Modified Citrus Pectin Prevents Blood-Brain Barrier Disruption in Mouse Subarachnoid Hemorrhage by Inhibiting Galectin-3.

Background and Purpose- Plasma levels of galectin-3-a matricellular protein-are increased after aneurysmal subarachnoid hemorrhage (SAH), but the functional significance remains undetermined. This study was conducted to evaluate whether modified citrus pectin (MCP; galectin-3 inhibitor) prevents post-SAH early brain injury, focusing on blood-brain barrier disruption. Methods- C57BL/6 male adult mice (n=251) underwent sham or filament perforation SAH modeling, followed by a random intracerebroventricular injection of vehicle or drug at 30 minutes post-modeling. First, vehicle-treated and 0.8, 4, 16, or 32 µg MCP-treated mice were assessed by neuroscore and brain water content at 24 and 48 hours post-modeling. Second, Evans blue extravasation, Western blotting, coimmunoprecipitation and immunostaining were performed in vehicle-treated or 4 µg MCP-treated mice at 24 hours post-modeling. Third, vehicle or R-galectin-3 (recombinant galectin-3) was administered to SAH mice simultaneously with vehicle or MCP, and neuroscore and Evans blue extravasation were evaluated at 24 hours post-modeling. Fourth, vehicle or R-galectin-3 was administered to MCP-treated SAH mice at 24 hours, and neuroscore and IgG immunostaining were evaluated at 48 hours post-SAH. Results- Among tested dosages, 4 µg MCP showed the best neuroprotective effects as to preventing neurological impairments and brain edema at 24 to 48 hours post-SAH. Four micrograms MCP attenuated post-SAH blood-brain barrier disruption and galectin-3 upregulation in brain capillary endothelial cells, associated with inactivation of ERK (extracellular signal-related kinase) 1/2, STAT (signal transducer and activator of transcription)-3, and MMP (matrix metalloproteinase)-9, and the consequent preservation of a tight junction protein ZO-1 (zonula occludens-1). Coimmunoprecipitation assay demonstrated physical interactions between galectin-3 and TLR (Toll-like receptor) 4. R-galectin-3 blocked the neuroprotective effects of MCP. Conclusions- MCP prevents post-SAH blood-brain barrier disruption possibly by inhibiting galectin-3, of which the mechanisms may include binding to TLR4 and activating ERK1/2, STAT-3, and MMP-9. This study suggests galectin-3 to be a novel therapeutic target against post-SAH early brain injury.

Tea catechins have dual effect on mast cell degranulation induced by compound 48/80.

Green tea catechins are emerging as one of the most efficient and safest ingredient in health promoting food. We investigated catechin's effects on intracellular ROS generation in mast cell activation and degranulation. Compound 48/80, receptor mimetic basic secretagogues for mast cell, induced ROS generation dose-dependently with bell-shaped degranulation pattern in canine cutaneous mastocytoma cells (CM-MC). When intracellular ROS level was relatively low, catechins decreased both ROS and the degranulation. However, when intracellular ROS level was remarkably high, catechins decreased ROS level but increased the degranulation paradoxically. Gallocatechins showed the stronger effects than non-gallated catechins. Exogenous H(2)O(2) also shows dual effect on degranulation dose-dependently. EGCG shows the dual effect on the tyrosine and threonine phosphorylation depending on the concentration of compound 48/80. Particularly, 60 kDa protein tyrosine-phosphorylated by EGCG with 3 microg/ml of compound 48/80 might be a negative regulator for the degranulation. Taken together, there is an optimal level of ROS for the degranulation, and the catechins have a dual function by controlling ROS level.

Inhibition of NADPH oxidase subunits translocation by tea catechin EGCG in mast cell.

