Nitric oxide inhibits endothelial cell apoptosis by inhibiting cysteine-dependent SOD1 monomerization.

Endothelial cell apoptosis is an important pathophysiology in many cardiovascular diseases. The gasotransmitter nitric oxide (NO) is known to regulate cell survival and apoptosis. However, the mechanism underlying the effect of NO remains unclear. In this research, by targeting cytosolic copper/zinc superoxide dismutase (SOD1) monomerization, we aimed to explore how NO inhibited endothelial cell apoptosis. We showed that treatment with the NO synthase (NOS) inhibitor nomega-nitro-l-arginine methyl ester hydrochloride (L-NAME) significantly decreased the endogenous NO content of endothelial cells, facilitated the formation of SOD1 monomers, inhibited dismutase activity, and promoted reactive oxygen species (ROS) accumulation in human umbilical vein endothelial cells (HUVECs); by contrast, supplementation with the NO donor sodium nitroprusside (SNP) upregulated NO content, prevented the formation of SOD1 monomers, enhanced dismutase activity, and reduced ROS accumulation in L-NAME-treated HUVECs. Mechanistically, tris(2-carboxyethyl) phosphine hydrochloride (TCEP), a specific reducer of cysteine thiol, increased SOD1 monomer formation, thus preventing the NO-induced increase in dismutase activity and the decrease in ROS. Furthermore, SNP inhibited HUVEC apoptosis caused by the decrease in endogenous NO, whereas TCEP abolished this protective effect of SNP. In summary, our data reveal that NO protects endothelial cells against apoptosis by inhibiting cysteine-dependent SOD1 monomerization to enhance SOD1 activity and inhibit oxidative stress.

Formononetin represses cervical tumorigenesis by interfering with the activation of PD-L1 through MYC and STAT3 downregulation.

A. membranaceus is a traditional Chinese medicine that regulates blood sugar levels, suppresses inflammation, protects the liver, and enhances immunity. In addition, A. membranaceus is also widely used in diet therapy and is a well-known health tonic. Formononetin is a natural product isolated from A. membranaceus that has multiple biological functions, including anti-cancer activity. However, the mechanism by which formononetin inhibits tumor growth is not fully understood. In this present study, we demonstrated that formononetin suppresses PD-L1 protein synthesis via reduction of MYC and STAT3 protein expression. Furthermore, formononetin markedly reduced the expression of MYC protein via the RAS/ERK signaling pathway and inhibited STAT3 activation through JAK1/STAT3 pathway. Co-immunoprecipitation experiments illustrated that formononetin suppresses protein expression of PD-L1 by interfering with the interaction between MYC and STAT3. Meanwhile, formononetin promoted PD-L1 protein degradation via TFEB and TFE3-mediated lysosome biogenesis. T cell killing assay revealed that formononetin could enhance the activity of cytotoxic T lymphocytes (CTLs) and restore ability to kill tumor cells in a co-culture system of T cells and tumor cells. In addition, formononetin inhibited cell proliferation, tube formation, cell migration, and promoted tumor cell apoptosis by suppressing PD-L1. Finally, the inhibitory effect of formononetin on tumor growth was confirmed in a murine xenograft model. The present study revealed the anti-tumor potential of formononetin, and the findings should support further research and development of anti-cancer drugs for cervical cancer.

Inhibitory role of ginsenoside Rb2 in endothelial senescence and inflammation mediated by microRNA­216a.

