PIAS调控PPARγ的SUMO化修饰在小鼠内毒素性急性肺损伤内源性保护机制中的作用

Role of PIAS-regulated SUMOylation of PPARγin endogenous protective mechanism against endotoxin-induced acute lung injury in mice

目的评价小泛素相关修饰物(SUMO)E3连接酶(PIAS)调控过氧化物酶体增殖物激活受体γ(PPARγ)的SUMO化修饰在小鼠内毒素性急性肺损伤(ALI)内源性保护机制中的作用。方法实验Ⅰ清洁级野生型雄性C57BL/6小鼠24只,6~8周龄,体质量18~22 g,采用随机数字表法分为4组(n=6):对照组(C组)、ALI组、ALI+PPARγ诱导剂TZD组(ALI+T组)、ALI+TZD+SUMO化抑制剂漆树酸组(ALI+T+A组)。尾静脉注射LPS 15 mg/kg制备内毒素性ALI模型。ALI+T+A组注射LPS前1 h时腹腔注射漆树酸5 mg/kg;ALI+T组和ALI+T+A组注射LPS前30 min时腹腔注射TZD 50 mg/kg。给予LPS 12 h后处死小鼠取肺组织,测定湿重/干重(W/D)比值,光镜下观察病理学结果,并行肺损伤评分;分别采用Western blot法和PCR法测定PIAS1、PIAS2、PIAS3和PIASy及其mRNA的表达。实验Ⅱ体外培养的小鼠肺泡巨噬细胞(MH-S细胞)采用随机数字表法分为4组(n=5):对照组(C组)、LPS组(L组)、LPS+PIAS2 siRNA组(L+P组)和LPS+Con siRNA组(L+C组)。C组常规培养,余3组给予10μg/ml LPS刺激MH-S细胞,制备内毒素攻击模型。L+P组和L+C组于加入LPS前48 h时分别转染50 nmol/L PIAS2 siRNA或Con siRNA。加入LPS孵育24 h时收集细胞,检测细胞活力;采用流式细胞术检测肺泡M1型巨噬细胞和M2型巨噬细胞水平,计算M1型/M2型比值;采用Western blot法检测PIAS2和PPARγ表达,采用免疫共沉淀法测定PPARγ-SUMO1共表达;采用PCR测定TNF-α和IL-10的mRNA表达。结果实验Ⅰ与C组比较,其余3组肺损伤评分和肺组织W/D比值升高,PIAS2及其mRNA表达上调(P<0.05);与ALI组比较,ALI+T组和ALI+T+A组肺损伤评分和肺组织W/D比值降低,PIAS2及其mRNA表达上调(P<0.05);与ALI+T组比较,ALI+T+A组肺损伤评分和肺组织W/D比值升高,PIAS2及其mRNA表达下调(P<0.05)。4组间肺组织PIAS1、PIAS3和PIASy及其mRNA表达差异无统计学意义(P>0.05)。实验Ⅱ与C组比较,其余3组细胞活力降低,PPARγ表达和PPARγ-SUMO1共表达上调,M1型和M2型巨噬细胞水平、M1/M2型比值升高,TNF-αmRNA表达上调,IL-10 mRNA表达下调,L组和L+C组细胞PIAS2表达上调(P<0.05);与L组比较,L+P组细胞活力下降,PIAS2、PPARγ表达和PPARγ-SUMO1共表达下调,M1型巨噬细胞水平和M1型/M2型比值升高,TNF-αmRNA表达上调,IL-10 mRNA表达下调(P<0.05),L+C组上述指标差异无统计学意义(P>0.05);与L+C组比较,L+P组细胞活力下降,PIAS2、PPARγ表达和PPARγ-SUMO1共表达下调,M1型肺泡巨噬细胞水平和M1型/M2型比值升高,TNF-αmRNA表达下调,IL-10 mRNA表达上调(P<0.05)。结论PIAS2调控PPARγ的SUMO化修饰是小鼠内毒素性ALI的内源性保护机制,可能与抑制巨噬细胞向M1型极化,减轻炎症反应有关。

