Autoactivation and calpain-1-mediated shedding of hepsin in human hepatoma cells.

Hepsin is a transmembrane serine protease implicated in many biological processes, including hepatocyte growth, urinary protein secretion, auditory nerve development, and cancer metastasis. Zymogen activation is critical for hepsin function. To date, how hepsin is activated and regulated in cells remains an enigma. In this study, we conducted site-directed mutagenesis, cell expression, plasma membrane protein labeling, trypsin digestion, Western blotting, and flow cytometry experiments in human hepatoma HepG2 cells, where hepsin was originally discovered, and SMMC-7721 cells. Our results show that hepsin is activated by autocatalysis on the cell surface but not intracellularly. Moreover, we show that hepsin undergoes ectodomain shedding. In the conditioned medium from HepG2 and SMMC-7721 cells, we detected a soluble fragment comprising nearly the entire extracellular region of hepsin. By testing protease inhibitors, gene knockdown, and site-directed mutagenesis, we identified calpain-1 as a primary protease that acted extracellularly to cleave Tyr52 in the juxtamembrane space of hepsin. These results provide new insights into the biochemical and cellular mechanisms that regulate hepsin expression and activity.

High-Mobility Group Box 1 From Hypoxic Trophoblasts Promotes Endothelial Microparticle Production and Thrombophilia in Preeclampsia.

OBJECTIVE: Thrombophilia is a major complication in preeclampsia, a disease associated with placental hypoxia and trophoblast inflammation. Preeclampsia women are known to have increased circulating microparticles that are procoagulant, but the underlying mechanisms remain unclear. In this study, we sought to understand the mechanism connecting placental hypoxia, circulating microparticles, and thrombophilia. APPROACH AND RESULTS: We analyzed protein markers on plasma microparticles from preeclampsia women and found that the increased circulating microparticles were mostly from endothelial cells. In proteomic studies, we identified HMGB1 (high-mobility group box 1), a proinflammatory protein, as a key factor from hypoxic trophoblasts in stimulating microparticle production in human umbilical vein endothelial cells. Immunodepletion or inhibition of HMGB1 in the conditioned medium from hypoxic human trophoblasts abolished the endothelial microparticle-stimulating activity. Conversely, recombinant HMGB1 stimulated microparticle production in cultured human umbilical vein endothelial cells. The microparticles from recombinant HMGB1-stimulated human umbilical vein endothelial cells promoted blood coagulation and neutrophil activation in vitro. Injection of recombinant HMGB1 in pregnant mice increased plasma endothelial microparticles and promoted blood coagulation. In preeclampsia women, elevated placental HMGB1 expression was detected and high levels of plasma HMGB1 correlated with increased plasma endothelial microparticles. CONCLUSIONS: Our results indicate that placental hypoxia-induced HMGB1 expression and release from trophoblasts are important mechanism underlying increased circulating endothelial microparticles and thrombophilia in preeclampsia.

The interaction of MMP-2/B7-H3 in human osteoporosis.

The immune costimulatory molecule B7-H3 has been shown to be involved in the regulation of murine bone formation. However, the role of B7-H3 in bone metabolic diseases remains unknown. In our study, matrix metalloproteinase 2 (MMP-2) and soluble B7-H3 (sB7-H3) were found to be correlatively up-regulated in the sera of osteoporosis patients. Furthermore, our results showed that MG63 cells treated with MMP-2 inhibitors produced lower amounts of sB7-H3 while cells with recombinant MMP-2 had an increased membrane B7-H3 (mB7-H3) shedding. Therefore, elevated MMP-2 levels resulted in an elevation of serum sB7-H3 and reduction of osteoblastic mB7-H3. B7-H3 knockdown in MG63 cells significantly decreased osteoblastic markers and substantially decreased the number of mineralized nodules after 21days. Thus, B7-H3-deficient MG63 cells exhibited impaired bone formation. These results suggest that mB7-H3 is required for the later phases of osteoblast differentiation and that MMP-2/B7-H3 plays a negative regulatory role in osteoporosis.