Targeting microRNAs (miRs) using small chemical molecules has become a promising strategy for disease treatment. miR­216a has been reported to be a potential therapeutic target in endothelial senescence and atherosclerosis via the Smad3/NF­κB signaling pathway. Ginsenoside Rb2 (Rb2) is the main bioactive component extracted from the plant Panax ginseng, and is a widely used traditional Chinese medicine. In the present study, Rb2 was identified to have a high score for miR­216a via bioinformatics analysis based on its sequence and structural features. The microscale thermophoresis experiment further demonstrated that Rb2 had a specific binding affinity for miR­216a and the dissociation constant was 17.6 µM. In both young and senescent human umbilical vein endothelial cells (HUVECs), as well as human aortic endothelial cells, Rb2 decreased the expression of endogenous miR­216a. Next, a replicative endothelial senescence model of HUVECs was established by infection with pre­miR­216a recombinant lentiviruses (Lv­miR­216a) and the number of population­doubling level (PDL) was calculated. Stable overexpression of miR­216a induced a premature senescent­like phenotype, whereas the senescent features and increased activity of senescence­associated ß­galactosidase (SA­ß­gal) were reversed after Rb2 treatment. The percentage of SA­ß­gal­positive cells in senescent PDL25 cells transfected with Lv­miR­216a was decreased 76% by Rb2 treatment compared with the Lv­miR­216a group without Rb2 treatment (P=0.01). Mechanistically, miR­216a inhibited Smad3 protein expression, promoted IκBα degradation and activated NF­κB­responsive genes, such as vascular cell adhesion molecule 1 (VCAM1), which promoted the adhesiveness of endothelial cells to monocytes. These pro­inflammatory effects of miR­216a were significantly suppressed by Rb2 treatment. When Smad3 was suppressed by small interfering RNA, the elevated expression levels of intercellular adhesion molecule 1 and VCAM1 induced by miR­216a were significantly reversed. Collectively, to the best of our knowledge, the present study demonstrated for the first time that Rb2 exerted an anti­inflammation effect on the process of endothelial cell senescence and could be a potential therapeutic drug by targeting miR­216a.

Tetrahydropalmatine triggers angiogenesis via regulation of arginine biosynthesis.

Over a short span of two decades, the central role of angiogenesis in the treatment of wound healing, diverse cancers, nerve defect, vascular injury and several ophthalmic diseases has become evident. Tetrahydropalmatine, as the index component of Corydalis yanhusuo W. T. Wang, is inseparable from protecting cardiovascular system, yet its role in angiogenesis has been poorly characterized. We have demonstrated the binding potential of THP and VEGFR2 using molecular docking based on the clinical experience of traditional Chinese medicine in the pretest study. Here, we identified tetrahydropalmatine (THP) as one proangiogenic trigger via regulation of arginine biosynthesis by pharmacological assays and DESI-MSI/GC-MS based metabolomics. First, the proangiogenic effects of THP were evaluated by quail chorioallantoic membrane test in vivo and multiple models of endothelial cells in vitro. According to virtual screening, the main mechanisms of THP (2/5 of the top terms with smaller p-value) were metabolic pathways. Hence, metabolomics was applied for the main mechanisms of THP and results showed the considerable metabolite difference in arginine biosynthesis (p < 0.05) altered by THP. Finally, correlated indicators were deteced using targeted metabolomics and pharmacological assays for validation, and results suggested the efficacy of THP on citrulline to arginine flux, arginine biosynthesis, and endothelial VEGFR2 expression sequentially, leading to the promotion of angiogenesis. Overall, this manuscript identified THP as the proangiogenic trigger with the potential to develop as pharmacological agents for unmet clinical needs.

Anti-angiogenic activity of ShengMaBieJia decoction in vitro and in acute myeloid leukaemia tumour-bearing mouse models.

Context: ShengMaBieJia decoction (SMBJD) is used to treat solid and hematological tumours; however, its anti-angiogenesis activity remains unclear.Objective: This study verified the anti-angiogenic effects of SMBJD in vitro and in tumour-bearing acute myeloid leukaemia (AML) mouse models.Materials and methods: In vivo, the chicken chorioallantoic membrane (CAM) and BALB/c null mouse xenograft models were treated with SMBJD (0, 2, 4, and 8 mg/mL) for 48 h and for 2 weeks, respectively. Anti-angiogenic activity was assessed according to microvessel density (MVD) and immunohistochemistry (IHC) targeting CD31 and VEGFR2. In vitro, proliferation viability, migratory activity and tube formation were measured. Western blots and polymerase chain reaction (PCR) assays were used to examine the levels of PI3K, Akt, and VEGF.Results: HPLC analyses revealed the active constituents of SMBJD such as liquiritin, cimifugin, ferulic, isoferulic, and glycyrrhizic acids. In vitro, SMBJD treatment decreased cellular migration, chemotaxis, and tube formation at non-cytotoxic concentrations (2, 4, and 8 mg/mL) in a time- and dose-dependent manner. The dosage of less than IC20 is considered safe. In vivo, CAM models exhibited a decrease in MVD, and the tissues of xenografted mice possessed reduced CD31 and VEGFR2 expression. Conditioned media (CM) from AML cells (HL60 and NB4 cells) treated with non-cytotoxic doses of SMBJD inhibited chemotactic migration and tube formation in vitro. Both CM (HL60) and CM (NB4) exhibited downregulated expression of PI3K, Akt, and VEGF.Discussion and conclusions: SMBJD inhibited angiogenesis in AML through the PI3K/AKT pathway, which might be combined with targeted therapy to provide more effective treatment.

