Supplementary Material for: Interleukin-11 Is Elevated in Patients with Atrial Fibrillation, Correlates with Serum Fibrosis Markers, and Represents a Therapeutic Target for Atrial Fibrosis

Introduction: Inflammatory cytokines are closely associated with developing cardiac fibrosis. This research aimed to explore the significant role of IL-11 in atrial fibrosis progression and potential therapeutic targets. Methods: 207 AF patients and 160 healthy subjects were included in the case-control study. Blood samples were analyzed for the level of IL-11 by enzyme-linked immunosorbent assay (ELISA). Angiotensin II (Ang II)-treated fibrosis mouse models were generated, and expression of IL-11 mRNA and protein was detected by RT-qPCR and Western blot. IL-11 antagonist was used to evaluating atrial fibrosis-related markers. Results: The persistent atrial fibrillation patients (n = 76) had significantly larger left atrial size, higher serum levels of hypertrophic protein BNP, proinflammatory cytokine high-sensitivity C-reactive protein (hs-CRP), and interleukin-6 (IL-6) compared to paroxysmal atrial fibrillation patients (n = 131), and healthy subjects (all p < 0.05). Pearson correlation analysis revealed significant positive correlation between serum IL-11 and cardiac fibrosis markers BNP (r = 0.394, p < 0.001), CTX-I (r = 0.418, p < 0.001), PICP (r = 0.306, p < 0.001), PIIINP (r = 0.335, p < 0.001), and TGF-β1 (r = 0.273, p < 0.001). In the fibrosis mouse model, Ang II infusion significantly upregulated IL-11 mRNA and protein expression in the left atrium of mice (p < 0.05), as well as staining intensity of Masson trichrome, the intensity of α-SMA, and it increased mRNA expression of collagen I and III in atrial tissue. IL-11 antagonist treatment significantly attenuated Masson trichrome, number of α-SMA-positive myofibroblasts in atrial tissue. Also, it significantly reduced the p-ERK1/2 in atrial tissue of mice infused with Ang II (p < 0.05). Conclusions: IL-11 is upregulated in the serum of AF patients, and IL-11 inhibitor significantly inhibited Ang II-induced atrial fibrosis, a key pathological feature of AF. Therefore, IL-11 could be a potential therapeutic target for AF.

引言：炎症细胞因子与心脏纤维化的发生发展密切相关。本研究旨在探讨白细胞介素-11（IL-11）在心房纤维化进展中的重要作用及其潜在治疗靶点。
方法：本病例对照研究纳入了207例心房颤动（Atrial Fibrillation, AF）患者及160名健康受试者。采用酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）检测血液样本中IL-11的水平。构建血管紧张素II（Angiotensin II, Ang II）诱导的纤维化小鼠模型，通过逆转录实时定量聚合酶链反应（RT-qPCR）和蛋白质印迹法（Western blot）检测IL-11的mRNA及蛋白表达水平。使用IL-11拮抗剂评估心房纤维化相关标志物的表达情况。
结果：与阵发性心房颤动患者（n=131）及健康受试者相比，持续性心房颤动患者（n=76）的左心房内径显著更大，血清肥厚标志物脑钠肽（B-type natriuretic peptide, BNP）、促炎细胞因子高敏C反应蛋白（high-sensitivity C-reactive protein, hs-CRP）及白细胞介素-6（IL-6）水平均显著升高（所有p<0.05）。Pearson相关分析显示，血清IL-11与心脏纤维化标志物BNP（r=0.394，p<0.001）、I型胶原C端肽（CTX-I，r=0.418，p<0.001）、I型前胶原羧基端肽（PICP，r=0.306，p<0.001）、III型前胶原氨基端肽（PIIINP，r=0.335，p<0.001）及转化生长因子β1（Transforming Growth Factor-β1, TGF-β1）均呈显著正相关。在纤维化小鼠模型中，Ang II输注可显著上调小鼠左心房组织中IL-11的mRNA及蛋白表达水平（p<0.05），同时升高心房组织的马松三色染色法（Masson trichrome）强度、α平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）染色强度，并上调I型及III型胶原的mRNA表达水平。IL-11拮抗剂治疗可显著减弱心房组织的马松三色染色强度，减少α-SMA阳性肌成纤维细胞数量。同时，该治疗可显著降低Ang II输注小鼠心房组织中的磷酸化细胞外调节蛋白激酶1/2（p-ERK1/2）水平（p<0.05）。
结论：心房颤动患者血清中IL-11表达上调，IL-11抑制剂可显著抑制Ang II诱导的心房纤维化——心房颤动的关键病理特征。因此，IL-11有望成为心房颤动的潜在治疗靶点。