Effects of 2-deoxy-D-glucose and oxaliplatin on proliferation and apoptosis of human hepatoma cell line SMMC-7721

AIM: To investigate the effects of 2-deoxy-D-glucose (2-DG) or oxaliplatin (L-OHP) alone and the combination of both on the proliferation and apoptosis of hepatoma cell line SMMC-7721 in vitro. METHODS: Human hepatoma cell line SMMC-7721 was treated with 2-DG or L-OHP alone, or both. The inhibitory effect on the proliferation of SMMC-7721 cells was estimated by MTT method. The q value, which represents synergistic effect, was determined. Apoptotic rate and cell cycle were assayed by flow cytometry. The activity of caspase-3 was detected by a caspase-3 activity assay kit. RESULTS: 2-DG or L-OHP at different concentrations inhibited the growth of SMMC-7721 cells obviously and the inhibitory effect on SMMC-7721 cell growth strongly depended on the exposure time and dose. When the 2 drugs worked together, the inhibitory effect was improved (P<0.05). 2-DG induced cell apoptosis and arrested the cells at G₂/M phase. When combined with L-OHP,the 2 drugs induced more severe apoptosis and arrested the cells at S and G₂/M phase. Meanwhile, the activity of caspase-3 increased when the 2 drugs used together. CONCLUSION: 2-DG inhibits the growth of hepatoma cell line SMMC-7721. The combination of 2-DG and L-OHP improves the ability of L-OHP to attack the tumor cells. The mechanism might be related to increasing the activity of caspase-3.     

Effect of ginsenoside Rh2 on cell proliferation and cytoskeleton in hepatocarcinoma SMMC-7721 cells

AIM: To investigate the changes of cell growth and cytoskeleton in hepatocarcinoma SMMC-7721 cells treated with ginsenoside Rh₂.METHODS: Cell viability and apoptosis under the conditions of ginsenoside Rh₂ exposure at different concentrations were measured by MTT test and flow cytometry,respectively. The morphological changes of F-actin labeled with FITC-phalloidin were observed under confocal laser scanning microscope. The structures of nuclear matrix-intermediate fibre system were observed under transmission electron microscope (TEM).RESULTS: Rh₂ at 40 mg/L for 4 days inhibited the proliferation and induced apoptosis in SMMC-7721 cells more than those in control group, 10 mg/L Rh₂ group and 20 mg/L Rh₂ group. The F-actin in the cells treated with Rh₂ was well-distributed, lined up in order and the number of fibers increased, while those in the control cells were in disorder and punctiform. The results of whole mount TEM indicated that the intermediate fiber was plentiful, well-distributed and interweaved into a regular network in Rh₂ treated cells.CONCLUSION: Rh₂ effectively inhibits the cell proliferation, increases the cell apoptosis and induces the change of the cytoskeleton alignment in SMMC-7721 cells.     

Role of autophagy inhibitor 3-methyladenine in the injury of U251 cells induced by H2O2

AIM: To investigate the role of autophagy inhibitor 3-methyladenine(3-MA) in the injury of U251 glioma cells induced by H₂O₂. METHODS: The following groups in this study were set up: control group, 10 mmol/L 3-MA group, 1 mmol/L H₂O₂ group and 1 mmol/L H₂O₂ +10 mmol/L 3-MA group. The viability of U251 cells in each group was detected by MTT assay. Autophagic vacuoles in the cells were observed by staining with MDC. The cells were stained with Hoechst 33342 to determine the chromatin condensation. Cell apoptotic ratio was measured by flow cytometry analysis. RESULTS: Compared with control group, no effect of 3-MA on the viability of U251 cells was observed. In H₂O₂ group, the cell viability decreased and cell apoptotic ratio increased.The autophagic vacuoles and nuclear chromatin condensation in the cells were also detected. Compared with H₂O₂ group, addition of 3-MA inhibited the increase in autophagic vacuoles but exacerbated the apoptosis. CONCLUSION: Autophagy inhibitor 3-MA inhibits autophagy partially, but exacerbates apoptosis in U251 cells, indicating that autophagy exerts protective effect in the process of injury in U251 cells induced by H₂O₂.     

