Black Soybean Seed Coat Extract Improves Endothelial Function and Upregulates Oxidative Stress Marker Expression in Healthy Volunteers by Stimulating Nitric Oxide Production in Endothelial Cells.

Black soybean seed coat extract (BE) contains multiple bioactive polyphenols, including flavan-3-ols and anthocyanins. BE improves endothelial function; however, it is unclear whether BE protects endothelial cells from senescence. In this study, we examined the effects of BE on endothelial cell senescence and vascular function in healthy individuals. High concentrations of glucose were used to induce senescence in bovine aortic endothelial cells incubated with BE. Senescence, vascular function, and oxidative stress markers were measured. Incubation with BE remarkably inhibited senescence-associated ß-galactosidase and lactate dehydrogenase activities and dose dependently reduced intracellular reactive oxygen species levels in bovine aortic endothelial cells. BE treatment increased the levels of endothelial nitric oxide synthase (eNOS) mRNA and endothelial nitric oxide (NO) metabolites and increased the mRNA expression of klotho, a gene associated with an antiaging phenotype. To examine the effects of BE in humans, we conducted a clinical study using the second derivative of the fingertip photoplethysmogram to investigate vascular function and aging in 24 healthy volunteers. The participants consumed BE supplements (100 mg/day) or a placebo for 2 weeks. When compared with the placebo group, the BE group showed considerably improved vascular function, NO metabolite levels, and oxidative stress. These results suggest that BE supplementation improves endothelial function, possibly through antioxidant activity and NO production, and may consequently reduce the cardiovascular risk associated with aging. BE supplementation may be an effective and safe approach to reduce the risk of atherosclerosis and cardiovascular disease; however, additional studies investigating chronic vascular inflammation are needed.

Danhong formula alleviates endothelial dysfunction and reduces blood pressure in hypertension by regulating MicroRNA 24 - Phosphatidylinositol 3-Kinase-Serine/Threonine Kinase- Endothelial Nitric Oxide Synthase axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Essential hypertension (EH) is one of the important risk factors of cardio-cerebrovascular diseases, and it can significantly increase the incidence and mortality of acute myocardial infarction, cerebral infarction and hemorrhage. Danhong Formula (DHF) was consisting of Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Honghua in Chinese) (Plant names have been checked with http://www.the plant list.org on June 28th, 2023) was approved by State Food and Drug Administration of China, that has been used for thousands of years in the treatment of cardiovascular diseases in China with proven safety and efficacy. Though our previous studies have found that DHF improved endothelial dysfunction (ED) and decreased high blood pressure (BP), the underlying mechanisms of its antihypertensive effect still remain unclear. AIM OF THE STUDY: This study investigated whether DHF regulated MicroRNA 24- Phosphatidylinositol 3-Kinase-Serine/Threonine Kinase- Endothelial Nitric Oxide Synthase (miR-24 - PI3K/AKT/eNOS) axis to produce antihypertensive effect and improve endothelial dysfunction. MATERIALS AND METHODS: Firstly, the chemical components of DHF were analyzed by UHPLC-MS. After that, BP was continuously monitored within the 1st, 3rd, and 4th week in SHR to evaluate the antihypertensive effect of DHF intraperitoneal injection. In addition, not only the contents of serum nitric oxide (NO), prostacyclin (PGI2), and angiotensin II (Ang II) were detected, but also the isolated aorta ring experiment was conducted to evaluate the vasomotoricity to evaluate of DHF on improving endothelial dysfunction. Key proteins or mRNA expression associated with miR-24 - PI3K/AKT/eNOS axis in aorta were detected by capillary Western blot, immunohistochemistry or RT-PCR to explore the underlying mechanisms. Index of NO, Ang II PGI2 and key proteins or mRNA expression were also conducted in miR-24-3p over-expression HUVECs model. RESULTS: Compared with SHR control group, DHF (4 mL/kg/day, 2 mL/kg/day, 1 mL/kg/day) treatment significantly reduced high BP in SHR and selectively increased acetylcholine (Ach) induced vasodilation, but not sodium nitroprusside (SNP) in a manner of concentration dependency in isolated aorta ring. DHF (4 mL/kg/day, 1 mL/kg/day) treatment was accompanying an increment of NO and PGI2, and lowering AngII in SHR. Moreover, DHF treatment significantly up-regulated expression of p-PI3K, p-AKT, mTOR, eNOS and p-eNOS, but down-regulated miR-24-3p expression in aorta. Compared with miR-24-3p over-expression HUVECs model group, DHF treatment inhibited miR- 24-3p expression and up-regulated p-PI3K, p-AKT, mTOR and eNOS mRNA expression. Similarly, DHF treatment increased PI3K, AKT, mTOR and eNOS protein expression in HUVECs by Western blot. CONCLUSIONS: These findings suggest that DHF alleviates endothelial dysfunction and reduces high BP in SHR mediated by down-regulating miR-24 via ultimately facilitating up-regulation of PI3K/AKT/eNOS axis. This current study firstly demonstrates a potential direction for antihypertensive mechanism of DHF from microRNA aspect and will promote its clinical applications.

