消退素D1对脂多糖诱导小鼠急性角膜炎症反应的调节作用及机制
发布时间：2019-03-20 19:27
【摘要】：目的探讨脂多糖(lipopolysaccharide,LPS)诱导小鼠急性角膜炎的临床表现和炎症因子的表达水平变化：研究消退素D1 (RvD1)在LPS诱导小鼠急性角膜炎症中发挥的作用,对细胞因子及趋化因子的影响,探索消退素D1促进炎症消退的分子机制,初步了解其可能参与的信号通路。方法本实验包括两个部分,第一部分通过建立LPS诱导小鼠急性角膜炎模型,观察急性期的临床表现,通过病理学及免疫组织化学的方法研究其炎症特点,然后使用实时荧光定量PCR方法检测LPS处理前后角膜组织内IL-1β、IL-6、MCP-1及MIP-2四种细胞因子及趋化因子的表达。第二部分使用不同浓度RvD1(50ng/d和500ng/d)对急性期角膜组织进行局部干预,观察1、3、5天时RvD1对角膜混浊程度及上皮愈合速度的影响；比较角膜组织炎症反应程度；应用荧光定量PCR及ELISA方法检测IL-1β、IL-6、MCP-1及MIP-2四种炎症因子在角膜组织中mRNA及蛋白水平上的表达差异；并通过western blot方法检测NF-κB信号途径中内源性NF-κB抑制因子IκBα在角膜组织中表达水平的变化,从而探索RvD1可能参与炎症反应的信号通路。结果1、LPS刺激后小鼠角膜混浊,HE染色及免疫组化显示角膜基质内中性粒细胞浸润,1-3天达高峰,3-5天逐渐减轻；2、LPS刺激后角膜基质细胞合成的细胞因子IL-1β和IL-6,趋化因子MCP-1和MIP-2的mRNA表达水平较正常小鼠明显增加(P0.05)；3、通过RvD1-50组(低剂量50ng/d)、RvD1-500(高剂量500ng/d)和对照组(PBS)进行比较,发现RvD1可明显减轻角膜炎症后混浊程度,加快上皮愈合速度;HE染色及免疫组化显示RvD1可减少中性粒细胞数量,防止中性粒细胞持续浸润；4、荧光实时定量PCR和ELISA检测发现RvD1可降低急性期角膜组织IL-1p、IL-6、MCP-1和MIP-2的在mRNA水平和蛋白水平的表达,且和剂量呈正相关；5、western blot显示在1天和3天时RvD1-500ng/d与对照组相比可增加LPS诱导后NF-κB抑制因子IKBα的表达,而在5d时差异不明显。结论LPS刺激后小鼠角膜组织内细胞因子IL-1p和IL-6,趋化因子MCP-1和MIP-2的mRNA表达水平较正常小鼠明显增加,提示IL-1β、IL-6、MCP-1和MIP-2可能参与了角膜炎症反应。RvD1减轻角膜炎症后混浊程度,促进角膜上皮愈合；减少中性粒细胞持续浸润；RvD1可降低角膜组织IL-1β、IL-6、MCP-1和MIP-2的表达,炎症早期可部分逆转LPS诱导后抑制因子IKBα的表达下降,提示RvDl可能是通过抑制NF-κB信号传导通路起作用,促进LPS诱导的小鼠角膜炎症的消退。
[Abstract]:Objective to investigate the clinical manifestation and expression of inflammatory factors in acute keratitis induced by lipopolysaccharide (lipopolysaccharide,LPS) in mice. To study the role of RvD1 in acute keratitis induced by LPS in mice. The effects on cytokines and chemokine were studied to explore the molecular mechanism of anti-retrogression D1 in promoting inflammatory regression and to understand the signal pathway in which it might be involved. Methods this experiment consists of two parts. In the first part, the acute keratitis model of mice induced by LPS was established, the clinical manifestation of acute phase was observed, and the inflammatory characteristics were studied by means of pathology and immunohistochemistry. Then the expression of IL-1 尾, IL-6,MCP-1 and MIP- 2 cytokines and chemokines in corneal tissues before and after LPS treatment were detected by real-time fluorescence quantitative PCR. In the second part, different concentrations of RvD1 (50ng/d and 500ng/d) were used for local intervention in acute corneal tissue. The effects of RvD1 on corneal opacity and epithelial healing rate were observed at 1, 3, 5 days, and the degree of corneal inflammation was compared. The expression of IL-1 尾, IL-6,MCP-1 and MIP- 2 in corneal tissue was detected by fluorescence quantitative PCR and ELISA. Western blot was used to detect the expression of endogenous NF- 魏 B inhibitor I 魏 B 伪 in corneal tissues in the NF- 魏 B signaling pathway, so as to explore the possible involvement of RvD1 in the signal pathway of inflammatory reaction. Results 1LPs-stimulated corneal opacity, HE staining and immunohistochemistry showed that neutrophil infiltration in corneal stroma reached its peak at 3 days and decreased gradually at 3 days. 2. The mRNA expression levels of IL-1 尾 and IL-6, chemokine MCP-1 and MIP-2 in corneal stromal cells stimulated by LPS were significantly higher than those in normal mice (P0.05). (3) by comparing RvD1-50 group (low dose 50ng/d), RvD1-500 (high dose 500ng/d) and control group (PBS), it was found that RvD1 could significantly reduce the degree of opacity after corneal inflammation and accelerate the healing rate of epithelium. HE staining and immunohistochemistry showed that RvD1 could decrease the number of neutrophils and prevent the persistent infiltration of neutrophils. 4. Real-time fluorescence quantitative PCR and ELISA showed that RvD1 could decrease the expression of IL-1p,IL-6,MCP-1 and MIP-2 at mRNA level and protein level in acute corneal tissue, and it was positively correlated with the dose. 5. Western blot showed that RvD1-500ng/d increased the expression of NF- 魏 B inhibitor IKB 伪 on day 1 and day 3 compared with the control group, but there was no significant difference on day 5. Conclusion the mRNA expression levels of cytokines IL-1p, IL-6, chemokine MCP-1 and MIP-2 in corneal tissue of LPS-stimulated mice were significantly higher than those of normal mice, suggesting that IL-1 尾 and IL-6, were significantly increased. MCP-1 and MIP-2 may be involved in corneal inflammation. RvD1 may reduce the degree of opacity after corneal inflammation and promote corneal epithelial healing. Decrease the persistent neutrophil infiltration; RvD1 decreased the expression of IL-1 尾, IL-6,MCP-1 and MIP-2 in corneal tissue, and partially reversed the decreased expression of inhibitor IKB 伪 after LPS induction in the early stage of inflammation, suggesting that RvDl may play a role in inhibiting the signal transduction pathway of NF- 魏 B. To promote the regression of LPS-induced corneal inflammation in mice.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R772.21
