Contraction-dependent TGF-ß1 activation is required for thrombin-induced remodeling in human airway smooth muscle cells.

AIMS: Thrombin is a serine proteinase that is not only involved in coagulation cascade, but also mediates a number of biological responses relevant to tissues repair, and induces bronchoconstriction. TGF-ß plays a pivotal role in airway remodeling due to its effects on airway smooth muscle proliferation and extracellular matrix (ECM) deposition. Recently, bronchoconstriction itself is found to constitute a form of strain and is highly relevant to asthmatic airway remodeling. However, the underlying mechanisms remain unknown. Here, we investigated the role of contraction- dependent TGF-ß activation in thrombin-induced remodeling in human airway smooth muscle (HASM) cells. MATERIALS AND METHODS: Primary HASM cells were treated with or without thrombin in the absence or presence of anti-TGF-ß antibody, cytochalasin D and formoterol. CFSE labeling index or CCK-8 assay were performed to test cell proliferation. RT-PCR and Western blotting were used to examined ECM mRNA level and collagen Iα1, α-actin protein expression, respectively. Immunofluorescence was also used to confirm contraction induced by thrombin in HASM cells. KEY FINDING: Thrombin stimulation enhanced HASM cells proliferation and activated TGF-ß signaling. Thrombin induced ECM mRNA and collagen Iα1 protein expression, and these effects are mediated by TGF-ß. Abrogation of TGF-ß activation by contraction inhibitors cytochalasin D and formoterol prevents the thrombin-induced effects. SIGNIFICANCE: These findings suggest that contraction-dependent TGF-ß activation could be a mechanism by which thrombin leads to the development of asthmatic airway remodeling. Blocking physical forces with bronchodilator would be an intriguing way in reducing airway remodeling in asthma.

[Red blood cell distribution width combined with lipoprotein-associated phospholipase A2 detection for improving diagnostic accuracy of coronary artery stenosis in patients with coronary artery disease].

OBJECTIVE: To study the association of red blood cell distribution width (RDW) and lipoprotein-associated phospholipase A2 (LP-PLA2) with the degree of coronary artery stenosis in patients with coronary artery disease (CAD) and the value of RDW combined with LP-PLA2 detection in accurate evaluation of coronary artery stenosis. METHODS: A total of 224 patients including 119 non-CAD cases and 105 CAD cases admitted in our hospital between June, 2013 and June, 2014 were enrolled in this study. The patients' baseline clinical data were collected and venous blood samples were obtained for detecting WBC, RDW-CV and LP-PLA2. The Gensini score of the CAD patients was calculated based on coronary angiographic findings. RESULTS: Compared with the non-CAD patients, CAD patients had significantly higher RDW-CV (P=0.009) and LP-PLA2 (P=0.004) levels. The CAD patients with high Gensini scores had also significantly higher RDW-CV (P=0.001) and LP-PLA2 (P<0.001) levels than those with low scores; RDW-CV and LP-PLA2 were significantly correlated with the Gensini score, and the area under curve of their combined detection was 0.931. CONCLUSION: Combination of RDW and LP-PLA2 can improve the diagnostic accuracy of the degree of coronary artery stenosis in patients with CAD.

Rosiglitazone attenuates atherosclerosis and increases high-density lipoprotein function in atherosclerotic rabbits.

Rosiglitazone has been found to have anti-atherogenic effects and to increase serum high-density lipoprotein (HDL) cholesterol (HDL-C) levels. However, in vivo studies investigating the regulation of adenosine triphosphate-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) by rosiglitazone are limited. Moreover, the effects of rosiglitazone on the function and levels of HDL are unclear. In the present study, we investigated the effects of rosiglitazone on HDL function and its mechanisms of action in atherosclerotic rabbits. Our results revealed that rosiglitazone induced a significant increase in serum HDL-C levels, paraoxonase 1 (PON1) activity, [(3)H]cholesterol efflux rates, and the expression of ABCA1 and SR-BI in hepatocytes and peritoneal macrophages. The expression of ABCA1 was also increased in aortic lesions. Rosiglitazone markedly reduced serum myeloperoxidase (MPO) activity, aortic intima-media thickness (IMT) and the percentage of plaque area in the aorta. It can thus be concluded that in atherosclerotic rabbits, rosigitazone increases the levels of HDL-C and hinders atherosclerosis. Thus, it improves HDL quality and function, as well as the HDL-induced cholesterol efflux, exerting anti-inflammatory and antioxidant effects.

