| Objective: To observe the changes of mechanical pain threshold and thermal pain threshold in(SNL)rats after spinal nerve ligation,and to detect the expression of autophagy related protein and inflammatory factor IL-1?in spinal dorsal horn.The mechanism of massage analgesia was explored by regulating autophagy.To explore the analgesic mechanism of massage on neuropathic pain at the molecular level,in order to provide scientific theoretical basis for massage analgesia.Methods: A total of 180 male SD rats of SPF grade were used as subjects.The main results were as follows:(1)90 rats were randomly divided into three groups: normal group,sham operation group and model group.There were 30 rats in each group,and the normal group was not treated.The model group was established by using Kim and other spinal nerve ligature method to establish SNL rat model.In the sham operation group,only the spinal nerve was exposed and not ligated.The thermal pain threshold(PWL)and mechanical pain threshold(PWT)of rats in each group were measured at 1 day before modeling and 1,3,5 and 7 days after modeling.The expression of autophagy factor p62 and LC3 in spinal dorsal horn of 6 rats in each group were observed by Western blot method and immunofluorescence method on the 1st,3rd,5th and 7th day after modeling.On the 7th day after modeling,6 rats in each group were used to observe the relationship between p62 and glial fibrillary acidic protein(GFAP)(GFAP)expression by immunofluorescence.(2)In addition,90 rats were randomly divided into normal group(n = 15),sham operation group(n = 15),model group(n = 15),rapamycin group(n = 15),trimethyl adenine group(n = 15)and massage group(n = 15).No treatment was given to the normal group,the exposed spinal nerve was not ligated in the sham operation group,and the SNL rat model was established after successful intrathecal catheterization in the other groups.After successful modeling,rapamycin group was intrathecally injected with rapamycin(Rap),an autophagy inducer of 10 ? l.The trimethyladenine group was intrathecally given 10 ? l autophagy inhibitor trimethyladenine(3-MA),the other groups were given the same dose of 5% DMSO,and the massage group was treated with massage.Take the manipulation simulator once a day,Each group was intervened continuously for 7 days.PWL and PWT of rats in each group were measured at 1 day before modeling and 1?3?5 and 7 days after modeling.The expression of autophagy factor p62 and LC3 in spinal dorsal horn of rats in each group was observed by Western blot method,and the expression of IL-1? in serum was measured by ELISA method.Results: 1.General conditions:(1)the rats in each subgroup of the normal group had good spirit,glossy fur and free movement of the limbs.The fur of rats in each subgroup of the sham operation group was bright,dense and uniform,and the surgical suture healed well.The average weight gain of the rats was 4 g / d.On the 5th day after operation,the rats in the model group were mentally poor,their fur was dull,the surgical sutures were bitten open to varying degrees,the severe rats showed ulcerative surface at the mouth of the operation,the claudication aggravated when they walked,the eating and water intake decreased,the stool was thin and rotten,and the smell smelled.Death began to appear.(2)the rats in the normal group,the sham operation group and the model group were basically the same as before,and the rats in the rapamycin group were generally stable and improved on the 3rd day after modeling,while the rats in the trimethyl adenine group were generally worse than those in the model group on the 3rd day after modeling.The self-maiming behavior of rats was more significant,and the self-maiming behavior of massage group was stable and stopped on the 5th day after modeling.2.The results of PWL and PWT showed that there was no significant difference in PWL and PWT in the affected limbs of rats in each group before modeling.The main results were as follows:(1)on the 1st day after modeling,the PWL and PWT of the affected limbs in the model group were significantly lower than those in the normal group and the sham operation group(P < 0 05),and the PWL and PWT in the model group were significantly lower than those in the normal group and the sham operation group(P < 0 05).(2)compared with the model group,the levels of PWL and PWT in rapamycin group were significantly higher than those in model group at 1?3?5?7 days after modeling(P < 0 05).In trimethyladenine group,PWL and PWT decreased significantly on the 5th and 7th day after modeling(P < 0 05),and there was significant difference in trimethyladenine group(P < 0 05).In the massage group,PWL and PWT increased significantly on the 5th day after modeling(P < 0 05),and the difference was statistically significant(P < 0 05).On the 7th day after modeling,there were significant differences in PWL and PWT between normal group,sham operation group and model group,rapamycin group,trimethyl adenine group and massage group.3.The results of Western blot method:(1)compared with the normal group and the sham operation group,the expression of LC3-? in the spinal dorsal horn of the model group was higher and the expression of p62 protein was lower on the 1st day after modeling.After that,the expression of LC3-? decreased and the expression of p62 increased gradually.Compared with normal group and sham operation group,the expression of p62 and LC3-? in model group was significantly higher than that in normal group and sham operation group.(2)there was significant difference in the expression of p62 protein among normal group,sham operation group and model group,rapamycin group and trimethyladenine group(P < 0.05).Compared with the model group,the expression of p62 protein in rapamycin group was significantly lower than that in model group(P < 0.05),while the expression of p62 protein in trimethyladenine group was significantly higher than that in model group(P < 0.05).The expression of p62 protein in rapamycin group was significantly lower than that in model group(P < 0.05).The expression of p62 protein in massage group was significantly higher than that in massage group(P < 0.05).There was significant difference in the expression of LC3 protein among normal group,sham operation group and model group,rapamycin group,trimethyl adenine group and massage group.Compared with the model group,the expression of LC3 protein in rapamycin group and massage group was significantly increased(P < 0 05),while the expression of LC3 protein in trimethyl adenine group was significantly decreased(P < 0 05).4.The results of immunofluorescence showed that p62 was not co-expressed with GFAP in normal group,sham operation group and model group within 7 days after modeling.5.The results of ELLSA method showed that there were significant differences between normal group,sham operation group and model group,rapamycin group,trimethyl adenine group and massage group(P < 0 05).It can be considered that model group,rapamycin group,trimethyl adenine group and massage group were significantly different from those in model group,rapamycin group,trimethyladenine group and massage group.The expression level of inflammatory factor IL-1? in the control group was higher than that in the sham operation group and normal group.There was significant difference between model group and massage group(P < 0.05).Conclusion:(1)autophagy is related to neuropathic pain.When autophagy occurs,the expression of p62 and LC3 in spinal dorsal horn is elevated,and autophagy is correlated with time.(2)astrocytes were not involved in autophagy within 7 days of SNL model.(3)Massage can relieve neuropathic pain,and its mechanism may be related to up-regulation of autophagy,inhibition of inflammatory reaction and analgesic effect. |
PDF Full Text Request