本文编号：2444556
[Abstract]:Objective to investigate the clinical manifestation and expression of inflammatory factors in acute keratitis induced by lipopolysaccharide (lipopolysaccharide,LPS) in mice. To study the role of RvD1 in acute keratitis induced by LPS in mice. The effects on cytokines and chemokine were studied to explore the molecular mechanism of anti-retrogression D1 in promoting inflammatory regression and to understand the signal pathway in which it might be involved. Methods this experiment consists of two parts. In the first part, the acute keratitis model of mice induced by LPS was established, the clinical manifestation of acute phase was observed, and the inflammatory characteristics were studied by means of pathology and immunohistochemistry. Then the expression of IL-1 尾, IL-6,MCP-1 and MIP- 2 cytokines and chemokines in corneal tissues before and after LPS treatment were detected by real-time fluorescence quantitative PCR. In the second part, different concentrations of RvD1 (50ng/d and 500ng/d) were used for local intervention in acute corneal tissue. The effects of RvD1 on corneal opacity and epithelial healing rate were observed at 1, 3, 5 days, and the degree of corneal inflammation was compared. The expression of IL-1 尾, IL-6,MCP-1 and MIP- 2 in corneal tissue was detected by fluorescence quantitative PCR and ELISA. Western blot was used to detect the expression of endogenous NF- 魏 B inhibitor I 魏 B 伪 in corneal tissues in the NF- 魏 B signaling pathway, so as to explore the possible involvement of RvD1 in the signal pathway of inflammatory reaction. Results 1LPs-stimulated corneal opacity, HE staining and immunohistochemistry showed that neutrophil infiltration in corneal stroma reached its peak at 3 days and decreased gradually at 3 days. 2. The mRNA expression levels of IL-1 尾 and IL-6, chemokine MCP-1 and MIP-2 in corneal stromal cells stimulated by LPS were significantly higher than those in normal mice (P0.05). (3) by comparing RvD1-50 group (low dose 50ng/d), RvD1-500 (high dose 500ng/d) and control group (PBS), it was found that RvD1 could significantly reduce the degree of opacity after corneal inflammation and accelerate the healing rate of epithelium. HE staining and immunohistochemistry showed that RvD1 could decrease the number of neutrophils and prevent the persistent infiltration of neutrophils. 4. Real-time fluorescence quantitative PCR and ELISA showed that RvD1 could decrease the expression of IL-1p,IL-6,MCP-1 and MIP-2 at mRNA level and protein level in acute corneal tissue, and it was positively correlated with the dose. 5. Western blot showed that RvD1-500ng/d increased the expression of NF- 魏 B inhibitor IKB 伪 on day 1 and day 3 compared with the control group, but there was no significant difference on day 5. Conclusion the mRNA expression levels of cytokines IL-1p, IL-6, chemokine MCP-1 and MIP-2 in corneal tissue of LPS-stimulated mice were significantly higher than those of normal mice, suggesting that IL-1 尾 and IL-6, were significantly increased. MCP-1 and MIP-2 may be involved in corneal inflammation. RvD1 may reduce the degree of opacity after corneal inflammation and promote corneal epithelial healing. Decrease the persistent neutrophil infiltration; RvD1 decreased the expression of IL-1 尾, IL-6,MCP-1 and MIP-2 in corneal tissue, and partially reversed the decreased expression of inhibitor IKB 伪 after LPS induction in the early stage of inflammation, suggesting that RvDl may play a role in inhibiting the signal transduction pathway of NF- 魏 B. To promote the regression of LPS-induced corneal inflammation in mice.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R772.21
【参考文献】
相关期刊论文 前1条
1 ;NF-κB and Its Regulation on the Immune System[J];Cellular & Molecular Immunology;2004年05期
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