Protective effect of alizarin on vascular endothelial dysfunction via inhibiting the type 2 diabetes-induced synthesis of THBS1 and activating the AMPK signaling pathway.

BACKGROUND: In this study, we investigated the protective effects of alizarin (AZ) on endothelial dysfunction (ED). AZ has inhibition of the type 2 diabetes mellitus (T2DM)-induced synthesis of thrombospondin 1 (THBS1). Adenosine 5'-monophosphate- activated protein kinase (AMPK), particularly AMPKα2 isoform, plays a critical role in maintaining cardiac homeostasis. PURPOSE: The aim of this study was to investigate the ameliorative effect of AZ on vascular injury caused by T2DM and to reveal the potential mechanism of AZ in high glucose (HG)-stimulated human umbilical vein endothelial cells (HUVECs) and diabetic model rats. STUDY DESIGN: HUVECs, rats and AMPK-/- transgenic mice were used to investigate the mitigating effects of AZ on vascular endothelial dysfunction caused by T2DM and its in vitro and in vivo molecular mechanisms. METHODS: In type 2 diabetes mellitus rats and HUVECs, the inhibitory effect of alizarin on THBS1 synthesis was verified by immunohistochemistry (IHC), immunofluorescence (IF) and Western blot (WB) so that increase endothelial nitric oxide synthase (eNOS) content in vitro and in vivo. In addition, we verified protein interactions with immunoprecipitation (IP). To probe the mechanism, we also performed AMPKα2 transfection. AMPK's pivotal role in AZ-mediated prevention against T2DM-induced vascular endothelial dysfunction was tested using AMPKα2-/- mice. RESULTS: We first demonstrated that THBS1 and AMPK are targets of AZ. In T2DM, THBS1 was robustly induced by high glucose and inhibited by AZ. Furthermore, AZ activates the AMPK signaling pathway, and recoupled eNOS in stressed endothelial cells which plays a protective role in vascular endothelial dysfunction. CONCLUSIONS: The main finding of this study is that AZ can play a role in different pathways of vascular injury due to T2DM. Mechanistically, alizarin inhibits the increase in THBS1 protein synthesis after high glucose induction and activates AMPKα2, which increases NO release from eNOS, which is essential in the prevention of vascular endothelial dysfunction caused by T2DM.

Electroacupuncture activates angiogenesis by regulating the PI3K/Pten/Thbs1 signaling pathway to promote the browning of adipose tissue in HFD-induced obese mice.

This study investigated the effect of electroacupuncture (EA) on the browning of white adipose tissue (WAT) via angiogenesis and its potential mechanism in obese mice. Four-week-old male C56BL/6 mice were randomly divided into a high-fat diet (HFD) and a normal chow diet (ND) group. After 12 weeks, HFD mice were randomly divided into two groups to receive or not receive EA for 3 weeks. After EA treatment, body weight, adipocyte size, serum glucose (GLU), triacylglycerol (TG), cholesterol (CHO), leptin (Lep), monocyte chemoattractant protein-1 (MCP-1), WAT browning-related genes, angiogenesis-related genes, and the PI3K/Pten/Thbs1 signaling pathway were evaluated. The results indicated that EA significantly reduced body weight, adipocyte size, and serum concentrations of GLU, TG, CHO, Lep and MCP-1 and promoted WAT browning. Angiogenesis and the PI3K/Pten/Thbs1 signaling pathway were all activated by EA intervention. The expression levels were consistent with the results of RNA-seq and confirmed via qRTPCR and WB. Our study showed that EA may activate angiogenesis via the PI3K/Pten/Thbs1 signaling pathway in WAT, thereby promoting the browning and thermogenesis of adipose tissue.

Enterolactone and trabectedin suppress epithelial ovarian cancer synergistically via upregulating THBS1.

