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: To assess the () recombinant gingivalis gingipain R2 (rRgpB)-induced Ca mobilization in human gingival fibroblast (HGF) mediated by protease-activated receptor (PAR) and its downstream signal transduction pathways. : Flow cytometry was used to detect the expression of PAR in HGF. The proliferation of HGF was measured by CCK-8. The dynamic changes of intracellular Ca concentration in HGF induced by rRgpB and the blocking effect of PAR-1 antagonist were observed by laser confocal microscopy. Western blot was performed to determine the phosphorylation levels of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (p38 MAPK) and p65 in HGF. : PAR-1 and PAR-3 were expressed in HGF, and the rRgpB could promote the proliferation of HGF. rRgpB caused a transient increase in [Ca], which could be completely suppressed by vorapaxar, a PAR-1 antagonist. The phosphorylation levels of JNK, ERK1/2 and p65 were significantly up-regulated after the induction of rRgpB for and (all <0.05), which was completely inhibited by vorapaxar. However, the phosphorylation level of p38 MAPK had no significant change after rRgpB stimulation. : rRgpB causes an increase in [Ca] in HGF mediated by PAR-1. JNK, ERK1/2 and nuclear factor-κB may be involved in intracellular signal transduction after PAR-1 activation.
Subject(s)
Humans , Fibroblasts , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling System , Phosphorylation , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Objective: To observe the effect of CoCl on the cisplatin sensitivity of human ovarian cancer SKOV3 cells, and to clarify the possible mechanism. Methods: The SKOV3 cells were Cultured in vitro and randomly divided into control group, CoCT group, cisplatin (DDP) group and CoCT combined with DDP (combination) group. The cells in CoCL group were Cultured in normal cell medium for 20 h after cultured in 200 pmol • L CoCL for 4 h, the cells in DDP group were cultured in normal cell medium containing 10 mg • L DDP for 24 h, and the cells in combination group were cultured in 10 mg • L DDP for 20 h after cultured in 200 //mol • L CoCl • for 4 h. The survival rates of SKOV3 cells in various groups were detected by MTT method, and the positive expression intensities of hypoxia-inducible factor-1 a ( HIF-la) and inducible nitric oxide synthase (iNOS) in the cells in various groups were detected by immunofluorescence method. Rhod 2-AM fluorescence probe was used to observe the levels of Ca in mitochondria in the cells in various groups. Western blotting method was used to observe the expression levels of cytochrome C (cyto C) cysteinyl aspartasc 3 (caspasc 3) and cleaved cysteinyl aspartasc 3 (cleaved caspase 3). Muse apoptosis assay kit was used to detect the apoptotic rates of cells in various groups. Results: Compared with control group, the survival rate of the cells in CoCI group had no significant change (P> 0.05). and the survival rates of the cells in DDP and combination groups were decreased ( P0. 05) . and the expression levels of cyto C. caspase 3 and cleaved caspase 3 in DDP group were increased significantly ( P < 0.05); comparexl with DDP group, they were lower than those in combination group ( P<0. 05). Comparexl with control group∗ the apoptotic rate of SKOV3 cells in DDP group was increased significantly (P<.0. 05); the apoptotic rate of SKOV3 cells in combination group was lowe'r than that in DDP group (P<0. 05). Conclusion: CoCI can redece the mitochondrial apoptosis of human ovarian cancer SKOV3 cells by inhibiting the DDP-inducexl enhancement of iNOS expression and dccrease the sensitivity of SKOV3 cells to cisplatin.
