The role of neuroplastin65 in macrophage against E. coli infection in mice.

BACKGROUND: Innate immune response constitutes the first line of defense against pathogens. Inflammatory responses involve close contact between different populations of cells. These adhesive interactions mediate migration of cells to sites of infection leading the effective action of cells within the lesions. Cell adhesion molecules are critical to controlling immune response mediating cell adhesion or chemotaxis, as well as coordinating actin-based cell motility during phagocytosis and chemotaxis. Recently, a newly discovered neuroplastin (Np) adhesion molecule is found to play an important role in the nervous system. However, there is limited information on Np functions in immune response. To understand how Np is involved in innate immune response, a mouse model of intraperitoneal infection was established to investigate the effect of Np on macrophage-mediated clearance of E. coli infection and its possible molecular mechanisms. METHODS: Specific deficiency mice with Nptn gene controlling Np65 isoform were employed in this study. The expression levels of mRNA and proteins were detected by qPCR and western blot, or evaluated by flow cytometry. The expression level of NO and ROS were measured with their specific indicators. Cell cycle and apoptosis were detected by specific detection kits. Acid phosphatase activity was measured by flow cytometry after labelling with LysoRed fluorescent probe. Bone marrow derived macrophages (BMDMs) were isolated from bone marrow of mice hind legs. Cell proliferation was detected by CCK8 assay. Cell migration was measured by wound healing assay or transwell assay. RESULTS: The lethal dose of E. coli infection in Np65-/- mice dropped to the half of lethal dose in WT mice. The bacterial load in the spleen, kidney and liver from Np65-/- mice were significantly higher than that from WT mice, which were due to the dramatic reduction of NO and ROS production in phagocytes from Np65-/- mice. Np65 gene deficiency remarkably impaired phagocytosis and function of lysosome in macrophage. Furthermore, Np65 molecule was involved in maturation and proliferation, even in migration and chemotaxis of BMDM in vitro. CONCLUSION: This study for the first time demonstrates that Np is involved in multi-function of phagocytes during bacterial infection, proposing that Np adhesion molecule plays a critical role in clearing pathogen infection in innate immunity.

The Dynamic Interactions between Nanoparticles and Macrophages Impact Their Fate in Brain Tumors.

Functional nanomaterials such as iron oxide nanoparticles have been extensively explored for the diagnosis and treatment of central nervous system diseases. However, an insufficient understanding of the comprehensive nanomaterial-biological interactions in the brain hinders the nanomaterials from meeting the medical requirements for translational research. Here, FDA-approved ferumoxytol, an iron oxide nanoparticle, is chosen as the model nanomaterial for a systematic study of the dynamic interactions between ferumoxytol and immune cells, including microglia and macrophages, in the brain tumors. Strikingly, up to 90% of intratumorally injected ferumoxytol nanoparticles are recognized and phagocytized by tumor-associated microglia and macrophages. The dynamic trafficking progress of ferumoxytol in microglia and macrophages, including scavenger receptor-mediated endocytosis, lysosomal internalization, and extracellular vesicle-dominated excretion, is further studied. Importantly, the results demonstrate that extracellular vesicle-encapsulated nanoparticles could be gradually eliminated from the brain along with cerebrospinal fluid circulation over 21 days. Moreover, ferumoxytol exhibits no obvious long-term neurological toxicity after its injection. The study suggests that the dynamic biointeractions of nanoparticles with immune cells in the brain exert a key rate-limiting impact on the efficiency of targeting tumor cells and their in vivo fate and thus provide a deeper understanding of the nanomaterials in the brain for clinical applications.

Metal-Phenolic Network Covering on Zein Nanoparticles as a Regulator on the Oil/Water Interface.