The antisense phosphorothioate oligodeoxynucleotides of bcl- 2 oncogene enhances the apoptotic effect of As2O3 on NB4 leukemic cells

AIM: To study and explore whether the antisense phosphorothioate oligodeoxynucleotides (ASODN) of bcl-2 oncogene would increase the apoptotic effect of As₂O₃ on NB4 leukemic cells. METHODS: The biological and morphological changes in NB4 cells from microculture with As₂O₃, bcl-2 ASODN or both were observed. The changes in DNA content and Bcl-2 protein of NB4 cells from microculture with As₂O₃, bcl-2 ASODN or both were determined by tissue chemistry and flowcytometry. RESULTS: There was much more apoptotic effect of As₂O₃ on NB4 cells while it combined with bcl- 2 ASODN (ASODN 10.0 μmol/L+ As₂O₃ 0.25 μmol/L) than alone(ASODN 10.0 μmol/L or As₂O₃ 0.25μmol/L),and so did inhibitory effect of Bcl-2 protein expression by flow cytometry. CONCLUSION:The bcl-2 ASODN can enhance the apoptotic effect of As₂O₃ on NB4 leukemic cells.     

Effect of ginsenoside Rh2 on cultured PC-3M cells in vitro

AIM:To explore the influence of ginsenoside Rh₂ on cultured PC-3M cell cycle in vitro. METHODS:DNA content was measured using [³H]-TdR incorporation assay. To analysis the cycle changes of PC-3M cells, flow cytometry was used in the experiment.RESULTS:The results indicated that the rate of [³H]-TdR incorporation was lower in Rh₂-treated cells than the control in a dose-dependent manner. Flow cytometry demonstrated that the pecent of G₁ phase treated with Rh₂ was higher than that of control. CONCLUSION:These results suggested that PC-3M cells cultured with Rh₂ for 24 h were blocked at G₁ phase, and Rh₂ inhibited the growth of PC-3M cells in vitro.     

外源NO和H2O2诱导不同颜色马铃薯花青素积累

以不同块茎颜色马铃薯品种陇薯8号、陇薯7号、LC310-2和山东彩肉为材料,研究外源NO、H2O2、NO+H2O2处理对淀粉积累期马铃薯块茎花青素合成及相关酶活性的影响。结果表明：经外源NO和H2O2处理后,4个基因型马铃薯薯皮和薯肉花青素含量均升高,PAL、PPO和CHI活性增强|诱导效果为0.1 mmol·L-1 SNP（NO的供体）+0.25 mmol·L-1 H2O2＞0.25 mmol·L-1 H2O2＞0.1 mmol·L-1 SNP＞清水（CK）,说明NO和H2O2处理对马铃薯花青素合成具有一定的促进作用,且表现为加性效应。     

Down-regulation of miR-153-3p attenuates H2O2-induced H9C2 cell injury by promoting Nrf2 expression

AIM To study the effect of microRNA-153-3p (miR-153-3p) knock-down on oxidative injury of H9C2 cells induced by H₂O₂ and its specific mechanism. METHODS The oxidative stress injury of H9C2 cell model was induced by H₂O₂, and then the cell viability and the expression of miR-153-3p were detected by MTT assay and RT-qPCR, respectively. The effects of miR-153-3p knock-down on the H9C2 cell injury under oxidative stress were studied by RNA interference technology. The targets of miR-153-3p were identified by Western blot and dual-luciferase reporter assay. RESULTS MTT assay showed that the viability of H9C2 cells was decreased with the increase in H₂O₂ concentration (P<0.05). The results of RT-qPCR showed that the expression of miR-153-3p was increased with the increase in H₂O₂ concentration (P<0.05). Knock-down of miR-153-3p increased the viability of H9C2 cells under oxidative stress, decreased the cell apoptosis and the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD). The expression of nuclear factor E2-related factor 2（Nrf2） and antioxidant response element（ARE） activity were increased with the increase in H₂O₂ concentration (P<0.01). TargetScan analysis and dual-luciferase reporter assay showed that Nrf2 was one of the potential target genes of miR-153-3p. The results of Western blot further showed that over-expression of miR-153-3p inhibited the expression of Nrf2 (P<0.01), while down-regulation of miR-153-3p increased the expression of Nrf2 (P<0.01). Dual interference with Nrf2 and miR-153-3p significantly reduced H9C2 cell viability, promoted the apoptosis, increased MDA content, and decreased SOD activity in the presence of H₂O₂ (P<0.01). CONCLUSION Inhibition of miR-153-3p expression attenuates the injury of H9C2 cells induced by H₂O₂ through up-regulating Nrf2/ARE signaling pathway.     