Epithelial ovarian cancer (EOC) is the most common and fatal subtype of ovarian malignancies, with no effective therapeutics available. Our previous studies have demonstrated extraordinary suppressive efficacy of enterolactone (ENL) on EOC. A chemotherapeutic agent, trabectedin (Trabe), is shown to be effective on ovarian cancer, especially when combined with other therapeutics, such as pegylated liposomal doxorubicin or oxaliplatin. Thrombospondin 1 (THBS1), a kind of matrix glycoprotein, plays important roles against cancer development through inhibiting angiogenesis but whether it is involved in the suppression of EOC by ENL or Trabe remains unknown. To test combined suppressive effects of ENL and Trabe on EOC and possible involvement of THBS1 in the anticancer activities of ENL and Trabe. The EOC cell line ES-2 was transfected with overexpressed THBS1 by lentivirus vector. We employed tube formation assay to evaluate the anti-angiogenesis activity of ENL and of its combined use with Trabe after THBS1 overexpression and established drug intervention and xenograft nude mouse cancer models to assess the in vivo effects of the hypothesized synergistic suppression between the agents and the involvement of THBS1. Mouse fecal samples were collected for 16S rDNA sequencing and microbiota analysis. We detected strong inhibitory activities of ENL and Trabe against the proliferation and migration of cancer cells and observed synergistic effects between ENL and Trabe in suppressing EOC. ENL and Trabe, given either separately or in combination, could suppress the tube formation capability of human microvascular endothelial cells, and this inhibitory effect became even stronger with THBS1 overexpression. In the ENL plus Trabe combination group, the expression of tissue inhibitor of metalloproteinases 3 and cluster of differentiation 36 was both upregulated, whereas matrix metalloproteinase 9, vascular endothelial growth factor, and cluster of differentiation 47 were all decreased. With the overexpression of THBS1, the results became even more pronounced. In animal experiments, combined use of ENL and Trabe showed superior inhibitory effects to either single agent and significantly suppressed tumor growth, and the overexpression of THBS1 further enhanced the anti-cancer activities of the drug combination group. ENL and Trabe synergistically suppress EOC and THBS1 could remarkably facilitate the synergistic anticancer effects of ENL and Trabe.

Uncovering pharmacological mechanisms of Phellinus linteus on focal segmental glomeruloscleosis rats through tandem mass tag-based quantitative proteomic analysis, network pharmacology analysis and experimental validation.

OBJECTIVE: To explore the underlying molecular mechanism of (). METHODS: We used a tandem mass tag-based quantitative proteomic method to determine the differentially expressed proteins. Network pharmacology analysis was used to analysis the main components of and construct the compound-target network. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to validate the analyses results. RESULTS: The expression levels of thrombospondin-1 (TSP-1) and transforming growth factor (TGF)-ß1/Smad3 signaling pathway proteins were significantly upregulated in focal segmental glomeruloscleosis (FSGS) rats. The reduced the expression levels of TSP-1 and TGF-ß1 signaling pathway proteins. Network pharmacology analysis revealed that protocatechualdehyde was the main active component. Subsequent and experiments validated the results of proteomic and network pharmacology analyses. CONCLUSIONS: Our results suggested that may inhibit renal sclerosis by inhibiting TSP-1-activated TGF-ß1 signaling and may have potential applications in the treatment of FSGS.

Depleted uranium induces thyroid damage through activation of ER stress via the thrombospondin 1-PERK pathway.

Depleted uranium (DU) can cause damage to the body, but its effects on the thyroid are unclear. The purpose of this study was to investigate the DU-induced thyroid damage and its potential mechanism in order to find new targets for detoxification after DU poisoning. A model of acute exposure to DU was constructed in rats. It was observed that DU accumulated in the thyroid, induced thyroid structure disorder and cell apoptosis, and decreased the serum T4 and FT4 levels. Gene screening showed that thrombospondin 1 (TSP-1) was a sensitive gene of DU, and the expression of TSP-1 decreased with the increase of DU exposure dose and time. TSP-1 knockout mice exposed to DU had more severe thyroid damage and lower serum FT4 and T4 levels than wild-type mice. Inhibiting the expression of TSP-1 in FRTL-5 cells aggravated DU-induced apoptosis, while exogenous TSP-1 protein alleviated the decreased viability in FRTL-5 cells caused by DU. It was suggested that DU may caused thyroid damage by down-regulating TSP-1. It was also found that DU increased the expressions of PERK, CHOP, and Caspase-3, and 4-Phenylbutyric (4-PBA) alleviated the DU-induced FRTL-5 cell viability decline and the decrease levels of rat serum FT4 and T4 caused by DU. After DU exposure, the PERK expression was further up-regulated in TSP-1 knockout mice, and the increased expression of PERK was alleviated in TSP-1 over-expressed cells, as well as the increased expression of CHOP and Caspase-3. Further verification showed that inhibition of PERK expression could reduce the DU-induced increased expression of CHOP and Caspase-3. These findings shed light on the mechanism that DU may activate ER stress via the TSP 1-PERK pathway, thereby leading to thyroid damage, and suggest that TSP-1 may be a potential therapeutic target for DU-induced thyroid damage.

Construction of tea tree oil/salicylic acid/palygorskite hybrids for advanced antibacterial and anti-inflammatory performance.

