Baicalein attenuates rotenone-induced SH-SY5Y cell apoptosis through binding to SUR1 and activating ATP-sensitive potassium channels.

Dopaminergic neurons in the substantia nigra (SN) expressing SUR1/Kir6.2 type ATP-sensitive potassium channels (K-ATP) are more vulnerable to rotenone or metabolic stress, which may be an important reason for the selective degeneration of neurons in Parkinson's disease (PD). Baicalein has shown neuroprotective effects in PD animal models. In this study, we investigated the effect of baicalein on K-ATP channels and the underlying mechanisms in rotenone-induced apoptosis of SH-SY5Y cells. K-ATP currents were recorded from SH-SY5Y cells using whole-cell voltage-clamp recording. Drugs dissolved in the external solution at the final concentration were directly pipetted onto the cells. We showed that rotenone and baicalein opened K-ATP channels and increased the current amplitudes with EC50 values of 0.438 µM and 6.159 µM, respectively. K-ATP channel blockers glibenclamide (50 µM) or 5-hydroxydecanoate (5-HD, 250 µM) attenuated the protective effects of baicalein in reducing reactive oxygen species (ROS) content and increasing mitochondrial membrane potential and ATP levels in rotenone-injured SH-SY5Y cells, suggesting that baicalein protected against the apoptosis of SH-SY5Y cells by regulating the effect of rotenone on opening K-ATP channels. Administration of baicalein (150, 300 mg·kg-1·d-1, i.g.) significantly inhibited rotenone-induced overexpression of SUR1 in SN and striatum of rats. We conducted surface plasmon resonance assay and molecular docking, and found that baicalein had a higher affinity with SUR1 protein (KD = 10.39 µM) than glibenclamide (KD = 24.32 µM), thus reducing the sensitivity of K-ATP channels to rotenone. Knockdown of SUR1 subunit reduced rotenone-induced apoptosis and damage of SH-SY5Y cells, confirming that SUR1 was an important target for slowing dopaminergic neuronal degeneration in PD. Taken together, we demonstrate for the first time that baicalein attenuates rotenone-induced SH-SY5Y cell apoptosis through binding to SUR1 and activating K-ATP channels.

Immunity and inflammation in pulmonary arterial hypertension: From pathophysiology mechanisms to treatment perspective.

Pulmonary arterial hypertension (PAH) is a severe cardiopulmonary dysfunctional disease, characterized by progressive vascular remodeling. Inflammation is an increasingly recognized feature of PAH, which is important for the initiation and maintenance of vascular remodeling. High levels of various inflammatory mediators have been documented in both PAH patients and experimental models of PAH. Similarly, multiple immune cells were found to accumulate in and around the wall of remodeled pulmonary vessels and in the vicinity of plexiform lesions, respectively. On the other hand, inflammation is also a bridge from autoimmune diseases to PAH. Autoimmune diseases always lead to chronic inflammation, characterized by the low-level persistent infiltration of immune cells, and elevated levels of several pro-inflammatory cytokines and chemokines. In addition, circulating autoantibodies are found in the peripheral blood of patients, indicating a possible role of autoimmunity in the pathogenesis of PAH. Thus, anti-inflammatory and immunotherapy might be new strategies to prevent or even reverse the process of PAH. Many anti-inflammatory agents and immunotherapies have been confirmed in animal models while some clinical trials employing immunotherapies are completed or currently underway. Here, we review pathological mechanisms associated with inflammation and immunity in the development of PAH, and discuss potential interventions for the treatment of PAH.

Puerarin-V prevents the progression of hypoxia- and monocrotaline-induced pulmonary hypertension in rodent models.