Myocardial angiogenesis induced by concurrent vitamin D supplementation and aerobic-resistance training is mediated by inhibiting miRNA-15a, and miRNA-146a and upregulating VEGF/PI3K/eNOS signaling pathway.

This study aimed to investigate the effects of co-treatment of aerobic-resistance training (ART), vitamin D3 (VD3) on cardiovascular function considering the involvement of microRNA-15a and microRNA-146a, vascular endothelial growth factor (VEGF), phosphatidylinositol-3 kinase (PI3K), and endothelial nitric oxide synthase (eNOS) after myocardial infarction (MI) in rats. To induce MI, male Wistar rats subcutaneously received isoproterenol for 2 days, then MI was confirmed by echocardiography. MI rats were divided into six groups (n = 8/group). MI + VD3, MI + sesame oil (Veh), MI + ART, MI + VD3 + ART, and MI + Veh + ART, and received the related treatments for 8 weeks. Exercise tests, echocardiography, real-time quantitative polymerase chain reaction (qRT-PCR), western blotting, and histological staining were performed after the end of treatments. The highest ejection fraction (EF%), fractional shortening (FS%), exercise capacity (EC), and maximal load test (MLT) amounts were observed in the groups treated with VD3, ART, and VD3 + ART (P < 0.05). These were accompanied by a significantly increased angiogenesis post-MI. Furthermore, the levels of circulating microRNA-15a and microRNA-146a were significantly decreased in these groups compared to MI rats that were together with a significant upregulation of cardiac VEGF, PI3K, and eNOS expression. Overall, the best results were observed in the group treated with VD3 + ART. Concurrent VD3 supplementation and ART attenuated microRNA-15a and microRNA-146a and induced angiogenesis via VEGF/PI3K/eNOS axis. This data demonstrate that concurrent VD3 supplementation and ART is a more efficient strategy than monotherapy to improve cardiac function post-MI.

Influence of electroacupuncture on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase signaling pathway in spontaneously hypertensive rats.

OBJECTIVE: To investigate the influence of electroacupuncture (EA) on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathway in spontaneously hypertensive rats (SHRs). METHODS: Eight Wistar-Kyoto rats were used as the healthy blood pressure (BP) control (normal group), and 32 SHRs were randomized into model group, EA group, EA plus ghrelin group (EA + G group), and EA plus PF04628935 group (a potent ghrelin receptor blocker; EA + P group) using a random number table. Rats in the normal group and model group did not receive treatment, but were immobilized for 20 min per day, 5 times a week, for 4 continuous weeks. SHRs in the EA group, EA + G group and EA + P group were immobilized and given EA treatment in 20 min sessions, 5 times per week, for 4 weeks. Additionally, 1 h before EA, SHRs in the EA + G group and EA + P group were intraperitoneally injected with ghrelin or PF04628935, respectively, for 4 weeks. The tail-cuff method was used to measure BP. After the 4-week intervention, the rats were sacrificed by cervical dislocation, and pathological morphology of the abdominal aorta was observed using hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ghrelin, nitric oxide (NO), endothelin-1 (ET-1) and thromboxane A2 (TXA2) in the serum. Isolated thoracic aortic ring experiment was performed to evaluate vasorelaxation. Western blot was used to measure the expression of PI3K, Akt, phosphorylated Akt (p-Akt) and eNOS proteins in the abdominal aorta. Further, quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to measure the relative levels of mRNA expression for PI3K, Akt and eNOS in the abdominal aorta. RESULTS: EA significantly reduced the systolic BP (SBP) and diastolic BP (DBP) (P < 0.05). HE staining showed that EA improved the morphology of the vascular endothelium to some extent. Results of ELISA indicated that higher concentrations of ghrelin and NO, and lower concentrations of ET-1 and TXA2 were presented in the EA group (P < 0.05). The isolated thoracic aortic ring experiment demonstrated that the vasodilation capacity of the thoracic aorta increased in the EA group. Results of Western blot and qRT-PCR showed that EA increased the abundance of PI3K, p-Akt/Akt and eNOS proteins, as well as expression levels of PI3K, Akt and eNOS mRNAs (P < 0.05). In the EA + G group, SBP and DBP decreased (P < 0.05), ghrelin concentrations increased (P < 0.05), and the concentrations of ET-1 and TXA2 decreased (P < 0.05), relative to the EA group. In addition, the levels of PI3K and eNOS proteins, the p-Akt/Akt ratio, and the expression of PI3K, Akt and eNOS mRNAs increased significantly in the EA + G group (P < 0.05), while PF04628935 reversed these effects. CONCLUSION: EA effectively reduced BP and protected the vascular endothelium, and these effects may be linked to promoting the release of ghrelin and activation of the PI3K/Akt/eNOS signaling pathway.