Oxymatrine inhibits lipopolysaccharide-induced inflammation by down-regulating Toll-like receptor 4/nuclear factor-kappa B in macrophages.

Oxymatrine (OMT) is the quinolizidine alkaloid extracted from the Chinese herb Sophora flavescens Ait. that has many pharmacological effects and is used for the treatment of some inflammatory diseases. In this study, RAW264.7 cells and THP-1 differentiated macrophages were pretreated with various concentrations of OMT at 2 h prior to treatment with lipopolysaccharide (LPS) (1.0 µg/mL) for different durations. We detected the anti-inflammatory effect of OMT in LPS-stimulated macrophages and investigated the molecular mechanism. We showed that OMT pretreatment significantly inhibited the LPS-induced secretion of nitric oxide (NO), interleukin-1 beta (IL-1ß), and tumor necrosis factor-alpha (TNF-α) in supernatant, attenuated the mRNA levels of inducible nitric oxide synthase (iNOS), IL-1ß, TNF-α, and Toll-like receptor 4 (TLR4), increased TLR4 and phosphorylation of inhibitor of kappa B-alpha (p-IBα) in cytosol, and decreased the nuclear level of nuclear factor-κB (NF-κB) p65 in macrophages. In conclusion, OMT exerts anti-inflammatory properties in LPS-stimulated macrophages by down-regulating the TLR4/NF-κB pathway.

The role of Toll-like receptor 4 on inflammation and Aß formation in cortex astrocytes.

To investigate the role and possible molecular mechanism of astrocytes in inflammation and amyloid ß-protein (Aß) formation, in this research, by using LPS to stimulate cultured rat astrocytes in vitro with or without anti-Toll-like receptor 4 (TLR4) antibody pretreatment, we first detected the TLR4, TNF-α, IL-1ß, ß-amyloid precursor protein (ß-APP) and ß-site APP clearing enzyme 1 (BACE1) mRNA with real-time PCR, and TLR4, NF-κB/P65 protein in cultured astrocytes by Western blot, and then further probed the translocation of NF-κB/P65 using immunofluorescence and the contents of TNF-α, IL-1ß and Aß in culture supernatant through ELISA. We found that all of these indexes increased at different degrees after LPS-stimulation. However, if pretreatment with anti- TLR4 antibody, such stimulating effects of LPS on the nuclear translocation of NF-κB/P65 and TNF-α, IL-1ß, Aß contents in astrocytic culture supernatant were reduced significantly or disappeared in comparison with the group with only LPS-administration. Our results suggest that TLR4 in astrocytes might play an important role in the inflammation and Aß formation through the TLR4/NF-κB signaling pathway, thus providing new knowledge and understanding of the inflammatory hypothesis of AD pathogenesis.

Apolipoprotein A-I Binding Protein Inhibits the Formation of Infantile Hemangioma through Cholesterol-Regulated Hypoxia-Inducible Factor 1α Activation.

Infantile hemangioma (IH) is the most frequent vascular tumor of infancy with unclear pathogenesis; disordered angiogenesis is considered to be involved in its formation. Apolipoprotein A-I binding protein (AIBP)-also known as NAXE (NAD [P]HX epimerase)-a regulator of cholesterol metabolism, plays a critical role in the pathological angiogenesis of mammals. In this study, we found that AIBP had much lower expression levels in both tissues from patients with IH and hemangioma endothelial cells (HemECs) than in adjacent normal tissues and human dermal vascular endothelial cells, respectively. Knockout of NAXE by CRISPR-Cas9 in HemECs enhanced tube formation and migration, and NAXE overexpression impaired tube formation and migration of HemECs. Interestingly, AIBP suppressed the proliferation of HemECs in hypoxia. We then found that reduced expression of AIBP correlated with increased hypoxia-inducible factor 1α levels in tissues from patients with IH and HemECs. Further mechanistic investigation demonstrated that AIBP disrupted hypoxia-inducible factor 1α signaling through cholesterol metabolism under hypoxia. Notably, AIBP significantly inhibited the development of IH in immunodeficient mice. Furthermore, using the validated mouse endothelial cell (ie, EOMA cells) and Naxe-/- mouse models, we demonstrated that both endogenous AIBP from tumors and AIBP in the tumor microenvironment limit the formation of hemangioma. These findings suggested that AIBP was a player in the pathogenesis of IH and could be a potential pharmacological target for treating IH.

