Cao Tong-Tong, Chen Hui-Hua, Dong Zhiwei, Xu Yan-Wu, Zhao Pei, Guo Wei, Wei Hong-Chang, Zhang Chen, Lu Rong
Department of Pathology, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, Third Military Medical University, Chongqing, China.
Cell Physiol Biochem. 2017;42(1):103-114. doi: 10.1159/000477119. Epub 2017 May 11.
Autophagy is required for the maintenance of cardiomyocyte homeostasis. However, excessive autophagy plays a maladaptive role in pressure overload-induced heart failure. To identify mechanisms by which Stachydrine inhibits pressure overload-induced cardiac hypertrophy, we determined inhibitory activities against activation of NADPH oxidase, reactive oxygen species(ROS) production and excessive activation of autophagy.
Stachydrine was administered intragastrically to Wistar rats after Transverse aortic constriction(TAC) and H9c2 cells were treated with Stachydrine after Angiotension II stimulation. The activation of NADPH oxidase2 required the membrane translocation of p47phox and p67phox. Cell membrane fraction was isolated by ultracentrifuge in sucrose. The expression of p67phox, p47phox, gp91phox subunit in the cell membrane were determined by western blot. The combination of p67phox and gp91 phox subunit was detected by immunofluorescence staining. The expression of phosphorylated p47phox subunit was determined by western blot. The intracellular ROS were measured with DCF-DA fluoresence. The autophagic flux was measured by recording the fluorescence emission of the fusion protein mRFP-GFP-LC3 by dynamic live-cell imaging. Reuslts: We report here that stachydrine, a major constituent of Leonurus heterophyllus Sweet, inhibited AngII-induced excessive autophagy within H9c2 cells. Stachydrine blocked the over phosphorylation of the p47phox subunit, decreased the translocation of p47phox and p67phox to the membrane, inhibited the activity of NOX2, and reduced the generation of ROS. We also demonstrated that stachydrine ameliorated TAC-induced cardiac hypertrophy, dysfunction and excessive autophagy in vivo.
Our study highlights the importance of regulating NOX2 when autophagy is obviously activated. By inhibiting NOX2, Stachydrine inhibits ROS production, thus exerting a remarkable activity of inhibiting hypertrophy, which could have considerable effect on clinical practice.
自噬是维持心肌细胞稳态所必需的。然而，过度自噬在压力超负荷诱导的心力衰竭中起不良适应作用。为了确定水苏碱抑制压力超负荷诱导的心脏肥大的机制，我们测定了其对NADPH氧化酶激活、活性氧（ROS）产生和自噬过度激活的抑制活性。
在横断主动脉缩窄（TAC）后对Wistar大鼠进行水苏碱灌胃给药，在血管紧张素II刺激后用其处理H9c2细胞。NADPH氧化酶2的激活需要p47phox和p67phox的膜转位。通过在蔗糖中进行超速离心分离细胞膜组分。通过蛋白质免疫印迹法测定细胞膜中p67phox、p47phox、gp91phox亚基的表达。通过免疫荧光染色检测p67phox和gp91phox亚基的结合。通过蛋白质免疫印迹法测定磷酸化p47phox亚基的表达。用DCF-DA荧光法测量细胞内ROS。通过动态活细胞成像记录融合蛋白mRFP-GFP-LC3的荧光发射来测量自噬通量。结果：我们在此报告，益母草中的主要成分水苏碱可抑制H9c2细胞中AngII诱导的过度自噬。水苏碱可阻断p47phox亚基的过度磷酸化，减少p47phox和p67phox向膜的转位，抑制NOX2的活性，并减少ROS的产生。我们还证明水苏碱可改善TAC诱导的体内心脏肥大、功能障碍和过度自噬。
我们的研究强调了在自噬明显激活时调节NOX2的重要性。通过抑制NOX2，水苏碱抑制ROS产生，从而发挥显著的抑制肥大活性，这可能对临床实践产生重大影响。