[Involvement of ET-1/eNOS in the ameliorating effect of electroacupuncture on cardiac dysfunction in rats with spontaneously hypertensive].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Neiguan" (PC 6) on cardiac function of ventriculus sinister in rats with spontaneously hypertensive (SHR), and to explore the mediation effect of endothelin-1 (ET-1)/endothelial nitric oxide synthase (eNOS). METHODS: Six 12-week-old male Wistar Kyoto (WKY) rats were taken as the normal group. Eighteen 12-week-old SHR were randomly divided into a model group, an EA group and a sham EA group, 6 rats in each group. The rats in the EA group were treated with EA (disperse-dense wave, 2 Hz/15 Hz in frequency, 1 mA in current intensity) at "Neiguan" (PC 6), 30 min each time, once a day for 8 weeks. The rats in the sham EA group were treated with superficial needling at "Neiguan" (PC 6) with no electrical stimulation applied. After treatment, the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were tested by echocardiographic analysis. The left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), heart rate (HR), the maximum rate of increase/decrease of left ventricular pressure (±dp/dtmax) were detected. The serum content of ET-1 was detected by ELISA. Western blot was used to evaluate the expression of ETAR, eNOS in myocardial tissue of left ventricular. RESULTS: Compared with the normal group, LVEF, LVFS, +dp/dtmax/LVSP and -dp/dtmax/LVSP were decreased (P<0.01, P<0.05), while LVSP, LVEDP, +dp/dtmax and -dp/dtmax were increased (P<0.01) in the model group. Compared with the model group, LVEF, LVFS, +dp/dtmax/LVSP and -dp/dtmax/LVSP were increased (P<0.01, P<0.05), and LVSP and LVEDP were decreased (P<0.01) in the EA group. Compared with the normal group, the serum content of ET-1 and the expression of ETAR in myocardial tissue were increased (P<0.01), whereas expression of eNOS was decreased (P<0.01) in the model group. Compared with the model group, the serum content of ET-1 and the expression of ETAR in myocardial tissue were decreased (P<0.05), whereas expression of eNOS was increased (P<0.05) in the EA group. CONCLUSION: EA intervention may alleviate hypertensive cardiac function damage by up-regulating the expression of eNOS protein in myocardial tissue, down-regulating the serum content of ET-1 and the expression of ETAR protein in myocardial tissue.

A Ternary Synergistic eNOS Gene Delivery System Based on Calcium Ion and L-Arginine for Accelerating Angiogenesis by Maximizing NO Production.

Purpose: This study aimed to construct a delivery system based on L-arginine-modified calcium phosphate (CaP) to load eNOS plasmids (peNOS), which could amply nitric oxide (NO) to repair endothelial damage, promote angiogenic activities and alleviate inflammation. Methods: pDNA-loaded CaP nanocomplex (CaP/pDNA) were prepared by co-precipitation method, subsequently modified by L-arginine. The gene transfection efficiency, pro-angiogenic and anti-inflammatory ability were investigated in vivo and in vitro. The therapeutic effect on ischemic hindlimb in vivo was assessed. Results: L-arginine modification augmented the transfection efficiency of CaP/peNOS to elevate the eNOS expression, and then served as NO substrate catalyzed by eNOS. At the same time, calcium ions produced by degradation of CaP carriers enhanced the activity of eNOS. In vitro experiments, the loading capability and transfection performance of R(L)-CaP were confirmed to be superior to that of CaP. Additionally, HUVECs treated with R(L)-CaP/peNOS showed the strongest NO release, cell migration, tube formation and the lowest inflammatory levels compared to the CaP/peNOS and R(D)-CaP/peNOS groups. We also demonstrated the advantages of R(L)-CaP/peNOS in increasing blood reperfusion in hindlimb ischemia mice by accelerating angiogenesis and reducing inflammation, which can be attributed to the highest eNOS-derived NO production. Conclusion: The combination strategy of peNOS transfection, L-arginine supplement and calcium ions addition is a promising therapeutic approach for certain vascular diseases, based on the synergistic NO production.

Virtual Screening and Molecular Docking to Study the Mechanism of Chinese Medicines in the Treatment of Coronavirus Infection.

BACKGROUND Heat-clearing and detoxifying herbs (HDHs) play an important role in the prevention and treatment of coronavirus infection. However, their mechanism of action needs further study. This study aimed to explore the anti-coronavirus basis and mechanism of HDHs. MATERIAL AND METHODS Database mining was performed on 7 HDHs. Core ingredients and targets were screened according to ADME rules combined with Neighborhood, Co-occurrence, Co-expression, and other algorithms. GO enrichment and KEGG pathway analyses were performed using the R language. Finally, high-throughput molecular docking was used for verification. RESULTS HDHs mainly acts on NOS3, EGFR, IL-6, MAPK8, PTGS2, MAPK14, NFKB1, and CASP3 through quercetin, luteolin, wogonin, indirubin alkaloids, ß-sitosterol, and isolariciresinol. These targets are mainly involved in the regulation of biological processes such as inflammation, activation of MAPK activity, and positive regulation of NF-kappaB transcription factor activity. Pathway analysis further revealed that the pathways regulated by these targets mainly include: signaling pathways related to viral and bacterial infections such as tuberculosis, influenza A, Ras signaling pathways; inflammation-related pathways such as the TLR, TNF, MAPK, and HIF-1 signaling pathways; and immune-related pathways such as NOD receptor signaling pathways. These pathways play a synergistic role in inhibiting lung inflammation and regulating immunity and antiviral activity. CONCLUSIONS HDHs play a role in the treatment of coronavirus infection by regulating the body's immunity, fighting inflammation, and antiviral activities, suggesting a molecular basis and new strategies for the treatment of COVID-19 and a foundation for the screening of new antiviral drugs.

