Mixed Lineage Kinase Domain-like Protein MLKL Breaks Down Myelin following Nerve Injury.

Successful regeneration of severed peripheral nerves requires the breakdown and subsequent clearance of myelin, tightly packed membrane sheaths of Schwann cells that protect nerve fibers and harbor nerve growth-inhibitory proteins. How Schwann cells initiate myelin breakdown in response to injury is still largely unknown. Here we report that, following sciatic nerve injury, MLKL, a pseudokinase known to rupture cell membranes during necroptotic cell death, is induced and targets the myelin sheath membrane of Schwann cells to promote myelin breakdown. The function of MLKL in disrupting myelin sheaths requires injury-induced phosphorylation of serine 441, an activation signal distinct from the necroptosis-inducing phosphorylation by RIP3 kinase. Mice with Mlkl specifically knocked out in Schwann cells showed delayed myelin sheath breakdown. Lack of MLKL reduced nerve regeneration following injury, whereas overexpression of MLKL accelerated myelin breakdown and promoted the regeneration of axons.

Comparative genomic analysis reveals expansion of the DnaJ gene family in Lagerstroemia indica and its members response to salt stress.

DnaJs/Hsp40s/JPDs are obligate co-chaperones of heat shock proteins (Hsp70), performing crucial biological functions within organisms. A comparative genome analysis of four genomes (Vitis vinifera, Eucalyptus grandis, Lagerstroemia indica, and Punica granatum) revealed that the DnaJ gene family in L. indica has undergone expansion, although not to the extent observed in P. granatum. Inter-genome collinearity analysis of four plants indicates that members belonging to Class A and B are more conserved during evolution. In L. indica, the expanded members primarily belong to Class-C. Tissue expression patterns and the biochemical characterization of LiDnaJs further suggested that DnaJs may be involved in numerous biological processes in L. indica. Transcriptome and qPCR analyses of salt stressed leaves identified at least ten LiDnaJs that responded to salt stress. In summary, we have elucidated the expansion mechanism of the LiDnaJs, which is attributed to a recent whole-genome triplication. This research laid the foundation for functional analysis of LiDnaJs and provides gene resources for breeding salt-tolerant varieties of L. indica.

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.

Three-Year Follow-Up Study Investigating Changes in Retinal Vascular Morphology and the Relationship with Diabetic Retinopathy in Children with Type 1 Diabetes Mellitus.

INTRODUCTION: This study aimed to investigate changes in retinal microvascular morphology and associated factors, and their relationship with diabetic retinopathy (DR) in children with type 1 diabetes mellitus (T1DM). METHODS: Thirty-eight children enrolled in this 3-year follow-up study underwent complete ophthalmic examinations including fundus photography. Retinal vascular parameters were measured automatically and compared between baseline and follow-up. Multiple linear regression was used to investigate factors affecting changes in vascular parameters. Binary logistic regression was used to analyze the relationship between retinal microvascular morphology and DR. RESULTS: The caliber of all retinal vessels (within 1-1.5 papillary diameter [PD] from the center of the optic disc, p = 0.030; 1.5-2 PD, p = 0.003), arterioles, and venules (1.5-2 PD, p = 0.001) was narrower in nearly all regions in the follow-up group compared with the baseline group. Vascular tortuosity increased in the central part of the retina and decreased in the periphery. The density (1-1.5 PD, p = 0.030) and fractal dimension (p = 0.037) of retinal vessels were increased at the end of the follow-up compared with baseline. Retinal vascular caliber was independently correlated with DR (odds ratio 0.793 [95% confidence interval 0.633-0.993]; p = 0.044). CONCLUSION: Retinal microvascular morphology in children with T1DM varied with the disease course. Narrower retinal vessels may be an independent risk factor for DR. Results of this study emphasized the importance of regular follow-up of fundus vascular morphology for the detection of early DR in children with T1DM.

YTHDF2-Mediated m6A methylation inhibition by miR27a as a protective mechanism against hormonal osteonecrosis in BMSCs.

