Lee Eun Jung, Song Kee Jae, Kwon Jin Ho, Park Ah Young, Jo Kwang-Hee, Kim Kyung-Su
Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, Korea.
Am J Rhinol Allergy. 2014 May-Jun;28(3):e125-9. doi: 10.2500/ajra.2014.28.4037.
We recently showed that acute cholesterol depletion in the plasma membrane of NCI-H292 cells by methyl-β-cyclodextrin suppressed IL-1beta-induced MUC5AC gene expression. Because cholesterol depletion is clinically used as an antihypersecretory method, chronic cholesterol depletion by lovastatin is more rational and safe than acute depletion. Therefore, we sought to investigate whether chronic cholesterol depletion by lovastatin is feasible and, if so, suppresses the expression of GMUC5AC in NCI-H292 cells. We also considered whether this alteration of MUC5AC expression is related to IL-1 receptor and mitogen-activated protein kinase (MAPK) activity.
After NCI-H292 cells were pretreated with 10 μM of lovastatin for 1 hour, 10 ng/mL of IL-1β was added and cotreated with lovastatin for 24 hours. MUC5AC mRNA expression was then determined by real-time polymerase chain reaction. Cholesterol depletion by lovastatin was measured by modified microenzymatic fluorescence assay and filipin staining. The phosphorylation of IL-1 receptor, ERK, and p38 MAPK was analyzed by Western blot.
Cholesterol in the plasma membrane was significantly depleted by lovastatin treatment for 24 hours. IL-1beta0-induced MUC5AC mRNA expression was decreased by lovastatin and this decrease occurred IL-1 receptor specifically. Lovastatin suppressed the activation of p38 MAPK but not ERK1/2 in cells activated with IL-1beta. This result suggests that lovastatin-mediated suppression of IL-1beta-induced MUC5AC mRNA operated only viathe p38 MAPK-dependent pathway.
Chronic cholesterol depletion in the plasma membrane of NCI-H292 cells may be considered an antihypersecretory method, because it effectively inhibits mucin gene expression of human airway epithelial cells.
我们最近发现，用甲基-β-环糊精使NCI-H292细胞膜中的胆固醇急性耗竭，可抑制白细胞介素-1β（IL-1β）诱导的MUC5AC基因表达。由于胆固醇耗竭在临床上被用作一种抗分泌过多的方法，与急性耗竭相比，洛伐他汀导致的慢性胆固醇耗竭更合理、更安全。因此，我们试图研究洛伐他汀导致的慢性胆固醇耗竭是否可行，以及如果可行的话，是否能抑制NCI-H292细胞中GMUC5AC的表达。我们还考虑了MUC5AC表达的这种改变是否与IL-1受体和丝裂原活化蛋白激酶（MAPK）活性有关。
用10 μM洛伐他汀预处理NCI-H292细胞1小时后，加入10 ng/mL的IL-1β，并与洛伐他汀共同处理24小时。然后通过实时聚合酶链反应测定MUC5AC mRNA的表达。用改良的微酶荧光测定法和制霉菌素染色法检测洛伐他汀导致的胆固醇耗竭情况。通过蛋白质印迹法分析IL-1受体、细胞外信号调节激酶（ERK）和p38 MAPK的磷酸化情况。
洛伐他汀处理24小时后，细胞膜中的胆固醇显著耗竭。洛伐他汀可降低IL-1β诱导的MUC5AC mRNA表达，且这种降低具有IL-1受体特异性。在IL-1β激活的细胞中，洛伐他汀抑制p38 MAPK的激活，但不抑制ERK1/2的激活。这一结果表明，洛伐他汀介导的对IL-1β诱导的MUC5AC mRNA的抑制仅通过p38 MAPK依赖性途径起作用。
NCI-H292细胞膜中的慢性胆固醇耗竭可被视为一种抗分泌过多的方法，因为它能有效抑制人气道上皮细胞的黏蛋白基因表达。
