<br>RNA-seq揭示了苏拉明通过抑制LPS诱导的MH-S肺泡巨噬细胞中的NLRP3/caspase-1/GSDMD通路来抗焦亡作用,Gene

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RNA-seq揭示了苏拉明通过抑制LPS诱导的MH-S肺泡巨噬细胞中的NLRP3/caspase-1/GSDMD通路来抗焦亡作用
Gene ( IF 2.6 ) Pub Date : 2023-10-13 , DOI: 10.1016/j.gene.2023.147888
Yuhui Zhu ₁ , Zhen Wang ₁ , Jungang Zheng ₁ , Jun Wang ₁ , Yijun Chen ₁ , Changshun Huang ₁ , Haidong Zhou ₁

Affiliation

背景

急性肺损伤（ALI）/急性呼吸窘迫综合征（ARDS）是一种常见的脓毒症相关器官损伤，会引起不受控制和自我放大的肺部炎症。由于临床上缺乏有效的治疗方法，其死亡率仍然很高。苏拉明（SUR）最初作为一种抗寄生虫药物，在我们之前的工作中被发现可以改善脓毒症相关的急性肺损伤。然而，其保护作用的潜在机制尚未阐明。焦亡被归类为程序性细胞死亡的炎症形式，可以通过诱导肺泡巨噬细胞（AM）焦亡来加剧肺部炎症反应。

方法

用或不用脂多糖（LPS）或苏拉明刺激MH-S AM细胞系，并使用RNA测序（RNA-seq）挖掘差异表达基因（DEG）。为了确定这些基因的调节作用，进行了细胞焦亡相关基因（PRG）、GO/KEGG 和 GSEA 分析。我们还进行了WB、qRTPCR和ELISA来验证RNA-seq结果并进一步阐明苏拉明的保护作用。

结果

在对照（CON）和脂多糖（LPS）组之间鉴定出 624 个 DEG，这些基因的富集分析揭示了与免疫系统和信号转导相关的显着富集的通路。同时，在LPS/SUR+LPS组中鉴定出500个DEG。除上述通路外，还丰富了IL-17通路和C型凝集素受体信号通路。所有 6 条途径均与细胞焦亡有关。同时，“DESeq2”R 包用于识别差异表达的 PRG。 Nod1、Nod2、白细胞介素 (IL)-1b、IL-6、肿瘤坏死因子 (TNF)、NLRP3 在 LPS 刺激下表达上调。然后，在SUR+LPS组中，Nod2、IL-6、IL-1b、NLRP3下调。 WB、qRT-PCR和ELISA验证结果显示：苏拉明和LPS处理后，NLRP3、caspase-1、GSDMD的蛋白和mRNA表达水平以及IL-1b、IL-18的浓度降低。

结论

Suramin 可以抑制 LPS 诱导的 MH-S 肺泡巨噬细胞中的 NLRP3/caspase-1/GSDMD 经典焦亡途径。

"点击查看英文标题和摘要"

RNA-seq revealed the anti-pyroptotic effect of suramin by suppressing NLRP3/caspase-1/GSDMD pathway in LPS-induced MH-S alveolar macrophages

Background

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), acting as one common sepsis-associated organ injury, induces uncontrolled and self-amplifies pulmonary inflammation. Given the lack of clinically effective approaches, the mortality rate of it still remains high. Suramin(SUR), as an antiparasitic drug initially, was found to ameliorate sepsis associated ALI in our previous work. However, the underlying mechanism of its protective effects has not been clarified. Pyroptosis, categorized as an inflammatory form of programmed cell death, could aggravate lung inflammatory responses via inducing alveolar macrophages (AM) pyroptosis.

Methods

MH-S AM cell line was stimulated with or without lipopolysaccharide (LPS) or suramin, and the differential expression genes (DEGs) were excavated using RNA sequencing (RNA-seq). To identify the regulatory roles of these genes, pyroptosis-related genes (PRGs), GO/KEGG and GSEA analysis were conducted. We also performed WB, qRTPCR and ELISA to validate the RNA-seq results and further expound the protective effect of suramin.

Results

624 DEGs were identified between control (CON) and lipopolysaccharide (LPS) groups, and enrichment analysis of these genes revealed significantly enriched pathways that related to immune system and signal transduction. Meanwhile, 500 DEGs were identified in LPS/SUR+LPS group. In addition to the pathways mentioned above, IL-17 pathway and C-type lectin receptor signaling pathway were also enriched. All 6 pathways were connected with pyroptosis. Concurrently, the “DESeq2” R package was used to identify differentially expressed PRGs. Nod1, Nod2, interleukin (IL)-1b, IL-6, tumor necrosis factor (TNF), NLRP3 were upregulated under LPS stimulation. Then, in SUR+LPS group, Nod2, IL-6, IL-1b, NLRP3 were downregulated. The validation results of WB, qRT-PCR, and ELISA showed: the protein and mRNA expression levels of NLRP3, caspase-1, GSDMD and the concentrations of IL-1b, IL-18 were decreased when treated with suramin and LPS.