BACKGROUND: With the increasing incidence of steroid-induced necrosis of the femoral head (SNFH), numerous scholars have investigated its pathogenesis. Current evidence suggests that the imbalance between lipogenesis and osteoblast differentiation in bone marrow mesenchymal stem cells (BMSCs) is a key pathological feature of SNFH. MicroRNAs (miRNAs) have strong gene regulatory effects and can influence the direction of cell differentiation. N6-methyladenosine (m6A) is a prevalent epigenetic modification involved in diverse pathophysiological processes. However, knowledge of how miRNAs regulate m6A-related factors that affect BMSC differentiation is limited. OBJECTIVE: We aimed to investigate the role of miR27a in regulating the expression of YTHDF2 in BMSCs. METHODS: We compared miR27a, YTHDF2, and total m6A mRNA levels in SNFH-affected and control BMSCs. CCK-8 and TUNEL assays were used to assess BMSC proliferation and apoptosis. Western blotting and qRT‒PCR were used to measure the expression of osteogenic (ALP, RUNX2, and OCN) and lipogenic (PPARγ and C/EBPα) markers. Alizarin Red and Oil Red O staining were used to quantify osteogenic and lipogenic differentiation, respectively. miR27a was knocked down or overexpressed to evaluate its impact on BMSC differentiation and its relationship with YTHDF2. Bioinformatics analyses identified YTHDF2 as a differentially expressed gene in SNFH (ROC analysis) and revealed potential signaling pathways through GSEA. The effects of YTHDF2 silencing on the lipogenic and osteogenic functions of BMSCs were assessed. RESULTS: miR27a downregulation and YTHDF2 upregulation were observed in the SNFH BMSCs. miR27a knockdown/overexpression modulated YTHDF2 expression, impacting BMSC differentiation. miR27a silencing decreased m6A methylation and promoted osteogenic differentiation, while YTHDF2 silencing exerted similar effects. GSEA suggested potential signaling pathways associated with YTHDF2 in SNFH. CONCLUSION: miR27a regulates BMSC differentiation through YTHDF2, affecting m6A methylation and promoting osteogenesis. This finding suggests a potential therapeutic target for SNFH.

Deciphering the Mechanism of Wogonin, a Natural Flavonoid, on the Proliferation of Pulmonary Arterial Smooth Muscle Cells by Integrating Network Pharmacology and In Vitro Validation.

Wogonin is one of the main active components of Scutellaria baicalensis, which has anti-inflammatory, anti-angiogenesis, and anti-fibrosis effects. Nevertheless, the effect of wogonin on pulmonary hypertension (PH) still lacks systematic research. This study aims to elucidate the potential mechanism of wogonin against PH through network pharmacology and further verify it through biological experiments in pulmonary arterial smooth muscle cells (PASMCs). The potential targets and pathways of wogonin against PH were predicted and analyzed by network pharmacology methods and molecular docking technology. Subsequently, the proliferation of PASMCs was induced by platelet-derived growth factor-BB (PDGF-BB). Cell viability and migration ability were examined. The method of Western blot was adopted to analyze the changes in related signaling pathways. Forty potential targets related to the effect of wogonin against PH were obtained. Based on the protein-protein interaction (PPI) network, gene-ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment, and molecular docking, it was shown that the effect of wogonin against PH is closely related to the proliferation of PASMCs and the hypoxia-inducible factor-1α (HIF-1α) pathway. A variety of results from biological experiments verified that wogonin can effectively inhibit the proliferation, migration, and phenotypic transformation of PDGF-BB-mediated PASMCs. In addition, the anti-proliferation effect of wogonin may be achieved by regulating HIF-1/ NADPH oxidase 4 (NOX4) pathway.

Ultrafast Early Warning of Heart Attacks through Plasmon-Enhanced Raman Spectroscopy using Collapsible Nanofingers and Machine Learning.

