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In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages (PAMs) infected with porcine circovirus type 2 (PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type I interferon (IFN-I) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-I increased significantly at 48 h after infection with PCV2 (P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection (P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection (P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection (P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-I was significantly decreased after PCV2 infection for 48 h (P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-I in BX795-treated group was significantly reduced than that of the PCV2 group (P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-I secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.
Subject(s)
Animals , Cells, Cultured , Circovirus , Interferon Type I/genetics , Macrophages, Alveolar/virology , Membrane Proteins/metabolism , Nucleotidyltransferases/metabolism , Signal Transduction , SwineABSTRACT
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.
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Objective To compare the clinical characteristics,and outcomes of patients with heart failure with different left ventricular ejection fractions (LVEF).Methods A total of 1 182 hospitalized patients with heart failure (HF) were enrolled and retrospectively studied in the present study.The patients were stratified by LVEF as reduced (HFrEF,LVEF ＜ 40％,n =313),mid-range (HFmrEF,40％ ≤ LVEF ＜50％,n =287) and preserved (HFpEF,LVEF≥50％,n =582) ejection fraction groups.Among the 1 182 cases,941 of them (81.3％,84.9％,and 84.0％ inHFrEF,HFmrEF and HFpEF groups,respectively) were followed up for an median duration of 27.3 months.Results (1) Among the study patients,26.5％ were in HFrEF,24.3％ in HFmrEF,and 49.2％ in HFpEF groups.(2) Ischemic heart disease with HFmrEF was more frequent than that in patients with HFrEF.The average age,percentage of female subjects,systolic blood pressure,uric acid,N terminal B-type natriuretic peptide precursor (NT-proBNP),hemoglobin,and the incidence of hypertensive heart disease,anemia,atrial fibrillation in patients with HFmrEF were higher than those in patients with HFrEF,but lower than those in patients with HFpEF (all P ＜0.01).(3) The all-cause cumulative mortality was 10.8％ at 1 year,20.6％ at 2 years and 35.9％ at 5 years.No difference was observed in the all-cause cumulative mortality at 1 year,2 years,5 years among the three groups (all P ＞ 0.05).Conclusions The HFmrEF patients,as a new and distinct group,were with many intermediate characteristics compared with HFrEF and HFpEF subjects.However,the all-cause mortality was not significantly differeut among HF patients with different LVEF.
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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.
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Objective: To explore the relationship between serum levels of immunoglobulin E (IgE) and calciifc aortic valve disease (CAVD) in relevant patients. Methods: A total of 394 patients were enrolled in our study. Based on echocardiography presentation, the patients were divided into 2 groups: CAVD group,n=169 and Non-CAVD group,n=225. Serum levels of IgE were examined by chemiluminescence method. The IgE levels were compared between 2 groups and the relationship between serum IgE level and CAVD was analyzed. Results: Serum levels of IgE in CAVD group was significantly higher than Non-CAVD group 113.30 IU/ml vs 63.76 IU/ml (P Conclusion: Serum IgE level is obviously increased in CAVD patients. IgE is an independent biochemical indicator of CAVD, it may play the important role in CAVD pathogenesis.
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<p><b>OBJECTIVE</b>To investigate the effect of anti-miR-145 on human airway smooth muscle cell (HASMC) proliferation and osteopontin systhesis in vitro and explore the mechanisms.</p><p><b>METHODS</b>HASMCs were treated with 10-100 nmol/L anti-miR-145, and the cell proliferation and apoptosis were investigated using a CCK-8 assay and flow cytometry, respectively. The changes in osteopontin synthesis after the treatment was quantified with Western blotting.</p><p><b>RESULTS</b>Treatment with 10 and 50 nmol/L anti-miR-145 significantly promoted the proliferation and osteopontin synthesis in HASMCs (P<0.05 or <0.01), and 50 nmol/L anti-miR-145 obviously inhibited the cell apoptosis (P<0.01).</p><p><b>CONCLUSION</b>Anti-miR-145 promotes HASMC proliferation and osteopontin synthesis and inhibits HASMC apoptosis in vitro, indicating the important role of anti-miR-145 in the pathogenesis of airway remodeling.</p>
Subject(s)
Humans , Airway Remodeling , Apoptosis , Cell Proliferation , Cells, Cultured , MicroRNAs , Myocytes, Smooth Muscle , Osteopontin , Respiratory System , Cell BiologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of curcumin on the expression of syndecan-4 protein and p44/42 mitogen- activated protein kinase(MAPK) phosphorylation in rat vascular smooth muscle cells (VSMCs) induced by tumor necrosis factor-α (TNF-α) in vitro.</p><p><b>METHODS</b>Rat VSMCs cultured in vitro were stimulated for 24 h by 20 ng/ml TNF-α, 20 µmol/L curcumin, or 20 ng/ml TNF-α plus 20 µmol/lL curcumin. /assay was adopted to evaluate the proliferation of the VSMCs, and the expression of syndecan-4 protein and phosphorylated p44/42 MAPK were determined by Western blotting.</p><p><b>RESULTS</b>Compared with the normal control cells, VSMCs exposed to TNF-α showed significantly enhanced proliferation (P/0.01). Curcumin treatment did not obviously affect the growth of otherwise untreated VSMCs(P>0.05), but could significantly suppress TNF-α-induced proliferation of VSMCs (P/0.01). TNF-α treatment also significantly increased the expression of syndecan-4 protein and phosphorylated p44/42 MAPK (P<0.01), which was markedly lowered by treatment with curcumin (P/0.01). Curcumin alone did not produce any obvious effects on the expression of syndecan-4 protein or phosphorylated p44/42 MAPK (P>0.05).</p><p><b>CONCLUSION</b>Curcumin can suppress the proliferation of rat VSMCs and lower the expression of syndecan-4 protein and phosphorylated p44/42 MAPK in TNF-α-induced VSMCs.</p>
Subject(s)
Animals , Rats , Cells, Cultured , Curcumin , Pharmacology , Mitogen-Activated Protein Kinase 3 , Metabolism , Muscle, Smooth, Vascular , Metabolism , Phosphorylation , Rats, Sprague-Dawley , Syndecan-4 , Metabolism , Tumor Necrosis Factor-alpha , PharmacologyABSTRACT
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.
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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.
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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
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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
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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
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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
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AIM: To investigate the use of therapy ultrasound to enhance nonviral gene delivery. METHODS: Endothelial cells (EC) and vascular smooth muscle cells (VSMC) were cultured in 6-well plates. Plasmid (pcDNA3.1/His/LacZ) with or without microbubbles at the different concentrations was transfected into the cells with the use of ultrasound for 1 min at 2 MHz, 1.8 mechanical index (MI). Additional controls included ultrasound alone, microbubble alone and microbubble plus plasmid. The rate of blue cells and the activities of ?-Gal were measured. In addition, cell viability was detected with different time from 1 to 30 min of ultrasound irradiation and the different concentrations of microbubbles. RESULTS: In the group of ultrasound with microbubble, the rate of blue cells and activity of ?-Gal markedly increased by 60% and 9-fold, respectively. Microbubbles at concentration of 10% led to the highest transfection effect. Ultrasoud exposure at 1 to 30 minute had no cell toxic effects, while microbubbles at the concentration of 50% had significant effect on cell survival. CONCLUSIONS: Albumin-coated microbubbles markedly enhance gene delivery by therapeutic ultrasound-mediated microbubble destruction, which can be used as a safe and practicality vectors in gene therapy.
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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
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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