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Objective To explore a simplified method of coagulation test for the individuals with erythrocytosis. Methods The antico-agulants, blood volume and fixed blood collecting volume were adjusted by the formula: anticoagulants (mL)＝(100－HCT×100)× blood (mL)×0.001 85. A total of 124 blood samples for coagulation testing in which the calcium ( Ca2+) interval was designated and hematocrit (HCT) was more than 55% were tested with calibrated anticoagulants, adjusted blood volume and fixed blood collection vol-ume [anticoagulant(mL)/0.055 5]. The results of plasma prothrombin time (PT), international standardization ratio (INR) and acti-vated partial thromboplastin time (APTT) before and after adjustment were compared. The results of the samples from 3 groups after adjustment were also compared. The relationship of HCT with unadjusted PT and APTT were simultaneously observed. Results The unadjusted results of PT, INR and APTT were significantly higher than those after anticoagulants adjustment (27.52±16.37 vs 12.49± 1.35, 2.31±1.47 vs 0.99±0.11 and 50.09±13.32 vs 33.37±5.05) with statistically significant difference in paired comparison (P＜0.05). No statistical difference was found in the comparison of the results for PT, INR and APTT after adjustment within the 3 groups ( PT: 12.49±1.35 vs 12.84±1.54 vs 12.82±1.76, INR:0.99±0.11 vs 1.02±0.13 vs 1.02±0.15, APTT: 33.37±5.05 vs 33.49±5.09 vs 32.83±5.06) (P＞0.05). HCT values of the patients were positively correlated with unadjusted PT (r＝0.461, P＜0.05) and APTT (r＝0.571, P＜0.05). Conclusion The coagulation test of the individuals with erythrocytosis may use to adjust the blood volume and the fixed blood collection volume provided calcium concentration in reference interval.
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Objective@#To investigate the effect of overexpression of wild-type phosphatase and tensin homolog (PTEN) deleted on chromosome 10 and its mutant G129E (exhibiting the activity of protein phosphatase and losing the activity of lipid phosphatase) on F-actin in activated hepatic stellate cells (HSCs) cultured in vitro.@*Methods@#The activated hepatic stellate cell-T6 (HSC-T6) cells were cultured in vitro, and activated HSCs were transfected with adenovirus that carried wild-type PTEN gene and G129E gene using transient transfection. The HSCs were divided into the following groups: control group, which was transfected with DMEM medium instead of virus solution; Ad-GFP group, which was transfected with the empty adenovirus vector with the expression of green fluorescent protein (GFP); Ad-PTEN group, which was transfected with the recombinant adenovirus with wild-type PTEN gene and GFP expression; Ad-G129E group, which was transfected with the recombinant adenovirus with G129E gene and GFP expression. Western blot and quantitative real-time PCR were used to measure the protein and mRNA expression of PTEN in activated HSCs; under a laser scanning confocal microscope (LSCM), phalloidine labeled with the fluorescein tetramethylrhodamine isothiocyanate (TRITC) was used to observe the morphology of HSCs, distribution and fluorescence intensity of F-actin, and changes in pseudopodia and stress fibers, and a calcium fluorescence probe (Rhod-2/AM) was used to measure the changes in Ca2+ concentration in HSCs. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference test was used for comparison between two groups.@*Results@#Wild-type PTEN and G129E genes were highly expressed in activated HSCs. In the control group and the Ad-GFP group, HSCs had a starlike or polygonal shape, F-actin was reconfigured and formed a large number of stress fibers which stretched across the whole cell, and layered pseudopodia were seen around the cell. In the Ad-PTEN group and the Ad-G129E group, the HSCs had a fusiform shape, F-actin was mainly seen around the cell, a small number of stress fibers were seen inside the cell, and layered pseudopodia around the cell disappeared. The Ad-PTEN group and the Ad-G129E group had significant reductions in the fluorescence intensity of F-actin compared with the control group and the Ad-GFP group (357.67±13.39/377.25±14.55 vs 961.87±27.33/954.68±20.71, F = 1783.486, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05). The Ad-PTEN group and the Ad-G129E group had significant reductions in the relative concentration of Ca2+ compared with the control group and the Ad-GFP group (251.60±90.88/352.18±146.01 vs 1953.95±132.99/1937.57±115.17, F = 834.988, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05).@*Conclusion@#The overexpressed wild-type PTEN and its mutant G129E can significantly inhibit the formation and reconfiguration of cytoskeletal protein F-actin and reduce the concentration of Ca2+ in activated HSCs in vitro. In addition, there are no significant differences in the above effects between wild-type PTEN and G129E.
