Diterpenoids Derived from Strophioblachia Fimbricalyx Regulate Cardiac Hypertrophy by Blocking Autophagic Flux
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摘要:
目的 从宿萼木中筛选抗心肌肥厚活性二萜单体化合物 C080914，探究其抗心肌肥厚作用及分子机制。 方法 分离SD乳鼠原代心肌细胞，分别采用苯肾上腺素（PE，n = 6-10）、血管紧张素 Ⅱ（AngⅡ，n = 6）、异丙肾上腺素（ISO，n = 6）构建体外心肌肥厚模型；CCK-8检测化合物细胞毒性；Western blot 检测心肌肥厚、自噬相关蛋白；F-actin 免疫荧光观察心肌细胞肥大表型（n = 10）；mRFP-GFP-LC3双标腺病毒示踪自噬流（n = 5）；网络药理学结合分子对接预测并验证候选靶点；采用自噬抑制剂3-MA、激动剂雷帕霉素开展功能干预实验（n = 6）；多组间比较采用单因素方差分析，Tukey 法进行两两比较。 结果 C080914 细胞毒性低，CC50>50 μM；可显著抑制 PE、AngⅡ、ISO诱导ANP、MYH7蛋白表达上调（P < 0.05 或P < 0.001），缩小心肌细胞表面积（P < 0.001）。C080914 上调 LC3B、P62，下调 ATG9A 蛋白表达，造成自噬流阻断；分子对接显示 C080914 与 ATG9A 结合活性强，Vina 评分为-7.660 kcal/mol；3-MA 可部分逆转 C080914 抗心肌肥厚效应（P < 0.01）；雷帕霉素同样发挥抗肥厚作用，但二者调控自噬模式存在明显差异（P < 0.05）。 结论 宿萼木来源二萜C080914体外可抑制原代心肌细胞病理性肥厚，该效应伴随ATG9A 下调与自噬流阻断，可作为心肌肥厚候选先导分子，ATG9A为潜在研究靶点。 Abstract:Objective To screen the diterpenoid monomer C080914 with anti-myocardial-hypertrophy activity from Strophioblachia fimbricalyx, and to investigate its anti-hypertrophic effect against myocardial hypertrophy as well as the underlying molecular mechanism. Methods Primary cardiomyocytes were isolated from neonatal SD rats. In-vitro myocardial hypertrophy models were induced with phenylephrine （PE, n = 6-10）, angiotensin Ⅱ （AngⅡ, n = 6）, and isoproterenol （ISO, n = 6）, respectively. CCK-8 assay was used to evaluate the cytotoxicity of the compound. Western blot was performed to detect proteins associated with myocardial hypertrophy and autophagy. F-actin immunofluorescence staining was applied to observe the hypertrophic phenotype of cardiomyocytes （n = 10）. mRFP-GFP-LC3 dual-labeled adenovirus was utilized to trace autophagic flux （n = 5）. Network pharmacology combined with molecular docking was adopted to predict and verify candidate targets. Functional intervention experiments were carried out using the autophagy inhibitor 3-MA and autophagy agonist rapamycin （n = 6）. One-way analysis of variance （ANOVA） was used for comparisons among multiple groups, followed by Tukey’ s post-hoc test for pairwise comparisons. P < 0.05 was considered statistically significant. Results C080914 exhibited low cytotoxicity with CC50>50 μM; it significantly suppressed the up-regulation of ANP and MYH7 protein expressions induced by PE, AngⅡ and ISO （P < 0.05 or P < 0.001） and decreased cardiomyocyte surface area （P < 0.001）. C080914 up-regulated LC3B and P62 but down-regulated ATG9A protein expression, leading to autophagic flux blockage. Molecular docking demonstrated strong binding affinity between C080914 and ATG9A , with a Vina score of −7.660 kcal/mol. 3-MA partially reversed the anti-hypertrophic effect of C080914 （P < 0.01）. Rapamycin also produced anti-hypertrophic effects, yet distinct differences existed in their autophagy-modulating modes. Conclusion The diterpenoid C080914 from Strophioblachia fimbricalyx inhibits pathological hypertrophy of primary cardiomyocytes in vitro, This effect is accompanied by ATG9A down-regulation and autophagic flux blockage. C080914 may be used as a candidate lead molecule against myocardial hypertrophy, with ATG9A as a potential therapeutic target. -
Key words:
- Strophioblachia fimbricalyx /
- Diterpenoids /
- Myocardial hypertrophy /
- Autophagy /
- ATG9A /