Increased serum 2-oxoglutarate associated with high myocardial energy expenditure and poor prognosis in chronic heart failure patients.

Myocardial energy expenditure (MEE) and 2-oxoglutarate are elevated in chronic heart failure (CHF) patients compared with healthy controls. To explore whether 2-oxoglutarate could reflect the levels of MEE and predict the prognosis of CHF, 219 CHF patients and 66 healthy controls were enrolled. 2-Oxoglutarate was assayed with Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC/MS/MS). CHF patients were divided into 4 groups according to interquartile range of MEE and followed for death or recurrent hospital admission due to CHF for the mean follow-up time 6.64±0.16months. 2-Oxoglutarate was increased in CHF patients compared with controls (P<0.01) and correlated with estimated glomerular filtration rate (r=0.142, P=0.036), age (r=-0.269, P<0.01) and MEE levels (r=0.307, P<0.01) in a multiple linear correlation analysis in CHF patients. Furthermore, 2-oxoglutarate (OR=3.470, 95% CI=1.557 to 7.730, P=0.002), N-terminal pro-B-type natriuretic peptide (OR=4.013, 95% CI=1.553 to 10.365, P=0.004), age (OR=1.611, 95% CI=1.136 to 2.283, P=0.007) and left ventricular ejection fraction (OR=7.272, 95% CI=3.110 to 17.000, P<0.001) were independently associated with MEE on multiple logistic regression analysis. Kaplan-Meier event curves showed that high 2-oxoglutarate levels were associated with adverse outcomes (Log Rank, Chi(2)=4.026, P=0.045). This study showed that serum 2-oxoglutarate is associated with MEE levels, which can be used as potential biomarkers for MEE, and it can reflect the clinical severity and short-term outcome of CHF.

Sclederma of Poria cocos exerts its diuretic effect via suppression of renal aquaporin-2 expression in rats with chronic heart failure.

ETHNOPHARMACOLOGICAL RELEVANCE: Sclederma of Poria cocos (Hoelen) has been used as a diuretic in traditional Asian medicine. However, the underlying mechanism by which Sclederma of Poria cocos (hoelen) exerts its diuretic effect has not been well identified. The aim of the present study was to evaluate the effects of Sclederma of Poria cocos (hoelen) in rats with chronic heart failure (CHF) induced by acute myocardial infarction and to investigate the underlying mechanisms. MATERIALS AND METHODS: An aqueous extract of Sclederma of Poria cocos (hoelen) (2.4 g/kg/d, 1.2 g/kg/d or 0.6 g/kg/d) or furosemide (20 mg/kg/d) was administered orally to male Sprague-Dawley rats starting on the day of coronary ligation. The urine output of all rats was quantified and collected every day for 1 or 4 weeks. The expression of aquaporin-2 (AQP2) was examined after treatment for 1 or 4 weeks. RESULTS: Urinary output increased significantly and urinary osmolality decreased after oral administration of Sclederma of Poria cocos (hoelen) for both 1 and 4 weeks. Sclederma of Poria cocos (hoelen) caused less electrolyte disorder than furosemide. Furthermore, Sclederma of Poria cocos (hoelen) reduced the levels of plasma BNP in CHF rats, whereas furosemide had no effect. Importantly, both mRNA and protein expression of AQP2 were down-regulated and urinary excretion of AQP2 was decreased after administration of Sclederma of Poria cocos (hoelen) to CHF rats. Similarly, Sclederma of Poria cocos (hoelen) reduced plasma arginine vasopressin (AVP) level and down-regulated vasopressin type 2 receptor (V2R) mRNA expression. CONCLUSIONS: Sclederma of Poria cocos (hoelen) exerts its diuretic effect and improves cardiac function in CHF rats via the AVP-V2R-AQP2 axis.

Acute coronary syndrome remodels the protein cargo and functions of high-density lipoprotein subfractions.