This study describes the construction of a tailor-made clay-based hybrid with advanced dermocompatibility, antibacterial and anti-inflammatory performance by incorporating tunable ratios of tea tree oil (TTO) and salicylic acid (SA) into the naturally occurring porous structure of palygorskite (Pal). Among the three TTO/SA/Pal (TSP) systems constructed, TSP-1 with a TTO : SA ratio of 1 : 3 demonstrated the lowest 3T3 NRU predicted acute oral toxicity and dermal HaCaT cytotoxicity as well as the most pronounced antibacterial activity with a selective inhibitory action against the pathogens (E. coli, P. acnes and S. aureus) over the beneficial (S. epdermidis) species inhabiting on the human skin. Also noticeable is that exposure of these skin commensal bacteria to TSP-1 prevented the antimicrobial resistance evolution compared to the conventional antibiotic ciprofloxacin. Mechanistic investigation of its antibacterial modes of action revealed a synergy between the TTO and SA loadings on the Pal supports in reactive oxygen production, causing oxidative damage to bacterial cell membranes and increased leakage of intracellular compounds. Additionally, TSP-1 significantly decreased the proinflammatory cytokines of IL-1ß, IL-6, IL-8, and TNF-α in a bacterial lipopolysaccharide-stimulated differentiated THP-1 macrophage model, showing the potential to inhibit inflammatory responses in bacterial infections. Overall, this is the first report exploring the potential of constructing clay-based organic-inorganic hybrids as alternatives to antibiotics to combat bacterial resistance with advanced compatibility and anti-inflammatory benefits that are desired for the development of topically applied biopharmaceuticals.

Baicalin inhibited both the Furin/TGFß1/Smad3/TSP-1 pathway in endothelial cells and the AKT/Ca2+/ROS pathway in platelets to ameliorate inflammatory coagulopathy.

Inflammatory coagulopathy is resulted from endothelial dysfunction and platelet hyperactivation in inflammatory diseases. In this study, the effects of baicalin, an active component of the traditional Chinese medicine Huangqin, on inflammatory coagulopathy were observed both in vivo and in vitro. In LPS-induced rats, baicalin ameliorated coagulation indexes, inhibited platelet hyperactivation and decreased the expression of thrombospondin-1 (TSP-1) in vessels. In cultured endothelial cells, baicalin decreased the expression of TSP-1 and collagen as well as the TNF-α-induced increase in the levels of TSP-1 and ICAM-1. Baicalin could significantly decrease the platelet adhesion on endothelial cells treated with TNF-α. Baicalin also could inhibit the increase of ROS level and the activation of the NLRP3/Caspase-1/GSDMD pathway in TNF-α-induced endothelial cells. Furin was found to be the direct target of baicalin in HUVECs. Knockdown of Furin using siRNA could ameliorate the effects of baicalin on the activation of TGFß1/Smad3 pathway, TSP-1 expression and the adhesion of platelets on TNF-α-treated endothelial cells. At the same time, baicalin inhibited platelet aggregation induced by collagen or combination of collagen and TSP-1 peptide. Collagen-induced Ca2+ mobilization, ROS level increase, AKT1 phosphorylation, platelet degranulation and TSP-1 release could be all inhibited by baicalin. In all, baicalin ameliorated endothelial dysfunction by inhibiting Furin/TGFß1/Smad3/TSP-1 pathway and also ameliorated platelet activation by inhibiting AKT-related pathway. Both the inhibiting effects of baicalin on endothelial dysfunction and platelet activation might contribute to its ameliorating effects on inflammatory coagulopathy.

Targeting the CD47/thrombospondin-1 signaling axis regulates immune cell bioenergetics in the tumor microenvironment to potentiate antitumor immune response.

