Effect of TMB-8 on the activation,proliferation and cell-cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of ［8-(diethylamino) octyl-3, 4, 5 -trimethoxybenzoate］ (TMB-8), an intracellular Ca2+ antagonist, on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (Con A) in vitro. METHODS: After stimulated with Con A, T cells were treated with different concentrations of TMB-8 alone and its combination with cyclosporine A (CsA). The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation-related index was determined by carboxyl fluorescin diacetate succinmidyl ester (CFDA-SE) flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining.RESULTS: After 6 h culture, the activation rate of CD69+ T cell in Con A group was (74.88±1.88)%. 10, 20 and 40 μmol/L of TMB-8 inhibited the expression of CD69 (P<0.01), especially in 40 μmol/L (52.55%±1.54%). After 48 h and 72 h culture, the PI of Con A group was 1.24±0.01, 2.05±0.07, respectively. TMB-8 with the concentration up to 5 μmol/L exerted a definite inhibitory effect on the proliferation with a maximal inhibition in 40 μmol/L(P<0.01). In the combination of 10 μmol/L of TMB-8 with 25 μg/L of CsA, an evident synergistic effect was observed (P<0.01). Moreover, the cell-cycle distribution analysis showed that after 48 h culture, the concentration of TMB-8 over 10 μmol/L showed an evident suppression in S phase.CONCLUSION: TMB-8 significantly inhibites the early steps of the Con A-induced T cell activation and proliferation, as well as the progression of T lymphocytes in S phase.     

Effect of shRNA-mediated survivin gene silencing on sensitivity of lymphoma cells to adriamycin

AIM: This study was designed to use RNA interference technique to down-regulate the expression of survivin gene in human Burkitts lymphoma cell line Daudi and to explore the effect on sensitivity of Daudi cells to adriamycin. METHODS: The survivin-shRNA expression vector was constructed and transfected into Daudi cells. Expression of survivin mRNA and protein were assessed by RT-PCR and Western blotting analysis, respectively. Apoptosis index of transfected Daudi cells was quantified by flow cytometry. The sensitivity of Daudi cells to adriamycin (ADR) before and after transfection was detected by MTT test. RESULTS: The mRNA and protein levels of survivin were down-regulated by 62.32% and 61.88%, respectively, compared to those in control-shRNA treated group and PBS treated group (P<0.05). Meanwhile, the apoptosis index was significantly increased (19.10%±2.15%), compared to that in control group (4.48%±1.54%) and PBS group (4.35%±1.37%, P<0.05). The 50% inhibition concentration (IC50) of ADM to Daudi cells was significantly decreased (0.25±0.43) μmol/L, compared to that in control group (0.87±0.21) μmol/L and PBS group (0.91±0.36) μmol/L, P<0.05. CONCLUSION: Down-regulation of survivin expression in Daudi cells by shRNA effectively induces apoptosis and increases the sensitivity of Daudi cells to ADR.     

Effect of proteasome inhibitor MG132 on proliferation and cell cycle distribution of NK/T cell lymphoma cells

AIM: To study the influence of MG132 on the proliferation and cell cycle distribution of NK/T cell lymphoma cells, and to investigate the potential role of proteasome inhibitor on the treatment of NK/T cell lymphoma. METHODS: NK/T cell lymphoma cells HANK1 were treated with proteasome inhibitor MG132, and the proliferation was evaluated by MTT assay. The morphological changes were observed under inverse microscope. The cell cycle distribution and apoptosis were detected using flow cytometry. RESULTS: The growth inhibitory rate of HANK1 cells was(57.72±7.44)% after cultured for 24 h with 1 μmol/L MG132 and was just(3.98±0.07)% after cultured for 24 h with 0.1 μmol/L MG132. The positive relationship between the concentration of MG132 and growth inhibitory rate was observed. On the other hand, after cultured for 24 h with 1μmol/L MG132, the cells in G1 and G2 phases were(72.33±3.44)% and(12.86±1.29)%, respectively, much higher than those in control group(63.63%±2.29% and 7.94%±1.91%, respectively). The early and late apoptosis rates in MG132 group were 33.57%±2.10% and 16.66%±0.47%, respectively, much higher than that in control group (7.18%±0.82% and 3.81%±1.06%, respectively). CONCLUSION: MG132 inhibits cell proliferation and induces cell cycle arrested at G1 and G2 phases, and cell apoptosis in NK/T cell lymphoma cells in a concentration dependent manner. Proteasome inhibitor may be a good drug to treat patients with advanced NK/T cell lymphomas.     

