Central blockade of NLRP3 reduces blood pressure via regulating inflammation microenvironment and neurohormonal excitation in salt-induced prehypertensive rats.

BACKGROUND: Inflammation has been implicated in the development of cardiovascular disease. We determined whether nod-like receptor with pyrin domain containing 3 (NLRP3) involved in the process of prehypertension, central blockade of NLRP3 decreased inflammation reaction, regulated neurohormonal excitation, and delayed the progression of prehypertension. METHODS: Prehypertensive rats were induced by 8% salt diet. The rats on high-salt diet for 1 month were administered a specific NLRP3 blocker in the hypothalamic paraventricular nucleus (PVN) for 4 weeks. ELISA, western blotting, immunohistochemistry, and flow cytometry were used to measure NLRP3 cascade proteins, pro-inflammation cytokines (PICs), chemokine ligand 2 (CCL2), C-X-C chemokine receptor type 3 (CXCR3), vascular cell adhesion molecule 1 (VCAM-1), neurotransmitters, and leukocytes count detection, respectively. RESULTS: NLRP3 expression in PVN was increased significantly in prehypertensive rats, accompanied by increased number of microglia, CD4+, CD8+ T cell, and CD8+ microglia. Expressions of PICs, CCL2, CXCR3, and VCAM-1 significantly increased. The balance between 67-kDa isoform of glutamate decarboxylase (GAD67) and tyrosine hydroxylase (TH) was damaged. Plasma norepinephrine (NE) in prehypertensive rats was increased and gamma-aminobutyric acid (GABA) was reduced. NLRP3 blockade significantly decreased blood pressure, reduced PICs, CCL2, VCAM-1 expression in PVN, and restored neurotransmitters. Blood pressure and inflammatory markers were upregulated after termination of central blockage NLRP3. CONCLUSIONS: Salt-induced prehypertension is partly due to the role of NLRP3 in PVN. Blockade of brain NLRP3 attenuates prehypertensive response, possibly via downregulating the cascade reaction triggered by inflammation and restoring the balance of neurotransmitters.

Silencing salusin ß ameliorates heart failure in aged spontaneously hypertensive rats by ROS-relative MAPK/NF-κB pathways in the paraventricular nucleus.

OBJECTIVE: Sustained hypertension is a major cause of heart failure in aging hypertensive patients. Salusin ß, a novel bioactive peptide of 20 amino acids, has been reported to participate in various cardiovascular diseases, including hypertension. We therefore hypothesized that central knockdown of salusin ß might be effective for hypertension-induced heart failure treatment. METHODS AND RESULTS: Eighteen-month-old male aged spontaneously hypertensive rats (SHR) with heart failure and WKY rats were microinjected with either a specific adenoviral vector encoding salusin ß shRNA (Ad-Sal-shRNA) or a scramble shRNA (Ad-Scr-shRNA) in the hypothalamic paraventricular nucleus (PVN) for 4 weeks. Radiotelemetry and echocardiography were used for measuring blood pressure and cardiac function, respectively. Blood samples and heart were harvested for evaluating plasma norepinephrine, tyrosine hydroxylase, and cardiac morphology, respectively. The mesenteric arteries were separated for measurement of vascular responses. The PVN was analyzed for salusin ß, proinflammatory cytokines (PICs), mitogen-activated protein kinase (MAPK), NF-κB, and reactive oxygen species (ROS) levels. Compared with normotensive rats, aging SHR with heart failure had dramatically increased salusin ß expression. Silencing salusin ß with Ad-Sal-shRNA attenuated arterial pressure and improved autonomic function, cardiac and vascular dysfunction in aging SHR with heart failure, but not in aging WKY rats. Knockdown of salusin ß significantly reduced paraventricular nucleus PICs levels, MAPK and NF-κB activity, and ROS levels in aging SHR with heart failure. CONCLUSION: These data demonstrate that in aging SHR, the heart failure that was developed during the end stage of hypertension could be ameliorated by silencing salusin ß.

The Antitumor Mechanism of Paeonol on CXCL4/CXCR3-B Signals in Breast Cancer Through Induction of Tumor Cell Apoptosis.