Interfacial self-assembly has become a powerful force for regulating the amphipathy of Pickering emulsions on the oil/water interface. Herein, metal-phenolic supramolecular coatings, acting as a regulator on the oil/water interface, were fabricated on the surface of zein nanoparticles (NPs), as a consequence of which the prepared Pickering emulsions stabilized by the decorated zein NPs exhibited diverse properties, decided by different concentrations of zein, tannic acid (TA), and metal ions (Fe3+). Metal-phenolic network-decorated zein NPs named ZTFex NPs (ZTFe NPs represented zein/TA/Fe3+ NPs, and x represented different concentrations of compounds) exhibited increasing diameters of 100-110 nm. Three-phase contact angles also showed that the strong hydrophobicity of zein NPs could be decreased as a result of the formation of metal-phenolic networks. As for corresponding Pickering emulsions, the covering of TA-Fe3+ networks on zein NPs could enhance the stability of zein NP-based emulsion obviously, which might be due to the fact that ZTFex NPs revealed the ability to form strong films on the oil/water interfaces. ZTFe4 was selected as an optimal concentration because of its minimum size and excellent storage stability. Besides, it was also found that the diameter of ZTFe4-based emulsion enhanced with the increase in the oil phase. The rheological measurement results showed that both G' and G″ increased with the increase of x and the oil phase. In general, our paper not only highlighted a straightforward method for the interfacial nanofabrication of solid particles but also provided a novel and potential strategy in Pickering emulsion applications.

Early secreted antigenic target of 6-kDa of Mycobacterium tuberculosis induces transition of macrophages into epithelioid macrophages by downregulating iNOS / NO-mediated H3K27 trimethylation in macrophages.

BACKGROUND: Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). Granuloma is a pathological feature of tuberculosis and is a tight immune cell aggregation caused by Mtb. The main constituent cells are macrophages and their derivative cells including epithelioid macrophages. However, the molecular mechanism of the transition has not been reported. The purpose of this study was to investigate whether early secreted antigenic target of 6-kDa (ESAT6) can induce the transition of bone marrow-derived macrophages (BMDMs) into epithelioid macrophages and its possible molecular mechanism. METHODS: The recombinant ESAT6 protein was obtained from E.coli carrying esat6 gene after isopropyl ß-d-thiogalactopyranoside (IPTG) induction. BMDMs were isolated from bone marrow of mice hind legs. Cells viability was detected by Cell Counting Kit 8 (CCK8) assays. The expression levels of mRNA and proteins were detected by qPCR and Western blot, or evaluated by flow cytometry. The expression level of nitric oxide (NO) was measured with a nitric oxide indicator. RESULTS: ESAT6 could significantly induce mRNA and protein expression levels of a group of epithelioid macrophages marker molecules (EMMMs), including E-cadherin, junction plakoglobin, ZO1, desmoplakin, desmoglein3 and catenin porteins, in BMDMs. These events could be abrogated in macrophage from TLR2 deficiency mice. ESAT6 could also markedly induce iNOS/NO production that could significantly inhibit trimethylation of H3K27 in the cells. ESAT6-induced expressions of epithelioid macrophages marker molecules were significantly inhibited in the presence of H3K27 histone demethylase inhibitor GSK J1. Furthermore, ROS scavenging agent N,N'-Dimethylthiourea (DMTU) could markedly inhibit the transition induced by ESAT6 in macrophages. CONCLUSION: This study demonstrates that ESAT6 bound with TLR2 can activate iNOS/NO and ROS signalings to reduce the trimethylation of H3K27 resulting in the increment of EMMMs expression that is beneficial to the transition of macrophages into epithelioid macrophages. However, hypoxia can inhibit this transition event. This study has provided new evidence of pathogenesis of granuloma caused by Mtb and also proposed new ideas for the treatment of TB.

UPLC-Q-TOF/MS Based Metabolomics Approach to Study the Hepatotoxicity of Cantharidin on Mice.