Fig (Ficus exasperata and Ficus asperifolia)-Supplemented diet improves sexual function, endothelial nitric oxide synthase and suppresses tumour necrosis factor-alpha genes in hypertensive rats.

We measured the effect of varieties of Fig leaves, Ficus exasperata Vahl. (FE) and Ficus asperifolia Miq (FA), commonly found in Sub-sahara Africa for managing hypertension on sexual performance in hypertensive rats, which is unknown. Hypertensive rats experienced erectogenic damage after exposure to 40 mg kg-1  bw-1 , N(G)-nitro-l-arginine-methyl-ester (L-NAME). Experimental rats were grouped into eight groups (n = 6) namely: control rats, hypertensive rats, hypertensive treatment groups with atenolol (10 mg kg-1  day-1 ) and sildenafil (5.0 mg/kg), rats treated with FE- and FA-formulated biscuits at proportions of 2.5 g and 5.0 g respectively. Furthermore, we measured the level of sexual performance (Intromission number, latency, Mounting number, and latency), hormonal levels, phosphodiesterase-5 enzyme (PDE-5) activity and genes expressed using real-time quantitative polymerase chain reaction (RT-qPCR) in hypertensive rats. The result deduced revealed that treated hypertensive rats showed significantly reduced follicle-stimulating hormone, luteinising hormone levels, PDE-5 enzyme activity, tumour necrosis factor-alpha (TNF-α) expression while having a marked increase in testosterone level, sexual performance and endothelial nitric oxide synthase (eNOS) expression. Therefore, this study revealed the comparative dietary enhancing effect of FE- and FA-formulated biscuit on sexual behaviour activity, hormonal levels and the level of eNOS and TNF-α genes expressed in hypertensive rats.

Involvement of ET-1/eNOS in the ameliorating effect of electroacupuncture on cardiac dysfunction in rats with spontaneously hypertensive / 中国针灸

OBJECTIVE@#To observe the effect of electroacupuncture (EA) at "Neiguan" (PC 6) on cardiac function of ventriculus sinister in rats with spontaneously hypertensive (SHR), and to explore the mediation effect of endothelin-1 (ET-1)/endothelial nitric oxide synthase (eNOS).@*METHODS@#Six 12-week-old male Wistar Kyoto (WKY) rats were taken as the normal group. Eighteen 12-week-old SHR were randomly divided into a model group, an EA group and a sham EA group, 6 rats in each group. The rats in the EA group were treated with EA (disperse-dense wave, 2 Hz/15 Hz in frequency, 1 mA in current intensity) at "Neiguan" (PC 6), 30 min each time, once a day for 8 weeks. The rats in the sham EA group were treated with superficial needling at "Neiguan" (PC 6) with no electrical stimulation applied. After treatment, the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were tested by echocardiographic analysis. The left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), heart rate (HR), the maximum rate of increase/decrease of left ventricular pressure (±dp/dtmax) were detected. The serum content of ET-1 was detected by ELISA. Western blot was used to evaluate the expression of ETAR, eNOS in myocardial tissue of left ventricular.@*RESULTS@#Compared with the normal group, LVEF, LVFS, +dp/dtmax/LVSP and -dp/dtmax/LVSP were decreased (P<0.01, P<0.05), while LVSP, LVEDP, +dp/dtmax and -dp/dtmax were increased (P<0.01) in the model group. Compared with the model group, LVEF, LVFS, +dp/dtmax/LVSP and -dp/dtmax/LVSP were increased (P<0.01, P<0.05), and LVSP and LVEDP were decreased (P<0.01) in the EA group. Compared with the normal group, the serum content of ET-1 and the expression of ETAR in myocardial tissue were increased (P<0.01), whereas expression of eNOS was decreased (P<0.01) in the model group. Compared with the model group, the serum content of ET-1 and the expression of ETAR in myocardial tissue were decreased (P<0.05), whereas expression of eNOS was increased (P<0.05) in the EA group.@*CONCLUSION@#EA intervention may alleviate hypertensive cardiac function damage by up-regulating the expression of eNOS protein in myocardial tissue, down-regulating the serum content of ET-1 and the expression of ETAR protein in myocardial tissue.

Arginase inhibition by rhaponticin increases L-arginine concentration that contributes to Ca2+-dependent eNOS activation.