BACKGROUND: Paeonol, a phenolic component from the root bark of Paeonia moutan, has been identified to possess antitumor effects. However, the effect of paeonol and the mechanism of CXCL4/CXCR3-B signals in paeonol-induced breast cancer cell remain unknown. MATERIALS AND METHODS: After MDA-MB-231 cells were pretreated with paeonol or DMSO, the proliferation activity was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Hoechst, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and Annexin-V/propidium iodide staining flow cytometry. Western blot and immunohistochemistry of human breast cancer and noncancerous tissues were performed to determine the molecular alteration of CXCL4/CXCR3-B signals. RESULTS: Compared with the control, paeonol-treated breast cancer cells had low proliferation activity and high apoptotic index, indicating that paeonol induces breast cancer cell apoptosis. Western blot and immunohistochemistry showed that paeonol increased CXCR3-B signal, downregulated CXCL4, heme oxygenase (HO-1) with a corresponding increased BACH1, and decreased nuclear factor E2-related factor 2 (Nrf2). CONCLUSIONS: Thus, CXCL4/CXCR3-B may be involved in the mechanism of apoptosis induced by paeonol in breast cancer cells by regulating the expression of BACH1 and Nrf2 to downregulating HO-1 and promote apoptosis. Therefore, the authors suggest paeonol has a significant growth inhibitory effect on breast cancer cells, which may be related to the induction of apoptosis.

Blockade of TLR4 Within the Paraventricular Nucleus Attenuates Blood Pressure by Regulating ROS and Inflammatory Cytokines in Prehypertensive Rats.

BACKGROUND: Toll-like receptor 4 (TLR4) has been implicated in the progression of cardiovascular disease, including hypertension. However, the role of TLR4 in the development of prehypertension is uncertain. METHODS: Prehypertensive rats were treated with 8% salt for 12 weeks to induce prehypertension. These rats were then given either TAK-242 selective TLR4 blocker, or vehicle by bilateral micro-injection to the paraventricular nucleus (PVN). Blood pressure (BP) and renal sympathetic nerve activity were recorded. PVN expression of TLR4, myeloid differentiation factor 88 (Myd88), nuclear factor-kappa B (NF-κB) p65, proinflammation cytokines (PICs), interleukin (IL)-1ß, IL-6, tumor necrosis factor-alpha (TNF-α), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), NADPH oxidase 4 (NOX4), Cu/Zn superoxide dismutase (SOD) level, tyrosine hydroxylase, and 67 kDa isoform of glutamate decarboxylase (GAD67) were tested to determine the influence of TLR4 blockade. RESULTS: TLR4 expression increased significantly in the PVN of high-salt groups with a corresponding increase in reactive oxygen species (ROS) and PICs. TLR4 blockade significantly reduced the signaling molecules downstream TLR4 and the expression of TNF-α, IL-6, IL-1ß, decreased ROS, NOX2, NOX4 level, increased Cu/Zn-SOD, re-balanced neurotransmitters, and regulated sympathetic nerve activity in the PVN of prehypertensive rats. CONCLUSIONS: Salt-induced prehypertension is partly due to the upregulation of TLR4 in PVN. Blockade of TLR4 in the brain reduced salt-induced prehypertension response, possibly through downregulation of ROS and PICs expression, and the restorage of neurotransmitter balance in the PVN.

Paraventricular Nucleus Infusion of Epigallocatechin-3-O-Gallate Improves Renovascular Hypertension.

