DBZ Ameliorates Pressure Over-Loaded Cardiac Hypertrophy Via MTOR/β-TrcP/Nrf2 Pathway

Background:Heart failure(HF)has become one of the patients with extremely high mortality rates in China and even the world.Danshen drugs are widely used in clinical treatment of cardiovascular diseases,but the mechanism of action is often difficult to explain clearly.Based on previous observations and studies on clinical samples,this subject explored the protective effects of a new compound DBZ on pressure overloaded-induced heart failure in rats,and further observed its effects on mTOR/β-TrcP/Nrf2 signaling pathway.This study is expected to elaborate on the precise mechanism of action of DBZ against heart failure,and may provide strong evidence support for the development and application of new clinical drugs.Objectives:To explore the protective effect of DBZ on pressure overloaded-induced heart failure and the signal regulation mechanism through which the protective effect may be achieved from the clinical indicators,animal and cell levels.Methods:(1)Clinical observation:Observe the difference in the expression of serum endoplasmic reticulum stress proteins GRP78 and CHOP in the heart failure patient group(50 cases)and the healthy control group(20 cases).(2)Animal experiment:A total of 60 male rats(180-200g)were selected and randomly divided into 4 groups,15 in each group:①sham+solvent group;②sham+DBZ group;③TAC+solvent group;④T AC+DBZ group.The model group used aortic arch constriction to prepare pressure-induced heart failure models,and the sham operation group only had ligation without constriction.Groups ② and ④ were given 20 mg/kg DBZ gavage every day,and groups ① and ③ were given the same amount of solvent gavage every day.The entire treatment lasted for 8 weeks.Ultrasound was used to detect changes in cardiac function and the changes in related molecular indicators such as oxidative stress,endoplasmic reticulum stress and autophagy were detected after sampling.(3)Cell experiment:Angiotensin II(Ang II)and hydrogen peroxide(H2O2)were used to establish a model of myocardial hypertrophy and oxidative damage.Cardiomyocytes were induced by Ang II and H2O2 for 24h and 12h respectively.Electrophoresis and immunofluorescence were used to observe changes in oxidative stress and the changes in endoplasmic reticulum stress and intracellular autophagy levels,and at the same time observe the regulation of DBZ on the molecular changes of mTOR/β-TrcP/Nrf2 signaling pathway.Results:(1)The expression of GRP78 and CHOP protein in the blood circulation of patients with heart failure increased(p<0.05),and the results of Western blot also showed that the content of GRP78 and CHOP protein in failure patient group was higher(p<0.05).(2)After 8 weeks of treatment,DBZ significantly reduced the rat HW/BW ratio,HW/TL ratio and LVM weight(p<0.01);at the same time,it reduced the heart size and ANF,BNP and other heart failure indicators(p<0.01).(3)Echocardiography showed that DBZ improved the remodeling of the heart ventricular wall,including IVSD,LVID,LVPW and other indicators(p<0.01).Moreover,compared with the model group,the DBZ treatment group improved the pumping function of the heart(p<0.01).(4)The treatment of DBZ for 8 weeks significantly reduced the production of cardiac fibrosis compared with the model group in improving cardiac remodeling(P<0.05),and at the same time reduced the expression of procollagen I and procollagen Ⅲ expression(p<0.05).(5)In addition to the verification of the effect of DBZ in in vivo experiments,it was found in cell experiments that the DBZ treatment group significantly reduced the cell size(p<0.01)and the expression of BNP protein(p<0.01)compared with the AngⅡ treatment group.0.05),but also reduced the related genes expression of heart failure(p<0.01).(6)DBZ significantly reduced the production of ROS in myocardial tissue(p<0.01).The results of Western blot showed that DBZ reduced the expression of NOX2 and NOX4 in myocardial tissue,and increased the expression of antioxidant protein HO-1 expression(p<0.05).