| Objective: Through the experimental study on the effects of jiaji electroacupuncture on motor function and expression of P2X7R/NLRP3 signaling pathway in rats with acute spinal cord injury model,to explore the effects of jiaji electroacupuncture on expression of P2X7R/NLRP3 signaling pathway related factors in rat motor function and spinal cord tissue,and to clarify the mechanism of jiaji electroacupuncture on treatment of spinal cord injury.Methods:This study was divided into two parts.Experiment 1: The effect of jiaji electroacupuncture on behavioral change in ASCI rats.Seventy-two female SD rats were selected for the experiment,and were divided into three groups according to the random number table method:Sham group,Model group and EA group.Each group was further divided into four subgroups according to the time of acupuncture intervention,namely 1d,3d,7d and 21 d,with 6 rats in each subgroup.Rats in Sham group exposed spinal cord only by surgical removal of lamina.Model group rats were prepared ASCI Model by Allen's method.The rats in the Model group were given acupuncture treatment on the basis of preparation.At the end of each treatment period and before the surgery,BBB score was used to evaluate the changes of limb motor function in each group.Experiment 2: The effect of jiaji electroacupuncture on P2X7R/NLRP3 signaling pathway in spinal cord of ASCI rats.According to the random number table,120 female SD rats were divided into Sham operation group(Sham),Model group(Model),jiaji electroacupuncture group(EA),NLRP3 interference group(NLRP3si RNA)and control group(Controlsi RNA).Each group was divided into four subgroups(1d,3d,7d and 21d)according to different time points,with 6 rats in each subgroup.Spinal cords were exposed in Sham group.The Model group used improved Allen's method to prepare rat SCI Model.EA group in the model on the basis of preparation for jiaji electroacupuncture therapy,NLRP3 interference group injected in the successful spinal cord after injury NLRP3 si RNA interfere with the virus,interfere with the control group in the model based on the preparation of injection by the same means negative control virus,at each point in time,after the treatment executed based,IHC method was applied to detect each group in the rat spinal cord tissue NLRP3,P2X7 R,IL-1? and IL-18;Immunofluorescence double staining was used to observe the co-localization of NLRP3/P2X7 Rand OX42.Western-blot was used to detect NLRP3 and P2X7 R protein expression in spinal cord.NLRP3 m RNA and P2X7 Rm RNA expression in spinal cord tissues of rats in each group was detected by Realtime-PCR.Results:1.Comparison of BBB scores: Compared with Sham group,BBB scores in Model group were significantly reduced at each time point,and the difference was statistically significant(P< 0.05).Compared with the Model group,the BBB scores of the rats in the 3d,7d and 21 d,EA groups were all increased after operation,and the difference was statistically significant(p<0.05).Over time,THE BBB scores of the EA group and the Model group showed an increasing trend over time,and the increase rate of the EA group was higher than that of the Model group.2.Immunohistochemistry was used to detect the expressions of NLRP3,IL-1? and IL-18 positive cells in the spinal cord of rats in each group: The expressions of NLRP3,P2X7 R,IL-1? and IL-18 positive cells in the Model group continued to increase 1,3 and 7 days after surgery,and decreased at 21 days after surgery,with statistically significant differences compared with sham group(P < 0.05).The expression of NLRP3 and P2X7R positive cells in the spinal cord tissues of the EA group was significantly decreased on 3,7,and 21 days after surgery,and the difference was statistically significant compared with that of the Model group(P < 0.05).The expressions of IL-1?and IL-18 positive cells in the spinal cord of EA group were significantly decreased 7 and 21 days after the operation,and the difference was statistically significant(P < 0.05).The expression of NLRP3,P2X7 R,IL-1?and IL-18 positive cells decreased in the si RNA group at 3,7,and 21 days after surgery,and the differences were statistically significant when compared with the Model group and the Controlsi RNA group(P < 0.05).3.Immunofluorescence double-staining was used to detect the localized expression of NLRP3 and OX42 in the spinal cord of rats in each group: in the spinal cord of the model group and the control group,the expression of OX42 increased on 3d,7d and 21 d after surgery,and the co-expression of NLRP3 and OX42 increased with stronger fluorescence.In the spinal cord tissues of the EA group and the NLRP3 interference group,OX42 was less expressed on3 d,7d,21 d after surgery,while the total expression of NLRP3/OX42 was less and the fluorescence was weaker.The tendency of P2X7 R and OX42 is similar.4.Western blot detection of NLRP3 and P2X7 R protein expression in the spinal cord of rats in each group: 1,3 and 7 days after surgery,the expression of NLRP3/P2X7 Rprotein in the spinal cord of rats in the Model group continued to increase,and decreased at 21 days.At time points 3,7 and 21 days after surgery,the difference between Model group and Sham group was statistically significant(P < 0.05).The expression of NLRP3 and P2X7 R protein in the spinal cord of EA group was significantly decreased 7 and 21 days after operation,and the difference was statistically significant compared with that of Model group(P < 0.05).On 3,7,and 21 days after surgery,NLRP3/P2X7 Rprotein expression in the spinal cord tissues of the si RNA interference group decreased,with statistically significant differences compared with the Model group and the Controlsi RNA group(P < 0.05).5.RT-PCR was used to detect the expression of NLRP3 and P2X7 R m RNA in the spinal cord of rats in each group: 1,3,7,and 21 days after surgery,the expression of NLRP3 and P2X7 R gene was increased in the spinal cord of the Model group,and the difference was statistically significant compared with that of the Sham group(P < 0.05).At 3,7,and 21 days after surgery,the expression of NLRP3 and P2X7 Rgene in the spinal cord of EA group decreased,and the difference was statistically significant compared with that of the Model group(P < 0.05).On 1d,3d,7d and 21 d after surgery,the expression of NLRP3 and P2X7 R gene in the spinal cord tissues of the si RNA interference group was significantly reduced,and the difference was statistically significant compared with the Model group and the Controlsi RNA group(P < 0.05).Conclusion:1.Jiaji electroacupuncture can reduce spinal cord inflammation and improve SCI motor function in rats after ASCI;2.Jiaji acupuncture can inhibit the expression of NLRP3 protein and reduce the release of pro-inflammatory factors IL-1? and IL-18;3.Plasmid virus loaded with si RNA interfered with the expression of NLRP3,reduced the level of NLRP3 m RNA,inhibited the expression of NLRP3 protein in microglia cells,reduced the secretion of pro-inflammatory factors,and improved the inflammatory injury of SCI rat spinal cord;4.Jiaji electroacupuncture may reduce the secretion of pro-inflammatory factors by inhibiting the expression of NLRP3 protein and gene in microglia cells,thus improving the inflammatory injury of SCI rat spinal cord tissues. |
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