Experimental Study On The Effect Of "Tiaoshenchangzhi" Acupuncture Method On CaMKⅡ,PP-2A And Ca²⁺ In Striatal Of Rats With Levodopa-induced Dyskinesias

Objective:To observe the effects of"Tiaoshenchangzhi"acupuncture method on abnomal involuntary movements scores,striatal expressions of Ca MKⅡand PP-2A and Ca2+concentration in levodopa-induced dyskinesia rat model,and to explore the possible mechanism of"Tiaoshenchangzhi"acupuncture method in treating levodopa-induced dyskinesia.Methods:Among 90 male SD rats,10 rats were randomly selected and divided into a blank group and 10 rats were randomly selected and divided into a sham operation group.The remaining70 rats were used to establish a unilateral Parkinson’s disease（PD）model by intracerebral stereotactic injection of 6-hydroxydopamine（6-OHDA）.After operation,apomorphine（APO）induced rotation test and forelimb function evaluation test screened out PD model rats,which were injected with levodopa methyl ester（10mg/kg）and benserazide（2.5mg/kg）to prepare the rat model of levodopa-induced dyskinesia（LID）required for this experimental study.During the medication period,behavioral evaluation of abnomal involuntary movements（AIM）was performed twice a week.Four weeks later,the ultimately modeled LID model rats were randomly divided into the LID model group,western medicine group,routine acupuncture group,and the Tiaoshenchangzhi group,10 rats in each group.Rats in each group were given corresponding intervention treatments.Behavioral changes of rats in each group were comparatively observed after treatment.The expression of Ca MKII and PP-2A protein in rat striatum was detected by immunohistochemistry and Western Blot,and the Ca²⁺concentration in striatal nerve cells was detected by Fura-2/AM fluorescence probe method.The obtained data were statistically analyzed by SPSS26.0 and the results were visualized by Graph Pad Prism9.0.Results:1.Behavioral aspects:After PD modeling,rats were induced by APO to show obvious rotation behavior,which was significantly different from that in the blank control group（P<0.01）.Besides,the forelimb function score on the affected side of rats in the modeling was significantly lower than that on the healthy side（P<0.01）.During the establishment of LID model,the AIM score of rats showed an increasing trend with time,and entered the plateau after 21 days,when the AIM score peaked about 80min after dosing on the last day.Before treatment,the AIM scores of rats in Tiaoshenchangzhi group,routine acupuncture group,western medicine group and LID model group were significantly different from those of the blank control group（P<0.01）.After treatment,the AIM scores of rats in the Tiaoshenchangzhi group,the routine acupuncture group and western medicine group were lower than those in the LID model group（P<0.01）.Among them,the AIM score of the western medicine group was better than that of the routine acupuncture group（P<0.01）,and the AIM score of the Tiaoshenchangzhi group was also better than that of the routine acupuncture group（P<0.05）.There was no significant difference between western medicine group and Tiaoshenchangzhi group（P>0.05）.2.The expressions of Ca MKII and PP-2A was detected by immunohistochemistry:Compared with the blank group,there was no significant difference in the level of Ca MKII positive cells in the sham operation group（P>0.05）.The level of Ca MKII positive cells in the LID model group was significantly increased（P<0.01）.Compared with the LID model group,the levels of Ca MKⅡpositive cells in the Tiaoshenchangzhi group,the routine acupuncture group,and the western medicine group were all reduced（P<0.01）.The levels of Ca MKⅡpositive cells in the Tiaoshenchangzhi group and western medicine group were lower than that in the routine acupuncture group（P<0.01）,and those in the western medicine group were lower than that in the Tiaoshenchangzhi group（P<0.01）.The differences were statistically significant.There was no significant difference in the level of PP-2A positive cells among different groups（P>0.05）.3.The expressions of Ca MKII and PP-2A was detected by Western Blot:Compared with the blank control group,there was no significant difference in the expression level of Ca MKII protein in the sham operation group（P>0.05）.The expression level of Ca MKII protein in the LID model group was significantly increased（P<0.01）.Compared with the LID model group,the expression levels of Ca MKⅡprotein in the Tiaoshenchangzhi group,the routine acupuncture group,and the western medicine group were all reduced（P<0.01）.The expression levels of Ca MKⅡprotein in the Tiaoshenchangzhi group and western medicine group were lower than that in the routine acupuncture group（P<0.01）,and that in the western medicine group was lower than that in the Tiaoshenchangzhi group（P<0.01）.The differences were statistically significant.The differences were statistically significant.There was no significant difference in the expression level of PP-2A among different groups（P>0.05）.4.Detection of Ca²⁺concentration using Fura-2/AM fluorescent probe method:Compared with the blank group,the Ca²⁺concentration of striatum neurons in sham-operated group had no significant change（P>0.05）,and Ca²⁺concentration was significantly increased in the LID model group（P<0.01）.Compared with the LID model group,Ca²⁺concentrations in the Tiaoshenchangzhi group,the routine acupuncture group,and the western medicine group were all reduced（P<0.01）.Ca²⁺concentrations in the Tiaoshenchangzhi group and western medicine group were lower than those in the routine acupuncture group（P<0.01）,and those in the western medicine group were lower than those in the Tiaoshenchangzhi group（P<0.01）.The differences were statistically significant.Conclusion:1.LID model rats exhibited time-course AIM symptoms similar to"peak-dose dyskinesia"in humans,with increased Ca²⁺concentration in striatal nerve cells,as well as increased Ca MKⅡpositive cell level and protein expression.2.The acupuncture treatment of"Tiaoshenchangzhi"can well improve the AIM symptoms in the contralateral upper limb,orofacial region,axial region and motility in LID model rats.3.The acupuncture treatment of"Tiaoshenchangzhi"can reduce the concentration of Ca²⁺in striatal neurons and the level of Ca MKⅡpositive cells and protein expression in LID model rats.4.One of the mechanisms of"Tiaoshenchangzhi"acupuncture method in treating LID may be achieved through the regulation of Ca²⁺concentration and related regulatory enzymes such as Ca MKⅡto improve calcium homeostasis.