Based On The MiR-200a And MAPKs Signaling Pathways, The Effects Of Tangluoning On Rat Dorsal Root Neurons Under High Glucose Were Studied

Objective:This project validates and explores the possible mechanism of Tang-luo-ning(TLN)for the treatment of DPN from the perspective of in vitro experiments,miR-200a and MAPKs signaling pathways,explores the mechanism of traditional Chinese medicine for diabetic peripheral neuropathy,and provides new ideas for DPN treatment.Methods:1.Sixty male SD rats at 6-8 weeks were randomly divided into 4 groups according to the random number table classification:high-concentration TLN group(15),medium-concentration TLN group(15),low-concentration TLN group(15)and normal group(15),respectively,according to the TLN group prescription crude drugs 5 g/kg·d,2.5 g/kg·d,1.25 g/kg·d and equal amount of distilled water;Drug-containing serum and normal serum were prepared 10 days after the administration.DRGn was extracted from neonatal SD fetal rats.2.DRGn was inoculated into 96-well plates and randomly divided into 4 groups:normal group,50,75,100 mmol/L glucose group.The effects of each glucose concentration on DRGn cell activity were determined by CCK-8 method after cultured for 24,and 48 hours,respectively.3.DRGn was inoculated into 96-well plates and randomly divided into 6 groups:normal group,2.5%,5%,10%,20%,30%TLN containing serum group,and the effects of each TLN concentration on DRG cell activity were determined by CCK-8 method after cultured for 24,and 48 hours,respectively.4.DRGn was inoculated into 6-well plates and randomly divided into 5 groups:normal group,High glucose group,high-concentration TLN group,medium-concentration TLN group,low-concentration TLN group,and cultured for 24 hours.The mRNA expression levels of miR-200a,Jun,Mapk8,Map3k8 were detected by qRT-PCR method.The protein expression levels of Jun,JNK,and Map3k8 were detected by Western Blot method,and apoptosis was detected by flow cytometryResults:1.CCK-8 test results:Time comparison:Compared with 24 h,different concentrations of glucose in each group,the decrease of cell activity in 48 h was lower than 24 h(P<0.01 or P<0.05).Compared with 24 h,the cell viability in 10%TLN-containing serum group was significantly decreased compared with 48 h(P<0.01).Comparison between groups:At the same time point,with the increase of high glucose concentration,the activity of DRGn cells gradually decreased(P<0.01).After 24 hours,with the increase of TLN containing serum,the activity of rat DRGn cells showed a trend of increasing first and then decreasing.The concentration of the TLN-containing serum in 10%reached its peak(P>0.05).After 48 hours,the overall trend was consistent with 24 hours.The peak serum concentration of TLN-containing serum is 10%(P>0.05).Analyze the growth curve of glucose and TLN-containing serum at different concentrations after intervention,and select 75 mmol/L glucose concentration and TLN-containing serum stock solution(10%)as the intervention concentration.Observation is performed on the 24th hour after drug action point.2.qPCR test results:Compared with the normal group,the miR-200a,Jun,Mapk8,Map3k8 mRNA values in the high glucose group were significantly increased(P<0.01).Compared with the high glucose group,the miR-200a,Jun,Mapk8,Map3k8 mRNA in each group of the TLN group was significantly reduced(P<0.01).Compared with the medium-concentration TLN group,the miR-200a,Jun,Map3k8,and Mapk8 mRNA values in the high-concentration TLN group were significantly increased(P<0.01);the low-concentration TLN group miR-200a,Jun,Mapk8mRNA values increased significantly(P<0.01).3.Western blot test results:Compared with the normal group,the expression and phosphorylation level of JNK1,c-JUN,and Map3k8 phosphorylation protein in the high glucose group were significantly increased(P<0.01),the total protein level of Map3k8 was significantly increased(P<0.01),and JNK1,c-JUN total protein expression did not change significantly(P>0.05).Compared with the high glucose group,JNK1,c-JUN,Map3k8 phosphorylated protein expression and phosphorylation levels of each TLN group were significantly reduced(P<0.01),no significant change in total protein expression(P>0.05).Compared with high and low concentration TLN group,JNK1,c-JUN,Map3k8 phosphorylated protein expression and phosphorylation levels of medium concentration TLN group were significantly reduced(P<0.01).4.Apoptosis results:Compared with the normal group,the apoptosis rate of the high glucose group was significantly increased(P<0.01),which was statistically significant.Compared with the high glucose group,the apoptosis rate of each group in the TLN group was significantly reduced(P<0.01),which was statistically significant Significance.Compared with the high-concentration TLN group and the low-concentration TLN group,the apoptosis rate of the medium-concentration TLN group was significantly reduced(P<0.01),which was statistically significant.Conclusion:1.High glucose stimulation can inhibit the activity of rat DRGn cells,TLN-containing serum can significantly improve the inhibition of high glucose stimulation on rat DRGn cell activity.2.High glucose stimulation can up-regulate the level of miR-200a in rat DRGn cells,activate the MAPKs signaling pathway to promote apoptosis.TLN-containing serum,especially medium-concentration TLN-containing serum can inhibit Upregulation of miR-200a and inhibit the MAPKs signaling pathway,significantly reducing apoptosis,thereby reducing high glucose damage to DRGn cells.TLN may inhibit the expression of miR-200a,affect its MAPK signaling pathway,reduce the level of apoptosis,reduce nerve damage,and prevent the occurrence and progress of DPN.