MAPK通路参与鼻息肉上皮细胞中糖皮质激素受体的体外调控研究

Involvement of MAPK signal transduction pathways in regulating the expression of glucocorticoid receptor isoforms in nasal polyp epithelia in vitro

目的通过体外研究初步探讨鼻息肉黏膜中糖皮质激素受体（glucocorticoid receptor，GR）亚型GRα／GRβ比例降低的上游信号转导机制。方法以细菌脂多糖（lipopolysaccharide，LPS）体外诱导人鼻息肉黏膜上皮细胞（human nasal epithelia，HNE）构建糖皮质激素抵抗的细胞模型。以Western Blot及荧光定量反转录聚合酶链反应技术检测梯度浓度的LPS诱导前后及MAPK家族信号通路特异性阻断剂干预前后GR仪、GRβ和p38MAPK、ERK、JNK信号通路关键酶在蛋白质及核酸水平的表达变化。以SPSS16．0软件对诱导和干预前后的表达情况采用单因素方差分析。结果LPS呈浓度和时间依赖性诱导HNE中GRα／GRβ降低，成功建立GRa／GRβ降低的细胞模型。相同浓度梯度的LPS可呈相同规律地诱导HNE中p38MAPK、ERK、JNK信号通路激活。5、10、15mg／LLPS诱导的p38MAPK、ERK和JNK在mRNA水平的相对表达量（17．14±1．50、22．34±2．78、30．12±1．07，2．51±0．13、3．79±0．67、4．41±0．83，25．62±1．77、31．33±1．97、37．25±2．46）均明显高于对照组（7．39±0．31、2．04±0．34、2．38±0．35），差异有统计学意义（χ2值分别为15．347、18．331、14．671，P值均〈0．01）。p38MAPK通路的特异性阻断剂SB203580和JNK通路的特异性阻断剂SP600125均可明显抑制各自信号通路，且同时出现GRα／GRβ增高，SB203580干预后，GRa／GRβ增加更为明显。ERK通路特异性阻断剂PD98059干预后，GRα／GRβ未发现变化。结论LPS可能通过激活p38MAPK和JNK信号通路诱导体外培养的HNE中GRoVGRβ降低，其中p38MAPK通路较为主要，针对上述信号通路的调节可能有助于改善糖皮质激素抵抗性鼻息肉的治疗效果。

Objective To explore the upstream signal transduction mechanism responsible for the decrease of the ratio of the two glucocorticoid receptor （GR） subunits （ GRα and GRβ） in nasal polyp in vitro. Methods The GRα/GRβ decrease cell model was established by lipopolysaccharide （LPS）-induced human nasal epithelia （HNE） of nasal polyp in vitro. Changes in the protein and mRNA expression of GRα, GRβ and the key enzymes in the p38MAPK, ERK and JNK signal pathways were measured, respectively, before and after being induced with different doses of LPS and specific inhibitors of p38MAPK, JNK and ERK. SPSS 16.0 software （Analysis of variance, ANOVA） was used to analyze the data. Results With the LPS induction, the GRa/GR[3 ratio declined in both a time-dependent manner and a concentrationdependent manner in HNE, which demonstrated the successful establishment of a GRα/GRβ decrease model in vitro. After cultured HNE were induced with the same set of LPS, the p38MAPK, ERK and JNK signal pathways were also activated. The mRNA expression of p38MAPK and JNK in each LPS-induced group （17. 14±1.50, 22.34±2.78, 30.12±1.07; 2.51±0.13, 3.79±0.67, 4.41 ±0.83; 25.62±1.77, 31.33 ±1.97, 37.25± 2.46 ） was significantly higher than that （ 7.39 ± 0.31,2.04 ± 0.34, 2.38±0.35）in the control group （χ2 value was 15. 347, 18. 331, 14. 671, all P 〈 0.01 ）. Either a specific inhibitor （SB203580） of the p38MAPK pathway or a specific inhibitor （SP600125） of the JNK pathway increased the GRα/GRβ ratio at the meantime of inhibiting their pathways. SB203580 exhibited a much stronger increase effect on GRα/GRβ ratio than SP600125. The specific inhibitors （PD98059） of ERK had no influence on the expression of GR isoforms. Conclusions The above results demonstrated that the decrease of GRα/GRβ ratio in HNE induced by LPS in vitro is mediated through the p38MAPK and JNK signal pathways. It is possible to improve the treatment effect of GC resistance in nasal polyp by targeting these specific signal pathways.