Quercetin regulates Fas expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To investigate the role of 1, 4, 5- trisphosphate inositol (IP3) and Fas gene expression in apoptosis of HepG2 cells induced by quercetin. METHODS: HepG2 cells were treated with quercetin at different concentrations (including 20, 40, 60, 80 μmol/ L) for 72 h and treated with 60 μmol/ L quercetin for 6 h, 12 h, 24 h, 48 h and 72 h. IP3, Fas mRNA, Fas protein and apoptosis rate were assayed by IP3 - ［3H］ Birtrak assay, RT-PCR, Western blotting and flow cytometry, respectively. RESULTS: When HepG2 cells were incubated with different concentrations of quercetin for 72 h, the IP3 content was lower than those in control. Fas mRNA expression, Fas protein expression and the apoptosis rate were higher than those in control. When HepG2 cells were incubated with quercetin for 6 h, 12 h, 24 h, 48 h, 72 h, the IP3 contents were lower than those in control incubated with 60 μmol/L quercetin for 12 h. Fas mRNA expression was higher than that in control incubated with 60 μmol/L quercetin for 12 h . Fas protein expression was higher than that in control. The apoptosis rate was significantly higher than that in control incubated with 60 μmol/L quercetin for 24 h (P<0.01). CONCLUSION: Quercetin induces apoptosis of HepG2 cells by reducing IP3 production and upregulating Fas gene expression.     

Cyclooxygenase-2 inhibitors enhance the antileukemia activity of STI571

AIM：To investigate whether celecoxib,a cyclooxygenase-2 (COX-2) inhibitor,potentiates the anti-leukemia activity of STI571 in K562 cells.METHODS：K562 cells were treated with STI571,celecoxib or combination of both at different concentrations in suspension culture.Cell proliferation was documented by MTT assay,and cell apoptosis was determined by flow cytometry and morphology.Meanwhile,RT-PCR was applied to analyze the probable mechanism underlying the effects of the drugs.RESULTS：The combination of STI571 and celecoxib dramatically suppressed the proliferation of K562 cells,in which 0.25 μmol/L STI571 and 40.0 μmol/L celecoxib enhanced the inhibiting rate to 76.1%±1.6%.Furthermore,the combining administration of drugs significantly promoted the apoptosis induced by STI571，which showed characteristic changes of morphologic features and increase in sub-G1 cells.By using RT-PCR technique,the expression of COX-2 had no decline by single administration of celecoxib or STI571.However,a progressive down-regulation was caused by coadministration of two drugs.In contrast with COX-2,the expression of VEGF had no changes at any time.CONCLUSION：The administration of celecoxib alone only inhibits the proliferation of K562 cells.Combination treatment with STI571 and celecoxib promotes the apoptosis induced by STI571.     

Effects of recombinant human CD40 ligand on biological behavior of ovarian cancer SKOV3 cells in vitro

