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目的 研究中药814对肺泡巨噬细胞(AMs)分泌肿瘤坏死因子(TNF—α)的影响进而探讨814对肺气肿的作用机理。方法 支气管肺泡灌洗(BAL)收集金黄地鼠AMs,调整细胞浓度为5×10~5/ml,接种于24孔板培养,在脂多糖(LPS)刺激前后加入中药814分别收集培养细胞上清液,通过ELISA测定上清液TNF—α含量,以及运用TNF—α敏感的小鼠L929细胞株,测定上清液中TNF—α的细胞毒活性;此外,选取对人重组TNF—α(rhTNF—α)特异性的单克隆抗体(MAb)做中和培养上清实验。结果 (1) ELISA检测显示:加814各组TNF—α量均明显低于LPS刺激组和单纯AMs上清组(P<0.001),且814在LPS刺激后和与LPS共孵育时有剂量依赖性(P<0.05)。(2) L929细胞杀伤活性显示:加814各组TNF—α活性均低于LPS刺激组(P<0.001),且814在LPS刺激前、后和与LPS共孵育时有剂量依赖性(P<0.05)。(3) 中和实验显示:培养上清液的细胞毒是由于TNF—α造成的。结论 中药814可能会抑制AMs分泌TNF—α从而减轻TNF—α介导的肺组织损伤。
Objective To study the effect of Chinese medicine 814 on the secretion of tumor necrosis factor (TNF-α) in alveolar macrophages (AMs) and explore the mechanism of action of 814 on emphysema. Methods AMs from golden hamsters were collected by bronchoalveolar lavage (BAL). The cell concentration was adjusted to 5×10~5/ml. The cells were inoculated in 24-well plates and cultured before and after stimulation with lipopolysaccharide (LPS). Liquid, TNF-α content in the supernatant was determined by ELISA, and TNF-α-sensitive mouse L929 cell line was used to determine the cytotoxic activity of TNF-α in the supernatant; in addition, human recombinant TNF-α (rhTNF) was selected. - α) specific monoclonal antibody (MAb) was used as a neutralization culture supernatant assay. Results (1) ELISA showed that the amount of TNF-α in each group was significantly lower than that of LPS-stimulated group and pure AMs-supernagged group (P<0.001), and 814 was dose-dependent after LPS stimulation and co-incubation with LPS. Sex (P<0.05). (2) L929 cell killing activity showed that the activity of TNF-α in each group was significantly lower than that in LPS group (P<0.001), and 814 was dose-dependent before and after LPS stimulation and co-incubated with LPS (P< 0.05). (3) Neutralization experiments showed that the cytotoxicity of the culture supernatant was due to TNF-α. Conclusion Traditional Chinese medicine 814 may inhibit the secretion of TNF-α by AMs and reduce TNF-α-mediated lung injury.