GLP-2对LPS活化巨噬细胞的调节作用及其机制
发布时间：2018-11-10 14:51
【摘要】：胰高血糖素样肽-2（glucagon-like peptide2，GLP-2）是由肠道L内分泌细胞分泌的由33个氨基酸组成的单链多肽，主要通过GLP-2R发挥生物学作用。GLP-2能增加肠道血流量，促进小肠生长、营养物质吸收，抑制胃肠动力和胃酸分泌，降低肠道通透性等作用。在非甾体类抗炎药物诱导的小鼠肠炎模型中，GLP-2具有降低肠炎小鼠的死亡率，降低炎性因子表达等作用。 巨噬细胞是先天免疫的重要组成细胞，，通过表面模式识别受体识别结合、进而吞噬和清除入侵的病原体或凋亡细胞。当炎症发生时，巨噬细胞分泌不同功能的细胞因子参与炎症反应。然后，过度释放促炎细胞因子会导致多种疾病的发生，如感染性休克、风湿性关节炎和其他慢性炎症性疾病。LPS为革兰氏阴性细菌细胞壁的主要成分，与LBP、CD14结合形成LPS-LBP-CD14三联复合物作用于巨噬细胞表面的TLR4，从而活化MAPKs和NF-κB两条信号通路，诱导相关靶基因的转录，释放IL-1β、TNF-α、IL-6等促炎细胞因子。 研究报道中指出GLP-2具有减轻炎症的作用，而巨噬细胞在炎症反应中扮演着重要的角色，本研究探讨了外源性GLP-2对LPS活化的小鼠腹腔巨噬细胞的调节作用及其调节机制。本实验首先通过腹腔注射巯基乙酸盐肉汤的方法，分离培养BALB/c小鼠腹腔巨噬细胞；再通过qRT-PCR、Western blot和ELISA方法检测GLP-2对LPS活化的巨噬细胞中促炎酶（iNOS和COX-2）和促炎细胞因子（IL-1β、TNF-α和IL-6）mRNA和蛋白表达水平的影响；然后，进一步通过Western blot方法检测了GLP-2对LPS活化的巨噬细胞中相关信号通路蛋白ERK、p38MAPK、JNK和IκB-α的磷酸化水平和NF-κB核转位的影响，从而研究GLP-2对LPS活化巨噬细胞的调节作用及其机制。 本研究结果显示：1）GLP-2（10-9、10-8、10-7、10-6M）能降低LPS活化的巨噬细胞中iNOS、COX-2、IL-1β、TNF-α和IL-6mRNA表达水平，以及细胞中iNOS、 COX-2蛋白表达水平和细胞培养上清中TNF-α和IL-6的蛋白表达水平，GLP-2浓度为10-6M时，抑制作用最显著。表明所选浓度的GLP-2可以有效降低由LPS介导活化的腹腔巨噬细胞促炎酶和促炎细胞因子的表达，具有明显减轻炎症的作用。2）Western blot结果显示GLP-2能够显著抑制LPS介导活化巨噬细胞中ERK和IκB-α的磷酸化，抑制NF-κB的核转位。本研究表明，GLP-2通过抑制LPS活化巨噬细胞MAPKs信号通路中ERK的磷酸化和抑制NF-κB信号通路，从而抑制促炎酶和促炎细胞因子的表达。
[Abstract]:Glucagon like peptide-2 (glucagon-like peptide2,GLP-2) is a 33 amino acid single chain polypeptide secreted by intestinal L endocrine cells, which plays a biological role mainly through GLP-2R. GLP-2 can increase intestinal blood flow. Promoting intestinal growth, absorption of nutrients, inhibition of gastrointestinal motility and gastric acid secretion, reducing intestinal permeability and so on. In the mouse model of enteritis induced by non-steroidal anti-inflammatory drugs, GLP-2 can reduce the mortality and the expression of inflammatory factors in mice with enteritis. Macrophages are important components of innate immunity. They are recognized by surface pattern recognition receptors and then phagocytosis and purging of invading pathogens or apoptotic cells. When inflammation occurs, macrophages secrete cytokines of different functions to participate in the inflammatory response. However, excessive release of pro-inflammatory cytokines can lead to the development of many diseases, such as septic shock, rheumatoid arthritis, and other chronic inflammatory diseases. LPS is a major component of the cell wall of Gram-negative bacteria and is associated with LBP,. CD14 binds to TLR4, on macrophage surface, which activates MAPKs and NF- 魏 B signaling pathway, induces transcription of related target genes, and releases IL-1 尾, TNF- 伪, IL-6 and other pro-inflammatory cytokines. It is pointed out that GLP-2 can attenuate inflammation and macrophages play an important role in inflammatory response. This study investigated the regulatory effect of exogenous GLP-2 on peritoneal macrophages activated by LPS and its mechanism. In this experiment, the peritoneal macrophages of BALB/c mice were isolated and cultured by intraperitoneal injection of mercaptoacetate broth. The effects of GLP-2 on the expression of iNOS and COX-2, IL-1 尾, TNF- 伪 and IL-6 mRNA and protein in LPS activated macrophages were detected by qRT-PCR,Western blot and ELISA. Then, the effects of GLP-2 on the phosphorylation of ERK,p38MAPK,JNK and I 魏 B- 伪 in LPS activated macrophages and the nuclear translocation of NF- 魏 B were detected by Western blot assay. To study the regulatory effect of GLP-2 on LPS activated macrophages and its mechanism. The results showed that: 1) GLP-2 (10-9 ~ (-8) -10 ~ (-7) ~ (-6) M) could reduce the expression levels of iNOS,COX-2,IL-1 尾, TNF- 伪 and IL-6mRNA in LPS activated macrophages and iNOS, in cells. The expression level of COX-2 protein and the expression of TNF- 伪 and IL-6 in the supernatant of cell culture were the most significant when the concentration of GLP-2 was 10-6 M. The results showed that the concentration of GLP-2 could effectively reduce the expression of pro-inflammatory enzymes and pro-inflammatory cytokines in peritoneal macrophages mediated by LPS. 2) Western blot results showed that GLP-2 could significantly inhibit the phosphorylation of ERK and I 魏 B- 伪 mediated by LPS and inhibit the nuclear translocation of NF- 魏 B in macrophages. This study suggests that GLP-2 inhibits the expression of proinflammatory enzymes and pro-inflammatory cytokines by inhibiting the phosphorylation of ERK and NF- 魏 B signaling pathway activated by LPS in macrophages MAPKs signaling pathway.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.3