AIM: To investigate the effect of recombinated human CD40 ligand (rhCD40L) on the biological behavior of ovarian cancer SKOV3 cell line in vitro.METHODS: After the SKOV3 cells were incubated with different concentrations of rhCD40L for various times, the cell proliferation was determined by MTT assay.The expression of the co-stimulatory molecules or adhesion molecules on SKOV3 cells and the changes of tumor necrosis factor receptor associated factor (TRAFs) inside the cells were measured by flow cytometry and direct immunofluorescence.Annexin V and PI dual color label assay were used to detect cell apoptosis or death in culture contained with rhCD40L.RT-PCR assay was employed to determine the change of apoptosis related gene c-myc, bcl-2 and bcl-xl expression in SKOV3 cells.RESULTS: rhCD40L inhibited proliferation of SKOV3 cells at concentration of 100 μg/L (0.65±0.10 vs 0.81±0.05) and reached a peak at concentration of 10 mg/L (0.13±0.12 vs 0.83±0.15, P＜0.01).The inhibitory effects showed a dose dependent manner.Cell cycle analysis showed that cell division was blocked in G1 phase.Increasing proportion of apoptosis of SKOV3 cells was related to up-regulation of CD95 expression (42.4% vs 59.2%, P＜0.05) and down-regulation of anti-apoptosis genes such as bcl-2 and bcl-xl expressions after incubation with rhCD40L.TRAF 2, 5 and 6 expressed highly in SKOV3 cells.The expression of TRAF 2 (81.3%±9.2% vs 50.4%±5.3%，P＜0.05), TRAF5 (47.2%±7.2% vs 7.2%±2.1%, P＜0.01) and TRAF6 (44.5%±6.3% vs 5.1%±1.1%, P＜0.01) was down-regulated and expression of TRAF 3 (25.2%±6.2% vs 68.8%±5.3%, P＜0.01) was up-regulated after co-culture with rhCD40L, but there was no effects found on the expression of TRAF 1 (4.3%±1.2% vs 5.1%±1.4%) and TRAF4 (7.4%±1.2% vs 8.1%±1.4%).CONCLUSION: By down-regulating expression of bcl-2, bcl-xl and changing expression profile of TRAF, rhCD40L inhibits the growth of SKOV3 cells by blocking the cell cycle progress in G1 and promotes the cells to apoptosis.     

Oxidative stress plays an important role in acetaldehyde-induced cardiomyocytes apoptosis

AIM: To elucidate the mechanism of alcoholic myocardiopathy (AHMD) by exploring the role of ROS mediated oxidative stress in acetaldehyde-induced cardiomyocytes apoptosis. METHODS: Cultured rat cardiomyocytes were stimulated with acetaldehyde (100 μmol/L) for 24 h, then the cell apoptotic index were examined and compared to that with alcohol (100 μmol/L) stimulation. The changes of ROS levels and SOD activities were explored by a time-dependent model in acetaldehyde-induced cardiomyocytes. Meanwhile, the phosphorylation changes of ROS mediated MAPK signaling pathway correlated proteins were also detected, with which compared that changes both in pre-adding NAC acetaldehyde-induced cardiomyocytes, and in H2O2 (100 μmol/L) induced cardiomyocytes, respectively. RESULTS: Acetaldehyde had more obvious apoptotic effect than alcohol through caspase 3 activating (P<0.05, vs control), both cellular ROS level and SOD activity increased in a time-dependent way, and reached the peak value of (78.84%±4.33%) for ROS and (0.55±0.02) for SOD among 18 to 24 h, respectively. Meanwhile, JNK and ERK protein phosphorylation upregulated in acetaldehyde-induced cardiomyocytes, and was reversed by NAC anti-oxidative effect. However all the phosphorylation levels were weaker than that in H2O2-induced group. CONCLUSION: Acetaldehyde causes oxidative damage in cardiomyocytes through enhancing cellular ROS level, and induces cardiocytes apoptosis by activating ROS mediated JNK pathway. The novel way of depressing cellular ROS level or blocking the special apoptotic pathway may have effects on AHMD preservation and therapy.     