Pro-angiogenic activity of Tongnao decoction on HUVECs in vitro and zebrafish in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Tongnao Decoction (TND) is a Chinese decoction approved and used in Jiangsu Province Hospital for the treatment of ischemic stroke. It shows conclusive efficiency in the improvement of neurologic impairment and activities of daily living of the patients. AIM OF THE STUDY: Recently, angiogenesis has been recognized as a potential therapeutic strategy for treating cerebral ischemia. This study was aimed to provide comprehensive evidence for the pro-angiogenic effect of TND and characterize the underlying mechanism. MATERIALS AND METHODS: We firstly established the chemical fingerprinting of TND. Then, the in vitro pro-angiogenic activities of TND were tested on human umbilical vein endothelial cells (HUVECs) through cell viability, wound healing and tube formation assays. The in vivo pro-angiogenic effects were evaluated on transgenic zebrafish embryos [Tg (fli-1: EGFP)] through the formation of intersegmental vessels (ISVs), subintestinal vessels (SIVs) and central arteries (CtAs). Lastly, the potential mechanisms of TND were analyzed by a blocking assay with eight pathways-specific kinase inhibitors. RESULTS: TND promoted the proliferation, migration and tube formation of HUVECs. TND also rescued the impairment of ISVs, SIVs and CtAs caused by VRI in a dose-dependent manner in zebrafish embryos. TND could activate vascular endothelial growth factor receptor-2 (VEGFR-2), phosphoinositide 3-kinase (PI3K) - protein kinase B (Akt) and Raf - mitogen-activated protein kinase1/2 (MEK1/2) - extracellular regulated kinase 1/2 (ERK1/2) signaling pathways. CONCLUSION: Our study firstly demonstrated the pro-angiogenic activities of TND. Our work provided evidences for the clinical usage of TND in restoring neurovascular function through promoting angiogenesis in the ischemic cerebral microvascular.

Liuwei Dihuang prevents postmenopausal atherosclerosis and endothelial cell apoptosis via inhibiting DNMT1-medicated ERα methylation.

ETHNOPHARMACOLOGICAL RELEVANCE: The classical and traditional Chinese medicine prescription, Liuwei Dihuang (LWDH), has been commonly used to treat the menopausal syndrome. It has been reported that LWDH could improve estrogen receptor α (ERα) expression to prevent atherosclerosis (AS), while the mechanism of LWDH on regulating ERα expression was still unknown. AIM OF THE STUDY: To reveal the mechanism of LWDH on regulating the ERα expression. MATERIALS AND METHODS: The protective effect of LWDH on Hcy-induced apoptosis of human umbilical vein endothelial cells (HUVECs) was examined. The expression of ERα and DNA methyltransferases 1 (DNMT1) were detected by Western blot and real-time polymerase chain reaction (RT-PCR). The methylation rate of the ERα gene was assayed by the bisulfite sequencing PCR (BSP). High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) was applied to determine the level of S-Adenosyl methionine (SAM) and S-Adenosyl homocysteine (SAH). In vivo, the ApoE-/- mice were ovariectomized to establish postmenopausal atherosclerosis (AS) model. RESULTS: In vitro study showed that LWDH protects HUVECs from Hcy-induced apoptosis. Treatment with LWDH significantly increased the ERα expression and reduced the methylation rate of the ERα gene by inhibiting the DNMT1 expression. The level of main methyl donor SAM and the ration of SAM/SAH were reduced by LWDH. In vivo, LWDH prevented the formation of plaque and reduced the concentration of Hcy. In addition, LWDH upregulated the ERα expression, as well as inhibiting the expression of DNMT1 in atherosclerotic mice. CONCLUSIONS: LWDH exerted protective effects on postmenopausal AS mice, and HUVECs treated with Hcy. LWDH increased of ERα expression via inhibiting DNMT1-dependent ERα methylation.

