钙调激酶Ⅱ抑制剂在异动症发生中的分子机制研究

摘要

目的 探讨钙调激酶Ⅱ(CaMKⅡ)抑制剂在异动症发生中的分子机制.方法 采用6-羟多巴胺立体定向注射至小鼠右侧内侧前脑束制备偏侧帕金森病(PD)小鼠模型,将造模成功的PD小鼠分为PD组(n=11)、KN-92组(n=11)和KN-93组(n=11),并设假手术组(n=10)(在相同部位注射生理盐水作为对照).KN-92组和KN-93组给予左旋多巴(15 mg/kg)和苄丝肼(12 mg/kg)腹腔注射,假手术组及PD组注射等量的生理盐水,1次/d,持续14 d.在第11 d、12 d左旋多巴给药前,KN-92组纹状体给予KN-92处理,KN-93组纹状体给予K-93处理(2μl,浓度0.1μg/μl),假手术组及PD组给予等量生理盐水处理.在给药的第1d、3d、5d、8d、10d、13d行异常不自主运动(AIM)评分.在给药的第4d、9d、14d行左前肢使用率的评价.采用Western blot法检测损伤侧纹状体区环磷腺苷效应元件结合蛋白(CREB)、多巴胺和环磷腺苷调节的磷酸化蛋白-32(DARPP-32)及其磷酸化水平变化情况.结果 假手术组治疗前后左前肢使用率差异无统计学意义.PD组给药后左前肢使用率逐渐下降,与治疗前比较差异有统计学意义(P<0.01),KN-92组和KN-93组给药后左前肢使用率逐渐升高,与治疗前比较差异有统计学意义(P<0.01).PD组、KN-92组和KN-93组给药前左前肢使用率两两比较差异无统计学意义.PD组、KN-92组和KN-93组小鼠治疗前及治疗第4 d、9 d左前肢使用率均显著低于假手术组,差异有统计学意义(P<0.01),PD组、KN-92组治疗第14 d左前肢使用率均低于假手术组,差异有统计学意义(P<0.01),KN-93组治疗第14 d左前肢使用率与假手术组比较差异无统计学意义.与PD组比较,KN-92组治疗第4 d、9 d、14 d及KN-93组治疗第4 d、9 d左前肢使用率显著升高(均P<0.01).与KN-92组比较,KN-93组第14 d左前肢使用率显著升高(P<0.01).KN-93组、KN-92组小鼠治疗后第1 d、3 d、5 d、8 d、10 d AIM评分呈现逐渐增高趋势,两两比较差异有统计学意义(均P<0.01).治疗后第14 d,KN-93组AIM评分明显低于KN-92组(P<0.01),治疗后第1 d、3 d、5 d、8 d、10 d,KN-93组AIM评分与KN-92组差异无统计学意义.与假手术组比较,PD组及KN-93组p-DARPP-32及p-CREB水平显著降低(均P<0.01),KN-92组p-DARPP-32及p-CREB水平显著升高(均P<0.01).与PD组比较,KN-92组p-DARPP-32及p-CREB水平显著升高(均P<0.01),KN-93组比较差异无统计学意义.KN-93组p-DARPP-32及p-CREB水平明显低于KN-92组(均P<0.01).结论 左旋多巴诱发的异动症可能与CaMKⅡ有关,其抑制剂KN-93可能是治疗PD引发的运动并发症的一种新的治疗方式.%Objective To investigate the molecular mechanism of calmodulin kinase Ⅱ ( CaMK Ⅱ) inhibitor on the occurrence of dyskinesia. Methods 6-hydroxy dopamine was injected into the right medial forebrain bundle to establish the model of Parkinson's disease (PD) mice. The established PD mice were divided into PD group(n=11), KN-92 group(n=11) and KN-93 group(n=11). Another group of sham-operated mice was involved as control(n=10). KN-92 group and KN-93 group received intraperitoneal injection of L-DOPA (15 mg/kg) and Benserazide (12 mg/ kg) , mice in sham-operated group and PD group were injected with the same volume of physiological saline, once daily for 14 d. At 11 d and 12 d, KN-92 or KN-93 (2 μl, concentration 0. 1 μg/μl) was intrastriatumly injected into mice before L-doap treatment. Mice in sham -operated group and PD group received normal saline treatment. At 1 d, 3 d, 5 d, 8 d, 10 d and 13 d of treatment, abnormal involuntary movement scale ( AIM) was performed in sham -operated group, PD group,KN-92 group and KN-93 group. At 4 d, 9 d and 14 d of treatment,the rate of usage of left fore was evaluated. The levels of cAMP-response element binding protein ( CREB) , dopamine and cAMP-regulated phosphoprotein of Mr 32000 ( DARPP-32 ) , phosphorylated levels of CREB and DARPP-32 were determined by western blot. Results There was no difference in the rate of usage on left fore after operation when compared with original condition in sham-operated group. Surprisingly the rate of usage on left fore decreased clearly after treatment and there was significantly difference in using rate when compared with it before treatment in PD treatment group ( P<0. 01 ) . The rate of usage on left fore increased clearly after treatment and there was significantly difference in KN-92 and KN-93 treating group when compared with it before treatment ( P<0. 01 ) . Before treatment there was no difference in rate of usage on left fore among all groups. At 4 d and 9 d after treatment the rate of usage on left fore in PD, KN-92, and KN-93 group was lower than that in sham-operated group ( P<0. 01). At 14 d, there was no difference between KN-93 group and sham-operated group but in both PD and KN-92 treating group rate of usage still lower than that in sham-operated group (P<0. 01). The rate of usage on left fore at 4 d, 9 d and 14 d in KN-92 group and at 4 d and 9 d in KN-93 group were significantly higher than those in PD group in corresponding days ( all P<0. 01 ) . Especially at 14 d, the rate of usage on left fore in KN-93 group was significantly higher than that in KN-92 group (P<0. 01). The AIM score in mice increased gradually in 1 d, 3 d, 5 d, 8 d and 10 d after treatment with KN-92 and KN-93 when compared with PD treatment (P<0. 01). Except at 14 d, the AIM score was higher after KN-92 treatment there was no difference on all other observing days between KN-92 and KN-93 treatment. The phosphorylated levels of CREB and DARPP-32 were both higher in mice of KN-92 group than those in PD group, and the differences were statistically significant (P<0. 01). Moreover, the phosphorylated levels of CREB and DARPP-32 in mice of KN-93 group were significantly reduced, and the differences were statistically significant (P<0. 01). Conclusion L-DOPA induced dyskinesia may be associated with CaMKⅡand its inhibitor KN-93 may be used in the treatment of PD motor complications as a new option.