The First Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510405, PR China; Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510405, PR China; Guangzhou Key Laboratory of Chinese Medicine for Prevention and Treatment of Chronic Heart Failure, Guangzhou 510405, PR China.
The First Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510405, PR China; Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510405, PR China; Guangzhou Key Laboratory of Chinese Medicine for Prevention and Treatment of Chronic Heart Failure, Guangzhou 510405, PR China; Huizhou Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510405, PR China.
Phytomedicine. 2022 Jul;101:154093. doi: 10.1016/j.phymed.2022.154093. Epub 2022 Mar 30.
Heart failure (HF) is a leading cause of death worldwide. Nuanxinkang (NXK) is an effective Chinese herbal formula used in treating HF, but its underlying potential mechanisms have not been fully elucidated.
To explore the protective activities of NXK in ischemia/reperfusion (IR)-induced HF through modulating the ratio of proinflammatory (M1) and anti-inflammatory (M2) macrophage populations and leading to the alleviation of inflammation.
In vivo, mice were subjected to myocardial IR to generate HF mouse models. Mice in the NXK group were treated with NXK for 28 days. Cardiac function was detected by echocardiography. Major lesions on mouse hearts were determined by hematoxylin-eosin (HE) staining, Masson staining, and TUNEL staining. Inflammatory cytokines were determined by enzyme-linked immunosorbent assay (ELISA) and qPCR examination. Flow cytometric analyses and qPCR examination were utilized for monitoring the temporal dynamics of macrophage infiltration following IR. In vitro, two polarized models were established by stimulating RAW264.7 cells with 200 ng/ml lipopolysaccharide (LPS) or 20 ng/ml interleukin-4 (IL-4). The RAW264.7 cells with nuclear factor-κB (NF-κB) overexpression was generated by transient transfection of NF-κB plasmids, and NXK intervention was conducted on this cell model to further clarify the involvement of NF-κB signaling in the NXK-mediated HF process.
In the present study, NXK was found to significantly contribute the cardiac function and ameliorate cardiac fibrosis and apoptosis after myocardial IR injury in vivo, which may be partially due to a decrease in inflammation. We therefore hypothesized that NXK reduced inflammatory damage by modulating subtypes of macrophages. And the results demonstrated that the percentage of proinflammatory macrophages infiltrated in the post-IR period was reduced with NXK treatment, and thereby blunting the wave of proinflammatory response and shifting the peak of the anti-inflammatory macrophage-mediated wound healing process towards an earlier time point. The further investigation showed that macrophage polarization was mediated by NXK through inhibiting the phosphorylation and the nuclear translocation of NF-κB. Besides, the phosphorylated IKKβ and IκBα, upstream mediators of the NF-κB pathway, also decreased by NXK. Moreover, the overexpression of NF-κB partially reversed the NXK-induced favorable activities; and successfully compensated the suppressive effect on inflammation and the phosphorylation of NF-κB.
In conclude, our results demonstrated that NXK induced the cardioprotective effects against IR injury through a regulatory axis of IKKβ/IκBα/NF-κB-mediated macrophage polarization. The information gained from this study provide a possible natural strategy for anti-inflammatory treatment of HF.
心力衰竭（HF）是全球范围内主要的死亡原因。 参附康（NXK）是一种用于治疗 HF 的有效的中药方剂，但它的潜在机制尚未完全阐明。
通过调节促炎（M1）和抗炎（M2）巨噬细胞群体的比例，减轻炎症，探讨 NXK 在缺血/再灌注（IR）诱导的 HF 中的保护作用。
体内，用 IR 使小鼠产生 HF 模型，NXK 组小鼠用 NXK 治疗 28 天。用超声心动图检测心功能。用苏木精-伊红（HE）染色、Masson 染色和 TUNEL 染色确定小鼠心脏的主要病变。用酶联免疫吸附试验（ELISA）和 qPCR 检测炎症细胞因子。流式细胞术分析和 qPCR 检测用于监测 IR 后巨噬细胞浸润的时间动态。体外，用 200ng/ml 脂多糖（LPS）或 20ng/ml 白细胞介素 4（IL-4）刺激 RAW264.7 细胞建立两种极化模型。通过瞬时转染 NF-κB 质粒生成核因子-κB（NF-κB）过表达的 RAW264.7 细胞，并在该细胞模型上进行 NXK 干预，以进一步阐明 NF-κB 信号在 NXK 介导的 HF 过程中的作用。
本研究发现，NXK 可显著改善体内心肌 IR 损伤后的心脏功能，减轻心肌纤维化和细胞凋亡，这可能部分归因于炎症的减轻。因此，我们假设 NXK 通过调节巨噬细胞亚群减少炎症损伤。结果表明，用 NXK 治疗后，IR 后浸润的促炎巨噬细胞比例降低，从而减弱了促炎反应波，将抗炎巨噬细胞介导的愈合过程的峰值推向更早的时间点。进一步的研究表明，巨噬细胞极化是通过 NXK 抑制 NF-κB 的磷酸化和核转位来介导的。此外，NF-κB 通路的上游介质磷酸化 IKKβ 和 IκBα 也被 NXK 降低。此外，NF-κB 的过表达部分逆转了 NXK 诱导的有利作用，并成功补偿了对炎症的抑制作用和 NF-κB 的磷酸化。
总之，我们的研究结果表明，NXK 通过 IKKβ/IκBα/NF-κB 介导的巨噬细胞极化调节轴诱导对 IR 损伤的心脏保护作用。本研究获得的信息为 HF 的抗炎治疗提供了一种可能的天然策略。