Cantharidin is the major bioactive compound extracted from the blister beetle, a traditional Chinese medicine, and has been proved to be a natural component with widely antitumor activity. However, clinical application of cantharidin is relatively restricted due to its potential toxic effects, especially hepatotoxicity. Although cantharidin-induced liver injury has been reported, the underlying molecular mechanisms remain unclear. In the present study, an UPLC-Q-TOF/MS based metabolomics approach combined with blood biochemical analysis, histopathological examination, and cell apoptosis assay were used to investigate the mechanisms of cantharidin-induced hepatotoxicity. A total of 54 significantly changed metabolites and 14 disturbed metabolic pathways were identified in the cantharidin exposed groups. Among them, four metabolites (oxidized glutathione, glutathione, 3-sulfinoalanine, and deoxycholic acid 3-glucuronide) were selected based on their high impact value and potential biological function in the process of liver injury post cantharidin treatment. Our study provides a deeper understanding of the mechanisms of cantharidin-induced hepatotoxicity and may contribute to reduce the liver injury and gain more effective and safe clinical use of cantharidin. In addition, our results also demonstrated that cantharidin could impair multiple biological processes in liver, and future studies will be necessary to reveal the detailed molecular mechanisms of cantharidin-induced hepatotoxicity.

Early secreted antigenic target of 6-kDa of Mycobacterium tuberculosis promotes caspase-9/caspase-3-mediated apoptosis in macrophages.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that triggers several survival mechanisms against the host immune system. Many studies show that the diverse components of Mtb can modulate apoptosis in various types of cells differently. So far, apoptosis induced by ESAT-6, an early secreted antigenic target of 6-kDa of Mtb, has been studied but the details of molecular mechanism and signaling pathway remain incompletely defined. This study investigated the role of recombinant ESAT-6 in inducing apoptosis in primary bone marrow-derived macrophages (BMDMs) of mice using Annexin V/PI assay with FACS analysis and Western blotting technique. It has been found that ESAT-6-induced apoptosis in BMDMs in a dose- and time-dependent pattern. Apoptosis induced by ESAT-6 was mainly via the intrinsic pathway with elevated protein levels of cleaved caspase-9 and -3. Furthermore, ESAT-6 also induced Bim activation during this process. Interestingly, this event was TLR2-dependent since the effect of ESAT-6 on apoptosis vanished in BMDM from mice with TLR2 deficiency. Furthermore, ROS generation and MAPKs phosphorylation induced by ESAT-6 were also involved in caspase-9 and caspase-3 activation. Taken together, these data suggest that ESAT-6-mediated apoptosis is involved in ROS-MAPKs signaling and further activating the intrinsic pathway, which provides new insights into the basic physiology of macrophage death in tuberculosis.

Downregulation of B-myb promotes senescence via the ROS-mediated p53/p21 pathway, in vascular endothelial cells.

OBJECTIVES: To reveal whether B-myb is involved in preventing senescence of vascular endothelial cells, and if so, to identify possible mechanisms for it. MATERIALS AND METHODS: C57/BL6 male mice and primary human aortic endothelial cells (HAECs) were used. Bleomycin was applied to induce stress-related premature senescence. B-myb knockdown was achieved using an siRNA technique and cell senescence was assessed using the senescence-associated ß-galactosidase (SA-ß-gal) assay. Intracellular reactive oxygen species (ROS) production was analysed using an ROS assay kit and cell proliferation was evaluated using KFluor488 EdU kit. Capillary tube network formation was determined by Matrigel assay. Expressions of mRNA and protein levels were detected by real-time PCR and western blotting. RESULTS: B-myb expression significantly decreased, while p53 and p21 expressions increased in the aortas of aged mice. This expression pattern was also found in replicative senescent HAECs and senescent HAECs induced by bleomycin. B-myb knockdown resulted in upregulation of p22phox , ROS accumulation and cell senescence of HAECs. Downregulation of B-myb significantly inhibited cell proliferation and capillary tube network formation and activated the p53/p21 signalling pathway. Blocking ROS production or inhibiting p53 activation remarkably attenuated SA-ß-gal activity and delayed cell senescence induced by B-myb-silencing. CONCLUSION: Downregulation of B-myb induced senescence by upregulation of p22phox and activation of the ROS/p53/p21 pathway, in our vascular endothelial cells, suggesting that B-myb may be a novel candidate for regulating cell senescence to protect against endothelial senescence-related cardiovascular diseases.

