胡椒碱通过调控mTORC1信号增强巨噬细胞固有免疫功能的机制研究

发布时间：2018-01-26 03:21

本文关键词： 胡椒碱腹腔巨噬细胞(pMΦs) 哺乳动物雷帕霉素靶蛋白(mTOR) 白细胞介素(IL)-6 肿瘤坏死因子(TNF)-α　出处：《暨南大学》2017年博士论文　论文类型：学位论文

【摘要】：目的胡椒碱是从胡椒中分离获得的一种生物碱,具有抗肠胃炎、抗抑郁、抗癫痫和神经保护等多种药理学效应,但其确切的作用机制尚不清楚。本论文重点研究胡椒碱对巨噬细胞(特别是腹腔定居的巨噬细胞)功能的影响及机制。巨噬细胞是重要的固有免疫细胞,与许多慢性疾病包括动脉粥样硬化、哮喘、炎症性肠病(IBD)、类风湿性关节炎和纤维化等密切相关。巨噬细胞内富含溶酶体及线粒体,具有吞噬杀菌、清除凋亡细胞碎片的能力,并在组织修复中起着重要作用。巨噬细胞的功能受到多种信号通路的精细调控以维持其自稳态。其中mTOR不仅是细胞代谢的调控枢纽,而且能够影响固有免疫细胞(包括单核细胞/巨噬细胞)的免疫应答,调控炎症相关信号通路及细胞因子表达,还能影响巨噬细胞的代谢、活化和分化过程。mTOR与其他蛋白结合所构成的两个复合物——mTORC1和mTORC2,拥有不同的调节活性。其中mTORC1能够被胰岛素信号和氨基酸(如亮氨酸和谷氨酰胺)代谢所激活。研究表明,肠道黏膜固有免疫稳态的维持,依赖于肠上皮细胞对氨基酸的吸收和代谢功能,后者通过激活mTORC1调控抗菌肽的表达。有意思的是,早期的研究表明胡椒碱能促进肠上皮细胞对亮氨酸等氨基酸的吸收,提示胡椒碱可能通过增强氨基酸代谢,上调mTORC1信号,从而影响固有免疫细胞(包括巨噬细胞)的功能。为此,本论文首先利用mTOR抑制剂INK128,研究mTOR信号对巨噬细胞活化和功能的影响,同时探讨胡椒碱是否通过干预mTOR信号而调节腹腔定居巨噬细胞(p MΦs)的固有免疫功能,从而阐明胡椒碱影响巨噬细胞功能的机制。该机制的阐明将为胡椒碱在炎症性疾病等方面的临床应用以及相关新药的开发提供实验依据。方法在细胞水平上,通过WST-1试剂检测mTOR抑制剂INK128对小鼠巨噬细胞系RAW 264.7的细胞毒性,免疫印迹法检测细胞mTOR和NF-κB通路相关蛋白的变化,通过流式微球阵列(CBA)试剂盒检测细胞培养上清中IL-6、TNF-α等细胞因子的含量,以及通过实时定量PCR(q PCR)技术检测IL-6、TNF-α的mRNA表达水平。同时,以血清饥饿的He La和p MΦs细胞为体外模型,研究胡椒碱对亮氨酸、谷氨酰胺诱导的mTORC1活化的影响,分别通过免疫印迹法检测mTORC1底物p70S6K的磷酸化,采用细胞免疫荧光显微术观察细胞内mTOR与溶酶体蛋白LAMP2的共定位(mTORC1的激活在溶酶体表面进行)、以及细胞内p70S6K底物p-S6的表达;胡椒碱对氨基酸转运蛋白SLC7A5/SLC3A2在细胞内分布的影响也采用免疫荧光显微术进行观察,并采用小干扰RNA(si RNA)沉默SLC7A5基因后以免疫印迹法检测p70S6K磷酸化水平。此外,采用CBA试剂盒检测胡椒碱对p MΦs细胞分泌炎症因子(包括IL-6、TNF-α)的影响,并用PI3K/AKT/mTOR通路抑制剂干扰细胞因子的表达。在小鼠败血症模型中,研究胡椒碱对腹腔注射大肠杆菌(E.coli)小鼠存活的影响,以及采用苏木精-伊红(HE)染色观察肝脏和结肠组织变化,CBA试剂盒检测腹腔灌洗液中细胞因子的表达,LB琼脂板计数灌洗液中菌落形成单位(CFU),流式细胞仪检测腹腔巨噬细胞对荧光标记的细菌的吞噬作用和细胞内Caspase-3的活化情况。结果当mTOR的活性被INK128抑制时,其底物p70S6K、4E-BP1和AKTSer473的磷酸化水平下调,LPS刺激巨噬细胞RAW 264.7分泌IL-6、TNF-α的能力也被抑制,表明mTOR能够调控巨噬细胞中炎症因子的表达。胡椒碱上调细胞内mTORC1底物p70S6K和4E-BP1的磷酸化水平,表明胡椒碱增强mTORC1的活性,这种增强效应依赖于亮氨酸和谷氨酰胺的存在。胡椒碱能促进细胞内氨基酸转运蛋白SLC7A5/SLC3A2募集到细胞膜上;敲低SLC7A5的表达能明显降低胡椒碱、亮氨酸和谷氨酰胺激活的p70S6K磷酸化水平。胡椒碱增强亮氨酸和谷氨酰胺诱导细胞内mTOR和LAMP2的共聚集(即mTORC1活化),而该效应也能被氨基酸转运蛋白抑制剂BCH所拮抗。这些结果表明,胡椒碱能增强氨基酸代谢所诱导的mTORC1信号。胡椒碱促进LPS刺激的p MΦs分泌IL-6、TNF-α,该促进作用可被PI3K/AKT/mTORC1通路的抑制剂所拮抗。胡椒碱灌胃能提高E.coli感染小鼠的存活率,减少细菌引起的肝脏和结肠炎症性损伤。与溶剂组相比,在E.coli感染小鼠6 h时胡椒碱增强腹腔内IL-6、TNF-α的表达,而感染24 h时腹腔内IL-6、TNF-α的表达和细菌载量明显下降。胡椒碱能阻止细菌引起的F4/80high p MΦs和F4/80highCFSE+(E.coli)p MΦs比率减少,促进对细菌的吞噬作用,抑制细菌引起的CD11bhigh/GATA6bright p MΦs的减少,并抑制CD11b+p MΦs和F4/80+p MΦs细胞内cleaved Caspase-3的表达,而mTORC1的间接抑制剂二甲双胍能明显拮抗胡椒碱的上述作用。这些结果表明,胡椒碱通过调控mTORC1信号增强巨噬细胞功能,从而发挥抗感染作用。结论胡椒碱可能通过上调氨基酸转运蛋白SLC7A5/SLC3A2在细胞膜上的分布,促进亮氨酸和谷氨酰胺的吸收和代谢,从而上调mTORC1的活化,增强腹腔定居巨噬细胞在感染初期分泌炎症因子IL-6和TNF-α,抑制腹腔定居巨噬细胞死亡,增强其吞噬功能,从而提高小鼠抵抗细菌感染和败血症组织损伤的能力。总之,胡椒碱通过促进氨基酸代谢依赖性的mTORC1活性而增强巨噬细胞的固有免疫功能,发挥其免疫调节和抗感染的药理效应。
[Abstract]:The purpose of piperine is an alkaloid obtained from the separation of pepper, with anti gastroenteritis, antidepressant, multiple antiepileptic and neuroprotective pharmacological effects, but the exact mechanism is not clear. This paper focuses on the study of piperine on macrophages (especially in peritoneal macrophages) function effect and mechanism. Macrophages are important innate immune cells, and many chronic diseases including atherosclerosis, asthma, inflammatory bowel disease (IBD), rheumatoid arthritis and fibrosis are closely related. Rich in lysosomes and mitochondria in macrophages, phagocytosis ability is, the clearance of apoptotic cell fragments, and in tissue repair plays an