Stargazin在切口痛大鼠脊髓背角GluR1-AMPA受体胞浆至胞膜转运中的作用

Role of Stargazin in trafficking of GluR1-containing AMPA receptors from cytoplasm to cell membrane in spinal dorsal horn in rats with incisional pain

目的 评价Stargazin在切口痛大鼠脊髓背角含谷氨酸受体1亚基的使君子酸（GluR1-AMPA）受体胞浆至胞膜转运中的作用.方法 成年雄性清洁级SD大鼠45只,体重280 ～ 300 g,6～8周龄,采用随机数字表法,将其分为5组：正常对照组（C组）、假手术组（S组）、切口痛＋生理盐水组（P组）、切口痛＋ Stargazin小干扰RNA（siRNA）组（I组）和切口痛＋Stargazin无意义siRNA组（N组）.P组、I组和N组分别鞘内注射生理盐水10μl、20 μmol/L siRNA、20μmol/L无意义siRNA 10μl,2次/d,连续3d,4d后制备右足底切口痛模型.切口痛术后3h时测定大鼠累计痛评分（CPS）和机械缩足阈（PWT）.然后处死大鼠,取L3-6节段脊髓背角,采用Westem blot法检测胞浆和胞膜GluR1和GluR2亚基表达,采用免疫共沉淀技术检测脊髓背角Stargazin与GluR1或GluR2亚基的共表达.结果 与C组比较,P组和N组CPS评分升高,PWT降低,脊髓背角胞浆GluR1表达下调,脊髓背角胞膜GluR1表达上调,I组CPS评分升高（P＜0.05或0.01）；与P组比较,I组CPS评分降低,PWT升高,脊髓背角胞浆GluR1表达上调,脊髓背角胞膜GluR1表达下调,脊髓背角Stargazin和Stargazin与GluR1共表达下调（P＜ 0.05或0.01）.结论 Stargazin介导了切口痛大鼠GluR1-AMPA受体从胞浆至胞膜转运.

Objective To evaluate the role of Stargazin in trafficking of GluR1-containing AMPA receptors from cytoplasm to cell membrane in the spinal dorsal horn in rats with incisional pain.Methods Forty-five adult male Sprague-Dawley rats,aged 6-8 yr,weighing 280-300 g,were randomly divided into 5 groups （n =9 each）：control group （group C）,sham operation group （group S）,incisional pain ＋ normal saline group （group P）,incisional pain ＋ Stargazin small interference RNA （siRNA） group （group Ⅰ）,and incisional pain ＋ non-sense siRNA group （group N）.In P,I and N groups,normal saline 10 μl,20 μmol/L siRNA and 20 μmol/L non-siRNA 10 μl were injected intrathecally,respectively,twice a day for 3 consecutive days.A 1 cm long incision was made in the plantar surface of right hindpaw.Cumulative pain score （CPS） and paw-withdrawal threshold to yon Frey stimuli （PWT） were measured at 3 h after incision.The animals were sacrificed after pain behavior assessment.Their lumbar segments of the spinal cord （L3-6） were removed.The expression of GluR1 and GluR2 in cell membrane and cytoplasm in spinal cord dorsal horn was determined by Western blot analysis.The co-expression of Stargazin with GluR1 and GluR2 in the spinal dorsal horn was examined by co-immuno-precipitation.Results Compared with group C,the CPS was significantly increased,the PWT was decreased,the expression of GluR1 in cytoplasm was down-regulated and the expression of GluR1 in cell membrane was up-regulated in N and P groups,and the CPS was increasedin group Ⅰ （P ＜ 0.05 or 0.01）.Compared with group P,the CPS was significantly decreased,the PWT was increased,the expression of GluR1 in cytoplasm was up-regulated,the expression of GluR1 in cell membrane was down-regulated,and the expression of Stargazin and co-expression of Stargazin with GluR1 and GluR2 in the spinal dorsal horn was down-regulated in group Ⅰ （P ＜ 0.05 or 0.01）.Conclusion Stargazin mediates trafficking of GluR1-containing AMPA receptors from cytoplasm to cell membrane in spinal dorsal horn and is involved in the development of hyperalgesia in rats with incisional pain.