Endothelial VWF is critical for the pathogenesis of vaso-occlusive episode in a mouse model of sickle cell disease.

Vaso-occlusive episode (VOE) is a common and critical complication of sickle cell disease (SCD). Its pathogenesis is incompletely understood. von Willebrand factor (VWF), a multimeric plasma hemostatic protein synthesized and secreted by endothelial cells and platelets, is increased during a VOE. However, whether and how VWF contributes to the pathogenesis of VOE is not fully understood. In this study, we found increased VWF levels during tumor necrosis factor (TNF)-induced VOE in a humanized mouse model of SCD. Deletion of endothelial VWF decreased hemolysis, vascular occlusion, and organ damage caused by TNF-induced VOE in SCD mice. Moreover, administering ADAMTS13, the VWF-cleaving plasma protease, reduced plasma VWF levels, decreased inflammation and vaso-occlusion, and alleviated organ damage during VOE. These data suggest that promoting VWF cleavage via ADAMTS13 may be an effective treatment for reducing hemolysis, inflammation, and vaso-occlusion during VOE.

Megakaryocyte/platelet-derived TGF-ß1 inhibits megakaryopoiesis in bone marrow by regulating thrombopoietin production in liver.

Transforming growth factor ß1 (TGF-ß1) regulates a wide variety of events in adult bone marrow (BM), including quiescence of hematopoietic stem cells, via undefined mechanisms. Because megakaryocytes (MKs)/platelets are a rich source of TGF-ß1, we assessed whether TGF-ß1 might inhibit its own production by comparing mice with conditional inactivation of Tgfb1 in MKs (PF4Cre;Tgfb1flox/flox) and control mice. PF4Cre;Tgfb1flox/flox mice had ∼30% more MKs in BM and ∼15% more circulating platelets than control mice (P < .001). Thrombopoietin (TPO) levels in plasma and TPO expression in liver were approximately twofold higher in PF4Cre;Tgfb1flox/flox than in control mice (P < .01), whereas TPO expression in BM cells was similar between these mice. In BM cell culture, TPO treatment increased the number of MKs from wild-type mice by approximately threefold, which increased approximately twofold further in the presence of a TGF-ß1-neutralizing antibody and increased the number of MKs from PF4Cre;Tgfb1flox/flox mice approximately fourfold. Our data reveal a new role for TGF-ß1 produced by MKs/platelets in regulating its own production in BM via increased TPO production in the liver. Additional studies are required to determine the mechanism.

Falcarindiol inhibits LPS-induced inflammation via attenuating MAPK and JAK-STAT signaling pathways in murine macrophage RAW 264.7 cells.

Falcarindiol (FAD) is a natural polyacetylene compound found rich in many plants of the Umbelliferae family. Previously, we isolated FAD from the rhizome of Cnidium officinale Makino, which belongs to the Umbelliferae family and found it to have a significant inhibitory effect on lipopolysaccharide (LPS)-induced production of nitric oxide, a pro-inflammatory molecule in murine macrophage RAW 264.7 cells. In this study, we investigated its effect on the expression of other major pro-inflammatory molecules as well as the mechanism underlying these effects. Pre-treatment of RAW 264.7 cells with FAD suppressed LPS-stimulated mRNA expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and interleukin-1 beta (IL-1ß) and thereby reduced the respective protein levels. Mechanistic studies demonstrated that FAD attenuated the LPS-induced activation of JNK, ERK, STAT1, and STAT3 signaling molecules. Moreover, we found that FAD did not influence LPS-induced activation of p38 and NFκB signaling pathways. Collectively, this study provides evidence that FAD inhibits the production of major pro-inflammatory molecules in LPS-challenged murine macrophages via suppression of JNK, ERK, and STAT signaling pathways.

Pinus densiflora needle supercritical fluid extract suppresses the expression of pro-inflammatory mediators iNOS, IL-6 and IL-1ß, and activation of inflammatory STAT1 and STAT3 signaling proteins in bacterial lipopolysaccharide-challenged murine macrophages.