Objective To evaluate the role of small ubiquitin-associated modifier(SUMO)E3 ligase(PIAS)-regulated SUMOylation of peroxisome proliferator-activated receptorγ(PPARγ)in the endogenous protective mechanism against endotoxin-induced acute lung injury(ALI)in mice.Methods ExperimentⅠTwenty-four clean-grade wild type male C57BL/6 mice,aged 6-8 weeks,weighing 18-22 g,were divided into 4 groups(n=6 each)using a random number table method:control group(C group),ALI group,ALI+PPARγinducer TZD group(ALI+T group)and ALI+TZD+SUMOylation inhibitor anacardic acid group(ALI+T+A group).Lipopolysaccharide(LPS)15 mg/kg was injected into the tail vein to develop the ALI model.In ALI+T+A group,anacardic acid 5 mg/kg was intraperitoneally injected at 1 h before LPS administration.In ALI+T group and ALI+T+A group,TZD 50 mg/kg was intraperitoneally injected at 30 min before LPS administration.The mice were sacrificed at 12 h after LPS administration,and the lung tissues were obtained to examine the pathological changes which were scored and to determine the wet/dry(W/D)weight ratio,and expression of PIAS1,PIAS2,PIAS3 and PIASy protein and mRNA(by Western blot or polymerase chain reaction).ExperimentⅡMouse alveolar macrophages(MH-S cells)were cultured in vitro and divided into 4 groups(n=5 each)using a random number table method:control group(C group),LPS group,LPS+PIAS2 siRNA group(L+P group)and LPS+Con siRNA group(L+C group).Cells were routinely cultured in group C.Cells were stimulated with 10μg/ml LPS to develop the model of endotoxin challenge.PIAS2 siRNA 50 nmol/L and Con siRNA 50 nmol/L were transfected at 48 h before LPS was added in L+P group and L+C group,respectively.The cells were collected at 24 h of incubation with LPS to determine the cell viability,levels of M1 and M2 alveolar macrophages(by flow cytometry),expression of PIAS2 and PPARγ(by Western blot),co-expression of PPARγ-SUMO1(by immunoprecipitation)and expression of tumor necrosis factor-alpha(TNF-α)and interleukin-10(IL-10)mRNA(by polymerase chain reaction).The ratio of M1/M2 was calculated.Results ExperimentⅠCompared with C group,the lung injury scores and W/D ratio were significantly increased,and the expression of PIAS2 protein and mRNA was up-regulated in the other three groups(P<0.05).Compared with ALI group,the lung injury scores and W/D ratio were significantly decreased,and the expression of PIAS2 protein and mRNA was up-regulated in ALI+T group and ALI+T+A group(P<0.05).Compared with ALI+T group,the lung injury scores and W/D ratio were significantly increased,and the expression of PIAS2 protein and mRNA was down-regulated in ALI+T+A group(P<0.05).There was no significant difference in the expression of PIAS1,PIAS3 and PIASy protein and mRNA in lung tissues among the four groups(P>0.05).ExperimentⅡCompared with C group,the cell viability was significantly decreased,the expression of PPARγand co-expression of PPARγ-SUMO1 was up-regulated,the levels of M1 and M2 macrophages and M1/M2 ratio were increased,the expression of TNF-αmRNA was up-regulated,and the expression of IL-10 mRNA was down-regulated in the other three groups,and PIAS2 expression was significantly up-regulated in L group and L+C group(P<0.05).Compared with L group,the cell viability was significantly decreased,the expression of PIAS2 and PPARγand PPARγ-SUMO1 co-expression were down-regulated,the M1 macrophage level and M1/M2 ratio were increased,TNF-αmRNA expression was up-regulated,and the expression of IL-10 mRNA was down-regulated in L+P group(P<0.05),and no significant change was found in the parameters mentioned above in L+C group(P>0.05).Compared with L+C group,the cell viability was significantly decreased,the expression of PIAS2 and PPARγand co-expression of PPARγ-SUMO1 were down-regulated,the level of M1 alveolar macrophages and M1/M2 ratio were increased,the expression of TNF-αmRNA was down-regulated,and the expression of IL-10 mRNA was up-regulated in L+P group(P<0.05).Conclusions PIAS2-regulated SUMOylation of PPARγis the endogenous protective mechanism against endotoxin-induced ALI in mice,which may be related to inhibition of macrophage polarization into M1 type and alleviation of inflammatory responses.