Cardioprotection And Molecular Mechanisms Of Flavonoids From Dracocephalum Rupestre Hance

Backgrounds:Cardiovascular diseases (CVD) are harmful diseases to the health of humans with characteristics such as high incidence of mortality and morbidity, high recurrence rate and many complications. The mortality of CVD remains in the forefront of the world, the number of deaths amount to 20% of the world. Men over the age of 45 and women over the age of 65 are easily in cardiovascular symptoms, such as myocardial infarction, hypertension, congestive heart failure, coronary heart disease especially. In recent years the patients become younger and younger, which has immense threat on people's health. CVD is considered belonging to the disorderly category of haemodynamics. For the past few years, cure policy has changed to controlling the development of the diseases. Some studies have demonstrated that reactive oxygen species (ROS) may be the important participant of occurrence and development of CVD. ROS may lead to the cardiovascular functional disturbance, cell apoptosis and necrosis, endogenous antioxidation system destruction, metabolism and function change of lipid and protein, signal transduction change, and gene expression alteration. Therefore it is very valuable for CVD to treatment with antioxidation.Doxorubicin is an anthracycline antibiotic with a broad spectrum of activity and high potency against human malignant neoplasms such as leukemia and leucoma. However, its clinical use is limited by its severe cumulative dose-related cardiotoxicity, which leads to the development of cardiomyopathy and heart failure. When used with other antineoplastic agents such as cyclophosphamide, bleomycetin, and cisplatin, cardiotoxicity becomes more serious. Once cardiac failure happens, the fatality rate may amount to 33%～70%, which restricts its application in clinic greatly.In recent years it has been demonstrated that cardiomyopathy induced by doxorubicin is connected with the following aspects mainly: (1) free radical metabolism abnormality; (2) calcium overload; (3) inhibition of nucleic acid and protein synthesis; (4) energy metabolism obstacle. These mechanisms influence each other and act in common; especially free radical metabolism abnormality and calcium overload have close relation to other mechanisms, which play an important role in doxorubicin-induced-cardiotoxicity especially in the occurrence and development of cardiomyoxyte apoptosis.ROS is generated by mitochondria electron transfer, immunocyte infiltration, and oxidase (Xanthine oxidase, NADH/NADPH oxidase, Nitric oxide synthesis) induction. ROS and oxidative stress play a crucial role in the pathophysiology of diverse cardiovascular disease. ROS may lead to the cardiomyocyte apoptosis and necrosis, endogenous antioxidation system destruction, metabolism and function change of lipid and protein, signal transduction change, gene expression alteration, and intracellular calcium (Ca²⁺) overload, which form infernal circle. Intracellular Ca²⁺ overload is considered as initiating agent of cell apoptosis. Furthermore Ca²⁺ overload can increase ROS synthesis and cause cell injury induced by ROS. Therefore lipid peroxidation of cardiomyocytes and intracellular Ca²⁺ overload correlate each other, which are the key mechanisms for CVD and cardiotoxicity induced by doxorubicin.The traditional Chinese medicine action range is broad; the poisonous side effect is few, which can protect CVD and cardiotoxicity induced by doxorubicin by eliminating ROS and inhibiting the formation of lipid peroxidation and regulating intracellular Ca²⁺ homeostasis. Therefore, it is still one of the prior roles for pharmacy researchers to seek new, high effective, low poisonous, and many targeted drugs from natural plant.There are rich plant resources in our country. Dracocephalum rupestre Hance is a perennial herb found throughout the northern and western China, i.e. Liaoning, Neimeng, Shanxi and Qinghai Province. Its aerial parts were commonly employed in relieving headache, soothing sore throat, subsiding cough, and preventing icterohepatitis in traditional Chinese medicine. It has been demonstrated that the total flavonoids of Dracocephalum rupestre Hance can relieve acute