The Role Of Autophagy In Postherpetic Neuralgiaand Its Mechanism

Objectives : To investigate the effects of different levels of autophagy on GABAergic interneurons in the dorsal horn of the spinal cord in a mouse model of postherpetic neuralgia.Methods:A.36 Kunming mice were randomly divided into group normal saline,group solvent and group PHN by using SPSS19.0 random number generator,and 12 mice in each group.Establishment of PHN model by intraperitoneal injection of 0.2ug/g Resiniferotoxin.Group solvent and group NS were injected with equal volume of solvent and normal saline.Von-Frey wire was used to detect the paw mechanical thresholds.The hot plate method was used to detect the shrinkage time threshold to the numerical stability at the same time every day.The expression of LC3 and beclin-1 was detected by real-time fluorescence quantitative PCR,immunohistochemistry and Western-blot in the spinal cord segment of L4~L6,and the autophagic bodies were observed by transmission electron microscope when mice were sacrificed after anesthesia.B.48 Kunming mice were randomly divided into group PHN+Rapa,group PHN,group PHN+3-MA and group control by using SPSS19.0 random number generator,and 12 mice in each group.PHN+Rapa group received intraperitoneal injection of 1μg/(kg.d)and autophagy inducers rapamycin,PHN+3-MA group were injected with 2μg/(kg.d)autophagy inhibitor 3-methyladenine for 14 consecutive days,but the PHN group was injected with normal saline,Control group without any treatment,after copying the PHN model.The expression of LC3,beclin-1 and SQSTM1/P62 was detected by Western-blot in the spinal cord segment of L4~L6;Detection of beta endorphin and substance P in spinal cord homogenate and arterial blood by ELSIA;and the autophagic bodies were observed by transmission electron microscope when mice were sacrificed after anesthesia.And the daily changes of PMWT and HPPT values in each group were statistically analyzed.C.48 Kunming mice were randomly divided into group PHN+Rapa,group PHN,group PHN+3-MA and group control by using SPSS19.0 random number generator,and 12 mice in each group.Group PHN+Rapa received intraperitoneal injection of 1μg/(kg.d)and autophagy inducers rapamycin,group PHN+3-MA were injected with 2μg/(kg.d)autophagy inhibitor 3-methyladenine for 14 consecutive days,but the group PHN was injected with normal saline,group control without any treatment,after copying the PHN model.he expression of bcl-2 and bax were detected by Western-blot in the spinal cord segment of L4~L6,and detection of apoptotic cells in spinal cord by fluorescence Tunel,Immunofluorescence labeling of GABAergic interneurons in dorsal horn of spinal cord when mice were sacrificed after anesthesia.And the daily changes of PMWT and HPPT values in each group were statistically analyzed.Results:A.The PMWT value decreased significantly,while HPPT increased significantly in group PHN after intraperitoneal injection of 0.2ug/g Resiniferotoxin(p<0.05),while the level of m RNA and protein,the relative expression of LC3,beclin-1 was significantly increased(p<0.05);B.After intraperitoneal injection of rapamycin,the relative expression level of LC3 II and beclin-1 protein in the group PHN+Rapa was significantly increased,and SQSTM1/P62 was significantly lower(p<0.05);The relative expression level of LC3 II and beclin-1 protein in the group PHN+3-MA was significantly lower,and the SQSTM1/P62 was significantly increased;Compared with group C,the levels of beta endorphin and substance P in serum and spinal cord homogenate of group PHN+Rapa,group PHN and group PHN+3-MA were significantly increased(P < 0.05).Compared with group PHN,the serum levels of and the spinal cord homogenate decreased significantly in group PHN+Rapa,while the PHN+3-MA group was significantly increased(P < 0.05).The concentration of substance P in serum and spinal cord homogenate of group PHN+Rapa was significantly higher than that of group PHN,but the group PHN+3-MA was significantly lower(P < 0.05);Compared with group PHN,PMWT values were significantly decreased,HPPT values significantly increased in the group PHN+Rapa after intraperitoneal injection of rapamycin(p<0.05).The PMWT value was significantly increased and HPPT was significantly lower in the group PHN+3-MA after when the autophagy inhibitor3-MA(p<0.05).C.The relative expression level of Bcl-2 and Bax protein was significantly increased,the number of apoptotic cells was significantly increased,and the number of GABAergic interneurons in the dorsal horn of spinal cord was significantly decreased after rapamycin induced autophagy in PHN spinal cord (p<0.05).The relative expression level of Bcl-2 expression was significantly increased,while Bax was significantly lower,the number of apoptotic cells was significantly decreased and the number of GABAergic interneurons in the dorsal horn of spinal cord was significantly increased after 3-MA decreasing autophagic activity in PHN spinal cord(p<0.05).Conclusion:A.Intraperitoneal injection of 0.2ug/g RTX can replicate PHN mice model;B.Increased levels of autophagy in PHN mice;C.The over activation of spinal cord promotes the development of PHN;D.The activation of PHN may be one of the important factors that lead to the reduction of the number of GABA neurons in the spinal Cord.