Effects of PKC and ROCK on vasodilation induced by nifedipine

AIM: To investigate the effects of Rho-associated kinase (ROCK) and protein kinase C (PKC) on the relaxation of isolated rat aortic rings induced by nifedipine and the mechanisms. METHODS: The changes of tension in vascular rings induced by nifedipine under the basic condition and pre-contracted by norepinephrine (NE, 10^-6 mol/L) or KCl (60 mmol/L) were observed. The effects of ROCK and PKC on the vasodilation induced by nifedipine were studied using the vascular ring perfusion device. RESULTS: Nifedipine (10^-10 mol/L, 10^-9 mol/L, 10^-8 mol/L, 10^-7 mol/L, 10^-6 mol/L and 10^-5 mol/L) had no significant relaxation effect on isolated aortic rings under basic condition. Nifedipine induced dose-dependent relaxation in both endothelium-intact and endothelium-denuded aortic rings pre-contracted by 10^-6 mol/L NE and 60 mmol/L KCl (P<0.05). No obvious difference between endothelium-intact group and endothelium-denuded group was observed. After incubation of the PKC inhibitor staurosporine (STA, 10^-8 mol/L) and PKC agonist phorbol 12-myristate 13-acetate (PMA, 10^-7 mol/L), STA increased the relaxation induced by nifedipine, while PMA reduced the effect of nifedipine on blood vessels (P<0.05). After the incubation of the ROCK inhibitor fasudil (10^-6 mol/L) and ROCK agonist angiotensin Ⅱ (Ang-Ⅱ, 10^-9 mol/L), fasudil increased the relaxation induced by nifedipine, while Ang-Ⅱ reduced the effect of nifedipine on blood vessels (P<0.05). The relaxation induced by nifedipine was not statistically inhibited by BaCl₂ (10^-4 mol/L), tetraethylammonium (10^-3 mol/L), glibenclamide (10^-5 mol/L) and 4-aminopyridine (10^-3 mol/L). In calcium-free and high-potassium solution, pre-treatment with nifedipine (10^-9 mol/L, 5×10^-8 mol/L and 10^-6 mol/L) inhibited calcium-induced contraction of the aortic rings (P<0.05). However, nifedipine pre-treatment did not affect the contraction induced by NE in Ca²⁺-free medium. CONCLUSION: Nifedipine exhibits vasodilatation effect in a dose-dependent manner and the vasodilatation activity is endothelium-independent. The vasodilatation effect of nifedipine may be related to the inhibition of extracellular calcium influx, and inhibition of PKC and ROCK enhances the vasodilatation effect of nifedipine.     

Effect of inhaled nitric oxide on adhesion molecule CD11b expression in lung neutrophils of meconium aspiration rabbits

AIM:To evaluate effects of inhaled nitric oxide(iNO) on adhesion molecule CD11b expression on lung neutrophils in experimental meconium aspiration syndrome(MAS) rabbits treated with conventional mechanical ventilation under room air or 100%O₂. METHODS:Animals were randomly allocated to 8 groups(n=48) of 6 each: two MAS model groups(under room air or 100%O₂ without iNO treatment), 6 treatment groups were treated with continuous NO inhalation at a dose of 0.2×10^-6mol/L, 0.33×10^-6mol/L or 0.67×10^-6mol/L respectively for 12 hours under room air or 100%O₂. Mean systemic arterial pressure(SAP) and methemoglobin (MeHb) were performed at basement time, 0, 2, 4, 12 hours. Expression of CD11b on neutrophils in the bronchoalveolar lavage fluid(BALF) was detected with flow cytometry. RESULTS:SAP, MeHb at different time among different groups were within the normal scale. CD11b expression on the neutrophils in the BALF significantly decreased in groups of inhalation 0.33×10^-6 mol/L or 0.67×10^-6 mol/L NO, compared with the two MAS model groups. (x±s: under 21%O₂, 0.33×10^-6 mol/L NO, 121±20 υs 392±204; 0.67×10^-6 mol/L NO, 112±30 υs 392±204;under 100%O₂, 0.33×10^-6 mol/L NO, 113±24υs293±65; 0.67×10^-6 mol/L 102±114 υs293±65, P<0.05). 0.2×10^-6mol/L NO inhalation did no effect on CD11b expression. (x±s:21%O₂, 190±101 υs 392±204; 100%O₂, 222±85 υs 293±65; P>0.05). No statistic difference was observed between groups inhaled 0.33×10^-6 mol/L NO and 0.67×10^-6mol/L NO. CONCLUSION:0.33×10^-6 mol/L or 0.67×10^-6 mol/L NO inhalation down-regulated the CD11b expression on the neutrophils in BALF to reduce the sequestration of neutrophils in rabbit lung.     

