Expression of Bim, Bax and Bak in the process of gingipain-induced osteoblast apoptosis / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To establish osteoblast apoptosis model induced by gingipains, and to examine the expression of pro-apoptotic protein Bcl-2 interacting mediator (Bim), Bcl-2 associated X protein (Bax) and Bcl-2 antagonist/killer (Bak).</p><p><b>METHODS</b>Gingipain and gingipain acticity were extracted and measured. Mouse osteoblast cell line MC3T3-E1 cells were cultured in the presence of 0.453, 0.906, 1.812 U/L gingipains for 0, 16, 24 and 48 h. Apoptosis was examined by 4',6-diamidino-2-phenylindole (DAPI) staining or annexin V/propidine iodide (PI) staining.Protein expression of Bim, Bax and Bak was determined by Western blotting after osteoblasts were cultured with 1.812 U/L gingipain for 0, 4, 8, 16, 24 and 48 h. Osteoblasts were cultured with 1.812 U/L gingipain which had been inhibited with N-alpha-tosyl L-lysyl-chlorom ethylketone (TLCK). Western blotting was used to detect Bim expression and DAPI staining to measure apoptosis.</p><p><b>RESULTS</b>Arginine-specific proteinases (Rgp) activity was (18.11 ± 2.11) U/L and specific proteinases (Kgp) was (1.02 ± 0.25) U/L. Percentage of osteoblast apoptosis induced by 1.812 U/L gingipain rose to (6.31 ± 0.37)% after 16 h, and reached (11.20 ± 0.35)% at 24 h and (10.80 ± 0.46)% after 48 h with DAPI staining. Annexin V/PI staining supported the result from DAPI staining.Bim protein level increased during osteoblast apoptosis, the relative fold rose to (0.31 ± 0.03) after 4 h (about 2 fold compared to control), peaking at 24 h (0.57 ± 0.05, 3-4 fold compared to control). Proteinase inhibitor TLCK effectively blocked the activity of gingipain and inhibited up-regulation of Bim induced by gingipains from (0.58 ± 0.04) to (0.14 ± 0.03). The percentage of osteoblast apoptosis decreased from (11.20 ± 0.35)% to (4.31 ± 0.38)% in the presence of TLCK. Expression of Bax remained unchanged when cells were cultured with or without gingipains. Bak was under the detectable level in MC3T3-E1.</p><p><b>CONCLUSIONS</b>1.812 U/L gingipains induced osteoblast apoptosis. Protein expression of Bim was up-regulated during cell apoptosis and was down-regulated when gingipain inhibited with TLCK, suggesting that Bim was involved in osteoblast apoptosis induced by gingipain. Inhibition of Bim protein expression protected osteoblast from apoptosis.</p>

Expression of integrin α5 and β1 in osteoblast in the process of gingipains-induced apoptosis / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the regulatory mechanisms of integrin α5 and β1 in osteoblast in the process of gingipains-induced apoptosis.</p><p><b>METHODS</b>Gingipains were isolated and purified from supernatants of Porphyromonas gingivalis W83 which was cultured under standard anaerobic conditions. MC3T3-E1 was challenged with or without 8.3480 U/L gingipains for 48 h and apoptosis was examined by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (TUNEL-DAPI) staining. The expression of integrin α5 and β1 was analyzed by Western blotting after MC3T3-E1 was treated under different conditions.</p><p><b>RESULTS</b>Arginine-specific proteinases(Rgp) activity was (41.74 ± 2.11) U/L and lysine-specific proteinase(Kgp) was (1.02 ± 0.25) U/L.Gingipains induced MC3T3-E1 cells apoptosis after 48 h. Compared with control group, expression of integrin α5 and β1 was down-regulated by gingipains in a time-dependent manner within short periods ( ≤ 72 h), integrin α5 and β1 relative expression was (0.485 ± 0.039),(0.504 ± 0.002) at 48 h,(0.398 ± 0.058),(0.179 ± 0.001) at 72 h respectively (P < 0.05). After 72 h, integrin α5 expression in MC3T3-E1 cells was stable compared with control group while integrin β1 was still lower(control group:1.000 ± 0.000, 96 h:0.604 ± 0.003, 120 h: 0.357 ± 0.002) (P < 0.05). Proteinase inhibitor tosyl- L- lysine-chloromethyl-ketone(TLCK) effectively blocked the activity of gingipain and inhibited down-regulation of integrin α5 and β1 induced by gingipains from (0.398 ± 0.058,0.179 ± 0.001 ) to (0.781 ± 0.012, 0.857 ± 0.060) (P < 0.05). TLCK alone did not have any effect on integrin α5 and β1(P > 0.05). Gingipains also decreased integrin α5 and β1 in a dose-dependent manner.When cells were treated with 20.8700 U/L gingipains, integrin α5 and β1 relative expression reached to the lowest(0.105 ± 0.004,0.020 ± 0.000) (P < 0.05).</p><p><b>CONCLUSIONS</b>Gingipains inhibited the expression of integrin α5 and β1 in a time- and dose- dependent manner in osteoblasts in the process of apoptosis, which may not be mediated by direct proteolytic effect.</p>

