Stachydrine Ameliorates Cardiac Fibrosis Through Inhibition of Angiotensin II/Transformation Growth Factor β1 Fibrogenic Axis
Stachydrine Ameliorates Cardiac Fibrosis Through Inhibition of Angiotensin II/Transformation Growth Factor β1 Fibrogenic Axis
复制标题
水苏碱通过抑制血管紧张素 II/转化生长因子 β 1 纤维形成轴改善心脏纤维化
DOI：
10.3389/fphar.2019.00538
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发表时间：
2019-05-22
影响因子：
5.6
通讯作者：
Lu, Rong
中科院分区：
文献类型：
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作者：
Liu, Xiao;Shan, Xiaoli;Lu, Rong
心血管疾病是世界范围内的主要死亡原因，与病理性心肌纤维化密切相关。水苏碱（Stachydrine，Sta）是益母草中的主要活性成分，有研究表明其具有抗心肌纤维化的作用，但其抗心肌纤维化的细胞和分子机制尚不清楚。本研究采用压力超负荷和血管紧张素Ⅱ刺激的小鼠心脏成纤维细胞（CFs）模型，探讨Sta的抗纤维化作用及其机制。将小鼠随机分为假手术组、生理盐水组（TAC C Sal）、替米沙坦组（TAC C Tel）和Sta组（TAC C Sta）。通过超声心动图和组织学方法评价心脏形态学和功能变化，并通过蛋白质印迹法检测分子改变。原代培养的新生小鼠CFs分别加入或不加入血管紧张素II（AngII，10（-7）M）、转化生长因子β 1（TGF β 1，10 ng/mL）和不同剂量的Sta（10（-6）-10（-4）M）处理96 h，检测细胞增殖、细胞毒性、形态学及相关信号。体内结果显示，TAC显著诱导心功能不全、左心室扩张、心肌肥大和心肌胶原沉积增加，伴随纤维化标志物包括α-平滑肌肌动蛋白（α-SMA）和骨膜蛋白增加。然而，Sta治疗部分逆转了心脏形态和功能的恶化，并显着钝化心脏纤维化以及Tel。心肌血管紧张素原（AGT），血管紧张素转换酶（ACE），血管紧张素Ⅱ 1型受体（AT 1 R），和TGF β 1转录的增加，以及ACE和血管紧张素Ⅱ蛋白水平的增加，TAC后显着下调Sta治疗。巧合的是，体外实验表明，AngII刺激CFs导致AT 1 R和TGF β 1的上调，因此促进CFs转分化为过度活化的心肌成纤维细胞（MF），如增加的细胞增殖、胶原和纤维化标记所证明的。相反，Sta有效下调但不直接抑制AT 1 R，抑制TGF β 1的产生，以及AngII在CF中的促纤维化作用。此外，在TAC模型和AngII刺激的CF中均观察到纤维化过程中TGF β 1/Smads信号的激活，其也被Sta显著钝化。然而，Sta未能消除由TGF β 1触发的CF的激活。综上所述，本研究表明，Sta抑制ACE/AngII/AT 1 R-TGF β 1促纤维化轴，特别是通过下调AGT/ACE和AT 1 R抑制AngII的从头产生，因此使CF失活并使MF转变钝化，最终防止心脏纤维化。
Cardiovascular diseases, the leading cause of death worldwide, are tightly associated with the pathological myocardial fibrosis. Stachydrine (Sta), a major active compound in Chinese motherwort Leonurus heterophyllus, was reported to effectively attenuate cardiac fibrosis, but the cellular and molecular mechanism remains unclear. In this study, the anti-fibrotic effect of Sta and mechanism underlying were explored in a mouse model of pressure overload and AngII stimulated cardiac fibroblasts (CFs). Mice were randomly divided into sham, transverse aorta constriction with saline (TAC C Sal), TAC with telmisartan (TAC C Tel), and TAC with Sta (TAC C Sta) groups. Cardiac morphological and functional changes were evaluated by echocardiography and histological methods, and the molecular alterations were detected by western blotting. Primary cultured neonatal mouse CFs were treated with or without angiotensin II (AngII, 10(-7) M), transformation growth factor beta 1 (TGF beta 1, 10 ng/mL), and different dosage of Sta (10(-6)-10(-4) M) for up to 96 h, and cell proliferation, cytotoxicity, morphology and related signals were also detected. The in vivo results revealed that TAC prominently induced cardiac dysfunction, left ventricular dilation, myocardial hypertrophy, and elevated myocardial collagen deposition, accompanied with increased fibrotic markers including alpha-smooth muscle actin (alpha-SMA) and periostin. However, Sta treatment partially reversed cardiac morphological and functional deteriorations, and significantly blunted cardiac fibrosis as well as Tel. Increments of myocardial angiotensinogen (AGT), angiotensin converting enzyme (ACE), AngII type 1 receptor (AT1R), and TGF beta 1 transcripts, together with increased protein levels of ACE and AngII, after TAC were dramatically down-regulated by Sta treatment. Coincidently, in vitro experiments demonstrated that AngII stimulation in CFs led to up-regulation of AT1R and TGF beta 1, and therefore promoted CFs trans-differentiating into hyper-activated myocardial fibroblasts (MFs) as evidenced by increased cell proliferation, collagen and fibrotic makers. On the contrary, Sta potently down-regulated but not directly inhibited AT1R, suppressed TGF beta 1 production, and the pro-fibrotic effect of AngII in CFs. Moreover, activation of TGF beta 1/Smads signal in the fibrotic process were observed both TAC model and in AngII stimulated CFs, which were also notably blunted by Sta. However, Sta failed to abolish the activation of CFs triggered by TGF beta 1. Taken together, it was demonstrated in this study that Sta suppressed ACE/AngII/AT1R-TGF beta 1 profibrotic axis, especially on the de novo production of AngII via down-regulating AGT/ACE and AT1R, and therefore inactivated CFs and blunted MFs transition, which ultimately prevented cardiac fibrosis.