Effect of curcumin on the proliferation and apoptosis in LNCap cells

AIM:To observe the effect of curcumin on the proliferation and apoptosis of prostate cancer cell line LNCap. METHODS:LNCap cells were treated with different doses (10 μmol/L, 20 μmol/L, 30 μmol/L, 40 μmol/L) of curcumin and its effects were analyzed in cell growth and apoptosis by microscope, MTT colorimetric assay and flow cytometry. The expression of prostate specific antigen (PSA) was measured by AXSYMTM system-chemical luciferase methods and expression of androgen receptor (AR) was detected by Western blotting. RESULTS:The results showed that curcumin inhibited the proliferation of LNCap cells. The cell growth was inhibited by curcumin in a dose dependent manner and the optimal dose and time was 40 μmol/L, 24 h. Curcumin induced apoptosis in LNCap cells, the most dramatic dose was 40 μmol/L curcumin, at this dose the apoptosis rate was 9.23%. Curcumin inhibited the expression of PSA in LNCap cells and the most dramatic dose and time was 40 μmol/L, 24 h. The PSA in this group was 20% of the control group. Curcumin inhibited the expression of AR on prostate cancer cells. CONCLUSION:Curcumin decreases proliferation and induces apoptosis in LNCap cells in a dose-dependent manner. Curcumin also inhibites the expression of PSA and AR on LNCap cells.     

C*HSDGIC* from cyclization of PACAP (1-5) attenuates UVB-induced apoptosis of human retinal pigment epithelial cells

AIM: To investigate the influence of C^*HSDGIC^* (CHC), a cyclopeptide from the cyclization of with disulfide, on the proliferation and apoptosis of cultured human retinal pigment epithelial (RPE) cells induced by ultraviolet B(UVB). METHODS: The expression of PACAP type 1 (PAC1) receptor in human RPE cells was identified by Western blotting. The cells were exposed to UVB irradiation and cultured in fresh medium with or without gradient concentrations (1 nmol/L to 1 mmol/L) of CHC. The viability of the cells was determined by CCK-8 assay. The early apoptosis of the cells was detected by flow cytometry with annexin V and propidium iodide staining.The mitochondrial menbrane potential was detected by flow cytometry with JC-1 staining. RESULTS: The PAC1 receptor in human RPE cells was identified by Western blotting. The best results of CHC on the proliferation and anti-apoptosis of human RPE cells were achieved at the concentration of 100 μmol/L, which increased the viability by (34.23±3.39)% and (20.10±1.48)%, respectively. The percentage of apoptotic cells was decreased by (5.63±1.49)% with CHC treatment (100 μmol/L) after UVB irradiation,and the percentage of mitochondrium-depolarizing cells was decreased by (5.2±0.5)%. CONCLUSION: PAC1 receptor exists in human RPE cells. C^*HSDGIC^* increases the viability of RPE cells and attenuates UVB-induced apoptosis.     

Effect of camptothecin on the activation,proliferation and cell cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of camptothecin (CPT) on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (ConA) in vitro. METHODS: A model of T cell activation and proliferation was established by stimulated the cells with Con A. T cells were treated with different concentrations of CPT. The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation index was determined by carboxyl fluorescin diacetate succinmidyl ester by flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining. RESULTS: After stimulation with Con A for 6 h, the activation rate of CD69+ T cell in Con A group was (58.88±0.55)%. The percentages of CD69 positive cells were (55.48±0.98)%, (54.67±1.05)%, (50.40±0.82)%, (42.47±1.32)%, correspond to the treatments with different concentrations of CPT (10 nmol/L, 20 nmol/L, 50 nmol/L, 100 nmol/L), respectively. After 48 h treatment with Con A, the proliferation index in different concentrations of CPT treatment (10 nmol/L, 20 nmol/L, 50 nmol/L and 100 nmol/L) exerted a definite inhibitory effect on the proliferation (P<0.01). Moreover, the cell-cycle distribution analysis showed that apoptosis peak was observed in different concentrations of CPT treatment after 48 h cultured with Con A. CONCLUSION: CPT significantly inhibits the early stages of the Con A-induced T cell activation and proliferation, and detents the T lymphocytes in G₀/G₁ phase.     

