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作者	单位	E-mail
王梅芬	上海交通大学附属儿童医院中医科上海 200040	wangmf94@163.com
邹亚	1 上海交通大学附属儿童医院中医科上海 200040；2 上海中医药大学附属龙华医院上海 200030
郑培永	上海中医药大学附属龙华医院上海 200030
景晓平	上海交通大学附属儿童医院中医科上海 200040
何丽	上海交通大学附属儿童医院中医科上海 200040
郭盛	上海交通大学附属儿童医院内分泌遗传代谢科上海 200040

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中文摘要:

摘要目的：探讨白介素17A（interleukin-17A, IL-17A）对脂多糖（lipopolysaccharide, LPS）诱导的人支气管上皮细胞（16-HBE）炎症损伤的影响及其可能机制。方法：体外培养16-HBE细胞系，予LPS、IL-17A进行干预，分为空白对照组、LPS组、IL-17A组、IL-17A+LPS组。采用酶联免疫吸附法（Enzyme linked immunosorbent assay, Elisa）测定细胞培养液上清中IL-4、IFN-?酌、IL-6、IL-8等炎症因子的水平，蛋白印迹法（Western Blot, WB）检测细胞中丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPKs)信号通路相关蛋白：细胞外调节蛋白激酶（ERK）、P38蛋白激酶（P38）、c-Jun氨基末端激酶（JNK）的表达及其相应磷酸化蛋白（P-ERK、P-P38、P-JNK）的表达。体外培养16-HBE细胞系，予LPS、IL-17A以及ERK1/2抑制剂（U0126）、p38抑制剂（SB203580）和JNK抑制剂（SP600125），分为空白对照组、LPS+IL-17A组、IL-17A+LPS+UO126组、IL-17A+LPS+SB203580组、IL-17A+LPS+SP600125组。采用酶联免疫吸附法测定细胞培养液上清中IFN-?酌、IL-4、IL-6、IL-8等炎症因子水平。结果：与空白对照组比较，LPS、IL-17A组，细胞上清中IL-6、IL-8的表达明显升高（P<0.01），IL-4的表达降低（空白组vs LPS组P<0.01，空白组vs IL-17A组P<0.05），细胞内磷酸化ERK、P38、JNK蛋白的表达明显增加（空白组vs LPS组P<0.05，空白组vs IL-17A组P<0.01）。IL-17A+LPS组细胞上清中IL-6、IL-8、IL-4水平及细胞内P-ERK、P-P38、P-JNK的表达较LPS、IL-17A组更高（P<0.05）。添加ERK、P38和JNK抑制剂后，与LPS+IL-17A组对比，IL-17A+LPS+U0126组、IL-17A+LPS+SB203580组和IL-17A+LPS+SP600125组细胞上清中IL-6、IL-8、IL-4水平下降（P<0.05）。结论：IL-17A可能通过上调IL-6、IL-8的表达加重LPS诱导的16-HBE细胞炎症损伤，MAPKs可能是这一过程中的重要信号转导通路。

英文摘要:

ABSTRACT Objective: To investigate the effects of interleukin-17A (IL-17A) on lipopolysaccharide (LPS) -induced human bronchial epithelial cells (16-HBE) and their possible mechanisms. Methods: The 16-HBE cell line was cultured in vitro and intervened with LPS and IL-17A, and divided into blank control group, LPS group, IL-17A group and IL-17A+LPS group. The concentrations level of IL-4, IFN-γ, IL-6, IL-8 and other inflammatory factors in the supernatant of cell culture medium were determined by Enzyme linked immunosorbent assay(Elisa). The expression and activation of Mitogen-activated protein kinase(MAPKs) pathway related proteins: Phosphorylated extracellular regulated protein kinase (P-ERK), Extracellular regulated protein kinase (ERK), Phosphorylated P38 protein kinase (P-P38), P38 protein kinase (P38), Phosphorylated c-Jun N-terminal kinase (P-JNK), c-Jun N-terminal kinase (JNK) were detected by Western Blotting (WB). The 16-HBE cell line was cultured in vitro and intervened with LPS, IL-17A, and ERK inhibitor(U0126), p38 inhibitor(SB203580), and JNK inhibitor (SP600125), and divided into blank control group, LPS + IL-17A group, IL-17A + LPS + UO126 group, IL-17A + LPS + SB203580 group, IL-17A + LPS + SP600125 group. Enzyme-linked immunosorbent assay was used to determine the levels of inflammatory factors such as IFN-γ, IL-4, IL-6, and IL-8 in the cell culture supernatant. Results: Compared with the blank control group, the concentration level of IL-6 and IL-8 were significantly increased in the cellular supernatant (P<0.01), while the concentration level of IL-4 was decreased(con vs LPS P<0.01, con vs IL-17A P<0.05) in the supernatant with either IL-17A or LPS stimulation. The expression of P-ERK, P-P38 and P-JNK protein was significantly increased (con vs LPS P<0.05, con vs IL-17A P<0.01). the concentration level of IL-6 and IL-8 were significantly increased (P<0.01). The levels of IL-6, IL-8, IL-4 and the expression of P-ERK, P-P38, and P-JNK in the cell supernatant of IL-17A + LPS group were higher than those in LPS and IL-17A group (P<0.05). After adding ERK, P38, and JNK inhibitors, compared with the LPS + IL-17A group, IL-17A + LPS + U0126 group, IL-17A + LPS + SB203580 group, and IL-17A + LPS + SP600125 group, the expression of IL-6, IL-8, IL-4 in cell supernatants has decreased (P<0.05). Conclusion: IL-17A might aggravate LPS-induced 16-HBE inflammatory injury by up-regulating IL-6 and IL-8 concentration level, and MAPKs may be an important signal transduction pathway in the process.

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