益肾养髓方调控NLRP3/Caspase-1/GSDMD通路抑制脊髓型颈椎病神经元焦亡的作用机制研究OA

Objective To investigate the mechanism of Yishen Yangsui Formula(益肾养髓方,YSYSF)in inhibiting neuronal pyropto-sis via A2 astrocyte-derived exosomes based on the NOD-like receptor protein 3(NLRP3)/caspase-1/gasdermin D(GSDMD)signaling pathway.Methods A2-reactive astrocytes were induced and subjected to oxygen-glucose deprivation(OGD),followed by MTT assay for cell viability assessment.Exosomes were then extracted from the culture supernatant of A2 astrocytes in the control,OGD,and YSYSF groups via ultracentrifugation.The extracted exosomes were characterized by transmission electron microscopy,nanoparticle tracking analysis(NTA),and Western blot(WB).PKH67-labeled exosomes were co-cultured with neurons.The uptake of A2 astrocyte-derived exosomes(A2-exos)by neurons was observed using fluorescence microscopy.Neuronal cell viability was analyzed by MTT assay,and the expression levels of pyroptosis-related proteins(NLRP3,ASC,caspase-1,GSDMD)were detected by WB.Results Compared with the control group,the cell viability of A2 astrocytes was significantly decreased in the OGD group(P<0.001).Immuno-fluorescence(IF)results indicated that neurons in all groups(control,OGD,YSYSF)were capable of taking up A2-exos.Compared with the control group,the OGD group exhibited reduced neuronal cell viability(P<0.001)and increased expression of pyroptosis-related proteins(NLRP3,ASC,caspase-1,GSDMD;P<0.001).In contrast,the YSYSF group showed increased neuronal cell viabil-ity(P<0.001)and decreased expression of these pyroptosis-related proteins(P<0.001).Conclusion YSYSF may inhibit neuronal pyroptosis by modulating the NLRP3/caspase-1/GSDMD pathway via A2 astrocyte-derived exosomes,thereby potentially alleviating the progression of cervical spondylotic myelopathy(CSM).