Guanxin V alleviates ventricular remodeling after acute myocardial infarction with circadian disruption by regulating mitochondrial dynamics.

PURPOSE: Circadian disruption has been a common issue due to modern lifestyles. Ventricular remodeling (VR) is a pivotal progressive pathologic change after acute myocardial infarction (AMI) and circadian disruption may have a negative influence on VR according to the latest research. Whether or not Guanxin V (GXV) has a positive effect on VR after AMI with circadian disruption drew our interest. METHODS: Rats were randomly divided into a sham group, an AMI group, an AMI with circadian disruption group, and an AMI with circadian disruption treated with the GXV group according to a random number table. RNA sequencing (RNA-Seq) was utilized to confirm the different expressed genes regulated by circadian disruption. Cardiac function, inflammation factors, pathological evaluation, and mitochondrial dynamics after the intervention were conducted to reveal the mechanism by which GXV regulated VR after AMI with circadian disruption. RESULTS: RNA-Seq demonstrated that NF-κB was up-regulated by circadian disruption in rats with AMI. Functional and pathological evaluation indicated that compared with the AMI group, circadian disruption was associcataed with deteriorated cardiac function, expanded infarcted size, and exacerbated fibrosis and cardiomyocyte apoptosis. Further investigation demonstrated that mitochondrial dynamics imbalance was induced by circadian disruption. GXV intervention reversed the inflammatory status including down-regulation of NF-κB. Reserved cardiac function, limited infarct size, and ameliorated fibrosis and apoptosis were also observed in the GXV treated group. GXV maintained mitochondrial fission/fusion imbalance through suppressed expression of mitochondrial fission-associated proteins. CONCLUSION: The study findings suggest that identified mitochondrial dysfunctions may underlie the link between circadian disruption and VR. GXV may exert cardioprotection after AMI with circadian disruption through regulating mitochondrial dynamics.

Animal model of coronary microembolization under transthoracic echocardiographic guidance in rats.

The aim of the study was to develop a model of coronary microembolization (CME) in rats at a lower cost. We developed a novel rat model without thoracotomy and ventilation under the guidance of echocardiography. Rats were sacrificed at 3 h, 24 h and 1 month postoperatively in both the Echo-CME and Open-chest CME groups for the comparison of the modeling accuracy, mortality, cardiopulmonary circulation, pleural adhesion and ventilation-induced lung injury (VILI). Results showed that the coronary microthrombus formed at 3 h and reached its peak at 24 h postoperatively, which included platelet aggregation and fibrin web. The Echo-group increases success rates, decreased mortality, postoperative complications including pleural adhesion, cardiopulmonary dysfunction and VILI postoperatively than the Open-chest group at 1month postoperatively. The ejection fraction of the CME group decreased to 50% and obvious cardiac fibrosis formed at 3 months postoperatively. Our unique surgical method provided a platform to study molecular mechanisms and potential new pathways for CME treatment.

The circular RNA hsa_circ_0007623 acts as a sponge of microRNA-297 and promotes cardiac repair.

Circular RNAs (circRNAs) are a kind of closed loop endogenous non-coding RNAs have attracted increasing interest in recent years. However, the mechanism of circRNAs in the pathogenesis of multiple cardiovascular diseases, particularly myocardial ischemia, is rarely reported. In the present study, we examined a circular RNA, hsa_circ_0007623, which is highly expressed in hypoxia-induced human umbilical vein endothelial cells (HUVECs) and can act as a sponge for miR-297, which is involved in cardiac repair after acute myocardial ischemia. In hypoxia-stimulated HUVECs, the inhibition of hsa_circ_0007623 expression was found to reduce cell proliferation, migration, and angiogenesis. Further in vivo experiments confirmed the cardioprotective effect of hsa_circ_0007623 expression in isoproterenol-induced acute ischemia mice. Bioinformatics analysis predicted hsa_circ_0007623, sponge miR-297 and miR-297 directly target VEGFA, which was validated by dual-luciferase assay. Subsequently, functional experiments revealed hsa_circ_0007623 silencing could up-regulate miR-297 and down-regulate VEGFA expression, and reduce cell proliferation, migration, and angiogenesis. We concluded that hsa_circ_0007623 can bind to miR-297, promote cardiac repair after acute myocardial ischemia, and protect cardiac function.