Thiamme2-G, a Novel O-GlcNAcase Inhibitor, Reduces Tau Hyperphosphorylation and Rescues Cognitive Impairment in Mice.

BACKGROUND: Abnormal hyperphosphorylation of microtubule-associated protein tau plays a pivotal role in Alzheimer's disease (AD). We previously found that O-GlcNAcylation inversely correlates to hyperphosphorylation of tau in AD brain, and downregulation of brain O-GlcNAcylation promotes tau hyperphosphorylation and AD-like neurodegeneration in mice. OBJECTIVE: Herein we investigated the effect of increasing O-GlcNAcylation by using intermittent dosing with low doses of a potent novel O-GlcNAcase (OGA) inhibitor on AD-like brain changes and cognitive function in a mouse model of sporadic AD (sAD) induced by intracerebroventricular (ICV) injection of streptozotocin (STZ). METHODS: STZ was injected into the lateral ventricle of C57BL/6J mice. From the second day, Thiamme2-G (TM2G) or saline, as a vehicle control, was orally administered to the ICV-STZ mice three times per week for five weeks. A separate group of ICV-saline mice treated with saline was used as a baseline control. Behavioral tests, including open field and novel object recognition, were conducted three weeks after the first dose of the TM2G or saline. Protein O-GlcNAcylation, tau hyperphosphorylation, synaptic proteins, and neuroinflammation in the mouse brain were assessed by western blotting. RESULTS: ICV-STZ caused decreased protein O-GlcNAcylation. Enhancement of O-GlcNAcylation to moderate levels by using low-dose OGA inhibitor in ICV-STZ mice prevented STZ-induced body weight loss, rescued cognitive impairments, and restored AD-like pathologies, including hyperphosphorylation of tau and abnormalities in synaptic proteins and neuroinflammation. CONCLUSION: These findings suggest that moderately increasing protein O-GlcNAcylation by using low doses of OGA inhibitor may be a suitable therapeutic strategy for sAD.

The CXCL12-CXCR4 Signaling Axis Plays a Key Role in Cancer Metastasis and is a Potential Target for Developing Novel Therapeutics against Metastatic Cancer.

Metastasis is the main cause of death in cancer patients; there is currently no effective treatment for cancer metastasis. This is primarily due to our insufficient understanding of the metastatic mechanisms in cancer. An increasing number of studies have shown that the C-X-C motif chemokine Ligand 12 (CXCL12) is overexpressed in various tissues and organs. It is a key niche factor that nurtures the pre-metastatic niches (tumorigenic soil) and recruits tumor cells (oncogenic "seeds") to these niches, thereby fostering cancer cell aggression and metastatic capabilities. However, the C-X-C motif chemokine Receptor 4 (CXCR4) is aberrantly overexpressed in various cancer stem/progenitor cells and functions as a CXCL12 receptor. CXCL12 activates CXCR4 as well as multiple downstream multiple tumorigenic signaling pathways, promoting the expression of various oncogenes. Activation of the CXCL12-CXCR4 signaling axis promotes Epithelial-Mesenchymal Transition (EMT) and mobilization of cancer stem/progenitor cells to pre-metastatic niches. It also nurtures cancer cells with high motility, invasion, and dissemination phenotypes, thereby escalating multiple proximal or distal cancer metastasis; this results in poor patient prognosis. Based on this evidence, recent studies have explored either CXCL12- or CXCR4-targeted anti-cancer therapeutics and have achieved promising results in the preclinical trials. Further exploration of this new strategy and its potent therapeutics effect against metastatic cancer through the targeting of the CXCL12- CXCR4 signaling axis may lead to a novel therapy that can clean up the tumor microenvironment ("soil") and kill the cancer cells, particularly the cancer stem/progenitor cells ("seeds"), in cancer patients. Ultimately, this approach has the potential to effectively treat metastatic cancer.

