西格列汀对3T3-L1细胞NALP3炎症复合体的作用及机制研究

发布时间：2018-03-06 04:34

本文选题：NALP3炎症体　切入点：西格列汀　出处：《北京协和医学院》2015年博士论文　论文类型：学位论文

【摘要】：研究背景：2型糖尿病(type 2 diabetes mellitus, T2DM)严重威胁着人类健康,然而T2DM的发病机制尚未明确。研究表明,以NALP3炎症体为中心的慢性炎症反应参与2型糖尿病的发生。NALP3炎症体是核苷酸结构域样受体(NALPs)家族成员,是新近发现的活化caspase-1、调控IL-1β成熟和分泌的分子平台；IL-1β是目前已知促炎活性的细胞因子之一,文献提示其能够促进胰岛素抵抗的发展。目前以干预代谢性炎症通路、降低炎症因子水平为目标的抗炎治疗在T2DM的降糖治疗仍处于起步阶段。本实验组的早期观察研究发现,西格列汀可能对NALP3炎症体具有一定抑制作用。本研究旨在本实验组已有的研究基础上进一步探讨西格列汀对3T3-L1前脂肪细胞内NALP3炎症体的抑制作用,寻找西格列汀可能存在的药理新机制,为抗炎治疗在T2DM的降糖治疗提供理论依据。方法：将3T3-L1细胞进行常规培养,设定分组为空白对照组、西格列汀药物处理组,分别设定浓度梯度及时间梯度。待细胞密度90%后48h进行诱导分化,同时按分组设定予以各组药物作用。用酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)测定细胞培养基上清IL-1β、IL-18、IL-6、TNF-α的浓度。用western blot方法测定细胞中NALP3复合体各组分蛋白的表达情况。用real-time PCR测定细胞中NALP3复合体各组分NALP3. ASC. caspase-1 RNA表达情况。用Western Blot方法测定细胞中NF-κB及IκBα蛋白表达水平情况。此外将3T3-L1细胞进行常规培养,设定分组为空白对照组、lOnM西格列汀药物处理组,10nM西格列汀+10nM Exendin 9-39药物处理组,lOnM西格列汀+50nM Exendin 9-39药物处理组。待细胞密度90%后48h进行诱导分化,同时按分组设定予以各组药物作用。用酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)测定细胞培养基上清IL-lβ、IL-18、IL-6、TNF-α的浓度。结果：1.5-100nmol/L西格列汀能明显抑制3T3-L1细胞诱导分化期IL-1β、TNF-α、IL-18、IL-6分泌,呈剂量依赖关系。2. 110nmol/L西格列汀能明显抑制3T3-L1细胞诱导分化期IL-1β、TNF-α、 IL-18、IL-6分泌,呈时间依赖关系。3.5-100nmol/L西格列汀处理3T3-L1细胞诱导分化期一定时间后,细胞内NALP3炎症体ASC、Caspasel组分表达减少, pro-caspasel组分表达增加。4. 10nmol/L西格列汀处理3T3-L1细胞诱导分化期不同的时间均观察到细胞内NALP3炎症体ASC、Caspasel组分表达减少,pro-caspasel组分增加。5.5-100nmol/L西格列汀处理3T3-L1细胞诱导分化期一定时间后,细胞内NALP3炎症体ASC、Caspasel、NLRP3组分mRNA表达减少。6.50nM Exendin9-39能拮抗10nM西格列汀对3T3-L1细胞诱导分化期IL-1βTNF-α、IL-6、IL-18炎症因子的抑制效应；10nM Exendin9-39能部分拮抗lOnM西格列汀对3T3-L1细胞诱导分化期炎症因子的抑制效应。7. 10nmol/L西格列汀处理3T3-L1细胞诱导分化期2天后观察到细胞内NF-κB组分p65表达减少,IκBα蛋白表达增加。结论：西格列汀能够抑制3T3-L1细胞诱导分化期细胞炎症因子的表达,且呈现剂量依赖关系和时间依赖关系；同时西格列汀能够抑制3T3-L1细胞诱导分化期NALP3炎症体蛋白的表达,NALP3炎症体各组分mRNA合成减少。我们的实验提示西格列汀可能依赖GLP-1通路,通过抑制细胞内NF-κB通路抑制NALP3炎症体,从而抑制炎性介质IL-1β、IL-18、IL-6、TNF-α的分泌,这可能是西格列汀降糖作用的药理机制之一。
[Abstract]:Background: type 2 diabetes (type diabetes 2 mellitus, T2DM) is a serious threat to human health, but the pathogenesis of T2DM is not clear. The study shows that inflammation.NALP3 body chronic inflammatory reaction with NALP3 as the center of the body inflammation in type 2 diabetes is nucleotide domain like receptor (NALPs) family members, is the activation of Caspase-1 found recently, molecular platform IL-1 beta maturation and secretion of cytokines; IL-1 beta is one of the currently known proinflammatory activity, suggesting that the literature can promote the development of insulin resistance. The intervention of metabolic pathway, decrease the level of inflammatory factors as the target for anti-inflammatory treatment in the hypoglycemic treatment of T2DM is still in the initial study on the early stage. The experimental group found that sitagliptin may have certain inhibitory effect on inflammation. NALP3 basic research progress of this study is to the existing experimental group A further study on Sig Leo Dean inhibitory effect on fat cells NALP3 inflammatory 3T3-L1 precursors, find new pharmacological mechanism of sitagliptin may exist, and provide a theoretical basis for the treatment of T2DM in hypoglycemic anti-inflammatory treatment. Methods: 3T3-L1 cells were cultured, set divided into blank control group, sitagliptin treatment group. Set concentration and time gradient respectively. The cell density of 90% 48h after induction of