OBJECTIVES: This study examined alterations in the functions and proteome of high-density lipoprotein (HDL) subfractions (HDL2 and HDL3) isolated from patients with acute coronary syndrome (ACS) compared with control subjects. METHODS: We measured HDL subfraction cholesterol efflux capacity, inflammatory index (HII), paraoxonase-1 (PON1) activity, and lipid hydroperoxide (LOOH) levels in both male age-matched controls and the ACS group (n = 40/group). Additionally, proteomic analysis was used to monitor changes in the HDL subfraction proteome between controls and ACS subjects. RESULTS: Both HDL2 and HDL3 from ACS patients had greater HII and LOOH levels compared with controls (P<0.001); PON1 activity and cholesterol efflux capacity in both HDL2 and HDL3 from the ACS group were significantly less than those of controls (P<0.001). Using proteomic analysis, we demonstrated that, compared with the control group, nine proteins were selectively enriched in HDL3 from subjects with ACS, and ras-related protein Rab-7b was decreased in HDL3. Additionally, in the ACS subjects, 12 proteins were decreased in HDL2 and 4 proteins were increased in HDL2. CONCLUSIONS: Functional HDL subfractions shifted to dysfunctional HDL subfractions during ACS, and the functional impairment was linked to remodeled protein cargo in HDL subfractions from ACS patients.

[Effect of dexamethasone on expressions of TWEAK and Fn14 in the lung of asthmatic mice].

AIM: To investigate the effect of dexamethasone (Dex) on the expressions of TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible immediate-early response protein 14 (Fn14) in the lung of asthmatic mice. METHODS: Ovalbumin (OVA) was used to induce asthma in BALB/c mice. Thirty-six female mice were randomly divided into control group (n=12), asthmatic group (n=12) and Dex treated group (n=12). The airway inflammation was evaluated by HE staining. The expressions of TWEAK and Fn14 at mRNA and protein levels were detected by RT-PCR and immunohistochemistry, respectively. RESULTS: Both mRNA and protein levels of TWEAK and Fn14 in the asthmatic model group were significantly higher than those of control group (P<0.01), and both mRNA and protein levels of TWEAK and Fn14 in the Dex treated group were significantly lower than those of asthmatic group (P<0.01). CONCLUSION: Dex can reduce the airway inflammation through inhibiting the expressions of TWEAK and Fn14.

Probucol alleviates atherosclerosis and improves high density lipoprotein function.

BACKGROUND: Probucol is a unique hypolipidemic agent that decreases high density lipoprotein cholesterol (HDL-C). However, it is not definite that whether probucol hinders the progression of atherosclerosis by improving HDL function. METHODS: Eighteen New Zealand White rabbits were randomly divided into the control, atherosclerosis and probucol groups. Control group were fed a regular diet; the atherosclerosis group received a high fat diet, and the probucol group received the high fat diet plus probucol. Hepatocytes and peritoneal macrophages were isolated for [(3)H] labeled cholesterol efflux rates and expression of ABCA1 and SR-B1 at gene and protein levels; venous blood was collected for serum paraoxonase 1, myeloperoxidase activity and lipid analysis. Aorta were prepared for morphologic and immunohistochemical analysis after 12 weeks. RESULTS: Compared to the atherosclerosis group, the paraoxonase 1 activity, cholesterol efflux rates, expression of ABCA1 and SR-BI in hepatocytes and peritoneal macrophages, and the level of ABCA1 and SR-BI in aortic lesions were remarkably improved in the probucol group, But the serum HDL cholesterol concentration, myeloperoxidase activity, the IMT and the percentage plaque area of aorta were significantly decreased. CONCLUSION: Probucol alleviated atherosclerosis by improving HDL function. The mechanisms include accelerating the process of reverse cholesterol transport, improving the anti-inflammatory and anti-oxidant functions.

[Activation of Rho-kinase pathway is involved in angiotensin II-induced contraction of human airway smooth muscle cells].

OBJECTIVE: To investigate of the regulatory effect of Rho-kinase pathway activation on angiotensin II (Ang II)-induced contraction of human airway smooth muscle cells (HASMCs) in vitro. METHODS: Cultured primary HASMCs were divided into control group, AngII group, AngII + irbesartan group and AngII + Y-27632 group with corresponding treatment. AngII-induced contraction of HASMCs was evaluated using collagen gel lattices and observed morphologically using immunofluorescence assay. Western Blotting was significantly performed to examine the protein expression of Rho-kinase signal pathway. RESULTS: AngII-induced HASMC contraction was inhibited by treatments with irbesartan and Y-27632 as shown by gel contraction assay (P<0.001). Y-27632 treatment produced a stronger inhibitory effect than irbesartan on the expression of phosphorylated moesin, a substrate of Rho kinase (P<0.05). CONCLUSION: AngII induces the contraction of HASMCs partially as a result of activation of Rho-kinase pathway.

