| ObjectiveTo investigate the protective effect of electroacupuncture on motor f unction of Parkinson' s mice,and to investigate the mechanism of electroac upuncture on the effects of Parkinson's model mice based on mitochondria 1 autophagy.Method1.MPTP-induced Parkinson's Disease ModelC57BL/6 mice were injected intraperitoneally with 30 mg/kg 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)for 5 days to establish a PD mo use model.The mice were sacrificed on the 7th day and the 14th day after the injecton.The content of DA in the striatum was detected by high perf ormance liquid chromatography(HPLC).To investigate the effect of natural recovery on MPTP in PD mice by the expression of TH.2.Filter electroacupuncture parametersAccording to the frequency,intensity and time of the electroacupuncture instrument,the mice were treated with different parameters for 7 days.The behavioral indexes and the expression of TH positive cells were observed.The appropriate electroacupuncture parameters were screened.3.Effect of electroacupuncture on motor function and nerve function of PD model miceThe established PD model mice were randomly divided into control group,model group,electroacupuncture group and positive control group.Control group and model group were not treated,the electroacupuncture group was acupuncture for 15 min/d,the positive drug group was given methadonna 112.5 mg/(kg·d)for 7 days.The mitochondrial respiratory chain complex,mitochondrial membrane potential,ATPase,reactive oxygen species(ROS),superoxide dismutase(SOD)and malondialdehyde(MDA)were measured after 7 days of continuous treatment.The number of TH-positive cells was detected by immunohistochemistry.The ultrastructure of brain tissue mitochondria was observed by transmission electron microscopy.Western-Blot was used to detect a-synuclein(a-Syn)in brain tissue.4.Mechanism of Electroacupuncture on PD Mouse ModelWestern blot was used to detect the expression of mitochondrial dynamics related proteins(Mfn2,Fisl,Drpl)and autophagy-related proteins(Beclinl,LC3?/?,P62).The PD model mice were randomly divided into control group,model group,rapamycin group,rapamycin+electroacupuncture group,3-MA group and 3-MA+electroacupuncture group.Control group and model group were not treated,rapamycin group and rapamycin+electroacupuncture group were intraventricular injection of rapamycin,3-MA group and 3-MA+electroacupuncture group were injected intraperitoneally with autophagy inhibitor 3-MA.After 24 h,electroacupuncture group,rapamycin+electroacupuncture group and 3-MA+electroacupuncture group were treated with electroacupuncture,15 min/d.The mitochondrial respiratory chain complex,mitochondrial membrane potential,ATPase and reactive oxygen species were measured in the brain tissue of mice after 7 days of continuous treatment.The expression of TH was detected by immunohistochemistry,and the mitochondria and autophagy were observed by transmission electron microscopy(Mfn2,Fisl,Drpl),autophagy-related proteins(Beclinl,P62,LC3 II)and autophagy upstream pathway proteins(AMPK,AKT,MAPK).Result1.MPTP-induced stable PD model miceThe model mice showed weight loss,slow action and tremor.The result s showed that the climbing time was prolonged,and the time of turning ro d was shortened in 2 min.(P<0.01),indicating that the MPTP replication model was successful.The level of DA in the brain tissue of mice was dec reased by 80%and the expression of TH was decreased by about 60%(P<0.01).The expression of DA and the expression of TH-positive cells in the m ices were gradually increased after 14 days of model replication.Compare d with the control group,the DA content of the striatum was reduced by 60%and the TH in the brain was reduced by 50%(P<0.05),that is,DA and T H,although slightly rebound,but still below the normal level of 50%,in dicating that MPTP induced PD model mice during the 14 days of the experi ment can maintain a stable state.2.Screening the appropriate electro-acupuncture treatment parameters The results showed that the motor function and the expression of TH posit ive cells were the most obvious in the PD model mice under the electroacu puncture parameters of 2 Hz,continuous wave and 0.2 mA.Therefore,follo w-up experiment selected 2 Hz,continuous wave,0.2 mA as the PD model mi ce head acupuncture electroacupuncture treatment parameters.3.Effect of electroacupuncture on motor function and nerve function of PD model miceCompared with the model group,the electroacupuncture group can significa ntly shorten the climbing time and prolong the first drop time and reduce the number of drops within 2 min(P<0.01),suggesting that electroacupunc ture can effectively improve the motor function of PD mice.The number of TH-positive cells in the model mice was significantly increased by elect ro-acupuncture group(P<0.05),and the expression of a-syn was significan tly decreased(P<0.05),Suggesting that electroacupuncture can help to pr omote the body to clear the brain tissue abnormal aggregation of ?-syn,enhance the expression of TH-positive cells.The effect of electroacupunc ture on mitochondrial function was studied.The results showed that the m itochondrial complex I decreased significantly(P<0.01)and the ROS produ ction increased significantly(P<0.01)compared with the control group,w hile the MDA content increased and the ATPase activity was significantly decreased(P<0.05)Mitochondrial membrane potential decreased(P<0.05),i ndicating that MPTP induced mitochondrial dysfunction in mice.Compared w ith the model group,the activity of mitochondrial complex I of the electr oacupuncture group was significantly increased(P<0.01),but there was no significant difference in the activity of mitochondrial complex ??? ine ach groups(P<0.05),EA group can significantly reduce the ROS production(P<0.05),reduce the MDA content(P<0.05),while increasing the content of SOD(P<0.05),with increased activity of ATPase activity,increased mi toc hondrial