Based On MiRNA-200a To Explore The Mechanism Of Tangluoning Improving The Oxidative Stress Of Rat Dorsal Root Neurons Under High Glucose

OBJECTIVEIn rat DRGn induced by high glucose to investigate the effects of tangluoning(TLN)on the miR-200a and oxidative stress,to partially elucidate the possible mechanism of TLN in the clinical treatment of DPNMETHODSSPF grade SD rats were administered by intragastrical gavage for 10 days to prepare TLN-containing serum.DRGn cells were extracted and identified.DRGn cells were cultured with different glucose concentrations(50mmol/L,75mmol/L,100mmol/L)and different concentrations of TLN-containing serum(2.5%,5%,10%,20%,30%).The cell activity was detected by CCK-8 method to determine the optimal high glucose concentration and the optimal serum concentration of TLN.The ROS levels of DRGn cells were detected by fluorescence probe technology,the activity of SOD was determined by xanthine oxidase method,and the content of MDA was determined by thiobarbital method.The expression levels of miR-200a mRNA and PI3K mRNA were detected by real-time fluorescence quantitative PCR.The expression levels of PI3K,p-PI3K,AKT and p-AKT were detected by Western BlotRESULTS1.The effect of different concentrations of glucose and TLN-containing serum on DRGn cell activityWith the increase of glucose concentration,the activity of DRGn cells decreased significantly(P<0.01).Cell activity in the 50mmol/L glucose concentration group and the 75mmol/L glucose concentration group、in the 75mmol/L glucose concentration group and the 100mmol/L glucose concentration group decreased significantly,with statistical differences(P<0.01).Compared with 24h,the cell activity of DRGn in each glucose concentration group decreased significantly at 48h,with a statistical difference(P<0.05 or P<0.01).With the increase of serum concentration of TLN,the activity of DRGn cells showed a trend of increasing first and then decreasing,and the cell activity was the maximum when the concentration was 10%.There was no statistical difference in cell activity between the 2.5%concentration group and the 5%concentration group(P>0.05),and there were statistical differences between the 5%concentration group and the 10%concentration group,the 10%concentration group and the 20%concentration group,the 20%concentration group and the 30%concentration group(P<0.05 or P<0.01).Compared with 24h,the cell activity of DRGn under the same concentration of TLN-containing serum decreased at 48h,and the decrease of cell activity under the 10%TLN-containing serum showed statistical difference(P<0.05).2.The effect of TLN on the oxidative stress level of DRGn cells in high glucose cultureCompared with the control group,ROS and MDA in the high glucose group were obviously improved(P<0.01)and SOD was reduced(P<0.01).Compared with the high glucose group,ROS and MDA were remarkably decreased(P<0.01),and SOD was significantly increased(P<0.01)in TLN group.3.The effect of TLN on the expression levels of miR-200a mRNA and PI3K mRNA in DRGn cells under high glucose cultureCompared with the control group,miR-200a mRNA in high glucose group was significantly improved(P<0.01)and PI3K mRNA was reduced obviously(P<0.01).Compared with the high glucose group,miR-200a mRNA in TLN group remarkably decreased(P<0.01)and PI3K mRNA significantly increased(P<0.01).4.The effect of TLN on the expression levels of PI3K/AKT pathway-related proteins in DRGn cells under high glucose cultureCompared with the control group,the expressions of PI3K,p-PI3K,p-PI3K/PI3K,p-AKT,p-AKT/AKT in high glucose group were significantly reduced(P<0.01).Compared with the high glucose group,the expression levels of PI3K,p-PI3K,p-PI3K/PI3K,p-AKT,p-AKT/AKT in TLN group were significantly increased(P<0.01)CONCLUSIONS1.TLN is preferred to improve the oxidative stress level of DRGn cells in high glucose environment.2.The improvement of oxidative stress in DRGn cells under high glucose culture may be achieved by regulating the miR-200a and PI3K/AKT signaling pathway.