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.