RESUMEN
Upregulation of P2X3 receptor (P2X3R) has been strongly implicated in nociceptive signaling including bone cancer pain (BCP). The present study, using rat bone cancer model, aimed to explore the role of P2X3R in regulating rat pain behavior under the intervention of electroacupuncture (EA). The BCP model was successfully established by injection with MRMT-1 breast cancer cell into the medullary cavity of left tibia for 3 × 104 cells/3 µL PBS in rats as revealed by obvious bone destruction, decreased paw withdrawal thresholds (PWTs), and reduced paw withdrawal latencies (PWLs). Western blot analyses showed that P2X3R expression was significantly upregulated in ipsilateral lumbar 4-6 (L4-6) dorsal root ganglia (DRG), but the difference not seen in spinal cord dorsal horn (SCDH). With the in-depth study of P2X3R activation, we observed that intrathecal injection of P2X3R agonist α,ß-meATP aggravated MRMT-1 induced BCP, while injection of P2X3R inhibitor A-317491 alleviated pain. Subsequently, we demonstrated that BCP induced mechanical allodynia and thermal hyperalgesia were attenuated after EA treatment. Under EA treatment, total P2X3R protein expression in ipsilateral DRGs was decreased, and it is worth mentioning that decreased expression of P2X3R membrane protein, which indicated that both the expression and membrane trafficking of P2X3R were inhibited by EA. The immunofluorescence assay showed that EA stimulation exerted functions by reducing the expression of P2X3R-positive cells in ipsilateral DRGs of BCP rats. Ca2+ imaging analysis revealed that the EA stimulation decreased the percentage of α,ß-meATP responsive neurons in DRGs and inhibited calcium influx. Notably, the inhibitory effect of EA on mechanical allodynia and nociceptive flinches was abolished by intrathecal injection of α,ß-meATP. These findings demonstrated EA stimulation ameliorated mechanical allodynia and thermal hyperalgesia in rat model of MRMT-1-induced BCP. EA exerts analgesic effect on BCP by reducing the overexpression and functional activity of P2X3R in ipsilateral DRGs of BCP rats. Our work first demonstrates the critical and overall role of P2X3R in EA's analgesia against peripheral sensitization of MRMT-1-induced BCP and further supports EA as a potential therapeutic option for cancer pain in clinic.
Asunto(s)
Neoplasias Óseas , Dolor en Cáncer , Electroacupuntura , Ratas , Animales , Hiperalgesia/metabolismo , Dolor en Cáncer/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Ratas Sprague-Dawley , Electroacupuntura/métodos , Dolor/metabolismo , Neoplasias Óseas/metabolismo , Analgésicos , Ganglios Espinales/metabolismoRESUMEN
OBJECTIVE: To observe the effect of electroacupuncture (EA) on pain behavior and expression of µ-opioid receptor (MOR) and Rab5 (an important protein molecule for internalization of MOR) in the locus coeruleus (LC) region in bone cancer pain (BCP) rats with morphine tolerance (MT), so as to explore its mechanisms underlying improvement of BCP and MT. METHODS: The present study included two parts. In the first part, 23 female SD rats were randomized into sham BCP (nï¼6), BCP (nï¼9) and BCPï¼MT (nï¼8) groups, and in the second part, 61 female SD rats were randomized into 5 groups: sham BCP (nï¼11), BCP (nï¼11), BCPï¼MT (nï¼13), BCPï¼MTï¼EA (nï¼13) and BCPï¼MTï¼sham EA (nï¼13). The BCP morphine tolerance (BCPï¼MT) model was established by injection of 10 µL of human Walker 256 breast cancer cells (MRMT-1 breast cancer cells, 1 x104 cells/µL) into the bone marrow cavity at the upper part of the left tibia and intraperitoneal injection of morphine hydrochloride (10 mg/kg, once per 12 h, for 11 successive days). On day 21 after inoculation, EA (2 Hz/100 Hz, 0.5ï¼1.5 mA, increasing 0.5 mA every 10 min) was began to applied to bilateral "Zusanli" (ST30) and "Kunlun" (BL60) immediately after the first intraperitoneal injection of morphine. The treatment was performed for 30 min every time, once daily for 7 successive days. The paw withdrawal threshold (PWT) was detected before and 10, 11, 21, 22, 24, 26 and 28 days after inoculation. The immunoactivity of MOR and Rab5 proteins in the LC region was detected by immunofluorescence histochemistry. RESULTS: In the first part of the study, at the 10th day after inoculation of cancer cells, the PWT of the BCP and BCPï¼MT groups was significantly lower than that of the sham BCP group (P<0.05), suggesting a success of BCP model. From day 11 to 19 after inoculation (during injection of morphine), the PWTs of the BCPï¼MT group were significantly higher than those of the BCP group (P<0.01), and on day 21, the PWT of the BCPï¼MT group was similar to that of the BCP group (P>0.05) but significantly lower than that of the sham BCP group (P<0.01), suggesting a success of MT. Hï¼E. staining showed a large quantity of MRMT-1 cancer cells in the bone marrow cavity in both BCP and BCPï¼MT groups. In the second part of the study, the decreased PWTs from 10th to 28th day after inoculation were significantly increased on day 22, 24, 26 and 28 in the BCPï¼MTï¼EA group relevant to the BCP, BCPï¼MT and BCPï¼MTï¼sham EA groups (P<0.01). The ratios of MOR and Rab5 positive (ï¼) cells and MORï¼/Rab5ï¼ of the left LC region were significantly lower in the BCP and BCPï¼MT groups than those of the sham BCP group (P<0.01), but were considerably higher in the BCPï¼MTï¼EA group than those in the BCP, BCPï¼MT and BCPï¼MTï¼sham EA groups (P<0.01). The ratios of Rab5ï¼ and MORï¼/Rab5ï¼ cells of the BCPï¼MT group were significantly lower than those of the BCP group (P<0.05). No significant changes were found in the ratios of MORï¼ and Rab5ï¼ cells and MORï¼/Rab5ï¼ cells after BCPï¼MTï¼sham EA in comparison with the BCPï¼MT group (P>0.05). CONCLUSION: EA intervention can relieve pain and MT in bone cancer pain rats with MT, which may be related to its effects in increasing MOR expression and promoting endocytosis of MOR in LC region.