Although arginase primarily participates in the last reaction of the urea cycle, we have previously demonstrated that arginase II is an important cytosolic calcium regulator through spermine production in a p32-dependent manner. Here, we demonstrated that rhaponticin (RPT) is a novel medicinal-plant arginase inhibitor and investigated its mechanism of action on Ca2+-dependent endothelial nitric oxide synthase (eNOS) activation. RPT was uncompetitively inhibited for both arginases I and II prepared from mouse liver and kidney. It also inhibited arginase activity in both aorta and human umbilical vein endothelial cells (HUVECs). Using both microscope and FACS analyses, RPT treatments induced increases in cytosolic Ca2+ levels using Fluo-4 AM as a calcium indicator. Increased cytosolic Ca2+ elicited the phosphorylations of both CaMKII and eNOS Ser1177 in a time-dependent manner. RPT incubations also increased intracellular L-arginine (L-Arg) levels and activated the CaMKII/AMPK/Akt/eNOS signaling cascade in HUVECs. Treatment of L-Arg and ABH, arginase inhibitor, increased intracellular Ca2+ concentrations and activated CaMKII-dependent eNOS activation in ECs of WT mice, but, the effects were not observed in ECs of inositol triphosphate receptor type 1 knockout (IP3R1-/-) mice. In the aortic endothelium of WT mice, RPT also augmented nitric oxide (NO) production and attenuated reactive oxygen species (ROS) generation. In a vascular tension assay using RPT-treated aortic tissue, cumulative vasorelaxant responses to acetylcholine (Ach) were enhanced, and phenylephrine (PE)-dependent vasoconstrictive responses were retarded, although sodium nitroprusside and KCl responses were not different. In this study, we present a novel mechanism for RPT, as an arginase inhibitor, to increase cytosolic Ca2+ concentration in a L-Arg-dependent manner and enhance endothelial function through eNOS activation. [BMB Reports 2021; 54(10): 516-521].

Endothelial dysfunction and transcriptome aberration in mouse aortas induced by black phosphorus quantum dots and nanosheets.

Black phosphorus (BP) nanomaterials have shown great potential in versatile applications including biomedicine and potentially interact with vessel walls following intravenous injection in biomedical usage or environmental exposure. However, it remains unknown whether the exposure to BP nanomaterials induces alterations of the endothelium and further vascular injury. Herein, the endothelial function of human umbilical vein endothelial cells (HUVECs) and the structure and transcriptome of C57BL/6 mouse aortas are evaluated after the exposure to BP quantum dots (BPQDs) and nanosheets (BPNSs). BPNSs with irregular shapes and larger lateral size are more prone to inhibit in vitro angiogenesis at non-cytotoxic concentrations and markedly trigger platelet adhesion to HUVECs compared to BPQDs. Decreased nitric oxide (NO) production resulting from endothelial NO synthase (eNOS) dysregulation is involved in the BP-induced endothelial dysfunction. Both BPQDs and BPNSs at 0.8 and 6.4 µg mL-1 inhibit eNOS enzymatic activity through dephosphorylation of eNOS-Ser1177 and phosphorylation of eNOS-Thr495, but unlike BPQDs, BPNSs also downregulate eNOS expression. Despite no pathological damage in the structure of mouse aortas, BPQDs and BPNSs trigger aberration of aortic transcriptome involved in vasoconstriction abnormality, metabolic disturbance, and immune perturbation. This study demonstrates the adverse effect of BP nanomaterials on vasculature, and suggests that the morphological attribute of BP plays a crucial role in the vascular risks.

Bufalin inhibits peritoneal dissemination of gastric cancer through endothelial nitric oxide synthase-mitogen-activated protein kinases signaling pathway.

Peritoneal dissemination threatens the survival of patients with gastric cancer (GC). Bufalin is an extract of traditional Chinese medicine, which has been proved to have anticancer effect. The target of bufalin in suppressing gastric cancer peritoneal dissemination (GCPD) and the underlying mechanism are still unclear. In this research, GC cell line MGC-803 and high-potential peritoneal dissemination cell line MKN-45P were treated with bufalin or L-NAME. Malignant biological behavior and protein level of GC cell lines were detected with MTT, wound healing, transwell, adhesion, and western blotting. Bioinformatics analysis and patient tissues were used to verify the role of endothelial nitric oxide synthase (NOS3) in GC. Mice model was used to assess the effect of bufalin and role of NOS3 in vivo. We found that bufalin inhibited the proliferation, invasion, and migration in GC cell lines. NOS3, which was an independent prognostic factor of GC patients, was predicted to be a potential target of bufalin. Further experiments proved that bufalin reduced the phosphorylation of NOS3, thereby inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway, and ultimately suppressed GCPD by inhibiting EMT process. In conclusion, NOS3 was a potential therapeutic target and prognostic biomarker of GC. Bufalin could suppress GCPD through NOS3-MAPK signaling pathway, which provided more evidence support for intraperitoneal perfusion of bufalin to treat GCPD.

Periplocymarin protects against myocardial fibrosis induced by ß-adrenergic activation in mice.

