5, 7, 2', 4', 5'-Pentamethoxyflavanone regulates M1/M2 macrophage phenotype and protects the septic mice.

Flavonoids have been reported to exert protective effect against many inflammatory diseases, while the underlying cellular mechanisms are still not completely known. In the present study, we explored the anti-inflammation activity of 5, 7, 2', 4', 5'-pentamethoxyflavanone (abbreviated as Pen.), a kind of polymethoxylated flavonoid, both in vitro and in vivo experiments. Pen. was showed no obvious toxicity in macrophages even at high dosage treatment. Our results indicated that Pen. significantly inhibited both mRNA and protein level of proinflammatory cytokines, IL-1ß, IL-6, TNF-α and iNOS, which was characteristic expressed on M1 polarized macrophages. These effects of Pen. were further confirmed by diminished expression of CD11c, the M1 macrophage surface marker. Further researches showed that the mechanism was due to that Pen. downregulated the activity of p65, key transcription factor for M1 polarization. On the other hand, Pen. also enhanced M2 polarization with upregulation of anti-inflammatory factors and increase of M2 macrophage surface markers, which lead to the balance of M1 and M2 macrophages. Moreover, in vivo research verified that Pen. treatment alleviated LPS-induced sepsis in mice by increasing survival rate, decreasing inflammatory cytokines and improving lung tissue damage. In summary, our results suggested that Pen. modulated macrophage phenotype via suppressing p65 signal pathway to exert the anti-inflammation activity.

Betaine Effects on Morphology, Proliferation, and p53-induced Apoptosis of HeLa Cervical Carcinoma Cells in Vitro.

OBJECTIVES: To investigate the effects of betaine on HeLa cell growth and apoptosis and molecular mechanisms. MATERIALS AND METHODS: Concentrations of 0.1, 1.0, 5.0, 20.0, 100.0 mg/ml of betaine were used to evaluate the anticancer efficacy for HeLa cells respectively, and MCF-10A was also detected as a normal diploid cell control. RESULTS: We found that proliferation of HeLa cells was inhibited significantly upon exposure to increasing betaine levels with the MTT test (p<0.05). The percentage of S phase cells in the low dose groups (< 5mg/ml) were distinctly higher than in high dose groups, and the rates of Sub-G1 phase were the opposite (p<0.01); A high concentration of betaine (>5.0mg/ml) significantly promoted the apoptosis of HeLa cells (p<0.01). SOD activities of the low dose groups were slightly higher than the control group (p<0.05) and there were obvious synchronicity and correlation among the expression of promoting apoptosis genes Bax, P53, Caspase 3 and apoptosis suppression gene Bcl-2. In response to an apoptosis-inducing stimulus, p53 and cyclin D1 could be activated with blockage of the cell cycle at G1/S or S/G2 checkpoints. CONCLUSIONS: Our data showed that betaine could promote HeLa cells proliferation in vitro at low concentrations.In contrast, high concentrations could significantly inhibit cell growth and migration, and induce apoptosis of HeLa cells through caspase 3 signaling and further promoted necrosis. This might imply that betaine exhibits tumoricidal effects and acts as a biological response modifier in cancer treatment by inducing apoptosis and cell cycle arrest in a dose and time-dependent manner.

In vitro study of nucleostemin as a potential therapeutic target in human breast carcinoma SKBR-3 cells.

Although nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis and expression has been observed in some types of human cancer and stem cells, the molecular mechanisms involved in mediation of cell proliferation and cell cycling remains largely elusive. The aim of the present study was to evaluate NS as a potential target for gene therapy of human breast carcinoma by investigating NS gene expression and its effects on SKBR-3 cell proliferation and apoptosis. NS mRNA and protein were both found to be highly expressed in all detected cancer cell lines. The apoptotic rate of the pcDNA3.1-NS-Silencer group (12.1-15.4 ± 3.8%) was significantly higher than those of pcDNA3.1-NS (7.2-12.0 ± 1.7%) and non-transfection groups (4.1-6.5 ± 1.8%, P<0.01). MTT assays showed the knockdown of NS expression reduced the proliferation rate of SKBR-3 cells significantly. Matrigel invasion and wound healing assays indicated that the number of invading cells was significantly decreased in the pcDNA3.1-NS-siRNA group (P<0.01), but there were no significant difference between non-transfected and over-expression groups (P>0.05). Moreover, RNAi-mediated NS down- regulation induced SKBR-3 cell G1 phase arrest, inhibited cell proliferation, and promoted p53 pathway-mediated cell apoptosis in SKBR-3 cells. NS might thus be an important regulator in the G2/M check point of cell cycle, blocking SKBR-3 cell progression through the G1/S phase. On the whole, these results suggest NS might be a tumor suppressor and important therapeutic target in human cancers.