Effects of GW1929 on macrophage TLR expression and inflammation induced by ox-LDL

AIM: To investigate the effects of ox-LDL on TLR2 and TLR4 expression and production of TNF-α, IL-10, IL-12, NO and MDA in macrophages and to observe intervention effect of GW1929 in above procedure. METHODS: The mouse peritoneal macrophages were pretreated with ox-LDL (50 mg/L, 100 mg/L) and GW1929 (20 μmol/L) respectively for 24 h. The concentrations of MDA, NO-2/NO-3, TNF-α, IL-10 and IL-12 in the culture fluid were detected. Flow cytometry was used to observe TLR2 and TLR4 expressions after the mouse peritoneal macrophages were pretreated with ox-LDL (50 mg/L) and GW1929 (20 μmol/L) respectively for 6 h, 12 h, and 24 h. RESULTS: The concentrations of MDA, NO-2/NO-3, TNF-α and IL-10 in ox-LDL (50 mg/L, 100 mg/L) group were higher than those in control and GW1929 group obviously, but the concentrations of above index in ox-LDL (50 mg/L, 100 mg/L)+GW1929 group were lower than those in ox-LDL (50 mg/L, 100 mg/L) group apparently. No IL-12 in every group was detected. Expressions of TLR-2 in ox-LDL+GW1929 (6 h, 12 h, 24 h) group were lower than those in ox-LDL (6 h, 12 h, 24 h) group respectively. TLR-4 expressions in ox-LDL+GW1929 (12 h) were lower than those in ox-LDL (12 h) apparently. CONCLUSION: ox-LDL up-regulates TLR2 and TLR4 expressions and promotes the production of ROX, NO, TNF-α and IL-10 in macrophages. GW1929 is capable of inhibiting the above ox-LDL effects.     

Inhibitory effects of triptolide on cell proliferation and metastasis in Raji cells in vitro

AIM:To investigate the inhibitory effects of triptolide on cell proliferation and metastasis in Burkitts lymphoma cell line Raji cells.METHODS:The effects of triptolide on the growth of Raji cells were studied by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium(MTT) assay. The effects of triptolide on the cell apoptosis of Raji cells were detected by using Annexin Ⅴ/PI double-labled cytometry. The effects of triptolide on CXCR4 expression on Raji cells were studied by flow cytometric analysis. Chemotaxis assays were performed to observe the effects of triptolide on migration of Raji cells towards recombinant human SDF-1α (rhSDF-1α）in vitro.RESULTS:Triptolide inhibited the proliferation of Raji cells in a dose- and time-dependent way with a 24 h IC50 value of 43.06 nmol/L and a 36 h IC50 value of 25.08 nmol/L. Following the treatment of triptolide, the cell apoptosis rate was increased as the treatment concentration increased and the culture time extended. The effects were dose- and time- dependent. Triptolide could downregulate the expression of CXCR4 on Raji cells in a dose-dependent manner. Moreover, chemotaxis assay suggested that triptolide could block the migration of Raji cells to rhSDF-1α in vitro, and the inhibition was dose-dependent.CONCLUSION:Triptolide could inhibit the cell proliferation and induce the cell apoptosis of Raji cells. Furthermore， it could block the cell metastasis of Raji cells in vitro and the underlying mechanism might be related to the inhibition of the SDF-1/CXCR4 axis.     

Synergistic effect of decitabine and valproic acid on apoptosis and cell cycle arrest at G0/G1 phase in gastric cancer MGC-803 cells

AIM: To investigate the synergistic effect of decitabine (DCA) and valproic acid (VPA) on apoptosis and cell cycle arrest at G₀/G₁ phase in gastric cancer MGC-803 cells. METHODS: Gastric cancer MGC-803 cells were used in the study and divided into the following groups according to the treatment with different drugs for 72 h: DCA 1.5 μmol/L,DCA 3.0 μmol/L, VPA 1.5 mmol/L, DCA 1.5 μmol/L+VPA 1.5 mmol/L and DCA 3.0 μmol/L+VPA 1.5 mmol/L. The early and late apoptotic rates were detected by annexin V and PI staining. The cell cycle was also determined by flow cytometry. The relative nm23-H1 mRNA expression level was measured by real-time quantitative PCR. RESULTS: The apoptotic rates in VPA 1.5 mmol/L+DCA 1.5 μmol/L group (early: 33.58%±3.88%; late: 31.52%±4.20%) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (early: 42.61%±4.23%; late: 38.01%±3.86%), the percentages of the cells in G₀/G₁ phase in VPA 1.5 mmol/L+DCA 1.5 μmol/L group (61.55%±2.38%) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (66.75%±2.48%), and the relative nm23-H1 mRNA expression levels in VPA 1.5 mmol/L +DCA 1.5 μmol/L group (1.84±0.46) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (3.02±0.36) were all significantly higher than those in the corresponding concentrations of single drug treatment groups (P<0.01). CONCLUSION: Synergistic effect of VPA and DCA on apoptosis and cell cycle arrest in gastric cancer MGC-803 cells is possibly via inactivation of nm23-H1 gene expression.     

