Abstract
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 μg/ml), and middle-dose ZSHX. High-dose ZSHX (400 μg/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 (n = 3, P < 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 (n = 3, P < 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) (n = 3, P < 0.01). The ROS value was increased significantly after rapamycin inhibited mTOR (66.18 (+4.03 vs. 52.31 (+6.01), n = 3, P < 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, n = 4, P < 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, n = 3, P < 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, n = 3, P < 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.
Highlights
Ischemic heart disease is currently the key cause of disease mortality worldwide [1]
In order to better elucidate the pathological mechanism of I/R and the specific mechanism of Zishen Huoxue Decoction in protecting ischemia/reperfusion, we used H9C2 cells to simulate hypoxia/reoxygenation model in vitro and used Western Blot, RT-PCR technology, and key molecular inhibitors of mTOR complex 1 (mTORC1) pathway to intervene to explore the protective effects of mTORC1 signaling pathway and 4E-BP regulation axis on hypoxia/reoxygenation. erefore, the mechanism of mitochondrial function of oxygen myocardial cells was studied, and the effects of different concentrations of Zishen Huoxue Decoction on mitochondrial function of hypoxia/ reoxygenation myocardial cells were examined by Seahorse energy measurement and laser confocal technique
Myocardial cells of the control group were cultured under normal conditions. e morphology of H9C2 myocardial cells under different H/R conditions was observed under an inverted microscope, as shown in Figure 1. e results showed that the normal H9C2 cardiomyocytes were spindle-shaped, arranged neatly, uniform in size, with clear cytoplasmic boundaries of the nucleus; after hypoxia, with the prolongation of time, the H9C2 cells have shown irregular size in varying degrees, the cell body became round and shriveled, the nucleus enlarged, and there were vacuoles in the cytoplasm, while after hypoxia for 4 h, the morphological changes of H9C2 myocardial cells were observed after 16 h of reoxygenation, especially after 12 h of hypoxia/16 h of reoxygenation
Summary
Ischemic heart disease is currently the key cause of disease mortality worldwide [1]. Erefore, this paper mainly discusses the main mechanism of Zishen Huoxue Decoction in regulating mitochondrial membrane potential and protecting mitochondrial function. It is suggested that Zishen Huoxue Decoction can regulate the energy metabolism of myocardial cells and protect the cytoskeleton [9, 10] by upregulating ATP synthase alpha and downregulating CPT-1. Many studies have shown that traditional Chinese medicine can improve the energy metabolism of ischemic myocardium by regulating the activity of mitochondrial respiratory enzyme complex and transmembrane potential of myocardial cells [13,14,15]. In order to better elucidate the pathological mechanism of I/R and the specific mechanism of Zishen Huoxue Decoction in protecting ischemia/reperfusion, we used H9C2 cells to simulate hypoxia/reoxygenation model in vitro and used Western Blot, RT-PCR technology, and key molecular inhibitors of mTORC1 pathway to intervene to explore the protective effects of mTORC1 signaling pathway and 4E-BP regulation axis on hypoxia/reoxygenation. erefore, the mechanism of mitochondrial function of oxygen myocardial cells was studied, and the effects of different concentrations of Zishen Huoxue Decoction on mitochondrial function of hypoxia/ reoxygenation myocardial cells were examined by Seahorse energy measurement and laser confocal technique
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
