Effect of electroacupuncture on microglial polarization and activity of α7nAChR-TLR4/MyD88/NF-κB signaling pathway in the anterior cingulate cortex of rats with chronic inflammatory pain-depression comorbidity

To observe the effect of electroacupuncture (EA) on the regulation of microglia polarization in anterior cingulate cortex (ACC) by α7 nicotinic acetylcholine receptor (α7nAChR)-Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway in chronic inflammatory pain-depression comorbidity (CIPDC) rats, so as to explore its central “analgesic-antidepressant” mechanism underlying improvement of CIPDC.

Thirty-six adult male SD rats were randomly divided into blank, CIPDC model and EA groups, with 12 rats in each group. The CIPDC model was established by plantar injection of complete Freund’s adjuvant. On the 14^th day after modeling, EA (1.5 Hz, 1 m A) was applied to bilateral “Hegu” (LI4) and “Taichong” (LR3) for 20 min, once a day for 14 consecutive days. The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were detected at the 0, 14^th, and 28th day after modeling. The open field test (OFT), forced swim test (FST) and sucrose preference test were used to evaluate the rats’ depression-like behavior. Hematoxylin-eosin (H. E.) staining was used to observe the histopathological changes of ACC. The contents of tumor necrosis factor-α(TNF-α), interleukin (IL)-1β, IL-6, IL-4 and IL-10 in the ACC tissue were detected by enzyme-linked immunosorbent assay (ELISA). Double immunofluorescence staining was used to detect the co-expression of M1 microglia marker CD86 and ionized calcium binding adapter molecule 1 (Iba-1), and M2 microglia marker CD206 and Iba-1 in the ACC tissue. The protein expression levels of α7nAChR, TLR4, myeloid differentiation primary response protein 88 (My D88), NF-κB p65 and p-NF-κB p65 in the ACC tissue were detected by Western blot.

Compared with the blank group, the MWT, TWL, sucrose preference rate, total distance traveled and time spent in the center zone of OFT were significantly decreased (P<0.05), and the immobility time of FST was obviously increased (P<0.05) in the model group. In comparison with the model group, the MWT, TWL, sucrose preference rate, total distance traveled and time spent in the center zone of OFT were apparently increased (P<0.05), while immobility time of FST was strikingly decreased (P<0.05) in the EA group, suggesting an amelioration of pain and depression-like behavior after EA. The contents of TNF-α, IL-1β and IL-6, immunofluorescence area of CD86/Iba-1 and the expression levels of TLR4, My D88, p-NF-κB p65, and the ratio of p-NF-κB p65/NF-κB p65 were significantly higher (P<0.05), and the contents of IL-4 and IL-10, CD206/Iba-1 immunofluorescence-positive area and the expression level of α7nAChR were significantly lower (P<0.05) in the model group than in the blank group. Following EA intervention, the increased levels of TNF-α, IL-1β and IL-6, CD86/Iba-1 immunofluorescence-positive area and the expression levels of TLR4, My D88, p-NF-κB p65, and the ratio of p-NF-κB p65/NF-κB p65 and the decreased levels of IL-4 and IL-10, immunofluorescence area of CD206/Iba-1 and the expression level of α7nAChR after modelling were all reversed (P<0.05). H. E. staining showed disordered arrangement of tissues, neuronal cells shrinking, reduced number and deeply stained pyramidal cells, with vacuoles and nerve fiber tangles appeared in the interstitial spaces in the ACC of model group, which was relatively milder in the injury degree in the EA group, including a small number of neurons being deeply stained and smaller space between neurons and tissues.

EA can exert central “analgesic-antidepressant” effect in CIPDC rats, which may be related to its functions in alleviating central inflammatory response, reducing neuronal damage and promoting microglial polarization mediated by α7nAChR-TLR4/My D88/NF-κB signaling in the ACC tissue.