槲皮素抑制TREM-1激活巨噬细胞炎症反应及减轻LPS诱导小鼠急性肺损伤的研究

Quercetin inhibits macrophage inflammatory response activated by TREM-1 and reduces LPS induced acute lung injury in mice

目的观察槲皮素(QUE)对骨髓细胞上表达的触发受体1(TREM-1)激活巨噬细胞炎症反应及脂多糖(LPS)诱导小鼠急性肺损伤(ALI)的治疗作用,并探讨其可能机制。方法体外细胞实验:腹腔注射3%硫醇乙酸钙收集小鼠原代腹腔巨噬细胞,将收集的细胞分为:空白对照组、DMSO溶媒组、TREM-1激动剂(anti-Trem-1)组(10μg/ml)、QUE组(10μmol/L)及TREM-1激动剂+QUE组(于加激动剂前,用10μmol/L QUE预处理细胞30 min)。采用ELISA法检测原代巨噬细胞培养上清液中IL-1β、TNF-α和IL-6的分泌;为观察QUE对LPS诱导巨噬细胞TREM-1表达的影响,将巨噬细胞分为:对照组、LPS组(100 ng/ml)及LPS+QUE组(10μmol/L QUE预处理后再加入100 ng/ml LPS),收集提取蛋白,蛋白质免疫印迹(Western blot)检测TREM-1蛋白的表达。动物实验:将80只雄性C57BL/6小鼠随机分为对照组、ALI模型组、QUE组、QUE治疗组,每组20只。ALI模型组气管内注射LPS 5 mg/kg构建小鼠ALI模型,QUE治疗组气管内注射LPS 5 mg/kg构建小鼠ALI模型,之后腹腔注射QUE 15 mg/kg;对照组气管内给予等量生理盐水,之后腹腔注射DMSO;QUE组气管内给予等量生理盐水,之后腹腔注射QUE 15 mg/kg;HE染色观察各组小鼠肺组织病理改变;计数各组小鼠支气管肺泡灌洗液(BLAF)中炎症细胞及IL-1β、TNF-α和IL-6含量;实时荧光定量PCR(qRT-PCR)和Western blot检测各组小鼠肺组织中TREM-1 mRNA和蛋白的表达。结果在体外细胞实验中,TREM-1激动剂组原代巨噬细胞上清液中IL-1β、TNF-α和IL-6的分泌高于DMSO溶解组(均P<0.001),而TREM-1激动剂+QUE组巨噬细胞IL-1β、TNF-α和IL-6分泌低于TREM-1激动剂组(均P<0.001)。LPS组TREM-1蛋白表达高于对照组(P<0.05),而LPS+QUE组TREM-1蛋白表达低于LPS组(P<0.05)。动物实验发现,与对照组相比,ALI模型组小鼠肺组织病理损伤评分,BALF中总细胞、巨噬细胞和中性粒细胞的数量,TNF-α、IL-6、IL-1β含量均明显增加(均P<0.001);QUE治疗组上述指标均低于ALI模型组(均P<0.001)。qRT-PCR及Western blot结果发现:与对照组相比,ALI模型组小鼠肺组织内TREM-1 mRNA和蛋白的表达均增加,而QUE治疗组小鼠肺组织内TREM-1 mRNA和蛋白的表达低于ALI模型组(均P<0.05)。结论QUE可抑制TREM-1激活减轻巨噬细胞炎症反应及LPS诱导的小鼠急性肺损伤。

Objective To observe the therapeutic effect of quercetin(QUE)on triggering receptor expressed on myeloid cells(TREM-1)activated macrophage inflammation and lipopolysaccharide(LPS)induced acute lung injury(ALI)in mice,and explore its possible mechanism.Methods In vitro cell experiment:The primary peritoneal macrophages of mice were collected by intraperitoneal injection of 3%calcium mercaptan acetate.The collected cells were divided into blank control group,dimethylsulfoxide(DMSO)vehicle group,TREM-1 agonist group(10μg/ml),QUE group(10μmol/L)and TREM-1 agonist+QUE group(cells were pretreated with 10μmol/L QUE for 30 min before adding agonist).Enzyme linked immunosorbent assay(ELISA)was used to detect the secretion of interleukin(IL)-1β,tumor necrosis factor(TNF)-αand IL-6 in the culture supernatant of primary macrophages;To observe the effect of QUE on LPS-induced TREM-1 protein levels,macrophages were divided into:normal control group,LPS group(100 ng/ml)and LPS+QUE treatment group[macrophages were pretreated with 10μmol/L QUE for 2 hours,and then incubated with LPS(100 ng/ml)for 16 hours].Western blot was used to detect the expression of TREM-1 protein.In animal experiments:80 male C57BL/6 mice were randomly divided into 4 groups(20 in each group):normal control group,ALI model group,QUE group and QUE treatment group(LPS+QUE).In the ALI model group,the ALI model was established by intratracheal injection of 5 mg/kg LPS;The mouse ALI model was established by intratracheal injection of LPS 5 mg/kg in the QUE treatment group,and then intraperitoneal injection of 15 mg/kg QUE.The control group was given the same amount of normal saline intratracheal followed by intraperitoneal injection of DMSO,and the QUE group was given the same amount of normal saline intratracheal followed by intraperitoneal injection of 15 mg/kg QUE.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of lung tissue in each group;Inflammatory cells including IL-1β,TNF-αand IL-6 in bronchoalveolar lavage fluid(BLAF)of mice in each group were counted;The expression of TREM-1 mRNA and protein in lung tissue of mice in each group was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and Western blot.Results In vitro cell experiment:the secretion of IL-1β,TNF-αand IL-6 in the supernatant of primary macrophages in TREM-1 agonist group was higher than those in DMSO vehicle group,while the secretion of IL-1β,TNF-αand IL-6 in the supernatant of primary macrophages in TREM-1 agonist+QUE group were lower than that of TREM-1 agonist group(all P<0.001).The expression of TREM-1 protein in LPS group was higher than that in control group(P<0.05),while the expression of TREM-1 protein in LPS+QUE group was lower than that in LPS group(P<0.05).Animal experiments showed that compared with the control group,the ALI model group had higher lung pathological injury score,more total cells,macrophages and neutrophils in BALF and increased TNF-α,IL-6,IL-1βcontent(all P<0.001).The above indexes in QUE group were lower than those in ALI model group(all P<0.001).The results of qRT-PCR and Western blot showed that compared with the control group,the expression of TREM-1 mRNA and protein in the lung tissue of ALI model group was increased,while the expression of TREM-1 mRNA and protein in the lung tissue of QUE group was lower than that of ALI model group(all P<0.05).Conclusions Quercetin can inhibit TREM-1 activation,reduce macrophage inflammatory response and LPS induced acute lung injury in mice.