Role of cyclin D1 in PKC regulating the proliferation of airway smooth muscle cells in asthmatic rat

AIM: To observe the expression of cyclin D1 following PKC activation and the correlation between PKC-α and cyclin D1 in asthmatic rat airway smooth muscle cells (ASMCs), and to discuss the effects of cyclin D1 on the proliferation of airway smooth muscle cells regulated by PKC in asthmatic rat. METHODS: Twelve SD rats were randomly divided into control group (group N) and asthmatic 2-week group (group A), and then subdivided into 4 groups based on the drug interventions: (1) control group; (2) PMA; (3) PMA+5 μmol/L Ro-31-8220 group; (4) 5 μmol/L Ro-31-8220 group. The proliferations of ASMCss were examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expressions of PKC-α and cyclin D1 were analyzed by RT-PCR and Western blotting. The data were subject to correlation analysis. RESULTS: (1) Compared to group A1, there were significant differences in the percentage of S+G2/M phase, absorbance A value of MTT and the rate of positive expression of PCNA protein in group A2 and A4 (P<0.01). The same tendency in group N was observed according with group A. (2) Compared with A1, the ratios of A values of PKC-α mRNA and protein in group A2 and A4 were significantly changed (P<0.01) as well as the ratios of A values of cyclin D1 mRNA and protein. (3) There were positive correlations between PKC-α and cyclin D1 in mRNA (r=0.476, P<0.05) and protein(r=0.899, P<0.01). CONCLUSION: The activation of PKC-α promotes ASMCs proliferation in asthmatic rats, and there are positive correlations between the PKC-α and cyclin D1 in mRNA and protein, indicating that the PKC-α signal pathway may be involved in the process of airway smooth muscle remodeling by regulating the expression of cyclin D1 in asthmatic rats.     

Effect of nuclear factor-κB on proliferation of airway smooth muscle cells regulated by protein kinase C in asthmatic rats

AIM: To investigate the effect of protein kinase C (PKC)- nuclear factor-κB (NF-κB) signal pathway on proliferation of airway smooth muscle cells (ASMCs) in asthmatic rats.METHODS: (1) 16 Wistar rats were divided into asthmatic group (8 rats) and control group (8 rats).ASMCs from asthmatic group and control group were treated with PKC agonist PMA and NF-κB inhibitor PDTC.The proliferation of ASMCs was examined by cell cycle analysis,MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining,respectively.NF-κB activity was detected by NF-κB p65 immunofluorescence staining and electrophoretic mobility shift assay (EMSA),respectively.RESULTS: The percentage of S phase,A value,the positive expression rate of PCNA,the positive expression rate of NF-κB p65 and EMSA value in asthmatic ASMCs treated with PMA were higher than those in asthmatic ASMCs without treatment (P<0.05).After asthmatic ASMCs previously treated with PDTC,then with PMA,the above figures were lower than those in asthmatic ASMCs only treated with PMA and without treatment (P<0.05).The above figures in asthmatic ASMCs only treated with PDTC were lower than those in asthmatic ASMCs without treatment (P<0.05).CONCLUSION: NF-κB may contribute to the proliferation of ASMCs in asthmatic rat,in which PKC－NF-κB signal pathway is involved.     

Effects of Wnt/β-catenin signal pathway on asthma airway remodeling

AIM: To explore the effect of Wnt/β-catenin signaling pathway in airway smooth muscle cells (ASMC) on asthmatic airway remodeling.METHODS: The asthmatic airway remodeling model in rats was established and the ASMC was isolated and cultured. The protein expression of β-catenin, glycogen synthase kinase-3β (GSK-3β), c-Myc and cyclin D1 in the ASMC was determined by Western blot. After depressing the interaction between β-catenin and p300/CBP, the cell activity was measured by CCK-8 assay and the change of cell cycle distribution was analyzed by flow cytometry. Meanwhile, the protein expression of c-Myc and cyclin D1 in the ASMC was determined by Western blot after inhibiting P38 mitogen-activated protein kinase (MAPK) activity.RESULTS: The protein levels of β-catenin, c-Myc and cyclin D1 were significantly increased in asthma group while the protein level of GSK-3β was decreased in the same group (P<0.05). After depressing the interaction between β-catenin and p300/CBP, the cell activity of ASMC was decreased in asthma group compared with control group (P<0.05), and the change of the cell cycle distribution in asthma group was also more obvious (P<0.05). After inhibiting P38 MAPK activity, the protein levels of c-Myc and cyclin D1 were all decreased compared with control group in ASMC asthma and control rats (P<0.05).CONCLUSION: Wnt/β-catenin signaling pathway may participates in airway remodeling in asthma by increasing the protein expression of c-Myc and cyclin D1, reacting with the P38 MAPK signaling pathway and regulating the growth of ASMC.     

Expression of PI3K in airway smooth muscle cell of asthmatic rats

AIM: To investigate the expression of PI3K in airway smooth muscle cell (ASMC) of asthmatic rats.METHODS: 16 Wistar rats were divided into two groups, asthma and normal control at random. After establishment of asthmatic model, flow cytometry, immunofluorescence and Western blotting were applied to detect the growth fraction of ASMC and the expression of PI3K in cultured ASMC from each rat.RESULTS: It was revealed from flow cytometry that the ratio of S + G2/M to total number of cells in asthma group ［ (27.90±3.44) % ］ was higher than that in normal control group ［ (13.00±1.56) %, P<0.05］. The expression of PI3K was observed in both asthma and normal control group. However, it was much higher in asthma group than that in normal control group. There was a positive correlation between the expression of PI3K and the growth fraction in ASMC. CONCLUSION: The increased expression of PI3K might play an important role in regulating the proliferation of ASMC in asthma.     

