Maresin 1促进小鼠急性肺损伤炎症消退及其机制的研究

发布时间：2018-06-24 00:38

本文选题：脂多糖 + 急性肺损伤　；参考：《华中科技大学》2014年博士论文

【摘要】：第一部分Maresin1抑制脂多糖诱导的急性肺损伤模型中性粒细胞的粘附目的：探讨Maresin1(MaR1)对脂多糖(lipopolysaccharide, LPS)诱导的小鼠急性肺损伤(acute lung injury, ALI)模型炎症反应的影响及其机制。方法：128只雄性SPF级BALB/c小鼠随机分为4组：(1)假手术组(sham)：小鼠经口气管插管滴入生理盐水,1小时后尾静脉注射生理盐水。(2)脂多糖组(LPS)：小鼠经口气管插管滴入LPS (3mg/kg),1小时后尾静脉注射生理盐水。(3)MaRl小剂量组(LPS+LD-MaRl)：小鼠经口气管插管滴入LPS (3mg/kg)1小时后尾静脉注射MaRl (O.lng/只)。(4)MaRl大剂量组(LPS+HD-MaRl):小鼠经口气管插管滴入LPS (3mg/kg),1小时后尾静脉注射MaRl (1ng/只)四组小鼠在气管给药后24小时处死。抽取动脉血监测血气。观察小鼠肺组织病理学改变,肺泡灌洗液(broncheoalveolar lavage fluid, BALF)中性粒细胞和巨噬细胞数目的变化,以及肺组织湿干重比。分光光度计法检测BALF总蛋白含量与肺组织髓过氧化物酶活性。ELISA法检测BALF炎症细胞因子TNF-α, IL-1β, IL-6, KC, IL-10, MCP-5, MIP-la和MIP-1γ。免疫组化法观察肺组织中性粒细胞浸润。流式细胞学检测中性粒细胞与血小板之间的粘附。Western blotting检测肺组织ICAM-1,P-选择素和CD24的表达。结果：大剂量MaRl能显著减轻LPS诱导的肺损伤和肺水肿,抑制中性粒细胞浸润,改善氧合功能。同时大剂量MaRl能下调促炎症细胞因子(TNF-α, IL-1β, IL-6, KC)和趋化因子(KC, MCP-5, MIP-la, MIP-1γ)的表达,上调抗炎症细胞因子IL-10的表达。流式细胞学结果证实MaRl能抑制中性粒细胞和血小板之间的相互作用。肺组织Western blotting证实MaRl能减少ICAM-1, P-选择素和CD24的表达。结论：大剂量的MaR1能显著改善LPS诱导的肺组织结构的破坏和氧合功能障碍,下调中性粒细胞的粘附作用,降低促炎症细胞因子的表达,从而减轻LPS诱导的ALI的炎症反应。第二部分Maresin1促进中性粒细胞凋亡及其机制研究目的：研究MaRl对人中性粒细胞凋亡的影响并探讨其可能机制。方法：通过Percoll非连续密度梯度离心法,分离人中性粒细胞进行体外离体培养。用含MaRl (1-100nM)或/和caspase广谱抑制剂(z-VAD-fink)处理30min后,加入LPS (100-500ng/ml)处理。给予LPS后24小时,流式细胞学检测中性粒细胞凋亡和中性粒细胞caspase-3的活性。Western blotting检测中性粒细胞p-ERK1/2, p-p38, p-AKT, Bcl-2和Mcl-1的表达。结果：MaRl (1-10nM)不影响中性粒细胞凋亡,而高浓度100nM MaRl能显著抑制中性粒细胞凋亡。LPS可呈剂量依赖性地抑制中性粒细胞凋亡,500ng/mlLPS抑制中性粒细胞凋亡最强。尽管MaRl1nM也能拮抗LPS抑制中性粒细胞凋亡的作用,但在MaRl10nM时拮抗作用最强。同时,z-VAD-fink可明显抑制MaRl促进中性粒细胞凋亡和上调caspase-3表达的作用。Western blotting结果显示,LPS可诱导中性粒细胞的ERK1/2,p38,AKT磷酸化,作用30min时最为显著；lOnM MaRl处理30min后,可明显抑制LPS诱导的ERK1/2, p38, AKT的磷酸化。