Effects of calcium ionophore A23187 on proliferation ,cycle and expression of caspase-3 in hepatic stellate cells stimulated by transforming growth factor-β1 / 西安交通大学学报(医学版)

Objective To study the effect of calcium ionophore A23187 on the proliferation , cycle and expression of caspase-3 in rat hepatic stellate cells by stimulated transforming growth factor Bata 1 (TGF-β1 ) . Methods Hepatic stellate cells were cultured in 37 ℃ and 50 mL/L CO2 incubator .The cells were divided into 5 groups:blank group ,TGF-β1 (5 ng/mL) group ,TGF-β1 +low-,medium-and high-dose calcium ionophore A23187 groups:5 ng/mL TGF-β1 stimulation for 24 h ,and then 1μmol/L ,2μmol/L and 4 μmol/L of calcium ion carrier A23187 was added and treated for 24 h .The cells proliferation was detected by MTT .The cell cycle was detected by flow cytometry and the expression of caspase-3 was detected by immunoblotting .Results Different concentrations of calcium ionophore A23187 could significantly inhibit the proliferation of cells ( P< 0 .05 ) . And the dose of calcium ionophore A23187 increased ;RGR in the low-,medium-and high-dose groups was 85 .93% ,61 .71% ,and 48 .43% (P<0 .05) .There was significant difference between the two groups (P<0 .05) .The higher the dose of calcium ionophore A23187 ,the higher the proportion of G1 phase cells ,the lower the ratio of S+ G2 cells ( P<0 .05) ,with significant difference (P<0 .05) .With the increase of calcium ionophore A23187 concentration ,the expression of caspase-3 protein increased significantly ( P< 0 .05 ) . Conclusion Calcium ionophore A23187 prevents hepatic stellate cells from G1 phase to S phase and G2 phase ,inhibits its proliferation and up-regulates the expression of caspase-3 .

Involvement of throm box aneA2 and tyrosine kinase in the synergistic interaction of platelet activating factor and calcium ionophore A23187 in human platelet aggregation

The present study was carried out to examine the mechanisms of the synergistic interaction of PAF and A23187 mediated platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of PAF (5 nM) and A23187 (1 micrometer) was inhibited by PAF receptor blocker (WEB 2086, IC50=0.65 micrometer) and calcium channel blockers, diltiazem (IC50=13 micrometer) and verapamil (IC50=18 micrometer). Pretreatment of platelets with PAF and A23187 induced rise in intracellular calcium and this effect was also blocked by verapamil. While examining the role of the down stream signaling pathways, we found that platelet aggregation induced by the co-addition of PAF and A23187 was also inhibited by low concentrations of phospholipase C (PLC) inhibitor (U73122; IC50 = 10 micrometer), a cyclooxygenase inhibitor (indomethacin; IC50=0.2 micrometer) and inhibitor of TLCK, herbimycin A with IC50 value of 5 micrometer. The effect was also inhibited by a specific TXA2 receptor antagonist, SQ 29548 with very low IC50 value of 0.05 micrometer. However, the inhibitors of MAP kinase, PD98059 and protein kinase C, chelerythrine had no effect on PAF and A23187-induced platelet aggregation. These data suggest that the synergism between PAF and A23187 in platelet aggregation involves activation of thromboxane and tyrosine kinase pathways.

Induction of acrosome reaction by calcium ionophore A23187 in sperm separated by two-layer percoll gradient method.

The aim of this study was to compare thepercentages of sperm with an acrosome reaction between those with and without calcium ionophore A23187 induction after two-layer Percoll gradient separation. Thirty normal semen samples were obtained from the male partners of infertile couples attending the Infertility Clinic at Siriraj Hospital. After the process of sperm separation by two-layer Percoll gradient technique, the final samples samples were divided into 2 portions. An aliquot of 10 ตM of calcium ionophore A23187 was added to one portion to induce an acrosome reaction, while the other portion was used as a control. Fluorescein isothiocyanate-conjugated Pisum sativam agglutinin (FITC-PSA) staining was performed on both specimens and the acrosome reated-sperm were evaluated. The percentage of acrosome-reated sperm in the calcium ionophore A23187 induced group was significantly higher than those of the control group (24.8+6.6vs 15.4+6.0;p < 0.001). It is concluded that calcium ionophore can significantly induce an acrosome reaction on sperm separated by two-layer Percoll gradient technique, and it may be beneficial to add calcium ionophore A23187 to sperm preparation for use in IUI or IVF.

Rescue activation of calcium ionophore A23187 on unfertilized human oocytes after conventional in-vitro fertilization and intracytoplasmic sperm injection / 中国病理生理杂志

AIM: To explore the activation effect of calcium ionophore A23187 on unfertilized human mature oocytes after conventional in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). METHODS: Thirty-seven unfertilized mature oocytes from IVF and 41 after ICSI were included in our experiment. They were incubated in 5 ?mol/L calcium ionophore A23187 for 5 minutes. Second polar body extrusion and pronuclear formation were recorded 12-16 hours later. The activated oocytes were cultured for another 2 days in vitro. RESULTS: Activation rate of unfertilized oocytes from conventional IVF and ICSI were 64.9%(24/37)and 73.2%(30/41), respectively. Among 41 unfertilized oocytes after ICSI treated with A23187, 30 were activated and 24 had 2 polar body (PB) and 2 pronuclear (PN). But for the unfertilized oocytes from conventional IVF only 20% activated oocytes had 2 PN and 2 PB. The percentage difference of oocytes containing 2 PB and 2 PN between the two groups was significant ( P