电针对帕金森病模型大鼠泛素-蛋白酶体系统的影响

Impacts of electroacupuncture on ubiquitin-proteasome system in rats with Parkinson's disease

目的:探讨电针防治帕金森病(Parkinson’s disease,PD)的作用机制。方法:清洁级SD大鼠50只,随机分为正常组、溶剂对照组、模型组、预处理组、治疗组,每组10只。以颈背部皮下注射鱼藤酮(2mg/kg,溶解于葵花油,浓度2mg/mL)制备PD模型。溶剂对照组在与模型组相同部位注射等量的不含鱼藤酮的葵花油。治疗组以电针刺激"风府""太冲"两穴,采用断续波,频率2Hz,强度1mA,持续20min,1次/d,连续28d。预处理组先对正常大鼠以与治疗组相同的方式电针治疗7d,然后造模,造模后不做任何处理,28d后处死。正常组、模型组及溶剂对照组不做电针治疗。观察各组大鼠行为学改变并评分,采用Western-blot法检测黑质部Parkin(一种帕金森病相关蛋白)、泛素羧基端水解酶-L1(ubiquitin C terminal hydrolase-L1,UCH-L1)、泛素激活酶E1(ubiquitin activating enzyme 1,UBE1)的表达,采用免疫组化法检测黑质部α-突触核蛋白(α-synuclein)、泛素(ubiquitin,UB)、酪氨酸羟化酶(tyrosine hydroxylase,TH)阳性细胞数量。结果:模型组大鼠出现毛色变黄粗糙、弓背竖毛、拒捕行为减弱、运动减缓等异常行为学表现,治疗组及预处理组上述表现程度明显减轻。模型组大鼠Parkin、UCH-L1、UBE1、UB、TH表达较正常组和溶剂对照组显著降低,α-synuclein表达显著升高(P<0.01)。经电针治疗或预处理后,治疗组和预处理组Parkin、UCH-L1、UBE1、UB、TH表达高于模型组,α-synuclein表达低于模型组(P<0.01)。结论:电针治疗或预处理不仅对帕金森病模型大鼠具有保护效应,并且能增强泛素-蛋白酶体系统功能,提示电针防治帕金森病的机制可能与改善泛素-蛋白酶体系统功能有关。

Objective To explore action mechanism of electroacupuncture （EA） on treatment and prevention of Parkinson＇s disease （PD）. Methods Fifty clean-grade SD rats were randomly divided into a normal group, a con- trol group, a model group, a pretreatment group and a treatment group, ten rats in each one. The PD model was established by subcutaneous injection of rotenone in neck-back skin （2 mg/kg, dissolved in sun-flower oil, 2 mg/ mL in density）. The equal-volume sun-flower oil that didn＇t include rotenone was applied in the control group at the same area as the model group. EA was applied in the treatment group at ＂Fengfu＂ （GV 16） and ＂Taichong＂ （LR 3） with interrupted wave, 2 Hz in frequency, 1 mA in density, for 20 rain. The treatment was given once day for conti-nuous 28 days. Rats in the pretreatment group received the same EA as the treatment group for 7 days, and then put into model establishment. After the model establishment, the rats received no treatment and were sacrificed after 28 days. No EA was given in the normal group, model group and control group. The ethology changes were observed and scored. The expression of Parkin, ubiquitin C terminal hydrolase-L1 （UCH-L1） and ubiquitin activating enzyme-1 （UBE1） in substantia nigra was tested by Western-blot method. The positive cell numbers of a-synuclein, ubiquitin （UB） and tyrosine hydroxylase （TH） in substantia nigra was tested by immuno- histochemical method. Results There were abnormal ethology manifestation such as yellow and coarse hair, arched back, weaken behavior of resisting arrest and slow movement, which was more relieved in the treatment group andpretreatment group. Compared with normal group and control group, the expression of Parkin, UCH-L1, UBE1, UB,TH in the model group was obviously decreased while α-synuclein was obviously increased （all P〈0.01）. Af- ter EA or pretreatment, the expression of Parkin, UCH-L1, UBE1, UB, TH in the treatment group and pretreat- ment group was higher than that in the model group while expression of α-synuclein in the treatment group and pretreatment group was lower than that in the model group （all P〈0.01）. Conclusion EA or pretreatment could not only have protective effect for rats with PD, but also increase function of ubiquitin-proteasome system, indica- ting action mechanism of EA on treatment and prevention of PD may be related with ubiquitin-proteasome system.