(7)Compared with the H2O2 group,DBZ reduced the production of ROS in cardiomyocytes(p<0.01).Intracellular mitochondria is the main place for ROS production.The mitoSOX fluorescence results showed that DBZ significantly reduced the production of mitochondrial ROS(p<0.01).At the same time,similar to the in vivo results,DBZ significantly reduced the expression of NOX2 and NOX4 in cells and increased the expression of the expression of antioxidant protein HO-1(p<0.01).(8)The endoplasmic reticulum stress protein XBP1,CHOP,ATF6,GRP78,and p-PERK protein were found to be elevated in the pressure-induced cardiac hypertrophy model group(p<0.05),and at the same time in the myocardial cells induced by hydrogen peroxide stimulation,similar results were also observed(p<0.05).But DBZ can significantly reduce the expression of endoplasmic reticulum stress protein(p<0.05)and reduce endoplasmic reticulum stress.Excessive endoplasmic reticulum stress increases the apoptosis of cardiomyocytes.We have observed that excessive endoplasmic reticulum stress increases the expression of apoptotic proteins bax,PARP and caspase 3 in both myocardial tissues and in vitro induced cell models.The expression of anti-apoptotic protein bcl-2 was reduced in cardiac hypertrophic models,but DBZ treatment can reverse the apoptosis caused by endoplasmic reticulum stress(p<0.05).(9)Autophagy was slightly increased in the 8-week pressure-induced TAC model in rats,which was manifested in the proportion of autophagy protein LC3 II,the increase of Beclin 1 protein,and the slight decrease of p62 protein(p<0.05).Transmission electron microscopy results also indicated that the number of autophagosomes in the myocardial tissue of the rat TAC model was significantly increased(p<0.05).However,in the DBZ treatment group,the expression of autophagy-related proteins was significantly suppressed,the number of autophagosomes was reduced,and the expression of autophagy was inhibited(p<0.05).Similarly,in the in vitro cell induction experiment,we also observed similar results of autophagy inhibition of DBZ(p<0.05).(10)In order to explore the main mechanisms of DBZ exerts its therapeutic effects on cardiac hypertrophy,we observed whether increasing or inhibiting ROS could have influence in the effects of DB.The results showed that increasing the level of intracellular ROS increased endoplasmic reticulum stress(p<0.05),increased autophagy(p<0.05),increased apoptosis(p<0.05),and weakened the anti-apoptotic effect of DBZ.Similarly,by inhibiting endoplasmic reticulum stress,we observed the reduced autophagy expression(p<0.05)and also reduced apoptosis(p<0.05).However,we did not observe that inhibiting autophagy can reduce apoptosis,and inhibiting autophagy did not further enhance the anti-apoptotic therapeutic effect of DBZ(p<0.05).(11)The concentration of 1μM,5μM,and 10μM DBZ can all increase the expression of Nrf2 in the nucleus(p<0.05),and in the hydrogen peroxide-induced damage model,DBZ can not only increase the expression of Nrf2 in the nucleus(p<0.05),but also increase the expression of total Nrf2 in cells(p<0.05).siRNA Nrf2 can eliminate the anti-endoplasmic reticulum stress and anti-apoptotic effects of DBZ(p<0.05).(12)DBZ inhibits the expression of β-TrcP and GSK3β(Ser 9)protein(p<0.05),while inhibiting the expression of phosphorylated mTOR protein(p<0.05),indicating that DBZ may exerts its therapeutic effect on cardiac hypertrophy via mTOR/β-TrcP/GSK3β(Ser 9)/Nrf2.Conclusions:Clinical data had proved that the expression of endoplasmic reticulum stress protein GRP78 and CHOP in the heart failure patient is higher than that in the normal control group,indicating that endoplasmic reticulum stress plays an important role in the occurrence and development of heart failure.Animal experiments have shown that DBZ has a significant antioxidant effect of endoplasmic reticulum stress and autophagy response caused by oxidative stress increasing.However,the inhibitory effect of DBZ on autophagy does not play a major role in the treatment of cardiac hypertrophy.The anti-oxidation and anti-apoptosis mechanism of DBZ is probably realized through the mTOR/β-TrcP/GSK3β(Ser 9)/Nrf2 pathway.