内质网三磷酸肌醇受体在fractalkine诱发BV-2小胶质细胞p38MAPK信号通路激活中的作用

Role of inositol triphosphate receptor in fractalkine-induced activation of p38MAPK signaling pathway in BV-2 microglial cells

目的探讨内质网三磷酸肌醇受体在fractalkine诱发BV-2小胶质细胞p38丝裂原活化蛋白激酶（p38MAPK）信号通路激活中的作用。方法将BV-2小胶质细胞以1×105个／ml浓度接种于3．5cm培养皿（5ml／Ⅲ）、50ml培养瓶（8ml／瓶）、24孔培养板（1ml/孔）或6孔培养板（2ml／孔），采用随机数字表法，将其随机分为5组（n=25）：正常对照组（C组）、fractalkine组（F组）、CX3C趋化因子受体1抗体anti—CX3CRl＋fractalkine组（cF组）、内质网三磷酸肌醇受体拮抗剂2-APB＋fractalkine组（AF组）及p38MAPK抑制剂SB203580＋fractalkine组（sF组）。除c组外，其他4组加入10nmol／Lfracta—lkine，CF组、AF组及sF组于加入fractalkine前1h分别加入15μmol／Lanti．CX3CRl、50μmol／L2-APB及10／zmol／LSB203580。测定fractalkine孵育10min期间细胞内ca2＋浓度（[ca2＋]i）的最大值作为[ca2＋]，于fractalkine孵育即刻、30、60、120、240min时测定p38MAPK磷酸化水平，于fractalkine孵育24h时测定细胞培养液白细胞介素．1p（IL-Iβ）和肿瘤坏死因子．（TNF．a）浓度。结果与C组比较，F组、cF组、AF组和sF组[ca2＋]i、p38MAPK磷酸化水平、IL-1β和TNF—a浓度升高（P〈0．05）；与F组比较，cF组和AF组[ca2＋]i降低，cF组、AF组及sF组p38MAPK磷酸化水平、Ⅱ厂1G和TNF—a浓度降低（P〈0．05）。结论内质网三磷酸肌醇受体参与了fraetalkine诱发BV-2小胶质细胞p38MAPK信号通路激活。

Objective To evaluate the role of inositol triphosphate receptor （IP3 R） in the fractalkine-induced activation of p38MAPK signaling pathway in BV-2 microglial ,cells. Methods BV-2 microglial cells were seeded in 3.5 cm diameter dishes （5 ml/dish）, 50 ml culture flasks （8 ml/flask） or 24-well plates （1 ml/hole） with a density of 1 x 105/ml and randomly divided into 5 groups （ n = 25 each） ： control group （group C）, fracta- lkine group （group F）, CX3C chemokine receptor 1 （CX3CR1） antibody anti-CX3CRi ＋ fractalkine group （group CF）, IP3R antagonist 2-APB ＋ fractalkine group（ group AF） and p38 mitogen-activated protease （p38MAPK） inhibitor SB203580 ＋ fractalkinc group （group SF）. Fractalkine 10 μmol/L was added to the culture medium in groups F, CF, AF and SF. The anti-CX3CR1 15μmol/L, 2-APB 50/μmol/L and SB203580 10 μmol/L were added to the culture medium in groups CF, AF and SF, respectively, 1 h before addition of fractalkine. Thecells were then cultured for 24 h. The intracellular Ca2＋ concentration （[ Ca2＋ ] i） was measured during the 10 rain incubation with fractalkine. The phosphorylation of p38MAPK was measured at 0, 30, 60, 120 and 240 min of incubation with fractalkine. The concentrations of interleukin-lβ （IL-1β） and tumor necrosis factor-a （TNF-a） in the culture medium were determined at 24 h of incubation with fractalkine. Results Compared with group C, [ Ca2＋ ] i, and the phosphorylation of p38MAPEh and concentrations of IL-1β and TNF-a were significantly increased in groups F, CF, AF and SF （P 〈 0.05）. [ Ca2＋ ]i was significant lower in groups AF and CF and phosphorylation of p38MAPK and concentrations of IL-1β and TNF-a were significantly lower in groups CF, AF and SF than in group F （P 〈 0.05）. Conclusion IP3 R is involve in the fractalkine-induced activation of p38MAPK signaling pathway in BV-2 microglial cells.