To investigate the regulatory effect of electroacupuncture on Piezo1 protein in rats with cervical spondylotic radiculopathy (CSR) and its impact on the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signaling pathway, in order to explore the analgesic mechanism of electroacupuncture in treating CSR.

Male SD rats were randomly divided into sham-operation, model, electroacupuncture (EA), and EA+Yoda1 groups, with 10 rats in each group. The CSR model was established by a surgical procedure involving thread insertion into the vertebral canal. Rats in the EA group received electroacupuncture at bilateral “Hegu” (LI4) and “Taichong” (LR3) acupoints for 20 min per session, continuously for 7 days. Rats in the EA+Yoda1 group received an intraperitoneal injection of the Piezo1 agonist Yoda1 (1.5 mg/kg) before electroacupuncture treatment. Gait score, mechanical paw withdrawal threshold, and thermal paw withdrawal latency were measured before and after the electroacupuncture intervention. Pathological changes in the spinal cord tissue at the lesion site were observed using hematoxylin-eosin (HE) staining. The mRNA expression level of Piezo1 in the spinal cord was detected by qPCR. The expression of Piezo1 and calmodulin (CaM) in the spinal cord was detected by immunofluorescence double staining. The protein expression levels of CaM, neuronal nitric oxide synthase (nNOS), soluble guanylate cyclase (sGC), and PKG1 in the spinal cord were detected by Western blot. The content of cGMP in the spinal cord was measured by ELISA. The nitrite colorimetric method was used to determine the NO content in the spinal cord.

Compared to the sham-operation group, the model group showed increased gait score (P<0.01), decreased mechanical withdrawal threshold, and shortened thermal withdrawal latency before intervention (P<0.01). After intervention, the model group exhibited significant increases in Piezo1 mRNA expression, Piezo1 and CaM immunofluorescence intensity and co-localization, protein expressions of CaM, nNOS, sGC and PKG1, as well as cGMP and NO contents in the spinal cord tissue (P<0.01). HE staining revealed shrunken neurons, deepened cytoplasmic staining, blurred nuclear-cytoplasmic boundaries, glial cell hyperplasia, increased cell density, and dilated microvessels with hemorrhage in the spinal cord of the model group. Compared to the model group, the electroacupuncture group showed decreased gait score (P<0.01), increased mechanical withdrawal threshold, and prolonged thermal withdrawal latency (P<0.01). Piezo1 mRNA expression, Piezo1 and CaM immunofluorescence intensity and co-localization, protein expressions of CaM, nNOS, sGC, PKG1, as well as cGMP and NO contents were all reduced (P<0.01). HE staining showed occasionally shrunken neurons in the electroacupuncture group, with reduced microvascular dilation and glial cell hyperplasia compared to the model group. Compared to the electroacupuncture group, the EA+Yoda1 group showed shortened thermal withdrawal latency (P<0.01), and increased Piezo1 mRNA expression , Piezo1 and CaM immunofluorescence intensity and co-localization, protein expressions of CaM, nNOS, sGC, PKG1, as well as cGMP and NO contents (P<0.05, P< 0.01). HE staining revealed neuronal shrinkage, increased glial cell numbers, and some microvascular dilation with hemorrhage in the EA+Yoda1 group.

Electroacupuncture can effectively alleviate pain in CSR rats, potentially by inhibiting Piezo1 protein activation and suppressing the NO/cGMP/PKG signaling pathway in the spinal cord to exert analgesic effects.

To observe the effect of wheat-grain moxibustion (WGM) at “Guanyuan” (CV4), “Mingmen” (GV4), and bilateral “Xuanzhong”(GB39) on the oxidative stress and pyroptosis, and the activity of the Sirtuin 1 (SIRT1)/nuclear factor erythroid-2-related factor 2(Nrf2) signaling pathway in the hypothalamus of perimenopausal insomnia (PI) rats, so as to explore its underlying mechanism in improving PI.

Thirty-two female SD rats were randomly divided into four groups: sham, model, WGM, and WGM+EX527 (n=8 per group). The PI model was established by bilateral ovariectomy combined with an intraperitoneal injection of 4-Chloro-DL-phenylalanine suspension (350 mg/kg) for 2 consecutive days. Rats in the WGM and WGM+EX527 groups received WGM at CV4, GV4 and GB39, with 6 cones for each acupoint. The rats in the WGM+EX527 group received additional intraperitoneal injection of the SIRT1 inhibitor EX527 (10 mg/kg) 30 min before each moxibustion session. The interventions were conducted once daily for 14 consecutive days. The sleep latency and sleep duration were assessed using the pentobarbital sodium-induced sleep test (righting reflex), and the spontaneous activities (total distance moved, center zone dwelling time, and rearing times in 5 min) were measured using the open field test (OFT). The hematoxylin-eosin (H.E.) staining was used to observe morphological changes in the hypothalamic tissue. Western blot was used to detect protein expression levels of SIRT1, Nrf2, Kelch-like ECH-associated protein1 (Keap1), heme oxygenase-1 (HO-1), superoxide dismutase (SOD), catalase (CAT), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cysteingl aspartate specific protease 1 (Caspase-1), apoptosis-associated speck-like protein containing a CARD (ASC), and gasdermin-D (GSDMD) in the hypothalamus. The qPCR was used to measure mRNA expression levels of SIRT1, Nrf2, Keap1, and HO-1 in the hypothalamus. The reactive oxygen species (ROS) assay kit was used to detect ROS level of hypothalamic tissue. The ELISA was used to measure the content of interleukin (IL)-1β and IL-18 in the hypothalamus.

