The Protective Effects Of Puerarin On Cardiac Hypertrophy By Activation Of Autophagy Via AMPK/mTOR Signal Pathway

BackgroundCardiac hypertrophy is a kind of adaptive response to increased neuroendocrine activation and hemodynamic load. During the early period, the increased myocardial tension is mainly balanced via an increased number of bulky sarcomere to maintain the normal cardiac function, body growth and metabolism. However, if the stimulators persist, myocardial remodeling involving maladaptive cellular hypertrophy, cell loss, and cardiac fibrosis will occur. Under the situation of increased neuroendocrine activation, cardiac remodeling would prompt cardiac systolic dysfunction and finally progress to heart failure. Therefore, exploring the relative molecular mechanism of cardiac hypertrophy, and seeking for underling target for therapy would facilitate to retard the development of cardiac hypertrophy, reduce heart failure incidence, and finally improve the prognosis.Autophagy, as a kind of non-injury response to stimulators, could swallow and degrade cytoplasm and injured organelle. By doing that, autophagy enables cell to recycle or expel degraded materials, which having a great significance in maintaining normal cellular structure, metabolism and function. Under physiological Conditions, there is always a low level of autophagy to remove misfolded protein and aging organelle. When varieties of stimulators exist, autophagy as a responsive actor will be remarkably activated to improve cellular function, maintain environmental stable and keep normal structure. Moreover, autophagy can also perform protective effects on cells through removing pathogenic microorganisms.Autophagy has been proved to be associated with the development of cardiac hypertrophy according to previous data. The role of autophagy differs according to the different period of hypertrophy. Current opinions support that baseline autophagy is necessary for cell survival while over-activated autophagy prompt cellular death. Tanaka found in lamp2-/- mouse, heart is remarkably hypertrophic with significant reduced systolic function, lots of autophagic vacuoles accumulate in mitochondria, and acinus of hepatic cells. Mice autophagic dysfunction model is developed by special knockout of Atg5 gene. When the mice with impaired autophagy receive aortic Contraction, left ventricular hypertrophy with severe dysfunction is promptly induced. A low level autophagy can be detected in hypertrophic myocardium caused by hypertension or other factors. The autophagic dysfunction observed in animals subjected to aortic banding procedure indicates that autophage could inhibit the cardiac hypertrophy through promotion of protein degradation. Injured organelles would accumulate to induce apoptosis if autophagy dysfunction occurs. The release of pro-apoptotic factors including cytochrome C and caspase could decrease significantly by induction of mitophagy. All in all, autophagy plays a protective role at least in a particular stage of cardiac hypertrophy, therefore, as an underline therapeutic targets, careful autophagy regulation would facilitate to retard the development of cardiac hypertrophy.Puerarin with a chemical name of 4’,7-dyhydroxyl-8-β-D-glucose ISOflavone is a flavonoid monosome extracted from pueraria lobata. Amounts of data demonstrate that pueararin owns therapeutic effects on cancer, diabetes mellitus, neurodegeneration and inflammation for its wide pharmacological functions. In clinical practice and laboratory experiments, puerarin has been associated with many biological functions such as iron channel obstruction, inhibited platelet activation, anti-apoptosis and hemangiectasis. The clinical use of puerarin injection could significant lower the blood pressure compared with the Control patients with hypertension. In vitro study, puerarin can inhibit the hypetrophic cells induced by angitensin Ⅱ through antioxidant. In published data about autophagy, puerarin could activate autophagy in injured hepatic cells induced by ethyl alcohol, and cellular model of Alzheimer’s disease in vitro. Furthermore, as a kind of