[Change of IL-22R1 expression in human airway smooth muscle cells in response to different stimulating agents].

OBJECTIVE: To explore the effects of serum of asthmatic patients, dexamethasone, interleukin-4 (IL-4), interferon-gamma (IFN-γ) and transforming growth factor-ß (TGF-ß) on the expression of interleukin-22 receptor 1 (IL-22R1) mRNA and protein in HASMCs in vitro. METHODS: IL-22R1 mRNA and protein expressions in HASMCs treated with different stimulating agents were measured by real-time PCR and Western blotting, respectively. RESULTS: IL-22R1 mRNA and protein expressions in HASMCs were significantly increased after stimulation by serum from asthmatic patients, but decreased after co-stimulation with dexamethasone. IL-22R1 mRNA and protein expressions in the cells both increased after stimulation by IL-4, IFN-γ and TGF-ß. CONCLUSION: IL-22R1 in HASMCs might be involved in the pathogenesis of asthma, and the therapeutic effect of dexamethasone on asthma is mediated, at least partially, by IL-22R1. The effects of IFN-γ, IL-4, and TGF-ß on asthma may also be attributed to their actions on HASMCs.

[Alteration of PTEN gene expression affects the migration of human airway smooth muscle cells in vitro].

OBJECTIVE: To investigate the changes in human airway smooth muscle cell (HASMC) migration and related signaling pathway after interference with PTEN gene expression. METHOD: HASMCs were infected with an adenovirus vector and RNA interference vector of human PTEN gene to establish the cell model with PTEN gene over-expression (Ad-GFP-PTEN-HASMC) and one with PTEN gene silencing (Ad-shPTEN-HASMC), using Ad-GFP-infected and a blank cells as the negative controls and LY294002 as the positive control. Fluorescence microscopy and flow cytometric analysis were used to evaluate the transfection efficiency, and Western blotting was performed to examine the expression of PTEN and the activation of AKT and ERK1/2 signal pathway. Transwell assay and wound healing assay were used to assess the migration of HASMCs. RESULTS: The adenovirus over-expression vector and RNA interference vector significantly affected the expression of human PTEN gene. Up-regulation of PTEN gene resulted in a slow-down of the HASMC migration, an inhibition of PI3K/AKT signal pathway at the protein level but no changes in Ras-Raf-MEK1/2-ERK1/2 signal pathway. Down-regulated PTEN gene expression, however, was not associated with an enhancement of HASMC migration, but activated PI3K/AKT signal pathway and inhibited Ras-Raf-MEK1/2-ERK1/2 signal pathway. CONCLUSION: Upregulation of PTEN gene can effectively inhibit airway smooth muscle cell migration, the effect of which is probably mediated by the PI3K/AKT pathway.

[Effect of physiological doses of testosterone on mitochondrial DNA deletion in castrated mice].

OBJECTIVE: To evaluate the effect of physiological doses of testosterone on mitochondrial DNA (mtDNA) deletion in the aortic vascular wall of castrated C57BL/6J mice. METHOD: Twenty-four male C57BL/6J mice were randomized into normal control group (n=8), castrated+placebo group (castrated group, n=8), and castrated+physiological doses (1 mg/kg every 3 days) of testosterone group (n=8). The mice were fed normally for 3 months along with 8 mice with natural aging (18 months old), after which blood samples were obtained from all the groups for measurement of testosterone concentrations. The aortic mtDNA was extracted to analyze the deleted fragments using nested PCR, and fragments with deletions were purified and identified by sequence analysis. RESULTS: Compared with the normal control group, the castrated group showed a significantly higher optical density ratio of the deletions [(18.1713 ∓ 2.4317)% vs (36.8475 ∓ 3.3365)%], but no significant difference was found between the castrated and natural ageing group [(42.3075 ∓ 3.6556)%]. The castrated+testosterone showed a lowered optical density ratio of (23.6488 ∓ 2.7634)% as compared with the castrated and natural ageing group, but a similar one with the normal control group. Sequence analysis identified 4 different types of deletions in the aging aorta at 3713, 3864, 4236, and 4415 bp, and the presence of direct repeats was confirmed to flank the deletions. CONCLUSIONS: Multiple mtDNA deletions occur in ageing mice at a higher rate than in young mice. Testosterone deficiency is associated with increased aortic mtDNA deletions, which can be decreased by physiological doses of testosterone.

