Calpeptin对丙烯酰胺致大鼠大脑微管损伤的干预及机制

Intervention and mechanism of Calpeptin on brain microtubules injury induced by acrylamide

目的建立大鼠丙烯酰胺(Acrylamide,ACR)神经中毒模型和钙蛋白酶抑制剂Calpeptin(CP)干预模型,探讨CP对ACR诱导的大脑微管(Microtubules,MT)损伤的干预作用及其机制,以期为ACR诱导的神经损伤提供预防与治疗的理论依据。方法将40只SPF级雌性Wistar大鼠随机分为对照组、CP组、ACR组和ACR+CP组,每组10只。采用腹腔注射进行ACR染毒和CP干预,对照组给予生理盐水;CP组给予200μg/kg CP;ACR组给予30 mg/kg ACR;ACR+CP组在给予30 mg/kg ACR后1 h,给予200μg/kg CP进行干预。每周3次,共4周。每周末进行大鼠体重、步态评分和转棒试验测定。处理结束后,迅速分离大脑组织,测定钙蛋白酶Calpain的活力,采用凝胶电泳(SDS-PAGE)和免疫印迹法(Western blot)分析α-tubulin、β-tubulin、CDK5、p25和p35蛋白的含量。结果与对照组相比,ACR组自第三周开始步态评分升高、转棒试验停留时间缩短(P<0.05);与ACR组相比,ACR+CP组上述变化均有所改善(P<0.05)。至实验结束时与对照组相比,ACR组Calpain活力升高了59.86%(P<0.05);与ACR组相比,ACR+CP组Calpain活力降低了32.80%(P<0.05)。与对照组相比,CP组CDK5和p35的含量呈现升高趋势,分别升高了31.01%和19.06%(P<0.05),ACR组α-tubulin、β-tubulin、CDK5、p25和p35分别升高了12.63%、21.14%、52.81%、40.58%和25.05%(P<0.05),ACR+CP组CDK5和p35的含量呈现升高趋势,分别升高了42.80%和22.60%(P<0.05)。与ACR组相比,ACR+CP组α-tubulin、β-tubulin、CDK5、p25和p35分别降低了13.10%、10.96%、6.55%、12.79%和25.65%(P<0.05)。结论 CP对ACR诱导的MT损伤具有干预作用,其机制可能是CP通过Calpain调节p35-CDK5/p25通路来维持MT的稳定性。

Objective To understand the intervention effect of Calpeptin(CP) on acrylamide(ACR)-induced brain microtubules(MT) injury and its mechanism by establishing rat models for ACR neurotoxicity and CP intervention,in order to provide a theoretical basis for prevention and treatment of ACR-induced nerve damage. Methods Forty female SPF-grade Wistar rats were randomly divided into control group,CP group,ACR group and ACR+CP group,10 rats in each group. Intra-abdominal injection was used for ACR exposure and CP intervention. The control group was given normal saline,CP group was given200 μg/kg CP,ACR group was given 30 mg/kg ACR,ACR+CP group was given 200 μg/kg CP 1 h after 30 mg/kg ACR intervention,three times per week for four weeks. Rat body weight,gait score and rotarod test were performed weekly. After the end of the exposure,the brain tissue was quickly separated,and the activity of Calpain was determined by kit. The levels of α-tubulin,β-tubulin,CDK5,p25 and p35 protein were analyzed by gel electrophoresis(SDS-PAGE) and Western blot. Results Compared with the control group,an increase in the gait score and a shorter residence time in the rotarod test were found in ACR group since the third week(P<0.05);Compared with the ACR group,the above variations in ACR+CP group were weakened(P<0.05). By the end of the experiment,compared with the control group,the Calpain activity in the ACR group was increased by 59.86%(P<0.05);Compared with the ACR group,the Calpain activity in the ACR+CP group was decreased by 32.80%(P<0.05).Compared with the control group,the expression levels of CDK5 and p35 in the CP group were increased by 31.01% and19.06% respectively(P<0.05);The expression levels of α-tubulin,β-tubulin,CDK5,p25 and p35 in the ACR group were increased by 12.63%,21.14%,52.81%,40.58% and 25.05% respectively(P<0.05);The expression levels of CDK5 and p35 in the ACR+CP group were increased by 42.80% and 22.60% respectively(P<0.05). Compared with the ACR group,the expression levels of α-tubulin,β-tubulin,CDK5,p25 and p35 in the ACR+CP group were decreased by 13.10%,10.96%,6.55%,12.79%and 25.65% respectively(P<0.05). Conclusion CP may intervene with ACR-induced MT injury,and the mechanism may be that CP regulates p35-CDK5/p25 pathways by Calpain to maintain MT stability.