- Natural medicines
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图 1 不同浓度的化合物对原代心肌细胞活性的影响[（$ \bar x \pm s $），n = 6]
Figure 1. Effects of different concentrations of the compound on the viability of primary cardiomyocytes[（$ \bar x \pm s $），n = 6]
图 2 C080914对ISO、AngⅡ诱导的心肌肥厚的抑制作用[（$ \bar x \pm s $），n = 6]
A：AngII/ISO 诱导心肌肥厚模型经C080914处理后，肥厚标志物 MYH7 蛋白的代表性免疫印迹图；B：MYH7蛋白相对定量统计图（目标蛋白灰度值与GAPDH灰度值的比值），竖线左侧为ISO诱导模型，右侧为 AngⅡ 诱导模型，与CTL组相比，*P < 0.05，****P < 0.0001；与AngⅡ、ISO组相比，###P < 0.001，####P < 0.0001。
Figure 2. Inhibitory effects of C080914 on ISO- and AngⅡ-induced myocardial hypertrophy [（$ \bar x \pm s $），n = 6]
图 3 C080914对心肌细胞肥大及细胞面积的影响[（$ \bar x \pm s $），n = 10]
A：心肌细胞表面积免疫荧光代表图（×40）；B：运用ImageJ计算细胞表面积；与CTL组相比，**P < 0.01；与PE组相比，###P < 0.001。
Figure 3. Effects of C080914 on cardiomyocyte hypertrophy and cell surface area [（$ \bar x \pm s $），n = 10]
图 5 相关性排名前10信号通路的富集气泡图
Figure 5. Bubble plot of enrichment for the top 10 correlated signaling pathways by correlation ranking
图 6 C080914减轻心肌细胞肥厚并调控自噬相关蛋白表达[（$ \bar x \pm s $），n = 6]
A：PE诱导心肌肥厚后加入C080914处理后自噬相关蛋白LC3-Ⅱ、P62蛋白水平的代表性免疫印迹图；B：自噬相关蛋白LC3-Ⅱ相对定量统计图（目标蛋白灰度值与β-actin 灰度值的比值）；C：MYH7相对定量统计图（目标蛋白灰度值与β-actin 灰度值的比值）；D：自噬相关蛋白LC3-Ⅱ相对定量统计图（目标蛋白灰度值与β-actin灰度值的比值）；与CTL组相比，*P < 0.05，***P < 0.001；与AngⅡ、ISO组相比，###P < 0.001。
Figure 6. C080914 alleviates cardiomyocyte hypertrophy and regulates the expression of autophagy-related proteins [（$ \bar x \pm s $），n = 6]
图 7 C080914使ATG9A表达下调[（$ \bar x \pm s $），n = 6]
A：MYH7、ATG9A、LC3B 蛋白的电泳条带；B：ATG9A 蛋白相对表达水平定量分析（目标蛋白灰度值与β-actin 灰度值的比值）；与 CTL 组比较，#P < 0.05；与 PE 组比较，*P < 0.05。
Figure 7. C080914 downregulates ATG9A expression [（$ \bar x \pm s $），n = 6]
图 8 mRFP-GFP-LC3双标腺病毒检测原代心肌细胞自噬流[（$ \bar x \pm s $），n = 5]
A：mRFP-GFP-LC3荧光代表图像（×40）； B：各组细胞GFP斑点、mRFP斑点、黄色共定位斑点（未融合自噬体）、红色斑点（自噬溶酶体）定量统计。
Figure 8. Detection of autophagic flux in primary cardiomyocytes using mRFP-GFP-LC3 dual-labeled adenovirus [（$ \bar x \pm s $），n = 5]
图 9 C080914与ATG9A对接结合相互作用图
注：C080914（青色）- ATG9A（绿色）；主要为疏水作用和范德华接触。
Figure 9. Molecular docking interaction between C080914 and ATG9A
图 10 加入自噬抑制剂3MA后ANP蛋白表达WB图和统计图[（$ \bar x \pm s $），n = 6]
A：ANP 蛋白的电泳条带;B:ANP 蛋白相对表达水平定量分析（目标蛋白灰度值与β-actin 灰度值的比值）；与PE组比较，**P < 0.01；与CTL组比较，#P < 0.05；与080914组比较，&&P < 0.01。
Figure 10. Western blot and statistical analysis of ANP protein expression after treatment with autophagy inhibitor 3MA [（$ \bar x \pm s $），n = 6]
图 11 细胞表面积免疫荧光[（$ \bar x \pm s $），n = 10]
A：心肌细胞表面积免疫荧光代表图（×40）；B：运用ImageJ计算细胞表面积；与CTL组比较，###P < 0.001。
Figure 11. Immunofluorescence analysis of cell surface area [（$ \bar x \pm s $），n = 10]
图 12 自噬激动剂Rapa可改善PE诱导的心肌肥厚[（$ \bar x \pm s $），n = 6]
A：ANP 蛋白的电泳条带；B:ANP 蛋白相对表达水平定量分析（目标蛋白灰度值与β-actin灰度值的比值）；与PE组比较，*P < 0.05；与CTL组比较，#P < 0.05。
Figure 12. Autophagy agonist Rapa ameliorates PE-induced cardiomyocyte hypertrophy [（$ \bar x \pm s $），n = 6]
表 1 抗体信息
Table 1. Antibody information
抗体名称 批号 品牌 稀释倍数 NPPA抗体 27426 -1-APproteintech 1∶ 1000 MYH7抗体 22280 -1-APproteintech 1∶ 1000 LC3B抗体 ab192890 Abcam 1∶ 2000 β-actin抗体 ab134964 Abcam 1∶ 5000 GAPDH抗体 5174 Cell Signaling Technology 1∶ 1000 ATG9A抗体 ab228525 Abcam 1∶ 1000 P62抗体 Ab109012 Abcam 1∶ 10000 辣根过氧化物酶（HRP）标记羊抗兔 IgG（H+L）抗体 65- 6120 Invitrogen 1∶ 5000 辣根过氧化物酶（HRP）标记羊抗鼠 IgG（H+L）抗体 62- 6520 Invitrogen 1∶ 5000 
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