Pulmonary hypertension (PH) is a cardiopulmonary disease characterized by a progressive increase in pulmonary vascular resistance. One of the initial pathogenic factors of PH is pulmonary arterial remodeling under various stimuli. Current marketed drugs against PH mainly relieve symptoms without significant improvement in overall prognosis. Discovering and developing new therapeutic drugs that interfere with vascular remodeling is in urgent need. Puerarin is an isoflavone compound extracted from the root of Kudzu vine, which is widely used in the treatment of cardiovascular diseases. In the present study, we evaluated the efficacy of puerarin in the treatment of experimental PH. PH was induced in rats by a single injection of MCT (50 mg/kg, sc), and in mice by exposure to hypoxia (10% O2) for 14 days. After MCT injection the rats were administered puerarin (10, 30, 100 mg · kg-1 · d-1, i.g.) for 28 days, whereas hypoxia-treated mice were pre-administered puerarin (60 mg · kg-1 · d-1, i.g.) for 7 days. We showed that puerarin administration exerted significant protective effects in both experimental PH rodent models, evidenced by significantly reduced right ventricular systolic pressure (RVSP) and lung injury, improved pulmonary artery blood flow as well as pulmonary vasodilation and contraction function, inhibited inflammatory responses in lung tissues, improved resistance to apoptosis and abnormal proliferation in lung tissues, attenuated right ventricular injury and remodeling, and maintained normal function of the right ventricle. We revealed that MCT and hypoxia treatment significantly downregulated BMPR2/Smad signaling in the lung tissues and PPARγ/PI3K/Akt signaling in the lung tissues and right ventricles, which were restored by puerarin administration. In addition, we showed that a novel crystal type V (Puer-V) exerted better therapeutic effects than the crude form of puerarin (Puer). Furthermore, Puer-V was more efficient than bosentan (a positive control drug) in alleviating the abnormal structural changes and dysfunction of lung tissues and right ventricles. In conclusion, this study provides experimental evidence for developing Puer-V as a novel therapeutic drug to treat PH.

RKC-B1 Blocks Activation of NF-κB and NLRP3 Signaling Pathways to Suppress Neuroinflammation in LPS-Stimulated Mice.

RKC-B1 is a novel fermentation product obtained from the marine micromonospora FIM02-523A. Thus far, there have been few reports about the pharmacological activity of RKC-B1. In our present study, we investigated the anti-neuroinflammatory effects and the possible mechanism of RKC-B1 in LPS-stimulated mice. After treatment with RKC-B1, RNA-seq transcriptome of the cerebral cortex tissue was conducted to find the differentially expressed genes (DEGs). Inflammatory cytokines and proteins were evaluated by ELISA and WB. In RNA-seq analysis, there were 193 genes screened as core genes of RKC-B1 for treatment with neuroinflammation. The significant KEGG enrichment signaling pathways of these core genes were mainly included TNF signaling pathway, IL-17 signaling pathway, NOD-like receptor signaling pathway, NF-κB signaling pathway and others. The corresponding top five KEGG enrichment pathways of three main clusters in PPI network of core genes were closely related to human immune system and immune disease. The results showed that RKC-B1 reduced the levels of pro-inflammatory factors (IL-6, IL-1ß, MCP-1, and ICAM-1) and the expression of COX2 in cerebral cortex tissue. Additionally, we found that the anti-neuroinflammation activity of RKC-B1 might be related to suppress activating of NF-κB and NLRP3/cleaved caspase-1 signaling pathways. The current findings suggested that RKC-B1 might be a promising anti-neuroinflammatory agent.

Formononetin ameliorates myocardial ischemia/reperfusion injury in rats by suppressing the ROS-TXNIP-NLRP3 pathway.