In vitro and in vivo characterization of silk fibroin/gelatin composite scaffolds for liver tissue engineering.

OBJECTIVE: To investigate the cytotoxicity of silk fibroin/gelatin (SF/G) composite scaffolds in vitro as well as their biocompatibility and degradation in vivo. METHODS: The proliferation and relative growth rate of human hepatic QZG cells grown on different blends of two-dimensional (2-D) SF/G scaffolds were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry was used to evaluate apoptotic rate of QZG cells on different blends of 2-D SF/G scaffolds. The effect of silk protein materials on cell growth was observed by scanning electron microscopy. Three-dimensional (3-D) SF/G scaffolds of three different ratios (diameter 10 mm, thickness 1 mm) were implanted into subcutaneous pockets on male Sprague-Dawley (SD) rats. On the 7th, 14th and 30th day post-implantation, the rats were sacrificed. The scaffold area including the surrounding tissues was retrieved. Hematoxylin and eosin staining was performed for observation under a light microscope. RESULTS: Significant cell attachment and proliferation on the SF/G scaffolds were observed. As the increased gelatin concentration, SF/G scaffolds became more amenable to cell adhesion. After the subcutaneous implantation of the SF/G scaffolds in SD rats, immunological rejection tests showed only slight inflammation, measured by the presence of inflamed cells on day 7 and 14. By day 30, each scaffold had been completely infiltrated and organized by fibroblasts and inflamed cells. The greater the gelatin concentration in the scaffold, the faster the degradation rate. CONCLUSION: Composite SF/G scaffolds are a promising candidate matrix for implantable bio-artificial livers.

Ameliorative effects of histamine on spatial memory deficits induced by scopolamine infusion into bilateral dorsal or ventral hippocampus as evaluated by the radial arm maze task.

1. The present study examined the role of the hippocampal histaminergic system in the regulation of spatial memory deficit in rats using the radial arm maze task after scopolamine injection into the bilateral dorsal (DH) or ventral (VH) hippocampus. 2. Bilateral injection of scopolamine (5 microg/site) into both the DH and VH impaired spatial memory in the retrieval memory process. Injection of histamine (50 or 100 ng/site) in the DH and intraperitoneal injection of histidine (100 mg/kg) markedly improved working memory and reference memory deficits induced by scopolamine injection into the DH. The histamine H(1) receptor antagonist pyrilamine (1 microg/site) abolished the ameliorative effects of histidine on working memory deficits, whereas both pyrilamine and the H(2) receptor antagonist cimetidine (0.5 microg/site) abolished the effect of histidine on reference memory. 3. Local injection of histamine (25 or 50 ng/site) into the VH and systemic injection of histidine (50 or 100 mg/kg) markedly improved working memory deficits induced by scopolamine injection into the VH, but did not improve the deficits in reference memory. Injection of both pyrilamine (0.2, 0.5 and 1 microg/site) and cimetidine (0.1 and 0.5 microg/site) into the VH reversed the effects of histidine. 4. The results of the present study indicate that histamine has different actions on cholinergic-related memory in the the DH and VH. Histamine in the DH ameliorates spatial working memory deficits by acting on histamine H(1) receptors and reference memory deficits through both H(1) and H(2) receptors. However, histamine in the VH ameliorates working memory deficits via an action on both H(1) and H(2) receptors.

Histamine ameliorates spatial memory deficits induced by MK-801 infusion into ventral hippocampus as evaluated by radial maze task in rats.