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Objective To investigate the effect of Terpinen-4-ol on the proliferation of lung adenocarcinoma A-549 cells and its related mechanism. Methods A-549 cells were treated with different concentrations of Terpinen-4-ol. The inhibitory effect of Terpinen-4-ol on A-549 cells was tested by MTT method. Cell grow ability was determined by CCK-8 colorimetry. The ultrastructure of A549 cells were observed by transmission electron microscopy before and after Terpinen-4-ol treatment. The changes of cell cycle, apoptosis, and the level of intracel-lular calcium were inspected by flow cytometry. Inoculated the lung adenocarcinoma A-549 cells on the nude mice to form transplantation tumor. The experimental nude mice with transplantation tumors were divided into three groups:negative control group,high dose positive con-trol group and low dose positive control group. The mice were given continuously intraperitoneal injection for 10 days, and then the transplan-tation tumors were taken and the size and weight of them were detected. Results After Terpinen-4-ol treatment for 24 h,MTT assay showed that the IC50 value of A549 cells was 0. 067% v/v. The growth curves of positive control groups were significantly smooth than the negative control group. The formation of autophagosome increased after treatment with Terpinen-4-ol. The results of flow cytometry showed that the cell cycle was arrested in S phase,Terpinen-4-ol could induce apoptosis of A549 cell, The intracellular calcium concentrations in positive control groups were significantly higher than the negative control group(P<0. 05). Low dose group and high dose group restrained the growth of the transplantation tumor obviously, and the tumor inhibitory rate were 53. 33% and 77. 76% respectively. Conclusion Terpinen-4-ol has inhibitory effect on the proliferation of A-549 cells in vitro and in vivo.
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Objective To investigate the change of intracellular calcium ion concentration in prostate smooth muscle cells of SD rats with chronic abacterial prostatitis under high potassium solution.Methods SD rats were divided into experiment group and control group.The CP model was set up by castration and estradiol injection.The PSMC was cultured and purified in vitro.Laser confocal scanning microscope was used after the ceils were incubated with Quest Fluo-8TM.The cells were treated with high potassium solution,and the change of fluorescence intensity was observed.Results The pathologic specimens of the experiment group showed typical pathologic characteristics of chronic prostatitis under light microscope,the control group without inflammation performance.Using immunocytochemistry method confirmed that the experiment group and the control group were prostate smooth muscle cells.The change of fluorescence intensity of [Ca2+] i in the experiment group and control group in the high potassium solution was 27.86 ± 9.88 and 7.61 ± 4.31.There were statistically significant differences between the two groups (P ＜ 0.01).Conclusions High potassium solution cause intracellular calcium ion concentration increased.
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Objective: To study the e ects of Heying Anxin Fang on cardiac function and intracellular Ca2+ concentration in cadiocyte of chronic congestive heart failure rats. Methods: Animal models of heart failure were induced by subcutaneous injection of a large dose of ISO, and were randomly divided into model group, Shenmai injection group, captopril group, high, middle and low dosages of Heyinganxin Fang group and normal group with 9 in each group. To determine the indexes of LVSP, LVEDP, +dp/dtmax ,-dp/dtmax and heart weight, and determine Ca2+ concentration in cardiomyocytes by ow cytometer. Results: Compared with normal group, the indexes of heart weight, Ca2+ concentration and LVEDP in model group increased, but indexes of LVSP, +dp/dtmax, -dp/dtmax decreased obviously(P