Protein expression of cyclin D2 and p16 in proliferation and differentiation of cultured cardiac myocytes

AIM:To investigate the protein expression of cyclin D₂ and p16 in proliferation and differentiation of cultured cardiac myocytes.METHODS:One-day-old Sparague-Dawley rats were used. Cardiac myocytes(CM) were collected by a trypsin-dispersal method and cultured. Cell growth line and fluorescence activated cell sorting (FACS) were used to investigate the proliferation of CM. Ultra-thin sections were made to observe the ultrastructure of CM under transmission electron microscope. The expression of cyclin D₂ and p16 in CM were measured using immunocytochemistry and image analysis.RESULTS:①Results of cell growth line and FACS analysis showed that cultured CM could proliferate in the first 3 cultured days, but the ability decreased quickly, concomitant with differentiation. CM was obseved quiescent in cell cycle three days later. The ultrastructure of CM showed the large amount of myofilaments and mitochondrion. ②The protein expression of cyclin D₂ in 3,4,5 day CM group was 0.89 times(P＜0.05),0.80 times (P＜0.05) and 0.56 times (P＜0.01) of that in 1 day group, respectively. The expression of p16 in CM was increased during the culture process, 2,3,4,5 day group were 1.63 times, 1.72 times, 1.99 times and 2.84 times (P＜0.01) of that in 1 day group, respectively.CONCLUSION:Cultured neonatal rat cardiac myocytes could proliferate during the first 3 days after incubation, but the ability of proliferation decreased, from the fourth day, concomitant with differentiation. Cyclin D₂ and p16 play the key roles in CM postnatal development. Downregulation of cyclin D₂ and upregulation of p16 may induce CM differentiation.     

Astragalosides inhibit autophagy and apoptosis of rat cardiomyocytes induced by H2O2 through mTOR signaling pathway

AIM: To investigate the effects of astragalosides on autophagy and apoptosis of rat cardiomyocytes induced by hydrogenperoxide (H₂O₂).METHODS: The injury model of H9c2 cells induced by H₂O₂ was established, and the cells in astragalosides group and rapamycin group were treated with 20 mg/L astragalosides and 0.1 mg/L rapamycin, respectively. The apoptotic rate was detected by flow cytometry. The autophagy was observed by acridine orange staining. Western blot was used to detect the protein levels of p-mTOR, P70S6K, LC3 and caspase-3. RESULTS: Compared with H₂O₂ group and rapamycin group, the viability of H9c2 cells in astragalosides group was significantly increased (P<0.05). The shape of the H9c2 cells in astragalosides group was complete, the nuclei were stained with yellow-green fluorescence, and the chromatin was distributed evenly. The protein levels of p-mTOR and P70S6K in the H9c2 cells of astragalosides group were significantly increased (P<0.05), whereas the protein levels of LC3, cleaved caspase-3 and caspase-3 in the H9c2 cells of astragalosides group were decreased significantly (P<0.05). CONCLUSION: Astragalosides enhance the viability, inhibit the apoptosis, increase the protein levels of p-mTOR and P70S6K, and decrease the protein levels of LC3, cleaved caspase-3 and caspase-3 in the H₂O₂-induced rat myocardial H9c2 cells. The mechanism is related to the mTOR signaling pathway.     

Lipoxin A4 reduces lipopolysaccharide-induced expression of cyclooxygenase 2 and prostaglandin E2 in human bronchial epithelial cells

AIM: To explore the effects of lipoxin A₄ on the expression of cyclooxygenase 2 (COX-2) in human bronchial epithelial cells (HBECs). METHODS: HBECs were incubated with various concentrations (0.1, 1 and 10 mg/L) of lipopolysaccharide(LPS) for 9 h, or 1 mg/L LPS for different time (3 h, 6 h and 9 h). The levels of COX-2 mRNA in HBECs and prostaglandin E₂ (PGE₂) in the culture supernatant were measured. In addition, the HBECs were exposed to lipoxin A₄ at concentration of 0, 100 and 400 μmol/L after stimulated with LPS at concentration of 1 mg/L for 9 h, and the supernatant of the culture cells was collected for determining the content of PGE₂ by ELISA. The cells were also harvested, and the mRNA and protein levels of COX-2 were analyzed by RT-PCR and Western blotting, respectively. RESULTS: LPS increased the mRNA expression of COX-2 and production of PGE₂ in a dose and time dependent manners in HBECs. Induction of COX-2 mRNA and protein by LPS were inhibited by lipoxin A₄ in a dose-dependent manner. Lipoxin A₄ also significantly decreased LPS-induced production of PGE₂. CONCLUSION: Lipoxin A₄ down-regulates LPS-induced expression of COX-2 and consequently inhibits the production of PGE₂ in HBECs.     