Periplocymarin is an effective component of Periplocae Cortex, which was wildly used as an ingredient in Traditional Chinese Medicine. Our group previously reported that periplocymarin exerted cardiotonic role via promoting calcium influx. However, its exact role in the pathogenesis of myocardial fibrosis has not been elucidated yet. The present study was aimed at determining the potential effect and underlying mechanism of periplocymarin in isoproterenol (ISO)-induced myocardial fibrosis. C57BL/6 mice were subcutaneously injected with ISO (5 mg/kg/day) or saline for 1 week. The early-to-atrial wave ratio (E/A ratio) measured by echocardiography revealed that ISO-induced heart stiffness was remarkably reversed by administration of periplocymarin (5 mg/kg/day). Masson trichrome staining exhibited that treatment of periplocymarin reduced the excessive deposition of extracellular matrix (ECM). Further investigations employing real-time PCR and western blot demonstrated that periplocymarin suppressed the expression of fibrosis related genes (Col1a1, Col3a1, Acta2 and Tgfb1) and proteins (Collagen I, Collagen III, α-SMA and TGF-ß1) induced by ISO. Metabolomics analysis demonstrated that periplocymarin ameliorated the disorders triggered by ISO and many of the differential metabolic substances were involved in amino acid, glucose and lipid metabolism. Further analysis using network pharmacology revealed that three key genes, namely NOS2, NOS3 and Ptgs2, may be the potential targets of periplocymarin and responsible for the disorders. Validation using heart tissues showed that the mRNA expression of NOS3 was decreased while Ptgs2 was increased upon ISO treatment, which were reversed by periplocymarin. Moreover, the expression of COX-2 (Ptgs2 encoded protein) was consistent with the aspect of Ptgs2 mRNA, while eNOS (NOS3 encoded protein) expression was unchanged. In vitro studies exhibited that periplocymarin exerts anti-fibrotic function via regulating at least eNOS and COX-2 in cardiomyocyte. Taken together, periplocymarin protects against myocardial fibrosis induced by ß-adrenergic activation, the potential mechanism was that periplocymarin targeted on, at least eNOS and COX-2, to improve the metabolic processes of cardiomyocyte and thus attenuated the myocardial fibrosis. Our study highlighted that periplocymarin is a potential therapeutic agent for the prevention of myocardial fibrosis.

Liuwei Dihuang formula protect endothelial cells from apoptosis by up-regulating expression of estrogen receptor-α.

OBJECTIVE: To evaluate the efficacy of Liuwei Dihuang formula ( LWDHF) on endothelial cells, and to study the mechanism behind the action of modulating expression of estrogen receptors. METHODS: Hydrogen peroxide (H2O2) was applied to induce the apoptosis of human umbilical vein endothelial cells (HUVECs). The concentration of nitric oxide (NO), endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) were measured by assay kits. Western blot and real-time polymerase chain reaction (RT-PCR) were used to detect the expression of iNOS, eNOS, b-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), estrogen receptor (ER) α and ERß. Also, small interfering RNA (siRNA) was involved to confirm whether the protective effects of LWDHF was medicated by ERs. In vivo, the female rats were ovariectomized to establish postmenopausal vascular injury model. Then the model rats were divided into three groups and treated with saline, estradiol and LWDHF respectively. The concentration of NO and NOS in serum were measured by assay kits, and the expression of Bax, Bcl-2, ERα and ERß were detected by western blot and immunohistochemistry. RESULTS: In vitro study, LWDHF significantly protected HUVECs from H2O2-induced apoptosis, with the increase of Bcl-2 and the decrease of Bax. The treatment with LWDHF inhibited concentration of NO and iNOS, and upregulated the expression of eNOS, ERα and ERß. In addition, ERα siRNA could block the protective effects of LWDHF, while ERß siRNA showed little influence. In vivo, the treatment with LWDHF suppressed the vascular injury and reduced the level of NO and NOS. LWDHF increased the expression of Bcl-2, ERα and ERß, as well as inhibiting the Bax expression. CONCLUSION: LWDHF could improve endothelial function and protect HUVECs from apoptosis via increasing the expression of ERα.

Danlou Fang reduces microvascular obstruction through the endothelial/inducible nitric oxide synthase pathway in a rat model.

OBJECTIVE: To investigate the efficacy of the Danlou Fang (DL) Traditional Chinese Medicine formula on microvascular obstruction (no-reflow) through the endothelial/inducible nitric oxide synthase (eNOS/iNOS) pathway in a rat model. METHODS: Sprague-Dawley rats were subjected to 60 min of coronary artery occlusion (or sham procedure) followed by 2 h of reperfusion and were then divided into treatment groups: sham, model, DL (500 mg/kg), DL (500 mg/kg) + eNOS inhibitor L-nitroarginine (L-NNA; 7.5 mg/kg), and sodium nitroprusside (SNP; 0.5 mg/kg). There were 16 per group. Areas of no-reflow were determined by thioflavin S staining of heart tissue. Cardiac function was assessed by echocardiography. Myocardial enzymes and antioxidants in serum were measured and analyzed. The relative protein expression levels of eNOS and iNOS were determined by western blotting. RESULTS: DL had a myocardial protective effect on myocardial reperfusion and reduced the area of no-reflow. The serum levels of creatine kinase (CK), myocardial CK isoenzyme CK-MB, and lactate dehydrogenase were significantly lower in the DL group than in the model (P < 0.05). DL treatment also decreased the serum content of malondialdehyde and reactive oxygen species (ROS), increased the activity of superoxide dismutase and nitric oxide, and promoted eNOS expression (P < 0.05) while lowering iNOS expression. CONCLUSION: DL reduced the area of no-reflow and had a myocardial protective effect that may be associated with the eNOS/iNOS pathway.