The inhibitory mechanism of tea catechins for allergy remains undefined. We studied the effect of catechins, mainly EGCG, on the activation of mast cell line canine cutaneous mastocytoma cells (CM-MC). Compound 48/80 induced the degranulation in CM-MC dose dependently, whereas its release of beta-hexosaminidase was inhibited by EGCG and O-methylated EGCG (EGCG-Me). Both catechins were found to inhibit intracellular ROS generation dose dependently together with DPI. Intracellular ROS generation in human polymorphonuclear leukocytes was also inhibited by EGCG. Neither L-NAME, ebeselen nor NAC inhibited ROS generation. From the Western blot analysis of the subunits components of NADPH oxidase, we detected cytosolic subunits; p47(phox), p67(phox), p40(phox), rac2 and membrane subunits; gp91(phox), p22(phox) in CM-MC. Cytosolic subunits were translocated from cytosol to membrane time dependently after stimulation with compound 48/80. EGCG and DPI inhibited cytosolic subunits from translocating into membrane. These data suggest that EGCG inhibits the activation of NADPH oxidase in CM-MC.

Cytotoxicity and radical modulating activity of Moxa smoke.

The biological activities of Moxa, used as moxibustion, have not been well documented. We investigated here Moxa smoke for its tumor-specific cytotoxicity, anti-HIV activity, radical intensity and radical scavenging activity, in comparison with previously published data of Moxa extract. Moxa smoke showed slightly higher cytotoxicity against human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, promyelocytic leukemia HL-60) than against normal oral cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), yielding a tumor specificity index of 1.29. Moxa smoke dose-dependently induced internucleosomal DNA fragmentation, activation of caspases 3, 8 and 9, and slightly modified the expression of apoptosis-related proteins (Bcl-2, Bad, Bax) in HL-60 cells, but to much lesser extents than attained by positive controls (UV irradiation, actinomycin D treatment). ESR spectroscopy showed that Moxa smoke generated semiquinone-type radicals under alkaline conditions, and scavenged O2(-), hydroxyl radical, singlet oxygen and NO. All Moxa smoke preparations showed no apparent anti-HIV activity. These data demonstrate the antitumor potential of Moxa smoke.

Cytotoxicty and radical modulating activity of isoflavones and isoflavanones from Sophora species.

We investigated 2 isoflavones and 9 isoflavanones from Sophora species for their cytotoxic activity against 3 normal human cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast) and 2 human tumor cell lines (squamous cell carcinoma HSC-2, submandibular gland carcinoma HSG). Compounds with 2 isoprenyl groups (one in A-ring and the other in B-ring) such as tetrapterol G [YS31] and isosophoranone [YS24], and those with alpha,alpha-dimethylallyl group at C-5' of B-ring [YS26 (secundifloran), YS27 (secundiflorol A), YS28 (secundiflorol D), YS29 (secundiflorol E)] showed relatively higher cytotoxic activity. When hydrophobicity was assessed by octanol-water partition coefficient (log P), the maximum cytotoxic activity was observed at a log P value around 4. Compounds with intermediate cytotoxic activity [YS27, genistein, YS28, YS29, YS30 (secundiflorol F)] showed relatively higher tumor specificity. All isoflavones and isoflavanones did not stimulate the nitric oxide (NO) production by mouse macrophage-like Raw 264.7 cells, but almost completely inhibited the NO production by lipopolysaccharide (LPS)-activated Raw 264.7 cells. ESR spectroscopy showed that YS26 and YS28, which are the most inhibitory for NO production, efficiently scavenged superoxide anion and NO radicals. These data suggest that the inhibition of macrophage NO production by these isoflavanones may, at least in part, be explained by their radical scavenging or reduction activity.

A pilot study on antiplaque effects of mastic chewing gum in the oral cavity.

BACKGROUND: Chemical plaque control is a useful aid in mechanical oral hygiene, and various chemical agents have been evaluated as antiplaque agents. It has been shown that mastic chewing gum has antibacterial effects on Helicobacter pylori. In this study, the antiplaque effect of mastic chewing gum was investigated. METHODS: Twenty dental students who were both systemically and periodontally healthy participated in this study. The effects of mastic gum were assessed from 2 double-blinded, randomized studies. In the first trial, after mechanical toothbrushing, the inhibitory effect of mastic gum on bacteria in saliva following its use was compared to a placebo gum. Saliva samples were collected at the end of 1, 2, 3, and 4 hours; diluted; inoculated onto 10% horse blood chocolate agar plates; and cultured anaerobically at 37 degrees C for 48 hours. The total number of bacterial colonies on each plate was calculated (n = 20). In the second trial, the effects of mastic gum on de novo plaque formation on tooth surfaces and gingival inflammation were evaluated over a 7-day period without mechanical oral hygiene following random use of either mastic or placebo chewing gum. The degree of plaque accumulation and gingival inflammation were compared between the 2 groups (n = 10). RESULTS: The total number of bacterial colonies was significantly reduced during the 4 hours of chewing mastic gum compared to the placebo gum (P < 0.05, Student t test). The mastic group showed a significantly reduced plaque index (2.69 +/- 0.29 versus 3.15 +/- 0.24; P = 0.001, Student t test) and gingival index (0.44 +/- 0.15 versus 0.66 +/- 0.23, P = 0.021, Student t test) compared to the placebo group. CONCLUSION: These results suggest that mastic chewing gum is a useful antiplaque agent in reducing the bacterial growth in saliva and plaque formation on teeth.

