| Objective:The effects of Jiaji electroacupuncture on the activation and motor function of NLRP3 inflammatory bodies in rats with spinal cord injury were investigated by experimental study.To elucidate the mechanism of P2X7R inhibition of inflammatory reaction after spinal cord injury by inhibiting the activation of NLRP3 inflammatory corpuscle by regulating the spinal cord acupuncture,to provide experimental basis for clinical application of Jiaji Electroacupuncture.Methods:The experimental study is divided into four parts.Research one:To observe the effect of Jiaji electroacupuncture on the motor function of SCI rats and the pathomorphology of spinal cord tissue.36 female SD rats were randomly divided into 3 groups:sham operation group(Sham),model group(Model),and Jiaji electroacupuncture group(EA).Each group was divided into 2 subgroups according to 3d and 7d time points,and 6 rats in each subgroup were given corresponding treatment or treatment.Observe the motor function of each group by BBB score,and observe the pathological changes of spinal cord tissue in each group by HE staining.To determine the effect of Jiaji electroacupuncture on the behavior and morphology of SCI rats.Research two:To observe the effect of Jiaji electroacupuncture on the expression of proinflammatory cytokines IL-18 and IL-1 beta in the spinal cord of SCI rats.36 female SD rats were randomly divided into 3 groups:sham operation group(Sham),model group(Model),and Jiaji electroacupuncture group(EA).Each group was divided into 2 subgroups according to 3d and 7d time points,and 6 rats in each subgroup were given corresponding treatment or treatment.The expression of IL-18 and IL-1 beta in the spinal cord of rats was detected by ELISA.To determine the effect of Jiaji electroacupuncture on the expression of proinflammatory cytokines in spinal cord tissue of SCI rats.Research three:To observe the effect of Jiaji electroporation on the activation of NLRP3 inflammasome in the spinal cord of SCI rats.36 female SD rats were randomly divided into 3 groups:sham operation group(Sham),model group(Model),and Jiaji electroacupuncture group(EA).Each group was divided into 2 subgroups according to 3d and 7d time points,and 6 rats in each subgroup were given corresponding treatment or treatment.The expression of NLRP3,ASC and cleaved caspase-1 in the spinal cord of rats was detected by IHC.Clear role of Jiaji electroacupuncture in inhibiting the activation of NLRP3 inflammasome in spinal cord of SCI rats.Research four:To observe the effect of Jiaji electro-acupuncture on the expression of P2X7 receptor in the spinal cord and the activation of NLRP3 inflammasome in SCI rats.48 rats were randomly divided into 4 groups:sham operation group(Sham),model group(Model),Jiaji electroacupuncture group(EA)and BBG injection group(BBG),each group was divided into 2 subgroups according to 3d and 7d time points,and 6 rats in each subgroup were given corresponding treatment or treatment.The expression of P2X7R in spinal cord tissue of rats was detected by IHC,and the expression of Caspase-1 protein of P2X7R,NLRP3,ASC and cleaved in spinal cord tissue of rats was detected by Western blot method.From the P2X7 receptor mediated NLRP3 inflammatory corpuscle activation angle,the mechanism of Jiaji electroacupuncture(pinch acupuncture)to improve the inflammatory response of SCI rats was revealed.Result:Research one:(1)The BBB scoring method was used to observe the motor function of rats in each group:the rats in group Sham had no obvious motor dysfunction after operation,and the scores of 3d and 7d BBB after operation were all 21 points.Model group had severe hindlimb motor dysfunction after operation,and 3d?7d BBB scores were significantly decreased after operation,compared with Sham group,the difference was statistically significant(P<0.05).In group EA,the score of 3d and 7d BBB increased after operation,and the difference was statistically significant compared with that of the Model group(P<0.05).(2)HE staining was used to observe the histopathological changes of spinal cord in each group:the spinal cord tissue of Sham group was intact,and the boundary of gray matter and white matter was clear.The nuclear morphology was normal,the nucleolus was large and clear,no inflammatory cell infiltration,hemorrhage necrosis,tissue edema and so on.Model group 3d rat spinal cord tissue showed patchy bleeding,loose tissue,vacuolation,only surviving nerve cells,cell morphological changes,cell body becomes small,pyknosis,visible inflammatory cells,visible plasma cells showed nuclear pyknosis,nuclear staining deepened,too the other side of the cell,glial cells increased.In group EA,the pathological changes of spinal cord tissue in group 3d were similar to those in group Model.There were spotty small bleeding foci,loose tissue and increased number of nerve cells compared with group Model,and inflammatory cell infiltration and glial cell proliferation were reduced compared with Model group.Model group 7d in spinal cord of severe osteoporosis,vacuolation,white matter vacuolar degenerative like changes,the