phytoestrogen, puerarin might play a pro-autophagy role through affecting estrogen or estrogen receptor. Nevertheless, limited data about the relationship between puerarin and autophagy in cardiac hypertrophy model has been reported. In order to clarify the autophagic regulation of puerarin and the underlying molecular mechanism, this study develops the cardiac hypertrophy model in vivo and in vitro respectively to observe the protective role against cardiac hypertrophy and apoptosis of puerarin.Chapter Ⅰ. The Impact of Puerarin on Autophagy in Cardiac HypertrophyObjective:to observe the change of autophagy in cardiac hypertrophy and the impact of puerarin treatment on autophagy.Methods:rats with cardiac hypertrophy were developed by abdominal aortic banding(AB) and heart tissues were harvested after 1w,2w,3w and 6w following AB procedure. LC3Ⅱ were tested by Western Blot. All animals were divided into 4 groups: Sham group, AB group, AB+puerarin treatment group (Pue, 100mg/kg.d, subcutaneous injection) and AB+ Rapamycin treatment group (Rapa,1.2mg/kg.d, subcutaneous injection). After treatment for 3 weeks, LC3II, P62, and Beclinl were tested respectively by western blot. ISOpreterenol (ISO, 1μM) were used to stimulate H9C2 cells for 3h,6h,12h and 24h respectively. Pue treatments with increased Concentration were performed perspectively. After that, LC3Ⅱ were tested. In order to observe the autophagic flux, CQ(10μM) were used to pretreat H9C2 cells for 16h, and then Pue co-cultured cells with ISO. The influence of puerarin on autophagy flux was observed through GFP-LC3 transfection.Results:Reduced LC3B-Ⅱ and increased p62 protein were observed in AB rats compared with Sham rats at week 2 (0.04±0.02 vs.0.10±0.02, P<0.01;0.37±0.06, vs.0.18±0.04,P><0.01)and 3 (0.05±0.03 vs.0.10±0.02, P<0.01; 0.31±0.06 vs. 0.18±0.04, P<0,01)post-operative, respectively, while at week 6, the expression of LC3-Ⅱ was much more increased in AB rats than in Sham rats(0.15±0.03 vs.0.10± 0.02, P<0.05). By Contrast, p62 was significantly less in AB rats than in SO rats(0.05 ±0.03 vs.0.18±0.04, P<0.01). Beclinl was similar in all groups before week 3 post-AB procedure, but it was significantly higher in AB rats at week 6 post-procedure compared with Sham rats(0.36±0.03 vs.0.19±0.02, P<0.05).The representative images of LC3-positive fluorescence (green) also indicated that the AB procedure reduced LC3 at week 3, and that puerarin therapy resulted in the restoration of LC3.Treatment with ISO 1 μM for at least 12h significantly induced an increase in LC3B-Ⅱ (0.13±0.04 vs.0.33±0.01, P<0.01 for 12h; 0.13±0.06 vs.0.33± 0.01, P<0.01 for 24h respectively). However, decreased LC3B-Ⅱ in H9c2 was detected within 6h post-ISO treatment (0.85± 0.04 vs.0.33±0.01, P<0.01 for 3h; 0.81±0.34 vs.0.33±0.01, P<0.01 for 6h respectively). Except for 1μM,Puerarin pretreatment (5μM, 10μM, and 20μM respectively) for 18h followed by co-culture with ISO for 6 h could dose-dependently increase LC3B-Ⅱ compared with ISO treatment alone(P<0.01). Pretreatment with CQ for 16h resulted in increased LC3B-Ⅱ accumulation in H9c2 cells without any other treatment(140.87±28.07), and this was attenuated by ISO treatment(11.35± 4.69, P<0.01). Cells pretreated with both puerarin (20μM) and CQ, followed by co-culture with ISO, showed an additional increase in the number of GFP-LC3 puncta (119.48±12.54 vs.45.50±6.81, P<0.01).Conclusion:The change of autophagy during cardiac hypertrophy varied with time. In the early period of cardiac hypertrophy, autophagy was inhibited while activatedin the late period. Puerarin could activate autophagy during cardiac hypertrophy.Chapter Ⅱ Puerarin performs protective effects on cardiac hypertrophy through regulation of autophagyObjective:To observe the protective effects of puerarin on cardiac hypertriohy and apoptosis. By inhibiting autophagy, the protective role of puerarin against cardiac hypertrophy and apoptosis was observed.Method:After Pue or Rapa treatment for 6 weeks, heart weight to body weight, left ventriclular mass to body weight, echocardiography, histological analysis and qRT-PCR were applied to evaluate