S100A8和S100A9在慢性阻塞性肺疾病大鼠肺泡巨噬细胞中的表达及促炎作用

The expression and pro-inflammatory effect of S100A8 and S100A9 in alveolar macrophages of rats with chronic obstructive pulmonary disease

目的 观察慢性阻塞性肺疾病(简称慢阻肺)大鼠肺泡巨噬细胞(alveolar macrophage,AM)中S100A8和S100A9的表达,探讨S100A8和S100A9对慢阻肺大鼠AM释放炎症介质的影响。方法 将12只成年雄性Wistar大鼠随机分成正常对照组和慢阻肺组。采用烟熏联合气管内注射内毒素处理大鼠1个月构建慢阻肺模型,光镜下观察大鼠肺组织病理形态。采用瑞氏染色法测定大鼠支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中AM、中性粒细胞、淋巴细胞的数量及细胞总数。分离、培养正常对照组和慢阻肺组大鼠的AM,分别给予不同剂量的S100A8、S100A9处理两组大鼠AM 6 h和12 h。采用ELISA法测量大鼠AM上清液中白细胞介素(interleukin,IL)-8、IL-6和肿瘤坏死因子α(tumour necrosis factor-α,TNF-α)的含量,原位杂交方法观察大鼠AM中S100A8、S100A9 mRNA的表达情况,免疫组化方法观察大鼠AM中S100A8/A9蛋白的表达情况。结果 烟熏联合气管内注射内毒素处理1个月后大鼠肺组织呈典型的慢阻肺病理改变。与正常对照组比较,慢阻肺组大鼠BALF中细胞总数及AM、中性粒细胞、淋巴细胞的绝对值明显增加(均P<0.05),其中增加最明显的是AM。与正常对照组比较,慢阻肺组大鼠AM中S100A8 mRNA、S100A9 mRNA和S100A8/A9蛋白表达均明显增加(均P<0.05)。S100A8、S100A9处理慢阻肺大鼠AM后,其上清液中IL-8、IL-6和TNF-α的含量呈时间和剂量依赖性增加。给予相同剂量的S100A8、S100A9处理AM相同时间时,慢阻肺组大鼠AM上清液中IL-8、IL-6和TNF-α的含量均高于正常对照组(均P<0.05)。结论 S100A8、S100A9在离体培养的慢阻肺大鼠AM中表达明显增加。S100A8、S100A9呈时间、剂量依赖性促进慢阻肺大鼠AM分泌和释放炎症因子IL-6、IL-8和TNF-α。S100A8、S100A9促慢阻肺大鼠AM分泌IL-6、IL-8和TNF-α作用较正常大鼠明显增强。

Objective To observe the expression of S100A8 and S100A9 in alveolar macrophages(AMs)of chronic obstructive pulmonary disease(COPD)rats,and explore the effect on the release of inflammatory mediators from AMs in COPD rats.Methods Twelve adult male Wistar rats were randomly divided into a normal control group and a COPD group.The COPD model was established by exposing the rats to cigarette smoke and intratracheal injection of endotoxin for 1 month.The pathological changes of lung tissue of rats were observed under light microscope.Total cells counts and the number of AMs,lymphocytes,neutrophils in bronchoalveolar lavage fluid(BALF)of two groups were examined by Wright's staining methods.Rat AMs from the control group and the COPD group were isolated and cultured,and then treated with different doses of S100A8 and S100A9 for 6 hours and 12 hours.The levels of interleukin(IL)-8,IL-6 and tumour necrosis factor-α(TNF-α)in the AMs supernatants were measured by enzyme linked immunosorbent assay.The expression of S100A8 and S100A9 mRNA in AMs of rats were observed by in situ hybridization.The immunohistochemical method was used to observed the expression of S100A8/A9 protein of AMs.Results After cigarette smoking combined with intratracheal injection of endotoxin for 1 month,the lung tissue of rats showed typical pathological changes of COPD.Total cell counts and the number of AMs,lymphocytes,neutrophils in BALF of the COPD rats were significantly higher than those of the normal rats(P<0.05).Among them,the increase in the number of AMs was the most obvious.Compared with the control group,the expression of S100A8 mRNA,S100A9 mRNA and S100A8/A9 protein in AMs of the COPD group were up-regulated significantly(P<0.05).After the AMs of COPD rats were treated with S100A8 and S100A9,the contents of IL-8,IL-6 and TNF-αin AMs supernatants increased significantly in a time-and dose-dependent manner.When the AMs were treated with the same dose of S100A8 and S100A9 for the same time,the levels of IL-8,IL-6 and TNF-αin the AMs supernatant of the COPD group were higher than those of the normal control group.Conclusions The expression of S100A8 and S100A9 in cultured COPD rat AMs is significantly increased.S100A8 and S100A9 can promote the secretion and release of inflammatory factors IL-6,IL-8 and TNF-αfrom AMs of COPD rats in a time and dose-dependent manner.The effects of S100A8 and S100A9 on the secretion of IL-6,IL-8 and TNF-αin AM of COPD rats are significantly enhanced compared with those of normal rats.