Meglumine cyclic adenylate induces differentiation of bone marrow mesenchymal stem cells into cardiomyocytes in vitro

AIM: To study the effect of meglumine cyclic adenylate (MCA) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocytes in vitro. METHODS: The whole bone marrow adherent culture method was used to isolate, culture and amplify the BMSCs. The surface markers of BMSCs were determined by flow cytometry analysis. MCA at concentrations of 10^-2 mol/L, 10^-3 mol/L, 10^-4 mol/L, 10^-5 mol/L, 10^-6 mol/L and 10^-7 mol/L was added to the culture medium containing the second generation of BMSCs.5-Azacytidine(5-Aza) was used as a positive control. The cell viability was measured by MTT method.The cAMP content in BMSCs was detected by ELISA. The mRNA expression of GATA-4, Cx43 and β-MHC in MCA group and MCA+H89 (a PKA inhibitor) group was measured by SYBR-RT-PCR. The differentiation effects of MCA and 5-Aza were compared by flow cytometry. RESULTS: Most of the BMSCs expressed CD44 and CD71, and did not express CD45. MCA inhibited the viability of BMSCs in a time-and dose-dependent manner, and MCA atthe concentration of 10^-2 mol/L showed particularly remarkable effect. MCA significantly increased intracellular cAMP level in BMSCs in a concentration-dependent manner. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group were significantly higher than that in blank group (P<0.05), and the highest effect was under the condition of MCA induction at the concentration of 10^-3 mol/L for 3 days. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group was higher than that in 5-Aza group and H89+MCA group (both P<0.05). Differentiation rate in MCA group was slightly higher than that in 5-Aza group (20.24%±1.02% vs 18.39%±0.58%, P<0.05). CONCLUSION: MCA stimulates BMSCs to increase intracellular cAMP production and inhibits the viability of BMSCs, thus promoting the mRNA expression of GATA-4, β-MHC and Cx43 through the cAMP/PKA signaling pathway.     

Inhibitory effect of ampelopsin combined with mitomycin on proliferation of gastric carcinoma cells

AIM: To investigate the synergistic effects of ampelopsin (AMP) and a chemotherapeutic drug mitomycin (MMC) on the proliferation of gastric cancer cell line SGC-7901.METHODS: SGC-7901 cells were cultured in vitro and divided into 4 groups: control group, AMP group, MMC group and AMP+MMC group. Cell proliferation was measured by MTT method. The apoptotic index was examined by flow cytometry. The expression of apoptotic proteins, Bcl-2 and survivin, was detected by Western blotting.RESULTS: AMP at the concentrations ranging from 2.2 mg/L to 14.84 mg/L exerted inhibitory effect on the growth of SGC-7901 cells. Cell proliferation in AMP (14.84 mg/L) group was inhibitory by (60.85±1.13) %, significantly higher than that in control group (P<0.05). The inhibitory rates of cell proliferation varied from (17.40±0.30) % to (72.23±1.36) % when the concentrations of MMC increased from 1×10^-3 g/L to 1×10^-2 g/L. AMP combined with MMC showed a synergistic effect on the growth of SGC-7901 cells. The inhibitory rates of cell proliferation varied from (21.83±2.50) % to (46.70±1.45) % when the concentrations of MMC increased from 1×10^-3g/L to 5×10^-3g/L. The inhibitory effect of AMP plus MMC was higher than that of MMC or AMP alone. The protein levels of Bcl-2 and survivin were inhibited in AMP group, MMC group and AMP+MMC group, and were significantly lower in AMP+MMC group than those in AMP group or MMC group.CONCLUSION: AMP enhances the inhibitory effect of MMC on the growth of SGC-7901 cells. The mechanism is related to the inhibition of apoptotic protein expression.     