cardiac hypertrophy in vivo. Detection of C-Casp3 by Western Blot, and of apoptotic cell by Tunel assay were performed. In vitro study, all treated cells were divided into Control(Con) group, ISO(1μM for 48h) treated group and Pue (1μM、5μM、20μM for 48h respectively)treated group. qRT-PCR, Hoechst33342 staining assay, detection of C-Casp3 and Alexa Fluor 555 phalloidin staining assay were performed. After inhibition of autophagy by either 3-MA (10mM) pre-treatment or Atg5 siRNA transfection, the protective effects of puerarin against hypertrophy and apoptosis in ISO stimulated H9c2 were observed.Results:Compared with Sham group, ANP、β-MHC mRNA in AB group were significantly elevated (8.21±0.48 vs.1.05±0.08, P<0.01; 11.26±0.71 vs.1.30±0.27, P<0.01). Similar with that in Rapa group, ANP、P-MHC mRNA in Pue group were significantly down-regulated respectively(1.40±0.28 vs.8.21±0.48, P<0.01; 1.47±0.10 vs.11.26±0.71, P<0.01). Impaired LV function indicated by reduced LVEF and LVFS was not observed in AB rats at week 6.However, hypertrophic indicators such as LVPWd, LVPWs, ⅣSd, and ⅣSs increased significantly in the AB group (234.95±9.53,333.62±9.80,199.23±7.42, and 313.64±16.64) compared with the Sham group (134.78±5.68,215.64±1.14,128.53±4.76, and 222.32±5.57). After 6 weeks of therapy with either Pue (152.66±8.37,230.20±3.47,133.89±3.01, and 246.28±4.71) or Rapa (164.39±6.03,237.87±4.57,149.55±3.66, and 251.99±28.937), those indicators decreased significantly compared with untreated AB rats.HW/BW and LVm/BW in AB group (4.53±0.44,3.18±0.29) were much higher than that in Sham group(2.69±0.16,1.84±0.08), and in Pue group(3.00±0.38,1.74±0.43) and Rapa group(2.82±0.12,1.97±0.23) were much lower than AB group (P<0.01) respectively. H&E staining analysis of cardiac cross-sections revealed that the increased myocyte area noted in AB rats was reduced by either puerarin or rapamycin treatment. Analysis with Masson’s stain demonstrated that both interstitial and perivascular fibrosis were increased in rats with AB, and both puerarin and rapamycin treatment significantly reduced myocyte area and fibrosis compared with AB rats. A marked increase in cardiomyocyte apoptosis, as indicated by increased C-Casp3 protein, was detected at week 6 in AB ratscompared with Sham rats(1.20±0.22 vs. 0.04±0.02, P<0.01). TUNEL-positive cells were seldom identified in Sham rats(0.002 ±0.001), but were markedly increased in AB rats(0.101±0.022), and thiswas blocked significantly in both Pue-treated(0.008±0.002) and Rapa-treated AB rats(0.008±0.002). In vitro study, ANP、β-MHC mRNA in ISO group (3.50±0.52, 3.98±0.30) were significantly higher than Con group (0.93±0.22,0.85±0.20), in Pue (1μM) group was similar with ISO group (3.00±0.18 vs.3.50±0.52, P=0.06; 3.73±0.33 vs.3.98±0.30, P=0.18 respectively). Pue (5μM 及 20μM) treatment significantly down-regulated ANP and β-MHC mRNA compared with ISO group. Cellular area in ISO group were much bigger than Con group (703.00±18.57 vs. 299.00±26.14, P<0.01), difference between Pue (1μM) and ISO group was not significant (683.50±18.72 vs.703.00±18.57, P=0.20), but Pue (5μM,20μM) can significantly reduce the area compared with ISO group. Apoptotic index indicated as Hoechst33342 staining was higher in ISO group than that in Con group (0.12±0.03 vs. 0.03±0.01,P<0.01). Difference between Pue (1μM) group and ISO group were not significant (0.10±0.02 vs.0.12±0.03, P=0.34). Pue(5μM,20μM) could significantly reduce the area. Compared with Pue,3-MA+Pue+ISO had significant lower level of LC3Ⅱ (0.28±0.17 vs.1.12±0.30, P<0.01), increased area(821.87±41.45 vs. 392.29±23.42, P<0.01), up-regulated ANP and β-MCH mRNA(3.48±0.24 vs. 1.36±0.08, P<0.01; 4.75±0.37 vs.1.48±0.09, P<0.01), and increased apoptosis(0.15 ±0.03 vs.0.05±0.03, P<0.01 for Hoechest33342 staining; 16.61±0.77 vs.5.82±0.27, P<0.01 for flow cytometry, and 1.56±0.01 vs.0.11±0.03, P<0.01 for C-Casp3 respectively).After suppression of autophagy by pre-transfecting cells withAtg5-siRNA, puerarin failed to reduce the cell surface area and the mRNA expression levels of ANP and β-MHC in the presence of ISO. In cells stimulated