Study On The Effect Components And Molecular Mechanism Of Simiao Yong'an Decoction Against Diabetic Cardiomyopathy

Diabetic cardiomyopathy is one of the main causes of heart failure in diabetic patients,and 19%of patients with heart failure have diabetes,but there is no effective treatment.Si-Miao-Yong-An decoction is composed of Lonicerae Japonicae Flos,Scrophulariae Radix,Angelicae Sinensis Radix,and Glycyrrhizae Radix et Rhizoma,which has the function of improving blood circulation,alleviating fever and detoxification and nourishing Yin.According to the theory of traditional Chinese medicine,the pathogenesis of diabetic cardiomyopathy is mainly hot and dry internal injury,blood stasis caused by long-term disease,heart blood stasis,accumulated for a long time into poison,mutual stagnation of blood stasis and toxin,and heart body and heart function all damaged.Si-Miao-Yong-An decoction targets the pathogenesis of the stasis toxin injuring heart in diabetic cardiomyopathy and has been frequently used in the treatment of diabetic cardiomyopathy after syndrome differentiation.Basic research has proved that myocardial lipotoxicity induced by abnormal myocardial substrate metabolism plays an important role in the occurrence and development of diabetic cardiomyopathy and glucagon is a key mediator in mediating cardiolipid toxicity.Cardiac lipid toxicity may be the biological basis of stasis toxicity in traditional Chinese medicine.Our previous studies have shown that Si-Miao-Yong-An decoction can improve the cardiac function of mice with heart failure induced by isoproterenol or aortic arch constriction.However,there has been no report on the effect of Si-Miao-Yong-An decoction against diabetic cardiomyopathy,its mechanism and effective components.In this study,the glucagon lipid toxicity pathway was used as the target to study the mechanism and material basis of Si-Miao-Yong-An decoction in improving diabetic cardiomyopathy through in vivo and in vitro experiments.This study will select the optimal effective components of Si-Miao-Yong-An decoction for intervention of lipotoxic signal transduction with glucagon as the core,and partially reveal the effective components and targets of Si-Miao-Yong-An decoction for intervention of diabetic cardiomyopathy.Provide support for the development of innovative drugs against diabetic cardiomyopathy.Part one:literature reviewThis paper reviews the research progress on the role of glucagon in diabetic cardiomyopathy,comb out the relationship between glucagon and heart failure related diseases and potential signaling pathways,and analyze the shortcomings of the current research,so as to provide a basis for the construction of experimental research ideas.To systematically discuss the mechanism of Si-Miao-Yong-An decoction and its main components in improving metabolic syndrome,and to provide experimental reference for the study on the effect and mechanism of Si-Miao-Yong-An decoction in improving diabetic cardiomyopathy.Part two:Study on the effect and mechanism of Si-Miao-Yong-An decoction in the prevention and treatment of diabetic cardiomyopathyThe diabetic mice were constructed by streptozotocin.It has been demonstrated that Si-Miao-Yong-An decoction improves glucolipid metabolism,insulin sensitivity and hyperglucagonemia through measuring blood sugar,blood fat,oral glucose tolerance test,serum insulin,glucagon,pancreas and liver pathological morphology,alpha and beta cell distribution.The diabetic cardiomyopathy model is established in the 18th week,which is confirmed by cardiac ultrasound.Si-Miao-Yong-An decoction improves cardiac function,inhibits myocyte hypertrophy,myocardial fibrosis,myocardial apoptosis,lipid accumulation,inflammatory cell infiltration,and mitochondrial morphology through myocardial tissue HE,WGA,Masson,oil red O,TUNEL,CD45 and transmission electron mcroscopy staining,among which,the obvious lipid-lowering,inhibiting myocardial cell apoptosis and maintaining mitochondrial morphology effects indicate that the improvement of diabetic cardiomyopathy by Si-Miao-Yong-An decoction was related to the inhibition of lipid toxicity.Then,it is found that Si-Miao-Yong-An decoction can downregulate the expression or activation of glucagon(GLC),glucagon receptor(GCGR),Peroxisome proliferators-activated receptor α(PPARα),Peroxisome proliferator-activated receptor coactivator 1α(PGC-1α),and Nuclear