AIM: To investigate the role of histamine in memory deficits induced by MK-801 infusion into the ventral hippocampus in rats. METHODS: An 8-arm radial maze (4 arms baited) was used to assess spatial memory. RESULTS: Bilateral ventral intrahippocampal (ih) infusion of MK-801 (0.3 microg/site), an N-methyl-D-aspartate (NMDA) antagonist, impaired the retrieval process in both working memory and reference memory. Intrahippocampal injection of histamine (25 or 50 ng/site) or intraperitoneal (ip) injection of histidine (25, 50 or 100 mg/kg) markedly ameliorated the spatial memory deficits induced by MK-801. Both the histamine H1 antagonist pyrilamine (0.5 or 1.0 microg/site, ih) and the H2 antagonist cimetidine (2.5 microg/site, ih) abolished the ameliorating effect of histidine (100 mg/kg, ip) on reference memory deficits, but not that on working memory deficits induced by MK-801. CONCLUSION: The results indicate that histamine in the ventral hippocampus can ameliorate MK-801-induced spatial memory deficits, and that histamine's effect on reference memory is mediated by postsynaptic histamine H1 and H2 receptors.

Histidine enhances carbamazepine action against seizures and improves spatial memory deficits induced by chronic transauricular kindling in rats.

AIM: To investigate whether histidine can enhance the anticonvulsant efficacy of carbamazepine (CBZ) and simultaneously improve the spatial memory impairment induced by transauricular kindled seizures in Sprague-Dawley rats. METHODS: Chronic transauricular kindling was induced by repeated application of initially subconvulsive electrical stimulation through ear-clip electrodes once every 24 h until the occurrence of 3 consecutive clonic-tonic seizures. An 8-arm radial maze (4 arms baited) was used to measure spatial memory, and histamine and gamma-amino-butyric acid levels were measured by high performance liquid chromatography (HPLC). RESULTS: Chronic transauricular kindling produced a significant impairment of spatial memory and a marked decrease in histamine content in the hypothalamus, the brainstem, and the hippocampus. Injection of histidine (1000 mg/kg or 1500 mg/kg, ip) significantly inhibited transauricular kindled seizures. Injection of histidine at lower doses (200 mg/kg or 500 mg/kg, ip) had no appreciable anticonvulsant effect when administered alone, whereas it significantly potentiated the protective effects of CBZ against kindled seizures. CBZ had no ameliorative effect on memory deficit, but, in contrast, histidine (200 mg/kg or 500 mg/kg, ip) alone or co-administered with CBZ significantly ameliorated the memory deficits induced by the seizures. CONCLUSION: Chronic transauricular kindling is a very useful animal model for evaluating memory deficits associated with epilepsy, and histidine has both a potentiate effect on the anticonvulsant efficacy of CBZ and an ameliorative effect on the spatial memory deficits induced in this model. Histidine at a specific dosage range might serve as a beneficial adjuvant for the clinical treatment of epilepsy, especially when accompanied by impaired spatial memory.

[Changes of brain mast cells after transient global ischemia in rats].

OBJECTIVE: To investigate changes of brain mast cells after transient global ischemia in rats. METHODS: Transient global ischemia damage was induced by four-vessel occlusion. After 1 h to 14 days of ischemia, rats were perfused intracardially by 4% paraformaldehyde. The brains were dissected to serial sections using freeze microtome, and then stained with toluidine blue. Brain mast cell was observed under microscope. RESULT: Most brain mast cells were located in thalamus. The number of mast cells in thalamus markedly decreased during reperfusion after transient global ischemia. However, the degranulation rate of thalamus mast cells showed reverse change after ischemia. CONCLUSION: Brain mast cells markedly degranulate after transient global ischemia, which may be involved in the pathological process after ischemia.

[Effect of alahistidine on brain histamine content and seizure development].

OBJECTIVE: To investigate the effect of alahistidine on brain histamine content and seizure development. METHODS: The kindling seizure was induced by ip injection with subconvulsant dose of pentylenetetrazole every 48 h. Monoamines and their metabolites were measured using a HPLC system and fluorometric assay. RESULT: Chronic low histamine feeding markedly decreased histamine content in cortex and hypothalamus, and promoted seizure development induced by pentylenetetrazole. However, alahistidine feed reversed the decreased histamine content and slowed seizure development caused by low histamine feed. Both low histamine and alahistidine feed had no effect on norepinephrine, dopamine and its metabolites. CONCLUSION: Alahistidine may affect histaminergic system and seizure development.