AIM To investigate weather and how aminophylline induces human trachea smooth muscle cells (TSMCs) to apoptosis. METHODS Human TSMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Passage 4～6 cell were used in experiment. The cells were cultured with aminophylline or 8 Br cAMP for 24 or 48 h. Light micros copy and electron microscopy were used to observe morphological change. DNA fragmentation was analyzed by agarose gels electrophoresis. SP Immunohistological staing method was performed to detect the changes of expressions of p53,bcl 2 and bax gene. The apoptosis cell percentage were detected by situ end labeling technique(TUNEL) of fragmental DNA. RESULTS ①Aminophylline or 8 Br cAMP decreased the number of viable cells in time and concentration dependent manner; ② Electron microscopic examination showed nuclear contraction, chromatin condensation and apoptotic bodies formation in aminophylline group or 8 Br cAMP group; ③ Agarose gel electrophoresis of fragmented DNA showed a ladder like pattern; ④ The expression of p53 or bax gene in apoptosis group was significantly higher than in control group, but the expression of bcl 2 gene was lower than in control group; ⑤ The positive rate of TUNEL in aminophylline or 8 Br cAMP group was significantly higher than in control group ( P

Object The purpose of this study was to determine the effects of tea polyphenols on rat vascular smooth muscle cells (VSMCs) proliferation. Methods Rat aortic VSMCs were cultured and treated with tea polyphenols for 24 h. Cell proliferation was quantified by colormetric assay and definited by MTS/PES. Cell cycle analysis was performed by flow cytometry. Results Compared with the control group, VSMCs growth was significantly inhibited by tea polyphenols (P

Objective To investigate the effects and anti-atherosclerotic mechanism of rosiglitazone on the expression of ATP-binding cassette transporter A1 (ABCA1) and reverse cholesterol transport (RCT) in atherosclerotic rabbits. Methods Twelve rabbits were randomly divided into two groups (6 each): control group (only high cholesterol diet for 6 weeks), rosiglitazone group [high cholesterol diet plus rosiglitazone 0.5mg/(kg?d) for 6 weeks]. ABCA1 expression and [3H] cholesterol efflux rates were evaluated by flow cytometry and liquid scintillation spectrometry, respectively. Enzymatic methods were used to assay serum lipids levels and cholesterol contents in tissues, and the atherosclerotic area of aorta was calculated by professional image analysis software. Results For the rabbits of both control and rosiglitazone group, the serum levels of high-density lipoprotein cholesterol (HDL-C), non-high-density lipoprotein cholesterol (NHDL-C) and apolipoprotein A1 (apoA1) significantly went up when they took their cholesterol rich diet for 6 weeks (P0.05). Compared with control group, the ABCA1 expressions in monocytes, peritoneal macrophages, adipocytes and hepatocytes, as well as the cholesterol efflux rates in peritoneal macrophages, adipocytes and hepatocytes increased significantly (P

Objective:To explore the expression of ROR?t in pulmonary tissue of asthmatic mice and to investigate the association between the expression of ROR?t and the airway inflammation.Methods:Thirty female BLAB/c mice were randomly divided into the control group,asthmatic group and dexamethasone (Dex)-treated group.The asthma model was induced by classical method with ovalbumin(OVA).The concentration of IL-17 in bronchoalveolar lavage fluid (BALF) and serum was measured by enzyme-linked immunosorbent assay(ELISA).Airway inflammation was evaluated by HE staining.The expressions of IL-17,ROR?t mRNA and protein was measured by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot respectively.Results:The level of IL-17,ROR?t mRNA and protein of asthmatic group was significantly higher than those of control group and Dexamethasone treated group (P

AIM:To clarify if interferon-?(IFN-?), tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?)can induce apoptosis of human airway smooth muscle cells (ASMCs) in vitro. METHODS: Human ASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Passage 4-6 cell was used in the experiment. IFN-?,TNF-? and IL-1?, were used separately or together in the treatment of human ASMCs. The effects of IFN-?,TNF-? and IL-1? on the growth of the cells was detected by MTT method at the hour 0,24,48 and 72. Light microscopy and electron microscopy were used to examine the morphological change. DNA fragmentation was analyzed by agarose gel electrophoresis. SP immunohistological staing method was performed to detect the change of expressions of p53 , bcl-2 and bax gene. The apoptosis cell percentage were detected by in situ end labeling technique (TUNEL)of fragmental DNA. RESULTS: (1)IFN-? or IFN-? together with TNF-? and IL-1? decreased the number of viable cells in a time dependent manner. (2) Light and electron microscopic examination showed cell shrinkage, membrane blebbing, nuclear contraction, chromatin condensation and nuclear fragmentation in human ASMCs. (3) Agarose gel electrophoresis showed a characteristic"ladder"of DNA bands representing integer multiples of the internucleosomal fragments (about 180-200 bp) in cytokine cotreated human ASMCs. (4)The expression of p53 and bax gene in cytokine cotreated group was significantly higher than in control group, but the expression of bcl-2 gene was lower than in control group. (5)Stimultaneous treatment with IFN-?(4?10 5 U/L),TNF-?(4?10 5 U/L)and /or IL-1? (10?10 4 U/L) induced apoptosis of human ASMCs. Apoptotic index of human ASMCs in cytokine co-treated group was significantly higher than in control group ( P