Targeting of prostate-specific membrane antigen for radio-ligand therapy of triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer has extremely high risk of relapse due to the lack of targeted therapies, intra- and inter-tumoral heterogeneity, and the inherent and acquired resistance to therapies. In this study, we evaluate the potential of prostate-specific membrane antigen (PSMA) as target for radio-ligand therapy (RLT). METHODS: Tube formation was investigated after incubation of endothelial HUVEC cells in tumor-conditioned media and monitored after staining using microscopy. A binding study with 68Ga-labeled PSMA-addressing ligand was used to indicate targeting potential of PSMA on tumor-conditioned HUVEC cells. For mimicking of the therapeutic application, tube formation potential and vitality of tumor-conditioned HUVEC cells were assessed following an incubation with radiolabeled PSMA-addressing ligand [177Lu]-PSMA-617. For in vivo experiments, NUDE mice were xenografted with triple-negative breast cancer cells MDA-MB231 or estrogen receptor expressing breast cancer cells MCF-7. Biodistribution and binding behavior of [68Ga]-PSMA-11 was investigated in both tumor models at 30 min post injection using µPET. PSMA- and CD31-specific staining was conducted to visualize PSMA expression and neovascularization in tumor tissue ex vivo. RESULTS: The triple-negative breast cancer cells MDA-MB231 showed a high pro-angiogenetic potential on tube formation of endothelial HUVEC cells. The induced endothelial expression of PSMA was efficiently addressed by radiolabeled PSMA-specific ligands. 177Lu-labeled PSMA-617 strongly impaired the vitality and angiogenic potential of HUVEC cells. In vivo, as visualized by µPET, radiolabeled PSMA-ligand accumulated specifically in the triple-negative breast cancer xenograft MDA-MB231 (T/B ratio of 43.3 ± 0.9), while no [68Ga]-PSMA-11 was detected in the estrogen-sensitive MCF-7 xenograft (T/B ratio of 1.1 ± 0.1). An ex vivo immunofluorescence analysis confirmed the localization of PSMA on MDA-MB231 xenograft-associated endothelial cells and also on TNBC cells. CONCLUSIONS: Here we demonstrate PSMA as promising target for two-compartment endogenous radio-ligand therapy of triple-negative breast cancer.

Screening of angiogenesis inhibitors using a 3D vascular microfluidic chip to achieve contraception.

Angiogenesis plays a vital role in the process of embryo implantation, as it improves endometrial receptivity and guides embryo implantation, thus creating a favorable environment for subsequent development of the embryo. Hence, a theory of achieving contraception by inhibiting angiogenesis was put forward. Here, we screened the drugs inhibiting angiogenesis using cell scratch wound assay and a 3D biomimetic vascular microfluidic chip, then observed the effect of them on contraception by injecting these drugs into fertilized mice and observing if the embryos were implanted. We preliminarily verify the feasibility of contraception by inhibiting angiogenesis and gives a new direction in the development of contraceptive pills.

Experimental study of blood pressure and its impact on spontaneous hypertension in rats with Xin Mai Jia.

AIM: The aim of this study was to evaluate the antihypertensive effect of Xin Mai Jia (XMJ) and to explore the mechanism of its hypotensive effect. METHODS: A total of 50 spontaneously hypertensive rats (SHR) were randomised into five groups. A total of 30 Wistar-Kyoto rats were randomised into three groups, comprising the control group. All of the rats were administered medicine through a gastrogavage once a day for 8 weeks. The tail-cuff method was applied to their monitor blood pressure. After 8 weeks of treatment, serum NO, SOD activity, MDA level, ET, ALD, AngII, RE, and CGRP in the serum were detected in all of the rats. Pathological changes in the aorta were observed via haematoxylin-eosin (HE) and immunohistochemical staining. Vasodilation function was assessed by measuring acetylcholine-induced vessel relaxation in the rats' organ chambers. The function of the mesenteric arteries was measured using DMT wire myography. Human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs) injury models were induced by hydrogen peroxide (H2O2). HASMCs and HUVECs were injured by H2O2 and then exposed to various drugs. HASMC and HUVEC migration was evaluated using the cell scratch test. The expression of the AT1 receptors (AT1R) in the HASMCs was detected via immunofluorescence (IFC) assay. RESULTS: After 8 weeks of treatment, XMJ reduced the systolic blood pressure of the SHR. XMJ significantly reduced the serum RE, AngII, ALD, and ET-1 levels and increased the content of CGRP and NO in the SHR, upregulated the SOD content, and downregulated MDA level of the SHR. XMJ improved pathological damage of the aorta to varying degrees, decreased the expression of AT1R in the SHR aortic vessels, and improved the mesenteric microvascular relaxation of the SHR. Cell experiments confirmed that XMJ inhibited the migration of the HUVECs and HASMCs induced by H2O2 and the expression of AT1R in the HASMCs. CONCLUSION: XMJ had satisfactory hypotensive action on the SHR in this study. Its mechanism may be associated with inhibiting RAAS activity and improving RAAS function, inhibiting hypertensive-induced vascular diastolic dysfunction, and improving vascular endothelial function.

