"夹脊"电针通过调控NLRP3炎性小体活化改善肌萎缩侧索硬化症小鼠肢体功能障碍OA

Objective To investigate the regulatory effect of"Jiaji"(EX-B2)electroacupuncture(EA)on the NOD-like receptor thermal protein domain-associated protein 3(NLRP3)inflammasome in the lumbar spinal cord and to explore its mechanism in improving limb dysfunction in amyotrophic lateral sclerosis(ALS)mice.Methods Fifty-four mice carrying the human superoxide dismutase 1 G93A(hSOD1G93A)gene were randomly divided into model,Jiaji EA,and inhibitor groups,with 18 mice per group.Eighteen mice not carrying the hSOD1G93A gene served as wild group.At 60 d of age,the Jiaji EA group was given EA at L1—L2 and L5—L6 EX-B2 points with continuous wave,1 mA,2 Hz.The needles retained for 20 min,twice every week.The inhibitor group received intraperitoneal injection of the NLRP3 inhibitor(10 mg/kg,twice every week).All treatments were administered for 4 consecutive weeks.The survival period of mice in each group was observed to assess disease progression;the rotarod test was conducted to evaluate motor coordination and limb motor function;HE staining was used to observe pathological changes in the gastrocnemius,and muscle atrophy was assessed by measuring the cross-sectional area of the gastrocnemius fiber;Nissl staining was performed to evaluate the number of motor neurons in the lumbar spinal cord anterior horns;the protein expressions of NLRP3,Caspase-1,apoptosis-associated speck-like protein(ASC),interleukin-1β(IL-1β),interleukin-18(IL-18),and tumor necrosis factor-α(TNF-α)in the lumbar spinal cord were detected by Western blot;the mRNA expressions of NLRP3,Caspase-1,ASC,IL-1β,IL-18,and TNF-α were detected by real-time quantitative PCR.Results Compared with the wild group,mice in the model group exhibited shortened survival time(P<0.01);reduced time spent on the rotating rod starting from the 13th week of age(P<0.05);significantly shorter and rounded gastrocnemius fibers with enlarged inter-fiber spaces,nuclear translocation and leakage.The cross-sectional area of the gastrocnemius fibers was reduced(P<0.01);the number of motor neurons in the spinal anterior horns was decreased(P<0.01);protein and mRNA expressions of NLRP3,Caspase-1,ASC,IL-1β,IL-18,and TNF-α were increased in the lumbar spinal cord(P<0.01).Compared with the model group,survival time was prolonged in the Jiaji EA and inhibitor groups(P<0.01);rotarod time was prolonged starting from the 13th week of age(P<0.05);gastrocnemius fibers exhibited more regular margins and larger morphology,with reduced inter-fiber spaces,and reduced number of rounded fibers and improvement in nuclear translocation and leakage;cross-sectional areas of gastrocnemius fiber were increased(P<0.01);the numbers of motor neurons in the spinal anterior horns were increased(P<0.01);protein and mRNA expressions of NLRP3,Caspase-1,ASC,IL-1β,IL-18,and TNF-α decreased in the lumbar spinal cord(P<0.05,P<0.01).Compared with the inhibitor group,mice in the Jiaji EA group exhibited prolonged rotarod time from the 17th week of age(P<0.05);the cross-sectional area of the gastrocnemius fiber increased(P<0.05);protein and mRNA expressions of NLRP3,Caspase-1 and IL-18 in the lumbar spinal cord were higher(P<0.05,P<0.01),while protein and mRNA expressions of TNF-α were lower(P<0.05,P<0.01).Conclusion EX-B2 EA improves limb function and prolongs survival in ALS mice,with its underlying mechanism potentially involving the alleviation of neuroinflammation through inhibition of NLRP3 inflammasome activation.