Department of Rheumatology and Immunology, Huadong Hospital Affiliated to Fudan University, Shanghai, China.
Clin Rheumatol. 2021 Oct;40(10):4143-4155. doi: 10.1007/s10067-021-05755-y. Epub 2021 May 5.
Intestinal Behçet's syndrome (IBS) has high morbidity and mortality rates with serious complications. The purpose of this study was to investigate the expression of pyroptosis-related proteins in the intestinal tissues of IBS patients and explore the role of plasma exosomes derived from IBS patients in the pyroptosis of intestinal epithelial cells.
Immunohistochemistry was used to investigate the expression of nucleotide-binding domain-like receptor protein 3 (NLRP3), caspase-1, and gasdermin D (GSDMD). Quantitative real-time PCR was employed to measure the mRNA levels of IL-1β and IL-18 in the intestinal tissues. Plasma exosomes were isolated and observed by transmission electron microscopy. The exosomes were co-cultured with intestinal epithelial cells in vitro. Western blot was used to measure the expression of pyroptosis-related proteins including NLRP3, full-length GSDMD, N-terminal GSDMD, pro-caspase-1, and cleaved caspase-1. The levels of IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay. Cell death was measured by using the lactate dehydrogenase (LDH) release assay.
Expression of NLRP3 (12.2% ± 1.2%, 8.1% ± 0.9%, t = 4.692, p = 0.009), caspase-1 (24.6% ± 2.1%, 4.2% ± 1.8%, t = 12.842, p = 0.000), and GSDMD (16.6% ± 1.9%, 9.8% ± 1.3%, t = 5.194, p = 0.007) were significantly increased in the intestinal tissues of patients with IBS compared with normal control (NC) group, respectively. The relative mRNA levels of IL-1β (t = 4.308, p = 0.005) and IL-18 (t = 3.096, p = 0.021) in the intestinal tissues were significantly higher in IBS patients than in NC group, while the protein levels of IL-1β (t = 3.873, p = 0.018) and IL-18 (t = 4.389, p = 0.012) were also significantly increased, which was consistent with the results of the relative mRNA levels. Moreover, we found that exosomes from IBS patients significantly induced pyroptosis of intestinal epithelial cells via the activation of NLRP3 inflammasome in vitro experiments.
Plasma exosomes derived from IBS patients may induce pyroptosis of intestinal epithelial cells via the activation of NLRP3 inflammasome. Key Points •The role of exosomes in IBS is first reported in this study. • In this study, we explored the mechanism that plasma exosomes derived from IBS patients may induce pyroptosis of intestinal epithelial cells via the activation of NLRP3 inflammasome.
肠型贝赫切特综合征（IBS）发病率和死亡率高，且并发症严重。本研究旨在探讨 IBS 患者肠组织中细胞焦亡相关蛋白的表达，并探索 IBS 患者血浆外泌体在诱导肠上皮细胞焦亡中的作用。
采用免疫组织化学法检测核苷酸结合寡聚化结构域样受体蛋白 3（NLRP3）、半胱氨酸天冬氨酸蛋白酶-1（caspase-1）和 Gasdermin D（GSDMD）的表达。采用实时定量 PCR 法检测肠组织中白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）的 mRNA 水平。采用透射电子显微镜观察并分离血浆外泌体。将外泌体与体外培养的肠上皮细胞共培养。采用 Western blot 法检测细胞焦亡相关蛋白 NLRP3、全长 GSDMD、N 端 GSDMD、原 caspase-1 和裂解 caspase-1 的表达。采用酶联免疫吸附试验检测 IL-1β和 IL-18 的水平。采用乳酸脱氢酶（LDH）释放试验检测细胞死亡情况。
与正常对照组（NC 组）相比，IBS 患者肠组织中 NLRP3（12.2%±1.2%比 8.1%±0.9%，t=4.692，p=0.009）、caspase-1（24.6%±2.1%比 4.2%±1.8%，t=12.842，p=0.000）和 GSDMD（16.6%±1.9%比 9.8%±1.3%，t=5.194，p=0.007）的表达明显升高。IBS 患者肠组织中 IL-1β（t=4.308，p=0.005）和 IL-18（t=3.096，p=0.021）的相对 mRNA 水平明显高于 NC 组，IL-1β（t=3.873，p=0.018）和 IL-18（t=4.389，p=0.012）的蛋白水平也明显升高，与相对 mRNA 水平结果一致。此外，我们发现 IBS 患者的外泌体在体外实验中通过激活 NLRP3 炎性小体显著诱导肠上皮细胞发生细胞焦亡。
IBS 患者的血浆外泌体可能通过激活 NLRP3 炎性小体诱导肠上皮细胞发生细胞焦亡。关键点• 本研究首次报道了外泌体在 IBS 中的作用。• 本研究探讨了 IBS 患者血浆外泌体可能通过激活 NLRP3 炎性小体诱导肠上皮细胞发生细胞焦亡的机制。
