Effects Of Shenmai Formula On Mitochondrial Function And Energy Matebolism In Ischemic Myocardium

Shenmai formula (SM), derived from ancient recipe Shengmaisan, is composed of two herbs red ginseng (RG,Panax ginseng) and Radix Ophiopogonis (OP, Ophiopogon japonicus (L. f.) Ker-Gawl, root). Pharmacological researches indicated that SM can exert anti-shock, anti-arrhythmic, anti-inflammatory activities as well as regulate blood pressure through reliefing lipid peroxidation and improve blood rheology. However, there still lacks of clear evidence about molecular targets of SM. It is necessary to investigate the mechanism of action of SM for its protective effects on ischemic myocardium.In this paper, we adopted isobaric tags for relative and absolute quantitation(iTRAQ) technology to study the differentially expressed proteins in rat ischemic heart after SM treatment. Furthermore, we preliminarily investigated the mechanism of SM on mitochondrial function and energy matebolism in ischemic myocardium. The main contents of this dissertation are as follows:1. Pharmacoproteomics study of Shenmai formulaAfter seven days intraperitoneal treated with RG, OP or SM, rats with myocardial infarction were sacrified to harvest the ischemic part of heart for protein extraction. iTRAQ technique was employed to trace differentially expressed proteins altered by SM and its components. About 100 differential expressed proteins were identified, and four differentially expressed proteins were validated by Western blot. The results indicated that myocardial infarction induced more than 60 significantly changed proteins, and there are some differentially expressed proteins regulated by SM, RG or OP respectively. About half of these proteins located in mitochondrial memberane and mitochondrial matrix, and functionally related to oxidative phosphorylation, tricarboxylic acid cycle, ATPase activity, and other biological functions. Ischemia induced down-regulation of these proteins were attenuated by RG, OP and SM, suggusting that the molecular mechanism of SM against myocardial ischemia involved in maintaining mitochondrial function and recovering energy matebolism. To validate the reliability of the proteomic data, four crucial proteins(ATP5D, NDUFB10, MDH1, TNNC1) were picked out for Western blot experiment. The data acquired from the above two different experiments were consistent.2. This study investigated the effect of SM on improving mitochondrial function in hypoxia-damaged neonatal rat ventricular myocytes(NRVMs).Laser scanning confocal microscopy was used to examinemitochondrial membrane potential of NRVMs after staining with JC-1. We also measured intracellular ATP content and evaluated bioenergetics of NRVMs exposure to hypoxia by the Seahorse XF24 extracellular flux analyzer. The results showed that SM, RG and OP can prevent the decrease of mitochondrial membrane potential of cardiomyocytes subjected to hypoxia. Treatment of SM or RG can recover intracellular ATP levels in cardiomyocytes exposure to hypoxia for 24 hours. According to the data of oxygen consumption rate(OCR), SM can significantly improve multiple mitochondrial functions, like ATP-related respiratory, maximal respiratory rate, and the reserve capacity of cardiomyocytes exposed to 6 hrshypoxia. All those results suggested that SM can protect cardiomyocytes through maintaining mitochondrial function under the anoxic circumstance.3. This study explored the mechanism of improving mitochondrial energy metabolism by SM in hypoxia-damaged neonatal rat ventricular myocytes(NRVMs)CellTiter-Glo chemiluminescence method was used to determine viability of NRVMs exposure to hypoxia after SM treatment. Moreover, the expressions of protein and mRNA associated with mitochondrial energy metabolism were examined by Weatern blot and RT-PCR. The results found that SM can prevent hypoxia-induced injury of NRVMs in a dose dependent manner, while RG but not OP chiefly contributed to the protective effect. WB results showed that SM can up-regulate the expression of p MDH1 and ATP5F1 proteins in different leval, however, the mRNA expressions of PGC-1, SirT1 and ATP5F1 were not significantly changed.In summary, we found that SM may exhibit potent cardioprotective effects through maintaining mitochondrial membrane potential, increasing ATP content, maximal respiratory rate and the reserve capacity. Mitochondia might be major targeted organelle of SM. This research provided new evidences for further comprehending the mechanisms of SM for treating ischemic cardiomyopathy.