Oxidative stress plays an important role in the pathogenesis of hypertension. Epigallocatechin-3-O-gallate (EGCG) is the main polyphenol present in green tea and is known for its potent antioxidant and anti-inflammatory properties. In the present study, we hypothesize that EGCG attenuates oxidative stress in the paraventricular nucleus of hypothalamus (PVN), thereby decreasing the blood pressure and sympathetic activity in renovascular hypertensive rats. After renovascular hypertension was induced in male Sprague-Dawley rats by the two-kidney one-clip (2K-1C) method, the rats received bilateral PVN infusion of EGCG (20 µg/h) or vehicle via osmotic minipump for 4 weeks. Our results were shown as follows: (1) Hypertension induced by 2K-1C was associated with the production of reactive oxygen species in the PVN; (2) chronic infusion of EGCG in the PVN decreased stress-related NAD(P)H oxidase subunit gp91(phox) and NOX-4 and increased the activity of antioxidant enzymes (SOD-1), also balanced the content of cytokines (IL-1ß, IL-6, IL-10 and MCP-1) in the PVN, and attenuated the level of norepinephrine in plasma of 2K-1C rats. Our findings provide strong evidence that PVN infusion of EGCG inhibited renovascular hypertension progression through its potent anti-oxidative and anti-inflammatory activity in the PVN.

Overexpression of CXCL3 can enhance the oncogenic potential of prostate cancer.

PURPOSE: CXCL3 and its receptor CXCR2 were considered to play particularly important roles in the progression of malignancies. However, the investigations about CXCL3/CXCR2 axis in prostate cancer have been poorly involved. Herein we firstly reported our studies on the expression and biological roles of CXCL3 and CXCR2 in prostate cancer. METHODS: Expression levels of CXCL3 and CXCR2 in prostate cancer cell lines (PC-3, DU145 and LNCaP), immortalized prostate stromal cell line (WPMY-1) and immortalized prostate epithelial cell line (RWPE-1) were investigated by RT-PCR, ELISA and western blot, whereas expression levels of CXCL3 in a prostate tissue microarray were detected by immunohistochemistry. Cell counting kit-8 and transwell assays were, respectively, utilized to determine the effects of exogenous CXCL3 on the cell proliferation and migration. We further examined whether CXCL3 could regulate the expression of genes correlated with prostate tumorigenesis by RT- PCR. RESULTS: Elevated expression of CXCR2 was detected in DU145, LNCaP and RWPE-1. Moreover, high-level CXCL3 can be secreted by PC-3 and RWPE-1, and CXCL3 protein expression level in tissue microarray is concordant with prostate cancer metastasis. Exogenous CXCL3 does not contribute to proliferation, but has a significant effect on migration of prostate cancer cells and RWPE-1. Finally, our data showed that exogenous CXCL3 can regulate the expression of genes including ERK, TP73, NUMB, BAX and NDRG3. CONCLUSION: Our findings suggest that CXCL3 and its receptor CXCR2 are overexpressed in prostate cancer cells, prostate epithelial cells and prostate cancer tissues, which may play multiple roles in prostate cancer progression and metastasis.

Construction of an allogenic chimeric mouse model for the study of the behaviors of donor stem cells in vivo.

BACKGROUND: It is essential to establish an animal model for the elucidation of the biological behaviors of stem cells in vivo. We constructed a chimeric animal model by in utero transplantation for investigation of stem cell transplantation. METHODS: This chimerism was achieved by injecting the stem cells derived from the bone marrow of green fluorescence protein (GFP)-transgenic mice into fetal mice at 13.5 days of gestation. Several methods such as polymerase chain reaction (PCR), real-time PCR, fluorescence-assisted cell sorting (FACS) and fluorescence in situ hybridization (FISH) were used for the observation of donor cells. RESULTS: Under a fluorescence microscope, we observed the GFP cells of donor-origin in a recipient. PCR, FACS analysis and FISH indicated chimerism at various intervals. Real-time PCR indicated that some donor cells existed in chimera for more than 6 months. CONCLUSIONS: Allogenic stem cells may exist in recipients for a long time and this allogenic animal model provides a useful tool for studying the behavior of hematopoietic stem cells and also offers an effective model system for the study of stem cells.

A chimeric mouse model established by allogenic in utero transplantation.

In utero stem cells transplantation is a promising approach to the prenatal treatment of diseases. In order to investigate the fate of the stem cells after in utero transplantation, we have established a chimeric mouse model with the method of in utero transplantation. Mononuclear cells (including stem cells/progenitor cells) derived from male mouse bone marrow were injected into fetal mouse peritoneal cavity during the pre-immune period. The donor cells in the circulatory blood of female recipients were identified by fluorescence in situ hybridization (FISH), and the Y-chromosome specific DNA was detected by PCR as well as real-time quantitative PCR after the recipient mice were born. The results showed that a total of 4 female recipient mice were chimeric in their peripheral blood. Significantly, the donor cells in three chimeric mice persisted up to six months.

