Mas相关G蛋白偶联受体C参与电针干预慢性炎性痛及其外周δ-阿片受体机制

Involvement of Mas-related G protein-coupled receptor C in intervention of electro-acupuncture on chronic inflammatory pain and mechanism of peripheric δ opioid receptor

目的:观察电针(EA)干预完全弗氏佐剂(CFA)所致慢性炎性痛的镇痛效应并探讨Mas相关G蛋白偶联受体C(MrgprC)参与EA调制背根神经节(DRG)δ-阿片受体(DOR)的机制。方法:选取健康雄性SD大鼠80只。鞘内置管成功的40只大鼠按照随机区组法分为电针+MrgprC小干扰RNA组、电针+对照小干扰RNA组、模型组、正常组、牛肾上腺髓质8-22肽组,每组8只。大鼠右后足底注射CFA 0.1mL以建立慢性炎性痛模型,分别测量CFA造模后1、2、3、4、5、6d大鼠热辐射撤足潜伏期(TWL)。采用免疫印迹法检测大鼠患侧DRG中DOR总蛋白表达和膜/浆比值,采用免疫荧光法检测大鼠患侧DRG中DOR阳性细胞表达率。结果:与正常组比较,CFA模后1d各组大鼠患侧痛阈显著降低(P<0.01)。与模型组比较,电针+对照小干扰RNA组、电针+MrgC小干扰RNA组、牛肾上腺髓质8-22肽组自CFA造模后3d起痛阈均显著上升(P<0.01)。CFA造模后5d、6d时,电针+对照小干扰RNA组痛阈明显高于同时点的电针+MrgprC小干扰RNA组(P<0.01,P<0.05)。与电针+对照小干扰RNA组比较,牛肾上腺髓质8-22肽组在CFA造模后2d鞘内给予BAM8-22后出现痛阈明显升高(P<0.01)。与模型组比较,电针+对照小干扰RNA组患侧DRG中DOR总蛋白表达、DOR膜/浆比值和DOR阳性细胞表达率均上升(P<0.05,P<0.01)。与电针+对照小干扰RNA组比较,电针+MrgC小干扰RNA组患侧DRG中DOR总蛋白表达、DOR膜/浆比值和DOR阳性细胞表达率显著下降(P<0.05,P<0.01)。结论:MrgprC参与EA对CFA诱导的慢性炎性痛的外周镇痛作用,其机制可能与MrgprC调节患侧DRG中DOR表达及膜转移相关。

Objective: To observe analgesic effect of electro-acupuncture(EA) on chronic inflammatory pain induced by complete Freund's adjuvant(CFA) and discuss the mechanism of Mas-related G protein-coupled receptor C(MrgprC) involving δ opioid receptor(DOR) in(dorsal root ganglia) DRG mediated by EA. Methods: 80 healthy male SD rats were adopted. After managing to be made intrathecal catheterization, 40 rats weredivided into EA+MrgC si RNA group, EA+control si RNA group, model group, normal group, and BAM8-22 group by randomized blocks method, with 8 rats in each group. Chronic inflammatory pain model was established by subcutaneously injecting CFA(0.1mL) in bottom of right hind paw of the rat, and the thermal withdraw threshold(TWL) was observed at time point of 1, 2, 3, 4, 5, 6d after CFA injection. The total proteinexpression and ratio of membrane/cytosolic of DOR in DRG of the infected side were detected by western blot method, and the expression ratio of DOR positive cells in DRG of the infected side was detected by immunofluorescence method. Results: Compared with normal group, pain threshold in the infected side of each group at 1d after CFA injection decreased significantly(P<0.01). Compared with model group, pain threshold of EA+control si RNA group, EA+MrgC si RNA group, BAM8-22 group increased significantly since 3d after CFA injection(P<0.01). At time point of 5d and 6d after CFA injection, pain threshold of EA+control si RNA group was significantly higher than EA+MrgC si RNA group at same time(P<0.01). Compared with EA+control si RNA, pain threshold of BAM8-22 group obviously increased(P<0.01) after administration of BAM8-22 at 2d after CFA injection. Compared with model group, total protein expression of DOR in DRG of the infected side of EA+MrgC si RNA group, ratio of membrane/cytosolic of DOR and ratio of expression of DOR positive cells raised(P<0.05, P<0.01). Compared with EA+control si RNA group, total protein expression of DOR in DRG of the infected side of EA+MrgC si RNA group, ratio of membrane/cytosolic of DOR and ratio of expression of DOR positive cells reduced significantly(P<0.05, P<0.01). Conclusion: MrgprC participated in the peripheral analgesic effect of EA on chronic inflammatory pain induced by CFA, and the mechanism might be related to regulation of MrgprC on expression and membrane transfer of DOR in DRG of the infected side.