transcription factor κB(NF-kB),and upregulate the activation of AMP-activated protein kinase(AMPK)by WB showing that the improvement of diabetic cardiomyopathy by Si-Miao-Yong-An decoction is related to the regulation of GLC/AMPK/NF-κB and GLC/PPARα/PGC-1α pathway.Part three:Study on the effective components of Si-Miao-Yong-An decoction against myocardial lipid toxicityThe lipid toxicity model of H9C2 cardiomyocytes was established.The activity of H9C2 cardiomyocytes was significantly decreased after treated with 250 μM palmitic acid(PA)for 24h.Chlorogenic acid,Neochlorogenic acid,Liquiritigenin,Isoliquiritin,Neoisoliquiritin,Liquiritin apioside,Isoliquiritin apioside,luteolin,and Ferulic acid were shown to significantly inhibit the decrease of myocardial cells activity and the increase of myocardial cells apoptosis induced by PA through analyzing the effects of 20 prototype components on the PA-induced myocardial cells activity or apoptosis,which indicates that the 9 ingredients are the active components of Si-Miao-Yong-An Decoction to improve myocardial cell lipid toxicity.Among 9 active ingredients,chlorogenic acid and ferulic acid show the strongest effect,which are used in the follow-up study.Part four:Study on the effect mechanism of active ingredients of Si-Miao-Yong-An decoction against myocardial lipid toxicityThe results of Annexin V/PI double staining by flow cytometry showed that chlorogenic acid and ferulic acid could inhibit PA-induced apoptosis of cardiomyocytes.The expression of lipid metabolism-related genes was detected by RT-PCR.The results of oil red O and RT-PCR showed that chlorogenic acid and ferulic acid could reduce the lipid accumulation and the expression of lipid metabolism-related genes PPARα,CD36 and UCP3 in PA-induced cardiomyocytes.The results of palmitic acid and glucose absorption indicated that chlorogenic acid and ferulic acid could inhibit PA-induced substrate utilization disorders in H9C2 cardiomyocytes.Mitotracker staining and Seahorse function analysis of mitochondria showed that chlorogenic acid and ferulic acid could improve mitochondrial division and respiratory function injury induced by PA.The results showed that the inhibition of chlorogenic acid and ferulic acid for the cardiomyocyte lipid toxicity was related to the regulation of cardiomyocyte glucolipid metabolism and mitochondrial function.GCGR inhibitor(Adomelivant)can inhibit PA-induced apoptosis of cardiomyocytes,and it has been preliminarily proved that PA induces lipotoxicity of cardiomyocytes by activating GCGR.The expression of GLC pathway key proteins was detected by WB and the results demonstrated that chlorogenic acid and ferulic acid could down-regulate the expressions of GCGR,PPARa and PGC-1α in PA-induced cardiomyocytes,and increase the activation of p-AMPK,suggesting that chlorogenic acid and ferulic acid could improve PA-induced apoptosis of H9C2 cardiomyocytes by inhibiting GCGR signaling pathway.In conclusion,in vivo and in vitro study demonstrated the improvement of Si-Miao-Yong-An decoction and its active components(chlorogenic acid and ferulic acid)could improve the myocardial glucose and lipid metabolism,inhibit myocardial lipotoxicity,and prevent diabetic cardiomyopathy through regulating GLC/PPARa and GLC/AMPK pathways.Characteristics and innovation1.Theoretical innovation:the activating blood and detoxifying therapy and its classic prescription Si-Miao-Yong-An decoction are used in the intervention of diabetic cardiomyopathy,which enriches and develops the blood stasis toxin pathogenesis of diabetic cardiomyopathy.2.Technological innovation:integrating plasma prototype library after oral administration of Si-Miao-Yong-An decoction and in vitro efficacy screening,establishing the optimization method of effective components of Si-Miao-Yong-An decoction in intervening diabetic cardiolipid toxicity,and improving the efficiency of innovative drug discovery.3.Mechanism innovation:regarding GLC/AMPK/NF-κB and GLC/PPARα/PGC-1α as the breakthrough point,through in vivo and in vitro experiment,firstly in-depth discuss the intervention for myocardial cell lipid metabolism of Si-Miao-Yong-An decoction and the effective components(chlorogenic acid and ferulic acid)during the development of diabetic cardiomyopathy,and provide reliable experimental basis for clinical treatment of diabetic cardiomyopathy and further investigation.