BACKGROUND: Regulation of a persistently-activated inflammatory response in macrophages is an important target for treatment of various chronic diseases. Pine needle extracts are well known to have potent immunomodulatory effects. The current study was designed to evaluate the effects of Pinus densiflora needle supercritical fluid extract (PDN-SCFE) on bacterial lipopolysaccharide (LPS)-induced inflammatory response in RAW 264.7 murine macrophages. METHODS: Cytotoxic effect of PDN-SCFE was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of nitric oxide (NO) and the corresponding enzyme, inducible nitric oxide synthase (iNOS), were quantified by Griess and immunoblotting methods, respectively. The levels of cytokines were quantified using commercial ELISA kits. Quantitative real-time PCR (qRT-PCR) analysis was performed to assess the mRNA expression of iNOS and cytokines. To elucidate the mechanism of action, the involvement of nuclear transcription factor-kappa B (NFκB), mitogen activated protein kinases (MAPKs) and Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathways were examined by an immunoblotting method. In addition, the cellular localization of NFκB was analyzed by immunofluorescence staining. RESULTS: MTT assay results indicated that PDN-SCFE is non-toxic to RAW 264.7 cells up to a maximum assayed concentration of 40 µg/mL. The PDN-SCFE exhibited a concentration-dependent inhibitory effect on LPS-induced NO production by down regulating the expression of iNOS. In addition, the extract suppressed the LPS-induced expression of interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) but not tumour necrosis factor-α (TNFα). Mechanistic studies revealed that PDN-SCFE does not influence the NFκB and MAPK pathways. However, it showed a significant inhibitory effect on LPS-induced activation of STAT1 and STAT3 proteins in macrophages. CONCLUSION: The present findings revealed that the anti-inflammatory activity of PDN-SCFE in LPS-challenged RAW 264.7 macrophages is probably caused by the suppression of the JAK-STAT signaling pathway.

Anti-inflammatory activity of Ternstroemia gymnanthera stem bark extracts in bacterial lipopolysaccharide-stimulated RAW264.7 murine macrophage cells.

CONTEXT: Ternstroemia gymnanthera Sprague (Theaceae) possesses various known pharmacological properties. However, its anti-inflammatory activity has not been reported. OBJECTIVE: The anti-inflammatory activity of Ternstroemia gymnanthera stem bark aqueous extract (TGSBE) was evaluated using LPS-stimulated RAW264.7 macrophages. MATERIALS AND METHODS: Cytotoxicity was assessed by MTT assay after 24 h with TGSBE (25-200 µg/mL). Further testing used TGSBE at 100 and 200 µg/mL. Griess and ELISA methods after 24 h with TGSBE determined NO and cytokine levels, respectively; then, mRNA levels (iNOS & cytokines) were analyzed by Quantitative-PCR after 12 h. NF-κB and MAPK were assessed by immunoblotting after TGSBE treatment for 12 h, followed by LPS for 30 min. Immunofluorescence assay was also performed for NF-κB. ROS and MMP, after 12 h with TGSBE, were determined by flow cytometry. The antioxidant potential of TGSBE was analyzed by ABTS assay. The Folin-Ciocalteu method determined the total phenolic content of TGSBE. LPS concentration was 0.5 µg/mL. RESULTS: TGSBE at 200 µg/mL showed about 96.2% viability while suppressing the production of NO (88.99%), TNFα (24.38%), IL-6 (61.70%) and IL-1ß (55.12%) and gene expression by 67.88, 45.24, 65.84, and 70.48%, respectively. TGSBE decreased ROS (79.26%) and improved MMP (48.01%); it inhibited translocation of NF-κB and MAPK activation. Radical scavenging activity was 50% at 402.17 µg/mL (ascorbic acid standard: 88.8 µg/mL). Total phenolic content was 240.9 mg GAE/g. DISCUSSION AND CONCLUSION: TGSBE suppresses the inflammatory response by inhibiting the NF-κB and MAPK cascades exhibiting therapeutic potential to treat inflammatory diseases associated with increased activation of macrophages.

Pinus radiata bark extract induces caspase-independent apoptosis-like cell death in MCF-7 human breast cancer cells.

