Molecular Mechanism Reaserch Of The Protective Of Yiqihuoxuetongluo Recipe On Dorsal Root Ganglion Cells Based On Regulatoin Of JNK Signal Pathway By NGF

Objective: To observe the effect of containing serum of Yiqihuoxuetongluo Recipe on dorsal root ganglion of the JNK signal pathway by binding to its receptor regulation of NGF,and to explore the protective mechanism of Yiqihuoxuetongluo Recipe on diabetic peripheral neuropathy.Methods: Select the SD neonatal rat within 48 h,extract the dorsal root ganglion from neonatal rats and then dorsal root ganglion cells(DRGn)were primary cultured and identify DRGn by immunofluorescence staining.Dorsal root ganglion cells were randomly divided into 6 groups: normal control group,model group,Yiqihuoxuetongluo Recipe high,medium and low dose group(the serum concentrations of 10%,5% and2.5%)and western medicine control group.Except the normal control group,the other groups were modeled with 100mmol/L high glucose concentration,and the damage model was established under high glucose environment.After 24 h,except for high glucose group,the other groups were added 10%,5% and 2.5% serum containing serum and western medicine and continue culturing with 24 h.ELISA was used to detect the expression of NGF in dorsal root ganglion cell supernatant;the protein expression of Trk A,P75 NTR,JNK,P-JNK,Bax and Bcl-2 in each group western bolt method;the gene expression of P53 and Bax were detected by real-time fluorescence quantitative PCR technology.Results:1 NSE immunofluorescence staining method showed that NSE marked with FITC resistance expressed in the cytoplasm of DRGn with green fluorescence,Hochest positive staining,emit blue fluorescence,dorsal root ganglion cells with a purity of more than 95%;2 The results of MTT showed that the concentration of DRGn at 100 mmol/L and the optimal concentration of 24 h induced by high glucose was the most suitable time;3 Microscopic observation of nerve cell morphology and growth status,with high glucose incubation DRGn had obvious morphological changes,there is some improvement in dorsal root ganglion neurons and Yiqihuoxuetongluo Recipe containing serum on the damage caused by high glucose;4 ELISA assay,high glucose induced DRGn secretion decreased the content of NGF(p<0.01),the content of Yiqihuoxuetongluo Recipe containing serum can significantly increase glucose induced DRGn secretion of NGF(p<0.01),has a protective effect on dorsal root ganglion cells;5 Western Blot results showed that(1)compared with the high glucose model group,serum containing high,medium and low dose group and Western medicine group the expression of Trk A had increased in different degrees(p<0.01,p<0.05),each group decreased the expression of P75 NTR in varying degrees(p<0.01,p<0.05);(2)compared with the model group,Yiqihuoxuetongluo Recipe containing serum high,medium and low dose group and Western medicine control group P-JNK decreased JNK and DRGn expression in varying degrees(p<0.01,p<0.05);(3)Yiqihuoxuetongluo Recipe containing serum high,low dose group and Western medicine control group decreased the expression of Bax in both DRGn different levels(p<0.01,p<0.05),the expression of Bcl-2 increased in varying degrees(p<0.01,p<0.05);6 Real time fluorescence quantitative PCR results showed that,compared with the model group,decreased Yiqi Huoxue Tongluo prescription containing serum high,low dose group and Western medicine control group Bax P53 and DRGn gene expression in varying degrees(p<0.01,p<0.05).Conclusions:1 Incubation in high glucose environment can lead to the change of cell morphology and growth of DRGn,resulting in the damage of neurons and the decrease of NGF level;2 Yiqihuoxuetongluo Recipe containing serum can make high glucose induced DRGn cell morphology tends to improve,can increase the content of NGF in the cell supernatant;3 Yiqihuoxuetongluo Recipe containing serum can be combined by regulating NGF receptor and apoptosis,thereby inhibiting excessive activation of JNK signaling pathway as a result of,to protect the dorsal root ganglion neurons.