Effect of urotensin II on cultured pulmonary arterial smooth muscle cells of rabbits

AIM: The effect of urotensin II (U-II) on proliferation of cultured pulmonary arterial smooth muscle cells (PASMCs) of rabbits and its mechanism are investigated. METHODS: PASMCs were isolated using explant technique. RPASMCs were incubated in serum-free medium with different concentrations of nicardipine, calcimodulin antagonist W₇, PKC inhibitor H₇ or MAPK inhibitor (PD₉₈₀₅₉), with or without U-II. RPASMC proliferation was examined by MTT assay and by the increase in [³H]-thymidine incorporation into DNA. RESULTS: U-II (10^-9 mol/L-10^-7 mol/L) increased A value of PASMCs by MTT assay and [³H]-thymidine incorporation in PASMCs in a dose-dependent manner. U-II induced a maximal effect at a concentration of 10^-7 mol/L. A value and [³H]-thymidine incorporation rose 42.9% and 68.5% (P<0.05), respectively. U-II had no effect at a concentration of 10^-10 mol/L. Nicardipine, W₇, H₇, PD₉₈₀₅₉ (10^-7 mol/L-10^-5 mol/L) inhibited the effect of U-II in inducing increase of A value and -thymidine incorporation in a dose-dependent manner, with the maximal inhibitory rate of 42.3%, 19.6%, 23.2%, 10.5% (P<0.05) in A value and 46.6%, 9.8%, 21.7%, 14.7% (P<0.05) in [³H]-thymidine incorporation at concentration of 10^-5 mol/L, respectively. CONCLUSION: Our results suggest that U-II may induce proliferation of PASMCs of rabbits by Ca²⁺, CaM, PKC and MAPK signal transduction pathway.     

Inhibitory effects of dexamethasone on the expression of integrin CD18 induced by PMA

AIM:To study the effect of dexamethasone(Dex) on the expression of CD18. METHODS:Quantitative RT-PCR analysis, Northern blotting technique were used to measure the expression of CD18 in U937 cells treated by PMA.RESULTS:Dex could significantly attenuated the effects of PMA in a dose-dependent manner (10^-6 mol/L-10^-10 mol/L). These effects of Dex (10^-7 mol/L) were completely aborted by RU-486 (10^-6 mol/L).CONCLUSION:Dex, via GR, could inhibit CD18 mRNA expression in U937 cells treated by PMA. The effects of Dex might be possibly depended on the counteracting action on the NF-κB.     

Purification and adipogenic differentiation of human yolk sac mesenchymal stem cells

AIM:To purify human yolk sac mesenchymal stem cells (hYS-MSC) and investigate its adipogenic differentiation potential. METHODS:hYS-MSC were separated from yolk sac and purified via passaged culture. Flow cytometric analysis was used to identify the phenotype of hYS-MSC and the alkaline phosphatase(AKP) expression of hYS-MSC was also tested. Adipogenic differentiation of hYS-MSCs was induced by 10 mg/L insulin, 10^-5mol/L indomethacin and 10^-6mol/L dexamethasone. Oil Red O was used for fat staining. RESULTS:hYS-MSCs were purified at passages 2 or 3. Flow cytometric analysis showed the phenotype of purified YS-MSCs was uniformly positive for CD29, CD44, CD105, and CD166, and negative for reactivity to antigens CD34, CD45, or CD86. hYS-MSCs were weakly but clearly positive in AKP. Adipogenic differentiation of YS-MSCs was induced by 10 mg/L insulin, 10^-5mol/L indomethacin and 10^-6mol/L dexamethasone. Accumulation of lipid-rich vacuoles positive in oil red O staining within the cells were appeared and nuclears were pushed to one side of the cells during the period of induction. CONCLUSION:The phenotype of hYS-MSC is coincident with adult human mesenchymal stem cells. hYS-MSC can be induced to differentiate into adipocytes in vitro.     