In the present study, we investigated the anticancer activity of Pinus radiata bark extract (PRE) against MCF-7 human breast cancer cells. First, we observed that PRE induces potent cytotoxic effects in MCF-7 cells. The cell death had features of cytoplasmic vacuolation, plasma membrane permeabilization, chromatin condensation, phosphatidylserine externalization, absence of executioner caspase activation, insensitivity to z-VAD-fmk (caspase inhibitor), increased accumulation of autophagic markers, and lysosomal membrane permeabilization (LMP). Both the inhibition of early stage autophagy flux and lysosomal cathepsins did not improve cell viability. The antioxidant, n-acetylcysteine, and the iron chelator, deferoxamine, failed to restore the lysosomal integrity indicating that PRE-induced LMP is independent of oxidative stress. This was corroborated with the absence of enhanced ROS production in PRE-treated cells. Chelation of both intracellular calcium and zinc promotes PRE-induced LMP. Geranylgeranylacetone, an inducer of Hsp70 expression, also had no significant protective effect on PRE-induced LMP. Moreover, we found that PRE induces endoplasmic reticulum (ER) stress and mitochondrial membrane depolarization in MCF-7 cells. The ER stress inhibitor, 4-PBA, did not restore the mitochondrial membrane integrity, whereas cathepsin inhibitors demonstrated significant protective effects. Collectively, our results suggest that PRE induces an autophagic block, LMP, ER stress, and mitochondrial dysfunction in MCF-7 cells. However, further studies are clearly warranted to explore the exact mechanism behind the anticancer activity of PRE in MCF-7 human breast cancer cells.

Deoxyrhapontigenin, a Natural Stilbene Derivative Isolated From Rheum undulatum L. Induces Endoplasmic Reticulum Stress-Mediated Apoptosis in Human Breast Cancer Cells.

Although current chemotherapeutic agents are active at the beginning of therapy, the most common risk is the development of resistance during later stages in almost all cancer types including breast cancer. Hence, investigation of novel drugs is still a priority goal for cancer treatment. The objective of the present study is to investigate the anticancer effect of a derivative of stilbene, deoxyrhapontigenin (DR) isolated from Rheum undulatum L. root extracts against the chemoresistant MCF-7/adr and its parental MCF-7 human breast cancer cells. The morphological images indicate that DR induces an extensive cytoplasmic vacuolation in breast cancer cells. Mechanistic investigations revealed that DR treatment causes endoplasmic reticulum (ER) dilation and upregulated the expression of ER stress markers GRP78, IRE1α, eIF2α, CHOP, JNK, and p38. Subsequently, we also identified that DR increases the levels of apoptotic fragment of PARP (89 kDa) in breast cancer cells. Blocking the expression of one of the components of the ER stress-mediated apoptosis pathway, CHOP using siRNA significantly decreased DR-induced apoptotic cleavage of PARP. In summary, the present study suggests that the induction of ER stress-mediated apoptosis by DR may account for its cytotoxic effects in human breast cancer cells.

Antidiabetic activity of gossypin, a pentahydroxyflavone glucoside, in streptozotocin-induced experimental diabetes in rats.

BACKGROUND: Most of the currently available oral hypoglycemic drugs for the treatment of diabetes mellitus elicit detrimental side effects. Hence, the search for plant-derived products for the treatment of diabetes continues. Gossypin, a pentahydroxy flavone glucoside found in the flowers of Hibiscus vitifolius, has many biological properties, including as an antioxidant, anti-inflammatory and anticancer agent. The aim of the present study was to evaluate the effect of gossypin in streptozotocin (STZ)-induced experimental diabetes in rats. METHODS: Diabetic rats were administered 20 mg/kg per day gossypin orally for 30 days. On the 28th day, rats were subjected to an oral glucose tolerance test. In addition, blood glucose, plasma insulin, hemoglobin, and HbA1c levels were determined, as was the glycogen content of the liver and muscles. Plasma protein and blood urea were also estimated. RESULTS: Oral administration of gossypin to diabetic rats resulted in improved glucose tolerance. Increased blood glucose and HbA1c levels and the reduced plasma insulin and hemoglobin levels in diabetic rats were significantly reversed to near normal after oral administration of gossypin. Furthermore, the glycogen content of the liver and muscles was significantly improved after gossypin treatment of diabetic rats, and plasma protein and blood urea levels were almost normalized. The data obtained in gossypin-treated rats were comparable with those obtained following gliclazide treatment of rats, a standard reference drug for diabetes. CONCLUSIONS: The results of the present study indicate that gossypin has potent antidiabetic activity in STZ-induced experimental diabetes in rats.