Effect of IL-10 on IL-1β-induced cyclooxygenase-2 expression and prostaglandin E2 release in human mesangial cells

AIM: To investigate the effect of IL-10 on IL-1β-induced prostaglandin E₂(PGE₂) release and cyclooxygenase-2(COX-2) expression in human mesangial cells and to examine whether IL-10 has effect on the biological function of IL-1β.METHODS: The PGE₂ concentration in supernatants of HMC was measured by radioimmunoassay. The COX-2 mRNA and protein were measured by RT-PCR and Western blot, respectively. RESULTS: PGE₂ and COX-2 were significantly increased after treatment with IL-1β(P＜0.01 for both) in cultured human mesangial cells. IL-10 had no effects on basical production of COX-2 and PGE₂(P＞0.05, respectively), while it inhibited IL-1β-elicited PGE₂ production, as well as COX-2 mRNA and protein expression in a concentration-dependent fashion. CONCLUSIONS: These results indicated that IL-10 depressed the IL-1β-induced release of PGE₂ and expression of COX-2. These data suggested that IL-10 could exert anti-inflammatory actions at several levels, not only by inhibiting the production of pro-inflammatory cytokines but also by suppressing their biological function.     

Changes of serum autoantibodies against β1 and M2 receptors in the patients of obstructive sleep apnea syndrome with chronic obstructive pulmonary disease

AIM: To study the changes of serum autoantibodies against β₁-adrenergic and M₂-muscarinic receptors in obstructive sleep apnea syndrome (OSAS) patients with chronic obstructive pulmonary disease (COPD), that is, overlap syndrome (OS). METHOD: Serum autoantibodies against β₁ and M₂ receptors in 26 cases with OS, 32 with OSAS, 30 with COPD and 28 normal subjects were determined by using enzyme-linked immunosorbent assay(ELISA). RESULTS: The positive rates and titer of β₁ and M₂ receptor autoantibodies are significantly increased in OS group (92.2％,57.7% and 1:98, 1:67), compared to OSAS (71.9%, 40.6% and 1:83, 1:30) or COPD group (70.0%, 36.7% and 1:79, 1:28) (P＜0.05), and they are higher in these groups than in the control (25.0%, 14.3% and 1:20, 1:20) (P＜0.01). CONCLUSION: Serum β₁-and M₂ receptor autoantibodies are significantly increased in the patients with COPD, OSAS or OS, compared to the control, and the highest is in OS.     

Induction of apoptosis with Salvia miltiorrhiza monomer IH764-3 via downregulating focal adhesion kinase in H2O2-stimulated hepatic stellate cells

AIM: To investigate the effect of IH764-3 on the apoptosis of H₂O₂-stimulated hepatic stellate cells(HSCs) and the alteration of focal adhesion kinase (FAK). METHODS: The proliferation of HSCs was examined by direct cell count and the apoptosis was determined by Annexin-V/PI labeling, while the morphological change was observed by light microscopy and transmission electron microscopy. In addition, FAK mRNA was detected by RT-PCR. RESULTS: H₂O₂ promoted the proliferation of HSCs. IH764-3 induced the apoptosis of HSCs in a dose-dependent manner. The HSC apoptotic rates of different groups were 6. 35%,9. 28%,15. 10%,19. 69%,respectively, after treated with different concentrations of IH764-3 for 48 h while H₂O₂ group showed 2. 30%. In 30 mg/L group, the apoptosis rates were 6. 73%、10. 34%、15. 10% for the indicated time periods(12 h, 24 h, 48 h). In the presence of IH764-3, FAK mRNA decreased. The FAK mRNA reduction began at 2 h after adding IH764-3. CONCLUSION: IH764-3 induced the apoptosis of HSCs. Down-regulating the expression of FAK mRNA may be one of its mechanisms.     