Phenytoin inhibited changes in nitric oxide of rat hippocampus induced by stress

AIM: To investigate the changes in nNOS and iNOS expression of hippocampal CA3 pyramidal neurons and NO₂^-/NO₃^- level of hippocampal homogenate of rats induced by stress, and to explore the effect of phenytoin on them. METHODS: Rats were subjected to forced-swimming stress, phenytoin was administered(ip) at 30 min before stress. Using the immunohistochemistry and the computerized image technique, the expression levels of nNOS and iNOS of rat hippocampal CA3 pyramidal neurons were assayed quantitatively, and the NO₂^-/NO₃^- level of hippocampal homogenate was also measured using nitric acid deoxidize enzyme method. RESULTS: The nNOS average grey degree of hippocampal CA3 pyramidal neurons was significantly lower in stress group (155.42±3.77)than that in control group(164.54±4.62)and in stress plus phenytoin group(164.27±2.55)(P<0.01); The iNOS relative sectional area proportion of hippocampal CA3 pyramidal neuron was significantly larger in stress group(5.87%±2.90%) than that in control group (0.90％±0.89%) and in strers plus phenytoin groups (0.90%±0.88%)(P<0.01); The NO₂^-/NO₃^- level of hippocampal homogenate was significantly higher in stress group(42.75 umol/L±14.49 umol/L)than that in control group(21.23 umol/L±6.99 umol/L)and in stress plus phenytoin group(18.40 umol/L±8.11 umol/L)(P<0.01). CONCLUSION: It is suggested that the stress could induce nNOS and iNOS expression in CA3 pyramidal neurons and excessive production of NO in hippocampus of rats, which could be inhibited by phenytoin.     

The relations among PAMP,ADM and AngⅡ on vascular tissue

AIM: To elucidate the relations among proadrenomedullin N terminal 20 peptide(PAMP), adrenomedullin(ADM)and angiotensin(Ang).METHODS: Tissue slices of rat aorta were incubated as follows:(I) increasing concentrations of AngⅡ(10^-9, 10^-8, 10^-7 mol/L); increasing concentrations of PAMP(10^-9,10^-8,10^-7mol/L).The tissue and incubation concentrations of PAMP,ADM and AngⅡ were measured by the radioimmunoassay (RIA).RESULTS: The tissue and incubation concentrations of PAMP and ADM were concentration-dependently increased by AngⅡ,but the tissue and incubation concentrations of AngⅡ can not effected by PAMP.CONCLUSION: AngⅡ markedly stimulate the release of ADM and PAMP.It may be one of the factors which regulate the synthesis and release of ADM and PAMP.The regulation may play an important role in homeostasis regulation of cardiovascular system.     

AngⅡ/AT1R pathway leads to down-regulation of endothelial nitric oxide synthase phosphorylation

AIM:To investigate the mechanism of angiotensinⅡ (AngⅡ)/angiotensinⅡ type 1 receptor (AT₁R) pathway activating protein phosphatase 2A (PP2A) which leads to down-regulation endothelial nitric oxide synthase (eNOS) phosphorylation level in mesenteric arteries of rats. METHODS:The mesenteric arteries of adult male SD rats (weighing 160~180 g; n=90) were isolated under aseptic conditions. Firstly, to determine the effect of angiotensinⅡ down-regulated eNOS (Ser1177) phosphorylation level, the mesenteric arteries were randomly divided into normal control (control) group and AngⅡ group. The mesenteric arteries in AngⅡ group were incubated with AngⅡ at 1×10^-7 mol/L, 1×10^-6 mol/L and 1×10^-5 mol/L for 6 h, 12 h and 24 h, respectively. Secondly, to investigate the molecular mechanism by which angiotensinⅡ activated PP2A leading to down-regulation eNOS (Ser1177) phosphorylation level, the mesenteric arteries were randomly divided into control group, AngⅡ group and candesartan (CAN; a specific AT₁R blocker)+AngⅡ group. The mesenteric arteries were pretreated with 1×10^-5 mol/L CAN for 1 h, then incubated with 1×10^-7 mol/L AngⅡ for 12 h in CAN+AngⅡ group. The protein levels of eNOS, p-eNOS (Ser1177), PP2Ac, p-PP2Ac (Tyr307) and protein phosphatase 2A inhibitor 2 (I₂^PP2A) in the arteries were determined by Western blot. The activity of PP2A in the arteries was detected by PP2A activity kit. RESULTS:Compared with the control group, the protein level of p-eNOS (Ser1177) in the mesenteric arteries was decreased after incubated with AngⅡ for 6 h, 12 h and 24 h (P<0.05). The decreasing tendency of p-eNOS (Ser1177) showed concentration-dependently, especially in 12 h and 24 h groups. The expression of eNOS protein showed no significant difference in each group. Compared with the control group, the mesenteric arteries of the rats were incubated with AngⅡ at 1×10^-7 mol/L for 12 h in vitro, the protein levels of p-eNOS (Ser1177) were down-regulated (P<0.05); pretreatment with CAN significantly increased the protein level of p-eNOS (Ser1177) (P<0.05); the protein levels of eNOS showed no significant difference in each group. Compared with the control group, the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A were decreased after the mesenteric arteries were treated with AngⅡ at 1×10^-7 mol/L for 12 h (P<0.05). Candesartan pretreatment restored the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A (P<0.05), however the expression of PP2Ac protein showed no significant difference in each group. Compared with the control group, the activity of PP2A was increased in the mesenteric arteries incubated with AngⅡ at 1×10^-7 mol/L for 12 h (P<0.05). Candesarten pretreatment inhibited the activity of PP2A significantly (P<0.05). CONCLUSION:AngⅡ increases PP2A activity via AT₁R pathway, thus leading to down-regulation eNOS (Ser1177) phosphorylation level in mesenteric arteries. The molecular mechanism of PP2A activation may be associated with decreasing the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A.     

