Effects Of Bisdemethoxycurcumin On Myocardium Apoptosis Induced By Staurosporine In Mice And Its Mechanism

?Background?Cardiovascular disease is the most common killer to greatestly harm the human health and life in clinical,which has become a major global public problem.How to reduce its morbidity,disability and lethality is the primary task of the majority of medical workers.Among the high blood pressure,coronary heart disease,cardiomyopathy and other diseases,the common pathological changes are myocardial remodeling and cardiomyocayte death and their progress.Therefore,saving dying cardiomyocytes and maintaining the heart's systolic function are the key measure to rescue lives and to improve the quality of the patients' life.The death of cardiomyocyte can be divided into necrosis and apoptosis after suffering from the damaged facters.The necrosis is an irreversible pathological process but myocardial cell apoptosis can be reversed by proper intervention and is the major way to save the damaged myocardium.So,timely intervention and reversal of myocardial cell apoptosis process is the significantly clinical treatment for various types of heart disease and the effective means to protect the cardiac function.Based on this,people continue to actively explore and develop all kinds of myocardial protective drugsBisdemethoxycurcumin(BDMC)is a extracted ingredients from the traditional Chinese medicine turmeric.Compared with curcumin,BDMC not only has a better bioavailability and stability but also has a good antioxidant,anti-inflammatory function.Studies have shown that BDMC can inhibit vascular smooth muscle migration and proliferation and can improve obesity caused by high-fat diet as well as reduce the cell aging.Because this compound has a good pharmacological effects and stable chemical properties,Has it a protective effect on the myocardial apoptosis induced by the various types of injury? If it has,how is the mechanism? This scientific research is designed to answer this question.The Nrf2 / ARE pathway is currently considered to be a key endogenous mechanism in cell to resist oxidative stress.NF-E2-associated factor 2(Nrf2)is an important nuclear transcription factor in endogenous antioxidant system.In the oxidative stress conditions,Nrf2 would be activated to tanslocate into the nuclues from the cytoplasm and combine with anti-oxidation element(ARE)to start the expression of the downstream antioxidant stress-related protein and enzyme which have a very important role in the antioxidant stress,anti-inflammatory and anti-apoptosis process in the heart and other organs.However,little research has been done on the relationship between BDMC and Nrf2 and it is unclear that whether the BDMC can protect myocardium via activiting the Nrf2.Therefore,the aim of this study was to investigate the effect of Nrf2 / HO-1 pathway in the myocardium protection of BDMC in myocardial apoptosis induced by staurosporine in mice.?Research Purposes? 1.The effect of BDMC on cardiomyocyte apoptosis was observed by using the model of myocardium apoptosis induced by staurosporine.2.To investigate the role of Nrf2 / HO-1 pathway in BDMC myocardial protection and its mechanism.?Research Methods?1.The culture of primary cardiomyocytes: get the C57 mice(1-2 days)heart,cutting,collagenase digesting,culturing myocardial cells after the differential adherent separation.2.Using the 4?M strocytosporine to estamblish myocardial cell apoptosis model.The cardiomyocyte apoptosis was identified by measuring the cell viability and the activity of caspase-3 and TUNEL positive cells.3.The experimental group names are: control group,STS group,BDMC + STS group,BDMC + STS + Sn PPIX group,BDMC + STS + LY294002 group,BDMC + STS + PD98059 group and BDMC group.4.The CCK-8 method was used to detect the viability of cardiomyocytes.5.The activity of caspase-3 was detected by fluorescence quantitative method.6.TUNEL staining was used to detect the apoptosis of cardiomyocytes in each group.7.The levels of ROS in cardiomyocytes were detected by fluorescence quantitative method.8.The expression levels of Nrf2,HO-1,p-Akt and p-ERK and the levels of Nrf2 were detected by Western-blo.t9.Immunofluorescence was used to observe the nuclear translocation of Nrf2 in the corresponding groups.10.Use of PI3K/Akt pathway inhibitor LY294002 and MEK / ERK pathway inhibitor PD98059 and Nrf2 / HO-1 pathway inhibitor Sn PP IX.?Research Results? 