Effects And Mechanisms Of Luteolin-7-O-glucoside Against Angiotensin ? Induced Cardiac Hypertrophy

Pathological cardiac hypertrophy develops in response to disorders such as hypertension,coronary myocardial infarction,artery disease,metabolic and diabetic cardiomyopathy,and valvular heart disease,which eventually give rise to ventricular remodeling and cardiac dysfunction.Pathological hypertrophy is a key risk factor for heart failure.But the pathological mechanism of cardiac hypertrophy is complex and not clear at present.Autophagy degrades dysfunctional organelles,aggregated proteins,and intracellular pathogens,provides energy for cellular homeostasis.Constitutive autophagy in the heart is a homeostatic mechanism for maintaining cardiomyocyte size and global cardiac structure and function.However,when severely triggered;the autophagic machinery may lead to cell death.Angiotensin ?(Ang ?)is the primary effector peptide of renin-angiotensin system(RAS).Chronic elevation of Ang ? can lead to pathological cardiac hypertrophy.A number of studies have shown that autophagy plays important roles in Ang ?-induced cardiac hypertrophy,the functions and mechanisms of autophagy during the Ang ?-induced cardiac hypertrophy is still not totally understood.As the pathological mechanism of cardiac hypertrophy is complex,the effective method for prevention and treatment of myocardial hypertrophy has not been found yet.Autophagy pathway research will be beneficial to investigate the developing process and mechanisms of cardiac hypertrophy induced by Ang ? from the view point of autophagy,to define the relationship of myocardial hypertrophy and myocardial cell autophagy and adjustment of cardiomyocyte autophagy which will find targets for developing new treatment or prevention of myocardial hypertrophy,heart failure and myocardial infarction.Luteolin-7-O-glucoside(LUTG)was one of flavonoid glycosides,isolated from Dracocephalum tanguticum Maxim(Labiatae).Previous studies have found that LUTG pretreatment has significant protective effect against cardiac toxicity induced by doxorubicin and had no effect on its antitumor activity.LUTG can also reduce myocardial injury induced by starvation through regulating autophagy.No study carries out whether LUTG have an effect on Ang ?-induced cardiac hypertrophy at present.The aim of this research is to explore the effects and mechanisms of LUTG on Ang ?-induced cardiac hypertrophy in vitro and in vivo,in order to provide new idea about therapies for cardiac hypertrophy.MTT assay showed that LUTG had no effects on cell proliferation at low dose,and could promote cardiomyocyte proliferation if its concentration was more than 40?mol/L in H9c2 cells in vitro.We established the cardiac hypertrophy model induced by Ang ? and used Image Pro Plus software to count the cell surface area.The result showed that LUTG could decrease cardiomyocytes surface area induced by Ang ?.We used BCA assay to test the total protein concentration,the result showed that LUTG could decrease protein concentration induced by Ang ?.The expression of ANP mRNA was tested measured by RT-PCR.The result showed that LUTG could decrease the expression of ANP mRNA induced by Ang ?.These above results suggested that LUTG can reverse Ang ?-induced cardiac hypertrophy.We used DCFH-DA staining and MDA kit to detect the generation of ROS and content of MDA.Results showed that LUTG decreased the Ang ?-induced ROS generation and MDA content.AO staining and LTG staining were used to detect the autophagy;results showed that LUTG inhibited Ang ?-induced autophagy.Western blotting showed that LUTG reduced the expression of DNA double strand damage protein?-H2AX and Nrf2 translocation into nucleus.LUTG also reduced the exoression of autophagy related protein Beclinl and LC3B2/LC3B1,increased the expression of P62,p-Akt,Akt,mTOR,and p-mTOR.These above results indicated that Ang?-induced excessive ROS caused cardiomyocyte autophagy which was ameliorated by LUTG through inhibiting Akt/mTOR pathway.Furthermore,LTG staining and Western blotting was used to test the change of autophagy with 3-MA addition,results showed that pretreatment with 3-MA decreased Ang ?-induced autophagy.3-MA and LUTG synergistically suppressed autophagy induced by Ang ?.We further explored the mechanism of LUTG on Ang?-induced cardiac hypertrophy.DCFH-DA staining showed that NAC reduced Ang?-induced ROS generation.NAC combined with LUTG could synergistically reduce the concentration of ROS.In addition,NAC had no effect on normal cells.Western blotting results showed that NAC or NAC combined with LUTG all reduced the expression of y-H2AX and Nrf2 translocation into nucleus.They also reduced the expression of autophagy related protein Beclinl and LC3B2/LC3B1 increased the expression of P62,Akt,p-Akt,mTOR,and p-mTOR.These results showed that LUTG might inhibit autophagy by reducing ROS generation.We established myocardial hypertrophy mice model through subcutaneous injection of Ang ?(1.44mg/kg/d)for 8 weeks,meanwhile,C57BL/6J mice were then daily treated with LUTG(40mg/kg/d)by gavage administration for 8 weeks.Heart morphology photographs showed that the heart volume of C57BL/6J mice in model group was bigger than solvent control group,and LUTG decreased heart volume compared with model group.HE staining showed that the cells in the model group became hypertrophic,cell nucleus increased,the cell color faded compared with solvent control group.LUTG treatment reduced cell surface area and the degree of pathological changes of myocardial cells.Conclusion:LUTG could reverse Ang ?-induced cardiac hypertrophy through inhibiting autophagy;Ang II-induced excessive ROS generation caused cardiomyocyte autophagy which was ameliorated by LUTG through inhibiting Akt/mTOR pathway.