Electroacupuncture Attenuates Neuroinflammation in PCPA-Induced Insomnia Rats by Suppressing the TLR4/MyD88/NF-κB p65 Pathway and M1 Microglial Activation.

BACKGROUND: Insomnia is closely associated with neuroinflammation, yet the therapeutic mechanism of electroacupuncture (EA) remains unclear. This animal study investigated whether EA attenuates hypothalamic neuroinflammation in para-chlorophenylalanine (PCPA)-induced insomnia rats by suppressing the TLR4/MyD88/NF-κB p65 pathway and M1 microglial activation, using TAK-242 (HY-11109), a selective TLR4 inhibitor, as a positive control. METHODS: Forty Sprague-Dawley rats (20 male, 20 female, sex-balanced across groups) were randomly allocated to Control, Model, EA, and TAK-242 groups (n=10 per group). Insomnia was induced in the model, EA, and TAK-242 groups by intraperitoneal injection of PCPA (500mg/kg) for 2 consecutive days. The TAK-242 group received daily intraperitoneal injection of TAK-242 (3 mg/kg). The EA group received acupuncture at Baihui (GV20), bilateral Benshen (GB13), Shenmen (HT7) and Sanyinjiao (SP6), with ipsilateral Shenmen (HT7) and Sanyinjiao (SP6) connected to an electroacupuncture device using a 2 Hz continuous wavefor 20 minutes daily over 7 days. Sleep latency and total sleep duration were recorded via the pentobarbital sodium righting reflex test. Serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels were assessed via the enzyme-linked immunosorbent assay (ELISA); Hypothalamic microglial M1 polarization was assessed by Iba-1/CD86 immunofluorescence co-localization. Protein and mRNA levels of TLR4, MyD88, NF-κB p65, TNF-α, and IL-6 were quantified by Western blot and RT-qPCR, respectively. All outcome assessments were performed by investigators blinded to group allocation. RESULTS: Compared with the model group, both electroacupuncture and TAK-242 significantly shortened sleep latency and prolonged total sleep time (P < 0.01), and reduced serum levels of TNF-α and IL-6 (P < 0.01). Both interventions also attenuated CD86 fluorescence intensity (P < 0.01) and Iba-1/CD86 co-expression (P < 0.05), and down-regulated the protein and mRNA expression of TNF-α, IL-6, TLR4, MyD88, and NF-κB p65 in the hypothalamus (P < 0.01). The effects of electroacupuncture and TAK-242 on sleep parameters, CD86 fluorescence intensity, and Iba-1/CD86 co-expression were comparable, whereas TAK-242 exerted a more pronounced inhibitory effect than electroacupuncture on the expression of inflammatory cytokines and pathway-related proteins in the serum and hypothalamus (P < 0.05, P < 0.01). CONCLUSION: Electroacupuncture effectively improved sleep disturbances in PCPA-induced insomnia rats. The underlying mechanism may be associated with inhibition of the TLR4/MyD88/NF-κB p65 signaling pathway, suppression of M1 microglial activation, and downregulation of pro-inflammatory cytokines, thereby partly alleviating hypothalamic neuroinflammation.