抗菌肽LL37对脂多糖诱导的大鼠肺泡巨噬细胞炎性损伤的保护作用

Protective effect of antibacterial peptide LL37 on lipopolysaccharide-induced inflammatory injury in rat alveolar macrophages

目的探讨抗菌肽LL37对脂多糖(LPS)诱导的大鼠肺泡巨噬细胞(NR8383)炎症损伤的保护作用。方法实验分为对照组、LPS组、LL37组、实验组和干预组。对照组给予正常培养液培养12h, LPS组给予含1mg/L LPS的培养液培养12 h, LL37组给予含10 mg/L LL37培养液培养12h,实验组在1 mg/L LPS剌激的基础上,同时加入10 mg/L LL37培养12 h,干预组在实验组的基础上,加入10mg/LLL37中和抗体培养12 h。用CCK-8 (CellCounting Kit-8)法检测细胞活性.流式细胞仪检测细胞凋亡情况.酶联免疫吸附法检测细胞上清液肿瘤坏死因子p ( TNF-a)利IL-6的分泌水平,用蛋白质印迹法检测细胞核因子kB(NF-kB) p65蛋白表达水平。结果与空白组比较,LPS组细胞活性下降.细胞凋亡增多,细胞上清液TNF-α. IL-6水平升高,细胞中NF-kBp65蛋白表达增加(P值均<0.05);与LPS组比较.实验组中LL37抑制细胞活性的下降及细胞的凋亡,细胞上清液TNF-α. IL-6水平降低,细胞中NF+Bp65蛋白表达下降(P值均<0.05);LL37中和抗体可以抑制LL37的作用(P值均< 0.05)。结论 LL37通过下调NF-kB信号通路分子的表达,从而抑制LPS诱导的细胞炎性因子的产生和释放,以发挥其抗炎保护作用.

Objective To investigate the protective effect of antimicrobial peptide LL37 on inflammatory damage of rat alveolar macrophage (NR8383) induced by lipopolysaccharide (LPS). Methods The experiment was divided into control group, LPS group, LL37 group, test group and intervention group. Control group was cultured in normal culture medium for 12 h. LPS group was cultured in medium containing 1 mg/L LPS for 12 h. LL37 group was cultured in medium containing 10 nig' L LL37 for 12 h. On the basis of stimulation of 1 mg/L LPS for 12 h, test group was given 10 mg/L LL37 at the same time, intervention group was given 10 mg/L anti-LL-37 antibody on the basis of test group. Cell viability was detected by cell counting Kit-8 assay, cell apoptosis was detected by flow cytometry, The levels of inflammatory factors TNF-a and IL 6 in the cell supernatant were detected by ELISA, The prot&n expression levels of P-NF kB p65 were detected by Western blotting. Results Compared with the control group, the cell viability was decreased and the cell apoptosis was increased in LPS group, furthermore, the levels of TNF-q and IL-6 in the supernatant increased,and the protein expression levels of P NF kB p65 increased in the LPS group (all P <0.05), Compared with the LPS group, LL37 suppressed the decrease of cell viability and cell apoptosis, and the levels of TNF-a and IL-6 in the supernatant decreased, and the protein expression levels of P-NF-kB p65 also decreased in the test group (all P <C0.05), anti-LL-37 antibody could inhibit the effect of LL37 (all P <0.05). Conclusions LL37 can inhibit the production and release of macrophage inflammatory factors induced by LPS and thus play its antiinflammatory effect by down-regulating the expression of inflammatory-associated NF-kB pathway signaling molecules.