As the leading cause of death, heart attacks result in millions of deaths annually, with no end in sight. Early intervention is the only strategy for rescuing lives threatened by heart disease. However, the detection time of the fastest heart-attack detection system is >15 min, which is too long considering the rapid passage of life. In this study, a machine learning (ML)-driven system with a simple process, low-cost, short detection time (only 10 s), and high precision is developed. By utilizing a functionalized nanofinger structure, even a trace amount of biomarker leaked before a heart attack can be captured. Additionally, enhanced Raman profiles are constructed for predictive analytics. Five ML models are developed to harness the useful characteristics of each Raman spectrum and provide early warnings of heart attacks with >98% accuracy. Through the strategic combination of nanofingers and ML algorithms, the proposed warning system accurately provides alerts on silent heart-attack attempts seconds ahead of actual attacks.

Insights into Endothelin Receptors in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a disease which affects the cardiopulmonary system; it is defined as a mean pulmonary artery pressure (mPAP) > 20 mmHg as measured by right heart catheterization at rest, and is caused by complex and diverse mechanisms. In response to stimuli such as hypoxia and ischemia, the expression and synthesis of endothelin (ET) increase, leading to the activation of various signaling pathways downstream of it and producing effects such as the induction of abnormal vascular proliferation during the development of the disease. This paper reviews the regulation of endothelin receptors and their pathways in normal physiological processes and disease processes, and describes the mechanistic roles of ET receptor antagonists that are currently approved and used in clinical studies. Current clinical researches on ET are focused on the development of multi-target combinations and novel delivery methods to improve efficacy and patient compliance while reducing side effects. In this review, future research directions and trends of ET targets are described, including monotherapy and precision medicine.

Puerarin Inhibits NLRP3-Caspase-1-GSDMD-Mediated Pyroptosis via P2X7 Receptor in Cardiomyocytes and Macrophages.

Diabetic cardiomyopathy (DCM) is a critical complication of long-term chronic diabetes mellitus, and it is characterized by myocardial fibrosis and myocardial hypertrophy. Previous studies have shown that the pyroptosis pathway was significantly activated in DCM and may be related to the P2X7 receptor. However, the role of the P2X7 receptor in the development of DCM with pyroptosis is still unclear. In this study, we aimed to explore the mechanism of puerarin and whether the P2X7 receptor can be used as a new target for puerarin in the treatment of DCM. We adopted systematic pharmacology and bioinformatic approaches to identify the potential targets of puerarin for treating DCM. Additionally, we employed D-glucose-induced H9C2 rat cardiomyocytes and lipopolysaccharide-treated RAW264.7 mouse mononuclear macrophages as the in vitro model on DCM research, which is close to the pathological conditions. The mRNA expression of cytokines in H9C2 cells and RAW264.7 macrophages was detected. The protein expressions of NLRP3, N-GSDMD, cleaved-caspase-1, and the P2X7 receptor were investigated with Western blot analysis. Furthermore, molecular docking of puerarin and the P2X7 receptor was conducted based on CDOCKER. A total of 348 puerarin targets and 4556 diabetic cardiomyopathy targets were detected, of which 218 were cross targets. We demonstrated that puerarin is effective in enhancing cardiomyocyte viability and improving mitochondrial function. In addition, puerarin is efficacious in blocking NLRP3-Caspase-1-GSDMD-mediated pyroptosis in H9C2 cells and RAW264.7 cells, alleviating cellular inflammation. On the other hand, similar experimental results were obtained by intervention with the P2X7 receptor antagonist A740003, suggesting that the protective effects of puerarin are related to the P2X7 receptor. The molecular docking results indicated key binding activity between the P2X7 receptor and puerarin. These findings indicate that puerarin effectively regulated the pyroptosis signaling pathway during DCM, and this regulation was associated with the P2X7 receptor.

Network pharmacology approach and experimental verification of Dan-Shen Decoction in the treatment of ischemic heart disease.

