Department of Emergency, Jiangsu Province Hospital, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Department of Emergency, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing, China.
Oxid Med Cell Longev. 2020 Jul 16;2020:7028947. doi: 10.1155/2020/7028947. eCollection 2020.
Paraquat (PQ), a widely used toxic herbicide, induces lung inflammation through mechanisms that remain incompletely understood. In a previous study, we found that the plasma MUC5B mucin level was implicated in PQ poisoning in patients. Here, we hypothesize that MUC5B is a critical mediator in PQ-induced cell inflammation.
A mouse model of PQ-induced lung injury was used to examine the MUC5B expression level. A549 cells (alveolar epithelial cells line) were exposed to PQ in dose-dependent and time-dependent manners. Cell viability was detected by CCK-8 assays. The expression levels of MUC5B were examined by dot blot enzyme-linked immunosorbent assay (ELISA) and RT-qPCR. Western blotting was used to detect the levels of proteins in the MAPK and NF-B pathways. Inflammatory factors in the cell culture medium were measured by ELISA. NF-B and MAPK pathway inhibitors and MUC5B siRNA (siMUC5B) were used to determine the function of MUC5B. Finally, N-acetyl-cysteine (NAC) was added and its regulatory effect on the MAPK-NF-B-MUC5B pathway was examined in PQ-induced cell inflammation.
MUC5B was significantly upregulated accompanying the increases in TNF- and IL-6 secretion following PQ treatment in mouse and also in A549 cells after treatment with 50 M PQ at 24 hours. Furthermore, MAPK and NF-B pathway inhibitors could dramatically decrease the expression of MUC5B and the secretion of TNF- and IL-6. Importantly, siMUC5B could significantly attenuate the secretion of TNF- and IL-6 induced by PQ. As expected, the addition of NAC efficiently suppresses the TNF- and IL-6 secretion stimulated from PQ and also downregulated ERK, JNK, and p65 phosphorylation (ERK/JNK MAPK and NF-B pathways) as well as MUC5B expression.
Our findings suggest that MUC5B participates in the process of PQ-induced cell inflammation and is downstream of the NF-B and MAPK pathways. NAC can attenuate PQ-induced cell inflammation at least in part by suppressing the MAPK-NF-B-MUC5B pathway. These results nominate MUC5B as a new biomarker and therapeutic target for PQ-induced lung inflammation.
百草枯（PQ）是一种广泛使用的有毒除草剂，其诱导肺炎症的机制尚不完全清楚。在之前的研究中，我们发现血浆 MUC5B 粘蛋白水平与 PQ 中毒患者有关。在这里，我们假设 MUC5B 是 PQ 诱导细胞炎症的关键介质。
使用 PQ 诱导的肺损伤小鼠模型来检测 MUC5B 的表达水平。用剂量和时间依赖性方式将 A549 细胞（肺泡上皮细胞系）暴露于 PQ 中。通过 CCK-8 测定法检测细胞活力。通过斑点酶联免疫吸附测定（ELISA）和 RT-qPCR 检测 MUC5B 的表达水平。用 Western blotting 检测 MAPK 和 NF-B 通路中的蛋白水平。通过 ELISA 测量细胞培养物中炎性因子的水平。使用 NF-B 和 MAPK 通路抑制剂和 MUC5B siRNA（siMUC5B）来确定 MUC5B 的功能。最后，在 PQ 诱导的细胞炎症中加入 N-乙酰半胱氨酸（NAC），并检测其对 MAPK-NF-B-MUC5B 通路的调节作用。
PQ 处理后，小鼠和 50μM PQ 处理 24 小时后的 A549 细胞中，MUC5B 明显上调，同时 TNF-α和 IL-6 的分泌也增加。此外，MAPK 和 NF-B 通路抑制剂可显著降低 MUC5B 的表达和 TNF-α和 IL-6 的分泌。重要的是，siMUC5B 可显著减轻 PQ 诱导的 TNF-α和 IL-6 的分泌。正如预期的那样，NAC 的添加可有效抑制 PQ 刺激的 TNF-α和 IL-6 的分泌，并下调 ERK、JNK 和 p65 磷酸化（ERK/JNK MAPK 和 NF-B 途径）以及 MUC5B 的表达。
我们的研究结果表明，MUC5B 参与了 PQ 诱导的细胞炎症过程，是 NF-B 和 MAPK 途径的下游。NAC 至少可以通过抑制 MAPK-NF-B-MUC5B 途径来减轻 PQ 诱导的细胞炎症。这些结果将 MUC5B 作为 PQ 诱导的肺炎症的新生物标志物和治疗靶标。
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