SOCS3 and its role in associated diseases.

Cell-cell communication depends on cytokine and growth factor network. Bound to their receptors on the surface of target cell, these glycoproteins initiate a range of intracellular events. Subsequent dissipation of receptor signaling is essential to ensure the response of the cell does not become pathogenic. The Suppressors of cytokine signaling (SOCS) proteins are a family of proteins induced to attenuate cytokine signal transduction in response to signals from a diverse range of cytokines and growth factors. Current evidence indicates that intracellular JAK-STAT (Janus kinase-signal transducer and activator of transcription) signaling not only governs cytokine-induced immunological responses but also rapidly initiates SOCS expression and its biological functions. This review focuses on current understanding of SOCS3, a member of SOCS family. SOCS3 binds to JAK, certain cytokine receptors in intracellular domain, and some signaling molecules, which results in suppressing further signaling events in the cell. Studies using conditional knockout mice have shown that SOCS3 protein is the key physiological regulator and plays an important pathological role in immune homeostasis. Dysregulation of SOCS3 functions can cause a variety of diseases, including allergy, autoimmune diseases, inflammation and cancer.

The involvement of NADPH oxidase-mediated ROS in cytokine secretion from macrophages induced by Mycobacterium tuberculosis ESAT-6.

The 6-kDa early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis is strongly correlated with subversion of innate immune responses against invading mycobacteria. To understand the role of ESAT-6 in macrophage response against M. tuberculosis, the effects of ESAT-6 on macrophage generation of reactive oxygen species (ROS) and production of cytokines were studied. ESAT-6-induced macrophage secretion of monocyte chemoattractant protein-1 and TNF-α was found in a time- and dose-dependent manner. Signaling inhibition experiments indicate that NF-κB activation mediated by p38/JNK mitogen-activated protein kinase (MAPK) was involved in ESAT-6-triggered cytokine production. Moreover, TLR2 was engaged in ESAT-6-stimulated macrophage activation via rapidly induced ROS production and regulated activation of JNK/p38 MAPKs and NF-κB. More importantly, NADPH oxidase-mediated ROS generation is required during this process. Our study has identified a novel signal transduction pathway involving NADPH-ROS-JNK/p38-NF-κB in ESAT-6-induced cytokine production from macrophages. These findings provide an important evidence to understand the pathogenesis of M. tuberculosis infection in the modulation of the immune response.

OxLDL-induced IL-1 beta secretion promoting foam cells formation was mainly via CD36 mediated ROS production leading to NLRP3 inflammasome activation.

OBJECTIVE: IL-1ß is a master switch of inflammation and plays an important role in the pathogenesis of vascular disease. During early atherosclerosis development, it is not clearly understood how oxidized low density lipoprotein (oxLDL)induced signaling pathways control NLRP3 inflammasome activation and produce IL-1ß and promote foam cells formation. METHODS: The study used THP-1 macrophage as cell model. Western blot quantified the oxLDL-induced NLRP3 inflammasome related proteins. The FACS detected the expression of SR-A and CD36 receptors on the cells, and caspase-1 activation in the cells. The DCFH-DA assayed the reactive oxygen species (ROS). Oil red O staining techniques examined the intracellular lipid droplet. RESULTS: The OxLDL remarkably increased not only IL-1ß mRNA transcription and pro-IL-1ß protein synthesis but also IL-1ß secretion in human macrophages. The activation of the NLRP3 inflammasome depended on oxLDL-induced generation of ROS, potassium efflux and cathepsin B activity. The OxLDL-induced ROS production that mediates IL-1ß maturation mainly depended on the scavenger receptor of CD36 but not SR-A. The secreted IL-1ß served as an autocrine function for promoting macrophage foam cells formation. CONCLUSIONS: These findings suggest that oxLDL-induced NLRP3 inflammasome activation mainly depends on CD36 involved in the progression of atherosclerosis by promoting oxLDL-mediated inflammation and foam cell formation.

