Zishen Huoxue Recipe Protecting Mitochondrial Function of Hypoxic/Reoxygenated Myocardial Cells through mTORC1 Signaling Pathway.

OBJECTIVE

This study focuses on the role of Zishen Huoxue Decoction (ZSHX) in reducing mitochondrial membrane potential and reducing the proportion of apoptosis through the mTORC1 signaling pathway.

METHODS

In our experiment, we first constructed an in vitro hypoxia/reoxygenation (H/R) model of H9C2 cells. Then, the cells were divided into control group, model group (hypoxia/reoxygenation, H/R), ZSHX, ZSHX + Rapa, low-dose ZSHX (100 g/ml), and middle-dose ZSHX. High-dose ZSHX (400 g/ml) group was treated with Zishen Huoxue Decoction (ZSHX). Western Blot was used to detect the expression of cell-related protein and RT-PCR was used to detect the expression of the cell-related gene in each group. Flow cytometry was used to assay for ROS content and the apoptotic ratio of H9C2 cells, Seahorse Live Cell Energy Meter was used to detect the Mitochondrial Respiratory Function in H9C2 Cells, and confocal laser scanning was used to detect the mitochondrial membrane potential of H9C2 cells.

RESULTS

Western Blot assay showed that the relative expression of mTOR and Raptor in the H/R group was significantly lower than that in the control group ( = 3, < 0.05). The expression of mTOR and Raptor was upregulated and the relative expression of 4E-BP1 was downregulated in the middle- and high-dose ZSHX groups ( = 3, < 0.05). In addition, the ROS content of H9C2 cells was detected by flow cytometry, showing the ROS synthesis in H/R group (78.31 + 6.14) higher than that in the control group (34.53 + 6.10) ( = 3, < 0.01). The ROS value was increased significantly after rapamycin inhibited mTOR (66.18 (+4.03 vs. 52.31 (+6.01),  = 3, < 0.05). The basal mitochondrial respiration and ATP production in H/R group were significantly lower than those in the control group (38.17 + 17.76); the mitochondrial leakage in H/R model group was significantly higher than that in the control group (H/R: 40.93 + 5.18 vs. Ctrl: 27.17 + 8.92,  = 4, < 0.05). The apoptotic rate of cardiomyocytes in the H/R model group (70.91 + 4.57) was significantly higher than that in the control group (14.52 + 2.37,  = 3, < 0.01), and Zishen Huoxue Decoction could decrease the apoptotic rate of hypoxic-reoxygenated cardiomyocytes (ZSHX: 18.24 + 4.17 vs. H/R: 78.91 + 3.48,  = 3, < 0.01).

CONCLUSION

ZSHX Decoction has the effects of activating mTORC1, inhibiting the overexpression of 4E-BP1, inhibiting fatty acid oxidation, protecting the respiratory function of mitochondria, reducing ROS and apoptosis, and thus protecting myocardial cells from injury.