Angiotensin II-induced matrix metalloproteinase-9 expression mediated by NF-κB pathway in human THP-1 cells

AIM: The present study was undertaken to investigate the effect of angiotensin II (AngⅡ) on expression of MMP-9 in THP-1 macrophages. METHODS: Macrophages converted from THP-1 monocytes by incubating with PMA (0.1 μmol/L) for 48 h were divided into PMA group; PMA+AngⅡ group (10-7mol/L, 1 h); PMA+AngⅡ+PDTC group (10 μmol/L, 30 min) and PDTC group. Western blotting was used to detect the MMP-9 and phosphorylation of NF-κB p65, and the expression of MMP-9 mRNA in THP-1 macrophages was measured by RT-PCR.RESULTS: Compared to control group, the expression of MMP-9 (1.06±0.11, P<0.05) and phosphorylation of NF-κB p65 (1.02±0.10, P<0.05) in THP-1 macrophages were expressed when treated with AngⅡ (10-7mol/L); and the expression of MMP-9 mRNA were upregulated (1.22±0.08, P<0.05). However, NF-κB inhibitor PDTC reduced the NF-κB p65 (0.99±0.12, P<0.01) and MMP-9 (1.04±0.14, P<0.01) expressions and decreased the expression of MMP-9 mRNA (0.90±0.06,P<0.01). CONCLUSION: NF-κB signaling pathway contributes to the expression of MMP-9 in THP-1 macrophage induced by AngⅡ.     

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.     

Role of caveolin-1 on down-regulation of LPS-induced monocyte chemotactic protein 1 by 17β-estradiol in vascular smooth muscle cells

AIM: To elucidate the effect of caveolin-1 on the down-regulation of LPS-induced monocyte chemotactic protein 1 (MCP-1) by 17β-estradiol (E₂) in vascular smooth muscle cells (VSMCs).METHODS: The primary-cultured VSMCs were exposed to E₂ at concentrations of 10^-9-10^-6 mol/L. LPS-induced MCP-1 production was assayed by ELISA. The protein expression of caveolin-1 was determined by Western blotting and was silenced by β-methyl cyclodextrin(β-MCD) or caveolin-1 specific siRNA. RESULTS: LPS significantly enhanced MCP-1 production. E₂ at concentrations of 10^-9-10^-6 mol/L inhibited LPS-induced MCP-1 production. The use of caveolin-1 inhibitor β-MCD or silencing the protein expression of caveolin-1 by specific siRNA largely impaired LPS-enhanced MCP-1 production, while E₂ markedly inhibited caveolin-1 expression. CONCLUSION: Inhibition of LPS-induced MCP-1 production by E₂ is related to the suppression of caveolin-1.     

Effect of homocysteine on expression of interleukin-8 mRNA and protein in THP-1 macrophages

AIM: To investigate the effect of homocysteine (Hcy) on expression of interleukin-8 (IL-8) mRNA and protein in THP-1-derived macrophages (THP-1 macrophages). METHODS: Cultured THP-1 monocytes were induced to macrophages by 0.1 μmol/L PMA treatment for 72 hours, then the differentiated THP-1 macrophages were incubated with homocysteine (0.01 mmol/L-0.20 mmol/L) for 24 hours, or with 0.10 mmol/L Hcy for various time up to 48 hours. IL-8 protein in THP-1 supernatants was measured by ELISA, and IL-8 mRNA expression was detected by semiquantitive RT-PCR. RESULTS: Compared with control, Hcy significantly increased the expression of IL-8 protein in a concentration-dependent manner. 0.05 mmol/L, 0.10 mmol/L and 0.20 mmol/L Hcy increased IL-8 production by 1.28 fold, 1.32 fold and 1.55 fold, respectively (P＜0.01). IL-8 production were elevated significantly 3 h after treatment with 0.10 mmol/L Hcy. In addition, Hcy also increased IL-8 mRNA expression in a concentration-and time-dependent manner. CONCLUSION: Hcy may contribute to atherogenesis by inducing IL-8 expression and secretion in THP-1 macrophages.     

