糖尿病创面中性粒细胞胞外诱捕网生成增加对巨噬细胞黑色素瘤缺乏因子2炎症体的影响

Effect of neutrophils extracellular trap on macrophage absent in melanoma 2 inflammasome in diabetic wounds

目的探讨糖尿病创面中增高的中性粒细胞胞外诱捕网(NET)对巨噬细胞黑色素瘤缺乏因子2(AIM2)炎症体表达和活化的影响。方法腹腔注射链脲佐菌素诱导SD大鼠I型糖尿病模型,制作背部全层皮肤缺损创面。将糖尿病大鼠随机分为糖尿病组和DNase组,以正常大鼠作为正常组。正常组和糖尿病组大鼠每日创面给予0. 9%氯化钠溶液(30 u L); DNase组创面给予DNase I30μL(10 mg/m L),连续给药8 d。伤后第1、5、9、14天,麻醉大鼠,拍摄创面照片。伤后第2、5、9、14天取创面组织,检测创面NET及AIM2炎症体相关蛋白表达。提取大鼠腹腔中性粒细胞和巨噬细胞。佛波酯(0. 1 g/m L)刺激中性粒细胞诱导其形成NET,NET可被DNase I降解。巨噬细胞设为对照、NET和DNase 3组,分别与未经刺激的中性粒细胞、NET以及DNase I降解的NET共培养12 h。蛋白质印迹法和免疫荧光染色检测巨噬细胞AIM2炎症体相关蛋白AIM2、白细胞介素-1β(IL-1β)、白细胞介素-1β前体蛋白(pro-IL-1β)、caspase-1 p20表达和p65磷酸化水平。数据采用独立样本t检验。结果蛋白质印迹检测糖尿病组第2、5天Cit-H3、AIM2和IL-1β蛋白表达量(0. 136±0. 080,1. 119±0. 186,0. 918±0. 163; 1. 022±0. 270,1. 047±0. 123,1. 442±0. 177)均高于正常组(0. 043±0. 010,0. 600±0. 060,0. 172±0. 176; 0. 215±0. 420,0. 747±0. 052,0. 556±0. 179),差异有统计学意义(t=-21. 153、-4. 562、-5. 367、-. 232、-3. 898、-6. 012,P值均小于0. 05)。佛波酯可诱导中性粒细胞生成NET,NET组巨噬细胞AIM2、pro-IL-1β、IL-1β、caspase-1 p20表达和p65磷酸化水平(1. 113±0. 132,1. 098±0. 170,1. 129±0. 114,1. 083±0. 162)较对照组(0. 251±0. 067,0. 068±0. 237,0. 105±0. 155,0. 314±0. 161)明显升高,差异有统计学意义(t=-10. 050、-10. 388、-15. 455、-5. 828,P值均小于0. 05)。DNase组(0. 729±0. 092,0. 549±0. 115,0. 701±0. 172,0. 496±0. 031)则明显下调以上蛋白水平,差异有统计学意义(t=4. 124、4. 633、3. 596、6. 155,P值均小于0. 05)。p65核转位阻断剂(JSH-23)显著降低巨噬细胞AIM2、pro-IL-1β和IL-1β水平,差异有统计学意义(P <0. 05)。免疫荧光染色检测显示,DNase组显著减少创面NET和AIM2荧光水平。蛋白免疫印迹显示DNase组降低糖尿病组创面中IL-1β、caspase-1 p20水平,差异有统计学意义(P <0. 05)。DNase组创面愈合较糖尿病组加快,伤后第9天和第14天创面愈合率比较,差异有统计学意义(P <0. 05)。结论糖尿病创面NET生成增加可能通过活化p65,促进巨噬细胞AIM2炎症体表达活化。外源性补充DNase I降解创面NET,可降低AIM 2炎症体活化水平,加速创面愈合。

Objective To investigate the effect of the over-produced neutrophils extracellular trap(NET)on the absent in melanoma 2(AIM2)inflammasome of macrophage in diabetic wounds.Methods Type 1 diabetes was induced by I.P.injection of streptozocin,with normal rats as normal group.The full-thickness skin injury model was prepared.Diabetic rats were randomly assigned into diabetic and DNase group.The wounds of normal group,diabetic group were given 0.9%sodium chloride solution(30μL).And the wounds of DNase group were administrated by DNase I 30 uL(10 mg/mL)every 24 h for 8 consecutive days.At day 1,5,9 and 14 after injury,rats were anaesthetized,the wound were photographed and wound closure were measured.The wound tissue was collected to exam the levels of NET and AIM2 inflammasome related proteins at day 2,5,9,14 after injury.Isolated intraperitoneal-derived primary neutrophils and macrophages from SD rats.NET was induced by exposing to Phorbol 12-myristate 13-acetate(0.1 g/mL)and can be eliminated by DNase I(0.1 mg/mL).Macrophage were grouped into control,NET and DNase,which were incubated with unstimulated neutrophils,NET stimulated by Phorbol 12-myristate 13-acetate and NET digested by DNase I for 12 h,respectively.The levels of AIM2 inflammasome relative proteins in macrophage,such as AIM2,interleukin-1β(IL-1β),pro-IL-1β,caspase-1 p20 and the phosphorylation of p65 were detected by western blotting and immunofluorescence.Data were processed with independent sample t test.Results Western blotting showed the expression of Cit-H3,AIM2 and IL-1βprotein in the diabetic group(0.136±0.080,1.119±0.186,0.918±0.163;1.022±0.270,1.047±0.123,1.442±0.177)on the 2,5 day after injury were higher than the normal group(0.043±0.010,0.600±0.060,0.172±0.176;0.215±0.420,0.747±0.052,0.556±0.179),the differences were statistically significant(t=-21.153,-4.562,-5.367,-5.232,-3.898,-6.012,with P values below 0.05).Phorbol 12-myristate 13-acetate induced NET production in vitro.The level of AIM2,pro-IL-1β,IL-1β,activated caspase-1 expression and p65 phosphorylation in NET group(1.113±0.132,1.098±0.170,1.129±0.114,1.083±0.162)were higher than the control group(0.251±0.067,0.068±0.237,0.105±0.155,0.314±0.161),and the differences were statistically significant(t=-10.050,-10.388,-15.455,-5.828,with P values below 0.05).The DNase group(0.729±0.092,0.549±0.115,0.701±0.172,0.496±0.031)decreased the above proteins level,and the differences were statistically significant(t=4.124,4.633,3.596,6.155,with P values below 0.05).p65 nuclear translocation blocker(JSH-23)significantly decreased the levels of AIM2,pro-IL-1βand IL-1βin macrophages,and the difference was statistically significant(P<0.05).Immunofluorescence staining showed that the DNase group significantly reduced the level of NET and AIM2 in wounds.Western blotting showed that the DNase group reduced the levels of IL-1βand caspase-1 p20 in the diabetic group,and the difference was statistically significant(P<0.05).The wound healing in DNase group was faster than the diabetic group,and the difference of wound healing rate on the 9,14 day after injury was statistically significant(P<0.05).Conclusion NET might induce AIM2 inflammasome activation in macrophage through p65.Degradation NET by DNase I reduce the activation of AIM 2 inflammasome and accelerate the wound healing of diabetic rats.