Efeito in vitro de inibidores de proteases sobre trofozoítos de Giardia duodenalis / In vitro effect of protease inhibotors on trophozoites of giardia duodenalis

Entre os alvos mais promissores para o desenvolvimento de novos agentes antiparasitários, destacam-se as proteases que nos protozoários participam de processos metabólicos e fisiológicos, atuando como importantes fatores de virulência. Como a atividade dessas moléculas pode ser controlada por inibidores específicos, essas substâncias têm sido avaliadas quanto ao potencial terapêutico em diferentes infecções parasitárias, inclusive por Giardia. O presente estudo foi desenvolvido com o objetivo de avaliar o efeito in vitro dos inibidores de cisteína (IAA e E-64) e serina-proteases (antipaina, leupeptina e TLCK) sobre o crescimento, aderência, viabilidade e ultraestrutura de trofozoítos de cepa de Giardia isolada e axenizada em Botucatu. Para isso, trofozoítos foram incubados em meio contendo os inibidores a diferentes concentrações durante 24, 48 e 72 horas. Nos ensaios de crescimento e aderência, o número de trofozoítos foi estimado a partir de contagens em hemocitômetro, enquanto que a viabilidade celular e as alterações ultraestruturais foram avaliadas, respectivamente, pelo método de redução do MTT e por microscopia eletrônica de transmissão. De acordo com as observações feitas no presente estudo, todos os inibidores de proteases apresentaram efeito sobre o crescimento, aderência e viabilidade dos trofozoítos. Entretanto, melhor desempenho quanto à capacidade de reduzir os parâmetros avaliados foi demonstrado nos ensaios com os inibidores de cisteína-proteases, especialmente a IAA. As maiores porcentagens de inibição do crescimento e aderência e as menores taxas de viabilidade foram observadas após o tratamento com IAA...

The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In light of this, the proteolytic enzymes or proteases have excited the researcher’s interest, once they have been identified as important virulence factors as well as potential chemotherapeutic targets in parasites. Considering that proteases are naturally regulated by specific inhibitors, these substances have been evaluated for their therapeutic potential in parasitic infections including Giardia. In this way, we proposed to evaluate the in vitro effect of inhibitors of cysteine (IAA and E-64) and serine proteases (antipain, leupeptin and TLCK) on growth, adherence, viability and ultrastructure of Giardia trophozoites of a strain isolated and axenized in Botucatu. For this, trophozoites were incubated in medium containing the inhibitors at various concentrations for 24, 48 and 72 hours. In growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability and ultrastructural changes were evaluated, respectively, by the method of MTT and transmission electron microscopy. In this study, all protease inhibitors showed effect on growth, adherence and viability of trophozoites. However, better performance in their ability to reduce the parameters assessed was demonstrated in experiments with cysteine proteases inhibitors, especially IAA...