[Effects of simvastatin on plasma SOD, MDA and 8-iso-PGF2α in patients with stable angina].

OBJECTIVE: To observe the effects of simvastatin on plasma superoxide dismutase (SOD), malonaldehyde (MDA) and 8-iso-prostaglandin F2α (8-iso-PGF2α) as well as uric acid (UA) and serum lipids in patients with stable angina. METHODS Eighty-five patients with stable angina were divided into 4 groups, including hyperlipemia treatment group (HLT), hyperlipemia control group (HLC), normolipemia treatment group (NLT), and normolipemia control group (NLC). All the patients received routine treatment according to the guideline of CHD treatment, and those in the treatment groups were given Simvastatin (40 mg) every night, whereas those in the control group received placebo for 3 months. Before and after the treatments, the levels of plasma 8-iso-PGF2α were measured by enzyme-linked immunosorbent assay, and the plasma levels of SOD and MDA were detected by colorimetric method. LDL, HDL, TC, TG, and UA were also measured biochemically. RESULTS Compared with the control group, both of the treatment groups showed significantly increased levels of SOD and decreased MDA, 8-iso-PGF2α, UA and plasma lipids after the treatments (P<0.05). CONCLUSION In patients with coronary heart disease, simvastatins can decrease plasma lipids, inhibit lipid peroxidations, and promote the clearance of free radicals, thereby alleviating the oxidative stress.

[Effects of a new houttuyfonate derivative on proliferation of NIH3T3 cell and expression of syndecan-4 induced by tumor necrosis factor alpha].

OBJECTIVE: To investigate the effect of a new houttuyfonate derivative (NHD) on proliferation of NIH3T3 cell and expression of Syndecan-4 induced by TNF-alpha in vitro. METHODS: NIH3T3 cells were cultured and exposed to TNF-alpha or NHD respectively, and then cotreated with TNF-alpha and NHD. All the groups were cultured for 24 hour in vitro, in addition to the untreated control group established for comparison. The ratio of proliferation of NIH3T3 cell was determined by non-radioactive MTS/PMS assay and the expression of Syndecan-4 was evaluated by western blot using anti-Syndecan-4 antibody. RESULTS: Statistical analysis showed that, compared with the control group, NHD had no effect on VSMCs growth, but significantly inhibited NIH3T3 cell proliferation while induced by TNF-alpha. It also showed that compared with control group, NHD had no effect on the expression of Syndecan-4, but significantly inhibited its expression while induced by TNF-alpha (P < 0.05). CONCLUSIONS: NHD can inhibit the proliferation of NIH3T3 cell and the expression of syndecan-4 protein induced by TNF-alpha in vitro.

ISO-1, a macrophage migration inhibitory factor antagonist, inhibits airway remodeling in a murine model of chronic asthma.