1.Compared with control group,the STS group had obvious lower cell viability group(P <0.05,n = 10).Compared with STS group,the survival rate of myocardial cells in BDMC + STS group was significantly increased(P <0.05,n = 10).Compared with BDMC + STS group,the survival rate of BDMC + STS + Sn PP ? group and BDMC + STS + LY294002 group all were significantly lowered(P <0.05,n = 10),but the survival rate of myocardial cells in BDMC + STS + PD98059 group was no significantly change(P> 0.05,n = 10).2.Compared with control group,the STS group had a obvious elavation in caspase-3 activity(P <0.05,n = 10).Compared with STS group,the activity of caspase-3 in myocardial cells of BDMC + STS group was significantly lowered(P <0.01,n = 10).Compared with BDMC + STS group,the activity of caspase-3 in BDMC + STS + LY294002 group was significantly increased(P <0.01,n = 10),but the activity of caspase-3 in cardiomyocytes of BDMC + STS + PD98059 group and BDMC + STS + Sn PP ? group did not change significantly(P> 0.05,n = 10).3.Compared with the control group,the number of TUNEL positive cells in the STS group was significantly increased(P <0.05,n = 5).Compared with the STS group,the number of TUNEL positive cells in the BDMC + STS group had significantly decreased(P <0.01,n = 5).Compared with BDMC + STS group,the number of apoptotic cardiomyocytes in BDMC + STS + Sn PP ? group failed to reduce.4.Compared with STS group,BDMC + STS group significantly reduced ROS levels in cardiomyocytes(P <0.05,n=8),Compared with control group,the levels of ROS in cardiomyocytes of STS group were significantly increased(P <0.05 n = 8).5.Compared with the STS group,BDMC group the protein level of HO-1,p-Akt and p-ERK significantly increased(P <0.05,n = 5),compared with the control group The protein levels of HO-1,p-Akt,p-ERK in BDMC group also significantly increased(P <0.05,n = 5).6.Compared with STS group and control group,immunofluorescence showed that the nuclear Nrf2 in BDMC + STS group and BDMC group increased significantly,and the cytoplasmic Nrf2 decreased.Western-blot showed that the nuclear Nrf2 levels in the BDMC + STS group and BDMC group were significantly higher than those in the STS group and the control group(P <0.05,n = 5),and the plasma Nrf2 was decreased(P <0.05,n = 5).7.Compared with BDMC + STS group,the level of nuclear Nrf2 in BDMC + STS + LY294002 group was decreased and the level of cytosolic Nrf2 was increased(P <0.05,n = 5),the BDMC + STS + PD98059 had no significant difference at the the cytoplasmic Nrf2 level(P> 0.05,n = 5)but had some decrease in nucleus Nrf2 level compared with the BDMC + STS group(P< 0.05,n = 5).Immunofluorescence showed that the level of the nuclear Nrf2 in BDMC + STS + LY294002 group was lower than that in BDMC + STS group,and the level of the nuclaer Nrf2 in BDMC + STS + PD98059 group had a tiny decrease but the cytoplasmic Nrf2 level was not significantly changed compared with BDMC + STS group.8.The expression of p-Akt in BDMC + STS + LY294002 group was significantly inhibited compared with BDMC + STS group,and the expression of HO-1 was significantly decreased(P <0.05,n = 5).Although the expression of p-ERK was significantly decreased by using PD98059(P <0.05,n = 5),the expression of HO-1 was inhibited in BDMC + STS + PD98059 group compared with BDMC + STS group but not significantly(P< 0.05,n = 5).?Conclusions? 1.BDMC can effectively inhibit the apoptosis induced by the STS in primary cardiomyocyte via promoting cell surviving and reducing the caspase-3 activitiy.2.BDMC activates Nrf2 / HO-1 signaling molecules to arrive the cardioprotective effect mainly through the mediation of PI3 K / Akt pathway.