BACKGROUND: CD47 is an integral membrane protein that alters adaptive immunosurveillance when bound to the matricellular glycoprotein thrombospondin-1 (TSP1). We examined the impact of the CD47/TSP1 signaling axis on melanoma patient response to anti-PD-1 therapy due to alterations in T cell activation, proliferation, effector function, and bioenergetics. METHODS: A syngeneic B16 mouse melanoma model was performed to determine if targeting CD47 as monotherapy or in combination with anti-PD-1 impacted tumor burden. Cytotoxic (CD8+) T cells from Pmel-1 transgenic mice were used for T cell activation, cytotoxic T lymphocyte, and cellular bioenergetic assays. Single-cell RNA-sequencing, ELISA, and flow cytometry was performed on peripheral blood mononuclear cells and plasma of melanoma patients receiving anti-PD-1 therapy to examine CD47/TSP1 expression. RESULTS: Human malignant melanoma tissue had increased CD47 and TSP1 expression within the tumor microenvironment compared with benign tissue. Due to the negative implications CD47/TSP1 can have on antitumor immune responses, we targeted CD47 in a melanoma model and observed a decrease in tumor burden due to increased tumor oxygen saturation and granzyme B secreting CD8+ T cells compared with wild-type tumors. Additionally, Pmel-1 CD8+ T cells exposed to TSP1 had reduced activation, proliferation, and effector function against B16 melanoma cells. Targeting CD47 allowed CD8+ T cells to overcome this TSP1 interaction to sustain these functions. TSP1 exposed CD8+ T cells have a decreased rate of glycolysis; however, targeting CD47 restored glycolysis when CD8+ T cells were exposed to TSP1, suggesting CD47 mediated metabolic reprogramming of T cells. Additionally, non-responding patients to anti-PD-1 therapy had increased T cells expressing CD47 and circulating levels of TSP1 compared with responding patients. Since CD47/TSP1 signaling axis negatively impacts CD8+ T cells and non-responding patients to anti-PD-1 therapy have increased CD47/TSP1 expression, we targeted CD47 in combination with anti-PD-1 in a melanoma model. Targeting CD47 in combination with anti-PD-1 treatment further decreased tumor burden compared with monotherapy and control. CONCLUSION: CD47/TSP1 expression could serve as a marker to predict patient response to immune checkpoint blockade treatment, and targeting this pathway may preserve T cell activation, proliferation, effector function, and bioenergetics to reduce tumor burden as a monotherapy or in combination with anti-PD-1.

Serum Cat S, TSP-1, IL-11, BNP and sST2 Diagnostic and Prognostic Value in Chronic Heart Failure.

Context: Objectives • This study aimed to assess the diagnostic value of serum brain natriuretic peptide (BNP), cathepsin S (Cat S), serum soluble ST2 receptor (sST2), platelet reactive protein-1 (TSP-1) and interleukin-11 (IL-11) in patients with chronic heart failure (CHF). Context: Materials and Methods • A total of 112 patients admitted to in our hospital with HF were enrolled as the HF group and 120 healthy people undergoing physical examination were assigned to the control group. The serum levels of Cat S, TSP-1, IL-11, sST2 and BNP were measured and compared in the subgroups categorized according to New York Heart Association (NYHA) Functional Classification. Pearson correlation was applied to analyze the correlation between serum Cat S, TSP-1, IL-11, sST2 and BNP and NYHA functional class. Moreover, multivariate logistic regression was used to analyze the HF influencing factors. Context: Results • No correlation was found between the 2 groups in terms of general information, such as age, gender, body mass index (BMI), systolic blood pressure, diastolic blood pressure, smoking and heart rate (P > .05). The left ventricular ejection fraction (LVEF) in the HF group was lower than in the control group, while the level of LV end diastolic dimension (LVEDD) and left ventricular end diastolic volume (LVEDV) was significantly higher (P < .05). The levels of Cat S, TSP-1, sST2, IL-11 and BNP in the HF group were higher than in the control group (P < .05). The levels of Cat S, TSP-1, sST2, IL-11 and BNP in the grade IV group were higher than those in the grade II and III groups (P < .05). Serum Cat S, TSP-1, IL-11, sST2 and BNP levels were positively correlated with NYHA functional class (R = 0.568, 0.409, 0.472, 0.547, 0.632, respectively) (P < .001). Multivariate logistic regression analysis demonstrated that LVEF, LVEDD, LVEDV, Cat S, TSP-1, IL-11, sST2 and BNP were independent markers of CHF. Context: Conclusion • Abnormal Cat S, TSP-1, IL-11, sST2 and BNP levels were found in patients with CHF, and were highly associated with the cardiac function grades of CHF. Therefore, serum Cat S, TSP-1, IL-11, sST2 and BNP levels can serve as independent markers for CHF.

Potential of thrombospondin-1 in treatment of polycystic ovary syndrome rat model: a preliminary study.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disease in reproductive women, and the endocrine levels are also affected by diseases. The aim of this study was to determine the effect of thrombospondin-1 (TSP-1) on PCOS rat model. METHODS: We established the PCOS rat model, the serum hormones including TSP-1 expression were determined and morphological characteristics were investigated to evaluate the model. These above endocrine and morphological features were investigated again to evaluate the effect of TSP-1 treatment. RESULTS: In the PCOS model group, the serum hormones change (higher luteinizing hormone, testosterone and estrogen) and decreased TSP-1 expression levels were found compared with the control group. Besides, the morphological characteristics of PCOS were also observed in the model group. After TSP-1 treatment, the higher TSP-1, ANGPT2, PDGFB and PDGFD expression levels, the lower LH and T levels, decreased vessel density as well as VEGFA and ANGPT1 expression levels were found compared with the control group, and the ovary morphological changes were also observed in the TSP-1 experimental group. CONCLUSIONS: TSP-1 delivery system might be an alternative therapy for PCOS treatment.

