Mechanism of new optimized Sheng-Mai-San Formula to regulate cardiomyocyte apoptosis through NMDAR pathway

Figures

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  Fig. 1. Left heart function of rats in each group after drug administration. M-mode ultrasound images of rats in each group after 4 weeks of treatment (A); LVEF values of rats in each group after 4 weeks of treatment (B); LVFS values of rats in each group after 4 weeks of treatment (C). (Compared with Sham, **P < 0.01; compared with Model, ##P < 0.01).

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  Fig. 2. Histopathological changes of myocardium of rats in each group after drug administration. HE staining: Sham group: clear myocardial transverse lines, dense and neatly arranged fibers, intact cell structure, uniform size, no hypertrophy, centered nucleus, uniform cytoplasmic coloration, no obvious interstitial edema, no obvious abnormalities. Compared with the Sham group, the Model group had broken myofilaments, disorganized fiber arrangement, a large number of myocardial cells were necrotic and disappeared, replaced by proliferating fibrous connective tissue, and there were obvious cell swelling and a large number of inflammatory cell infiltration. Compared with the Model group, NO-SMS, Ifenprodil, and Enalapril myocardial tissue lesions were reduced, and neatly arranged myocardial fibers were seen, with uneven nuclei size and significantly reduced cell swelling, accompanied by partial inflammatory cell infiltration (A); Masson staining showed that the transverse myelopathy was clearly visible in the Sham group, with neatly arranged cells and mild proliferation of scattered collagen fibers in the intervals. Compared with Sham, Model showed that the transverse myocardial striae…

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  Fig. 3. Apoptosis of rat cardiomyocytes in each group after drug administration. TUNEL staining of rats in each group after 4 weeks of treatment (A); Apoptosis rate of cardiomyocytes in rats in each group (B); Compared with Sham, **P＜0.001; compared with Model, ##P＜0.001; compared with Enalapril group, P＞0.05 in NO-SMS group and Ifenprodil group; compared with NO-SMS group, P＜0.05 in Ifenprodil group.

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  Fig. 4. Apoptosis of H9c2 cardiomyocytes in each group. Annexin V-FITC/PI staining of H9c2 cardiomyocytes in each group treated with H2O2 (A); Annexin V-FITC/PI staining of H9c2 cardiomyocytes in each group treated with NMDA (B); Apoptosis rate of H9c2 cardiomyocytes in each group treated with H2O2 (C); Apoptosis rate of NMDA-treated H9c2 cardiomyocytes in each group (D); Compared with Sham, **P < 0.01; compared with NMDA, ##P < 0.01; comparison of H2O2+NO-SMS group and H2O2+Ifenprodil group, P＞0.05; comparison of NMDA + NO-SMS group and NMDA + Ifenprodil group, P＜0.05.

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  Fig. 5. Expression of apoptosis-related proteins in myocardial tissues. Representative immunoblot bands of various proteins (a); Evaluation of caspase-3, caspase-9, caspase-12, cyt-c, and Bcl-2/Bax levels in each group (b–f); Compared with Sham, **P < 0.01; compared with Model, #P < 0.05, ##P < 0.01.

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  Fig. 6. Apoptosis-related mRNA expression in myocardial tissues. Evaluation of Caspase-3, Caspase-9, Caspase-12, Cyt-c, Bax, Bcl-2 mRNA levels in each group (a–f); Compared with Sham, **P < 0.01; compared with Model, ##P < 0.01.

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  Fig. 7. Expression of apoptosis-related proteins in H2O2-treated H9c2 cardiomyocytes. Representative immunoblot bands of various proteins (a); Evaluation of caspase-3, caspase-9, caspase-12, cyt-c, and Bcl-2/Bax levels in each group (b–f); Compared with Sham, **P < 0.01; compared with H2O2, #P < 0.05, ##P < 0.01.

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  Fig. 8. Apoptosis-related mRNA expression in H2O2-treated H9c2 cardiomyocytes. Evaluation of Caspase-3, Caspase-9, Caspase-12, Cyt-c, Bax, Bcl-2 mRNA levels in each group (a–f); Compared with Sham, **P < 0.01; compared with H2O2, ##P < 0.01.

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  Fig. 9. Expression of apoptosis-related proteins in NMDA-treated H9c2 cardiomyocytes. Representative immunoblot bands of various proteins (a); Evaluation of caspase-3, caspase-9, caspase-12, cyt-c, and Bcl-2/Bax levels in each group (b–f); Compared with NMDA, **P < 0.01; compared with NMDA, #P < 0.05, ##P < 0.01.

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  Fig. 10. Apoptosis-related mRNA expression in NMDA-treated H9c2 cardiomyocytes. Evaluation of Caspase-3, Caspase-9, Caspase-12, Cyt-c, Bax, Bcl-2 mRNA levels in each group (a–f); Compared with Sham, **P < 0.01; compared with NMDA, ##P < 0.01.

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  Fig. 11. Ca2+ concentration of myocardial tissue and H9c2 cardiomyocytes in each group. Ca2+ concentration of myocardial tissue in each group (a); Ca2+ concentration of H9c2 cardiomyocytes treated with H2O2 in each group (b); Ca2+ concentration of H9c2 cardiomyocytes treated with NMDA in each group (c). Compared with Sham, **P < 0.01; compared with Model (H2O2/NMDA), ##P < 0.01.

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  Fig. 12. ROS concentration of myocardial tissue and H9c2 cardiomyocytes in each group. ROS concentration of myocardial tissue in each group (a); ROS concentration of H9c2 cardiomyocytes treated with H2O2 in each group (b); ROS concentration of H9c2 cardiomyocytes treated with NMDA in each group (c). Compared with Sham, **P < 0.01; compared with Model (H2O2/NMDA), ##P < 0.01.