Effects of angiotensin II and losartan on collagen synthesis in rat hepatic stellate cells

AIM: To investigate the effects of angiotensin II (Ang II) and AT_1a receptor antagonist (losartan) collagen synthesis in rat hepatic stellate cells (HSCs). METHODS: ① Rat HSCs were isolated, cultured and identified. ② Rat HSCs were incubated in the medium with different concentrations of AngII or losartan, then the quantity of collagen was examined by -proline release assay. RESULTS: ① The yield of HSCs was 2×10⁷-3×10⁷/per rat, their viability and purity was more than 95% and 90%, respectively. ② The yield of collagen in HSCs significantly got a rise in a concentration-dependent manner when HSCs were incubated with AngII (10^－6mol/L-10^－10 mol/L) (P<0.05). While HSCs were influenced by the antagonist of AT_1a (10^－6 mol/L-10^－9 mol/L), the quantity of collagen dropped greatly (P<0.05). CONCLUSIONS: Ang II stimulates HSCs to produce more collagen. Losartan inhibits the cell to synthesize collagen via AT_1a receptor (P<0.05). The results indicate that Ang II may play an important role in the development of hepatic fibrosis, and using AT_1a antagonist may offer a new strategy to prevent hepatic fibrosis.     

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)     

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.     

AngiotensinⅡ activates autophagy pathway through elevating ROS level in vascular endothelial cells

AIM: To evaluate the effects of angiotensinⅡ (AngⅡ) on autophagy induction in vascular endothelial cells. METHODS: Human vascular endothelial EA.hy926 cells were used in the study. Intracellular reactive oxygen species (ROS) levels were detected by a microplate reader after the cells were treated with AngⅡ (10^-7 mol/L) or AngⅡ combined with antioxidant N-acetyl-L-cysteine (NAC,50 μmol/L) for 24 h. The protein levels of LC3-Ⅱ was detected by Western blotting after the cells were stimulated by different concentrations (10^-8, 10^-7, 10^-6 mol/L) of AngⅡ for 24 h or by AngⅡ (10^-7mol/L) for different time (0 h, 6 h, 12 h, 24 h, 36 h). The number of autophagosomes was evaluated by fluorescence microscopy after stained with acridine orange. Similarly, the protein level of LC3-Ⅱ and the number of autophagosomes were detected after treated with AngⅡ(10^-7mol/L), AngⅡ combined with autophagy inhibitor 3-methyladenine (3-MA) at concentration of 2 mmol/L or AngⅡ combined with NAC at concentration of 50 μmol/L. RESULTS: Intracellular ROS level and LC3-Ⅱprotein level were significantly increased (P<0.05) after the cells were treated with AngⅡ, accompanied by the significant increase in the number of autophagosomes. AngⅡ-induced autophagy (as showed both in LC3-Ⅱprotein level and autophagosomes) was dramatically down-regulated by the treatment with 3-MA or NAC in EA.hy926 cells (P<0.05). CONCLUSION: AngⅡ induces autophagy through elevating ROS levels in EA.hy926 cells.     

Effects of thyroid hormone on the myosin heavy chain mRNA expression in cardiac myocytes induced by angiotensin Ⅱ

AIM: To study the effect of thyroid hormone on the expressional change of myosin heavy chain(MHC) gene in cardiomyocyte induced by angiotensinⅡ(AngⅡ) and its potential mechanism. METHODS: Cardiac myocyte was cultured according to the method of Simpson. 10^-8 mol/L T₃ and 10^-7 mol/L AngⅡ were added to the culture medium, respectively or synchronously. After 48 h, the expression of α and β-MHC mRNA in myocytes were detected by RT-PCR. The protein kinase C activation were detected by PepTag non-radioactive PKC assay. The incorporation of -Leucine and [³H]-thymine to test the protein and DNA synthesis in myocytes were also performed. RESULTS: AngⅡalone increased the incorporation of [³H]-Leucine of myocytes while it had no effect on the incorporation of [³H]-thy mine. The expression of β-MHC mRNA was increased and the expression of α-MHC mRNA was decreased significantly at the condition of AngⅡ. The enhanced PKC activation was induced by AngⅡalso. When AngⅡand T₃ were added to the culture medium synchronously, though the incorporation of [³H]-leucine and [³H]-thymine were not changed compared with AngⅡ treated alone. The α-MHC mRNA expression was increased and the β-MHC mRNA expression was decreased significantly. The PKC activation of the myocytes also was decreased. CONCLUSIONS: T₃ inhibited the expressional change of myosin heavy chain gene in cardiac myocytes induced by AngⅡ. The effect of T₃ on the change of PKC activation in cardiac myocytes may be one of its mechanisms.     