Mild hyperbaric oxygen exposure attenuates rarefaction of capillary vessels in streptozotocin-induced diabetic soleus muscle in rats.

We examined the effects of mild hyperbaric oxygen (mHBO) exposure on capillary rarefaction in skeletal muscles of rats with diabetes. Streptozotocin (100 mg/kg) was administered to male Wistar rats via the tail vein to prepare a diabetic model. These rats were divided into 2 groups: the group with mHBO exposure (1.25 atmospheres absolute (ATA) with 36% oxygen; 3 h/day) and the group without mHBO exposure. Age-matched rats were used as the control group. Eight weeks later, the soleus of the rats was removed and then analyzed. With the onset of diabetes mellitus, capillary number, diameter, and volume in the soleus of the rats with diabetes decreased compared with those of the rats in the control group. In addition, increased anti-angiogenic thrombospondin-1 (TSP-1) and decreased pro-angiogenic murine double minute 2 (MDM-2) protein expressions were observed in the rats with diabetes. Alternatively, mHBO exposure attenuated the decrease in capillary diameter and volume in skeletal muscles of rats with diabetes, suppressed the overexpression of TSP-1, and restored the MDM-2 expression. These results indicate the exposure of mHBO partially attenuates capillary rarefaction in diabetic soleus muscle.

Preconditioning Effect of High-Intensity Interval Training (HIIT) and Berberine Supplementation on the Gene Expression of Angiogenesis Regulators and Caspase-3 Protein in the Rats with Myocardial Ischemia-Reperfusion (IR) Injury.

OBJECTIVE: It has been shown that angiogenesis is a desirable treatment for patients with ischemic heart disease. We set out to investigate the impact of high-intensity interval training (HIIT) and berberine supplementation on the gene expression of angiogenesis-related factors and caspase-3 protein in rats suffering from myocardial ischemic-reperfusion injury. METHODS: Fifty rats were divided into the following groups: (1) trained, (2) berberine supplemented, (3) combined, and (4) IR. Each cohort underwent five sessions of HIIT per week for a duration of 8 weeks followed by induction of ischemia. Seven days after completion of reperfusion, changes in the gene expression of angiogenesis-related factors and caspase-3 protein were evaluated in the heart tissue. RESULTS: We observed a significant difference between four groups in the transcript levels of vascular endothelial cell growth factor (VEGF), fibroblast growth factor-2 (FGF2), and thrombospondin-1(TSP-1) (p ≤ 0.05). However, the difference in endostatin (ENDO) levels was not significant among the groups despite a discernible reduction (p ≥ 0.05). Moreover, caspase-3 protein and infarct size were significantly reduced in the intervention groups (p ≤ 0.05), and cardiac function increased in response to these interventions. CONCLUSION: The treatments exert their effect, likely, by reducing caspase-3 protein and increasing the expression of angiogenesis-promoting factors, concomitant with a reduction in inhibitors of the process.

Selenium nanoparticles act as an intestinal p53 inhibitor mitigating chemotherapy-induced diarrhea in mice.

Selenium, at high-dose levels approaching its toxicity, protects tissues from dose-limiting toxicities of many cancer chemotherapeutics without compromising their therapeutic effects on tumors, there by allowing the delivery of higher chemotherapeutic doses to achieve increased cure rate. In this regard, selenium nanoparticles (SeNPs), which show the lowest toxicity among extensively investigated selenium compounds including methylselenocysteine and selenomethionine, are more promising for application. The key issue remains to be resolved is whether low-toxicity SeNPs possess a selective protective mechanism. p53 or p53-regulated thrombospondin-1 has each been confirmed to be an appropriate target for therapeutic suppression to reduce side effects of anticancer therapy. The present study demonstrated that SeNPs transiently suppressed the expression of many intestinal p53-associated genes in healthy mice. SeNPs did not interfere with tumor-suppressive effect of nedaplatin, a cisplatin analogue; however, effectively reduced nedaplatin-evoked diarrhea. Nedaplatin-induced diarrhea was associated with activation of intestinal p53 and high expression of intestinal thrombospondin-1. The preventive effect of SeNPs on nedaplatin-induced diarrhea was correlated with a powerful concomitant suppression of p53 and thrombospondin-1. Moreover, the high-dose SeNPs used in the present study did not suppress growth nor caused liver and kidney injuries as well as alterations of hematological parameters in healthy mice. Overall, the present study reveals that chemotherapeutic selectivity conferred by SeNPs involves a dual suppression of two well-documented targets, the p53 and thrombospondin-1, providing mechanistic and pharmacologic insights on low-toxicity SeNPs as a potential chemoprotectant for mitigating chemotherapy-induced diarrhea.