Author Correction: The Transmembrane Serine Protease HAT-like 4 Is Important for Epidermal Barrier Function to Prevent Body Fluid Loss.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ectopic expression of human airway trypsin-like protease 4 in acute myeloid leukemia promotes cancer cell invasion and tumor growth.

Transmembrane serine proteases have been implicated in the development and progression of solid and hematological cancers. Human airway trypsin-like protease 4 (HAT-L4) is a transmembrane serine protease expressed in epithelial cells and exocrine glands. In the skin, HAT-L4 is important for normal epidermal barrier function. Here, we report an unexpected finding of ectopic HAT-L4 expression in neutrophils and monocytes from acute myeloid leukemia (AML) patients. Such expression was not detected in bone marrow cells from normal individuals or patients with chronic myeloid leukemia, acute lymphocytic leukemia and chronic lymphocytic leukemia. In AML patients who underwent chemotherapy, persistent HAT-L4 expression in bone marrow cells was associated with minimal residual disease and poor prognostic outcomes. In culture, silencing HAT-L4 expression in AML-derived THP-1 cells by short hairpin RNAs inhibited matrix metalloproteinase-2 activation and Matrigel invasion. In mouse xenograft models, inhibition of HAT-L4 expression reduced the proliferation and growth of THP-1 cell-derived tumors. Our results indicate that ectopic HAT-L4 expression is a pathological mechanism in AML and that HAT-L4 may be used as a cell surface marker for AML blast detection and targeting.

Increased Neutrophil Activation and Plasma DNA Levels in Patients with Pre-Eclampsia.

Pre-eclampsia (PE) is a chronic inflammatory disease in pregnancy, which is associated with enhanced blood coagulation and high thrombotic risk. To date, the mechanisms underlying such an association are not fully understood. Previous studies reported high levels of plasma deoxyribonucleic acid (DNA) in PE women, but the cellular source of the circulation DNA remains unknown. In this study, we tested the hypothesis that activated neutrophils undergoing cell death, also called NETosis, may be responsible for the elevated plasma DNA levels in PE women. We analysed plasma samples from non-pregnant, normal pregnant and PE women and found high levels of double-stranded DNA, myeloperoxidase (an abundant neutrophil granular enzyme) and histones (the major nucleosome proteins) in PE-derived samples, indicating increased NETosis in the maternal circulation. The high plasma DNA levels positively correlated with enhanced blood coagulation in PE women. When isolated neutrophils from normal individuals were incubated with PE-derived plasma, an elevated NETosis-stimulating activity was detected. Further experiments showed that endothelial micro-particles, but not soluble proteins, in the plasma were primarily responsible for the NETosis-stimulating activity in PE women. These results indicate that circulating micro-particles from damaged maternal endothelium are a potent stimulator for neutrophil activation and NETosis in PE women. Given the pro-coagulant and pro-thrombotic nature of granular and nuclear contents from neutrophils, enhanced systemic NETosis may represent an important mechanism underlying the hyper-coagulability and increased thrombotic risk in PE.

[Inhibitory effects of anisodamine and pentoxifylline on the expression of lipopolysaccharide -induced intercellular adhesion molecule-1 in rat cardiac muscle].