Gualou-Xiebai-Banxia decoction protects against type II diabetes with acute myocardial ischemia by attenuating oxidative stress and apoptosis via PI3K/Akt/eNOS signaling.

Gualou-Xiebai-Banxia decoction has a long history of medical use for treating cardiovascular diseases in China. In this study, we investigated the protective effect and underlying mechanisms GXB in type II diabetes with acute myocardial ischemia (T2DM-AMI) rats. We hypothesized that GXB may display its protective effect on T2DM-AMI by reducing endothelial progenitor cells (EPCs) apoptosisviaactivating PI3K (phosphatidyl inositol 3-kinase)/Akt (serine/threonine protein kinase B)/eNOS (endothelial nitric oxide synthase) signaling. Rats were challenged with a high-fat diet and intraperitoneal injection of streptozotocin to induce a model of type II diabetes mellitus (T2DM) and coronary ligation to induce acute myocardial infarction (AMI). Changes in metabolites were assessed via enzyme-linked immunoassay and biochemical examination. The number and apoptosis rate of EPCs in peripheral blood were detected by flow cytometry. Target mRNAs and proteins in EPCs were analyzed by RT-PCR and Western blot analysis. The results demonstrated that GXB treatment decreased T2DM-AMI-associated changes in plasma fasting blood glucose, muscular enzymes, and blood lipids, and reduced oxidative stress. Furthermore, EPC apoptosis was increased in T2DM-AMI rats and was associated with decreased mRNA and protein levels of PI3K, Akt, and eNOS compared to the controls. Conversely, T2DM-AMI rats treated with GXB exhibited more circulating EPCs and downregulated levels of cell apoptosis, combined with increased mRNA and protein levels of PI3K, Akt, and eNOS compared to those of untreated T2DM-AMI rats. Our study showed that GXB treatment mitigated EPC apoptosis and promoted PI3K/Akt/eNOS signaling in T2DM-AMI rats.

Angiogenesis is Differentially Modulated by Anthocyanin and Phenolic Acid Extracts from Wild Blueberry (V. angustifolium) Through PI3K Pathway.

Wild blueberries rank very high in anthocyanins (ACNs) and phenolic acids (PA) among other berries. Previous work from our group has documented their beneficial effects. In this study, human umbilical vein endothelial cells were used, and ACNs, PAs, and their combination (ACNs:PAs) at concentrations of 0.002, 8, 15, 60, and 120 µg/mL were tested for endothelial tube formation. Treatment with ACNs decreased, while treatment with PAs and ACNs:PAs increased overall endothelial cell tube formation compared to control. Endothelial cells exposed to ACNs downregulated gene expression of AKT1 and endothelial nitric oxide synthase (eNOS), while PAs upregulated AKT1 and vascular endothelial growth factor (VEGF) gene expression. Combination of ACNs:PAs decreased gene expression of AKT1 and eNOS, while protein levels of AKT1 increased. In summary, based on the type of wild blueberry extract, angiogenesis is differentially modulated and is concentration dependent. Further experiments will delineate the mechanism(s) of the differential action of the aforementioned extracts on angiogenesis.

Comparative Analysis of the Rabbit Endothelial Progenitor Cells from Bone Marrow and Peripheral Blood Treated with Selenium Nanoparticles.

BACKGROUND: Selenium Nanoparticles (Se-NPs) are known for their antioxidant and anti-inflammatory activities, which are effective in preventing oxidative damage and improving physiological processes. OBJECTIVES: This study aimed at investigating the effects of biosynthesized Se-NPs on bone marrow-derived Endothelial Progenitor Cells (bone marrow-derived EPCs) and blood-derived endothelial progenitor cells (blood-derived EPCs) isolated from rabbits in vitro. METHODS: The cultured EPCs incubated with biosynthesized Se-NPs at the concentrations of 0.19, 0.38, 0.76, 1.71, 3.42, 7.03, 14.25, 28.50, 57, 114, and 228µg/ml for 48h. After screening the proliferative potential of the Se-NPs by the MTT assay, the best concentrations were selected for Real-Time quantitative Polymerase Chain Reaction (RT-qPCR). Real-time quantification of Vascular Cell Adhesion Molecule 1 (VCAM-1), lectin-like oxidized Low-Density Lipoprotein (LDL) receptor-1 (LOX-1), endothelial Nitric Oxide Synthase (eNOS), and Monocyte Chemoattractant Protein-1 (MCP-1) gene expressions were analyzed by normalizing with Glyceraldehyde- 3-Phosphate Dehydrogenase (GAPDH) as an endogenous reference gene. RESULTS: Blood-derived EPCs and bone marrow-derived EPCs showed morphological differences before treatment in vitro. Se-NPs treated EPCs indicated a significant dose-dependent proliferative activity (p<0.01). In general, the expression levels of VCAM-1, LOX-1, and MCP-1 mRNA were significantly decreased (p<0.01), whereas that of the eNOS expression was significantly increased at the concentrations of 7.3 and 14.25µg/ml (p<0.01). Although the expressions of MCP-1, LOX-1, and eNOS mRNA were decreased at certain concentrations of Se-NPs (p<0.01 and p<0.05, respectively) in the treated bone marrow-derived EPCs, no significant differences were observed in the VCAM-1 mRNA expression levels in bone marrow-derived EPCs compared with the control group (p>0.05). CONCLUSION: This was the first report to demonstrate the effects of Se-NPs on proliferative, anti-oxidative, and anti-inflammatory activities for bone marrow-derived EPCs and blood-derived EPCs. Our findings suggested that Se-NPs could be considered as an effective agent that may ameliorate vascular problems.