Formononetin (FN), a methoxy isoflavone abundant in many plants and herbs, has been evidently proven to possess multiple medicinal properties. Our study aimed to clarify the impact of FN on myocardial ischemia/reperfusion (I/R) injury (MIRI) and the involved mechanism. A rat model of MIRI was produced by ligation and loosening of the left anterior descending (LAD) branch of the coronary artery. Rats received 10 and 30 mg/kg of FN when the reperfusion started. At 24 h after surgery, cardiac function, infarct size, and sera levels of the cardiac markers and inflammatory mediators were measured. To mimic the inflammasome activation in cardiomyocytes, neonatal rat cardiomyocytes (NRCMs) were cultured and treated with lipopolysaccharide (LPS) plus nigericin. Cell death and reactive oxygen species (ROS) were determined. Myocardial expression and activation of the nucleotide-binding domain and leucine-rich repeat-containing protein 3 (NLRP3) inflammasome in rats were examined by western blotting. The level of thioredoxin interacting protein (TXNIP)-NLRP3 interaction was assessed. FN notably attenuated cardiac dysfunction, infarct size, release of cardiac markers, and elevation of TNF-α, IL-1ß, and IL-6. FN alleviated LPS plus nigericin-induced injury and ROS increase in NRCMs. Western blotting revealed that FN suppressed the activation of NLRP3 inflammasome and TXNIP-NLRP3 interaction in rats. These findings indicate that FN ameliorated MIRI in rats and inhibited the activation of the NLRP3 inflammasome, at least partially, attributable to suppression of the ROS-TXNIP-NLRP3 pathway.

Vasorelaxant effect of quercetin on cerebral basilar artery in vitro and the underlying mechanisms study.

The aim of this study is to investigate the vasorelaxant effect of quercetin on cerebral basilar artery in vitro and provide a preliminary discussion concerning the underlying mechanisms. Using a DMT-isolated micro vessel system, quercetin was found to exhibit a vasodilatory effect on basilar arteries contracted by potassium chloride (KCl), endothelin-1 (ET-1), and 5-hydroxytryptamine (5-HT). The vasorelaxant effect of quercetin was partially attenuated when endothelium cells were removed. L-NAME, indomethacin, and ODQ treatment also decreased the potency of quercetin. In endothelium-denuded rings, the vasorelaxant effect of quercetin was not influenced by K+ channel inhibitors. However, quercetin inhibited KCl induced extracellular calcium influx and ET-1 induced transient intracellular calcium release in a Ca2+-free solution. In conclusion, quercetin induced relaxation of the basilar artery in vitro is partially dependent on endothelium, which is mainly related to NO and COX pathways. It also induces relaxation through blockage of calcium channels.

DL0805-2, a novel indazole derivative, relaxes angiotensin II-induced contractions of rat aortic rings by inhibiting Rho kinase and calcium fluxes.

AIM: DL0805-2 [N-(1H-indazol-5-yl)-1-(4-methylbenzyl) pyrrolidine-3-carboxamide] is a DL0805 derivative with more potent vasorelaxant activity and lower toxicity. This study was conducted to investigate the vasorelaxant mechanisms of DL0805-2 on angiotensin II (Ang II)-induced contractions of rat thoracic aortic rings in vitro. METHODS: Rat thoracic aortic rings and rat aortic vascular smooth muscle cells (VSMCs) were pretreated with DL0805-2, and then stimulated with Ang II. The tension of the aortic rings was measured through an isometric force transducer. Ang II-induced protein phosphorylation, ROS production and F-actin formation were assessed with Western blotting and immunofluorescence assays. Intracellular free Ca(2+) concentrations were detected with Fluo-3 AM. RESULTS: Pretreatment with DL0805-2 (1-100 µmol/L) dose-dependently inhibited the constrictions of the aortic rings induced by a single dose of Ang II (10(-7) mol/L) or accumulative addition of Ang II (10(-10)-10(-7) mol/L). The vasodilatory effect of DL0805-2 was independent of endothelium. In the aortic rings, pretreatment with DL0805-2 (1, 3, and 10 µmol/L) suppressed Ang II-induced Ca(2+) influx and intracellular Ca(2+) mobilization, and Ang II-induced phosphorylation of two substrates of Rho kinase (MLC and MYPT1). In VSMCs, pretreatment with DL0805-2 (1, 3, and 10 µmol/L) also suppressed Ang II-induced Ca(2+) fluxes and phosphorylation of MLC and MYPT1. In addition, pretreatment with DL0805-2 attenuated ROS production and F-actin formation in the cells. CONCLUSION: DL0805-2 exerts a vasodilatory action in rat aortic rings through inhibiting the Rho/ROCK pathway and calcium fluxes.