Endothelial progenitor cell-derived exosomes facilitate vascular endothelial cell repair through shuttling miR-21-5p to modulate Thrombospondin-1 expression.

Background: Our previous studies observed that administration of exosomes from endothelial progenitor cells (EPC) facilitated vascular repair in the rat model of balloon injury. However, the molecular events underlying this process remain elusive. Here, we aim to interrogate the key miRNAs within EPC-derived exosomes (EPC-exosomes) responsible for the activation of endothelial cell (EC) repair. Methods: The efficacy of EPC-exosomes in re-endothelialization was examined by Evans Blue dye and histological examination in the rat model of balloon-induced carotid artery injury. The effects of EPC-exosomes on human vascular EC (HUVEC) were also studied by evaluating the effects on growth, migratory and tube formation. To dissect the underlying mechanism, RNA-sequencing assays were performed to determine miRNA abundance in exosomes and mRNA profiles in exosome-treated HUVECs. Meanwhile, in vitro loss of function assays identified an exosomal miRNA and its target gene in EC, which engaged in EPC-exosomes-induced EC repair. Results: Administration of EPC-exosomes potentiated re-endothelialization in the early phase after endothelial damage in the rat carotid artery. The uptake of exogenous EPC-exosomes intensified HUVEC in proliferation rate, migration and tube-forming ability. Integrative analyses of miRNA-mRNA interactions revealed that miR-21-5p was highly enriched in EPC-exosomes and specifically suppressed the expression of an angiogenesis inhibitor Thrombospondin-1 (THBS1) in the recipient EC. The following functional studies demonstrated a fundamental role of miR-21-5p in the pro-angiogenic activities of EPC-exosomes. Conclusions: The present work highlights a critical event for the regulation of EC behavior by EPC-exosomes, which EPC-exosomes may deliver miR-21-5p and inhibit THBS1 expression to promote EC repair.

Aureobasidium pullulans produced ß-glucan is effective to enhance Kurosengoku soybean extract induced Thrombospondin-1 expression.

Black yeast, Aureobasidium pullulans is extracellularly produced ß-(1,3), (1,6)-D-glucan (ß-glucan) under certain conditions. In this study, using Glycine max cv. Kurosengoku (Kurosengoku soybeans), the production of ß-glucan through fermentation of A. pullulans was evaluated, and the effects of A. pullulans cultured fluid (AP-CF) containing ß-glucan made with Kurosengoku soybeans (kAP-CF) on a human monocyte derived cell line, Mono Mac 6 cells were investigated. Concentration of ß-glucan in kAP-CF reached the same level as normal AP-CF. An anti-angiogenic protein, Thrombospondin-1 (THBS1) was effectively induced after the stimulation with kAP-CF for comparison with AP-CF. The THBS1 is also induced after stimulation with hot water extract of Kurosengoku soybeans (KS-E), while the combined stimulation of ß-glucan with KS-E more effectively induced THBS1 than that with KS-E alone. These results suggest effects of A. pullulans-produced ß-glucan on the enhancement of Kurosengoku soybean-induced THBS1 expression.

Vitamin D supplementation lowers thrombospondin-1 levels and blood pressure in healthy adults.