Qihuang Zhuyu formula alleviates coronary microthrombosis by inhibiting PI3K/Akt/αIIbß3-mediated platelet activation.

BACKGROUND: Coronary microembolism (CME) is commonly seen in the peri-procedural period of Percutaneous Coronary Intervention (PCI), where local platelet activation and endothelial cell inflammation crosstalk may lead to micro thrombus erosion and rupture, with serious consequences. Qihuang Zhuyu Formula (QHZYF) is a Chinese herbal compound with high efficacy against coronary artery disease, but its antiplatelet mechanism is unclear. HYPOTHESIS/PURPOSE: This study aimed to elucidate the effects and mechanisms of QHZYF on sodium laurate-induced CME using network pharmacology and in vitro and in vivo experiments. METHODS: We employed high-performance liquid chromatography mass spectrometry to identify the main components of QHZYF. Network pharmacology analysis, molecular docking and surface plasmon resonance (SPR) were utilized to predict the primary active components, potential therapeutic targets, and intervention pathways mediating the effects of QHZYF on platelet activation. Next, we pretreated a sodium laurate-induced minimally invasive CME rat model with QHZYF. In vivo experiments were performed to examine cardiac function in rats, to locate coronary arteries on heart sections to observe internal microthrombi, to extract rat Platelet-rich plasma (PRP) for adhesion assays and CD62p and PAC-1 (ITGB3/ITGA2B) flow assays, and to measure platelet-associated protein expression in PRP. In vitro clot retraction and Co-culture of HUVECs with PRP were performed and the gene pathway was validated through flow cytometry and immunofluorescence. RESULTS: Combining UPLC-Q-TOF/MS technology and database mining, 78 compounds were finally screened as the putative and representative compounds of QHZYF, with 75 crossover genes associated with CME. QHZYF prevents CME mainly by regulating key pathways of the inflammation and platelets, including Lipid and atherosclerosis, Fluid shear stress, platelet activation, and PI3K-Akt signaling pathways. Five molecules including Calyson, Oroxin A, Protosappanin A,Kaempferol and Geniposide were screened and subjected to molecular docking and SPR validation in combination with Lipinski rules (Rule of 5, Ro5). In vivo experiments showed that QHZYF not only improved myocardial injury but also inhibited formation of coronary microthrombi. QHZYF inhibited platelet activation by downregulating expression of CD62p receptor and platelet membrane protein αIIbß3 and reduced the release of von Willebrand Factor (vWF), Ca2+ particles and inflammatory factor IL-6. Further analysis revealed that QHZYF inhibited the activation of integrin αIIbß3, via modulating the PI3K/Akt pathways. In in vitro experiments, QHZYF independently inhibited platelet clot retraction. Upon LPS induction, the activation of platelet membrane protein ITGB3 was inhibited via the PI3K/Akt pathway, revealing an important mechanism for attenuating coronary microthrombosis. We performed mechanistic validation using PI3K inhibitor LY294002 and Akt inhibitor MK-2206 to show that QHZYF inhibited platelet membrane protein activation and inflammation to improved coronary microvessel embolism by regulating PI3K/Akt/αIIbß3 pathways, mainly by inhibiting PI3K and Akt phosphorylation. CONCLUSION: QHZYF interferes with coronary microthrombosis through inhibition of platelet adhesion, activation and inflammatory crosstalk, thus has potential in clinical anti-platelet applications. Calyson, Oroxin A, Protosappanin A, Kaempferol and Geniposide may be the major active ingredient groups of QHZYF that alleviate coronary microthrombosis.

Effect of Guanxin V in animal model of acute myocardial infarction.