Salvianolic acid A attenuates vascular remodeling in a pulmonary arterial hypertension rat model.

AIM: The current therapeutic approaches have a limited effect on the dysregulated pulmonary vascular remodeling, which is characteristic of pulmonary arterial hypertension (PAH). In this study we examined whether salvianolic acid A (SAA) extracted from the traditional Chinese medicine 'Dan Shen' attenuated vascular remodeling in a PAH rat model, and elucidated the underlying mechanisms. METHODS: PAH was induced in rats by injecting a single dose of monocrotaline (MCT 60 mg/kg, sc). The rats were orally treated with either SAA (0.3, 1, 3 mg·kg(-1)·d(-1)) or a positive control bosentan (30 mg·kg(-1)·d(-1)) for 4 weeks. Echocardiography and hemodynamic measurements were performed on d 28. Then the hearts and lungs were harvested, the organ indices and pulmonary artery wall thickness were calculated, and biochemical and histochemical analysis were conducted. The levels of apoptotic and signaling proteins in the lungs were measured using immunoblotting. RESULTS: Treatment with SAA or bosentan effectively ameliorated MCT-induced pulmonary artery remodeling, pulmonary hemodynamic abnormalities and the subsequent increases of right ventricular systolic pressure (RVSP). Furthermore, the treatments significantly attenuated MCT-induced hypertrophic damage of myocardium, parenchymal injury and collagen deposition in the lungs. Moreover, the treatments attenuated MCT-induced apoptosis and fibrosis in the lungs. The treatments partially restored MCT-induced reductions of bone morphogenetic protein type II receptor (BMPRII) and phosphorylated Smad1/5 in the lungs. CONCLUSION: SAA ameliorates the pulmonary arterial remodeling in MCT-induced PAH rats most likely via activating the BMPRII-Smad pathway and inhibiting apoptosis. Thus, SAA may have therapeutic potential for the patients at high risk of PAH.

Fluorescein Diacetate Microplate Assay in Cell Viability Detection.

Objective To investigate the application of the fluorescein diacetate (FDA) microplate assay in cell viability detection. Methods Cells were seeded in a 96-well culture plate until detection. After incubated with FDA,the plate was detected by fluorescence microplate analyzer. The effects of FDA incubation duration,concentration,and other factors on the assay's accuracy and stability were assessed. We also compared the results of FDA with methyl thiazolyl(MTT) in terms of cell numbers and H2O2 injury. Results Within 0-30 minutes,the fluorescence-cell number coefficient of FDA assay increased with duration and reached 0.99 in 27-30 minutes. The optimum concentration of final FDA in this study was 10-30 µg/ml. On cell viability detection,the result of FDA method was equivalent to MTT method. As to H2O2 injury assay,the sensitivity of FDA method was superior to MTT on the higher concentration H2O2 treatment due to a relative shorter duration for detection. Conclusion As a stable and reliable method,FDA is feasible for cell variability detection under varied conditions.

Brazilin isolated from the heartwood of Caesalpinia sappan L induces endothelium-dependent and -independent relaxation of rat aortic rings.