CONTEXT: Dan-Shen Decoction, which is composed of Danshen, Tanxiang and Sharen, has a good therapeutic effect on ischemic heart disease (IHD). However, systematic research on the exact mechanism of action of Dan-Shen Decoction is still lacking. The anti-IHD effect of Dan-Shen Decoction was examined in this study using a systematic pharmacological method. OBJECTIVE: This study validates the efficacy and explores the potential mechanisms of Dan-Shen Decoction in treating IHD by integrating network pharmacology analyses and experimental verification. MATERIALS AND METHODS: The active components, critical targets and potential mechanisms of Dan-Shen Decoction against IHD were predicted by network pharmacology and molecule docking. H9c2 cells were pretreated with various 1 µg/mL Dan-Shen Decoction for 2 h before induction with 1000 µmol/L CoCl2 for 24 h. The cell viability was detected by CCK8, and protein expression was detected by western blots. RESULTS: The network pharmacology approach successfully identified 69 active components in Dan-Shen Decoction, and 122 potential targets involved in the treatment of IHD. The in vitro experiments indicate that the anti-IHD effect of Dan-Shen Decoction may be closely associated with targets such as AKT1 and MAPK1, as well as biological processes such as cell proliferation, inflammatory response, and metabolism. CONCLUSIONS: This study not only provides new insights into the mechanism of Dan-Shen Decoction against IHD, but also provides important information and new research ideas for the discovery of anti-IHD compounds from traditional Chinese medicine.

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.

A Generic Pixel Pitch Calibration Method for Fundus Camera via Automated ROI Extraction.

Pixel pitch calibration is an essential step to make the fundus structures in the fundus image quantitatively measurable, which is important for the diagnosis and treatment of many diseases, e.g., diabetes, arteriosclerosis, hereditary optic atrophy, etc. The conventional calibration approaches require the specific parameters of the fundus camera or several specially shot images of the chess board, but these are generally not accessible, and the calibration results cannot be generalized to other cameras. Based on automated ROI (region of interest) and optic disc detection, the diameter ratio of ROI and optic disc (ROI-disc ratio) is quantitatively analyzed for a large number of fundus images. With the prior knowledge of the average diameter of an optic disc in fundus, the pixel pitch can be statistically estimated from a large number of fundus images captured by a specific camera without the availability of chess board images or detailed specifics of the fundus camera. Furthermore, for fundus cameras of FOV (fixed field-of-view), the pixel pitch of a fundus image of 45° FOV can be directly estimated according to the automatically measured diameter of ROI in the pixel. The average ROI-disc ratio is approximately constant, i.e., 6.404 ± 0.619 in the pixel, according to 40,600 fundus images, captured by different cameras, of 45° FOV. In consequence, the pixel pitch of a fundus image of 45° FOV can be directly estimated according to the automatically measured diameter of ROI in the pixel, and results show the pixel pitches of Canon CR2, Topcon NW400, Zeiss Visucam 200, and Newvision RetiCam 3100 cameras are 6.825 ± 0.666 µm, 6.625 ± 0.647 µm, 5.793 ± 0.565 µm, and 5.884 ± 0.574 µm, respectively. Compared with the manually measured pixel pitches, based on the method of ISO 10940:2009, i.e., 6.897 µm, 6.807 µm, 5.693 µm, and 6.050 µm, respectively, the bias of the proposed method is less than 5%. Since our method doesn't require chess board images or detailed specifics, the fundus structures on the fundus image can be measured accurately, according to the pixel pitch obtained by this method, without knowing the type and parameters of the camera.

Puerarin-V Improve Mitochondrial Respiration and Cardiac Function in a Rat Model of Diabetic Cardiomyopathy via Inhibiting Pyroptosis Pathway through P2X7 Receptors.

