活化蛋白C通过靶向VLA-3-中性粒细胞亚群减轻小鼠的严重炎症反应的机制

The Mechanism by Which Activated Protein C Alleviates Severe Inflammatory Responses in Mice by Targeting the Vla-3-Neutrophil Subgroup

研究活化蛋白C通过靶向VLA-3中性粒细胞亚群来减轻小鼠的严重炎症反应的机制。75只小鼠分为3组:对照组(健康小鼠,n=25)、严重炎症组(脓毒症小鼠,n=25)和活化蛋白C组(脓毒症小鼠通过尾静脉注射活化蛋白C,n=25);通过蛋白质免疫印迹检测整联蛋白VLA-3在中性粒细胞中上调表达;通过整联蛋白结合实验,确定与活化蛋白C以高亲和力相互作用整联蛋白;通过流式细胞仪验证活化蛋白C和VLA-3之间对中性粒细胞的相互作用;通过苏木精和曙红染色检测小鼠组织学变化;通过RT-qPCR检测炎症细胞因子(TNF-α、IL-6和IL-10)mRNA表达水平;检测小鼠在手术后静脉注射活化蛋白C后存活率。与对照组(1. 32±0. 21)相比,严重炎症组(4. 57±0. 56) VLA-3表达量升高(P <0. 05),而VLA-5(1. 25±0. 24)和αVβ3(1. 27±0. 13)与对照组(1. 24±0. 33,1. 29±0. 28)相比无统计学意义(P> 0. 05);与VLA-5 (0. 66±0. 05)和αVβ3 (0. 68±0. 08)整联蛋白相比,VLA-3(1. 76±0. 25)对活化蛋白C组表现出高亲和力,且与严重炎症组(0. 66±0. 12)相比升高(P <0. 05);活化蛋白C组与VLA-3发生特异性结合,VLA-5、αVβ3与活化蛋白C组几乎不发生反应结合。与严重炎症组相比,活化蛋白C组值升高,而阻断剂降低活化蛋白C与VLA-3间的结合(P <0. 05);与对照组相比,严重炎症组充血严重,中性粒细胞聚集和血管内微血栓形成,而活化蛋白C治疗后,减少脓毒症所造成的中性粒细胞增加;严重炎症组TNF-α、IL-6和IL-10 mRNA表达水平较对照组提高,与严重炎症组相比,活化蛋白C组TNF-α、IL-6和IL-10 mRNA表达水降低,有较好的抗炎作用(P <0. 05);活化蛋白C组,小鼠存活率(18只,72. 00%)较严重炎症组(7只,28. 00%)高(P <0. 05)。活化蛋白C通过靶向VLA-3-中性粒细胞亚群,降低炎症细胞因子相关基因的表达,从而减轻小鼠的严重炎症反应。

To investigate the mechanism of activated protein C to alleviate severe inflammatory response in mice by targeting VLA-3 neutrophil subpopulation,the experiment was divided into three groups: control group( healthy mice,n = 25),severe inflammation group( septic mice,n = 25) and activated protein C group( septic mice activated protein C by tail vein injection,n = 25);up-regulated expression of integrin VLA-3 in neutrophils by Western blotting;determination of integrin interaction with activated protein C by high affinity by integrin binding assay;The cytoplasmic instrument verified the interaction of activated protein C and VLA-3 on neutrophils;the histological changes of mice were detected by hematoxylin and eosin staining;the inflammatory cytokines( TNF-α,IL-6 and IL-10) were detected by RT-qPCR. mRNA expression levels;the survival rate of mice after intravenous administration of activated protein C was measured. The results showed compared with the control group( 1. 32 ± 0. 21),the expression of VLA-3 was increased in the severe inflammation group( 4. 57 ± 0. 56)( P < 0. 05). And VLA-5( 1. 25 ± 0. 24) and αVβ3( 1. 27 ± 0. 13) was not statistically significant compared with the control group( 1. 24 ± 0. 33,1. 29 ± 0. 28). Compared with VLA-5( 0. 66 ± 0. 05) andαVβ3( 0. 68 ± 0. 08) integrin,VLA-3( 1. 76 ± 0. 25) showed high affinity for activated protein C group,and increased compared with severe inflammation group( 0. 66 ± 0. 12)( P < 0. 05). The activated protein C group and VLA-3 occurred. With specific binding,VLA-5 and αVβ3 hardly reacted with the activated protein C group. Compared with the severe inflammation group,the activated protein C group value was increased,and the blocker reduced the binding between activated protein C and VLA-3( P < 0. 05).Compared with the control group,severe inflammation group was severely hyperemia,neutrophil accumulation( black arrow) and intravascular microthrombus formation,while activated protein C treatment reduced neutrophil increase caused by sepsis,severe inflammation group. The expression levels of TNF-α,IL-6 and IL-10 mRNA were higher than those of the control group. Compared with the severe inflammation group,the expression of TNF-α,IL-6 and IL-10 mRNA in the activated protein C group was decreased,and the anti-inflammatory effect was better( P < 0. 05). The survival rate of the activated protein C group( 18,72. 00%) was higher than that of the severe inflammation group( 7,28. 00%)( P < 0. 05). Activated protein C reduced the expression of inflammatory cytokine-related genes by targeting VLA-3-neutrophil subpopulations,thereby alleviating severe inflammatory responses in mice.