AIM: Brazilin is one of the major constituents of Caesalpinia sappan L with various biological activities. This study sought to investigate the vasorelaxant effect of brazilin on isolated rat thoracic aorta and explore the underlying mechanisms. METHODS: Endothelium-intact and -denuded aortic rings were prepared from rats. The tension of the preparations was recorded isometrically with a force displacement transducer connected to a polygraph. The phosphorylation levels of ERK1/2 and myosin light chain (MLC) were analyzed using Western blotting assay. RESULTS: Application of brazilin (10-100 µmol/L) dose-dependently relaxed the NE- or high K(+)-induced sustained contraction of endothelium-intact aortic rings (the EC50 was 83.51±5.6 and 79.79±4.57 µmol/L, respectively). The vasorelaxant effect of brazilin was significantly attenuated by endothelium removal or by pre-incubation with L-NAME, methylene blue or indomethacin. In addition, pre-incubation with brazilin dose-dependently attenuated the vasoconstriction induced by KCl, NE or Ang II. Pre-incubation with brazilin also markedly suppressed the high K(+)-induced extracellular Ca(2+) influx and NE-induced intracellular Ca(2+) release in endothelium-denuded aortic rings. Pre-incubation with brazilin dose-dependently inhibited the NE-stimulated phosphorylation of ERK1/2 and MLC in both endothelium-intact and -denuded aortic rings. CONCLUSION: Brazilin induces relaxation in rat aortic rings via both endothelium-dependent and -independent ways as well as inhibiting NE-stimulated phosphorylation of ERK1/2 and MLC. Brazilin also attenuates vasoconstriction via blocking voltage- and receptor-operated Ca(2+) channels.

Vasodilatory effect of a novel Rho-kinase inhibitor, DL0805-2, on the rat mesenteric artery and its potential mechanisms.

PURPOSE: In the present study, we investigated the vasodilatory effect of a novel scaffold Rho-kinase inhibitor, DL0805-2, on isolated rat arterial rings including mesenteric, ventral tail, and renal arteries. We also examined the potential mechanisms of its vasodilatory action using mesenteric artery rings. METHODS: A DMT multiwire myograph system was used to test the tension of isolated small arteries. Several drugs were employed to verify the underlying mechanisms. RESULTS: DL0805-2 (10(-7)-10(-4) M) inhibited KCl (60 mM)-induced vasoconstriction in three types of small artery rings (pEC50: 5.84 ± 0.03, 5.39 ± 0.03, and 5.67 ± 0.02 for mesenteric, renal, and ventral tail artery rings, respectively). Pre-incubation with DL0805-2 (1, 3, or 10 µM) attenuated KCl (10-60 mM) and angiotensin II (AngII; 10(-6) M)-induced vasoconstriction in mesenteric artery rings. The relaxant effect on the rat mesenteric artery was partially endothelium-dependent (pEC50: 6.02 ± 0.05 for endothelium-intact and 5.72 ± 0.06 for endothelium-denuded). The influx and release of Ca(2+) were inhibited by DL0805-2. In addition, the increased phosphorylation levels of myosin light chain (MLC) and myosin-binding subunit of myosin phosphatase (MYPT1) induced by AngII were blocked by DL0805-2. However, DL0805-2 had little effect on K(+) channels. CONCLUSIONS: The present results demonstrate that DL0805-2 has a vasorelaxant effect on isolated rat small arteries and may exert its action through the endothelium, Ca(2+) channels, and the Rho/ROCK pathway.

[Advance in the study of myocardial ischemic preconditioning and postconditioning and the clinical applications].

Myocardial ischemic preconditioning and postconditioning can reduce myocardial infarct size, improve myocardial contractility, protect coronary endothelial and myocardial cell ultrastructure, as well as reduce the incidence of arrhythmias. Clinical practice has confirmed the safety and efficacy of these two methods of myocardial protection. This paper reviewed about ischemic preconditioning and postconditioning protection mechanisms in myocardial ischemia reperfusion injury and clinical research literatures in recent years, to provide a theoretical basis for finding new treatment strategies on the prevention and treatment of ischemic cardiomyopathy.

[Advance in study on pharmacological effect of Eucommiae Folium].

