鹿红方通过下调lncRNA-Meg3抑制STAT3调控的心肌细胞旁分泌作用抗心肌纤维化的机制研究

Myocardial fibrosis is the key pathological process of chronic heart failure. Our clinical and basic studies have confirmed that Luhong formula (LHF) has an exact anti-myocardial fibrosis effect, and its mechanism is related to up-regulating the level of the transcription factor STAT3 which inhibiting the secretion of profibrotic factors by myocardial cells. However, the upstream mechanism of LHF 's upregulation of STAT3 is unknown. Recent studies have shown that lncRNA-Meg3 (Meg3 for short) plays an important role in the pathogenesis of myocardial fibrosis. Considering that Meg3 can reduce the level of STAT3 through decoy effection, and our preliminary experiments have confirmed that LHF can inhibit the expression of Meg3 in hypertrophic myocytes, a hypothesis is proposed: LHF can down-regulate the expression of Meg3 in myocytes and/or its decoy effect on STAT3, up-regulate the level of STAT3, and inhibit myocardial fibrosis in a paracrine manner by myocardial cells. In the present study, we plan to establish a myocardial fibrosis model in vivo and in myocyte hypertrophy model in vitro, and to observe LHF's Intervention effect on Meg3 level and its decoy effect on STAT3, STAT3 level, paracrine factors (CTGF, TSP1, TIMP1), and the lesions situation of myocardial fibrosis. We aim to verify and elucidate LHF's anti-myocardial fibrosis effect and its mechanism at the global, cellular and transcriptional level through this stuy, so as to provide theoretical basis for its clinical application.

心肌纤维化是慢性心衰发生、发展的关键病理环节。我们临床和基础研究证实，鹿红方（LHF）具有确切的抗心肌纤维化作用，其机制与上调转录因子STAT3水平，抑制心肌细胞分泌促纤维化因子有关。但LHF上调STAT3的上游机制不明。最新研究表明，lncRNA-Meg3（简称Meg3）在心肌纤维化发病中起重要作用。鉴于Meg3可通过诱捕作用降低STAT3水平，且我们预初实验证实LHF可抑制肥大心肌细胞表达Meg3，提出假说：LHF通过下调心肌细胞Meg3表达和/或其对STAT3的诱捕作用，上调STAT3水平，抑制心肌细胞以旁分泌方式诱导心肌纤维化。拟建立在体心肌纤维化模型和体外心肌细胞肥大模型，观察LHF对Meg3及其诱捕作用、STAT3水平、旁分泌因子（CTGF、TSP1、TIMP1）以及心肌纤维化的干预效应，从整体、细胞及转录水平验证和阐释LHF抗心肌纤维化的作用和机制，为其临床应用提供理论依据。

本研究围绕项目假说“鹿红方（LHF）通过下调心肌细胞lncRNA-Meg3（Meg3）表达，上调STAT3水平，抑制心肌细胞以旁分泌方式诱导心肌纤维化”开展机制研究。我们制备大鼠心梗模型和肥大心肌细胞模型，开展在体和细胞实验，比较LHF组与模型组数据，获得如下重要结果：①心超显示LHF可缩小左室内径和容积（8.51±0.61 vs 9.19±0.90mm，402.54±12.21 vs 509.22±17.07μL）；提高左室射血分数和左室短轴缩短率（52.36±5.13 vs 44.46±6.22%，37.41±2.12 vs 26.98±3.67%）；②LHF可显著改善心脏指数以及左室质量指数（2.73±0.03 vs 3.02±0.09，2.26±0.03 vs 2.46±0.03）；③病理学染色证实LHF显著抑制心肌纤维化；④LHF显著降低CTGF、TSP1、TIMP1水平（2.05±0.23 vs 3.85±0.29ug/L，209.22±18.38 vs 250.41±26.09 ng/ml，17.62±1.46 vs 24.17±2.47 ug/L）；⑤免疫组化显示LHF降低心肌组织I型和III型胶原合成，并降低两者比值；⑥PCR显示LHF显著降低心肌组织和心肌细胞Meg3表达；⑦WB显示LHF显著提高心肌组织和心肌细胞STAT3表达；⑧confocal显示LHF显著提高心肌细胞STAT3表达；⑨RIP证实Meg3与STAT3存在相互结合；⑩LHF显著降低心肌细胞培养液CTGF、TSP1、TIMP1水平（2.55±0.13 vs 4.85±0.21ug/L，201.32±15.28 vs 301.40±22.09ng/ml，16.69±1.33 vs 44.17±2.41ug/L）；⑪CCK8显示LHF显著抑制成纤维细胞（CF）增殖(OD值：0.76 vs 0.93)；⑫confocal显示LHF显著抑制CF的I型胶原合成。综上，本研究阐明了Meg3/STAT3/旁分泌因子轴是心肌纤维化的重要机制，证明了本项目的科学假说，为LHF的临床应用补充了生物学依据。

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