Effects Of Tangluoning On MiR-211 And IRE1α-CHOP Pathways In Rat Dorsal Root Neurons Under High Glucose Culture

ObjectiveThe purpose of this study was to explore the mechanism of Tangluoning in preventing and treating diabetic peripheral neuropathy(DPN)from the point of view of miR-211 and IRE 1α-CHOP pathway in vitro.The role of miR-211 and IRE1α-CHOP pathway in the pathogene-sis of DPN was verified by gene silencing and overexpression..MethodsSixty SD rats were randomly divided into 35 normal group and 25 Tangluoning group.They were given 2.5 g/kg·d Tangluoning crude drug and the same amount of distilled water.Gavage for 10 consecutive days to prepare normal serum and Tangluoning serum.Newborn rats extract dorsal root neuron cells(DRGn).The cells were divided into normal group,model group,inhibitor control group,inhibitor group,traditional Chinese medicine group,agonist traditional Chinese medicine control group and agonist traditional Chinese medicine group by Lipo2000 method.Use q-PCR to detect the gene expression levels of miR-211,IRE1α,CHOP and XBP1 in DRGn cells;Western Blot method was used to detect the expression of IRE1α,p-IRE1α,XBP1 and CHOP in DRGn cells;Fluorescence probe method was used to detect ROS expression in DRGn cells,xanthine oxidase technique was used to detect SOD activity in DRGn cells,and thiobarbital method was used to detect MDA level.Results1.QRT PCR results:Compared with the normal group,the mRNA values of miR-211,IRE1α,XBP1 and CHOP in the model group were obviously heightened(P<0.01);compar-ed with the model group,the mRNA values of miR-211,IRE1α,XBP1 and CHOP in the TCM group and inhibitor group were obviously cut down(P<0.01),but there was no difference in the agonist TCM control group(P>0.05);compared with the TCM group,the mRNA values of miR-211,IRE1α,XBP1 and CHOP in the agonist TCM group were obviously decreased(P<0.01).The mRNA levels of miR-211,IRE1α,XBP1 and CHOP in the control group were not obviously different(P>0.05).2.Western Blot analysis:Compared with the normal group,IRE1α,p-IRE1α,XBP1 and CHOP protein expressions in the model group were obviously enhanced(P<0.01);compared with the model group,IRE1α,p-IRE1α,XBP1 and CHOP protein expressions in the TCM group and inhibitor group were obviously cut down(P<0.01),but there was no difference in the inhibitor control group(P>0.05);Compared with the TCM group,the protein expressio-ns of IRE1α,p-IRE1α,XBP1 and CHOP in the agonist TCM group were obviously enhanced(P<0.01),but there was no difference in the agonist TCM control group(P>0.05).3.Test results of oxidation index:Compared with normal group,ROS and MDA in model group were heighteneded obviously(P<0.01),SOD reduced obviously(P<0.01);compared with model group,ROS and MDA in TCM group and inhibitor group cut down obviously(P<0.01),SOD heightened obviously(P<0.01),but there was no difference in the agonist TCM control group(P>0.05);compared with TCM group,ROS and MDA in agonist TCM group heightened(P<0.01),and the SOD activity was reduced(P<0.01),there was no significant difference in agonist TCM control group(P>0.05).Conclusions1.DRGn cells can construct effective models of low expression and overexpression of miR-211 by transfecting miR-211 inhibitors and agonists.MiR-211 inhibitors can decrease the activity of IRE1α-CHOP pathway and alleviate the oxidative damage of DRGn cells induced by high glucose.MiR-211 agonist can increase the activity of IRE1α-CHOP pathway and antagonize the antioxidant effect of Tangluoning on DRGn cells induced by high glucose.2.High glucose can increase the expression of miR-211 in DRGn cells,then activate IRE1α-CHOP pathway,trigger ER stress and oxidative stress,and cause cell,while Tangluoni-ng drug-containing serum can reduce the level of miR-211 and decrease the activity of IRE1α-CHOP pathway,so as to improve ER stress and oxidative stress,and alleviate cell injury.This study shows that Tangluoning can improve the oxidative damage of DRGn cells ind-uced by high glucose.This protective effect depends on that Tangluoning can downregulate the expression of miR-211,then inhibit the activity of IRE1α-CHOP pa-thway under high glu-cose,alleviate oxidative damage and improve DPN.