Eucommia ulmoides is a valuable traditional Chinese medicine, whose cortexes have long been used as medi cines. Due to the scarcity of its resources, people began using its leaves instead of cortexes in medicines. Eucommiae Folium and its leaves have many pharmacological effects and thereby being clinically applied as genuine traditional Chinese medicines. Modern pharmological studies have showed that Eucommiae Folium leaves have such effects as blood pressure reduction, blood lipid regulation, cardiovascular protection, anti-obesity, anti-inflammation, anti-virus, enhancement of immunologic function, resistance against senility and anti-fatigue. In clinic, Eucommiae Folium is mainly used to treat hypertention and obstetrical and gynecological disease. The essay summarizes the latest advance in domestic and foreign studie on pharmacological effeets and clinical applications of Eucommiae Folium leaves, and thus providing reference for studies on new drugs of Eucommiae Folium leave.

Salvianolic acid A improve mitochondrial respiration and cardiac function via inhibiting apoptosis pathway through CRYAB in diabetic cardiomyopathy.

Salvianolic acid A (SAA) is a traditional Chinese medicine that has a good therapeutic effect on cardiovascular disease. However, the underlying mechanisms by which SAA improves mitochondrial respiration and cardiac function in diabetic cardiomyopathy (DCM) remain unknown. This study aims to elucidate whether SAA had any cardiovascular protection on the pathophysiology of DCM and explored the potential mechanisms. Diabetes was induced in rats by 30 mg/kg of streptozotocin (STZ) treatment. After a week of stability, 5 mg/kg isoprenaline (ISO) was injected into the rats subcutaneously. 3 mg/kg SAA was orally administered for six weeks and 150 mg/kg Metformin was selected as a positive group. At the end of this period, cardiac function was assessed by ultrasound, electrocardiogram, and relevant cardiac injury biomarkers testing. Treatment with SAA improved cardiac function, glucose, and lipid levels, mitochondrial respiration, and suppressed myocardial inflammation and apoptosis. Furthermore, SAA treatment inhibits the apoptosis pathway through CRYAB in diabetic cardiomyopathy rats. As a result, this study not only provides new insights into the mechanism of SAA against DCM but also provides new therapeutic ideas for the discovery of anti-DCM compounds in the clinic.

Protection of salvianolic acid A on rat brain from ischemic damage via soluble epoxide hydrolase inhibition.

Epoxyeicosatrienoic acids (EETs) and their regulating enzyme soluble epoxide hydrolase (sEH) have been associated with ischemic stroke. Salvianolic acid A (SAA) is proved to display potent cerebroprotection. However, little information is available about the link between them. This study aimed to investigate whether SAA exhibits its protective effects in rats subjected to middle cerebral artery occlusion (MCAO) through sEH and EETs. The results showed that SAA treatment ameliorated neurological deficits and reduced infarct volume. Notably, the beneficial effects of SAA were attenuated by co-administration of (14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE)), a putative selective EETs antagonist. Furthermore, SAA increased the 14,15-EET levels in the blood and brain of sham and MCAO rats. Assay for hydrolase activity showed that 1 and 3 mg/kg of SAA significantly diminished brain sEH activity of MCAO rats. A fluorescent assay in vitro indicated that SAA could inhibit recombinant human sEH activity in a concentration-dependent manner (IC(50) = 1.62 µmol/l). Immunohistochemical analysis showed that SAA at the doses of 1 and 3 mg/kg significantly decreased sEH protein expression in hippocampus CA1 region of MCAO rats. In conclusion, cerebral protection of SAA is mediated, at least in part, via inhibiting sEH to increase EETs levels.

Dan-Shen-Yin Granules Prevent Hypoxia-Induced Pulmonary Hypertension via STAT3/HIF-1α/VEGF and FAK/AKT Signaling Pathways.

