Studies On The Active Substances And Pharmacological Mechanisms Of Rhodiola Wallichiana Extracts For Treating Heart Failure

Heart failure is the final stage of various heart diseases such as ischemic heart disease and hypertension,with high morbidity and mortality.The pathological process of heart failure involves many pathological factors such as calcium overload,endothelial dysfunction,atherosclerosis,apoptosis and oxidative stress.Clinical therapeutic agents include diuretics,?-blockers,angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers(ACEI/ARBS),etc.However,the existing therapeutic strategies can not meet the clinical needs.The integrated traditional Chinese medicine(TCM)and western medicine therapy for heart failure based on the method of benefiting Qi and activating blood circulation has some advantages,reflecting in improving symptoms and delaying the progression of the disease.It is necessary to use modern science and technology to scientifically understand the active substances and pharmacological mechanisms of TCM on heart failure.It is of great significance for exploring novel therapies in heart failure and promoting the modernization of TCM.Rhodiola wallichiana extracts(HJT)is a typical TCM with the efficacy of strengthening body resistance,activating blood and dissolving stasis,promoting coronary circulation and relieving pain.At present,Chinese medicines containing HJT are mainly used to treat coronary heart disease-stable angina pectoris.It has been clinically found that standard therapy combined with HJT preparation can improve cardiac function and obtain clinical benifites in patients with heart failure,but its active substances and the pharmacological mechanisms have not been clarified.Therefore,the present study focused on cardiac hypertrophy and myocardial hypoxia injury to investigate the active substances and mechanisms of HJT for treating heart failure.The main findings and academic contributions of this thesis are as follows:1.It was confirmed that the active substances of HJT,such as Herbacetin,could improve cardiac hypertrophy by inhibiting Serum/Glucocorticoid regulated kinase 1(SGK1).(1)In a mice model with hypertrophic cardiomyopathy,we showed that HJT could block TAC-mediated cardiac hypertrophic responses,improved heart structure and possessed the anti-apoptotic and anti-oxidation effects.The gene expression profile-Connectivity Map(CMAP)was used to predict the effect of HJT.It showed that HJT may have an antiadrenergic effect with the highest similarity to phenoxybenzamine and SGK1 was the key target of HJT.HJT was found to inhibit TAC-induced activation of SGK1 in cardiac tissue.(2)A novel mass-spectrometry based kinase inhibitor assay was first developed to search for SGK1 inhibitors from HJT.Three SGK1 inhibitors were found,including herbacetin(HBT),rhodiosin and p-coumaric acid.HBT displayed the best activity with an IC50 of 752 n M that has not previously been reported.(3)A direct binding between Herbacetin and SGK1 was proved in vitro,and the site-specific mutation of Asp177 in the N-terminal domain of SGK1 completely ablated the inhibitory activity of Herbacetin.A panel of 20 flavonoids structurally related to Herbacetin were tested for their inhibitory potency on SGK1.The results of structure-activity relationship showed that the presence of-OH groups at the C-3,C-8,C-4' positions of Herbacetin were suggested to be favorable for the protein interaction,whereas the absence of an-OH group at the C-5 position may further enhance SGK1 inhibitory activity.The above studies found that a variety of flavonoids represented by Herbacetin are novel SGK1 inhibitors,suggesting that they are the main active substances of HJT for treating cardiac hypertrophy.2.The mechanism of Herbacetin regulating Fox O1 pathway by inhibiting SGK1 for treating cardiac hypertrophy was confirmed in vitro and in vivo.(1)The results showed that Herbacetin significantly suppressed PE-induced cardiomyocyte hypertrophy,and decreased the phosphorylation of SGK1 and FoxO1.Herbacetin can also reduce ROS synthesis and calcium accumulation in hypertrophic cardiomyocytes.In the cardiomyocytes transfected with constitutively active SGK1 mutant SGK1(S422D),Herbacetin treatment could significantly inhibit Fox O1 phosphorylation and the transmission of Fox O1 from the nucleus to cytoplasm.(2)In an isoproterenol-induced heart failure mouse model,Herbacetin attenuated cardiac hypertrophy,myocardial fibrosis and improved cardiac function.(3)Targeted metabolomics analysis further showed that Herabcetin effectively regulated the alteration in lipid metabolism and improved cardiometabolic disorders in ISOinduced heart failure mice.3.The myocardial protective effect of HJT and its active substances were confirmed in myocardial hypoxia injury,and the mechanism was related to the improvement of mitochondrial function.(1)The hypoxia/reoxygenation(H/R)-induced profound elevation of oxidative stress was suppressed by HJT.HJT could attenuate oxidative stress,improve cell survival and increase intracellular ATP contents.The mechanisms were related to the improvement of mitochondrial respiration,as well as the regulation of autophagy-associated proteins LC3,Beclin and m TOR.(2)Histological and physiological evaluation revealed that HJT significantly decreased the infarct area of the heart,and improved cardiac function in a rat model of coronary occlusion.HJT could also increase the expression of LC3 B in ischemia myocardium.(3)It was found that salidroside and tyrosol were the main active substances of HJT against H/R injury in cardiomyocytes.Biotin labeled salidroside and tyrosol were synthesized for target fishing and proteomics studies.Functional analysis indicated that the potential targets were mainly related to mitochondrial function.It was further found that salidroside and tyrosol can up-regulate the expression of mitochondrial fusion proteins MFN2 and OPA1 in H/R-induced myocardial injury.In summary,it was found for the first time that SGK1 is the key target of HJT for treating cardiac hypertrophy,and the main active substance is Herbacetin.The study also described an antioxidation injury ability of HJT by regulation the mitochondrial function,and the active substances are salidroside and tyrosol.Transcriptomics,chemical proteomics,metabolomics and mass spectrometry probes were used to reveal the active substances and pharmacological mechanisms of HJT for treating heart failure.The study developed a new strategy to explore the active substances from complex system and identify the targets of TCM.