Guangdong Provincial Key Laboratory of Proteomics; School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China.
Department of Critical Care Medicine, General Hospital of Southern Theatre Command of PLA, Guangzhou, 510010, China.
BMC Cardiovasc Disord. 2020 May 24;20(1):242. doi: 10.1186/s12872-020-01529-7.
Cardiac fibroblasts, regarded as the immunomodulatory hub of the heart, have been thought to play an important role during sepsis-induced cardiomyopathy (SIC). However, the detailed molecular mechanism and targeted therapies for SIC are still lacking. Therefore, we sought to investigate the likely protective effects of rolipram, an anti-inflammatory drug, on lipopolysaccharide (LPS)-stimulated inflammatory responses in cardiac fibroblasts and on cardiac dysfunction in endotoxic mice.
Cardiac fibroblasts were isolated and stimulated with 1 μg/ml LPS for 6 h, and 10 μmol/l rolipram was administered for 1 h before LPS stimulation. mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in fibroblasts and their protein concentrations in supernatant were measured with real-time PCR (rt-PCR) and enzyme-linked immunosorbent assay, respectively. The expression of dual specificity phosphatase 1 (DUSP1), an endogenous negative regulator that inactivates MAPK-mediated inflammatory pathways, was also measured by rt-PCR and western blotting. DUSP1-targeted small interfering RNA (siRNA) was used to examine the specific role of DUSP1. To evaluate the role of rolipram in vivo, an endotoxic mouse model was established by intraperitoneal injection of 15 mg/kg LPS, and 10 mg/kg rolipram was intraperitoneally injected 1 h before LPS injection. mRNA and protein levels of inflammatory cytokines and DUSP1 in heart, inflammatory cell infiltration and cardiac function were all examined at 6 h after LPS injection.
The results showed that LPS could increase the expression and secretion of inflammatory cytokines and decrease the transcription and expression of DUSP1 in cardiac fibroblasts. However, rolipram pretreatment significantly reversed the LPS-induced downregulation of DUSP1 and inhibited LPS-induced upregulation and secretion of TNF-α and IL-6 but not IL-1β. Moreover, DUSP1-targeted siRNA experiments indicated that the protective effect of rolipram on inflammatory response was specific dependent on DUSP1 expression. Moreover, rolipram could further reduce inflammatory cell infiltration scores as shown by pathological analysis and increase the ejection fraction (EF) detected with echocardiography in the hearts of endotoxic mice.
Rolipram could improve endotoxin-induced cardiac dysfunction by upregulating DUSP1 expression to inhibit the inflammatory response in cardiac fibroblasts, which may be a potential treatment for SIC.
心脏成纤维细胞被认为是心脏的免疫调节中心，在脓毒症性心肌病（SIC）中发挥着重要作用。然而，SIC 的详细分子机制和靶向治疗方法仍有待研究。因此，我们试图研究罗利普兰（一种抗炎药物）对脂多糖（LPS）刺激的心肌成纤维细胞炎症反应和内毒素血症小鼠心脏功能障碍的可能保护作用。
分离培养心脏成纤维细胞，用 1μg/ml LPS 刺激 6h，在 LPS 刺激前 1h 给予 10μmol/L 罗利普兰。实时 PCR（rt-PCR）和酶联免疫吸附试验（ELISA）分别检测成纤维细胞中肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和白细胞介素-1β（IL-1β）的 mRNA 水平及其上清液中的蛋白浓度。还通过 rt-PCR 和 Western blot 检测双特异性磷酸酶 1（DUSP1）的表达，DUSP1 是一种内源性负调节因子，可使 MAPK 介导的炎症途径失活。用 DUSP1 靶向小干扰 RNA（siRNA）检测 DUSP1 的特异性作用。为了评估罗利普兰在体内的作用，通过腹腔内注射 15mg/kg LPS 建立内毒素血症小鼠模型，在 LPS 注射前 1h 腹腔内注射 10mg/kg 罗利普兰。注射 LPS 6h 后，检测心脏中炎症细胞因子和 DUSP1 的 mRNA 和蛋白水平、炎症细胞浸润和心脏功能。
结果表明，LPS 可增加心脏成纤维细胞中炎症细胞因子的表达和分泌，降低 DUSP1 的转录和表达。然而，罗利普兰预处理可显著逆转 LPS 诱导的 DUSP1 下调，并抑制 LPS 诱导的 TNF-α和 IL-6的上调和分泌，但不抑制 IL-1β。此外，DUSP1 靶向 siRNA 实验表明，罗利普兰对炎症反应的保护作用特异性依赖于 DUSP1 的表达。此外，罗利普兰还可以通过病理分析进一步降低内毒素血症小鼠心脏中的炎症细胞浸润评分，并增加超声心动图检测的射血分数（EF）。
罗利普兰可通过上调 DUSP1 表达抑制心肌成纤维细胞炎症反应，改善内毒素诱导的心脏功能障碍，可能是 SIC 的潜在治疗方法。