Traditional Chinese medicine (TCM) plays an important role in the treatment of complex diseases, especially cardiovascular diseases. However, it is hard to identify their modes of action on account of their multiple components. The present study aims to evaluate the effects of Dan-Shen-Yin (DSY) granules on hypoxia-induced pulmonary hypertension (HPH), and then to decipher the molecular mechanisms of DSY. Systematic pharmacology was employed to identify the targets of DSY on HPH. Furthermore, core genes were identified by constructing a protein-protein interaction (PPI) network and analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) analysis. Related genes and pathways were verified using a hypoxia-induced mouse model and hypoxia-treated pulmonary artery cells. Based on network pharmacology, 147 potential targets of DSY on HPH were found, constructing a PPI network, and 13 hub genes were predicted. The results showed that the effect of DSY may be closely associated with AKT serine/threonine kinase 1 (AKT1), signal transducer and activator of transcription 3 (STAT3), and HIF-1 signaling pathways, as well as biological processes such as cell proliferation. Consistent with network pharmacology analysis, experiments in vivo demonstrated that DSY could prevent the development of HPH in a hypoxia-induced mouse model and alleviate pulmonary vascular remodeling. In addition, inhibition of STAT3/HIF-1α/VEGF and FAK/AKT signaling pathways might serve as mechanisms. Taken together, the network pharmacology analysis suggested that DSY exhibited therapeutic effects through multiple targets in the treatment of HPH. The inferences were initially confirmed by subsequent in vivo and in vitro studies. This study provides a novel perspective for studying the relevance of TCM and disease processes and illustrates the advantage of this approach and the multitargeted anti-HPH effect of DSY.

Aesculin suppresses the NLRP3 inflammasome-mediated pyroptosis via the Akt/GSK3ß/NF-κB pathway to mitigate myocardial ischemia/reperfusion injury.

BACKGROUND: Aesculin (AES), an effective component of Cortex fraxini, is a hydroxycoumarin glucoside that has diverse biological properties. The nucleotide-binding domain leucine-rich repeat-containing receptor, pyrin domain-containing 3 (NLRP3) inflammasome has been heavily interwoven with the development of myocardial ischemia/reperfusion injury (MIRI). Nevertheless, it remains unclear whether AES makes a difference to the changes of the NLRP3 inflammasome in MIRI. PURPOSE: We used rats that were subjected to MIRI and neonatal rat cardiomyocytes (NRCMs) that underwent oxygen-glucose deprivation/restoration (OGD/R) process to investigate what impacts AES exerts on MIRI and the NLRP3 inflammasome activation. METHODS: The establishment of MIRI model in rats was conducted using the left anterior descending coronary artery ligation for 0.5 h ischemia and then untying the knot for 4 h of reperfusion. After reperfusion, AES were administered intraperitoneally using 10 and 30 mg/kg doses. We evaluated the development of reperfusion ventricular arrhythmias, hemodynamic changes, infarct size, and the biomarkers in myocardial injury. The inflammatory mediators and pyroptosis were also assessed. AES at the concentrations of 1, 3, and 10 µM were imposed on the NRCMs immediately before the restoration process. We also determined the cell viability and cell death in the NRCMs exposed to OGD/R insult. Furthermore, we also analyzed the levels of proteins that affect the NLRP3 inflammasome activation, pyroptosis, and the AKT serine/threonine kinase (Akt)/glycogen synthase kinase 3 beta (GSK3ß)/nuclear factor-kappa B (NF-κB) signaling pathway via western blotting. RESULTS: We found that AES notably attenuated reperfusion arrhythmias and myocardia damage, improved the hemodynamic function, and ameliorated the inflammatory response and pyroptosis of cardiomyocytes in rats and NRCMs. Additionally, AES reduced the NLRP3 inflammasome activation in rats and NRCMs. AES also enhanced the phosphorylation of Akt and GSK3ß, while suppressing the phosphorylation of NF-κB. Moreover, the allosteric Akt inhibitor, MK-2206, abolished the AES-mediated cardioprotection and the NLRP3 inflammasome suppression. CONCLUSIONS: These findings indicate that AES effectively protected cardiomyocytes against MIRI by suppressing the NLRP3 inflammasome-mediated pyroptosis, which may relate to the upregulated Akt activation and disruption of the GSK3ß/NF-κB pathway.

Synthesis and biological activity of flavanone derivatives.

