Based On JNK Signaling Pathway, The Mechanism Of Needle-knife Intervention In Rats With Parkinson's Disease Was Investigated

Objective:to observe the effect of acupotomy on the neuron inflammation,neuronal apoptosis and related factors of substantia nigra in PD model rats,and to explore the underling mechanism of JNK signaling pathway in PD treatment by acupotomy.Methods:72 male SD rats were divided into blank group,sham operation group,model group,drug group,electro-acupuncture group and acupotomy group.The PD model was established by injecting with 6-OHDA in unilateral striatum,and behavioral testing were performed after apomorphine(APO)induction to verify the success of modeling.Rats in blank group did not receive any processing.Rats in sham operation group were injected same amount of saline containing 0.2%vitamin C in unilateral striatum.After modeling,the drug group received intragastric administration with Madopar(50mg/kg),electro-acupuncture group received EA treatment,and acupotomy group was given acupotomy intervention.All rats were sacrificed four weeks after treatment.The indexes observed included:(1)behavioral test:recording the number of rotating circles in 60 min(2)morphological observation of substantia nigra cells by HE staining(3)double labeled immunofluorescence staining of COX-2/TH and in p-c-Jun/TH in the substantia nigra(4)detection of expression of JNK,P-JNK1,p-c-Jun,COX-2 and TH in the substantia nigra by Westernblot(5)immunohistochemical staining of the substantia nigra of TH(6)substantia nigra DAT content detection by PCR(7)detection of apoptotic cells by TUNEL staining(8)nigral Caspase-3/TH double labeled immunofluorescence staining(9)detection of Caspase-3,Bcl-2 and Beclin-1 expression in the substantia nigra by Western blot.Results:(1)The behavioral test:There was no significant difference in the number of revolutions of the model group before and after the treatment(P>0.05).The number of rotating decreased while activity increased significantly after intervention in the drug group,the EA group and the acupotomy group,compared with the model group(P<0.01).(2)Morphological detection:(2)Morphological detection:? HE staining:different intervention methods can all increase the number of nigra neurons and make morphological structure tends to normal.In the drug group neurons of substantia nigra arranged in order and the number of neurons increased compared with the model group.The cell degeneration alleviates and glial cell proliferation is not obvious.The number of nigra neurons in the EA group was significantly higher than that in the model group and the cell degeneration alleviates.The number of nigra neurons in the acupotomy group is significantly higher than that in the model group,and the arrangement is more dense and the cell degeneration is reduced.? TH immunohistochemistry staining in substantia nigra:in normal group and sham operation group,dense TH immunoreactive positive cells in the substantia nigra were observed,brown in color with large cell body.The shape was mainly round or ovoid,rarely polygonal.In model group,the number of TH immunoreactive positive neurons was small,the cells were sparse,narrowed and outline is not clear.The number of TH immunoreactive neurons in drug group,EA group and acupotomy group increased significantly,the outline of the cell could be seen clearly.Dying strength was also increased.? Double labeled immunofluorescence staining of COX-2/TH and in p-c-Jun/TH in the substantia nigra:In normal group and sham operation group,intensive TH positive cells in the substantia nigra were observed,green in color with large cell body.The shape was mainly round or ovoid.Meanwhile,the expression of COX-2,p-c-Jun and Caspase-3 was red fluorescence,weak in quantitative and staining intensity.In model group,sparse TH positive cells in the substantia nigra were observed,green in color with cell swelling and degeneration,or shrivel,small in number with staining intensity decreased.At the same time,the expression of COX-2,p-c-Jun and Caspase-3 was red in color,and staining intensity and number were increased.In drug group,EA group and acupotomy group,TH positive cells were green,arranged in order and cell degeneration was alleviated with increased staining intensity.At the same time,the expression of COX-2,p-c-Jun,Caspase-3/TH was red,the number and the dyeing intensity decreased.Compared with the EA group and the drug group,the number of TH neurons was increased and the cell degeneration reduced.The number of COX-2 and p-c-Jun in acupotomy group were less than that in EA group,but more than drug group.The number of Caspase-3 in the drug group was less than that in the EA group and the acupotomy group.(3)Western blot results:? No significant difference of JNK content was found between the blank group and the model group in the substantia nigra(P>0.05).Compared with the blank group,expression of P-JNK,P-JNK/JNK,p-c-Jun(P<0.05)and COX-2(P<0.01)were increased.Compared with the model group,there was no significant difference in the JNK content among the drug group,the EA group and the acupotomy group(P>0.05),while P-JNK and p-c-jun expression were increased(P<0.05)in drug group and the EA group,and significantly increased in acupotomy group(P<0.01).Compared with model group,the expression of P-JNK/JNK had difference(P<0.05)in three groups.The expression of COX-2 had difference in EA group(P<0.05),with significant difference(P<0.05)between the drug group and the acupotomy group(P<0.01).? Compared with the blank group,the expression of TH in the substantia nigra of the model group was significantly decreased(P<0.01).Compared with the model group,the expression of TH in the substantia nigra of the drug group and the EA group increased(p<0.05),and the TH expression in acupotomy group was significantly increased(P<0.01).? Compared with the blank group,the expression of Caspase-3 in model group increased(P<0.05),the expression of Beclin-1 was significantly increased(P<0.01)while the expression of Bcl-2 significantly decreased(P<0.01).Compared with the model group,the content of Caspase-3 decreased in the drug group and the EA group(P<0.05),significantly decreased in acupotomy group(P<0.01);the content of Beclin-1 decreased in EA group(P<0.05),significantly decreased in the drug group and acupotomy group(P<0.01);the expression of Bcl-2 in the three groups increased compared with the model group(P<0.05).(4)Real time-PCR test results:Compared with the blank group,the expression of DAT in model group significantly decreased(P<0.01).Compared with the model group,DAT expression in drug group was significantly increased(P<0.01).The expression of DAT in EA group and acupotomy group was increased(P<0.05).(5)TUNEL staining:Compared with the blank group,the number of neurons in the model group increased significantly(P<0.01).Compared with the model group,the neuronal apoptosis decreased significantly in drug group,EA group and acupotomy group(P<0.01).Conclusion:(1)Acupotomy intervention can effectively reduce the inflammatory response of substantia nigra neurons in PD rats.It may reduce the phosphorylation level of JNK and increase the antioxidant activity of TH by reducing the activity of COX-2/p-c-Jun in JNK pathway,thus protecting the neuronal cells.(2)Acupotomy intervention can improve the shape of TH neurons,increase the number of TH-positive cells and increase the expression of DAT to protect the neurons.(3)Acupotomy intervention can effectively reduce the apoptosis of neurons in the substantia nigra of PD rats,which may be by reducing the pro-apoptotic activity of caspase-3,activating anti-apoptotic activity of Bcl-2,inhibiting autophagy gene Beclin-1 activity and improving the antioxidant activity of TH.(4)The inhibition of the JNK signaling pathway may be one of the mechanisms through which acupotomy could treat PD.