The association of elevated 2',5'-oligoadenylate-dependent RNase L with lung cancer correlated with deficient enzymatic activity and decreased capacity of RNase L dimerization.

RNase L mediates critical cellular functions including antiviral, proapoptotic, antiproliferative and tumor suppressive activities. In this study, the expression and function of RNase L in lung cancer cells were examined. Interestingly we have found that the expression of RNase L in lung cancer cells was 3- and 9-fold higher in its mRNA and protein levels, but a significant decrease of its enzymatic activity when compared to that in corresponding normal lung cells. Further investigation revealed that 2-5A-induced dimerization of the RNase L protein, a necessary prerequisite for activation of RNase L, was inhibited, as a result of that RLI, a specific inhibitor of RNase L, was remarkably up-regulated in the cancer cells. Our findings provide new insight into how cancer cells escape normal growth-regulating mechanisms to form a tumor and the information may be useful for the design of novel strategies for treating lung cancer through regulating RNase L activity.

Lentivirus-mediated short-hairpin RNA targeting IGF-1R inhibits growth and lymphangiogenesis in breast cancer.

In this study, we investigated the effects of lentivirus (LV)-mediated short hairpin RNA (shRNA) targeting IGF-1R on the growth and lymphangiogenesis of breast cancer. The LV vector effectively delivered the IGF-1R shRNA to MDA-MB­231 cells, leading to significant reduction of IGF-1R mRNA and protein expression. Infection of MDA-MB-231 cells with LV-IGF-1R shRNA reduced cell growth and migration. Transplantation of MDA-MB-231 cells with suppressed IGF-1R expression in SCID mice reduced tumor growth and lymphangiogenesis. These data collectively suggest that LV-mediated shRNA is an effective way to suppress IGF-1R expression and to inhibit growth and lymphangiogenesis of breast cancer. Specific inhibition of IGF-1R expression with shRNA represents a promising approach for the treatment of breast cancer.

An essential function for MKP5 in the formation of oxidized low density lipid-induced foam cells.

The uptake of oxidized low density lipoprotein (ox-LDL) by macrophages usually leads to the formation of lipid-laden macrophages known as "foam cells," and this process plays an important role in the development of atherosclerosis. Ox-LDL activates mitogen-activated protein kinase (MAP) kinases and nuclear factor (NF)-κB, and activations of p38 and NF-κB are important for the formation of foam cells. MAP kinase phosphatase (MKP) 5 is a member of the dual specificity phosphatases (DUSPs) family that can selectively dephosphorylate activated MAPKs to regulate innate and adaptive immune responses. However, the role of MKP5 in the formation of foam cells remains unknown. Here, we found that stimulation of ox-LDL induces the expression of MKP5 in macrophages. MKP5 deficiency blocked the uptake of ox-LDL and the formation of foam cells. Further analysis revealed that deletion of MKP5 reduced the ox-LDL-induced activation of NF-κB. Also, MKP5 deficiency markedly inhibited the production of TNF-α, but enhanced the levels of TGF-ß1 in ox-LDL-stimulated macrophages. Moreover, inhibition of NF-κB by p65 RNAi significantly reduced foam cell formation in macrophages from WT mice relative to MKP5-deficient mice. Thus, MKP5 has an essential role in the formation of foam cells through activation of NF-κB, and MKP5 represents a novel target for the therapeutic intervention of atherosclerosis.

PI3K/Akt and MAPK/ERK1/2 signaling pathways are involved in IGF-1-induced VEGF-C upregulation in breast cancer.