Quercetin alleviates high glucose-induced damage on human umbilical vein endothelial cells by promoting autophagy.

BACKGROUND: Quercetin, a flavonoid antioxidant, has been found to exert therapeutic effects in diabetic condition. Autophagy represents a homeostatic cellular mechanism for the turnover of unfolds proteins and damaged organelles through a lysosome-dependent degradation manner. We speculated that quercetin could protect endothelial cells against high glucose-induced damage by promoting autophagic responses. METHODS: HUVECs viability was evaluated by MTT method. Griess and TBARS assays were used to monitor the levels of NO and MDA, respectively. Intracellular ROS generation was determined in DCFDA-stained cells analyzed by flow cytometry. To investigate the role of quercetin in endothelial cell migratory behavior, we used a scratch test. The level of autophagy proteins LC3, Beclin-1 and P62 were measured by western blotting technique. RESULTS: Our results showed that quercetin had the potential to increase cell survival after exposure to high glucose (P < 0.05). Total levels of oxidative stress markers were profoundly decreased and the activity of GSH was increased by quercetin (P < 0.05). High glucose suppressed HUVECs migration to the scratched area (P < 0.05). However, a significant stimulation in cell migration was observed after exposure to quercetin (P < 0.05). Based on data, autophagy was blocked at the late stage by high glucose concentration while quercetin enhanced autophagic response by reducing the P62 level coincided with the induction of Beclin-1 and LC3-II to LC3-I ratio (P < 0.05). All these beneficial effects were reversed by 3-methyladenine as an autophagy inhibitor. CONCLUSION: Together, our data suggest that quercetin could protect HUVECs from high glucose induced-damage possibly by activation of the autophagy response.

Eupatoriopicrin Inhibits Pro-inflammatory Functions of Neutrophils via Suppression of IL-8 and TNF-alpha Production and p38 and ERK 1/2 MAP Kinases.

During chronic inflammation, neutrophils acting locally as effector cells not only activate antibacterial defense but also promote the inflammatory response. Interleukin 8 (IL-8), the main cytokine produced by activated neutrophils, positively correlates with the severity of respiratory tract diseases. By screening European plants traditionally used for treating respiratory tract diseases, we found that extracts of aerial parts of Eupatorium cannabinum inhibit IL-8 release from neutrophils. Using bioassay-guided fractionation, we identified five sesquiterpene lactones, eupatoriopicrin (1), 5'-deoxyeupatoriopicrin (2), hiyodorilactone A (3), 3-hydroxy-5'- O-acetyleupatoriopicrin = hiyodorilactone D (4), and hiyodorilactone B (5), that efficiently (IC50 < 1 µM) inhibited IL-8 and TNF-α release in lipopolysaccharide (LPS)-stimulated human neutrophils. Moreover, all these sesquiterpene lactones suppressed the adhesion of human neutrophils to an endothelial monolayer by downregulating the expression of the ß2 integrin CD11b/CD18 on the neutrophil surface. Furthermore, eupatoriopicrin efficiently suppressed LPS-induced phosphorylation of p38 MAPK and ERK and attenuated neutrophil infiltration in the thioglycolate-induced peritonitis model in mice. Altogether, these results demonstrate the potential of the sesquiterpene lactone eupatoriopicrin as a lead substance for targeting inflammation.

Biocompatible graphene-based nanoagent with NIR and magnetism dual-responses for effective bacterial killing and removal.

Antibiotics have been widely used in clinical applications to treat pathogenic infections at present, but the problem of drug-resistance associated with the abuse of antibiotics has become a large threat to human beings. Herein, we developed an antibacterial nanoagent by coating quaternized chitosan (QCS) on the surface of Fe3O4 nanoparticles-anchored graphene oxide (GO), which enabled QCS and GO to achieve synergistic effects on killing the drug-resistant bacteria. Systematical antibacterial experiments showed that the prepared nanoagent had antibacterial ability, which was significantly enhanced after the introduction of near-infrared (NIR). Importantly, the nanoagent could be easily recycled and reused without the reduction of the antibacterial ability. During the test time, this nanoagent exhibited no obviously toxic side effect to cells. Given the above advantages, we anticipate that the nanoagent has a promising future in various applications such as wound disinfection, water purification, and surface sterilization of medical devices.