myocardial ischemia and minificate the area of myocardial ischemia simultaneously. The bood flow of coronary artery is increased obviously. For seeking the active constituent and making use of the plant resource, the chemical composition study of these herbs has been carried out systematically. By Silica gel chromarography, Sephadex LH-20 chromatography and Chiral HPLC, many flavonoids and glycosides were isolated from the ethanol extract of this plant.Oxidize injury is one of the mechanisms of cardiovascular disease and myocardial damage induced by doxorubicin, therefore it is very important to study the antioxidation molecular mechanism for treatment to CVD. There are some regulatory gene and protein of antioxidant in cardiomyocytes such as NF-E2-related factor 2 (Nrf2), antioxidation response element (AREs), and extracellular signal-regulated kinase1/2 (ERK1/2). The signal transduction pathways mediating oxidation-reduction reaction are mainly Nrf2/ARE and the MAP kinase pathways.Inactive Nrf2 is retained in the cytosol by associated with the cytoskeletal protein 1 (Keap1). Cytosolic Nrf2 is phosphorylated and translocates into the nucleus in response to protein kinase C activation and Map kinase pathways. In the nucleus, Nrf2 activate genes through AREs by interacting with transcription factors in the bZIP family, including CREB, ATF4 and fos or jun. Enzymes involved in the Phase II detoxification of xenobiotics to reduce cellular stress include glutathione transferases A2 (GST-A2), quinone reductase-l(NQOl), epoxide hydrolase, heme oxygenase-1(HO-1), UDP-glucuronosyl transferases, and gamma-glutamylcysteine synthetase (γ-GCS or GCL). Expression of these genes protects cells from oxidative damage and can prevent mutagenesis and cancer. Transcription of these enzymes is regulated through AREs. Nrf2 is transcription factors that bind to AREs and activate these genes. Nrf2 activation is opposed by small maf proteins, including MafG and MafK, maintaining a counterbalance between Nrf2 ;and the oxidation level of the intracellular environment.In addition, the cardiomyopathy induced by doxorubicin involves generation of ROS and cardiomyocyte apoptosis. Oxidize injury and Ca²⁺ overload can lead to cardiomyocyte apoptosis. Bcl-2 and the Caspase family play important role in apoptosis pathway. Therefore, protection or alleviation of CVD and doxorubicin cardiotoxicity can be achieved by administration of drugs via protecting oxidative damage, decreasing Ca²⁺ overload, and acting signal transduction pathway.In our experiments, we observed the effects of cardioprotection and molecular mechanisms of flavonoids from Dracocephalum rupestre Hance on cardiotoxicity induced by doxorubicin via culturing cardiomyocytes in vitro. Naringenin-7-O-glucoside (NARG), a major active flavonoid isolated from Dracocephalum rupestre Hance, has attracted our interest as a compound potentially and was detected the effects on cardiomyocyte injury induced by doxorubicin. Furthermore, we tried to analyze the relation between many mechanisms. It will be very significant to develop new drugs for treatment to CVD, decrease the cardiotoxicity induced by doxorubicin, and increase the safe of clinical application.Methods:1. In the present study, the pharmacological activities of five flavonoids extracted from Dracocephalum rupestre Hance such as eriodictyol, eriodictyol-7- O-glucoside, luteolin, compound six, and NARG were screened primarily by MTT assay. NARG was found to have more protective effect compared to others. Then the protective effects and possible mechanisms of NARG on cardiomyocyte injury induced by doxorubicin were evaluated. 1) Cells are stained by crystal violet first and then cell viability was detected by MTT assay. The effects on proliferation in normal and injured cardiomyocytes were detected in diferent doses (2.5,5,10,20,40,80μM). 