糖络宁对高糖环境下大鼠背根神经节细胞IRE1α-CHOP通路的影响观察

Effects of Tangluoning on IRE1α-CHOP pathway of dorsal root ganglion in peripheral neuropathy rats under high glucose environment

目的探讨糖络宁对高糖环境下大鼠背根神经元(DRGn)细胞肌醇酶1α(IRE1α)-转录因子C/EBP同源蛋白(CHOP)通路的影响。方法将60只清洁级雄性SD大鼠随机分为糖络宁组25只、正常对照组35只,分别以糖络宁组方生药2.5 g/(kg·d)和蒸馏水灌胃,用于制备糖络宁含药血清及正常对照血清。取新生SD胎鼠DRGn制备细胞悬液,将DRGn细胞在6孔板进行接种,随机分为正常(A)组(予空白血清培养)、模型(B)组(予75 mmol/L葡萄糖+空白血清培养)、中药(C)组(予75 mmol/L葡萄糖+糖络宁含药血清培养)、miR-211抑制剂(D)组(予75 mmol/L葡萄糖+空白血清培养+miR-211抑制剂)、miR-211抑制剂对照(d)组(予75 mmol/L葡萄糖+空白血清培养+miR-211抑制剂阴性对照)、激动剂中药(E)组(予75 mmol/L葡萄糖+糖络宁含药血清培养+miR-211激动剂)、激动剂中药对照(e)组(予75mmol/L葡萄糖+糖络宁含药血清培养+miR-211激动剂阴性对照)。培养24 h后,q-PCR检测miR-211、IRE1α、p-IRE1α、XBP1、CHOP基因表达;荧光探针法测定DRGn细胞ROS水平,黄嘌呤氧化酶技术检测SOD活性,硫代巴比妥法测定MDA含量。结果各组ROS、SOD、MDA、miR-211 mRNA、IRE1α、XBP1、CHOP比较,差异均有统计学意义(P<0.01)。与A比较,B组ROS、MDA及miR-211、IRE1α、XBP1、CHOP mRNA表达高,SOD水平低(P<0.01)。与B组比较,C组和D组ROS、MDA及miR-211、IRE1α、XBP1、CHOP mRNA表达低,C组SOD水平高于B组(P<0.01);d组ROS、SOD、MDA、miR-211、IRE1α、XBP1、CHOP mRNA与B组比较,差异均无统计学意义(P>0.05)。与C组比较,E组ROS、MDA及miR-211、IRE1α、XBP1、CHOP mRNA表达高,SOD水平低(P<0.01);e组ROS、SOD、MDA、miR-211、IRE1α、XBP1、CHOP mRNA与C组比较,差异无统计学意义(P>0.05)。结论糖络宁通过下调miR-211表达降低IRE1α-CHOP通路活性。

Objective To study the effects of Tangluoning on IRE1α-CHOP signaling pathway in rat dorsal root neurons(DRGn)under high glucose environment.Methods Sixty clean grade male SD rats were randomly divided into Tangluoning group(25 rats)and normal group(35 rats),and the crude drug of Tangluoning was given in the Tangluoning group with 2.5 g/(kg·d)and the same amount of distilled water was administered by gavage for the preparation of comparative serum.The dorsal root ganglion(DRG)used to prepare cell suspension was obtained from SD fetal rats.DRGn cells were inoculated into 6-well plate and randomly divided into 7 groups:normal group(group A cultured with blank serum),model group(group B cultured with 75 mmol/L glucose and blank serum),Tangluoning group(group C cultured with 75 mmol/L glucose+Tangluoning medicated serum),miR-211 inhibitor group(group D cultured 75 mmol/L glucose+blank serum culture+miR-211 inhibitor negative),miR-211 inhibitor control group(group d cultured with 75 mmol/L glucose+Tangluoning medicated serum culture+miR-211 inhibitor negative control),agonist traditional Chinese medicine group(group E cultured with 75 mmol/L glucose+Tangluoning medicated serum culture+miR-211 agonist)and agonist traditional Chinese medicine control group(group e cultured with 75 mmol/L glucose+Tangluoning medicated serum culture+miR-211 agonist negative control).After 24 hours of culture,q-PCR was used to detect the gene expression levels of miR-211,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.ResultsCompared the levels of ROS,SOD,MDA,miR-211 mrna,IRE1α,XBP1 and CHOP in each group,the differences were statistically significant(P<0.01).Compared with group A,the levels of ROS,MDA as well as miR-211,IRE1α,XBP1,CHOP mRNA in group B were obviously heightened and SOD reduced obviously(P<0.01).Compared with group B,the levels of ROS,MDA as well as miR-211,IRE1α,XBP1,CHOP mRNA in group C and D were obviously reduced and SOD level of group C was higher than that of group B.Compared the levels of ROS,MDA as well as miR-211,IRE1α,XBP1,CHOP mRNA in group d with B,the differences were not statistically significant(P>0.05);compared with group B,the levels of ROS,SOD,MDA,miR-211,IRE1α,XBP1,CHOP mRNA in group E were obviously heightened,SOD reduced(P<0.01);compared the levels of ROS,SOD,MDA,miR-211,IRE1α,XBP1,CHOP mRNA in group e with group C,the differences were not statistically significant(P>0.05).ConclusionTangluoning could reduce the activity of IRE1α-CHOP pathway by down-regulating the expression of miR-211.