GLP-2對LPS活化巨噬細胞的調節(jié)作用及其機制
發(fā)布時間：2018-11-10 14:51
【摘要】：胰高血糖素樣肽-2（glucagon-like peptide2，GLP-2）是由腸道L內分泌細胞分泌的由33個氨基酸組成的單鏈多肽，主要通過GLP-2R發(fā)揮生物學作用。GLP-2能增加腸道血流量，促進小腸生長、營養(yǎng)物質吸收，抑制胃腸動力和胃酸分泌，降低腸道通透性等作用。在非甾體類抗炎藥物誘導的小鼠腸炎模型中，GLP-2具有降低腸炎小鼠的死亡率，降低炎性因子表達等作用。 巨噬細胞是先天免疫的重要組成細胞，，通過表面模式識別受體識別結合、進而吞噬和清除入侵的病原體或凋亡細胞。當炎癥發(fā)生時，巨噬細胞分泌不同功能的細胞因子參與炎癥反應。然后，過度釋放促炎細胞因子會導致多種疾病的發(fā)生，如感染性休克、風濕性關節(jié)炎和其他慢性炎癥性疾病。LPS為革蘭氏陰性細菌細胞壁的主要成分，與LBP、CD14結合形成LPS-LBP-CD14三聯(lián)復合物作用于巨噬細胞表面的TLR4，從而活化MAPKs和NF-κB兩條信號通路，誘導相關靶基因的轉錄，釋放IL-1β、TNF-α、IL-6等促炎細胞因子。 研究報道中指出GLP-2具有減輕炎癥的作用，而巨噬細胞在炎癥反應中扮演著重要的角色，本研究探討了外源性GLP-2對LPS活化的小鼠腹腔巨噬細胞的調節(jié)作用及其調節(jié)機制。本實驗首先通過腹腔注射巰基乙酸鹽肉湯的方法，分離培養(yǎng)BALB/c小鼠腹腔巨噬細胞；再通過qRT-PCR、Western blot和ELISA方法檢測GLP-2對LPS活化的巨噬細胞中促炎酶（iNOS和COX-2）和促炎細胞因子（IL-1β、TNF-α和IL-6）mRNA和蛋白表達水平的影響；然后，進一步通過Western blot方法檢測了GLP-2對LPS活化的巨噬細胞中相關信號通路蛋白ERK、p38MAPK、JNK和IκB-α的磷酸化水平和NF-κB核轉位的影響，從而研究GLP-2對LPS活化巨噬細胞的調節(jié)作用及其機制。 本研究結果顯示：1）GLP-2（10-9、10-8、10-7、10-6M）能降低LPS活化的巨噬細胞中iNOS、COX-2、IL-1β、TNF-α和IL-6mRNA表達水平，以及細胞中iNOS、 COX-2蛋白表達水平和細胞培養(yǎng)上清中TNF-α和IL-6的蛋白表達水平，GLP-2濃度為10-6M時，抑制作用最顯著。表明所選濃度的GLP-2可以有效降低由LPS介導活化的腹腔巨噬細胞促炎酶和促炎細胞因子的表達，具有明顯減輕炎癥的作用。2）Western blot結果顯示GLP-2能夠顯著抑制LPS介導活化巨噬細胞中ERK和IκB-α的磷酸化，抑制NF-κB的核轉位。本研究表明，GLP-2通過抑制LPS活化巨噬細胞MAPKs信號通路中ERK的磷酸化和抑制NF-κB信號通路，從而抑制促炎酶和促炎細胞因子的表達。
[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- 偽, 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- 偽 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- 偽 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- 偽 and IL-6mRNA in LPS activated macrophages and iNOS, in cells. The expression level of COX-2 protein and the expression of TNF- 偽 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- 偽 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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.3
本文編號：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- 偽, 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- 偽 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- 偽 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- 偽 and IL-6mRNA in LPS activated macrophages and iNOS, in cells. The expression level of COX-2 protein and the expression of TNF- 偽 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- 偽 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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.3
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相關期刊論文 前2條
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2 趙陽;趙勇;;單核-巨噬細胞起源及發(fā)育分化的特征與分子調控[J];中國免疫學雜志;2014年01期
本文編號：2322761
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