Comparison of S-RNase,RNase T1, T2, and A effects on growth inhibition and RNA degradation of in vitro-cultured pear pollen

To obtain the basic information on fruit set regulation, effects of several RNases including S-RNase on pollen tube growth and RNA degradation in the tube were studied in the pear. Purified S-RNase from the Japanese pear ‘Kosui’ (S₄S₅) predominantly inhibited the growth of ‘Kosui’ pollen tubes (self) in vitro at 0.28 unit μL⁻¹, but it inhibited ‘Chojuro’ (S₂S₃) pollen (cross) only slightly. The same unit of RNase T₁ (EC 3.1.27.3) clearly inhibited the pollen tube growth, but the action was significantly weaker than that of the S-RNase against the self-pollen. Inhibitory effect of RNase T₂ (EC 3.1.27.1) and RNase A (EC 3.1.27.5) was only slight. The proteins other than the S-RNase extracted from pear style did not have any inhibitory action, though they possessed RNase activity 3.8 times higher than S-RNase. Thus, RNases tested here could not substitute for the S-RNase in specific inhibition against the self-pollen tube growth. Total RNA degradation by each RNase occurred in the pollen tubes as following order; S-RNase (self) ≥T₁ > T₂ ≥ A > S-RNase (cross). Degradation degree of 28S and 18S rRNA was as follows; S-RNase (self) > A > T₁ > T₂ > S-RNase (cross). The degradation of 5.8S and 5S rRNA was; S-RNase (self) > S-RNase (cross) > A > T₂ > T_1. The degree of rRNA degradation was, thus, not always in parallel with the degree of pollen growth inhibition. The S-RNase may degrade not only rRNA but also mRNA essential for pollen tube growth, and may be specifically adapted to inhibit the growth of self-pollen tubes. Therefore, controlling S-RNase amount in the style will produce self-thinning cultivars efficiently, which are unnecessary not only for hand-pollination but fruit-thinning practices in the pear. Practically, cultivar with weak self-incompatibility and small amount of S-RNase, such as ‘Okusankichi’, may be an expecting candidate for breeding self-thinning cultivars.     

Effects of proline on antioxidant system in leaves of grapevine (Vitis vinifera L.) exposed to oxidative stress by H2O2

Although proline is one of the major computable organic solutes that accumulate in many plant species in abiotic stresses, a hot debate continues about whether proline accumulation is a reaction to abiotic stress, or a plant's response is associated with stress tolerance. The effects of proline on antioxidative system in grape leaves of Vitis vinifera L. cv., ‘Öküzgözü’ exposed to oxidative stress by H₂O₂ was investigated. Endogenous proline, hydrogen peroxide (H₂O₂), malondialdehyde (MDA) concentrations, percentage of electrolyte leakage (EL), and some of the antioxidant enzyme activities; such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) were measured spectrophotometrically. Inhibitory effect of H₂O₂ on antioxidant enzyme activities, MDA, and EL was found. In the presence of proline, SOD and CAT activities decreased, while POD and APX activities increased. Proline pre-treatment resulted in a decrease in cellular H₂O₂ content, MDA, and EL, while cellular concentration of proline increased. Based on the finding, it was suggested that proline and H₂O₂ could play an important role in oxidative stress injury of grapevine leaves grown in vitro culture. Also, proline might have a direct positive effect on antioxidant enzyme system, membrane phase change, MDA, and EL.     

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.     

Regulation of LPS-induced elevation of Ca2+ intracellular level of alveolarmacrophages in chronic bronchitis by Angelica Sinensis and nifedipine

AIM: To explore regulation of lipopolysaccharide (LPS)-induced elevation of Ca²⁺ intracellular level in alveolar macrophages(AMs) from patients with chronic bronchitis by Angelica Sinensis and nifedipine.METHODS:AMs was obtained from 7 patients with chronic bronchitis and 6 normal controls by bronchoalveolar lavage and intracellular Calevel was detected after adding Angelica Sinensis, nifedipine or LPS to the supernatant of AMs loaded by Fura-2. RESULTS: In contrast with normal control group (99.65±32.21 nmol/L), intracellular Ca²⁺ level in AMs from chronic bronchitis group (189.47±23.69 nmol/L) was increased significantly in the absence of extracellular Ca²⁺ but not 1 mmol/L. Intracellular Ca²⁺ level in AMs from chronic bronchitis group were significantly increased by adding 10 μg/mL LPS to the supernatant of AMs. LPS-induced elevation of intracellular Ca²⁺ level in AMs from chronic bronchitis group was completely inhibited by Angelica Sinensis or nifedipine.CONCLUSION: Both Anelica Sinensis and nifedipine may inhibit activation of AMs from patients with chronic bronchitis by reducing LPS-induced elevation of intracellular Ca²⁺ level in AMs, suggested that these two medicines may inhibit non-specific inflammation of airways in chronic bronchitis.