内毒素耐受对内毒素诱导的葡萄膜炎炎症程度及虹膜睫状体中PI3K/AKT 信号通路的影响

doi:10. 13281/j. cnki. issn.1004-4469. 2020. 01. 010

眼科

内毒素耐受对内毒素诱导的葡萄膜炎炎症程度及虹膜睫状体中PI3K/AKT 信号通路的影响

王婧张楠余烁刘新丽卢弘

首都医科大学附属北京朝阳医院眼科 100020

收稿日期:2018-10-15 出版日期:2020-01-25 发布日期:2020-02-12
通讯作者: 卢弘，Email：13701220319 @163.com
基金资助:
国家自然科学基金（81471575；81273246）

Effects of endotoxin tolerance on endotoxin-induced uveitis and on the PI3K/AKT signaling pathway in the iris ciliary body

Wang Jing, Zhang Nan, Yu Shuo, Liu Xinli, Lu Hong

Department of Ophthalmology, Beijing Chao Yang Hospital, Capital Medical University, Beijing 100020, China

Received:2018-10-15 Online:2020-01-25 Published:2020-02-12
Contact: Lu Hong, Email: 13701220319@163.com
Supported by:
National Nature Science Fund of China (81471575; 81273246)

摘要/Abstract

摘要： 目的探讨低剂量脂多糖（lipopolysaccharide，LPS）预处理对内毒素诱导葡萄膜炎（endotoxin induced uveitis，EIU）动物模型炎症程度的影响，并分析虹膜睫状体组织中PI3K/AKT表达水平的改变。设计实验研究。研究对象 8-10周龄健康雄性Wistar大鼠90只。方法随机分为正常对照（normal control，NC）组、内毒素耐受（endotoxin tolerance， ET）组和内毒素诱导的葡萄膜炎（EIU）组，每组30只。通过皮下注射1 mg/kg LPS建立内毒素诱导的葡萄膜炎动物模型。ET组：EIU模型诱导之前连续5日腹腔注射0.1 mg/kg LPS诱导内毒素耐受。 EIU组及NC组连续5日注射等体积生理盐水。第6天相同时间ET组及EIU组接受200 μg LPS皮下注射，NC组接受等体积生理盐水注射。注射后 24小时，在裂隙灯显微镜下进行临床评分。眼球摘除行HE染色组织病理学观察。ELISA测量房水TNF-α浓度，蛋白印迹法检测虹膜睫状体PI3K和AKT蛋白的表达。主要指标眼前节炎症反应评分，虹膜切片HE染色观察，房水TNF-α浓度及虹膜睫状体PI3K/AKT蛋白表达。结果临床评分ET组、EIU组、NC组分别为（1.65±0.49）、（6.65±0.59）、（0.20±0.41）分（ ?字2= 53.261，P=0.001）；组织病理评分ET组、EIU组、NC组分别为（0.6±0.5）、（3.8±0.4）、（0.2±0.4）分（?字2= 46.137，P=0.001）。房水TNF-α浓度 ET组、EIU组、NC组分别为（10.58±0.67）、（30.96±1.55）、（10.32± 0.61）pg/ml（F=260.08，P=0.03）。蛋白印迹法显示虹膜睫状体组织中PI3K和AKT的蛋白水平ET组、EIU组、NC组分别为（0.197±0.019）、（0.539±0.017）、（0.453±0.014）（F=210.66，P=0.002）和（0.234±0.019）、（0.553±0.013）、（0.428±0.002）（F=275.35，P=0.001）。结论内毒素耐受可以降低大鼠内毒素诱导的葡萄膜炎的炎症反应程度，下调虹膜睫状体组织中PI3K和AKT的表达，揭示内毒素耐受对EIU的保护机制可能与PI3K/AKT信号通路有关。（眼科， 2020, 29： 50-54）

关键词: , 内毒素诱导的葡萄膜炎；内毒素耐受；PI3K/AKT

Abstract: Objective To explore the effects of lipopoly saccharide (LPS) pretreatment on endotoxin-induced uveitis (EIU) and the change of PI3K/AKT pathway. Design Experimental study. Participants Male pathogen-free Wistar rats (8-10 weeks old, weighing 180-200 g). Method Wistar rats were randomly divided into normal control (NC) group, endotoxin tolerance (ET) group and EIU group, with 30 rats in each group. EIU was induced by a subcutaneous injection of 200 μg LPS. For the ET group, induction of EIU was preceded by daily intraperitoneal injection of 0.1 mg/kg LPS for five days. EIU and NC groups were injected with saline of the same volume for 5 consecutive days. On day 6, ET group and EIU group received subcutaneous injection of 200 g LPS at the same time, and NC group received injection of saline. Clinical scores were graded 24 h after EIU under a slit lamp microscope. HE stain was performed to observe the histopathology. Aqueous humor TNF-α concentration was quantified with enzyme-linked immunosorbent assay. The expressions of PI3K and AKT in iris were detected through Western blot analysis. Main Outcome Measures Inflammation score of the animal models, HE stain, aqueous humor TNF-α, PI3K/AKT protein expression. Results Clinical score in ET group, EIU group , NC group was 1.65±0.49, 6.65±0.59, 0.20±0.41, respectively （?字2= 53.261, P=0.001）. The pathological score of in ET group, EIU group, NC group was 0.6±0.5, 3.8±0.4, 0.2±0.4, respectively (?字2= 46.137, P=0.001）. The concentration of TNF-α in aqueous humor of in ET group, EIU group, NC group was 10.58±0.67(pg/ml), 30.96±1.55(pg/ml), 10.32±0.61(pg/ml), respectively (F=260.08, P=0.03). PI3K of Western blot in ET group, EIU group, NC group was 0.197±0.019, 0.539±0.017, 0.453±0.014, respectively（F=210.66, P=0.002）. AKT of Western blot in ET group, EIU group, NC group was 0.234±0.019, 0.553±0.013, 0.428±0.002, respectively（F=275.35, P=0.001）. Conclusions ET can reduce the inflammatory response of EIU in rats, and down-regulate the expression of PI3K and AKT in iris ciliary body tissue. This protective mechanism of ET against EIU may be associated with PI3K/AKT signaling pathway. (Ophthalmol CHN, 2020, 29: 50-54)

Key words: endotoxin-induced uveitis, endotoxin tolerance, PI3K/AKT

王婧张楠余烁刘新丽卢弘. 内毒素耐受对内毒素诱导的葡萄膜炎炎症程度及虹膜睫状体中PI3K/AKT 信号通路的影响[J]. 眼科, doi: 10. 13281/j. cnki. issn.1004-4469. 2020. 01. 010.

Wang Jing, Zhang Nan, Yu Shuo, Liu Xinli, Lu Hong. Effects of endotoxin tolerance on endotoxin-induced uveitis and on the PI3K/AKT signaling pathway in the iris ciliary body[J]. Ophthalmology in China, doi: 10. 13281/j. cnki. issn.1004-4469. 2020. 01. 010.

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内毒素耐受对内毒素诱导的葡萄膜炎炎症程度及虹膜睫状体中PI3K/AKT 信号通路的影响

Effects of endotoxin tolerance on endotoxin-induced uveitis and on the PI3K/AKT signaling pathway in the iris ciliary body

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