降钙素基因相关肽下调肺炎克雷伯菌脂多糖所致炎症反应

Calcitonin gene-related peptide down-regulates the inflammatory response induced by lipopolysaccharide of Klebsiella pneumoniae

目的探讨降钙素基因相关肽(CGRP)对肺炎克雷伯杆菌脂多糖(LPS)诱导炎症反应的影响。方法体外培养肺炎克雷伯杆菌提取其LPS,不同浓度LPS刺激BEAS-2B细胞后,通过免疫荧光法检测细胞内人类β防御素2(hBD-2)的活化情况,并检测不同浓度CGRP处理后细胞内hBD-2的活性;MTT试验以及流式细胞术分别检测LPS刺激以及CGRP共同处理后BEAS-2B细胞的增殖以及凋亡水平;最后以中性粒细胞明胶酶相关载脂蛋白(NGAL)作为其特异性颗粒的标记物,检测CGRP处理后细胞内中性粒细胞颗粒的释放水平。结果免疫荧光检测结果显示,不同浓度LPS刺激均能够显著增加细胞内hBD-2的相对表达量,而用CGRP处理后则会显著抑制hBD-2的表达;50 ng/ml、100 ng/ml和200 ng/ml的LPS对BEAS-2B细胞的细胞活性无显著影响,而用400 ng/ml LPS处理24 h,则能够显著降低细胞活性,促进细胞凋亡。当用10 nmol/L CGRP与LPS共同处理时,能明显增加细胞活性,抑制细胞凋亡;不同浓度的LPS均能刺激中性粒细胞特异性颗粒的释放。而10 nmol/L CGRP则能够显著抑制LPS引起的中性粒细胞特异性颗粒释放。结论CGRP能够抑制hBD-2表达、促进细胞增殖并减少中性粒细胞的脱颗粒,进而下调肺炎克雷伯杆菌LPS诱导的炎症反应。

Objective To investigate the effects of calcitonin gene-related peptide (CGRP) on the inflammatory response induced by lipopolysaccharide (LPS) of Klebsiella pneumoniae. Methods Klebsiella pneumoniae was cultured in vitro to extracted LPS. Different concentrations of LPS were used to stimulate BEAS-2B cells. The activation of human β defensin 2 (hBD-2) in these cells was detected by immunofluorescence assay before and after adding different concentrations of CGRP. MTT assay and flow cytometry were respectively used to detect the proliferation and apoptosis of BEAS-2B cells after LPS stimulation with and without CGRP treatment. Neutrophil granules released in the cells after CGRP treatment were detected using neutrophil gelatinase-related apolipoprotein (NGAL) as the marker. Results Immunofluorescence assay results showed that LPS at different concentrations could significantly increase the relative expression of hBD-2 in BEAS-2B cells, which was significantly inhibit by CGRP intervention. LPS at 50 ng/ml, 100 ng/ml and 200 ng/ml had no significant effect on the activity of BEAS-2B cells, while treatment with 400 ng/ml of LPS for 24 h could significantly reduce the activity and promote the apoptosis of BEAS-2B cells. In addition, remarkedly increased cell activity and suppressed cell apoptosis were induced when BEAS-2B cells were treated with 10 nmol/L of CGRP in combination with LPS. LPS at different concentrations could induce the release of neutrophil-specific granules, while the LPS-induce release could be significantly inhibited by 10 nmol/L of CGRP. Conclusions CGRP could inhibit the expression of hBD-2, promote cell proliferation and reduce the degranulation of neutrophils to down-regulate the inflammatory response induced by LPS of Klebsiella pneumoniae.