INTRODUCTION: Vitamin D insufficiency, defined as 25-hydroxyvitamin D (25(OH)D) levels < 75nmol/L is associated with cardio-metabolic dysfunction. Vitamin D insufficiency is associated with inflammation and fibrosis, but it remains uncertain whether these anomalies are readily reversible. Therefore, we aimed to determine the effects of vitamin D supplementation on markers of: 1) nitric oxide (NO) signaling, 2) inflammation, and 3) fibrosis, in healthy volunteers with mild hypovitaminosis. METHODS: Healthy volunteers (n = 35) (mean age: 45 ± 11 years) with 25(OH)D levels <75nmol/L, received vitamin D supplementation (Ostelin ® capsules 2000IU) for 12 weeks. Resting systolic and diastolic blood pressures (BP) were assessed. Routine biochemistry was examined. Plasma concentrations of asymmetric dimethylarginine (ADMA), thrombospondin-1 (TSP-1), plasminogen activator inhibitor-1 (PAI-1), hs-CRP, activin-A, and follistatin-like 3 (FSTL3) were quantitated. RESULTS: Vitamin D administration for 12 weeks significantly increased 25-(OH)D levels (48.8 ± 16 nmol/L to 100.8 ± 23.7 nmol/L, p<0.001). There was significant lowering of systolic and diastolic BP, while there was no significant change in lipid profiles, or fasting insulin. Plasma concentrations of ADMA, hs-CRP, PAI-1, activin A, and FSTL-3 did not change with vitamin D supplementation. However, there was a marked reduction of TSP-1 (522.7 ± 379.8 ng/mL vs 206.7 ± 204.5 ng/mL, p<0.001). CONCLUSIONS: Vitamin D supplementation in vitamin D insufficient, but otherwise healthy individuals markedly decreased TSP-1 levels and blood pressure. Since TSP-1 suppresses signaling of NO, it is possible that the fall in BP is engendered by restoration of NO effect.

Preventive effects of nucleoprotein supplementation combined with intermittent loading on capillary regression induced by hindlimb unloading in rat soleus muscle.

Physical inactivity leads to muscle atrophy and capillary regression in the skeletal muscle. Intermittent loading during hindlimb unloading attenuates the muscle atrophy, meanwhile the capillary regression in the skeletal muscle is not suppressed. Nucleoprotein has antioxidant capacity and may prevent capillary regression. Therefore, we assessed the combined effects of intermittent loading with nucleoprotein supplementation on capillary regression induced by hindlimb unloading. Five groups of rats were assigned: control (CON), 7 days hindlimb unloading (HU), HU plus nucleoprotein supplementation (HU + NP), intermittent loading during HU (HU + IL), and intermittent loading combined with nucleoprotein supplementation during HU (HU + IL + NP). Seven days HU resulted in decrease in capillary number-to-fiber number (C/F) ratio accompanied with disuse-associated changes in fetal liver kinase-1 (Flk-1), a proangiogenesis factor, and thrombospondin-1 (TSP-1), an antiangiogenesis factor, in the soleus muscle. In addition, citrate synthase (CS) activity was decreased and protein level of superoxide dismutase (SOD)-2 was increased. Neither nucleoprotein supplementation nor intermittent loading prevented the decrease in the C/F ratio, whereas nucleoprotein supplementation combined with intermittent loading prevented the regression of capillary during unloading. Moreover, the levels of Flk-1, TSP-1, and SOD-2 protein and the CS activity were maintained up to control levels. These results suggested that nucleoprotein supplementation combined with intermittent loading was effective to prevent capillary regression induced by muscle atrophy.

Biochemical Pathways and Myometrial Cell Differentiation Leading to Nodule Formation Containing Collagen and Fibronectin.

Utilizing both primary myometrial cells and a myometrial cell line, we show here that myometrial cells undergo transition to a myofibroblast-like phenotype after a biological insult of 72 hours serum starvation and serum add-back (SB: 1% to 10% FBS). We also found that thrombospondin-1 was increased and that the transforming growth factor-beta (TGFB)-SMAD3/4 pathway was activated. This pathway is a key mediator of fibrosis and extracellular matrix (ECM) deposition. Applying the same insult supplemented with TGFB3 (1-10ng/ml) and ascorbic acid (100µg/ml) in the serum add-back treatment, we further demonstrated that cells migrated into nodules containing collagen and fibronectin. The number of cellnodules was inversely related to the percentage serum add-back. Using transmission electron microscopy we demonstrated myofibroblast-like cells and fibril-like structures in the extracellular spaces of the nodules. This study is the first direct evidence of induction of myofibroblast transdifferentiation in cultured myometrial cells which is related to the increase of thrombospondin-1 (THBS1) and the activation of TGFBSMAD 3 / 4 pathways. Combined, these observations provide biochemical and direct morphological evidence that fibrotic responses can occur in cultured myometrial cells. The findings are the first to demonstrate uterine healing mechanisms at a molecular level. Our data support the concept that fibrosis may be an initial event in formation of fibroid which exhibits signaling pathways and molecular features of fibrosis and grow by both cellular proliferation and altered extracellular matrix accumulation. Our data assists in further understanding of myometrium tissue remodeling during gestation and postpartum.

[The roles of TSP-1 and its receptor CD47 in pathogenesis of paraquat-induced pulmonary fibrosis in rats].