Insulin induced LPL mRNA expression in THP-1 macrophage and acceleration of transferring the macrophage to foam cell

AIM:To investigate the effect of insulin on ox-LDL transferring the THP-1 cells to foam cells and influencing the LPL mRNA expression in THP-1 cells.METHODS:THP-1 cells were incubated with 50 mg/Lox-LDL and insulin at concentrations of 10 mU/L, 100 mU/L, 1 000 mU/L and 10 000 mU/L, respectively. The expression of LPL mRNA in cells was detected by RT-PCR. Lipoprotein lipase of THP-1 cells was presented by no-specific lipase staining. THP-1 cells were stained with oil red O. Accumulation of total cholesterol (TC) in THP-1 cells was determined with oxidase assay.RESULTS:In 100 mU/L、1 000 mU/L、10 000 mU/L insulin groups, LPL mRNA expression increased 2 times, the average cell perilength was longer, the percentage of positive oil red O staining cells was significant higher, the content of cholesterol in THP-1 cells was higher than in ox-LDL control (P＜0.05).CONCLUSION:Insulin accelerates transferring of THP-1 cells to foam cell with exposed to ox-LDL because LPL mRNA expression increased in the cells.     

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.     

Lectin-like oxidized low density lipoprotein receptor-1 mediates expression of MCP-1 induced by ox-LDL in cultured human vascular endothelial cells

AIM: To investigate the effects of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) on the expression of MCP-1 in the cultured human unbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs was incubated with ox-LDL, or preincubated with carrageenan and polyinosinic acid. LOX-1 and MCP-1 mRNA and protein were determined by RT-PCR and Western blot. RESULTS: Incubation of HUVECs with ox-LDL (from 0-100 mg/L) for 24 h markedly increased the expression of LOX-1 and MCP-1 (mRNA and protien) in a concentration-dependent fashion. Preincubation of HUVECs with carrageenan and polyinosinic acid, the chemical inhibitors of LOX-1, for 2 h, ox-LDL-mediated upregulation of LOX-1 and MCP-1 was suppressed (P<0.05). CONCLUSION: These findings indicate that ox-LDL upregulates MCP-1 and its own endothelial receptor, and ox-LDL-induced MCP-1 is mediated by the action of LOX-1. LOX-1 plays a critical role in the pathogenesis of atherosclerosis.     

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.     

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.     

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.     

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.     

Effects of platelet-derived growth factor and angiotensinⅡ on the expression of cell cycle related genes in vascular smooth muscle cells

AIM: To investigate the different expressions of cell cycle related genes in hyperplastic and hypertrophic vascular smooth muscle cells caused by platelet-derived growth factor (PDGF-BB) and angiotensinⅡ(AngⅡ). METHODS: Rat aorta media smooth muscle cells were cultured. PDGF-BB and AngⅡ were added into serum-free medium at a concentration of 20 μg/L and 10^-6 mol/L , respectively. Vascular smooth muscle cells (VSMCs) were harvested after stimulated for 24 hours. The expression of cell cycle related genes was measured by DNA chips(Atlas cDNA Expression Arrays, Clontech Laboratories, Inc.). RESULTS: The expression of cyclin D3 mRNA ,cyclin G1 mRNA,p57 mRNA,p16 mRNA,E2F-3 mRNA and DP2 mRNA were higher in PDGF-BB than those in AngⅡstimulated VSMCs. p15 mRNA,p19 mRNA,E2F-1 mRNA, E2F-5mRNA,and N-myc mRNA were only detected in PDGF-BB stimulated group. But the expression of p53-associated protein mRNA were higher in AngⅡstimulation group. The expression of PCNA mRNA, c-myc binding protein mRNA,p53-dependent cell growth regulater mRNA,cyclinC mRNA, cyclinB1 mRNA, E2F-3mRNA were similar in the two groups. CONCLUSION: The procession of cell cycle relys on the coordination of many regulater molecules expressed in different phases. Our study preliminarily definite the genes that express during PDGF-stimulated VSMC's hyperplasia and Ang II-stimulated VSMC's hypertrophy.     

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.     

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.