BACKGROUND: Acute myocardial infarction (AMI) is the most serious and lethal manifestation of coronary heart disease worldwide, presenting extremely high disability and mortality. Our previous studies have shown that Guanxin V (GXV) could significantly improve the cardiac function and the blood flow dynamics, and reduce serum levels of inflammatory factors in AMI rats, thus triggering ventricular remodeling (VR) at post-AMI. METHODS: An in vivo AMI model was established in Syrian hamsters by performing the ligation of the left anterior descending coronary artery. Syrian hamsters were randomly divided into four groups, namely Sham operation group (n = 12), AMI group (n = 12), GXV group (GXV 6 g/Kg/d, n = 12), and Tranilast group (Tra 105 mg/Kg/d, n = 12). Drug intervention was conducted for consecutive 8 weeks. Relative biological indicators were measured in the 4th and 8th week, respectively. RESULTS: Cardiac functions were improved, and the infarcted size and heart weight index were limited in Syrian hamsters of GXV and Tra groups compared with those in AMI group. Furthermore, GXV was able to decrease the number of mast cells and chymase level in Syrian hamsters with AMI. Administration of GXV remarkably inactivated the renin-angiotension-aldosterone system, and alleviated myocardial fibrosis and cardiomyocyte apoptosis, thus slowing down VR at post-AMI. CONCLUSION: GXV slows down the process of VR at post-AMI by reducing chymase level and mast cells number, as well as inactivating the reninangiotension-aldosterone system..

Puerarin promotes the viability and differentiation of MC3T3-E1 cells by enhancing LC3B-mediated autophagy through downregulation of miR-204.

Puerarin is a bioactive substance extracted from Pueraria lobata. It is known to promote the viability, differentiation and mineralization of osteoblasts. However, the molecular mechanisms involved in these activities are not well understood. The present study was conducted with the aim of elucidating the effect of puerarin on osteoblasts and to explore the underlying mechanism. CCK-8 analysis showed that puerarin (0.1, 1 and 10 µM) promoted the viability of osteoblastic MC3T3-E1 cells, with 1 µM of puerarin exhibiting the strongest effect. Moreover, 1 µM puerarin significantly increased the activity of alkaline phosphatase (ALP) and the formation of mineralized nodules in the MC3T3-E1 cells. Treatment with 1 µM puerarin for 72 h led to a significant upregulation in the expression level of microtubule-associated light chain 3 (LC3)B and Beclin1 proteins. This treatment was more effective in promoting LC3B expression than what was observed following treatment with rapamycin (overexpression for autophagy). The bilayer membrane structure of autophagosomes was observed by electron microscopy. Conversely, 3-methyladenine (3-MA, inhibitor of autophagy) reduced the cell viability as well as the activity of alkaline phosphatase (ALP) in MC3T3-E1 cells, although, there was no significant influence on mineralization. Prediction results of the biological information showed that LC3B could be a direct target of microRNA-204 (miR-204). In the present study, the expression level of miR-204 was decreased by puerarin. miR-204 mimics significantly decreased LC3B expression and inhibited auotophagosome formation, while the miR-204 inhibitor had the opposite effects. To conclude, the results of the present study suggest that puerarin promotes the viability and differentiation of MC3T3-E1 cells through autophagy, which is possibly associated with miR-204-regulated LC3B upregulation.

Astragaloside IV exerts angiogenesis and cardioprotection after myocardial infarction via regulating PTEN/PI3K/Akt signaling pathway.