Diverse biological activities of fermented pine seed shell extract.

Intraperitoneal administration of fermented pine seed shell extract (PSSE) (up to 2 g/kg) induced no apparent acute toxicity to mice. Pretreatment of mice with PSSE protected them from the lethality of Escherichia coli infection. PSSE showed a very weak cytotoxic activity against both normal and tumor cells and no anti-HIV activity, but stimulated the mouse macrophage-like Raw 264.7 cells to produce nitric oxide (NO) and citrulline. ESR spectroscopy showed that PSSE produced no detectable radicals, but effectively scavenged O2- (generated by the hypoxanthine-xanthine oxidase reaction), hydroxyl radical (generated by the Fenton reaction) and NO (generated by NOC-7). Comparison of PSSE with other natural products, such as polyphenols and vitamins, further confirmed the close association between radical intensity and radical scavenging activity, suggesting the bimodal action of natural products. Although the biological activities of PSSE were relatively lower than those of other natural products, the present study suggests the possible medicinal efficacy of PSSE.

Tumor-specificity and radical scavenging activity of poly-herbal formula.

A total of 14 poly-herbal formula extracts were compared for their biological activities both in vivo and in vitro. Pretreatment of mice with the extracts protected them from E. coli infection to various extents. Among the extracts, the HD-12 and DLH-3073 extracts showed the highest cytotoxicity against both HIV-infected and mock-infected MT4 cells, without induction of any apparent anti-HIV activity. The extracts showed significantly higher cytotoxic activity against five human tumor cell lines (HSC-2, HSC-3, HSG, MT-4, HL-60) than against three normal human cell lines (HGF, HPC, HPLF). Agarose gel electrophoresis demonstrated that the HD-12 and DLH-3073 extracts induced intemucleosomal DNA fragmentation in HL-60 cells. ESR spectroscopy showed that all the extracts produced radicals and this was paralleled by their ability to scavenge the superoxide anion (produced by hypoxanthine-xanthine oxidase reaction), the hydroxyl radical (produced by Fenton reaction) and nitric oxide (produced by NOC- 7) in the presence of radical trapping agents. Higher and lower concentrations of extracts enhanced or reduced respectively, the radical intensity of sodium ascorbate, suggesting their bimodal actions. The tumor specificity and antioxidant properties of the herb extracts further suggest their medicinal efficacy.

Partial purification of cytotoxic substances from moxa extract.

The major cytotoxic activity of Moxa was extracted with CH2Cl2 and partially purified by three cycles of silica gel column chromatography. The active fractions showed higher cytotoxicity against six human tumor cell lines (two oral squamous cell carcinoma, one salivary gland tumor, one melanoma, two leukemia) than three normal oral human cells (gingival fibroblast, periodontal ligament fibroblast, pulp cell). All fractions failed to protect the cells from the cytopathic effect induced by HIV infection. ESR spectroscopy showed that all fractions produced little or no radical under alkaline conditions, while showing much lower O2- scavenging activity, generated by hypoxanthine-xanthine oxidase reaction, than antioxidants and polyphenols. Active fractions induced DNA fragmentation in HL-60 cells, but failed to modify the mobility and activity of mitochondrial Mn-containing superoxide dismutase (MnSOD), in contrast to Moxa smoke. These data suggest that the active principles in the Moxa extract might be different from that in Moxa smoke, which produced carbon radical and modified MnSOD mobility and activity.