Effects of tumor necrosis factor-α-induced exosomes on the endothelial cellular behavior, metabolism and bioenergetics.

OBJECTIVE: To investigate the effects of TNF-α-induced exosomes release on the biological behavior, metabolism, and bioenergetics of HUVECs. METHODS: Exosomes were isolated from conditioned media of HUVECs by ultracentrifugation after treatment with or without TNF-α. HUVECs were treated with or without TNF-α, or different concentrations of exosomes isolated from conditioned media with or without TNF-α induction (TExo and CExo , respectively). RESULTS: The results showed that TNF-α significantly inhibited migration, tube formation, and increased apoptosis rate of HUVECs compared with controls. Furthermore, TNF-α-induced exosomes (TExo ) rather than CExo , indicated similar effects to inhibit migration, tube formation and promote endothelial apoptosis. Although TNF-α treatment did not show a statistical difference, TExo significantly inhibited extracellular OCR compared with controls. TExo could significantly inhibited intracellular OCR in a hypoxia condition. TNF-α significantly increased L-ECA compared with control cells, and TExo showed similar stimulative effect on L-ECA. CONCLUSIONS: TNF-α-induced exosomes could significantly (a) change migration, tube formation, and apoptosis; (b) inhibit endothelial extracellular OCR and intracellular OCR (hypoxia); (c) increase glycolysis rate of the endothelial cells. These data provide new evidence for exploring endothelial behavior regulation using exosomes and their effects on endothelial metabolism and bioenergetics.

The effect of formononetin on the proliferation and migration of human umbilical vein endothelial cells and its mechanism.

Formononetin is one of the main active components of traditional Chinese medicine red clover (Trifolium pratense L). Prior studies have demonstrated that formononetin is a typical phytoestrogen and exhibits an estrogen-like effect that protects the cardiovascular system with minimal side effects. In this study, we further investigated the role of formononetin in human umbilical vein endothelial cells (HUVECs) and its potential molecular mechanisms. We founded that formononetin promotes proliferation and migration of HUVECs as assessed by the MTT and wound healing assays. Meanwhile, insulin-like growth factor 1 (IGF-1) and intercellular adhesion molecule 1 (ICAM-1) protein levels were increased in a dose-dependent manner. Our findings illustrated that formononetin exhibits a protective effect on HUVECs, and the molecular mechanisms may correlate with IGF-1R and ICAM-1, as well as upregulation of vascular endothelial growth factor (VEGF) and activation of extracellular signal-regulated kinase (ERK).

Rheum palmatum extract exerts anti-hepatocellular carcinoma effects by inhibiting signal transducer and activator of transcription 3 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatocellular carcinoma (HCC) is among the most common malignancies. Signal transducer and activator of transcription 3 (STAT3), with abnormal expression and constitutive activation, has been reported to promote proliferation, metastasis, survival and angiogenesis of HCC cells. Rheum palmatum (RP), a traditional Chinese medicinal herb, exhibited tumor-suppressing effects in multiple human cancers, but its potential functions in HCC remain unexplored. AIM OF THE STUDY: This study aimed to examine the involvement of STAT3 signaling in the anti-HCC effects of RP extract. MATERIALS AND METHODS: SMMC-7721 and HepG2 HCC cell lines were treated with RP extract for 24 h, and then viability, migration, and invasion of HCC cells and angiogenesis of human umbilical vein endothelial cells (HUVECs) were analyzed using MTS, wound-healing, Transwell invasion and tube formation assays, respectively. Western blotting and immunohistochemistry (IHC) were used to examine the activation of key molecules in STAT3 signaling, including STAT3, JAK2, and Src. Additionally, we explored the in vivo antitumor effects of RP extract in a xenograft tumor nude mouse model of HCC. RESULTS: The result showed that RP extract reduced viability, migration, and invasion of SMMC-7721 and HepG2 cells and angiogenesis of HUVECs. It suppressed the phosphorylation of STAT3 and its upstream kinases including JAK2 and Src. In addition, RP extract treatment downregulated STAT3 target genes, including survivin, Bcl-xL, Mcl-1, Bcl-2, MMP-2, MMP-9, Cyclin D1, CDK4, c-Myc, and VEGF-C. Furthermore, RP extract suppressed the xenograft tumor growth and activation of STAT3 in xenograft tumor mice. CONCLUSION: Collectively, the results showed that RP extract prevented HCC progression by inhibiting STAT3, and might be useful for the treatment of HCC.