OBJECTIVE: To investigate the signaling pathways involved in insulin-like growth factor-1 (IGF-1)-induced vascular endothelial growth factor C (VEGF-C) up-regulation and lymphatic metastasis in MDA-MB-231 breast cancer cells. METHODS: MDA-MB-231 breast cancer cells were exposed to IGF-1 with various concentrations. The expression level of VEGF-C was assessed by real-time PCR and Western blot. Akt and ERK1/2 phosphorylation was detected by Western blot. Signaling transduction inhibitors, LY294002 and PD98059, were used to block PI3K/Akt and MAPK/ERK1/2 signaling pathways, respectively. RESULTS: IGF-1 increased the level of VEGF-C expression in a dose-dependent manner in MDA-MB-231 breast cancer cells. In addition, phosphorylation of Akt and ERK1/2 was enhanced by IGF-1. Remarkably, inhibition of Akt phosphorylation by LY294002 completely blocked the effects on IGF-1-induced VEGF-C up-regulation. Inhibition of ERK1/2 phosphorylation by PD98059 reduced IGF-1-induced VEGF-C expression. CONCLUSION: This study identified that PI3K/Akt and MAPK/ERK1/2 signaling pathways were involved in IGF-1-induced VEGF-C up-regulation and suggested their important roles in lymphatic metastasis in breast cancer.

Effect of overexpression of human SR-AI on oxLDL uptake and apoptosis in 293T cells.

Type I class A macrophage scavenger receptor (SR)-AI plays an important role in foam cell formation and in apoptosis in atherosclerosis, however the mechanism remains unclear. Therefore, we generated a pEGFP-C1-SR-AI plasmid construct for transient transfection of 293T human embryonic kidney cells and observed if SR-AI expression led: (i) to foam cell formation or apoptosis; and (ii) to expression of apoptosis-related genes Bcl-2 and Bak-1 in cells treated with oxidized low-density lipoprotein (oxLDL). The pEGFP-C1 (empty vector) transfected cell line was used as a control. Transfection efficiency of each group was >90% and transfected cells expressed functional SR-AI protein. Binding and uptake of 3,3'-dioctadecylindocarbocyanine-labeled oxLDL (DiI-oxLDL) were verified by flow cytometry; increases in the rate of oxLDL binding and uptake were observed in pEGFP-C1-SR-AI transfected 293T cells and incubation with oxLDL also led to increased apoptosis (≈50%) compared with controls. A decrease in Bcl-2 and an increase in Bak-1 mRNA and protein expression were observed in pEGFP-C1-SR-AI transfected cells compared with controls. We conclude that transient over-expression of SR-AI leads to an increase in oxLDL uptake and binding in a non-macrophage cell line. In addition, over-expression of SR-AI induced non-macrophage cell apoptosis via downregulation of Bcl-2 and upregulation of Bak-1 expression. We conclude that the 293T cell expression described here is a model for foam cell formation. These results may form the basis of further research into SR-AI structure and function (including lipoprotein uptake, apoptosis modulation and adhesion), which may give an insight into the progression of atherosclerosis in vivo.

Interleukin-10 down-regulates oxLDL induced expression of scavenger receptor A and Bak-1 in macrophages derived from THP-1 cells.