important role in macrophages. By regulation of multiple signaling pathways to maintain its homeostasis. The mTOR is not only the hub regulation of cell metabolism, and can affect the innate immune Cells (including monocytes / macrophages) immune response, regulate the expression of inflammation related signaling pathways and cytokines can affect the metabolism of macrophages, activation and differentiation of.MTOR and other proteins with two compound formed by mTORC1 and mTORC2, have different regulatory activity. The mTORC1 can be insulin signaling and amino acid (such as leucine and glutamine metabolism activation). The study shows that the maintenance of intestinal mucosal innate immune homeostasis, dependent on intestinal epithelial cell absorption and metabolism of amino acids, the latter through activation of mTORC1 expression regulation of antimicrobial peptides. Interestingly, early studies showed that piperine can promote the absorption of intestinal epithelial cells of leucine other amino acids, suggesting that piperine may enhance the metabolism of amino acids, up regulation of mTORC1 signal, thus affecting the innate immune cells (macrophages). Can. For this reason, this paper first use of mTOR inhibitor INK128, the effects of mTOR signal on macrophage activation and function, and explore whether piperine through the intervention of mTOR signal regulated peritoneal resident macrophages (P M s) of the innate immune function, so as to clarify the function mechanism of piperine in macrophages. The influence mechanism of development for the clinical application of piperine in inflammatory diseases and the related drug and provide experimental basis. At the cellular level, by WST-1 reagent for detection of mTOR inhibitor INK128 on the cytotoxicity of murine macrophage cell line RAW 264.7, mTOR and NF- cells to detect the changes of B pathway related protein by Western blotting, by flow cytometric bead array (CBA) kit for detection of IL-6 in the supernatant of cell culture, the content of TNF- alpha and other cytokines, and by quantitative real-time PCR (Q PCR) technology to detect IL-6, TNF- alpha mRNA The level of serum starvation. At the same time, with He La and P M with s cells as in vitro models of piperine on leucine, effect of glutamine induced mTORC1 activation, were detected by mTORC1 blotting substrate p70S6K phosphorylation, CO localization by immunofluorescence microscopy of intracellular mTOR and lysosome protein LAMP2 (the activation of mTORC1 in lysosomes, and the expression of cell surface) p70S6K substrate p-S6; effect of piperine on amino acid transporter SLC7A5/SLC3A2 in the intracellular distribution by immunofluorescence microscopy were observed, and using small interfering RNA (Si RNA) after silencing the SLC7A5 gene to detect the phosphorylation of p70S6K by Western blot. In addition, using piperine CBA kit to detect the secretion of inflammatory cytokines of P M with s cells (including IL-6, TNF- alpha) effect, and PI3K/AKT/mTOR pathway inhibitors interfere with cytokine table As in a mouse model of sepsis, Bacillus of piperine on intraperitoneal injection of Escherichia coli (E.coli) affect the survival of mice, as well as by hematoxylin