| Objective:This study was designed to establish rat model of Parkinson's disease(PD)induced by rotenone,then they were treated with electroacupuncture(EA)treatment Fengfu(GV16),Taichong(LR3),therefore,to observe the effects of EA on the expression of HDAC6 and LC3-II in the substantia nigra of PD rats,and to explore the involved mechanism of EA therapy in the treatment of PD.Methods:60 SPF healthy male SD rats weighing 200±20g,after 2 weeks of adaptive feeding,24 rats were extracted by random number table method and then were randomly assigned to normal group and sham operation group,12 rats in each group.The remaining rats were used to establish the PD model and the rats were subcutaneously injected with rotenone which was dissolved into sunflower oil emulsion(the concentration was 2mg/ml)on the back of the rat's neck at the standard of 2mg/Kg/d specification,1 time/d,and for continuous 28 days.Rats in sham operation group were injected the sunflower oil emulsion without rotenone on the back of the rat's neck,and 1 time/d,continuous 28 days.No treatment was done in the normal group.After 28 days,we observed the appearance and behavior of all rats injected with rotenone,then scored according to the behavioral scoring standard formulated by Chen Xin.Behavioral scores between 2-8 of the rats were regarded as successful models.Finally,24 successful model rats were randomly divided into PD group and PD EA group,with 12 rats in each group.After the models were established,we adopted stainless steel filiform needle with 13 mm in length and 0.3 mm in diameter,choosed acupoints of Fengfu(GV16),Taichong(LR3)for rats in PD EA group,the acupoints of Fengfu(GV16)was penetrated 4 mm and Taichong(LR3)was puntured 1mm perpendicularly,alternate use of the left and right Taichong(LR3),then interconnected with HANS-200 EA apparatus and selected stimulus parameters as follows: continuous-wave at a frequency of 2Hz,and 1mA,a slight twitch in rat limb strength,20min/time,1 time /d,7d as a course of treatment,for 4 weeks.The rats in normal group,sham operation group and PD group were fixed at the same time every day,but they were not treated.After 4 weeks treatment,the behavior scores of each group was evaluated and the results were compared,and eventually,12 rats in each group were included in the behavior test.After the next day of the behavioral test,half of the rats in each group to section and half of the rats in each group to homogenate.Immunohistochemical staining were used to observe TH and ?-syn immune response in the substantia nigra of rats,and using image analysis to calculate the mean optical density of positive cells respectively.Wesrern blot was used to examine the expression of HDAC6 and LC3-II protein in substantia nigra of rats;RT-PCR were used to detecte HDAC6 mRNA,LC3-II mRNA relative expression levels in the substantia nigra of rats.Results:1.After 4 weeks treatment,the appearance and behavior of rats in PD EA group were significantly improved and the behavioral scores decreased significantly compared with that of PD group(P < 0.01);The appearance and behavior scores of rats in PD group increased significantly compared with that of normal group and sham operation group(P < 0.01);No obvious abnormal behavior were obseved in normal and sham operation group.2.The immune response of TH in substantia nigra of rats in PD EA group enhanced and the mean optical density of TH significantly increased compared with that of PD group(P < 0.01);The immune response of TH in substantia nigra of rats in the PD group weakened and the mean optical density of TH significantly decreased compared with that of normal group and sham operation group(P < 0.01).3.The immune response of ?-syn in substantia nigra of rats in PD EA group weakened and the mean optical density of ?-syn significantly decreased compared with that of PD group(P < 0.01);The immune response of ?-syn in substantia nigra of rats in the PD group enhanced and the mean optical density of ?-syn significantly increased compared with that of normal group and sham operation group(P < 0.01);No obvious ?-syn aggregation in normal group and sham operation group.4.The protein expression of HDAC6 and LC3-II in substantia nigra of rats in PD EA group significantly increased compared with that of PD group(P < 0.01);The protein expression of HDAC6 and LC3-II in substantia nigra of rats in PD group increased compared with that of normal group and sham operation group(P < 0.05);There was no significant difference between normal group and sham operation group in protein expression of HDAC6 and LC3-II(P > 0.05).5.The relative expression levels of HDAC6 mRNA and LC3-II mRNA in substantia nigra of rats in PD EA group significantly increased compared with that of PD group(P < 0.01);The relative expression levels of HDAC6 mRNA and LC3-II mRNA in substantia nigra of rats in PD group increased compared with that of normal group and sham operation group(P < 0.05);There was no significant difference between normal group and sham operation group in relative expression levels of HDAC6 mRNA and LC3-II mRNA(P > 0.05).Conclusions: 1.It was further verified that the PD rat model could be effectively replicated by the subcutaneous injection of rotenone on the back of the rat's neck.2.EA can obviously improve the appearance and behavior of PD rats.3.EA can significantly reduce the accumulation of ?-syn in the substantia nigra of PD rats,thus relieving the toxic effects on dopaminergic neurons.4.EA could significantly increase the content of TH in the substantia nigra of PD rats,and to a certain extent alleviated the decrease of DA level in the nigrostriatal pathway.5.EA can significantly increase the expression levels of HDAC6 mRNA,LC3-II mRNA and its protein in substantia nigra of PD rats,which may be the key to EA to promote the clearance of ?-syn,which may be related to upregulating HDAC6 level,enhancing autophagy activity and promoting the autophagic degradation of ?-syn. |
PDF Full Text Request