本文编号：2322761
[Abstract]:Glucagon like peptide-2 (glucagon-like peptide2,GLP-2) is a 33 amino acid single chain polypeptide secreted by intestinal L endocrine cells, which plays a biological role mainly through GLP-2R. GLP-2 can increase intestinal blood flow. Promoting intestinal growth, absorption of nutrients, inhibition of gastrointestinal motility and gastric acid secretion, reducing intestinal permeability and so on. In the mouse model of enteritis induced by non-steroidal anti-inflammatory drugs, GLP-2 can reduce the mortality and the expression of inflammatory factors in mice with enteritis. Macrophages are important components of innate immunity. They are recognized by surface pattern recognition receptors and then phagocytosis and purging of invading pathogens or apoptotic cells. When inflammation occurs, macrophages secrete cytokines of different functions to participate in the inflammatory response. However, excessive release of pro-inflammatory cytokines can lead to the development of many diseases, such as septic shock, rheumatoid arthritis, and other chronic inflammatory diseases. LPS is a major component of the cell wall of Gram-negative bacteria and is associated with LBP,. CD14 binds to TLR4, on macrophage surface, which activates MAPKs and NF- 魏 B signaling pathway, induces transcription of related target genes, and releases IL-1 尾, TNF- 伪, IL-6 and other pro-inflammatory cytokines. It is pointed out that GLP-2 can attenuate inflammation and macrophages play an important role in inflammatory response. This study investigated the regulatory effect of exogenous GLP-2 on peritoneal macrophages activated by LPS and its mechanism. In this experiment, the peritoneal macrophages of BALB/c mice were isolated and cultured by intraperitoneal injection of mercaptoacetate broth. The effects of GLP-2 on the expression of iNOS and COX-2, IL-1 尾, TNF- 伪 and IL-6 mRNA and protein in LPS activated macrophages were detected by qRT-PCR,Western blot and ELISA. Then, the effects of GLP-2 on the phosphorylation of ERK,p38MAPK,JNK and I 魏 B- 伪 in LPS activated macrophages and the nuclear translocation of NF- 魏 B were detected by Western blot assay. To study the regulatory effect of GLP-2 on LPS activated macrophages and its mechanism. The results showed that: 1) GLP-2 (10-9 ~ (-8) -10 ~ (-7) ~ (-6) M) could reduce the expression levels of iNOS,COX-2,IL-1 尾, TNF- 伪 and IL-6mRNA in LPS activated macrophages and iNOS, in cells. The expression level of COX-2 protein and the expression of TNF- 伪 and IL-6 in the supernatant of cell culture were the most significant when the concentration of GLP-2 was 10-6 M. The results showed that the concentration of GLP-2 could effectively reduce the expression of pro-inflammatory enzymes and pro-inflammatory cytokines in peritoneal macrophages mediated by LPS. 2) Western blot results showed that GLP-2 could significantly inhibit the phosphorylation of ERK and I 魏 B- 伪 mediated by LPS and inhibit the nuclear translocation of NF- 魏 B in macrophages. This study suggests that GLP-2 inhibits the expression of proinflammatory enzymes and pro-inflammatory cytokines by inhibiting the phosphorylation of ERK and NF- 魏 B signaling pathway activated by LPS in macrophages MAPKs signaling pathway.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.3
【参考文献】
相关期刊论文 前2条
1 王强;一氧化氮在炎症反应中的作用[J];医学综述;2002年04期
2 赵阳;赵勇;;单核-巨噬细胞起源及发育分化的特征与分子调控[J];中国免疫学杂志;2014年01期
本文编号：2322761
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