IL-8在人肺上皮细胞的MUC5B表达及调控通路

IL-8 Regulates the Expression and Pathway of MUC5B in Human Lung Epithelial Cells

目的:在肺上皮细胞模型中探讨IL-8是否对MUC5B的转录和合成产生影响,并探讨其是否通过JAK2/STAT6信号通路调节黏蛋白MUC5B分泌。方法:(1)体外培养人肺上皮细胞(HBE135-E6E7),无血清培养24h后分为不同浓度组IL-8(20ng/ml、40ng/ml、60ng/ml、80ng/ml、160ng/ml)、不同时间组(6h、12h、24h、48h),用ELISA测得各组MUC5B蛋白含量。(2)HBE135-E6E7无血清培养24h,以JAK2抑制剂AG490和MUC5B抑制剂NAC(乙酰半胱氨酸)为处理因素,分为以下六组:A组:空白对照组;B组:IL-8最适浓度组(160ng/ml);C组:IL-8+AG490(50μmol/L)组;D组:AG490组;E组:IL-8+NAC(10mmol/L);F组:NAC,处理48h,RT-PCR检测MUC5B mRNA水平,ELISA法检测MUC5B蛋白水平,WB法检测p-JAK2、p-STAT6、JAK2、STAT6蛋白水平。结果:(1)IL-8可刺激肺上皮细胞中MUC5B产生,且MUC5B的表达随着IL-8浓度的增加而增加。(2)在一定浓度IL-8的作用下,MUC5B蛋白相对表达量随着时间的增加而增加。(3)加入MUC5B抑制剂NAC后,RT-PCR检测MUC5B mRNA水平,与E组相比,B组增高,C组降低(P<0.05),与A组相比,B组、C组表达均升高且B组升高更明显(P<0.05);加入JAK2抑制剂AG490后发现,相比于A组,B组p-STAT6、p-JAK2蛋白含量表达增加,MUC5B蛋白含量、mRNA表达增加,且有统计学意义(P<0.05)。相比于B组,C组p-STAT6、p-JAK2蛋白含量表达降低,MUC5B蛋白含量、mRNA表达降低,且有统计学意义(P<0.05)。而各组非磷酸化的STAT6、JAK2表达无明显变化。结论:(1)IL-8可刺激肺上皮细胞中黏蛋白MUC5B的表达,且与浓度、时间均有关。其中最适IL-8浓度为160ng/ml,最适处理时间为48h。(2)IL-8可能通过JAK2/STAT6途径调控MUC5B分泌。

Objective:This paper focuses on the study of whether IL-8 has an effect on the transcription and synthesis of MUC5 B in the lung epithelial cell model,and whether it regulates MUC5 B secretion through the JAK2/STAT6 signaling pathway.Methods:(1)Culture human lung epithelial cells(HBE135-E6 E7) in vitro.After 24 hours of serum-free culture,cells were divide into several groups with different concentrations of IL-8(20 ng/ml,40 ng/ml,60 ng/ml,80 ng/ml,and 160 ng/ml) and under different time(6 hours,12 hours,24 hours,and 48 hours).The expression of MUC5 B protein was detected by ELISA.(2)HBE135-E6 E7 were cultured in vitro,after 24 hours of serum-free culture,based on the factor of the JAK2 inhibitor AG490 and MUC5 B inhibitor NAC(acetylcysteine) were used as treatment factors,and they were divided into the following six groups:group A:blank control;group B:IL-8 optimal concentration group(160 ng/ml);group C:IL-8+AG490(50μmol/L);group D:AG490;group E:IL-8+NAC(10 mmol/L);group F:NAC,were treated for 48 hours.The mRNA levels of MUC5 B in each group were measured by PT-PCR,the protein levels of MUC5 B measured by ELISA,and the protein levels of p-JAK2,p-STAT6,JAK2 and STAT6 were measured by WB.Results:(1)IL-8 can stimulate the production of MUC5 B in lung epithelial cells,and the expression of MUC5 B increased in response to the increase of IL-8 concentration.(2)Under the stimulation by IL-8 of a certain concentration,the relative expression of MUC5 B protein increases along with the increase of time period.(3)After adding MUC5 B inhibitor NAC,RT-PCR detected MUC5 B mRNA level.Compared with group E,group B increased and group C decreased(P<0.05).Compared with group A,the expression of group B and group C both increased and the increase in group B was more obvious(P<0.05).After adding JAK2 inhibitor AG490,it was found that the expression of p-STAT6 and p-JAK2 protein in group B increased compared with group A,and the protein content and mRNA expression of MUC5 B increased(P<0.05).Compared with group B,the protein content of p-STAT6 and p-JAK2 in group C decreased,and the protein content and mRNA expression of MUC5 B decreased(P<0.05).However,there was no significant change in the expression of non-phosphorylated STAT6 and JAK2 in each group.Conclusion:(1)IL-8 can stimulate the expression of MUC5 B in lung epithelial cells,which is related to the concentration and time.The optimal concentration of IL-8 is 160 ng/ml,and the optimal treatment time is 48 hours.(2)IL-8 may stimulate MUC5 B secretion through JAK2/STAT6 pathway.