Expression of bFGF mRNA in pulmonary artery of rat with chronic hypoxia and its effect on tension of rat pulmonary artery in vitro

AIM: To explore the role of basic-fibroblast growth factor (bFGF) in the development of pulmonary hypertension induced by hypoxia. METHODS: 1) The pulmonary arteries of SD rats with hypoxia for one and two weeks were isolated, from which the total RNA were extracted by acid guanidinium thiocyanate-phenol-chlorform .Then the levels of mRNA were measured by RT-PCR. 2) About 3mm-long arterial rings cut from SD rat pulmonary arterial stem were suspended between stainless steelhooks in chamber with warmed (37℃) Kreb's solution. Different concentrations of bFGF were added in a cumulative fashion into the chamber where the rings were suspended. The cumulative concentration response curve was obtained. RESULTS: 1)The levels of bFGF mRNA in pulmonary artery of rats with hypoxia were increased significantly compared with those that without hypoxia (2578±384 counts·min^-1 (control) vs 5303±756 (hypoxia) for 1 week and 4054±547 (hypoxia) for 2 weeks, P all ＜0.05). 2) bFGF at concentrations ranged from 5.56×10^-10~2.78×10^-7mol/L caused dose-dependent contraction of vessel rings of rat pulmonary artery (r=0.695,P＜0.05), with EC₅₀ being 2.62×10^-7mol/L. CONCLUSION: bFGF may play an important role in the hypoxic pulmonary hypertension.     

The effect of vasoactive substances on synthesis and release of C-type natriuretic peptide in cultured human endothelial cells

AIM:To investigate the effect of endothelin (ET), angiotensin II (AngII) and homocysteine (Hcy) on C-type natriuretic peptide (CNP) synthesis and release. METHODS: Human endothelial cell was cultured; CNP was measured by radioimmunoassay method. RESULTS: ET and AngII could augment CNP synthesis in human endothelial cells. Compared with control group, 10^-9,10^-8,10^-7 mol/L ET and Ang II increased CNP content of endothelial cells by 1%(P＞0.05), 49%(P＜0.05),117%(P＜0.01) and 137% (P＜0.01),165%(P＜0.01),201%(P＜0.01),respectively. A great dose of ET and Ang II also stimulated CNP release from cultured human endothelial cells. Hcy had no effect on CNP synthesis, but 10^-9,10^-8,10^-7 mol/L Hcy enhanced CNP release from cultured human endothelial cells by 17%(P＞0.05),84%(P＜0.01) and 555%(P＜0.01), respectively. CONCLUSION: ET, AngII and Hcy might be involved in the synthesis and release of human endothelial cell CNP.Fig 1 Time-course of CNP syntheis and release in cultured human endothelial cell ( ±s,n=6)     

