This study used a transgenic mouse model of amyotrophic lateral sclerosis (ALS) carrying the human superoxide dismutase 1 G93A (hSOD1G93A) mutation. A total of 54 hSOD1G93A mice were randomized into three experimental groups: model, Jiaji (EX-B2) electroacupuncture (EA), and inhibitor, with 18 mice per group. An additional 18 non-transgenic mice served as wild-type controls. The design compared the effects of Jiaji EA with pharmacologic inhibition of the NLRP3 inflammasome and with untreated disease model controls.
Interventions began when mice reached 60 days of age and continued for 4 consecutive weeks. The Jiaji EA group received electroacupuncture at bilateral lumbar EX-B2 (“Jiaji”) points corresponding to L1–L2 and L5–L6. Stimulation parameters were continuous wave, 1 mA, 2 Hz; needles were retained for 20 minutes per session, administered twice weekly. The inhibitor group received intraperitoneal injection of an NLRP3 inhibitor at 10 mg/kg, twice weekly, for the same 4-week period. The model group received no specific treatment beyond standard care; the wild group were non-transgenic controls.
The study measured survival time and motor coordination using the rotarod test. Compared with the wild group, hSOD1G93A model mice showed a shortened survival period (P < 0.01) and reduced rotarod performance starting from 13 weeks of age (P < 0.05). Both the Jiaji EA and the inhibitor groups had prolonged survival relative to the untreated model group (P < 0.01) and showed improved rotarod times beginning at 13 weeks (P < 0.05). When compared directly, the Jiaji EA group demonstrated longer rotarod times than the inhibitor group from 17 weeks of age (P < 0.05).
Gastrocnemius muscle morphology and motor neuron counts in the lumbar spinal cord anterior horns were evaluated. In the model group, gastrocnemius fibers were markedly shortened, rounded, and separated by enlarged inter-fiber spaces, with evidence of nuclear translocation and leakage; cross-sectional fiber area was reduced (P < 0.01). The number of motor neurons in the spinal anterior horns decreased relative to wild controls (P < 0.01).
Both Jiaji EA and inhibitor treatments improved gastrocnemius morphology: fibers had more regular margins, larger morphology, reduced inter-fiber spacing, fewer rounded fibers, and amelioration of nuclear translocation/leakage. Cross-sectional area of gastrocnemius fibers increased with both interventions (P < 0.01), and motor neuron counts in the anterior horns were higher than in the untreated model (P < 0.01). Compared with the inhibitor group, the Jiaji EA group had a greater increase in gastrocnemius fiber cross-sectional area (P < 0.05).
The investigators quantified protein expression by Western blot and mRNA expression by real-time quantitative PCR for key inflammasome components and proinflammatory cytokines in the lumbar spinal cord: NLRP3, Caspase-1, apoptosis-associated speck-like protein (ASC), interleukin-1β (IL-1β), interleukin-18 (IL-18), and tumor necrosis factor-α (TNF-α).
Compared with wild controls, model mice showed increased protein and mRNA expression of NLRP3, Caspase-1, ASC, IL-1β, IL-18, and TNF-α in the lumbar spinal cord (P < 0.01). Both Jiaji EA and pharmacologic NLRP3 inhibition reduced these protein and mRNA expression levels versus the model group (P < 0.05 or P < 0.01), indicating downregulation of NLRP3 inflammasome activation and associated cytokines after treatment.
When Jiaji EA was compared directly with the inhibitor group, several differences emerged: Jiaji EA-treated mice exhibited higher NLRP3, Caspase-1, and IL-18 protein and mRNA expression than inhibitor-treated mice (P < 0.05, P < 0.01), while TNF-α protein and mRNA expression were lower in the Jiaji EA group (P < 0.05, P < 0.01). These comparative findings indicate that while both interventions modulated inflammasome-related markers, the pattern of changes differed between electroacupuncture and pharmacologic inhibition.
Both Jiaji EA and direct NLRP3 inhibition produced broadly similar beneficial effects on survival, motor performance (rotarod), muscle morphology, motor neuron counts, and reduction of multiple inflammasome-related markers when compared with untreated hSOD1G93A mice. Jiaji EA produced a later but sustained improvement in rotarod performance relative to the inhibitor (significant from 17 weeks) and a larger gastrocnemius fiber cross-sectional area. At the molecular level, Jiaji EA decreased many inflammasome-associated proteins and transcripts versus model but remained higher than inhibitor treatment for selected markers (NLRP3, Caspase-1, IL-18) while achieving lower TNF-α expression.
The authors conclude that EX-B2 electroacupuncture improves limb function and prolongs survival in hSOD1G93A ALS mice. The proposed mechanism is mitigation of neuroinflammation via inhibition of NLRP3 inflammasome activation in the lumbar spinal cord. Both Jiaji EA and pharmacologic NLRP3 inhibition reduced inflammasome component expression and proinflammatory cytokines and yielded correlated improvements in muscle histology, motor neuron counts, and motor behavior. The study reports statistical significance levels for these comparisons in the abstract but does not detail absolute values or effect sizes in this summary. Conflict of interest: the authors declared none.