differentiation, at the same time by grouping to set each drug. Using enzyme-linked immunosorbent assay (enzyme-linked immunosorbent, assay, ELISA) measured cell culture supernatant of IL-1 beta, IL-18, IL-6, TNF- concentration of alpha. Determination of expression of NALP3 complex group protein in cells by Western blot method. Determination of cell NALP3 complex components of NALP3. ASC. caspase-1 RNA real-time PCR expression by Western was determined by Blot method. The expression of NF- K B and I kappa B alpha protein levels in cells. 3T3-L1 cells were cultured in addition, set the packet into the blank control group, lOnM sitagliptin treated group, 10nM sitagliptin +10nM Exendin 9-39 treatment group, lOnM +50nM Exendin 9-39 sitagliptin treatment group. The cell density 90% 48h after induction of differentiation, at the same time by grouping to set each drug. Using enzyme-linked immunosorbent assay (enzyme-linked immunosorbent, assay, ELISA) by cell culture supernatant of IL-l beta, IL-18, IL-6, TNF- alpha concentration. Results: 1.5-100nmol/L sitagliptin could obviously inhibit the differentiation of 3T3-L1 cells of IL-1 beta, TNF- alpha, IL-18, IL-6 secretion in a dose-dependent manner.2. 110nmol/L sitagliptin could obviously inhibit the differentiation of 3T3-L1 cells of IL-1 beta, TNF- alpha, IL-18, IL-6 secretion in a time dependent manner..3.5-100nmol/L sitagliptin at 3T3-L1 cell differentiation period after a certain period of time, the intracellular NALP3 inflammation ASC, Caspasel component decreased expression of pro-caspasel group increased.4. expression 10nmol/L sitagliptin treatment induced the differentiation of 3T3-L1 cells in different time period were observed in cells of NALP3 inflammation ASC, Caspasel component decreased expression of pro-caspasel group increased.5.5-100nmol/L sitagliptin treatment induced 3T3-L1 cell differentiation after a certain period of time, the intracellular NALP3 ASC Caspasel NLRP3, inflammation, component mRNA decreased expression of.6.50nM Exendin9-39 can antagonize 10nM sitagliptin on 3T3-L1 cell differentiation induced by IL-1 alpha beta TNF-, IL-6, IL-18 inhibitory effect on inflammatory factors; 10nM Exendin9-39 can antagonize the lOnM part of the West Glenn Ting differentiation of 3T3-L1 cells induced by inflammatory cytokines.7. 10nmol/L inhibitory effect of sitagliptin treatment induced 3T3-L1 cell differentiation stage was observed after 2 days of cell NF- K The B component p65 decreased expression of I kappa B alpha protein expression. Conclusion: the expression of Sig Leo Dean could inhibit 3T3-L1 cells induced by inflammatory cytokine differentiation stage, and showed a dose dependence and time dependence; at the same time, the expression of sitagliptin can inhibit the differentiation of 3T3-L1 cells NALP3 protein NALP3 inflammation, inflammation of body components the reduction of mRNA synthesis. Our experiments suggest that sitagliptin may depend on the GLP-1 pathway by inhibiting intracellular NF- B pathway inhibits NALP3 inflammation, thereby inhibiting inflammatory mediators IL-1 beta, IL-18, IL-6, TNF- secretion, which may be one of the pharmacological mechanism of hypoglycemic effect of sitagliptin.

【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】：R587.1

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