The Mechanism Of Plantago Asiatica L. Seeds Extract Against Cardiac Hypertrophy

Objective:Cardiac hypertrophy is the early stage of many heart diseases,such as coronary heart disease,hypertension,valvular dysfunction and cardiomyopathy.Cardiomyocyte autophagy and apoptosis play an important role in the process of cardiac hypertrophic response.Plantago asiatica L.seeds has a long history as a traditional Chinese medicine in China.Its traditional functions include promoting water circulation,removing dampness and stopping diarrhea,clearing liver and improving vision,clearing heat and resolving phlegm.According to the syndrome types of cardiovascular diseases,Plantago asiatica L.seeds is often used in the combination of Zhenwu Decoction,Buyang Huanwu Decoction and Yiqi Qiangxin Decoction.Modern studies have found that Plantago asiatica L.seeds has immunomodulatory,hypoglycemic,hypolipidemic,and anti-inflammatory effects,and can alleviate liver injury,recover renal function,regulate blood-pressure,and improve metabolic syndrome.Plantago asiatica L.seeds extract(PASE)is prepared from Plantago asiatica L.seeds and has shown tremendous pharmacological activities.Acteoside(AC)is the main monomer component in Plantago asiatica extract,and it is also considered as one of the main indexes to evaluate the quality of Plantago asiatica.Studies have found that AC also has rich pharmacological activities such as anti-inflammatory,antioxidant,anti platelet aggregation,anti-aging,anti anxiety,relieving fatigue,sedation and hypnosis,immune regulation,liver protection and neuroprotection.However,whether PASE could relieve cardiac hypertrophy has not been elucidated.The present study is aimed to investigate the effect of PASE on cardiac hypertrophy model and explore its potential underlying mechanism.Methods:1.Study on the effect and mechanism of PASE on isoproterenol(ISO)-induced cardiac hypertrophy in mice: Cardiac hypertrophy was induced in C57BL/6 mice by subcutaneous injection of ISO for two weeks.Meanwhile,the mice were intraperitoneally injected with PASE at dosages of 20,40 and 80 mg/kg/day.Cardiac hypertrophy was evaluated by echocardiographic examination,haematoxylin and eosin staining and quantitative real-time polymerase chain reaction.Expressions of proteins involved in autophagy and apoptosis such as Beclin1,P62,LC3 BII,Bax,Bcl-2 and Cleave-caspase3 were detected by western blotting analysis.2.Study on the effect of PASE on ISO-induced H9c2 cells: Western blot,transient transfection,acridine orange staining and autophagy inducer were used to observe the effect and mechanism of PASE on cardiomyocyte,H9c2 cell,induced by ISO with excessive autophagy and apoptosis.3.Screening of effective components of PASE for inhibiting myocardial hypertrophy:H9c2 cells induced by ISO were treated with the four main components of PASE.The protein and m RNA expressions of ANP,BNP and β-MHC were examined by Western blot and q PCR,respectively.4.Study on the effect and mechanism of AC on ISO-induced cardiac hypertrophy in mice: C57BL/6 mice were injected subcutaneously with ISO to induce cardiac hypertrophy.At the same time,mice were injected intraperitoneally with AC at doses of15,30 and 60 mg/kg/d.Echocardiography,hematoxylin-eosin staining and real-time quantitative polymerase chain reaction were used to evaluate myocardial hypertrophy.Western blot was conducted to detect the expressions of Beclin1,P62,LC3 BII,Bax,Bcl-2 and Cleaved-caspase-3 and other proteins involved in autophagy and apoptosis.5.Study on the effect of AC on ISO-induced H9c2 cells: Western blot,transient transfection,acridine orange staining and other methods were used to observe the effect of PASE on the excessive autophagy and apoptosis of ISO-induced cardiomyocyte H9c2 cells.Result:1.PASE significantly improved the cardiac function indexes,including EF,FS,SV and CO,in mice.It reduced the ratio of myocardial hypertrophy index HW/BW and myocardial cell size,and down-regulated the m RNA expressions of myocardial hypertrophy markers ANP,BNP and β-MHC.In addition,PASE also restored the abnormal expressions of ISO-induced autophagy and apoptosis markers,such as LC3 II,Beclin1,P62,Bcl2,Bax,and Cleaved-caspase-3.2.PASE suppressed the m RNA expressions of ANP,BNP and β-MHC in H9c2 cells induced by ISO.It restored the abnormally expressed autophagy and apoptosis markers such as LC3 BII,Beclin1,P62,Bcl2,Bax and Cleaved-caspase-3;reduced the accumulation of acidic vesicle organelles(AVO),and inhibited ISO-induced autophagosomes and autolysosomes;in addition,PASE could counteract the increase of autophagy induced by the autophagy inducer rapamycin.3.On ISO-induced H9c2 cells,acteoside,one of the four components of PASE,showed the best effect on the inhibition of ANP,BNP and β-MHC.4.AC significantly improved the cardiac function indicators,such as EF,FS,SV and CO,in mice.It reduced the ratio of HW/BW and myocardial cell size,and down-regulated the m RNA expressions of ANP,BNP and β-MHC.Moreover,PASE re reversed the abnormally expressed autophagy and apoptosis markers,such as LC3 II,Beclin1,P62,Bcl2,Bax,and Cleaved-caspase-3,induced by ISO.5.AC inhibited the m RNA expressions of ANP,BNP and β-MHC in H9c2 cells induced by ISO.It restored the abnormally expressed proteins involved in autophagy and apoptosis,including LC3 BII,Beclin1,P62,Bcl2,Bax and Cleaved-caspase-3;reduced the accumulation of AVO,and inhibited the increase of autophagosomes and autolysosomes,and decreased mitochondrial membrane potential.Conclusions:PASE attenuated the cardiac hypertrophy induced by ISO by inhibiting excessive autophagy and apoptosis of cardiomyocytes.Acteoside is the best effective ingredient of PASE to inhibit cardiac hypertrophy.