Bisphenol A vascular toxicity: Protective effect of Vitis vinifera (grape) seed extract and resveratrol.

The underlying mechanism of Bisphenol A (BPA)-induced vascular toxicity and the protective role of grape seed extract (GSE) and resveratrol were investigated in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) were exposed to different concentrations of GSE and resveratrol. Then, BPA was added to the cells and cell viability and effects on the protein level of cell adhesion molecules were measured through MTT and western blotting. Animals were randomly divided into control, GSE (3 and 12 mg·kg-1 ·day-1 ip), resveratrol (100 mg·kg-1 ·day-1 ip), BPA (35 mg·kg-1 ·day-1 , gavage), BPA plus GSE (3, 6, and 12 mg·kg-1 ·day-1 ip), BPA plus resveratrol (25, 50, and 100 mg·kg-1 ·day-1 ip), and BPA plus vitamin E (200 IU/kg per every other day ip). After 2 months, contractile and relaxant responses were evaluated on the isolated aorta. BPA increased the level of aorta malondialdehyde (p < 0.001) and decreased vascular responses to KCl (p < 0.01), phenylephrine (p < 0.001), and acetylcholine (p < 0.01). In HUVECs, BPA (IC50 : 220 µM) increased protein level of vascular cell adhesion molecule (p < 0.05) and cleaved capase3 (p < 0.001). GSE, resveratrol, and vitamin E cotreatment restored toxic effects of BPA in some levels. BPA vascular toxicity was attributed to lipid peroxidation and endothelial dysfunction. The protective role of GSE and resveratrol against BPA-endothelial dysfunction could be attributed to their potent antioxidant properties.

Passive heat therapy protects against endothelial cell hypoxia-reoxygenation via effects of elevations in temperature and circulating factors.

KEY POINTS: Accumulating evidence indicates that passive heat therapy (chronic use of hot tubs or saunas) has widespread physiological benefits, including enhanced resistance against novel stressors ('stress resistance'). Using a cell culture model to isolate the key stimuli that are likely to underlie physiological adaptation with heat therapy, we showed that both mild elevations in temperature (to 39°C) and exposure to serum from human subjects who have undergone 8 weeks of heat therapy (i.e. altered circulating factors) independently prevented oxidative and inflammatory stress associated with hypoxia-reoxygenation in cultured endothelial cells. Our results elucidate some of the mechanisms (i.e. direct effects of temperature vs. circulating factors) by which heat therapy seems to improve resistance against oxidative and inflammatory stress. Heat therapy may be a promising intervention for reducing cellular damage following ischaemic events, which has broad implications for patients with cardiovascular diseases and conditions characterized by 'chronic' ischaemia (e.g. peripheral artery disease, metabolic diseases, obesity). ABSTRACT: Repeated exposure to passive heat stress ('heat therapy') has widespread physiological benefits, including cellular protection against novel stressors. Increased heat shock protein (HSP) expression and upregulation of circulating factors may impart this protection. We tested the isolated abilities of mild heat pretreatment and serum from human subjects (n = 10) who had undergone 8 weeks of heat therapy to protect against cellular stress following hypoxia-reoxygenation (H/R), a model of ischaemic cardiovascular events. Cultured human umbilical vein endothelial cells were incubated for 24 h at 37°C (control), 39°C (heat pretreatment) or 37°C with 10% serum collected before and after 8 weeks of passive heat therapy (four to five times per week to increase rectal temperature to ≥ 38.5°C for 60 min). Cells were then collected before and after incubation at 1% O2 for 16 h (hypoxia; 37°C), followed by 20% O2 for 4 h (reoxygenation; 37°C) and assessed for markers of cell stress. In control cells, H/R increased nuclear NF-κB p65 protein (i.e. activation) by 106 ± 38%, increased IL-6 release by 37 ± 8% and increased superoxide production by 272 ± 45%. Both heat pretreatment and exposure to heat therapy serum prevented H/R-induced NF-κB activation and attenuated superoxide production; by contrast, only exposure to serum attenuated IL-6 release. H/R also decreased cytoplasmic haemeoxygenase-1 (HO-1) protein (known to suppress NF-κB), in control cells (-25 ± 8%), whereas HO-1 protein increased following H/R in cells pretreated with heat or serum-exposed, providing a possible mechanism of protection against H/R. These data indicate heat therapy is capable of imparting resistance against inflammatory and oxidative stress via direct heat and humoral factors.