remaining nerve cells to further reduce the infiltration of inflammatory cells and the proliferation of glial cells.In group EA,the structure of 7d tended to be intact.The gray matter and white matter boundary were faint.Vacuoles in tissues were rare,inflammatory cells and glial cells were reduced,and a large number of irregular cells were normal.Research two:ELISA method was used to detect the expression of IL-18 and IL-1 beta in spinal cord tissues of rats in each group.There was a small amount of IL-18 and IL-1 beta expression in spinal cord tissue of Sham group,and 3d?7d remained stable after operation.After 3d and 7d,the expression of IL-18 and IL-1 beta in the spinal cord tissue of the model group increased,and the difference was statistically significant compared with the Sham group(P<0.05).After giving Jiaji electroacupuncture,the expression of IL-18 and IL-1 IL-1 in spinal cord tissue of EA group decreased,and there was a significant difference between 7d group and Model group(P<0.05).Research three:IHC was used to detect the expression of NLRP3,ASC and cleaved caspase-1 in spinal cord tissues of every group.Sham group showed a small amount of NLRP3,ASC and cleaved caspase-1 in the spinal cord tissue of the Sham group,showing a positive expression in brown cytoplasm,and the expression of 3d and cleaved remained stable after operation.At 3d and 7d time after operation,the average optical density of NLRP3,ASC and cleaved caspase-1 positive cells in spinal cord tissue of model group increased,and the difference was statistically significant compared with Sham group(P<0.05).After giving Jiaji electroacupuncture,the average optical density of NLRP3,ASC and cleaved caspase-1 positive cells in EA group decreased,and there was a significant difference between 3d group and 7d group compared with Model group(P<0.05).Research four:(1)IHC was used to detect the expression of P2X7R in spinal cord of rats in each group.In Sham group,a small amount of P2X7R was found in the spinal cord of the Sham group,showing a brown yellow positive expression in the cytoplasm,and 3d and 7d remained stable after operation.At postoperative 3d and 7d time points,the average optical density of P2X7R positive cells in spinal cord tissue of Model group increased,and the difference was statistically significant compared with Sham group(P<0.05).The mean light density of P2X7R positive cells in the spinal cord tissue of group EA rats decreased after 7d time,and the difference was statistically significant(P<0.05)compared with that of the group Model.At postoperative 3d and 7d time points,the average optical density of P2X7R positive cells in spinal cord tissue of BBG group decreased,and the difference was statistically significant compared with Model group(P<0.05).(2)Western blot detection of the expression of P2X7R,NLRP3,ASC and cleaved caspase-1 protein in the spinal cord of ratsTo detect the expression of P2X7R protein in the spinal cord tissue of rats by western blot:the visible expression of a small amount of P2X7R protein in spinal cord of rats in group sham,and after 3d,7d remained at a stable level.After 3d,7d time points,increased expression of model P2X7R protein in spinal cord of rats,compared with the sham group,with statistical significance the difference(P<0.05).The postoperative time point of 7d,reduce the expression of EA P2X7R protein in spinal cord of rats,compared with model group,the difference was statistically significant(P<0.05).After 3d,7d time point,reduce the expression of BBG P2X7R protein in spinal cord of rats,compared with group model,with statistically significant difference(P<0.05).Western blot was used to detect the expression of NLRP3,ASC and cleaved caspase-1 in spinal cord tissues of rats.Sham group showed a small amount of NLRP3,ASC and cleaved caspase-1 protein expression in the spinal cord tissue of Sham group and maintained stable level after operation.At 3d and 7d time after operation,the expression of NLRP3,ASC and cleaved caspase-1 in spinal cord tissue of Model group increased,and the difference was statistically significant compared with Sham group(P<0.05).At 3d and 7d time after operation,the expression of NLRP3,ASC and cleaved caspase-1 protein in spinal cord tissue of group EA and BBG decreased,and the difference was statistically significant compared with Model group(P<0.05).Conclusion:(1)Jiaji electroacupuncture can improve the inflammatory injury of spinal cord in SCI rats and promote the recovery of motor function;(2)Jiaji electroacupuncture can down-regulate the expression of interleukin-18 and interleukin-1? in spinal cord of SCI rats and inhibit micro-environmental inflammation;(3)Jiaji electroacupuncture can inhibit the over-activation of NLRP3 inflammasome in spinal cord of SCI rats and down-regulate the expression of pro-inflammatory factors;(4)Jiaji electroacupuncture may improve the expression of P2X7R in the spinal cord of SCI rats,inhibit the over-activation of NLRP3 inflammasome,and improve the inflammatory injury in SCI rats;... |
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