Objective: To establish a rat model of paraquat (PQ) -induced pulmonary fibrosis and observe the changes in thrombospondin-1 (TSP-1) and its receptor CD47 in lung tissue, and to investigate their roles in the pathogenesis of PQ-induced pulmonary fibrosis. Methods: Fifty-four clean adult male Sprague-Dawley rats were randomly divided into normal control group (n=6) and 2 h, 12 h, 1 d, 3 d, 7 d, and 14 d PQ poisoning groups (n=8 per group). A rat model of PQ poisoning was established by a single gavage of 20 wt.% PQ solution (50 mg/kg). Flow cytometry was used to determine the concentration of reactive oxygen species (ROS) in blood and lung tissue. Enzyme-linked immunosorbent assay was used to determine the concentrations of hydroxyl radicals, malondialdehyde, and hydroxyproline in lung tissue. HE staining and Masson staining were used to observe the pathological damage of lung tissue after PQ poisoning. The expression of TSP-1 and CD47 in lung tissue was measured by Immunohistochemistry. Results: Compared with the normal control group, the 2 h to 7 d PQ poisoning groups showed significant increases in ROS fluorescence intensity in red blood cells and lung tissues and the concentrations of malondialdehyde and hydroxyl radicals in lung tissue (P<0.05) , and the 14 d PQ poisoning group had a significant increase in the concentration of hydroxyproline in lung tissue (P<0.05). HE staining showed that the 2 h to 7 d PQ poisoning groups had significantly higher semiquantitative pathological scores of pulmonary alveolitis than the normal control group (P<0.05). The Masson staining showed that the 7 d and 14 d PQ poisoning groups had significantly higher semiquantitative pathological scores of pulmonary fibrosis than the normal control group (P<0.05). Compared with the normal control group, all PQ poisoning groups (except the 12 h group) had significantly increased expression of TSP-1 in lung tissue (P<0.05) , and all PQ poisoning groups (except the 1 d group) had significantly increased expression of CD47 in lung tissue (P<0.05). Within 2 h after PQ poisoning, the expression of TSP-1 and CD47 was positively correlated with the concentrations of ROS, hydroxyl radicals, and malondialdehyde and the degree of pulmonary alveolitis (P<0.01) ; at 1 d after PQ poisoning, the expression of TSP-1 and CD47 was positively correlated with the concentration of hydroxyproline in lung tissue (P<0.01) . Conclusion: The expression of TSP-1 and CD47 is closely related to oxidative stress and subsequent pulmonary fibrosis, and they may be involved in the development and progression of pulmonary alveolitis and subsequent pulmonary fibrosis in rats with PQ poisoning.

[Antitumor Effect of Ganoderma lipsiense Extract on Triple-negative Breast Cancer Model Mice and Mechanism Study].

OBJECTIVE: To study the inhibitory effect and mechanism of Ganoderma lipsiense extract (GLE) on the growth of triple-negative breast cancer (TNBC) cell line MDA-MB-231-HM in a mouse model. METHODS: The mouse model of TNBC was established by subcutaneous injection of 1.5 x 10(6) of MDA-MB-231-HM cells into BALB/c-nu mouse. Twenty successfully modeled mice were divided into the GLE group and the negative control group according to random digit table, 10 in each group. GLE (0.2 mL 100 mg/mL) was peritoneally injected to mice in the GLE group, while equal dose of normal saline was peritoneally injected to mice in the negative control group. The medication was administered once per 3 days and discontinued after 45 days. The CD34 expression was detected using immunohistochemical assay for counting microvessels. Meanwhile, expressions of thrombospondin 1 (TSP-1) and cyclin D1 were detected using immunohistochemical assay. RESULTS: The average weight was obviously lower in the GLE group than in the negative control group [(0.33 ± 0.16) g vs (0.68 ± 0.37)g, P < 0.05]. The tumor inhibition rate was 51.4% in the GLE group. The volume of transplanted tumor was obviously lesser in the GLE group than in the negative control group (P < 0.05). Results of immunohistochemical staining showed, the microvessel density (MVD) under every field was (20.7 ± 2.1), TSP-1 positive cell count was (66.2 ± 9.2), cyclin D1 positive cell count was (33.8 ± 16.4) in the GLE group, and they were 34.0 ± 2.0, 24.0 ± 6.6, and 168.2 ± 32.6, respectively in the negative control group. There was statistical difference in all indices between the two groups (P < 0.05). CONCLUSION: GLE could inhibit malignant proliferation of tumor cells by suppressing angiogenesis of blood vessels in tumor tissues and regulating cell cycles, thereby inhibiting TNBC.