OBJECTIVE: To investigate the inhibitory effects of anisodamine (654-2) and pentoxifylline (PTX) on the expression of lipopolysaccharide (LPS)-induced intercellular adhesion molecule-1 (ICAM-1) in rat cardiac muscle in vivo. METHODS: The animals were randomly divided into five groups (each n=6): (1) normal control group, (2) model group, (3) 654-2 treated group, (4) PTX treated group and (5) 654-2+PTX treated group. The endotoxemia model was reproduced by intravenous injection LPS 5 mg/kg. The expression of ICAM-1 protein in rat cardiac muscle was assayed by Western blotting at 0, 2, 4, 6, 8, 10 hours after intravenous LPS injection. Then the expression of ICAM-1 protein in different groups was assayed at different time points. RESULTS: The changes in expression of ICAM-1 in rat cardiac muscle after LPS injection were in a time-dependent pattern, gradually elevating to approach the peak at 6 th, then it lowered, but it still appeared at 10 hours (P<0.05). Western blotting also showed that ICAM-1 protein with decreased with pre-treatment of 654-2 or PTX respectively (both P<0.01). It was reduced to a much lower level when the animals were pretreated with a combination of 654-2 and PTX, compared with the group of 654-2 alone or PTX alone (both P<0.05). CONCLUSION: The combination of 654-2 and PTX may play a protective role in rat against injury to cardiac muscle induced by LPS in vivo via inhibiting the production of ICAM-1 protein.

The Transmembrane Serine Protease HAT-like 4 Is Important for Epidermal Barrier Function to Prevent Body Fluid Loss.

Membrane-bound proteases are essential for epidermal integrity. Human airway trypsin-like protease 4 (HAT-L4) is a type II transmembrane serine protease. Currently, its biochemical property, cellular distribution and physiological function remain unknown. Here we examined HAT-L4 expression and function in vitro and in vivo. In Western analysis, HAT-L4 expressed in transfected CHO cells appeared as a 48-kDa protein. Flow cytometry confirmed HAT-L4 expression on the cell surface with the expected membrane topology. RT-PCR and immunostaining experiments indicated that HAT-L4 was expressed in epithelial cells and exocrine glands in tissues including skin, esophagus, trachea, tongue, eye, bladder, testis and uterus. In the skin, HAT-L4 expression was abundant in keratinocytes and sebaceous glands. We generated HAT-L4 knockout mice by disrupting the Tmprss11f gene encoding HAT-L4. HAT-L4 knockout mice were viable and fertile. No defects were found in HAT-L4 knockout mice in hair growth, wound healing, water repulsion and body temperature regulation. Compared with wild-type controls, HAT-L4-deficient newborn mice had greater body fluid loss and higher mortality in a trans-epidermal body fluid loss test. In metabolic studies, HAT-L4-deficient adult mice drank water more frequently than wild-type controls did. These results indicate that HAT-L4 is important in epidermal barrier function to prevent body fluid loss.

[Expression of tumor suppressor PTEN in hypertrophy cardiomyocyte].

OBJECTIVE: To investigate the role of tumor suppressor PTEN in cardiac hypertrophy, the expression of PTEN mRNA and protein was analyzed in the tissue of left ventricle in abdominal aorta constricted-induced cardiac hypertrophic rats which treated with and without captopril. The expression of PTEN mRNA and protein in cultured neonatal rat cardiomyocyte treated with AngII was studied. METHODS: SD rats were divided into control group, hypertrophy group and captopril group. The expression of PTEN in different groups at 2 and 4 weeks after operation as well as in cultured neonatal rat cardiomyocyte treated with AngII was detected by RT-PCR and Western blot. The localization of PTEN in left ventricle and cultured cardiomyocyte was determined by immunohistochemistry. RESULTS: (1) Compared with control group, the expressions of PTEN mRNA and protein in left ventricle of hypertrophy group as well as in cultured cardiomyocyte treated with AngII were reduced. (2) Compared with hypertrophy group, the expressions of PTEN mRNA and protein in left ventricle of captopril group were upregulated, which were similar to those of control group. (3) Positive immunohistochemical staining of PTEN was located in the nucleus of cardiomyocytes. CONCLUSION: PTEN may play a negative regulation role in the process of cardiac hypertrophy, and the role of PTEN may be closely related with renin-angiotensin system.

