糖尿病对大鼠胫骨前肌烟碱型乙酰胆碱受体的影响

Effects of diabetes on nicotinic acetylcholine receptors in tibial anterior muscle of rats

目的探讨糖尿病对大鼠胫骨前肌烟碱型乙酰胆碱受体(nAChR)表达和功能的影响。方法SPF级成年雄性SD大鼠48只,6~8周龄,体重200~250 g。采用随机数字表法将大鼠分为四组:正常对照组(NC组)、糖尿病2周组(D2组)、糖尿病4周组(D4组)和糖尿病8周组(D8组),每组12只。取大鼠胫骨前肌,采用Western blot法检测ε-nAChR、γ-nAChR及α7-nAChR蛋白含量,RT-PCR法检测ε-nAChR、γ-nAChR及α7-nAChR mRNA表达量。采用I^(125)-α-银环蛇毒素对运动终板nAChR进行放射性标记,根据放射性强度值计算运动终板nAChR的数目、密度和终板面积(ESA)。检测罗库溴铵预处理的胫骨前肌运动终板,记录运动终板nAChR与罗库溴铵浓度为2.5、5.0、7.5、10.0μg/ml时的结合力。结果与NC组比较,D2组γ-nAchR和α7-nAchR mRNA表达量明显升高,ESA明显增大(P<0.05);D4组γ-nAchR、α7-nAchR蛋白含量明显升高,ε-nAchRh、γ-nAchR和α7-nAchR mRNA表达量均明显升高,ESA明显增大,罗库溴铵浓度为2.5、5.0μg/ml时nAChR与罗库溴铵结合力均明显降低(P<0.05);D8组ε-nAchR、γ-nAchR、α7-nAchR蛋白含量和mRNA表达量均明显升高,胫骨前肌运动终板nAChR数目明显增多、密度明显增大、ESA明显增大,罗库溴铵浓度为2.5、5.0、7.5、10.0μg/ml时nAChR与罗库溴铵结合力均明显降低(P<0.05)。与D2组比较,D4组ε-nAchR和γ-nAchR蛋白含量均明显升高,ε-nAchR和α7-nAchR mRNA表达量明显升高,nAChR数目明显增多、ESA明显增大,罗库溴铵浓度为5.0μg/ml时nAChR与罗库溴铵结合力均明显降低(P<0.05);D8组ε-nAchR、γ-nAchR、α7-nAchR蛋白含量和mRNA表达量均明显升高,nAChR数目明显增多、密度明显增大、ESA明显增大,罗库溴铵浓度为2.5、5.0、7.5、10.0μg/ml时nAChR与罗库溴铵结合力均明显降低(P<0.05)。与D4组比较,D8组ε-nAch、γ-nAchR蛋白含量和mRNA表达量均明显升高,nAChR数目明显增多、密度明显增大,罗库溴铵浓度为7.5μg/ml时nAChR与罗库溴铵结合力明显降低(P<0.05)。结论糖尿病可影响nAChR的表达和功能,使胫骨前肌γ-nAChR和α7-nAChR蛋白含量升高,ε-nAChR蛋白含量先降低后逐渐升高,ε-nAChR、γ-nAChR和α7-nAChR mRNA表达量升高,运动终板nAChR数目增多、密度增大、与罗库溴铵结合力降低。

Objective To investigate the effect of diabetes on the expression and function of nicotinic acetylcholine receptors(nAChR)in the tibial anterior muscle of rats.Methods Forty-eight specific pathogen free(SPF)male SD rats,aged 6-8 weeks,weighing 200-250 g,were divided into four groups by using a random number table:the normal control group(group NC),diabetic for 2 weeks group(group D2),diabetic for 4 weeks group(group D4),and diabetic for 8 weeks group(group D8),12 rats in each group.Tibialis anterior muscles tissue was sampled for molecular biology.Western blot was used to detect the expression of nAChR subtypes,includingε-nAChR,γ-nAChR andα7-nAChR.The mRNA levels of nAChR subtypes were assessed using real-time polymerase chain reaction(RT-PCR).The tibialis anterior muscle nAChR was labeled with I^(125)-α-bungarotoxin,and the quantity,density of nAChRs on each motor endplate and the endplates surface area(ESA)were obtained by measuring the radioactivitiy of motor endplates.The muscles pretreated with rocuronium was used to explore the affinity of nAChR on endplates to rocuronium concentrations of 2.5,5.0,7.5,and 10.0μg/ml.Results Compared with group NC,the mRNA levels ofγ-nAchR andα7-nAchR were increased significantly,and the ESA was increased significantly in group D2(P<0.05);theγ-nAChR andα7-nAChR protein expression were increased significantly,the mRNA levels ofε-nAchRh,γ-nAchR andα7-nAchR were increased significantly,ESA was increased significantly,the affinity of motor endplate nAChR to rocuronium was reduced significantly at rocuronium concentrations of 2.5 and 5.0μg/ml in group D4(P<0.05);the expression of protein and mRNA levels ofε-nAchRh,γ-nAchR andα7-nAchR were increased significantly,the quantity and density of nAChRs on motor endplates were increased significantly,the ESA was increased significantly,the affinity of motor endplate nAChR to rocuronium was reduced significantly at rocuronium concentrations of 2.5,5.0,7.5,and 10.0μg/ml in group D8(P<0.05).Compared with group D2,the expression ofε-nAchRh andγ-nAchR protein were increased significantly,the mRNA levels ofε-nAchRh andα7-nAchR were increased significantly,the quantity of nAChRs and ESA were increased significantly,the affinity of motor endplate nAChR to rocuronium was reduced significantly at rocuronium concentration of 5.0μg/ml in group D4(P<0.05);the expression of protein and mRNA levels ofε-nAchRh,γ-nAchR andα7-nAchR were increased significantly,the quantity and density of nAChRs on motor endplates were increased significantly,ESA was increased significantly,the affinity of motor endplate nAChR to rocuronium was reduced significantly at rocuronium concentrations of 2.5,5.0,7.5,and 10.0μg/ml in group D8(P<0.05).Compared with group D4,the expression of protein and mRNA levels ofε-nAchRh andγ-nAchR were increased significantly,the quantity and density of nAChRs on motor endplates were increased significantly,the affinity of motor endplate nAChR to rocuronium was reduced significantly at rocuronium concentration of 7.5μg/ml(P<0.05).Conclusion In diabetic rats,the expression ofγ-nAChR andα7-nAChR increases,while the expression ofε-nAChR had a tendency of decreasing first and then increasing to a high level.Diabetes may lead to the increased quantity and density of AChRs on motor endplate,and the dcreased affinity of motor endplate nAChR to rocuronium which may affect the expression and function of nAChR.