There is a new form of puerarin, puerarin-V, that has recently been developed, and it is unclear whether puerarin-V has a cardioprotective effect on diabetic cardiomyopathy (DCM). Here, we determined whether puerarin-V had any beneficial influence on the pathophysiology of DCM and explored its possible mechanisms. By injecting 30 mg/kg of STZ intraperitoneally, diabetes was induced in rats. After a week of stability, the rats were injected subcutaneously with ISO (5 mg/kg). We randomly assigned the rats to eight groups: (1) control; (2) model; (3) metformin; (4-6) puerarin-V at different doses; (7) puerarin (API); (8) puerarin injection. DCM rats were found to have severe cardiac insufficiency (arrythmia, decreased LVdP/dt, and increased E/A ratio). In addition, cardiac injury biomarkers (cTn-T, NT-proBNP, AST, LDH, and CK-MB), inflammatory cytokines (IL-1ß, IL-18, IL-6, and TNF-α), and oxidative damage markers (MDA, SOD and GSH) were markedly increased. Treatment with puerarin-V positively adjusts these parameters mentioned above by improving cardiac function and mitochondrial respiration, suppressing myocardial inflammation, and maintaining the structural integrity of the cardiac muscle. Moreover, treatment with puerarin-V inhibits the P2X7 receptor-mediated pyroptosis pathway that was upregulated in diabetic hearts. Given these results, the current study lends credence to the idea that puerarin-V can reduce myocardial damage in DCM rats. Furthermore, it was found that the effect of puerarin-V in diabetic cardiomyopathy is better than the API, the puerarin injection, and metformin. Collectively, our research provides a new therapeutic option for the treatment of DCM in clinic.

Systems Pharmacology-Based Strategy to Investigate Pharmacological Mechanisms of Total Flavonoids in Dracocephalum moldavica on Chronic Heart Failure.

Heart failure (HF) is a clinical syndrome of cardiac insufficiency caused by abnormalities in cardiac structure and function that arise for various reasons, and it is the final stage of most cardiovascular diseases' progression. Total flavonoid extract from Dracocephalum moldavica L. (TFDM) has many pharmacological and biological roles, such as cardioprotective, neuroprotective, anti-atherogenic, antihypertensive, anti-diabetic, anti-inflammatory, antioxidant, etc. However, its effect on HF and its molecular mechanism are still unclear. In this study, we used systems pharmacology and an animal model of HF to investigate the cardioprotective effect of TFDM and its molecular mechanism. Eleven compounds in TFDM were obtained from the literature, and 114 overlapping genes related to TFDM and HF were collected from several databases. A PPI network and C-T network were established, and GO enrichment analysis and KEGG pathway analysis were performed. The top targets from the PPI network and C-T network were validated using molecular docking. The pharmacological activity was investigated in an HFpEF (heart failure with preserved ejection fraction) mouse model. This study shows that TFDM has a protective effect on HFpEF, and its protective mechanism may be related to the regulation of proinflammatory cytokines, apoptosis-related genes, fibrosis-related genes, etc. Collectively, this study offers new insights for researchers to understand the protective effect and mechanism of TFDM against HFpEF using a network pharmacology method and a murine model of HFpEF, which suggest that TFDM is a promising therapy for HFpEF in the clinic.

Systematic Elucidation of the Mechanism of Genistein against Pulmonary Hypertension via Network Pharmacology Approach.

: Numerous studies have shown that genistein has a good therapeutic effect on pulmonary hypertension (PH). However, there has been no systematic research performed yet to elucidate its exact mechanism of action in relation to PH. In this study, a systemic pharmacology approach was employed to analyze the anti-PH effect of genistein. Firstly, the preliminary predicted targets of genistein against PH were obtained through database mining, and then the correlation of these targets with PH was analyzed. After that, the protein-protein interaction network was constructed, and the functional annotation and cluster analysis were performed to obtain the core targets and key pathways involved in exerting the anti-PH effect of genistein. Finally, the mechanism was further analyzed via molecular docking of genistein with peroxisome proliferator-activated receptor γ (PPARγ). The results showed that the anti-PH effect of genistein may be closely related to PPARγ, apoptotic signaling pathway, and the nitric oxide synthesis process. This study not only provides new insights into the mechanism of genistein against PH, but also provides novel ideas for network approaches for PH-related research.