Reduced serum B7-H3 levels in patients with ankylosing spondylitis.

This study aimed to investigate molecule B7-H3 expression profiles of patients with ankylosing spondylitis (AS) and the clinical significance of B7-H3 in the pathogenesis of AS. Serum B7-H3 levels were measured by ELISA in patients with AS and healthy controls. The expression of B7-H3 protein and mRNA on CD14+ monocytes of peripheral blood mononuclear cells (PBMCs) and serum levels of T cell-associated cytokines were also analyzed. The serum B7-H3 levels in AS patients were significantly lower than in healthy controls. The expression of B7-H3 protein and mRNA on CD14+ monocytes of PBMCs and serum levels of TNF-α, IL-6, and IL-17A in AS patients were significantly higher than in controls. The reduced serum B7-H3 level was highly negatively correlated with AS Disease Activity Score (ASDAS), TNF-α, and IL-17A. Upregulated B7-H3 protein may play a role in the pathogenesis of AS by binding its receptor on T cells.

[Effect of captopril on expression of PTEN in aorta of aortic-induced hypertensive rats].

This study inquired about the role of tumor suppressor PTEN in the arterial remodeling of Ang II induced hypertension. The expression of PTEN of aorta was examined in the aortic-constricted hypertensive rats (hypertension group), in the aortic-constricted hypertensive rats treated with captopril(hypertension and captopril group), and in the rats having undergone sham operation (control group). At day 28 after surgery, the aortas were collected from the groups. The expression of PTEN mRNA was detected by RT-PCR. The expression and location of PTEN protein were determined by immunohistochemistry. The results showed that the expression of PTEN in aorta of the hypertension group was significantly lower than that of the hypertension and captopril group, and similarly lower than that of the control group. The intensity of PTEN-positive immunohistochemical production in aorta of the hypertension group was weaker than that of the hypertension and captopril group, and likewise, it was weaker than the control. PTEN-positive immunohistochemical production was located in VSMC of aorta. The findings indicated that the expression of PTEN is reduced in hypertensive aorta, that the reduced PTEN experession can be reversed by captopril treatment, that AngII and the increased mechanical strain may participate in regulating expression of PTEN, and that PTEN may play a role in the arterial remodeling induced by hypertension.

Role of Toll-like receptor 4 in inflammatory reactions of hippocampal neurons.

Lipopolysaccharide stimulates Toll-like receptor 4 on immune cells to produce immune mediators. Toll-like receptor 4 is also expressed by non-immune cells, which can be stimulated by lipopolysaccharide. However, whether Toll-like receptor 4 is expressed by primary cultured hippocampal neurons and its specific role in lipopolysaccharide-induced neuroinflammation is currently undefined. In this study, Toll-like receptor 4 antibody blocking was used to analyze the Toll-like receptor 4 signaling pathway and changes in inflammation of lipopolysaccharide stimulated hippocampal neurons. Immunofluorescence showed that Toll-like receptor 4 protein was mainly located in the membrane of hippocampal neurons. Quantitative reverse transcription-PCR and western blot assay showed that after stimulation of lipopolysaccharide, the mRNA and protein levels of Toll-like receptor 4 and the mRNA levels of interleukin-1ß and tumor necrosis factor-α were significantly increased. In addition, there was increased phosphorylation and degradation of kappa B α inhibitor in the cytosol and increased nuclear factor-κB p65 expression in the nuclei. Pretreatment with Toll-like receptor 4 antibody could almost completely block this increase. These experimental findings indicate that lipopolysaccharide participates in neuroinflammation by stimulating Toll-like receptor 4/nuclear factor-κB pathway in hippocampal neurons, which may be both "passive victims" and "activators" of neuroinflammation.

[The role of TLR4-mediated MyD88-dependent pathway in neuroinflammation in hippocampal neurons of rats].

