Zhang Xiaoqing, Qu Huiyan, Yang Tao, Liu Qian, Zhao Dandan, Liu Wenrui, Wang Tian, Zhou Hua
Institute of Cardiovascular Disease of Integrated Traditional Chinese and Western Medicine, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Department of Cardiovascular Disease, ShuGuang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Evid Based Complement Alternat Med. 2022 Mar 11;2022:5867987. doi: 10.1155/2022/5867987. eCollection 2022.
Myocardial fibrosis caused by myocardial infarction (MI) is the key factor leading to cardiac remodeling; nod-like receptor family pyrin domain-containing 3 (NLRP3) plays an important role in regulation of myocardial injury; however, its relationship with TLR4/MyD88/NF-B signaling pathway is largely unreported. In recent years, traditional Chinese medicine (TCM) prevention and treatment of cardiovascular diseases has shown its unique advantages and broad application prospects. LuQi Formula (LQF) has been used for more than 20 years in Shuguang Hospital (Shanghai, China), and it was confirmed that it can improve the clinical symptoms of patients after MI. Here, we investigated the mechanism of LQF by suppressing NLRP3 inflammasome activation and TLR4/MyD88/NF-B pathway in mice with MI.
The purpose of this study was to verify the positive effects of the LQF in ameliorating myocardial fibrosis and inflammasome infiltration in the MI mice in vivo.
Forty mice were randomized into four groups: the sham group, the MI group, the LQF group, and the perindopril group ( = 10 per group). Left anterior descending (LAD) coronary artery ligation was performed in all groups except the sham group. The mice were treated with LQF after MI. After 4 weeks, LDH, cTnI, IL-1, and IL-18 were measured by enzyme-linked immunosorbent assay (ELISA) kit, and cardiac function was evaluated by echocardiography. Hematoxylin and eosin (H&E) and Masson staining were used to evaluate the myocardial injury and fibrosis. Western blot was used to evaluate the expression of collagen I, -SMA, NLRP3 inflammasome, and TLR4/MyD88/NF-B signaling pathway. Immunohistochemical analysis was used to further detect the expression of Fibronectin, -SMA, collagen I, collagen III, NLRP3, and NF-B in myocardial tissue.
Compared with the MI group, the ejection fraction (EF) and fractional shortening (FS) in the LQF group were significantly improved, while the left ventricular end diastolic diameter (LVEDd) and left ventricular internal dimension systole (LVIDs) were significantly decreased. The representative staining of H&E and Masson showed that treatment with LQF could effectively reduce myocardial injury and fibrosis. ELISA results showed that serum LDH, cTnI, TNF-, IL-18, and IL-1 in LQF group were significantly lower than those in MI group. The western blot results showed that the expressions of collagen I and -SMA were decreased significantly in the LQF group. Moreover, the expressions of NLRP3 inflammasome and TLR4/MyD88/NF-B signaling pathway were downregulated in the LQF treatment group.
Our results suggested that LQF could significantly improve cardiac function and ameliorate myocardial fibrosis. In addition, we found that LQF could downregulate the TLR4/MyD88/NF-B signaling pathway and then inhibit the activation of NLRP3 inflammasome, suggesting that LQF alleviated cardiac fibrosis by decreasing the TLR4/MyD88/NF-B signaling pathway and then inhibited NLRP3 inflammasome activation in MI mice, which indicates potential therapeutic effect of LQF on patients with MI.
心肌梗死（MI）所致心肌纤维化是导致心脏重塑的关键因素；含吡啶结构域的NOD样受体家族3（NLRP3）在心肌损伤调控中起重要作用；然而，其与Toll样受体4（TLR4）/髓样分化因子88（MyD88）/核因子κB（NF-κB）信号通路的关系在很大程度上尚未见报道。近年来，中医药防治心血管疾病已显示出独特优势和广阔应用前景。鹿芪方（LQF）在上海曙光医院应用20余年，已证实其可改善心肌梗死后患者的临床症状。在此，我们研究LQF通过抑制MI小鼠NLRP3炎性小体激活和TLR4/MyD88/NF-κB通路的机制。
本研究旨在验证LQF在改善MI小鼠体内心肌纤维化和炎性小体浸润方面的积极作用。
40只小鼠随机分为四组：假手术组、MI组、LQF组和培哚普利组（每组n = 10）。除假手术组外，其余各组均行左冠状动脉前降支（LAD）结扎术。MI后对小鼠进行LQF治疗。4周后，采用酶联免疫吸附测定（ELISA）试剂盒检测乳酸脱氢酶（LDH）、心肌肌钙蛋白I（cTnI）、白细胞介素-1（IL-1）和白细胞介素-18（IL-18），并通过超声心动图评估心功能。采用苏木精-伊红（H&E）染色和Masson染色评估心肌损伤和纤维化。采用蛋白质印迹法评估I型胶原、α-平滑肌肌动蛋白（α-SMA）、NLRP3炎性小体及TLR4/MyD88/NF-κB信号通路的表达。采用免疫组织化学分析进一步检测心肌组织中纤连蛋白、α-SMA、I型胶原、III型胶原、NLRP3和NF-κB的表达。
与MI组相比，LQF组射血分数（EF）和缩短分数（FS）显著改善，而左心室舒张末期内径（LVEDd）和左心室内径收缩期（LVIDs）显著减小。H&E和Masson染色结果显示，LQF治疗可有效减轻心肌损伤和纤维化。ELISA结果显示，LQF组血清LDH、cTnI、肿瘤坏死因子-α（TNF-α）、IL-18和IL-1水平显著低于MI组。蛋白质印迹结果显示，LQF组I型胶原和α-SMA表达显著降低。此外，LQF治疗组NLRP3炎性小体及TLR4/MyD88/NF-κB信号通路表达下调。
我们的结果表明，LQF可显著改善心功能并减轻心肌纤维化。此外，我们发现LQF可下调TLR4/MyD88/NF-κB信号通路，进而抑制NLRP3炎性小体激活，提示LQF通过降低TLR4/MyD88/NF-κB信号通路，进而抑制MI小鼠NLRP3炎性小体激活来减轻心脏纤维化，这表明LQF对MI患者具有潜在治疗作用。
