Effect of electroacupuncture of"Jiaji"(EX-B2)on NOD-like receptor protein 3 mediated pyroptosis in spinal cord tissue of rats with acute spinal cord injury
Objective To observe the effect of electroacupuncture(EA)stimulation of"Jiaji"(EX-B2)on motor function,histomorphology,and expression of NOD-like receptor protein 3(NLRP3)and N-terminal domain of gasdermin D(GSDMD-N)in the spinal cord tissue of rats with spinal cord injury(SCI),so as to explore its mecha-nism underlying improvement of SCI.Methods Forty eight female SD rats were randomly divided into sham surgery(sham),SCI model(model),EA,and NLRP3 agonist(monosodium urate,MSU)combined with Jiaji EA(MSU+EA)groups,with 12 rats in each group which were further divided into 3 d and 7 d subgroups,with 6 rats at each time point.Two EA groups received EA stimulation of EX-B2 with a frequency of 100 Hz,electrical current of 1-2 mA for 30 min,once a day for 3 or 7 days.After 5 min,6 h,and 24 h of modeling,rats of the MSU+EA group received intraperitoneal injection of MSU(200 μg/kg,200 μg/mL).The motor function was evaluated using Basso-Beattie-Bresnahan(BBB)scale,the morphological structure of rat spinal cord tissue was observed by H.E.staining.The expression of pyroptosis related factors NLRP3,cleaved Caspase-1 and GSDMD-N of the spinal cord was observed by using immunohisto-chemistry and Western blot separately,the expression and localization of Iba-1 and GSDMD-N in the spinal cord tissue were observed using immunofluorescence double staining method.Results Compared with the sham group,the BBB scores after modeling and on day 3 and 7 were decreased(P<0.05),while the average OD values(immunoactivity)and expression levels of NLRP3,cleaved Caspase-1 and GSDMD-N proteins,and the immunofluorescence intensity of Iba-1/GSDMD-N(co-expression)of the spinal cord tissues on day 3 and 7 were significantly increased in the model group(P<0.05).In comparison with the model group,the BBB scores on day 3 and 7 were obviously increased(P<0.05),while the immunoactivity and expression levels of NLRP3,cleaved Caspase-1 and GSDMD proteins,and the immunofluorescence intensity of Iba-1/GSDMD-N on day 3 and 7 significantly down-regulated in the EA group(P<0.05)but not in the MSU+EA group(P>0.05),suggesting an elimination of the effects of EA after administration of NLRP3 agonist(MSU).H.E.staining showed obvious bleeding area in the spinal cord tissue,loose tissue and inflammatory cell infiltration on day 3 after modeling,and basic absorption of the bleeding,loose tissue,obvious vacuolar changes of the white matter area,loss and contraction of neurons with infiltration of a large number of inflammatory cells,which was milder in the EA group but not in the MSU+EA group.Conclusion EA of EX-B2 can improve the motor function of SCI rats,which may be related to its functions in inhibiting pyroptosis of microglia mediated by NLRP3/Caspase-1 signaling pathway.
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