Department of Physiology & Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA.
Int J Mol Sci. 2023 Oct 31;24(21):15816. doi: 10.3390/ijms242115816.
Airway inflammation and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) underlie the pathophysiology of respiratory diseases, including asthma. Previously, we showed that TNFα activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 spliced (XBP1s) endoplasmic reticulum (ER) stress pathway in human airway smooth muscle (hASM) cells. The ER stress pathway is activated by the accumulation of unfolded proteins in the ER. Accordingly, chemical chaperones such as 4-phenylbutyric acid (4-PBA) may reduce ER stress activation. In the present study, we hypothesized that chemical chaperone 4-PBA mitigates TNFα-induced ER stress in hASM cells. hASM cells were isolated from bronchiolar tissue obtained from five patients with no history of smoking or respiratory diseases. The hASM cells' phenotype was confirmed via the expression of alpha-smooth muscle actin and elongated morphology. hASM cells from the same patient sample were then separated into three 12 h treatment groups: (1) TNFα (20 ng/mL), (2) TNFα + 4-PBA (1 μM, 30 min pretreatment), and (3) untreated control. The expressions of total IRE1α and phosphorylated IRE1α (pIRE1α) were determined through Western blotting. The splicing of mRNA was analyzed using RT-PCR. We found that TNFα induced an increase in pIRE1α phosphorylation, which was mitigated by treatment with chemical chaperone 4-PBA. We also found that TNFα induced an increase in mRNA, which was also mitigated by treatment with chemical chaperone 4-PBA. These results support our hypothesis and indicate that chemical chaperone 4-PBA treatment mitigates TNFα-induced ER stress in hASM cells.
气道炎症和促炎细胞因子，如肿瘤坏死因子 α（TNFα），是包括哮喘在内的呼吸道疾病的病理生理学基础。此前，我们已经证明 TNFα 在人气道平滑肌（hASM）细胞中激活肌醇需求酶 1α（IRE1α）/X 框结合蛋白 1 剪接（XBP1s）内质网（ER）应激途径。该 ER 应激途径是由 ER 中未折叠蛋白的积累而激活的。因此，化学伴侣如 4-苯基丁酸（4-PBA）可以减轻 ER 应激的激活。在本研究中，我们假设化学伴侣 4-PBA 可以减轻 TNFα 诱导的 hASM 细胞 ER 应激。从五个无吸烟史或呼吸道疾病史的患者的细支气管组织中分离出 hASM 细胞。通过表达α-平滑肌肌动蛋白和伸长的形态来确认 hASM 细胞的表型。然后，将同一患者样本的 hASM 细胞分为三组，每组 12 小时：（1）TNFα（20ng/mL），（2）TNFα+4-PBA（1μM，30 分钟预处理）和（3）未处理的对照。通过 Western blot 测定总 IRE1α 和磷酸化 IRE1α（pIRE1α）的表达。使用 RT-PCR 分析 mRNA 的剪接。我们发现，TNFα 诱导 pIRE1α 磷酸化增加，而化学伴侣 4-PBA 处理减轻了这种增加。我们还发现，TNFα 诱导 mRNA 增加，而化学伴侣 4-PBA 处理也减轻了这种增加。这些结果支持我们的假设，并表明化学伴侣 4-PBA 处理减轻了 TNFα 诱导的 hASM 细胞 ER 应激。