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.

Cardioprotective Effects of Puerarin-V on Isoproterenol-Induced Myocardial Infarction Mice Is Associated with Regulation of PPAR-Υ/NF-κB Pathway.

Puerarin is a well-known traditional Chinese medicine which has been used for the treatment of cardiovascular diseases. Recently, a new advantageous crystal form of puerarin, puerarin-V, has been developed. However, the cardioprotective effects of puerarin-V on myocardial infarction (MI) heart failure are still unclear. In this research, we aim to evaluate the cardioprotective effects of puerarin-V on the isoproterenol (ISO)-induced MI mice and elucidate the underlying mechanisms. To induce MI in C57BL/6 mice, ISO was administered at 40 mg/kg subcutaneously every 12 h for three times in total. The mice were randomly divided into nine groups: (1) control; (2) ISO; (3) ISO + puerarin injection; (4⁻9) ISO + puerarin-V at different doses and timings. After treatment, cardiac function was evaluated by electrocardiogram (ECG), biochemical and histochemical analysis. In vitro inflammatory responses and apoptosis were evaluated in human coronary artery endothelial cells (HCAECs) challenged by lipopolysaccharide (LPS). LPS-induced PPAR-Υ/NF-κB and subsequently activation of cytokines were assessed by the western blot and real-time polymerase chain reaction (PCR). Administration of puerarin-V significantly inhibits the typical ST segment depression compared with that in MI mice. Further, puerarin-V treatment significantly improves ventricular wall infarction, decreases the incidence of mortality, and inhibits the levels of myocardial injury markers. Moreover, puerarin-V treatment reduces the inflammatory milieu in the heart of MI mice, thereby blocking the upregulation of proinflammatory cytokines (TNF-α, IL-1ß and IL-6). The beneficial effects of puerarin-V might be associated with the normalization in gene expression of PPAR-Υ and PPAR-Υ/NF-κB /ΙκB-α/ΙΚΚα/ß phosphorylation. In the in vitro experiment, treatment with puerarin-V (0.3, 1 and 3 µM) significantly reduces cell death and suppresses the inflammation cytokines expression. Likewise, puerarin-V exhibits similar mechanisms. The cardioprotective effects of puerarin-V treatment on MI mice in the pre + post-ISO group seem to be more prominent compared to those in the post-ISO group. Puerarin-V exerts cardioprotective effects against ISO-induced MI in mice, which may be related to the activation of PPAR-γ and the inhibition of NF-κB signaling in vivo and in vitro. Taken together, our research provides a new therapeutic option for the treatment of MI in clinic.

Discovery of Novel N-Substituted Prolinamido Indazoles as Potent Rho Kinase Inhibitors and Vasorelaxation Agents.

Inhibitors of Rho kinase (ROCK) have potential therapeutic applicability in a wide range of diseases, such as hypertension, stroke, asthma and glaucoma. In a previous article, we described the lead discovery of DL0805, a new ROCK I inhibitor, showing potent inhibitory activity (IC50 6.7 µM). Herein, we present the lead optimization of compound DL0805, resulting in the discovery of 24- and 39-fold more-active analogues 4a (IC50 0.27 µM) and 4b (IC50 0.17 µM), among other active analogues. Moreover, ex-vivo studies demonstrated that 4a and 4b exhibited comparable vasorelaxant activity to the approved drug fasudil in rat aortic rings. The research of a preliminary structure-activity relationship (SAR) indicated that the target compounds containing a ß-proline moiety have improved activity against ROCK I relative to analogues bearing an α-proline moiety, and among the series of the derivatives with a ß-proline-derived indazole scaffold, the inhibitory activity of the target compounds with a benzyl substituent is superior to those with a benzoyl substituent.

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.