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The purpose of this study was two-fold. First was to evaluate whether the molecular sieving model was appropriate for ionic dissociation experiment. Second was to compare the dissociation of calcium and hydroxyl ions from five types of calcium hydroxide pastes (Pure calcium hydroxide paste, DT temporary dressing(R), Metapaste(R), Chidopex(R), Metapex(R)) in three vehicles (aqueous, viscous and oily) and the antibacterial effect. Each calcium hydroxide pastes was placed into 0.65ml tube with cap and then 15% polyacrylamide gel was placed onto calcium hydroxide pastes. After the gel was hardened, the tubes were filled with tridistilled water (pH 7.14) and closed with cap. The tubes were stored in 37degrees C, 100% incubator. The pH reading and the concentration of calcium ions were taken at 1, 4, 7, 10, and 14 days. The brain heart infusion agar plates with S. mutans and A. actinomycetemcomitans were used for antibacterial activity test. Middle of agar plate was filled with the calcium hydroxide pastes. The plates were incubated at 37degrees C and observations were made to detect the zones of inhibition. These data were evaluated statistically by use of the analysis of variance and duncan test. The results were as follows. 1. In fresh mixing state, the pH of five types of calcium hydroxide pastes were measured between 12.5 and 12.8. 2. The pH was increased in all five types of calcium hydroxide pastes compared with control group. In 14 days, Pure calcium hydroxide paste (11.45) and DT temporary dressing(R) (11.33) showed highest pH, followed by Metapaste(R) (9.49), Chidopex(R) (8.37) and Metapex(R) (7.59). 3. Calcium was higher in all five types of calcium hydroxide pastes compared with control group. In 14 days, Pure calcium hydroxide paste (137.29 mg%) and DT temporary dressing(R) (124.6 mg%) showed highest value, followed by Metapaste(R) (116.74 mg%), Chidopex(R) (111.84 mg%) and Metapex(R) (60.22 mg%). 4. The zones of bacterial inhibition were seen around all five types of calcium hydroxide pastes. Chidopex(R) and Metapex(R) groups which include iodoform were observed significantly larger zone of inhibition in A. actinomycetemcomitans compared with the other calcium hydroxide groups (p<0.05). However, Metapex(R) showed the least antibacterial effect on S. mutans compared with other groups (p<0.05). The molecular sieving model was found to be acceptable in dissociation experiment of hydroxyl and calcium ions when compared with the previous tooth model study. But this model was not appropriate for the antibacterial test.
Subject(s)
Acrylic Resins , Agar , Brain , Calcium , Calcium Hydroxide , Dissociative Disorders , Heart , Hydrocarbons, Iodinated , Hydrogen-Ion Concentration , Hydroxides , Incubators , Ions , Ointments , Tooth , WaterABSTRACT
Extensive use of tributyltin (TBT) has caused serious environmental pollution, which is harmful to human and other organisms. As a cytotoxic chemical, an important mode for TBT effects on the cells is to induce apoptosis. The present paper mainly reviewed the mechanism by which TBT induces apoptosis at low concentration.
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Fura—2 was used as a Ca~(2+) indicator to determine the intracellular calcium ion concentra-tion(〔Ca~(2+)〕i)of rat peritoneal macrophages(RPM?s),and APAAP enzyme immnoassay was ap-plied to detect the expression of Ia antigens on RPM?s.The results showed that norepinephrine(NE,10~(-9)mol/L)could markedly increase the〔Ca~(2+)〕i of the RPM?s(p
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Objective To study the mechanism of myocardical aging. Methods Wistar rats used were divided aging group (22 months old) and adult group (7 months old). Qualitative morphological changes of organelles of myocardium were observed by TEM. Quantitative morphological changes of organelles of myocardium were determined by stereological method, Ca 2+ regulation of organelles of myocardium were analysed by EDS. Results Compared with adult group, aging group:(1) The nuclei were indented, myofibril were arranged irregularly, intercalated disk were separated, mitochondria and sarcoplasmic reticulum were swelled, lipofuscin and residual body were increased. (2) The volume of myocardium not occupied by myocyte were increased, the volume density of mitochondria and sarcoplasmic reticulum were reduced, specific surface of outer membrance of mitochondria inter membrane plus cristae of mitochondria and the membrane of sarcoplasmic reticulum were also reduced. (3) Ca 2+ in myofibril and mitochondria were increased, but Ca 2+ in sarcoplasmic reticulum were decreased. Conclusion The contractility of aging myocardium were declined, the morphological changes and Ca 2+ regulation of mitochondria and sarcoplasmic reticulum might be directly related to myocardical aging.