with ISO, puerarin treatment reduced apoptotic cells significantly. Importantly, the protective role of puerarin against apoptosis was blocked by treatment with Atg5-siRNA.Conclusion:Puerarin could active autophagy to prevent cardiac hypertrophy and inhibit cellular apoptosis, which failed when autophagy inhibition were pre-performed, indicating that puerarin could play a protective role in cardiac hypertrophy through activation of autophagy.Chapter Ⅲ Puerarin Regulates Autophagy through AMPK/mTOR Signaling PathwayObjective:to observe the impact of puerarin on AMPK/mTOR signal pathway in cardiac hypertrophy, and to further observe the regulation of puerarin on autophagy after inference of AMPK.Method:ISO (1μM) stimulated H9c2 cells in vitro for 3h,6h,12h, and 24h respectively. Puerarin (1μM,5μM, 10μM, and 20μM respectively) treated H9c2 cells followed by co-culture with ISO, and the detection of p-AMPK, LC3B, C-Casp3 were performed by western blot. Cell apoptosis were analysed by Hoeehst33342 stain. After inference of AMPK by either si-AMPK transfection or Dor administration, the regulation of autophagy by puerarin was observed. The change of p-AMPK was observed with time after AB procedure in vivo. The related protein involved in AMPK/mTOR pathway were observed and compared among Sham, AB, Pue and Rapa group.Results:The Thr172 phosphorylation of AMPK showed similar changes to those of LC3B-II in AB rats over time. Decreased phosphorylation of AMPK at Thr172 was also observed at week 2 (0.18±0.04) and 3 (0.22±0.05) post-procedure respectively compared with Sham group (0.49±0.16, P<0.01 respectively). ISO(1μM) stimulated H9C2 cells for 3h,6h respectively, which could down-regulated P-AMPK compared with Con group (0.18±0.06 vs.0.93±0.04, P<0.01; 0.20±0.05 vs. 0.93±0.04.,P<0.01), but for 12h and 24h respectively could significantly up-regulated P-AMPK (2.22±0.25 vs.0.93±0.04, P<0.01; 2.26±0.48 vs.0.93±0.04,P<0.01). The difference of P-AMPK expression between Pue (1μM) group and ISO group was not significant (0.21±0.03 vs.0.22±0.03, P=0.87). P-AMPK in Pue (5μM, 10μM, and 20μM respectively) compared with that in ISO group were much higher (0.40±0.01 vs.0.22±0.03,P<0.01; 0.48±0.03 vs.0.22±0.03,P<0.01; 0.48±0.07 vs.0.22±0.03, P<0.01). Puerarin treatment for 3w in AB rats could significantly up-regulated P-AMPK (0.87±0.06) compared with AB rats without any treatment (P<0.01). Compared with Sham group, P-mTOR (1.55±0.09 vs.0.54±0.02, P<0.01), P-4EBP1 (2.19±0.34 vs.1.09±0.02, P<0.01), and P-p70S6K (0.97±0.05 vs. 0.33±0.01, P<0.01) up-regulated remarkably. Puerarin treatment for 3w could significantly down-regulate P-mTOR (0.12±0.02 vs.1.55±0.09, P<0.01), P-4EBP1 (0.89±0.10 vs.2.19±0.34, P<0.01), P-p70S6K (0.09±0.05 vs.0.97±0.05, P<0.01) compared with AB group. Compared with Con group (1.07±0.19), ISO group had significantly lower level of LC3Ⅱ (0.14±0.11, P<0.01). Compared with ISO group, Met group or Pue (20μM) could respectively up-regulate LC3Ⅱ (1.03±0.23 vs.0.14±0.11,P<0.01; 1.20±0.35 vs. 0.14±0.11,P<0.01). Compared with Pue group, Dor group had much lower level of LC3Ⅱ (0.09±0.08 vs.1.20±0.35,P<0.01), bigger area (745.75±41.06 vs. 325.00±12.91, P<0.01), higher relative level of ANP(4.08±0.44 vs.1.07±0.29,P<0.01) and β-MHC mRNA(3.99±0.25 vs.0.97±0.19,P<0.01) and greater apoptosis (0.15±0.03 vs.0.05±0.03, P<0.01 for Hoechst33342 staining; 0.46±0.12 vs. 0.09±0.03, P<0.01 for C-Casp3). RNAi was applied to specifically knockdown AMPK gene, LC3II was much lower in Si-AMPK transfected cells than that in C-siRNA cells. The difference of LC3II between Pue (C-siRNA) and ISO (C-siRNA) was not significant. Compared with Pue (C-AMPK) group, Pue (Si-AMPK) had much lower level of LC3Ⅱ (0.05±0.02 vs.1.51±0.07,P<0.01), bigger area (727.75±71.37 vs.342.50±74.92, P<0.01), higher level of ANP(4.18±0.49 vs.0.98±0.15, P<0.01) and P-MCH mRNA (4.03±0.22 vs.0.95±0.24, P<0.01) and greater apoptosis(0.13±0.02 vs.0.05±0.03, P<0.01 for Hoechst33342 staining; 0.36±0.10 vs.0.09±0.02, P<0.01 for C-Casp3 protein).Conclusion:AMPK/mTOR signal pathway could be partly involved in the puerarin’s regulation of autophagy.