Study On The Role Of GDNF And PI3K-Akt-mTOR Signaling Pathway In Acupuncture Promoting The Recovery Of Facial Nerve Injury Based On Autophagy

Objective:To explore the role of autophagy,GDNF and PI3K-Akt-mTOR signaling pathway in the acupuncture promoting the recovery of facial nerve injury,based on the exact therapeutic effect of acupuncture on peripheral facial paralysis,in order to reveal the autophagy mechanism of acupuncture treatment,and to provide scientific basis for its clinical treatment.Method:In the experiment one,60 Sprague Dawley?SD?male rats were randomly divided into normal group,sham surgery group,surgery group,acupuncture group and acupuncture plus 3-MA group,12 rats in each group.The facial paralysis model was made by crushing the main trunk of facial nerve.The rats underwent acupuncture treatment at dicang?ST 4?,jiache?ST 6?,yifeng?SJ 17?and hegu?LI 4?for 10 min,and then retained for 20 min after the surgery.Acupuncture stimulation was performed once daily for 10 days with two rest day every 5 days.Simone 10 score method was used to evaluate the function recovery of facial nerve,and operated respectively on the 1^st,4^th,7^th,10^thand 13^thday after surgery.The injured facial nerve was harvested at the end of treatment.The morphological changes were observed by HE staining.The immunohistochemistry,immunofluorescence and RT-PCR were used to evaluate the levels of autophagy.In the experiment two,60 SD male rats were randomly divided into normal group,sham surgery group,surgery group,acupuncture group and acupuncture plus anti-GDNF antibody group,12 rats in each group.The model preparation and acupuncture treatment were the same as the experiment one.Simone10 score method was used to evaluate the function recovery of facial nerve.The GDNF and PI3K-Akt-mTOR signaling pathway were detected by immunohistochemistry.Results:1. Behavioral scoreThere was no significant difference in behavioral score between the sham surgery group and normal group?all P>0.05?,and that of the surgery group was obviously decreased compared with the sham surgery group?all P<0.01?.On the 10^thand 13^thday after surgery,the scores of the acupuncture group were higher compared with the surgery group?P<0.05,P<0.01?,and the scores of acupuncture plus 3-MA group and acupuncture plus anti-GDNF antibody group were decreased compared with the acupuncture group?all P<0.05?.2. Morphological observation of facial nerveIn the normal group and sham surgery group,the morphological structure of facial nerve were normal without obvious damage under the microscope.In the surgery group,the morphological structure of facial nerve was damaged,and the intrinsic fascicular structure of nerve fibers disintegrated and disappeared,which was silk network structure or even empty ring structure under the microscope.In the acupuncture group,the facial nerve fibers were swollen and degenerated,but arranged in order.In the acupuncture plus 3-MA group,the morphological structure of facial nerve was incomplete,and some axons and myelin sheath lost in flakes under the microscope.3. The level of autophagyThere was no significant difference in the expression of Beclin1 protein,LC3protein and LC3 mRNA between the sham surgery group and normal group?all P>0.05?.Compared with the sham surgery group,the expression of Beclin1 protein,LC3protein and LC3 mRNA in the surgery group were increased significantly?all P<0.01?,while the P62 protein and P62 mRNA expression in the surgery group were decreased significantly?all P<0.01?.The expression of Beclin1 protein and LC3protein in the acupuncture group were obviously decreased compared with the surgery group?all P<0.01?,while the P62 protein and P62 mRNA expression in the acupuncture group were obviously increased?all P<0.01?.There was no significant difference between the surgery group and acupuncture group in the expression of LC3mRNA?P>0.05?.Compared with the acupuncture group,the expression of Beclin1protein,LC3 protein and LC3 mRNA in the acupuncture plus 3-MA group were decreased?P<0.05,P<0.05,P<0.01?.4. GDNF and PI3K-Akt-mTOR signaling pathwayThere was no significant difference in the expression of GDNF protein,Rai protein,PI3K protein and mTOR protein between the sham surgery group and normal group?all P>0.05?.Compared with the sham surgery group,the expression of GDNF protein,Rai protein,PI3K protein and mTOR protein in the surgery group were decreased significantly?all P<0.01?.The expression of GDNF protein,Rai protein,PI3K protein and mTOR protein in the acupuncture group were obviously increased compared with the surgery group?all P<0.01?.Compared with the acupuncture group,the expression of GDNF protein,Rai protein,PI3K protein and mTOR protein in the acupuncture plus anti-GDNF antibody group were decreased significantly?all P<0.01?.Conclusion:1.Acupuncture treatment can improve the motor function and tissue structure of the facial nerve,so as to promote the recovery of facial nerve injury.2.Acupuncture treatment may inhibit autophagy by promoting the expression of GDNF protein,activating PI3K-Akt-mTOR signaling pathway,down-regulating the autophagy related molecules Beclin1 and LC3,and up-regulating P62,so as to improve the motor function of facial nerve.After inhibiting the expression of GDNF,the improvement effect on motor function of facial nerve by acupuncture was not obvious.