membrane potential(P<0.05),suggested that electroacupuncture ca n improve the mitochondrial dysfunction of PD mice,increase the activity of mitochondrial complex I,enhance the ability of anti-oxidative stress,restore mitochondrial membrane potential and increase ATPase activity.Th e ultrastructure of mitochondria was observed by transmission electron mi croscopy(TEM).The mitochondrial structure of the blank control group wa s complete and showed long column and oval shape.The mitochondrial outer membrane was damaged,the rupture of the ridge structure,the outerchamb er swelling and the void degeneration.Swelling of the situation improved,degeneration of mitochondria was significantly reduced,further indicati ng that the head acupuncture can effectively protect the PD mice mitochon drial morphology,to maintain its function.4.Mechanism of Electroacupuncture on PD Mouse ModelThe intrinsic molecular mechanism was further studied by detecting autoph agy pathway-associated protein and mitochondrial splitting fusion-related proteins.The results showed that the expression of mitochondrial divisio n protein(DRpl,Fisl)was decreased(P<0.05),and the expression of mitoc hondrial fusion protein(Mfn2)increased(P<0.05),Suggesting that electr oacupuncture can inhibit mitochondrial division,promote mitochondrial fu sion in PD model mice.The expression of Belinl and LC3II in EA group was significantly higher than that in model group(P<0.05),and the expressi on of P62 was significantly decreased(P<0.05),suggesting that EA may pl ay an important role in the development of PD model mice by autophagy.Further study,combined with autophagy-induced rapamycin and autophagy in hibitor 3-MA,confirmed whether electroacupuncture was improved by autoph agy pathway in PD model mice with motor dysfunction.The results showed th at compared with the model group,after the rapamycin intervention,the t ime of mice climbing rods was shortened,the first drop time of the rods was prolonged,and the number of drops was decreased within 2 min(P<0.01),Showed that promote autophagy improve motor dysfunction in PD mice,r apamycin+EA better therapeutic effect than rapamycin alone.The time of 3-MA intervention was prolonged obviously,and the time of the first drop of the rod was shortened and the number of drops decreased in 2 min(P<0.01),And 3-MA+EA treatment,the situation improved significantly,which indicated that the inhibition of autophagy pathway could aggravate the mo tor dysfunction of PD mice.TH immunohistochemistry showed that the numbe r of TH cells in rapamycin group and rapamycin+electroacupuncture group was significantly higher than that in model group(P<0.01),and the numb er of TH cells in 3-MA group was significantly(P<0.05).Mitochondrial te st results showed that the activity of mitochondrial complex I increased,the antioxidant capacity increased,the membrane potential increased,and the activity of ATPase in rapamycin group and rapamycin+electroacupunct ure group compared with model group(P<0.05).While the activity of mitoc hondrial complex I decreased,the antioxidant capacity decreased,the memb rane potential decreased,the activity of ATPase decreased in 3-MA group,3-MA+EA treatment was significantly improved,suggesting that inductio n of PD body autophagy to improve its motor function and mitochondrial fu nction has a good effect.Compared with the model group,the expression o f a-Syn protein was down-regulated by rapamycin and rapamycin+EA(P<0.05),rapamycin+electroacupuncture combined with the effect is better th an rapamycin alone.The expression of a-Syn protein was significantly up-regulated in 3-MA group(P<0.05),and significantly decreased after 3-MA+electro-acupuncture treatment,Suggesting that electroacupuncture can p romote induced autophagy,effective removal of a-Syn protein.Mitochondr ial division and fusion protein test showed that the expression of mitoch ondrial division protein Drpl and Fisl in rapamycin group and rapamycin+electroacupuncture group was significantly decreased,the expression of fusion protein Mfn2 increased(P<0.05),The expression of Drpl and Fisl i n the 3-MA group was significantly increased,and the expression of Mfn2 was decreased(P<0.05).It was further verified that electroacupuncture c ould inhibit mitochondrial division and promote mitochondrial fusion by i nducing autophagy,so as to improve the motor function and mitochondrial function of PD mice.To further study the effects of EA on AMPK,AKT and MAPK pathway in the u pstream initiation mechanism of autophagy.The results showed that AMPK pr otein expression was down-regulated and p-AMPK protein was up-regulated i n EA group compared with model group,while AKT and MAPK protein did not c hange significantly,suggesting that the acupuncture point may interfere w ith the AMPK signal pathway to control the occurrence and development of autophagic process.Further inhibit the mitosis of mitochondria,promote i ts integration,to maintain the integrity of mitochondrial morphology,an d thus play a role in improving motor function and mitochondrial function of PD mice.Conclusion1 MPTP induced PD mouse model in the short term can be stable express ion of PD pathological features,the model meets the requirements,the me thod is simple.2 Low voltage and high current continuous wave electroacupuncture tre atment of PD model mice to improve the motor function is more obvious.3 The effect of acupoint penetration on the motor function of PD mice was significantly improved.By observing the effect of autophagy on PD mo del mice,it was found that electroacupuncture could induce autophagy and remove a-Syn aggregation,Splitting,promoting mitochondrial fusion,im proving mitochondrial function,and thus play a role in improving neurolo gical function protection.4 After mTOR upstream protein pathway screening,we found that electro acupuncture induced autophagy and AMPK pathway is closely related. |
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