Metallothionein inhibits homocysteine-induced proliferation of rat vascular smooth muscle cells

AIM:To investigate the effect of metallothionein(MT) on proliferation of rat vascular smooth muscle cells (VSMCs) stimulated by homocysteine and its mechanism. METHODS:VSMCs proliferation was measured by [³-H]-TdR incorporation, mitogen-activated protein kinase(MAPK)activity were determined by immunoprecipitation method, the intracellular contents of MT and malondialdehyde (MDA)were assayed by -hemoglobin saturation method and TBA reaction, respectively, and lactate dehydrogenase (LDH) leakage was measured by NADH oxidation. RESULTS:Hcy(10^-6-10^-4 mmol/L) stimulated [³-H]-TdR incorporation by the VSMCs in a concentration-dependent manner. Compared with control, [³-H]-TdR incorporation in VSMCs treated with 0.1 mmol/L Hcy was increased by 4.2 fold (P＜0.01). Meanwhile, Hcy enhanced MAPK activity, MDA formation and LDH release (P＜0.01)in a concentration-dependent manner. Treatment of VSMCs with MT alone did not change above parameters, compared with control. However, MT (10^-6-10^-4 mol/L)attenuated significantly Hcy-stimulated proliferation of VSMCs (P＜0.01)in a concentration-dependent manner. And MT inhibited obviously Hcy-induced activation of MAPK activity, MDA formation and LDH release. Preincubation of VSMCs with 0.5 mmol/L ZnCl₂ for 6 h induced an increase cellular MT content by 5.7-fold (P＜0.01). The MT-overexpressed VSMCs resisted Hcy-stimulating action on MAPK activity, MDA formation and LDH leakage (P＜0.01). CONCLUSION:These results show that MT has an inhibitory effect on Hcy-induced VSMCs proliferation, and that MT could inhibit Hcy-stimulated MAPK activity and lipid peroxidation.     

Metallothionein protects rat hepatic nuclear nucleoside triphosphatase from hydroxyl radical-induced suppression

AIM:Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotection during metal exposure and oxidative stress. The present study was designed to investigate whether MT can directly protect NTPase on nuclear envelope from damage induced by hydroxyl radical.METHODS:Isolated hepatic nuclei from rat liver were exposed to Fe²⁺/H₂O₂ with or without MT, and the NTPase activity on nuclei was assayed using ATP and GTP as substrate, respectively.RESULTS:Incubation of rat hepatic nuclei with the Fe²⁺/H₂O₂ (in μmol·L^-1/μmol·L^-1 : 0.1/0.5, 0.5/2.5, 1/5, 5/25) resulted in a concentration-dependent decrease in nuclear NTPase activities (P＜0.01). Incubation of hepatic nuclei with different concentrations of MT (10^-9-10^-4mol·L^-1)and Fe²⁺/H₂O₂ (1 μmol·L^-1/5 μmol·L^-1) for 10 min, nuclear NTPase activities were increased in a MT concentration-dependent fashion as compared with that of incubation with Fe²⁺/H₂O₂(1 μmol·L^-1/5 μmol·L^-1) alone. When MT was at 10^-4 mol·L^-1, TNPase activities reversed to (102±10) nmol·mg^-1 protein·min^-1(for ATP as substrated) and (131±12) μmol·g^-1 protein·min^-1(for GTP as substrate), which had no significant defferences from that of the controls (112±8 and 142±10 μmol·g^-1 protein·min^-1, respectively) (P＞0.05). In addition, incubation of hepatic nuclei with only MT had no effect on nuclear NTPase activity. CONCLUSION:These data demonstrate that hydroxyl radical generated from Fe²⁺/H₂O₂ might attack nuclear NTPase. MT antagonistically reduces toxicity of Fe²⁺/H₂O₂ system to the NTPase.     