[Stem cells and prenatal therapy].

The special biological properties of stem cells bring us new hope for the research and application of prenatal therapy. The basic research of prenatal therapy utilizing stem cells developed quickly,providing reference and direction for clinical therapy. This review is mainly related to the progress in this field in recent years.

ER-α variant ER-α36 mediates antiestrogen resistance in ER-positive breast cancer stem/progenitor cells.

Accumulating evidence indicates that cancer stem cells (CSC) play important roles in breast cancer occurrence, recurrence and metastasis as well as resistance to therapy. However, the roles of breast cancer stem cells in antiestrogen resistance and the underlying molecular mechanisms have not been well established. Previously, we identified and cloned a novel variant of estrogen receptor α, ER-α36, with a molecular weight of 36kDa. ER-α36 mediates rapid antiestrogen signaling and is highly expressed in ER-positive breast cancer stem/progenitor cells. In this study, we investigated the function and the underlying mechanism of ER-α36-mediated antiestrogen signaling in ER-positive breast cancer stem/progenitor cells. ER-positive breast cancer cells MCF7 and T47D as well as variants with different levels of ER-α36 expression were used. The effects of antiestrogens tamoxifen and ICI 182, 780 on breast CSC's ability of growth, self-renewal, differentiation and tumor seeding were examined using tumorsphere formation, flow cytometry, indirect immunofluorences and in vivo xenograft assays. The underlying mechanisms were also analyzed with Western blot analysis. We found that the cancer stem/progenitor cells enriched from ER-positive breast cancer cells were more resistant to antiestrogens than the bulk cells. Antiestrogens increased the percentages of the stem/progenitor cells from ER-positive breast cancer cell through stimulation of luminal epithelial lineage specific ER-positive breast cancer progenitor cells while failed to do so in the cells with knocked-down levels of ER-α36 expression. Our results thus indicated that ER-α36-mediated antiestrogen signaling such as the PI3K/AKT plays an important role in antiestrogen resistance of ER-positive breast cancer stem/progenitor cells.

ER-α36-mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells.

The breast cancer stem cells (BCSC) play important roles in breast cancer occurrence, recurrence and metastasis. However, the role of estrogen signaling, a signaling pathway important in development and progression of breast cancer, in regulation of BCSC has not been well established. Previously, we identified and cloned a variant of estrogen receptor α, ER-α36, with a molecular weight of 36 kDa. ER-α36 lacks both transactivation domains AF-1 and AF-2 of the 66 kDa full-length ER-α (ER-α66) and mediates rapid estrogen signaling to promote proliferation of breast cancer cells. In this study, we aim to investigate the function and the underlying mechanism of ER-α36-mediated rapid estrogen signaling in growth regulation of the ER-positive breast cancer stem/progenitor cells. ER-positive breast cancer cells MCF7 and T47D as well as the variants with different levels of ER-α36 expression were used. The effects of estrogen on BCSC's abilities of growth, self-renewal, differentiation and tumor-seeding were examined using tumorsphere formation, flow cytometry, indirect immunofluorence staining and in vivo xenograft assays. The underlying mechanisms were also studied with Western-blot analysis. We found that 17-ß-estradiol (E2ß) treatment increased the population of ER-positive breast cancer stem/progenitor cells while failed to do so in the cells with knocked-down levels of ER-α36 expression. Cells with forced expression of recombinant ER-α36, however, responded strongly to E2ß treatment by increasing growth in vitro and tumor-seeding efficiency in vivo. The rapid estrogen signaling via the AKT/GSK3ß pathway is involved in estrogen-stimulated growth of ER-positive breast cancer stem/progenitor cells. We concluded that ER-α36-mediated rapid estrogen signaling plays an important role in regulation and maintenance of ER-positive breast cancer stem/progenitor cells.