AIMS AND METHODS: Acute myocardial infarction (AMI) is a common cardiovascular disease with high mortality. Astragaloside IV (AS-IV) was reported to have cardioprotective effect after AMI. We hypothesize that the cardioprotective role of AS-IV is exerted by enhancing angiogenesis via regulating PTEN/PI3K/Akt signaling pathway. To valid our hypothesis, AMI rats and human umbilical vein endothelial cells (HUVECs) were employed in our study. KEY FINDINGS: After treatment, cardiac function, survival rate, infarct size, pathological changes and fibrosis, cell apoptosis, ultrastructural changes, angiogenesis and expression of PTEN/PI3K/Akt signaling pathway were evaluated, respectively. In vitro study we detected proliferation, tube formation and signaling pathway activation of HUVECs treated with AS-IV, lentivirus overexpressed PTEN was employed to elucidate the potential mechanism. The results indicated that AS-IV administration significantly improved cardiac function and survival rate, limited infarct size, ameliorated pathological changes and fibrosis deposition, inhibited apoptosis, relieved ultrastructure injury and enhanced angiogenesis, PTEN/PI3K/Akt signaling pathway was activated simultaneously compared to the model group. In vitro study suggested that AS-IV treatment promoted cell proliferation and tube formation, and induced PTEN/PI3K/Akt signaling pathway activation. Importantly, overexpression of PTEN by lentivirus abolished AS-IV-induced angiogenesis. SIGNIFICANCE: Our study indicated that AS-IV could promote angiogenesis and cardioprotection after myocardial infarction. The mechanisms involve activation of PTEN/PI3K/Akt signaling pathway.

Effect of Kangshuanyihao Formula on the Inflammatory Reaction and SIRT1/TLR4/NF-κB Signaling Pathway in Endothelial Injury.

Endothelial injury plays an important role in atherosclerosis (AS). Kangshuanyihao formula uses therapeutic principles from Chinese medicine to supplement Qi, thereby promoting blood circulation, and remove blood stasis. The mechanism by which the formula inhibits endothelial injury was examined in a rat model of 1,25-dihydroxyvitamin D3 (VD3) intraperitoneal injection and high-fat-induced endothelial injury. Rats were randomly divided into the model, high-dose, middle-dose, low-dose, positive drug (rosuvastatin), and combination (positive drug + middle-dose) groups; 10 Sprague-Dawley rats served as the blank group. The aortic endothelium was stained with hematoxylin and eosin and the levels of blood lipids and inflammation markers (mRNA and protein) were measured. Endothelial injury, lipid levels, and inflammation were increased in the model. Kangshuanyihao formula reduced endothelial injury, improved lipid levels, and downregulated inflammation, as shown by significant reduction of the protein levels of SIRT1, TLR4, and NF-κB and mRNA levels of SIRT1, TLR4, NF-κB, IL-1ß, IL-6, and IL-12. Thus, we conclude that Kangshuanyihao formula can inhibit the inflammatory reaction in the rat model of high-fat-induced endothelial injury after intraperitoneal injection of VD3. This mechanism may be attributed to regulating the SIRT1/TLR4/NF-κB signaling pathway.

Astragaloside IV enhances cardioprotection of remote ischemic conditioning after acute myocardial infarction in rats.

BACKGROUND: Remote ischemic conditioning (RIC) has been shown to be a practical method for protecting the heart from ischemic/reperfusion (I/R) injury. In the present study, we investigated whether or not the combination of RIC and Astragaloside IV (AS-IV) could improve cardioprotection against acute myocardial infarction (AMI)-induced heart failure (HF) when compared with individual treatments. MATERIAL AND METHODS: A rat model of AMI was established via permanent ligation of the left anterior descending coronary artery (LAD). Postoperatively, the rats were randomly grouped into a sham group (n=10), a model group (n=15), an AS-IV alone group (n=15), an RIC alone group (n=15) and a combined treatment group (AS-IV+RIC; n=15). All treatments were administered for 2 weeks. RESULTS: After treatment for 2 weeks, the survival rate was improved, the cardiac function was preserved and the infarcted size was limited in AS-IV alone and RIC alone treatment groups compared to the model group, whereas the combined treatment yielded the most optimal protective effects. Additional studies suggested that AS-IV enhanced the cardioprotective effects of RIC by alleviating myocardial fibrosis, suppressing inflammation, attenuating apoptosis and ameliorating impairment of the myocardial ultrastructural. CONCLUSION: AS-IV enhances the cardioprotective effects of RIC against AMI-induced HF and ventricular remodeling, which represents a potential therapeutic approach for preserving cardiac function and improving the prognosis of AMI.