Objective To investigate the influence of benzopyrene on extracellular matrix(ECM) protein deposition of human airway smooth muscle cells(HASMC) and the related pathway mechanism.Methods HASMC were primarily cultured and the 2-6 generations cells were applied in this experiment.The expression amount of ECM gene and protein was detected by real time PCR and Western Blot the phosphorylation level was analyzed by using the Western Blot method.Results Benzopyrene could to increase the expression of HASMC collagen Ⅰ α1 protein (P＜0.01) and ECM protein (including collagen Ⅰ α1,versican,fibronectin,laminin α2) mRNA(P＜0.05).Benzopyrene could induce the rapid increase of ERK1/2 phosphorylation level (P＜0.01).Furthermore,the ERK pathway inhibitor PD98059 could significantly inhibit the increase of benzopyrene-induced collagen Ⅰ α1 (P＜0.01)and ECM protein(including collagen Ⅰ α1,versican,fibronectin,laminin α2) mRNA expression(P＜0.01).Conclusion Benzopyrene induces the ECM protein deposition of HASMC by activating the ERK1/2 pathway,blocking the ERK1/2 signal pathway can inhibit the benzopyrene-induced airway remodeling.

Objective To observe the onset of vascular smooth muscle cell (VSMC) senescence induced by tert-butyl hydroperoxide (t-BHP) and the intervention effect of dehydroepiandrosterone (DHEA). Methods The VSMCs were divided into four groups: blank control group, t-BHP group (incubated with 80 μmol/L t-BHP for 72 h), 10 nmol/L DHEA intervention group (pretreated with 10 nmol/L DHEA 30 min before t-BHP) and 100 nmol/L DHEA intervention group (pretreated with 100nmol/L DHEA 30 min before t-BHP). Two ageing markers of ageing associated β-galactosidase activity and cell proliferation activity were adopted as main indexes. β-galactosidase activity was measured with immunocytochemical method and cell proliferation activity was measured with flowcytometry. Results After continuous treatment with 80 mmol/L t-BHP for 72 h, the ratios of G0/G1 phase cells and SA-β-galactosidase staining positive cells increased as compared with blank controlgroup [(89.4±3.4)% vs. (49.5±5.5)%, (3.5±1.2)% vs. (75.3±4.3)%], which indicated that VSMCs senescence were successfully induced by t-BHP. While the above changes were smaller in 100 nmol/L DHEA intervention group than in t-BHP group. Conclusions With ageing,accumulation of damage produced by reactive oxygen species may be an important mechanism causing the onset of VSMCs senescence. DHEA may be able to retard the progression of VSMCs senescence through antioxidant effect.

AIM: To investigate the influence and mechanism of recombinant macrophage migration inhibitory factor(rMIF) on fibroblasts.METHODS: MRC-5 fibroblasts were divided into two groups: the treated group was treated with rMIF(25-100 ?g/L,12 h,24 h or 48 h) and the control was non-rMIF treatment.The activity of proliferation in both groups was investigated and compared by CCK-8 means.Synthesis of collagen in the culture supernatants was detected by the hydroxyproline.The expression of collagen type I mRNA was examined using RT-PCR analysis.The level of collagen type I protein induced by rMIF was quantified by Western blotting.RESULTS: The production of proliferation ratio of fibroblasts treated with 50 ?g/L and 100 ?g/L rMIF at 24 h or 48 h were increased obviously(P

Objective To investigate of the effect and mechanisman of SERCA2 on the phenotype modulation of HASMCs. Methods HASMCs were starved for 5 days and divided into different groups ,then we observed morphology change of the cells from the microscope and detected a-actin、SERCA2 and p-ERK by Western Blot,cells proliferation was observed by CCK-8 method. Results Compared with the control group,PDGF could reduce a-actin of HASMCs and increased the cells proliferation ability ,TSG could significantly inhibit the effect (P<0.01), PDGF could also significantly inhibit SERCA2 protein and increased the expression p-ERK (P<0.01), while U0126 significantly inhibited the effect (P < 0.01). Conclusion PDGF may induce HASMCs phenotype modulation through the regulation of SERCA2 and p-ERK.

Objective The current study aimed to evaluate whether the induction of macrophage inflammatory cytokines by Ox-LDL is related to the expression of ABCA1 pathway. Methods After THP1/PMA macrophages were transfected with ABCA1 antisense oligonucleotides (100nmol/L) followed by treatment with Ox-LDL (30mg/L), the expressions of ABCA1, ICAM-1 and MCP-1 mRNA and protein were determined by real-time fluorescent quantitative RT-PCR, Western blot or ELISA. Results Ox-LDL induced expressions of ABCA1, ICAM-1, and MCP-1 at both mRNA and protein levels from THP1/PMA macrophages. Transfection with ABCA1 antisense oligonucleotides reduced ABCA1 mRNA levels after 3 and 6 hours and protein levels after 12 and 24 hours. The expression of ICAM-1 and MCP-1 induced by Ox-LDL was also decreased after inhibition of ABCA1 protein expression by ABCA1 antisense oligonucleotide decreased. Conclusion The induction of macrophage inflammatory cytokines by Ox-LDL is partially dependent on expression of ABCA1. Our studies disclose new functions of ABCA1 in macrophages.