Here, we investigated the therapeutic potential of IL-10 by testing its effects on oxLDL-induced lipoprotein uptake and apoptosis by flow cytometry in THP-1-derived macrophages. The mRNA and protein expressions of lipid scavenger receptors (SR-A, CD36) and apoptosis-related proteins (Bcl-2, Bak-1) were also detected. Co-incubation of oxLDL with IL-10 reduced DiI-oxLDL uptake by 16.1±3.8%, 35.2±3.8% and 28.9±1.8% at 6, 12 and 24h of treatment, respectively. Furthermore, treatment with oxLDL for 24h enhanced the SR-A mRNA and protein expressions by 89.3±17.1% and 70.1±17.6%, respectively. IL-10 abrogated the oxLDL-induced SR-A mRNA expression by 50.2±3.9% and its protein by 45.6±1.9%. Meanwhile IL-10 had no effect on the oxLDL-induced increase of CD36 expression. IL-10 inhibited the oxLDL-induced cell apoptosis in a time-dependent manner by 17.3±3.3%, 36.4±2.8% and 31.0±4.3% at 6, 12 and 24h, respectively. OxLDL increased Bak-1 mRNA and protein expressions by 38.4±13.3% and 36.9±12.1%, respectively. However co-stimulation of oxLDL with IL-10 for 24h inhibited Bak-1 expression to 28.4±7.2% (mRNA) and 25.7±6.3% (protein). Meanwhile, IL-10 had no effect on the oxLDL-induced decrease of Bcl-2 expression. Our findings suggested that IL-10 reduced the oxLDL-induced lipoprotein uptake and apoptosis partly via down-regulating the oxLDL-induced expression of SR-A and Bak-1 in THP-1-derived macrophages.

Inhibitory effects of genistein on metastasis of human hepatocellular carcinoma.

AIM: To investigate the inhibitory effects of genistein on metastasis of MHCC97-H hepatocellular carcinoma cells and to explore the underlying mechanism. METHODS: MHCC97-H hepatocellular carcinoma cells were exposed to genistein. A cell attachment assay was carried out in a microculture well pre-coated with fibronectin. The invasive activity of tumor cells was assayed in a transwell cell culture chamber, and cell cycle and apoptosis were evaluated by a functional assay. In addition, the expression and phosphorylation of FAK were detected by Western blotting. In situ xenograft transplantation of hepatocellular carcinoma was performed in 12 nude mice and lung metastasis of hepatocellular carcinoma was observed. RESULTS: Genistein significantly inhibited the growth of MHCC97-H cells in vitro. Adhesion and invasiveness of MHCC97-H cells were inhibited in a concentration-dependent fashion, and the inhibitory effect of genistein was more potent in the 10 microg/mL and 20 microg/mL genistein-treated groups. Genistein caused G(0)/G(1) cell cycle arrest, an S phase decrease, and increased apoptosis. The expression and phosphorylation of FAK in MHCC-97H cells were significantly decreased. In situ xenograft transplantation of hepatocellular carcinoma was also significantly suppressed by genistein. The number of pulmonary micrometastatic foci in the genistein group was significantly lower compared with the control group (12.3 +/- 1.8 vs 16.6 +/- 2.6, P < 0.05). CONCLUSION: Genistein appears to be a promising agent in the inhibition of metastasis of hepatocellular carcinoma.

[Effects of Astragalus membranaccus on cardiac function and SERCA2a gene expression in myocardial tissues of rats with chronic heart failure].

OBJECTIVE: To investigate the effects of Astragalus membranaccus (As) on cardiac function and SERCA2a gene expression in left ventricular tissues of rats with chronic heart failure. METHODS: Heart failure was induced by clipping the abdominal aorta 60 male SD rats were divided into four groups: sham-operated (Sham), aortic stenosis (Model), Model + As (20 g/kg) and Model + Captopril (0.05 g/kg). The drugs were administered orally from the 13th week after surgery. Rats were examined after 12 weeks' treatment with drugs. The parameters of hemodynamics including LVSP, LVEDP, and +/- LVdp/dt(max) were measured. SERCA2a mRNA and protein expressions in left ventricular tissues were determined by half-quantitative RT-PCR and Western blot normalized to abundance of GAPDH mRNA and portein, respectively. RESULTS: LVSP and LVEDP were obviously enhanced (P < 0.01 or P < 0.001) in model rats in vivo. Both Captopril and As prevented the increase of LVSP (P < 0.05 or P < 0.01) and LVEDP (P < 0.05 or P < 0.01). RT-PCR and Western blot results demonstrated that SERCA2a gene expression was downregulated (P < 0.05) significantly in model group compared with sham group. As upregulated SERCA2a gene expression (P < 0.05), whereas Captopril had no effect on that. CONCLUSION: As can ameliorate abnormity of cardiac function, especially diastoilc function in rats with pressure overload-induced heart failure, and that may be partly related to its up-regulation of SERCA2a gene expressions in left ventricular tissues.