Airway remodeling is the process of airway structural change that occurs in patients with asthma in response to persistent inflammation and leads to increasing disease severity. Drugs that decrease this persistent inflammation play a crucial role in managing asthma episodes. Mice sensitized (by intraperitoneal administration) and then challenged (by inhalation) with ovalbumin (OVA) develop an extensive eosinophilic inflammatory response, goblet cell hyperplasia, collagen deposition, airway smooth muscle thickening, and airway wall area increase, similar to pathologies observed in human asthma. We used OVA-sensitized/challenged mice as a murine model of chronic allergic airway inflammation with subepithelial fibrosis (i.e., asthma). In this OVA mouse model, mRNA and protein of macrophage migration inhibitory factor (MIF) are upregulated, a response similar to what has been observed in the pathogenesis of acute inflammation in human asthma. We hypothesized that MIF induces transforming growth factor-ß1 (TGF-ß1) synthesis, which has been shown to play an important role in asthma and airway remodeling. To explore the role of MIF in the development of airway remodeling, we evaluated the effects of an MIF small-molecule antagonist, (S,R)3-(4-hy-droxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), on pathologies associated with the airway-remodeling process in the OVA mouse model. We found that administration of ISO-1 significantly mitigated all symptoms caused by OVA treatment. In addition, the treatment of OVA-sensitized mice with the MIF antagonist ISO-1 significantly reduced TGF-ß1 mRNA levels in pulmonary tissue and its protein level in bronchial alveolar lavage fluid supernatants. We believe the repression of MIF in the ISO-1 treatment group led to the significant suppression observed in the inflammatory responses associated with the allergen-induced lung inflammation and fibrosis in our murine asthma (OVA) model. Our results implicate a possible function of MIF in the pathogenesis of chronic asthma and suggest that MIF might be an important therapeutic target for airway remodeling.

[Pravastatin inhibits the expression of syndecan-4 protein in tumor necrosis factor-alpha-induced rat vascular smooth muscle cells in vitro].

OBJECTIVE: To investigate the effect of pravastatin on the proliferation of rat vascular smooth muscle cells (VSMCs) and expression of syndecan-4 protein induced by tumor necrosis factor-alpha (TNF-alpha). METHODS: VSMCs cultured in vitro were exposed to 20 ng/ml TNF-alpha, 10 micromol/ml pravastatin, 20 micromol/ml pravastatin, 10 micromol/ml pravastatin with 20 ng/ml TNF-alpha, or 20 micromol/ml pravastatin with 20 ng/ml TNF-alpha for 24 h. The proliferation of the VSMCs was determined by non-radioactive MTS/PMS assay and the expression of syndecan-4 protein was detected by Western blotting using anti-syndecan-4 antibody. RESULTS: Compared to the control group, TNF-alpha at 20 ng/ml significantly stimulated the proliferation of rat VSMCs (P<0.05). Pravastatin alone produced no obvious effect on VSMCs growth (P>0.05), but significantly inhibited TNF-alpha-induced VSMC proliferation (P<0.05). The expression of syndecan-4 protein in the VSMCs was significantly enhanced by 20 ng/ml TNF-alpha (P<0.01). Pravastatin alone did not affect the expression of syndecan-4 protein (P>0.05), but significantly inhibited TNF-alpha-induced enhancement of syndecan-4 protein expression (P<0.01). CONCLUSION: Pravastatin can inhibit the proliferation and syndean-4 protein expression in rat VSMCs induced by TNF-alpha in vitro.

[Effects of dexamethasone on expression of Th17 transcription factor RORgammat mRNA in experimental asthma mice.].

AIM: To study the effects of dexamethasone (Dex) on expression of Th17 transcription factor retinoic acid-related orphan receptor gamma t(RORgammat)in asthma. METHODS: The BALB/c mice asthma model was induced by ovalbumin(OVA) with classic method.Thirty female mice were randomly divided into control group, asthmatic group and Dex treated group. The level of IL-17 in mice bronchoalveolar lavage fluid (BALF) and serum was measured by enzyme-linked immunosorbent assay(ELISA). Airway Responsiveness to acetylcholine chloride(Ach) was measured by a modified non-invasive method; The airway inflammation was evaluated by HE staining.The expression of RORgammat mRNA was measured by reverse transcription-polymerase chain reaction(RT-PCR). RESULTS: The level of RORgammat mRNA, IL-17 of asthmatic group were significantly higher than those of control group(P<0.01), which were significantly reduced by Dex compared with the asthmatic group(P<0.05). CONCLUSION: Dex can inhibit the release of IL-17, whose mechanism may be the blockade of TH17 differentiation in asthmatic models by inhibit the RORgammat expression.

[Effect of recombinant adenovirus carrying PTEN gene on the proliferation of human airway smooth muscle cells in vitro and the mechanisms].