Compared to the sham group, the model group showed a striking decrease in the total distance traveled, central zone dwelling time and rearing times in the OFT (P<0.01), and significantly shortened sleep duration and prolonged sleep latency (P<0.01). In comparison with the model group and the WGM+EX527 group, the WGM group showed an obvious increase in the total distance traveled, central zone dwelling time and rearing times (P<0.01), and shortened sleep latency, prolonged sleep duration (P<0.01). Additionally, in comparison with the sham group, the model group had a significant down-regulation in the expression levels of hypothalamic SIRT1, Nrf2, Keap1, and HO-1 proteins and mRNAs, and SOD and CAT proteins (P<0.01), and an obvious up-regulation in the expression levels of NLRP3, Caspase-1, ASC, GSDMD proteins, ROS level, and contents of IL-1β and IL-18 (P<0.01) in the hypothalamus. In comparison with the model group and the WGM+EX527 group, both the down-regulation and up-regulation of expression levels of the proteins and mRNAs mentioned above, and ROS level and the contents of IL-1β and IL-18 were reversed in the WGM group (P<0.01,P<0.05). H.E. staining showed that in comparison with the sham group, more hypothalamic neurons were wrinkled with unclear cell structures, varying sizes and uneven distribution in the model and WGM+EX527 groups, which was alleviated in the degree of cell injury in the WGM group, for instance clearer cell structure and uniform distribution.

WGM can improve the sleep and spontaneous activity of PI rats, which may be related to its functions in alleviation of hypothalamic oxidative stress and pyroptosis by regulating the SIRT1/Nrf2 signaling pathway.

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.

To observe effects of electroacupuncture (EA) on the activation of astrocytes and high mobility group protein B1 (HMGB1)/Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling pathway, as well as related cytokines in rats with cervical spondylosis radiculopathy (CSR), so as to explore the analgesic mechanism of EA in treating CSR.

Twenty-four male SD rats were randomly divided into blank, sham surgery, model, and EA groups, with 6 rats in each group. CSR rat model was established by using cervical spinal cord canal puncture method. On the 7^th day after successful modeling, EA was applied to rats in the EA group at bilateral “Hegu” (LI4) and “Taichong” (LR3) for 20 minutes (1.5 Hz, 1 mA), once daily for 7 consecutive days. Before and after intervention, gait impairment scores and mechanical pain thresholds were assessed. HE staining was used to observe pathological changes in spinal cord tissue. Western blot was used to detect the expression of HMGB1, TLR4, MyD88, and glial fibrillary acidic protein (GFAP) in the spinal cord. ELISA was used to measure the contents of CXC chemokine ligand 1 (CXCL1), chemokine ligand 2 (CCL2), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β in spinal cord. Immunofluorescence staining was used to observe GFAP protein positive expression in spinal cord tissue.

There was no significant difference of all indexes between the blank group and the sham surgery group.Compared with the sham surgery group, mechanical pain threshold of rats in the model group was decreased (P<0.01), while gait impairment score, the contents of CXCL1, CCL2, TNF-α, IL-1β, protein expressions of HMGB1, TLR4, MyD88 and GFAP, and positive expression of GFAP in spinal cord tissue were increased (P<0.01); HE staining indicated severe overall morphological damage in the spinal cord of rats in the model group, with significant shrinkage of gray matter neurons, reduced number of Nissl bodies, and increased inflammatory cell infiltration. Compared with the model group, mechanical pain threshold in the EA group was increased (P<0.01), while gait impairment score, the contents of CXCL1, CCL2, TNF-α, IL-1β, protein expressions of HMGB1, TLR4, MyD88 and GFAP, and positive expression of GFAP in spinal cord were reduced (P<0.01); HE staining showed more intact neuronal cell bodies, increased number of Nissl bodies, and reduced shrinkage of gray matter neurons, inflammatory cell infiltration, and microvascular dilation in the spinal cord of rats in the EA group.

EA can effectively alleviate pain in CSR rats, which is possibly by inhibiting astrocyte activation, HMGB1/TLR4/MyD88 signaling pathway, and reducing the release of related inflammatory cytokines, thus alleviating central sensitization in spinal segments.