A series of new flavanone derivatives of farrerol was synthesized by a convenient method. The in vitro anti-tumor activity of these compounds was evaluated against human Bel-7402, HL-60, BGC-823 and KB cell lines, the protein tyrosine kinase (PTK) inhibitor activity was also tested. Their cytoprotective activity was tested using hydrogen peroxide (H2O2)-induced injury in human umbilical vein endothelial cells. Their in vitro anti-atherosclerosis activity was tested on vascular smooth muscle cells by the MTT method using tetrandrine as a positive contrast drug. The structures of all compounds synthesized were confirmed by 1H, 13C NMR and ESI-MS. Most of the compounds exhibited good pharmacological activity and the preliminary structure-activity relationships were described.

Glucose consumption assay discovers coptisine with beneficial effect on diabetic mice.

Many drugs with anti-diabetic effects regulate glucose consumption in peripheral tissues. Via cellular glucose consumption assays, we identified that coptisine, a main effective constituent from the plant Coptis chinensis, enhanced hepatic and skeletal muscle glucose consumption. We further explored its effects on glucose metabolism in diabetic animals to elucidate its mechanism of action. Our results showed that coptisine did not show cytotoxicity. Intragastric administration of coptisine for ten days in normal ICR mice markedly decreased fasting blood-glucose levels without significant effects on body weight. In alloxan-induced type 1 diabetic mice, intragastric administration of coptisine for 28 days decreased fasting and non-fasting blood-glucose levels as well. In type 2 diabetic KKAy mice, intragastric administration of coptisine for nine weeks improved glucose tolerance. It decreased fasting/non-fasting blood-glucose and fructosamine levels. Coptisine decreased low-density lipoprotein and total cholesterol levels, however, had no significant effect on triglyceride levels. Coptisine increased AMPK phosphorylation while decreasing Akt phosphorylation in HepG2 hepatic cells and C2C12 myotubes. Coptisine also reduced mitochondrial respiration in isolated and cellular mitochondria, suggesting that coptisine lowered cellular energy levels. In particularly, coptisine administration (10-6 M) decreased the mitochondrial oxygen consumption rate (OCR) with a greater extracellular acidification rate (ECAR), resulting in an oxidative-to-glycolysis phosphorylation shifted for cellular energy generation. Our results demonstrate that coptisine acts as an enhancer of peripheral glucose consumption could improve glucose metabolism in diabetic animals. Coptisine may serve as a novel anti-diabetic agent and warrant further evaluation.

A novel hypertensive crisis rat model established by excessive norepinephrine infusion and the potential therapeutic effects of Rho-kinase inhibitors on it.

Hypertension crisis is a severe disease and needs emergency treatment in clinic. It is an important task to discover novel drugs which could lower the blood pressure steadily and quickly. However, animal models for screening anti-hypertensive crisis agents are unsatisfactory. The present study aimed to establish a new hypertensive crisis rat model and then explore the therapeutic effects of three Rho-kinase inhibitors including Fasudil, DL0805-1 and DL0805-2 on such a disease model. The hypertensive crisis symptoms were developed on male Wistar rats by subcutaneously injecting small doses of norepinephrine (NE) for 10 days in the initial stage. A sudden increase in blood pressure (BP) was then induced by excessive NE infusion. Compounds to be tested were intravenously injected into the rats immediately when the rapidly increased systolic blood pressure appeared. The results have shown that after small dose administration with NE, the rats could obtain acute BP increase to a high level without sudden death when a large dose of NE was injected. Fasudil, DL0805-1 and DL0805-2 could lower the blood pressure quickly in a dose dependent manner and improve the survival rate. The compounds also prevent the animals from organ damage. In conclusion, we established a novel hypertensive crisis animal model which could evaluate agents within a short time. In this model, we found that three Rho-kinase inhibitors have potential therapeutic effects on hypertensive crisis. This work might contribute to the discovery and development of new anti-hypertensive crisis drugs.