Tubeimoside I promotes angiogenesis via activation of eNOS-VEGF signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Tubeimoside I (TBM) is a triterpenoid saponin purified from tubeimu (tuber of Bolbostemma paniculatum (Maxim.) Franquet). In traditional Chinese medicine, tubeimu had been used to treat acute mastitis, snake bites, detoxication, inflammatory diseases, and tumors for over 1000 years. AIM OF THE STUDY: This study aimed to investigate whether TBM could promote angiogenesis and how to promote angiogenesis. MATERIALS AND METHODS: In vivo, the pro-angiogenic effects of TBM were examined using the hindlimb ischemia model. After the ischemia operation, 1 mg/kg/day TBM was given via intraperitoneal injection for 28 days and the recovery of blood flow was monitored by Doppler scanner every 7 days. The capillary density in gastrocnemius muscle was detected by immunofluorescence. Expression of related proteins were determined by western blotting. In vitro, the pro-angiogenic effects of TBM on HUVECs were examined by Cell Counting Kit-8, scratch assay, endothelial cell tube formation assay and western blotting. RESULTS: TBM improved recovery from hindlimb ischemia in C57BL/6 mice. TBM promoted endothelial cell viability, migration and tube formation in HUVECs. TBM could activate eNOS-VEGF signaling pathway by enhancing expression of eNOS. And TBM's pro-angiogenesis effects could be abolished by L-NAME (an inhibitor of eNOS). CONCLUSIONS: TBM promoted angiogenesis via the activation of eNOS-VEGF signaling pathway and TBM could be a novel agent for therapeutic angiogenesis in ischemic diseases.

Beetroot juice lowers blood pressure and improves endothelial function in pregnant eNOS-/- mice: importance of nitrate-independent effects.

KEY POINTS: Maternal hypertension is associated with increased rates of pregnancy pathologies, including fetal growth restriction, due at least in part to reductions in nitric oxide (NO) bioavailability and associated vascular dysfunction. Dietary nitrate supplementation, from beetroot juice (BRJ), has been shown to increase NO bioavailability and improve cardiovascular function in both preclinical and clinical studies. This study is the first to investigate effects of dietary nitrate supplementation in a pregnant animal model. Importantly, the effects of nitrate-containing BRJ were compared with both 'placebo' (nitrate-depleted) BRJ as well as water to control for potential nitrate-independent effects. Our data show novel, nitrate-independent effects of BRJ to lower blood pressure and improve vascular function in endothelial nitric oxide synthase knockout (eNOS-/- ) mice. These findings suggest potential beneficial effects of BRJ supplementation in pregnancy, and emphasize the importance of accounting for nitrate-independent effects of BRJ in study design and interpretation. ABSTRACT: Maternal hypertension is associated with adverse pregnancy outcomes, including fetal growth restriction (FGR), due in part to reductions in nitric oxide (NO) bioavailability. We hypothesized that maternal dietary nitrate administration would increase NO bioavailability to reduce systolic blood pressure (SBP), improve vascular function and increase fetal growth in pregnant endothelial NO synthase knockout (eNOS-/- ) mice, which exhibit hypertension, endothelial dysfunction and FGR. Pregnant wildtype (WT) and eNOS-/- mice were supplemented with nitrate-containing beetroot juice (BRJ+) from gestational day (GD) 12.5. Control mice received an equivalent dose of nitrate-depleted BRJ (BRJ-) or normal drinking water. At GD17.5, maternal SBP was measured; at GD18.5, maternal nitrate/nitrite concentrations, uterine artery (UtA) blood flow and endothelial function were assessed, and pregnancy outcomes were determined. Plasma nitrate concentrations were increased in both WT and eNOS-/- mice supplemented with BRJ+ (P < 0.001), whereas nitrite concentrations were increased only in eNOS-/- mice (P < 0.001). BRJ- did not alter nitrate/nitrite concentrations. SBP was lowered and UtA endothelial function was enhanced in eNOS-/- mice supplemented with either BRJ+ or BRJ-, indicating nitrate-independent effects of BRJ. Improvements in endothelial function in eNOS-/- mice were abrogated in the presence of 25 mm KCl, implicating enhanced EDH signalling in BRJ- treated animals. At GD18.5, eNOS-/- fetuses were significantly smaller than WT animals (P < 0.001), but BRJ supplementation did not affect fetal weight. BRJ may be a beneficial intervention in pregnancies associated with hypertension, endothelial dysfunction and reduced NO bioavailability. Our data showing biological effects of non-nitrate components of BRJ have implications for both interpretation of previous findings and in the design of future clinical trials.