[Effect of astragalus on calcium accumulation and SERCA2a gene expression in myocardial tissues in rats with pressure overload-induced left ventricular hypertrophy].

OBJECTIVE: To investigate the effect of astragalus (As) on calcium accumulation and SERCA2a gene expression in left ventricular tissues in rats with pressure overload-induced cardiac hypertrophy. METHOD: cardiac hypertrophy was induced by clipping the abdominal aorta in rats. Male SD rats were allocated to six groups: sham-operrated (Sham), aortic stenosis (Model), model +As-L (5 g x kg(-1) x d(-1)), model+As-M (10 g x kg(-1) x d(-1)), model+As-H (20 g x kg(-1) x d(-1)) and model + captopril (0.05 mg x kg(-1) x d(-1), a positive control). The drugs were administered orally from the 13 th week after surgery. Rats were examined after 12 week treatment with drugs. The cardiac hypertrophy was evaluated by left ventricular mass index (LVMI, left ventricular weight/ body weight). The calcium content in left ventricular tissue was measured by atomic absorption spectrometry. SERCA2a mRNA and protein expressions in left ventricular tissues were determined by half-quantitative RT-PCR and Western blot normalized to abundance of GAPDH mRNA and protein, respectively. RESULT: The increase of LVMI was dose-dependently lessened by As (P < 0.01, P < 0.001). The effect of As-H was similar to that of Captopril. As markedly attenuated calcium accumulation in myocardial tissure (P < 0.01). RT-PCR and Western blot results demonstrated that SERCA2a gene expressions were downregulated (P < 0.05) significantly in model group compared with sham group. As-H upregulated SERCA2a gene expressions (P < 0.05), whereas Captopril had no effect on that. CONCLUSION: The inhibition of As on left ventricular hypertrophy induced by pressure overload in rats may partly contribute to its attenuation of calcium accumulation and up-regulation of SERCA2a gene expressions in left ventricular tissues.

TGFbeta inhibits LPS-induced chemokine mRNA stabilization.

The mechanisms involved in anti-inflammatory action of transforming growth factor beta (TGFbeta) have been examined by evaluating its effect on chemokine gene expression in mouse macrophages. Lipopolysaccharide (LPS)-stimulated expression of the CXC chemokines KC and MIP-2 was selectively reduced by TGFbeta in a time- and protein synthesis-dependent process. While TGFbeta had a modest effect on transcription of the KC and MIP-2 mRNAs as measured by nuclear run-on, it had no effect on LPS-stimulated luciferase expression driven by the KC promoter nor on the activation of nuclear factor kappaB (NFkappaB) DNA-binding activity and transactivation function. Interestingly, KC mRNA levels were markedly reduced by TGFbeta treatment in cells transfected with KC genomic or cDNA constructs driven from either the KC or cytomegalovirus (CMV) promoters, demonstrating the importance of sequences within the mature mRNA and suggesting that suppression may involve a posttranscriptional mechanism. In support of this possibility, LPS stimulation prolonged the half-life of KC mRNA and this stabilization response was blocked in cells treated with TGFbeta. Examination of KC mRNA expressed under control of a tetracycline-responsive promoter demonstrated that TGFbeta prevented stabilization of KC mRNA, in response to LPS but did not alter KC mRNA half-life directly. KC mRNA stabilization by LPS was dependent on activation of p38 mitogen-activated protein kinase (MAPK) activity, and TGFbeta treatment inhibited p38 MAPK activation. These findings support the hypothesis that TGFbeta-mediated suppression of chemokine gene expression involves antagonism of LPS-stimulated KC mRNA stabilization via inhibition of p38 MAPK.