Effect of pioglitazone on pre-adipocytes differentiation and GILZ expression

AIM: To investigate the roles of pioglitazone on differentiation and expression of GILZ in 3T3-L1 pre-adipocytes. METHODS: The morphological changes during 3T3-L1 cell differentiation were observed. The cells were treated with pioglitazone at concentrations of 1×10^-4～1×10^-2 mmol/L for 48 h, then the relative content of triglyceride were analyzed by oil red O staining at 2nd, 4th and 6th day during adipogenesis. The mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and lipoprotein lipase (LPL) was measured by real-time PCR. GILZ protein expression was determined by Western blot after the cells were treated with pioglitazone at concentrations of 1×10^-4 ～1×10^-2 mmol/L for 48 h.RESULTS: Oil-red O staining showed that the relative contents of triglyceride in adipocytes were increased with the increase in the pioglitazone concentration. Compared with the control, the relative contents of triglyceride in group 1×10^-3 mmol/L and group 1×10^-2 mmol/L were significantly increased (P < 0.05). The mRNA expression of PPARγ2 and LPL was also increased with the increase in the pioglitazone concentration. When pioglitazone concentration was more than 1×10^-3 mmol/L, compared with the control, the mRNA expression of PPARγ2 and LPL significantly increased (P < 0.01). The protein expression of GILZ was decreased with the increase in the pioglitazone concentration.CONCLUSION: Pioglitazone down-regulates GILZ expression, and up-regulate PPARγ2 expression and the downstream functional factor such as LPL.     

Effects of nicotine on activation of PMNs and the ICAM-1 mRNA expression of HUVEC

AIM: To investigate the effects of nicotine on activation of PMNs, adhesion of PMNs-HUVEC and expression of ICAM-1 mRNA in HUVEC. METHODS: Activation of PMNs was measured by detecting the activity of β-glucuronidase and lysozym of PMNs. Adhesion of PMNs and HUVEC was observed. Northern blot was conducted for quantitating ICAM-1 mRNA. RESULTS: Nicotine could increase the activity of β-g [(8.76± 1.01)μg/10⁷·h vs(14.87±2.00)μg/10⁷·h,P<0.05]and Lysozym [(20.0±1.5)μg/10⁷·h vs(36.5±4.4)μg/10⁷·h,P<0.05], and also could promote adhesion of PMNs-HUVEC(38.5±9.8 vs 61.0±4.4,P＜0.05). The expression of ICAM-1 mRNA was induced by nicotine in dose-dependent fashion (10^-5-10^-3mol/L).After a 2 h treatment of HUVEC with nicontine(10^-4mol/L), the level of ICAM-1 mRNA is above the control(1.23 vs 1.63) and the highest level (2.03) is at a 12 h treatment. 764-3 can obviously counteract the above effect of nicotine. CONCLUSIONS: Nicotine could activate PMNs, enhance adhesion of PMNs-HUVEC and increase the expression of ICAM-1 mRNA in HUVEC.     

Rapid effects of dexamethasone on activation of ERK and p38 in HO-8910cells

AIM: To investigate the effects of synthetical glucocorticoid dexamethasone(Dex) on the activation of two members of mitogen-activated protein kinase (MAPK) family, extracellular signal-regulated protein kinase1/2(ERK1/2) and p38 MAPK (p38) in human ovarian cancer cell line HO-8910. METHODS: The activation of ERK1/2 and p38 was determined by Western blot. RESULTS: Inhibition of activation of ERK1and ERK2 by10^-7 mol/L Dex occurred at 5 min, with maximum up to 41% and 54% respectively at 30min (P<0.01), and sustained until 4 h. On the contrary, p38 activity was rapidly stimulated by 10^-7 mol/L Dex, with maximum to 84% at15 min (P<0.01), and sustained till1h. Furthermore, these effects increased with the concentration of Dex(10^-10-10^-6 mol/L). RU486, an antagonist of glucocorticoid receptor (GR), did not affect these effects. CONCLUSION: Dex can rapidly inhibit ERK1/2 and stimulate p38 activation in a GR-independent manner in HO-8910cells, which might play a role in Dex-mediated growth inhibition in these cells.     