Selenium dioxide induces leukemia cell apoptosis and changes of intracellular reactive oxygen species (ROS) and Ca2+ levels

AIM: The effects of selenium dioxide (SeO2) on proliferation, apoptosis, intracellular reactive oxygen species (ROS) and Ca2+ levels in three leukemia cell lines NB4, K562 and HL-60 were investigated. METHODS: Three leukemia cell lines were treated with 3-30 μmol/L SeO2. Flow cytometry was used to detect apoptosis rate, and analyze the changes of ROS and Ca2+ level within cells. RESULTS: SeO2 at 10 and 30 μmol/L inhibited proliferation in three leukemia cell lines. Treatment with 30 μmol/L SeO2 for 48 h induced 54.0%, 46.5%, 49.6% apoptosis in NB4, K562, and HL-60 cells, respectively, and also markedly decreased ROS and Ca2+ levels among three cell lines. The rate of ROS positive cells in NB4 and HL-60 decreased with the increase in SeO2 concentrations. ROS was clearly reduced with 30 μmol/L SeO2 in K562. Ca2+ levels were tardily declined with 10, 30 μmol/L SeO2 in NB4 and HL-60 cells. Ca2+ levels were clearly reduced with 30 μmol/L SeO2 in K562. CONCLUSION: SeO2 induces apoptosis in three leukemia cells. The declines of intracellular ROS and Ca2+ levels are involved in apoptosis induced by SeO2.     

Effects of low-dose mifepristone on apoptosis in human ovarian luteinized granulosa cells

AIM: To investigate the effect of different low-dose mifepristone on apoptosis in granulosa cells and to test low-dose mifepristone as an orally contraceptive drug. METHODS: By using immunofluorescence, terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL) and flow cytometry technique, the nuclear morphologic features and ratio of apoptosis and fluorescent intensity of caspase-3 in granulosa cells cultured in vitro treated with different low-doses of mifepristone were observed, respectively. RESULTS: By the display of immunofluorescence, the granulosa cells in treatment group were classified as apoptotic cells on the basis of their morphologic features contained a single condensed chromatin, multiple nuclear fragments. The results of TUNEL showed significant difference between control group and groups treated with different concentration of mifepristone (P<0.01). A significant difference (P<0.01) was also observed between the treatment groups with 1.25 μmol/L and 2.50 μmol/L mifepristone. The fluorescent intensity of caspase-3, observed by flow cytometry showed significant difference (P<0.01) between control group and treatment groups. CONCLUSION: Granulosa cells are induced to apoptosis by low-dose mifepristone, which may be regulated by the activation of caspase-3.     

Effect of activated protein C on apoptosis in human umbilical vein endothelial cells induced by lipopolysaccharide

AIM:To study the effect of activated protein C (APC) at different concentrations on apoptosis of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS).METHODS:The HUVECs were induced by LPS (1.0 mg/L) as apoptotic model that was administered by different concentration of APC (10 μg/L or 50 μg/L). Meanwhile, the control group and induced apoptosis group induced by LPS (1.0 mg/L) stimulation were also set up. The changes of cellular ultrastructures were observed under electron microscope. The DNA ladder and TUNEL fluorescent staining were measured in cells. Annexin-Ⅴ/PI double staining was used to measure the cell apoptosis rate by flow cytometry. Cell survival rate was measured by MTT assay. The proliferating cell nuclear antigen (PCNA) expression levels in cells were also measured by Western blotting to reflect the proliferation of the cells.RESULTS:There were significant apoptotic changes in the cells induced by LPS, but the apoptotic changes were reduced and apoptosis rates were decreased in the cells treated with APC. Meanwhile, cell survival rate and the protein levels of PCNA were increased after APC treatment, particularly at the concentration of 50 μg/L, which showed difference when compared with those induced apoptosis group by LPS (P<0.05).CONCLUSION:APC can inhibit HUVECs apoptosis induced by LPS and promote cell proliferation, thus protect the cells from injury.     