OBJECTIVE: To investigate weather there is a toll-like receptor 4 (TLR4)-mediated myeloid differentiation factor 88 (MyD88)-dependent pathway in hippocampal neurons of rats and the probable role of the pathway in neuroinflammation. METHODS: To establish the proper model, primarily cultured hippocampal neurons were treated with lipopolysaccharides (LPS), or pretreated with TLR4 antibody then co-treated with LPS. The expression of mRNA of MyD88 and TNF-alpha receptor associated factor 6 (TRAF6) were tested by RT-qPCR. The content of MyD88 and TRAF6 were tested by Western blot. The nuclear translocation of nuclear factor-kappaB/P65 (NF-kappaB/p65) was tested by immunofluorescence. The content of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and nitric oxide (NO) were tested by ELISA. RESULTS: LPS could increase MyD88 and TRAF6 mRNA, upregulate protein level of MyD88 and TRAF6 and increase the level of TNF-alpha, IL-1beta and NO in cell culture supernatant. LPS also could promote NF-kappa B/p65 translation to the nucleus. The pretreatment with TLR4 antibody reduced the translocation to nucleus for NF-kappaB/P65 and the contents of TNF-alpha, IL-1beta and NO in the culture supernatant. CONCLUSION: There is a TLR4-mediated MyD88-dependent pathway in hippocampal neurons. The activation of this pathway can increase the level of TNF-alpha, IL-1beta and NO in cell culture supernatant. TLR4-mediated MyD88-dependent pathway in hippocampal neurons participate in neuroinflammation, that means neurons are not passive in inflammation.

Oxymatrine reduces neuroinflammation in rat brain: A signaling pathway.

Cerebral neuroinflammation models were established by injecting 10 µg lipopolysaccharide into the hippocampus of male Sprague-Dawley rats. The rats were treated with an intraperitoneal injection of 120, 90, or 60 mg/kg oxymatrine daily for three days prior to the lipopolysaccharide injection. Twenty-four hours after model induction, the hippocampus was analyzed by real-time quantitative PCR, and the cerebral cortex was analyzed by enzyme-linked immunosorbent assay and western blot assay. The results of the enzyme-linked immunosorbent assay and the real-time quantitative PCR showed that the secretion and mRNA expression of the pro-inflammatory cytokines interleukin-1ß and tumor necrosis factor-α were significantly decreased in the hippocampus and cerebral cortex of model rats treated with oxymatrine. Western blot assay and real-time quantitative PCR analysis indicated that toll-like receptor 4 mRNA and protein expression were significantly decreased in the groups receiving different doses of oxymatrine. Additionally, 120 and 90 mg/kg oxymatrine were shown to reduce protein levels of nuclear factor-κB p65 in the nucleus and of phosphorylated IκBα in the cytoplasm of brain cells, as detected by western blot assay. Experimental findings indicate that oxymatrine may inhibit neuroinflammation in rat brain via downregulating the expression of molecules in the toll-like receptor 4/nuclear factor-κB signaling pathway.

[The effect of meloxicam on the inflammatory reaction induced by beta amyloid protein in Alzheimer's disease rats].

OBJECTIVE: To investigate the effect and mechanism of meloxicam on the inflammatory reaction induced by beta amyloid protein (AB) in Alzheimer's disease (AD) rats. METHODS: The rat model was established by microinjection of Abeta(1-40) into hippocampus. The expression of NF-kappaB p65 and glial fibrillary acidic protein (GFAP) in hippocampus were detected by immunohistochemistry. The content of GFAP in cortex was tested by Western-blot. The content of TNF-alpha in cortex was tested by ELISA. The expression of IL-1beta mRNA was tested by RT-PCR. RESULTS: The expression of NF-kappaB p65, GFAP and TNF-alpha as well as IL-1beta mRNA were decreased by meloxicam. CONCLUSION: Meloxicam can reduce the proliferation of astrocyte by decreasing the expression of GFAP in AD model rat's hippocampus and cortex. And the depression of NF-kappaB p65 may significantly decrease the expression of TNF-alpha1 and IL-1beta to lessen the inflammatory reaction in cerebral tissue.