Effects of leptin on BSEP protein expression and signaling pathway in HepG2 cells

AIM: To investigate the effects of leptin on the expression of bile salt export pump (BSEP) and signaling pathway in human hepatocellular carcinoma cell line HepG2. METHODS: HepG2 cells were cultured in vitro. Leptin at concentrations of 10^-8, 10^-7 and 10^-6 mol/L was used as a stimulating factor. The protein levels of adenosine monophosphate-activated protein kinase alpha subunit (AMPKa), phosphorylated AMPKa (p-AMPKa) and BSEP in the HepG2 cells at 24 h, 48 h and 72 h were detected by Western blotting. The optimal culture time and leptin concentration were selected, and compound C at concentration of 10 μmol/L was added to this group. The protein expression of BSEP was detected by Western blotting. RESULTS: Intervention of HepG2 cells with leptin for 72 h increased the protein expression of AMPKa gradually in a concentration-dependent manner, and leptin at concentration of 10^-6 mol/L induced the strongest AMPKa expression (P<0.01). Intervention of HepG2 cells with leptin for 24 h increased the phosphorylation level of AMPKa gradually in a dose-dependent manner (P<0.01). The effect of leptin on the increase in the protein expression of p-AMPKa was also in a time-dependent manner (P<0.01). After intervention with different concentrations of leptin for 24 h, the protein expression of BSEP in the HepG2 cells was gradually increased by the stimulation of leptin in a concentration- and time-dependent manner (P<0.01). Compared with NC group, the protein expression of BSEP in 10^-6 mol/L leptin group and 10^-6 mol/L leptin+10 μmol/L compound C group was increased at 72 h (P<0.01), and that in 10^-6 mol/L leptin+10 μmol/L compound C group was lower than that in 10^-6 mol/L leptin group at 72 h (P<0.01). CONCLUSION: Leptin promotes the protein expression of BSEP in HepG2 cells by leptin-AMPK-BSEP signaling pathway. Leptin promotes the increases in AMPKa protein and the level of phosphorylation of AMPKa in HepG2 cells.     

Inhibitory effects of gossypol on the activation of human T-lymphocytes stimulated with polyclonal activators

AIM: Peripheral blood mononuclear cells (PBMC) were cultured in vitro to study the effect of gossypol, a polyphenolic antifertility agent, on the activation of normal human T cells. METHODS: Double fluorescent staining together with flow cytometry was adopted to analyze the influence of gossypol on expression of the early activation antigen CD69 on T-lymphocytes under stimulation of mitogen or phorbol ester. RESULTS:Analysis of T cell activation in vitro revealed that preincubation of PBMC with 100 μmol/L gossypol could completely inhibit the expression of early activation marker CD69 on CD3⁺ T cells in response to 10mg/L PHA, and block T cell activation by 10^-7 mol/L PDB as well. The suppression of CD69 expression was dose-dependent and IC₅₀ of gossypol on PDB and PHA were (35.7±2.9) μmol/L and (32.8±1.5) μmol/L(x ±s), respectively. Besides, gossypol had similar inhibitory effect on CD69 expression of CD3^- lymphocytes. However, it did not have any significant effect on T cell surface molecule CD3 down-regulation. CONCLUSION: Gossypol could inhibit T cell activation in vitro in response to polyclonal activators, both PHA and PDB, suggesting that its action site may be at PKC or its downstream and that gossypol possessed potential immuno-regulatory effect.     

Changes in platelet-activating factor receptor binding characteristics in cerebral thrombotic core and penumbra of tree shrews

AIM: To observe the changes in platelet-activating factor (PAF) receptor binding characteristics and explore the action of PAF on formation of thrombotic core and penumbra following local cerebral ischemia. METHODS: Neuron's membrane protein was abstracted, and the local cerebral ischemia model were induced by photochemistry in tree shrews. The PAF binding sites on central neuron membrane were studied by-PAF binding assay. RESULTS: There were two different affinities of PAF receptors on tree shrew's brain cell membrane, with kD₁=(3.61 ±0.72) nmol/L and kD₂=(17.04±2.41) nmol/L, corresponding respectively to maximum number of binding sites: B_max1=(1 457.94±168.01) pmol/g protein and B_max2=(5 017.40±742.16) pmol/g protein. The binding sites decreased in ischemic core, penumbra and contralateral regions at 4,24 and 72 h after ischemia (P<0.01), with those of 24 h reaching the minimum levels. CONCLUSION: PAF receptors play an important role in cerebral ischemia, may be related to the secondary damage in ischemic penumbra, and also are molecular bases of brain injury induced by PAF.