2) Lipid peroxidation measurement: the content of reduced glutathione (GSH) and MDA, the activities of superoxide dismutase (SOD), glutathine peroxidase (GSH-Px ), and catalase(CAT) were also detected according to manufacture kit.2. The effects of NARG on cardiomyocyte apoptosis induced by doxorubicin were detected: 1) To determine the effects of NARG on apoptosis induced by doxorubicin in H9C2 cells, we examined cell morphology after Wright's Giemsa and Hoechst 33258 staining under inversion and fluorescence microscope; 2 ) Chromosomal DNA fragmentation was analyzed by agarose gel electrophoresis; 3) Then cellular fluorescence was measured by flow cytometry analysis after FITC-Annexin V/propidium iodide (PI) double staining; 4) The expression of caspase-3, caspase-9, Bcl-2 and HO-1 mRNA was detected by using RT-PCR; 5) The level of Bcl-2 and HO-1 protein was detected by using Western blotting; 6) Effect of NARG on intracellular Ca²⁺ overload in H9c2 cardiomyocytes was detected by using fluorescent probe Fura-2/AM.3. Effects of NARG on endogenous antioxidant enzymes against doxorubicin toxicity: 1) The expression of glutamate-cysteine ligase modifier subunit (GCLM),glutamate-cysteine ligase catalytic subunit (GCLC) and NQO1 mRNA was detected by using RT-PCR; 2) The level of NQO1 protein was detected by using Western blotting; 3 ) The potential role of ERK1/2 in regulation of NARG-induced Nrf2-dependent gene expression in H9c2 cardiomyocytes was detected by using Western blotting.Results:1. Five flavonoids extracted from Dracocephalum rupestre Hance at each of low doses (2.5,5,10,20,40μM) alone did not cause any apparent cytotoxicity. The inhibitory effect increased at doses higher than 80μM. NARG (10, 20, 40μM) pretreatment had a significant protective effect against doxorubicin-mediated cytotoxicity in a dose-dependent manner at low doses. The protective effect decreased at doses higher than 80μM. Compared with the control, doxorubicin treated cells possessed significantly less GSH levels and activities of SOD, GSH-Px, and CAT, whereas NARG (10, 20 and 40μM) pretreatment could effectively prevent doxorubicin-induced these reduction. The intracellular GSH level was significantly increased and MDA was decreased after treatment with NARG compared to the control group. Incubation of H9C2 cells with NARG resulted in significant upregulation in SOD, GSH-Px, and CAT activities in a concentration-dependent manner at low doses.2. NARG (10, 20, and 40μM) pretreatment provided a significant protective effect against doxorubicin-mediated apoptosis:Doxorubicin induced rapid changes in the nuclear morphology of H9C2 cells, with heterogeneous intensity and chromatin condensation under fluorescence microscope. Normal cells were seen as round-shaped nuclei with homogeneous fluorescence intensity. Pretreatment with NARG (10, 20, and 40μM) significantly protected the cells from the morphological changes induced by doxorubicin. The effect decreased at doses higher than 80μM. Chromosomal DNA fragmentation was shown that chromatin of H9C2 cells were undergone mternucleosomal cleavage after treatment with doxorubicin, as demonstrated by the formation of characteristic ladder pattern of DNA migration. NARG (10, 20, and 40μM) pretreatment significantly prevented DNA fragmentation, further confirmed by flow cytometric detection. Furthermore, NARG increased the protein levels of HO-1 and Bcl-2 in cardiomyocytes and suppressed the mRNA expression of caspase-3 and caspase-9. NARG (10, 20, and 40μM) pretreatment significantly decreased intracellular Ca²⁺ overload in H9c2 cardiomyocytes.3. The effect of NARG on induction of endogenous antioxidant enzymes: The mRNA expression of GCLM and GCLC was upregulated by NARG as detected by RT-PCR. NARG (10, 20, and 40μM) pretreatment increased NQO1 mRNA and protein levels, phosphorylation of ERK1/2, and nuclear translocation of Nrf2 in cardiomyocytes as detected by Western blotting.Conclusions:The development of CVD and doxorubicin cardiomyopathy involves generation of ROS and cardiomyocyte apoptosis. Since cardiomyocyte apoptosis contributes to the development of myocardial dysfunction in heart failure, inhibition of cardiomyocyte apoptosis may provide new opportunities for the prevention and treatment of CVD and doxorubicin-induced heart failure. These results suggest that NARG has protective effects against doxorubicin-induced apoptosis by induction of endogenous antioxidant enzymes via phosphorylation of ERK1/2 and nuclear translocation of Nrf2, effects which could underlie the use of NARG therapeutic agent for treating CVD or preventing cardiomyopathy associated with doxorubicin.