and flow cytometry were used to investigate the influence of various concentrations of glucose on the survival rate of cells at different time points.2.A total of 30 Sprague-Dawley rats were treated with Yiqi Huoxue Tongluo prescription by gavage to prepare the serum containing medicine.HUVECs incubated in vitro were divided into different groups: the normal control group,the model group,the groups treated with high-glucose media containing different concentrations of YHTP medicated serum,and the positive control(alpha lipoic acid)group.The levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were measured using ELISA kits and Western blotting.3.Hoechst 33258 staining and flow cytometry(annexin V/PI double staining)were used to examine the apoptosis of HUVECs.Expression of JNK family proteins and RNA were examined by Western blot and real-time PCR(RT-PCR).Meantime,the expression levels of Bax,Bcl-2 and Caspase-3 were measured by Western blot and RT-PCR.Results:1.MTT assay showed that the 33.3 mmol/L high glucose group showed a significant reduction in cell viability after 48 hours of cell culture(P<0.01).Chromatin gathered to one side and cell nucleus was dyed densely and compact,presenting blue-white.The apoptosis rates increased significantly(P<0.01).2.Compared with the expression in the normal control group,the expression of IL-1β and TNF-α in the model group was obviously increased(P<0.05).The YHTP medicated serum obviously down-regulated the expression of IL-1β and TNF-α in HUVECs treated with high glucose for 24 h(P<0.05).3.Compared with that in the control group,JNK and P-JNK expression in the model group was significantly increased(P< 0.01).Compared with that in the model group,JNK and P-JNK expression in all treated groups was noticeably down-regulated(P<0.01).Compared with that observed in the normal group,the high-glucose-treated cells displayed apoptotic morphology,the apoptosis rate significantly increased(P<0.01).The expression levels of Bax and Caspase-3 increased significantly(P<0.05),and the expression level of Bcl-2 decreased dramatically(P<0.05).The YHTP medicated serum significantly decreased apoptosis,and obviously down-regulated the expression of Bax and Caspase-3,and the expressions of Bcl-2 depressed dramatically(P<0.05,P<0.01).Conclusion:1.Human umbilical vein endothelial cells were cultured with 33.3 mmol/L glucose for 48 h,the cell viability was significantly decreased,the inflammatory factor was increased,the JNK pathway was activated and the cell apoptosis rate was significantly increased.2.Yiqi Huoxue Tongluo prescription can inhibit the high expression of inflammatory factors and JNK,and inhibit the apoptosis of HUVECs.3.These findings suggested that YHTP can regulate the activation of JNK pathways and prevent inflammatory injury and apoptosis in high-glucose-stimulated endothelial cells,so as to protect the peripheral nerve of diabetes,the mechanism may be related to JNK signal pathway.