Fraxinellone has anticancer activity in vivo by inhibiting programmed cell death-ligand 1 expression by reducing hypoxia-inducible factor-1α and STAT3.

Dictamnus dasycarpus is a traditional Chinese medicine thathas been commonly used in the treatment of cancer. Fraxinellone is a natural product isolated from the D. dasycarpus plant, which has been shown to exhibit neuroprotective and anti-inflammatory activities. However, whether fraxinellone exerts anticancer effects and the mechanisms by which it may inhibit tumor growth remain unknown. Here, we found that fraxinellone, in a dose-dependented manner, inhibited the expression of programmed cell death ligand-1 (PD-L1), which plays a pivotal role in tumorigenesis. It was subsequently shown that fraxinellone reduced HIF-1α protein synthesis via the mTOR/p70S6K/eIF4E and MAPK pathways. It also inhibited activation of STAT3 via the JAK1, JAK2, and Src pathways. Immunoprecipitation and western blotting assays showed that fraxinellone inhibited PD-L1 expression by reducing STAT3 and HIF-1α cooperatively. Flow cytometry, colony formation, and EdU incorporation assays demonstrated that fraxinellone inhibited cell proliferation through suppression of PD-L1. Tube formation, migration, and invasion assays showed that fraxinellone inhibits angiogenesis by suppressing PD-L1. In vivo studies further supported anticancer role for fraxinellone, demonstrating that fraxinellone treatment inhibited the growth of tumor xenografts. We concluded that fraxinellone inhibits PD-L1 expression by downregulating the STAT3 and HIF-1α signaling pathways, subsequently inhibiting proliferation and angiogenesis in cancer cells. These studies reveal previously unknown characteristics of fraxinellone and provide new perspectives into the mechanism of cancer inhibition of the compound.

Jianpi Jiedu decoction, a traditional Chinese medicine formula, inhibits tumorigenesis, metastasis, and angiogenesis through the mTOR/HIF-1α/VEGF pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine has been utilized for the treatment of cancer. Jianpi Jiedu decoction (JPJD), a traditional Chinese medicine formula, has been used for the treatment of colorectal cancer for decades. However, the underlying molecular mechanistic basis for the effect of JPJD on colorectal cancer is poorly understood. AIM OF THE STUDY: The aim of this study was to identify the effects of JPJD on human colon cancer cells in vitro as well as in vivo and to investigate the mechanistic basis for the anticancer effect of JPJD. MATERIALS AND METHODS: The in vitro antitumor activity of JPJD was assessed by MTT assay, flow cytometric analysis, wound-healing assay, transwell assays, and tube formation assays in order to assess cell activity, apoptosis, migration, invasion, and angiogenesis, respectively. The anticancer properties of JPJD in vivo were assessed by immunohistochemistry in a nude mouse xenograft model of HCT116 cells. In addition, the level of mTOR/HIF-1α/VEGF signaling pathway proteins in HCT116 cells and tumor tissue was evaluated by immunoblotting. RESULTS: In vitro, JPJD significantly inhibited colorectal cancer cell lines viability and proliferation. Flow cytometry analysis demonstrated JPJD to induce HCT116 cell apoptosis. Additionally, JPJD effectively suppressed tumor cell migration, invasion, and angiogenesis by inhibiting the mTOR/HIF-1α/VEGF signaling pathway. In vivo, JPJD significantly inhibited HCT116 tumor growth in athymic nude mice, decreased the levels of CD34 as well as VEGF, and downregulated the mTOR/HIF-1α/VEGF pathway. CONCLUSIONS: JPJD treatment produced anti-colorectal tumor effects by inhibiting tumorigenesis, metastasis, as well as angiogenesis through the mTOR/HIF-1α/VEGF pathway. Thus, these results provide a strong rationale for the therapeutic use of JPJD in cancer treatment. Further studies are required to investigate the mechanisms underlying anti-CRC effect of JPJD.