eosin (HE) staining was used to observe the changes of liver and colon tissue, the expression of cytokines in peritoneal lavage fluid CBA detection kit, LB agar plate count by colony formation unit lotion in (CFU), activation of Caspase-3 cells and phagocytosis of peritoneal macrophages by flow cytometry for fluorescence labeling. The results when mTOR activity was inhibited by INK128, its substrate p70S6K, phosphorylation of 4E-BP1 and downregulation of AKTSer473, LPS stimulated the secretion of RAW 264.7 IL-6, TNF- alpha also is suppressed, show that mTOR can control the expression of inflammatory cytokines in macrophages. MTORC1 substrate p70S6K and 4E-BP1 piperine increased the intracellular phosphorylation of piperine showed enhanced mTORC1 activity, this enhanced The effect depends on the presence of leucine and glutamine. Piperine can promote the amino acid transporter SLC7A5/SLC3A2 cells recruited to the cell membrane; knockdown SLC7A5 protein can significantly reduce the piperine, phosphorylation of p70S6K leucine and glutamine. Activation of piperine enhanced copolymerization of leucine and glutamine induced intracellular mTOR and LAMP2 (set mTORC1), and the effect of activation can be antagonized by amino acid transporter inhibitor BCH. These results indicate that piperine can enhance mTORC1 signal induced by the metabolism of amino acids. Piperine enhanced LPS stimulated secretion of IL-6 P M with s, TNF- alpha, the role of PI3K/AKT/mTORC1 pathway inhibitor can be antagonized by pepper. Alkali gavage can improve the survival rate of E.coli infected mice, reduce bacterial and liver inflammatory injury. Compared with the solvent group, in mice infected with E.coli 6 h piperine Enhanced abdominal IL-6, expression of TNF-, and 24 h intraperitoneal infection of IL-6, expression of TNF- alpha and bacterial load decreased. Piperine can prevent bacteria induced by F4/80high P M with s and F4/80highCFSE+ (E.coli) P M s diameter decreased. The phagocytosis promoting to bacteria, reduce bacteria the CD11bhigh/GATA6bright P M s in diameter, and inhibit the expression of CD11b+p s and F4/80+p M M Phi Phi s cleaved in cells Caspase-3, and indirect inhibitor mTORC1 can the effect of metformin significantly antagonize piperine. These results show that piperine enhanced macrophage function by regulating mTORC1 signal, which play a role of anti infection. Conclusion pepper alkali may regulate amino acid transporter SLC7A5/SLC3A2 on cell membrane distribution, promote the absorption and metabolism of leucine and glutamine, activation and upregulation of mTORC1, enhanced peritoneal resident macrophages The secretion of inflammatory cytokines IL-6 and TNF- alpha in the early stage of infection, inhibition of peritoneal resident macrophages death, enhances phagocytic function, so as to improve the ability to resist bacterial infection and sepsis tissue injury in mice. In short, piperine and enhance the innate immune function of macrophages by promoting the metabolism of amino acid dependent mTORC1 activity and pharmacological effects of its immunomodulatory and anti infection.

【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285

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