OBJECTIVE: To observe the effect of exogenous phophatase and tensin homolog deleted on chromosone 10 (PTEN) gene transfer via recombinant adenoviruses on the proliferation of human airway smooth muscle cells (HASMCs) in vitro and investigate the possible mechanisms. METHODS: With a recombinant adenovirus vector containing PTEN (Ad-PTEN) constructed using the pAdxsi system, PTEN gene was transiently transfected into HASMCs and the transfection efficiency was determined by fluorescence microscope. RT-PCR and Western blotting were performed to detect the expression of PTEN mRNA and protein in the infected cells. MTS/PMS assay was used to analyze the proliferation of HASMCs, and the cell cycle changes of the transfected cells were evaluated by flow cytometry with PI staining. The expression levels of Akt and p-A kt proteins were detected by Western blotting, and P21 mRNA expression determined by RT-PCR. RESULTS: The recombinant adenovirus Ad-PTEN showed a wild-type PTEN gene transfer efficiency of 98% at the multiplicity of infection (MOI) of 100. RT-PCR and Western blotting showed that infection with the recombinant adenovirus resulted in PTEN overexpression in the HASMCs, causing also increased ratio of G(0)/G(1) cells and proliferation inhibition of the ASMCs. The overexpression of PTEN significantly decreased the expression level of p-Akt but increased P21 mRNA expression. CONCLUSION: The recombinant adenovirus containing PTEN can be successfully transfected into HASMCs cultured in vitro, resulting in PTEN overexpression at both the mRNA and protein levels. PTEN overexpression can efficiently inhibit the proliferation of HASMCs possibly through the PI3K/PKB/AKt and P21 pathways.

[Effects of dexamethasone on intracellular expression of Th17 cytokine interleukin 17 in asthmatic mice].

OBJECTIVE: To study the effects of dexamethasone on intracellular expression of Th17 cytokine interleukin 17 and the mechanisms in asthmatic mice. METHODS: Experimental asthma was induced by ovalbumin (OVA) sensitization in 20 in female Balb/c mice with (dexamethasone group, n=10) or without dexamethasone treatment (model group, n=10), with another 10 serving as the control group. The levels of IL-17 in the bronchoalveolar lavage fluid (BALF) and serum of the mice were measured by enzyme-linked immunosorbent assay (ELISA), and the airway inflammation was evaluated by HE staining. The expressions of IL-17 and RORgammat mRNA were measured by reverse transcription-polymerase chain reaction (RT-PCR), and the expression of RORgammat protein was measured by immunohistochemical staining. RESULTS: The levels of RORgammat and IL-17 mRNA and protein in the asthmatic model group were significantly higher than those in the control group (P<0.01), and the increased expressions of RORgammat and IL-17 mRNA and protein in the asthmatic mice were significantly reduced by dexamethasone treatment (P<0.05). CONCLUSION: Dexamethasone can inhibit the release of IL-17 probably by inhibiting RORgammat expression and blocking Th17 differentiation in asthmatic mice.

[Construction of a recombinant adenovirus vector containing PTEN and its expression in airway smooth muscle cells].

OBJECTIVE: To construct a recombinant adenovirus vector containing phosphatase and tensin homolog deleted on chromosome 10 (PTEN) using the pAdxsi system. METHODS: PTEN cDNA from plasmid pcDNA3-PTEN was cloned into the shuttle plasmid pShuttle-GFP-CMV. The shuttle vector was transformed into competent DH5alpha strain with the vector pAdxsi to achieve the homologous recombination. The recombinant construct was subsequently linearized with PacI and transfected into HEK293 cells via Lipofectamine 2000. The recombinant adenovirus particles were collected, and after titration, the recombinant adenovirus was traced by monitoring GFP expression under fluorescence microscope. The expression of PTEN mRNA and protein in the recombinant adenovirus vector and airway smooth muscle cells were detected by PCR and Western blotting, respectively. RESULTS: GFP was expressed in HEK293 cells infected by recombinant adenovirus, and the expression intensity increased gradually with the passage of time, with obvious cytopathic effect (CPE) noted in the cells. After 3 cycles of amplification, the titer of adenovirus containing PTEN reached an appropriate level. The viral titer of pAdxsi-GFP-PTEN was 2x10(10) pfu/ml, and PTEN mRNA expression was detected by PCR. The homologous protein expressed in the infected human airway smooth muscle cells significantly increased in comparison with that in the control cells. CONCLUSION: The recombinant adenovirus containing PTEN is constructed successfully, which provides an experimental basis for studying the role of PTEN gene in asthma therapy.