此外,lOnM MaR1处理2小时后,可明显抑制LPS上调的Bcl-2和Mcl-1的表达。结论：MaRl能拮抗LPS抑制中性粒细胞凋亡的作用,其作用机制可能是通过阻断LPS诱导ERK1/2, p38, AKT磷酸化,和Bcl-2和Mcl-1的表达,最终激活中性粒细胞caspase-3活性来实现。第三部分Maresin1通过促进中性粒细胞凋亡加速急性肺损伤炎症消退目的：探讨MaR1对LPS诱导的小鼠ALI炎症消退的影响及其机制的研究。方法：实验分为两部分。第一部分探讨MaRl是否能促进LPS诱导的小鼠ALI炎症消退。80只雄性SPF级BALB/c小鼠随机分为两组：LPS组和LPS+MaRl组。经口气管内滴注LPS (3mg/kg)制备小鼠ALI模型。24小时后尾静脉注射MaRl (1ng/只)或生理盐水。分别于不同的天数处死小鼠：0、1、2、4、7天(每次处死8只)。观察小鼠肺组织病理学改变,BALF中性粒细胞和巨噬细胞数目的变化,以及肺组织湿干重比。分光光度计法检钡BALF,总蛋白的含量。第二部分探讨MaRl能否通过促进中性粒细胞凋亡,从而发挥促进ALI炎症消退。128只雄性SPF级BALB/c小鼠随机分为四组：假手术组,LPS组,LPS+MaRl治疗组,LPS+MaRl+caspase广谱抑制剂组。除假手术组,每只小鼠气管内滴注LPS(3mg/kg),24小时后尾静脉注射MaRl (1ng/只)或生理盐水。同时腹腔注射caspase广谱抑制剂：z-VAD-fink (10μg/kg),分别于4小时后和8小时后再腹腔追加z-VAD-fmk,其余组给予等体积的生理盐水。给予MaRl24小时后,流式细胞学检测中性粒细胞凋亡和中性粒细胞caspase-3的活性。观察小鼠肺组织病理学改变,BALF中性粒细胞和巨噬细胞细胞数目的变化,以及巨噬细胞吞噬凋亡小体；分光光度计法检测BALF,总蛋白含量和肺组织髓过氧化物酶活性;ELISA法检测BALF炎症因子TNF-α, IL-1β, IL-10, MCP-5,MIP-1γ。结果：第一部分结果显示气管内滴注LPS后,肺组织结构紊乱,中性粒细胞浸润以及肺水肿在24小时达炎症高峰。24小时后肺损伤逐渐减轻,至第7天肺损伤基本恢复正常。在炎症高峰期(24小时),尾静脉注射MaRl明显减轻了LPS诱导的肺损伤。肺损伤在第4天基本恢复正常。第二部分结果显示MaRl能促进BALF中性粒细胞凋亡并上调中性粒细胞caspase-3的表达。同时MaRl能减轻肺组织病理学损伤,减少中性粒细胞浸润,降低肺组织髓过氧化物酶的活性和抑制BALF炎症因子TNF-α, IL-1β, MCP-5, MIp-1γ的产生,促进抗炎症细胞因子IL-10的产生。相反,z-VAD-fmk可部分拮抗MaRl的促凋亡作用,同时显著抑制MaRl对急性肺损伤的保护作用。结论：MaRl通过促进中性粒细胞凋亡,从而加速LPS诱导的小鼠ALI炎症消退。
[Abstract]:first part maresinl inhibits lipopolysaccharide - induced adhesion of lipopolysaccharide - induced acute lung injury model neutrophils

Objective : To investigate the effect and mechanism of Maresinl ( MaR1 ) on the inflammatory response of lipopolysaccharide ( LPS ) induced acute lung injury ( ALI ) model in mice .

