Background Human airway smooth muscle cells (ASMCs) contribute to bronchial contraction and airway hyperresponsiveness in patients with bronchial asthma. They also generate cytokines, chemokines, and matricellular proteins. Ovarian cancer G protein-coupled receptor 1 (OGR1) senses extracellular protons and mediates the production of interleukin-6 (IL-6) and connective tissue growth factor (CTGF) in ASMCs. Methods ASMCs were stimulated for the indicated time by pH 6.3 or pH 7.4-adjusted Dulbecco's Modified Eagle Medium (DMEM) containing 0.1% bovine serum albumin (BSA) (0.1% BSA-DMEM). As a control stimulant, pH 7.4-adjusted 0.1% BSA-DMEM containing 10 ng/mL tumor necrosis factor-α (TNF-α) was used. Interleukin-8/C-X-C motif chemokine ligand 8 (CXCL8) mRNA expression in ASMCs was quantified by RT-PCR using real-time TaqMan technology. CXCL8 secreted from ASMCs was measured by enzyme-linked immunosorbent assay (ELISA). Phosphorylation at serine 536 of NF-κB p65 and binding of p65 to oligonucleotide containing an NF-κB consensus binding site were analyzed by Western blotting and an ELISA-based kit. Results Acidic pH induced a significant increase of CXCL8 mRNA expression and CXCL8 protein secretion in ASMCs. ASMCs transfected with small interfering RNA (siRNA) targeted for OGR1 produced less CXCL8 compared with those transfected with non-targeting siRNA. Protein kinase C (PKC) inhibitor, MEK1/2 inhibitor, and the inhibitor of IκB phosphorylation reduced acidic pH-stimulated CXCL8 production in ASMCs. Dexamethasone also inhibited acidic pH-stimulated CXCL8 production of ASMCs in a dose-dependent manner. Dexamethasone did not affect either phosphorylation or binding to the consensus DNA site of NF-κB p65. Conclusions CXCL8 released from ASMCs by extracellular acidification may play a pivotal role in airway accumulation of neutrophils. Glucocorticoids inhibit acidic pH-stimulated CXCL8 production independent of serine 536 phosphorylation and the binding to DNA of NF-κB p65, although NF-κB activity is essential for CXCL8 production in ASMCs.

中文翻译：

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细胞外酸化通过人气道平滑肌细胞中的质子感应受体 OGR1 诱导 CXCL8 产生：地塞米松抑制的反应。

背景 人气道平滑肌细胞 (ASMC) 有助于支气管哮喘患者的支气管收缩和气道高反应性。它们还产生细胞因子、趋化因子和基质细胞蛋白。卵巢癌 G 蛋白偶联受体 1 (OGR1) 感知细胞外质子并介导 ASMC 中白细胞介素 6 (IL-6) 和结缔组织生长因子 (CTGF) 的产生。方法 用含有 0.1% 牛血清白蛋白 (BSA) (0.1% BSA-DMEM) 的 pH 6.3 或 pH 7.4 调节的 Dulbecco's Modified Eagle 培养基 (DMEM) 刺激 ASMCs 指定的时间。作为对照兴奋剂，使用含有 10 ng/mL 肿瘤坏死因子-α (TNF-α) 的 pH 7.4 调整的 0.1% BSA-DMEM。使用实时 TaqMan 技术通过 RT-PCR 定量 ASMC 中的白细胞介素 8/CXC 基序趋化因子配体 8 (CXCL8) mRNA 表达。通过酶联免疫吸附试验（ELISA）测量从 ASMC 分泌的 CXCL8。通过蛋白质印迹和基于 ELISA 的试剂盒分析 NF-κB p65 丝氨酸 536 处的磷酸化和 p65 与含有 NF-κB 共有结合位点的寡核苷酸的结合。结果酸性pH诱导ASMC中CXCL8 mRNA表达和CXCL8蛋白分泌显着增加。与用非靶向 siRNA 转染的相比，用靶向 OGR1 的小干扰 RNA (siRNA) 转染的 ASMC 产生的 CXCL8 较少。蛋白激酶 C (PKC) 抑制剂、MEK1/2 抑制剂和 IκB 磷酸化抑制剂降低了 ASMC 中酸性 pH 刺激的 CXCL8 产生。地塞米松还以剂量依赖性方式抑制酸性 pH 刺激的 CXCL8 产生 ASMC。地塞米松不影响磷酸化或与 NF-κB p65 共有 DNA 位点的结合。结论 ASMCs 胞外酸化释放的 CXCL8 可能在气道中性粒细胞积聚中起关键作用。糖皮质激素抑制酸性 pH 刺激的 CXCL8 产生，独立于丝氨酸 536 磷酸化和与 NF-κB p65 的 DNA 结合，尽管 NF-κB 活性对于 ASMC 中 CXCL8 的产生是必不可少的。