耳蜗螺旋动脉上连接蛋白43与老年性耳聋的关系研究

Correlation between Connexin 43 on Spiral Modiolar Artery and Presbycusis

背景老年性耳聋是指随着年龄的增长而缓慢出现听力阈值增加的疾病,耳蜗螺旋动脉(SMA)在维持耳蜗血供和听力方面发挥重要的作用。连接蛋白43(Cx43)在血管壁上广泛表达,对血管的舒缩功能有着重要的影响,但Cx43在SMA上的表达与老年性耳聋的关系尚不清楚。目的以D-半乳糖(D-gal)诱导的老年性耳聋豚鼠为研究对象,研究SMA上Cx43与老年性耳聋的关系。方法2018年5月,将无耳疾3月龄SPF级豚鼠36只随机分为Control组、NS组、D-gal组,各12只。D-gal组在正常饲养的基础上,颈背部皮下注射D-gal500mg·kg^-1·d^-1;NS组在正常饲养的基础上,颈背部皮下注射0.9%氯化钠溶液(体积等同D-gal组);对照组正常饲养,不做任何处理;共6周。取Control组豚鼠交配产下的3只新生豚鼠,处死分离SMA,贴壁法培养原代SMA血管平滑肌细胞(VSMCs),鉴定后取第3代SMAVSMCs制备细胞衰老模型:对照组为正常培养48h;衰老组为在正常培养基础上加入30g/LD-gal培养48h;AAP10组为在正常培养的基础上加入100nmol/LAAP10预处理,而后加入30g/LD-gal培养48h。采用Morris水迷宫测试实验检测Control组、NS组、D-gal组豚鼠测试第1、2、3、4、5天逃避潜伏期及第6天穿越平台的次数;比较Control组、NS组、D-gal组豚鼠听性脑干反应(ABR)阈值,肝组织、脑组织、血清超氧化物歧化酶(SOD)活性、丙二醛(MDA)水平,SMA上Cx43mRNA及Cx43表达水平。比较对照组、衰老组、AAP10组VSMCs内SOD活性、MDA水平,VSMCs中Cx43表达水平。结果组别与时间在豚鼠逃避潜伏期中存在交互作用(F交互=6.99,P交互<0.05);组别与时间在豚鼠逃避潜伏期中主效应显著(F组间=53.43,F时间=38.15;P组间<0.05,P时间<0.05)。D-gal组豚鼠测试第3、4、5天逃避潜伏期长于Control组和NS组(P<0.05);D-gal组豚鼠测试第6天穿越平台次数少于Control组和NS组(P<0.05)。D-gal组豚鼠ABR阈值高于Control组和NS组,肝组织、脑组织、血清SOD活性低于Control组和NS组,肝组织、脑组织、血清MDA水平高于Control组和NS组(P<0.01)。衰老组VSMCs内SOD活性低于对照组和AAP10组,MDA水平高于对照组和AAP10组(P<0.01);AAP10组VSMCs内SOD活性低于对照组,MDA水平高于对照组(P<0.01)。D-gal组豚鼠SMA上Cx43mRNA及Cx43表达水平低于Control组和NS组(P<0.01)。衰老组VSMCs中Cx43表达水平低于对照组和AAP10组(P<0.05);AAP10组VSMCs中Cx43表达水平低于对照组(P<0.05)。结论 D-gal致衰老情况下豚鼠SMA上Cx43表达水平的变化可能与老年性耳聋有关,为临床后续的研究方向奠定了基础。

Background Presbycusis is a kind of disease in which hearing threshold increases slowly with age.Spiral modiolar artery(SMA)plays an important role in maintaining cochlear blood supply and hearing production.Connexin 43(Cx43)is widely expressed in the vascular wall and has an important effect on the vasomotor function,but the relationship between its expression in the cochlear spiral artery and presbycusis is still unclear.Objective The senile deafness guinea pigs induced by D-galactose(D-gal)were studied to explore the relationship between Cx43 on SMA and presbycusis.Methods In May 2018,36 specific pathogen free(SPF)guinea pigs aged three months without ear diseases were randomly divided into the Control group,NS group and D-gal group with 12 guinea pigs in each group.On the basis of normal feeding,guinea pigs in the D-gal group and NS group were subcutaneously injected with D-gal 500 mg·kg^-1·d^-1 and 0.9%sodium chloride solution(the same volume as the D-gal group)respectively in the neck and back,while guinea pigs in the Control group were raised normally without any treatment.All guinea pigs in three groups were raised for six weeks in total.Three neonatal guinea pigs born in the Control group were taken and sacrificed to isolate SMA.The primary SMA vascular smooth muscle cells(VSMCs)were cultured by adherence method.After identification,the 3rd generation of SMA VSMCs was used to prepare cell senescence model.SMA VSMCs in control group were normally cultured for 48 h.SMA VSMCs in aged group were normally cultured with 30 g/L D-gal for 48 h.SMA VSMCs in the AAP10 group were pretreated with 100 nmol/L AAP10 and then cultured with 30 g/L D-gal for 48h.Morris water maze test was used to detect the escape latency of guinea pigs on day 1,2,3,4 and 5 and the number of times of crossing the platform on day 6 in the Control group,NS group and D-gal group.The threshold values of auditory brainstem response(ABR),superoxide dismutase(SOD)activity and malondialdehyde(MDA)level of liver tissue,brain tissue and serum,and Cx43 mRNA on SMA and protein were compared among the Control group,NS group and D-gal group.SOD activity,MDA level and Cx43 expression in VSMCs were compared among the control group,aged group and AAP10 group.Results There was an interaction between group and time in the escape latency of guinea pigs(Finteraction=6.99,Pinteraction<0.05)and the main effects of group and time were significant(Fintergroup=53.43,Ftime=38.15;Pintergroup<0.05,Ptime<0.05).The escape latency in D-gal group was longer than that in Control group and NS group on day 3,4,and 5(P<0.05).The number of times of crossing the platform in D-gal group was less than that in Control group and NS group on day 6(P<0.05).ABR threshold and MDA level of liver tissue,brain tissue and serum in D-gal group was higher and SOD activity of liver tissue,brain tissue and serum was lower than that in Control group and NS group(P<0.01).SOD activity in VSMCs in aged group was lower and MDA level was higher than that in control group and AAP10 group(P<0.01).SOD activity in VSMCs in AAP10 group was lower and MDA level was higher than that in control group(P<0.01).Cx43 mRNA on SMA and protein expression in D-gal group was lower than that in Control group and NS group(P<0.01).The expression of Cx43 in VSMCs in aged group was lower than that in control group and AAP10 group(P<0.05)and the expression of Cx43 in VSMCs in AAP10 group was lower than that in control group(P<0.05).Conclusion The change of Cx43 expression on SMA in guinea pigs induced by D-gal may be related to presbycusis,which lays a foundation for future clinical follow-up research.