["Jiaji" (EX-B2) electroacupuncture modulates NLRP3 inflammasome activation to improve limb dysfunction in...

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.

Fifty-four mice carrying the human superoxide dismutase 1 G93A (hSOD1^G93A) gene were randomly divided into model, Jiaji EA, and inhibitor groups, with 18 mice per group. Eighteen mice not carrying the hSOD1^G93A 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.

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 13^th 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 13^th 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 17^th 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).

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.