Genistein downregulates survivin gene expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To probe into the role of 1, 4, 5-trisphosphate inositol (IP3) and survivin protein in apoptosis of HepG2 cells induced by genistein. METHODS: HepG2 cells were treated with 60 μmol/L genistein for 12 h, 24 h, 48 h and 72 h. IP3, survivin and apoptosis rate were assayed by IP3-［3H］ Birtrak assay, Western blotting and flow cytometry, respectively. RESULTS: IP3 in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein were significantly lower than that in control (P<0.01) ［(12.0±1.4) pmol/106cells, (7.5±0.8) pmol/106 cells, (5.6±0.5) pmol/106cells, (3.3±0.6) pmol/106 cells， vs (29.2±0.6) pmol/106 cells］. V-survivin/ V-β-actin, which was the gray degree multiply area of survivin/the gray degree multiply area of β-actin in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein, were significantly lower than that in control (P<0.01) ［（0.36±0.13, 0.33±0.03, 0.23±0.04, 0.18±0.04）， vs 0.63±0.06］. The apoptosis rate in groups incubated with 60 μmol/L genistein for 24 h, 48 h and 72 h was significantly higher than that in control （P<0.01） ［(7.4%±0.5%, 20.5%±2.0%, 30.7%±1.6%) vs 2.6%±0.1%］. CONCLUSION: Genistein induces apoptosis in HepG2 cells by reducing IP3 production and survivin protein expression.     

Effect of preconditioning with pioglitazone on ischemia reperfusion/hypoxia reoxygenation-induced mitochondrial structure and membrane potential in rats

AIM: To observe the effect of preconditioning with pioglitazone on ischemia reperfusion/hypoxia reoxygenation-induced mitochondrial ultramicro-structure and membrane potential in rats. METHODS: Sprague-Dawley rats were randomly divided into four groups: sham-operated (SO) group, ischemia reperfusion (IR) group, pioglitazone preconditioning group (Pio-P) and 5-HD+pioglitazone (5-HD+Pio) group. Apart from the SO group, IR, Pio-P and 5-HD+Pio groups were subjected to 30 min ischemia and 4 h reperfusion. The heart was quickly removed for observing the structure of mitochondria and measurement of the apoptosis index (AI) by TUNEL. Primary cultured cardiomyocytes of Sprague-Dawley rats were divided into control, hypoxic reoxygenation (HR) and different concentrations of Pio-P group. JC-1 staining flowcytometry was adopted to examine mitochondrial membrane potential (ΔΨm). RESULTS: The injury of mitochondrial structure in IR group was severer than that in Pio-P group, while the difference between 5-HD+Pio group and IR group was not evident. Flameng score in Pio-P group(1.62±0.60) was significantly lower than that in IR group (2.75±1.09), P<0.01. AI in Pio-P group (28.19%±4.93%) was lower than that in IR group (55.44%±6.63%),P<0.05. The rates of low ΔΨm cells in (5 μmol/L,10 μmol/L and 15 μmol/L) Pio-P group were (45.89±3.63)%, (17.13±1.37)% and (18.43±2.44)%, significantly lower than that in HR group (56.52%±2.87%),P<0.05, while the difference between 10 μmol/L group and 15 μmol/L group was not significant (P>0.05). CONCLUSION: Pioglitazone protects the heart from ischemia reperfusion/ hypoxia reoxygenation injury evidenced by improving mitochondrial ultrastructure and lessening the loss of mitochondrial membrane potential, and decreasing apoptosis. The cardioprotective effects can be inhibited by the blocker of mitochondrial ATP-sensitive potassium channels.