Methods : 128 male SPF BALB / c mice were randomly divided into four groups : ( 1 ) sham operation group ( sham ) : mice were injected with normal saline at the end of 1 hour after tracheal intubation .

Results : Large dose of MaR1 can significantly reduce LPS - induced lung injury and pulmonary edema , inhibit neutrophil infiltration and improve oxygenation . At the same time , the large dose of MaR1 can downregulate the expression of pro - inflammatory cytokines ( TNF - 伪 , IL - 1尾 , IL - 6 , KC ) and chemokine ( KC , MCP - 5 , MIP - la , MIP - 1 纬 ) . The results of flow cytometry confirm that MaR1 can inhibit the interaction between neutrophils and platelets . The expression of ICAM - 1 , P - selectin and CD24 can be reduced by Western blotting .

Conclusion : The large dose of MaR1 can significantly improve the damage and oxygenation of LPS - induced lung tissue structures , regulate the adhesion of neutrophils , decrease the expression of pro - inflammatory cytokines , and reduce the inflammatory response of LPS - induced ALI .

Study on the Effect of the Second Part Maresinl on Neutrophils Apoptosis and Its Mechanism ;

Objective : To study the effect of MaR1 on human neutrophil apoptosis and to explore its possible mechanism .

Methods : In vitro ex vivo culture of human neutrophils was carried out by means of the continuous density gradient centrifugation . After treated with MaR1 ( 1 - 100 nM ) or / and caspase - wide - spectrum inhibitor ( z - VAD - fink ) for 30 min , LPS ( 100 - 500 ng / ml ) was added . 24 hours after LPS administration , the activity of neutrophil apoptosis and neutrophil caspase - 3 was detected by flow cytometry . Western blotting was used to detect the expression of neutrophil p - 1 / 2 , p - p38 , p - p38 , Bcl - 2 and Mcl - 1 .

Results : MaR1 ( 1 - 10 nM ) did not influence the apoptosis of neutrophils , while the high concentration of 100 nM MaR1 could significantly inhibit the apoptosis of neutrophils .
After treated with lOnM MaR1 for 30 min , LPS - induced phosphorylation of 1 / 2 , p38MAPK was significantly inhibited . In addition , the expression of Bcl - 2 and Mcl - 1 up - regulated by LPS could be significantly inhibited after lOnM MaR1 treatment for 2 hours .

Conclusion : MaR1 can inhibit LPS from inhibiting neutrophil apoptosis . The mechanism of action may be by blocking LPS - induced phosphorylation of 1 / 2 , p38 , the phosphorylation of p38 , and the expression of Bcl - 2 and Mcl - 1 , and finally activating the activity of caspase - 3 .

The third part Maresin1 accelerates the inflammation of acute lung injury by promoting neutrophil apoptosis .

Objective : To investigate the effect of MaR1 on LPS - induced ALI inflammation and its mechanism .

Methods : The mice were divided into two groups : LPS group and LPS + MaR1 group . The rats were sacrificed at 0 , 1 , 2 , 4 , 7 days after intravenous injection of LPS ( 3 mg / kg ) .
TNF - 伪 , IL - 1尾 , IL - 10 , MCP - 5 , MIP - 1 纬 were detected by ELISA .

Results : In the first part , LPS - induced lung injury was observed in the lungs after LPS injection . The lung injury was gradually reduced after 24 hours . The lung injury was reduced gradually to the 7th day . The results showed that MaR1 could promote the apoptosis of BALF neutrophils and increase the expression of TNF - 伪 , IL - 1尾 , MCP - 5 , MIp - 1 纬 .

Conclusion : MaR1 accelerates LPS - induced ALI inflammation by promoting neutrophil apoptosis .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R563.8

【共引文献】