Mechanism of electroacupuncture alleviating pain in rats with cervical spondylotic radiculopathy by regulating the NO/cGMP/PKG signaling pathway via Piezo1 protein

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.