Nandeshi: a powerful inhibitor of human acrosin activity / 中华男科学杂志

<p><b>OBJECTIVE</b>To evaluate the inhibitory effect of Nandeshi, an acrosin inhibitor, on human acrosin activity.</p><p><b>METHODS</b>We collected sperm samples from 10 healthy fertile men and cultured them with Nandeshi at 30 degrees C for 5 minutes at the concentrations of 0. 100, 0.120, 0.144, 0.173, 0.207, 0.249, 0.299, 0.358 and 0.430 mmol/L, with the controls treated with a well-known acrosin inhibitor N-alpha-p-tosyl-L-lysine chloromethylketone (TLCK) at 150.0, 189.8, 213.6, 240.3, 270.3, 304.1 and 342.1 mmol/L. Then we determined the residual activity of human acrosin by improved Kennedy assay.</p><p><b>RESULTS</b>The residual activity of acrosin was negatively correlated with the Nandeshi concentration, and Nandeshi exhibited an inhibition rate about 800 times that of TLCK.</p><p><b>CONCLUSION</b>Nandeshi has a powerful inhibitory effect on human acrosin, and improved Kennedy assay is a simple, practical and highly sensitive technique for the detection of human acrosin activity.</p>

Modulation of tryptase and histamine release from human lung mast cells by protease inhibitors.

Inhibition of IgE dependent histamine release from human mast cells by protease inhibitors has been observed in skin, tonsil and synovial tissues. However, little is known about the actions of protease inhibitors on tryptase release from human lung mast cells. We therefore examined the ability of protease inhibitors to modulate tryptase and histamine release from human lung mast cells. IgE dependent tryptase release from dispersed lung mast cells was inhibited to a maximum of approximately 53.8% and 44.5% by N-a-tosyl-L-lysine chloromethyl ketone (TLCK) and N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), respectively. A similar degree of inhibition of calcium ionophore A23187 (CI) induced tryptase release was also observed with these two inhibitors. Preincubation of TLCK or TPCK with the mast cells at 37 degrees C for 20 minutes before addition of anti-IgE or CI did not improve their ability to inhibit anti-IgE and CI induced tryptase release. At a concentration of 10 microg/ml, protamine inhibited anti-IgE or CI induced tryptase release; but at 100 microg/ml, it increased anti-IgE and CI induced release of tryptase from lung mast cells. A concentration dependent inhibition of anti-IgE and CI induced release of histamine from lung mast cells was also observed with TLCK, TPCK and protamine. The maximum inhibition of anti-IgE induced histamine release was approximately 40.7%, 40.2% and 33.4% with TLCK, TPCK and protamine, respectively. At the concentrations tested, TLCK and TPCK by themselves did not stimulate tryptase and histamine release from lung mast cells. A specific inhibitor of aminopeptidase, amastatin, had no effect on anti-IgE induced tryptase and histamine release and was used as control. In conclusion, it was demonstrated that protease inhibitors are able to inhibit IgE dependent tryptase and histamine release from human lung mast cells, which suggested that they could be developed to a novel class of anti-inflammatory drugs to treat allergic conditions in man.

Effect of eucalyptus globulus oil on activation of nuclear factor-kappaB in THP-1 cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study the effect of eucalyptus globulus oil on the activity of nuclear factor-kappaB(NF-kappaB) in THP-1 cell line.</p><p><b>METHODS</b>THP-1 cells were cultured with or without eucalyptus globulus oil at different concentrations (1, 10, 100 mg x L(-1), 30 min) before being stimulated with lipopolysaccharide (LPS, 1 mg x L(-1), 30 min). The location of NF-kappaB p65 subunit (NF-kappaB/p65) in THP-1 cells was detected by indirect immunofluorescence and laser scanning confocal microscope. The expression of NF-kappaB/p65 in nuclei was measured by Western-blot analysis.</p><p><b>RESULT</b>The FITC-label NF-kappaB/p65 was mainly located in the nuclei after THP-1 cells were stimulated with LPS. Whereas, no fluorescence were seen in the nuclei of cells pretreated with eucalyptus globulus oil. This effect on NF-kappaB/p65 nuclear translocation was in a concentration dependent manner.</p><p><b>CONCLUSION</b>Eucalyptus globulus oil inhibits the nuclear translocation of NF-kappaB induced by LPS in THP-1 cells.</p>