| In traditional Chinese medicine(TCM) theory, "obstruction", "heartburn" is in the process of the pathological changes of blood stasis. The cerebral ischemia is induced by circulation of blood stasis in the head and myocardial infarction is caused by stasis in the heart. For thousands of years, TCM in the treatment of blood stasis syndrome of cardio-cerebro-vascular diseases that result has made great progress, effective, and bright prospects. Carthamustinctorius L – Boswelliaserrate(B. serrata) combination is widely used for treatment and prevention of cerebrovascular and cardiovascular diseases, a well-known TCM formula. According to the culmination of Surgery(Wai Ke Da Cheng), Carthamustinctorius L and B. serrata are commonly used in combination, famous as Huoxue Dingtong Decoction in clinical practice. Carthamus tinctorius L and B. serrata are two traditional Chinese medicinal herbs popularly used in China for their activating blood removing stasis effects. Scientific connotation of relations among TCMs was still unclear. Little is known about their synergism biochemical mechanism of Carthamustinctorius L and B. serrata in treating myocardial ischemia injury by antioxidant action. We identified that hydroxy safflower yellow A(HSYA) and acetyl-11-keto-beta-boswellic acid(AKBA) were one of main active components of Carthamustinctorius L and B. serrata by using differential efficacy of serum chromatographic analysis(DESCA).( National Natural Science Foundation of China. No.81373947). In this paper, the effects of HSYA and AKBA on myocardial infarction with blood stasissyndrome were studied and their synergistic effect was assessed. The study would reveal scientific connotation of relations between HSYA and AKBA and provide novel approaches for improving quality of Chinese Potent Medicine and theories of TCMs.Objective1. We explore whether the effects of HSYA and AKBA on cardioprotection is related to ameliorating the myocardial ischemia injury. The synergistic effects of them are also assessed.2. We explore whether synergistic effects of HSYA and AKBA on cardioprotection is related to reducing the oxidative stress.3. To explore the expression of PGC-1α/Nrf2 signaling pathway in the myocardial ischemia injury and to determine the role of PGC-1α/Nrf2 signaling pathway in coping with oxidative stress.MethodsThe myocardial injury was produced in Sprague-Dawley rats via injection with isoproterenol for 2 days. To model ischemia-like conditions in vitro, H9C2 cells were exposed to transient oxygen and glucose deprivation(OGD). The levels of creatine kinase-MB(CK-MB), lactate dehydrogenase(LDH), superoxide dismutase(SOD) and malondialdehyde(MDA) were examined. The apoptotic cell death was determined by TUNEL and Hoechst Staining. Mitochondrial reactive oxygen species(ROS) production and mitochondrial potential were measured using the fluorescent dyes Mito-sox and JC-1. Expressions of PGC-1αand Nrf2 were quantified by immunoblotting and immunohistochemistry.Results1. HSYA and AKBA protect against myocardial injury(MI). Marked myofibrillar degeneration, necrosis, edema and infiltration with neutrophil granulocytes were found in ISO treated group. Both HSYA and AKBA protect against the MI, and combination of HSYA and AKBA appears to be more effective. Pretreatment with HSYA or AKBA significantly reduced the ISO mediated increases of CK-MB and LDH levels(P<0.05). HSYA and AKBA also dramatically decreased CK-MB and LDH levels in H9C2 cells upon OGD treatment. Combination of HSYA and AKBA showed synergistic effects both in vivo and in vitro. Furthermore, the protective effects of HSYA and AKBA against myocardial damages were confirmed by TUNEL staining on sections from myocardium at 48 h after ISO induced ischemia in rats. In ISO treated group, TUNEL-positive cells were densely distributed in the myocardium. The HSYA and AKBA-treated groups exhibited smaller amount of TUNEL-positive cells. The percentages of TUNEL-positive cells in the ischemic myocardium were decreased from 34.57% to 19.47% or 19.61% by HSYA or AKBA treatment respectively(n=6 per group; P<0.05). Combination of HSYA and AKBA showed further decrease of TUNEL-positive cells compared to HSYA or AKBA group(P<0.05). Most of H9C2 cells in OGD treated group were shrunken with a triangulated pycnotic nucleus. In contrast, cell damages were substantially reduced in the HSYA and AKBA treated groups.2. HSYA and AKBA attenuated oxidative stress. Assessment of mitochondrial ROS level showed that HSYA and AKBA effectively reduced OGD-induced increase of mitochondrial ROS level. OGD-induced decrease of mitochondrial potential was restored by HSYA or AKBA. Combination of HSYA and AKBA showed further decrease of ROS and increase of mitochondrial potential compared to HSYA or AKBA in OGD treated H9C2 cells. The MDA level, which is an index of lipid peroxidation was significantly increased in ISO or OGD treated group compared with the sham group. Reduction of the MDA level was observed in HSYA or AKBA group(n=6 per group; P<0.05). SOD activity was decreased in the ISO or OGD treated group compared with the sham group, and was restored by HSYA or AKBA(n=6 per group; P<0.05). Combination of HSYA and AKBA showed further decrease of MDA level and further increase of SOD activity both in ISO or OGD treated group.3. HSYA and AKBA increased the expressions of PGC-1α and Nrf2. To identify whether PGC-1α/Nrf2 signaling is involved in the cardioprotective effect of HSYA or AKBA, myocardium was analyzed by immunohistochemistry and H9C2 cell was measured by western blot. Immuhistochemistry analysis of myocardium showed that HSYA or AKBA increased expressions of PGC-1α and Nrf2 after ISO treatment. Consistently, western blot analysis showed that the expressions of PGC-1α and Nrf2 were upregulated by HSYA or AKBA after OGD treatment. Combination of HSYA and AKBA enhanced expressions of PGC-1α and Nrf2 in H9C2 cells compared to HSYA or AKBA respectively(n=6 per group; P<0.05).ConclusionsIn this study, we demonstrated that HSYA and AKBA would be a safe and effective in protecting against myocardial injury through alleviating mitochondria-dependent oxidative stress by enhancing expressions of PGC-1α and Nrf2. In addition, HSYA and AKBA appear to have synergistic effects on cardio-protection. |
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