Effect of L-arginine on intimal proliferation and expression of related cell cycle regulatory factors after vascular injury in rats

AIM:To investigate the effect of L-arginine (L-Arg) on intimal proliferation and expression of related cell cycle regulatory factors after vascular injury in rats. METHODS:Rats were divided into three groups :sham operation group, balloon injury group(this group included balloon 48 h,7 d and 14 d subgroup) and balloon+L-Arg group. Neointima area were calculated morphologiocally. The expression of cyclin dependent kinase-2(CDK2),cyclin E and proliferation cell nuclear antigen (PCNA) were measured by means of immunohistochemical technique and computer image analyzer. RESULTS:After vascular balloon injury, the level of plasma NO decreased, CDK2、cyclin E and PCNA expressed in the media at 48 h and in the neointima at 7 d and 14 d but with low and undetected expression in the media, the expression of CDK2, cyclin E and PCNA increased with the intima thickening. Compared with balloon 14 d group, the plasma NO level increased (P＜0.01), the neointima area reduced by 59.1%(P＜0.01) and the positive expression indexes of CDK2, cyclin E and PCNA decreased by 36.1%, 46.3% and 76.2% respectively in balloon+L-Arg group (P all＜0.01). CONCLUSION:L-Arg can effectively repress intima proliferation after vascular injury, which may be associated with its inhibiting the proliferation of vascular smooth muscle cell through downregulating the excessive expression of CDK2, cyclin E and PCNA.     

Effects of glucocorticoid inhalation on the levels of CD4+CD25+ regulatory T cells in peripheral blood of asthmatic children

AIM: To investigate the effect of glucocorticoid inhalation on the levels of CD4+CD25+ regulatory T cells in peripheral blood of asthmatic children. METHODS: Glucocorticoid inhalator was inhaled by 70 children with attack asthma. The levels of CD4+CD25+Tr in peripheral blood of asthmatic children were tested by flow cytometry (FCM). RESULTS: The CD4+CD25+Tr levels in peripheral blood of asthmatic children were (5.62%±1.29%) and (7.05%±1.61%) before and after of regulated glucocorticoid inhalation, respectively (P<0.01). The Tr levels were (7.56%±1.88%), (7.09%±1.23%) and (6.11%±1.96%) in the complete control group, part control group and poor control group, respectively (P<0.05). The Tr level in formal treatment group (7.05%±1.61%) was higher than that in irregular treatment group (5.91%±1.76%), P<0.01. CONCLUSION: The level of CD4+CD25+Tr is remarkable increased by regulated glucocorticoid inhalation, and the level of Tr can reflect the effects of glucocorticoid inhalation.     

Overexpression of integrin-linked kinase improves survival and proliferation of cardiac c-Kit+ cells

AIM: To investigate the effects of integrin-linked kinase (ILK) overexpression on survival and proliferation of cardiac c-Kit⁺ cells, and the role of ILK-overexpressing c-Kit⁺ cell transplantation in cardiac function in a rat myocardial infarction (MI) model.METHODS: Cardiac c-Kit⁺ cells were isolated from the hearts of neonatal Sprague-Dawley (SD) rats and cultured to prepare the ILK-c-Kit⁺ cells by infected with recombinant adenoviral vector harboring human wild-type ILK cDNA. The survival and proliferation of cardiac c-Kit⁺ cells were detected by cell counting and CCK-8 assay at 48 h after infection, respectively. The protein levels of cyclin D1 and proliferating cell nuclear antigen (PCNA) in the cardiac c-Kit⁺ cells were examined by Western blot. MI was induced by coronary artery ligation in 40 adult rats. After 15 min, ILK-c-Kit⁺ cells were transplanted into the hearts by myocardial injection at 3 different sites in the infracted zone and border zone. All rats were randomly divided into 4 groups:sham group, MI plus saline injection group (MI group), MI plus null vector-infected cardiac c-Kit⁺ cell injection group (Ad-null-c-Kit⁺ cell group), and MI plus ILK-overexpressing cardiac c-Kit⁺ cells injection group (ILK-c-Kit⁺ cell group), with 10 rats in each group. At 2 weeks after MI, the protein levels of c-Kit in MI hearts were investigated by immunohistochemical assay. At 4 weeks, left ventricular function was examined by hemodynamic measurement.RESULTS: The survival and proliferation of cardiac c-Kit⁺ cells and the protein levels of cyclin D1 and PCNA were enhanced by ILK overexpression compared with Ad-null group. In MI rat model, the number of c-Kit⁺ cells was increased by ILK-c-Kit⁺ cell injection compared with Ad-null-c-Kit⁺ cell group at 2 weeks after MI. Cardiac function was significantly improved in ILK-c-Kit⁺ cell-transplanted rats.CONCLUSION: ILK overexpression improves survival and proliferation of cardiac c-Kit⁺ cells by increasing the protein levels of cyclin D1 and PCNA. ILK-c-Kit⁺ cell transplantation enhances the therapeutic efficiency of cardiac c-Kit⁺ cells in the post-MI hearts of rats.     

PAF promotes proliferation of smooth muscle cells of rat airway

AIM: To study the effect of platelet-activating factor(PAF) on proliferation of cultured rat airway smooth muscle cells(ASMCs).METHODS: The cells were divided into control group and PAF group. The cells in PAF group were subdivided into four small groups by concentrations of PAF 10-6, 10-7, 10-8, 10-9 mol·L-1, MTT assay was used not only to investigate the effects of PAF on proliferation of ASMC but also to confirm the optimal concentration. Flow cytometry and immuneohistochemistry for proliferating cell nuclear antigen (PCNA) were also used to analyse its function on proliferation of ASMC.RESULTS: PAF (10-6-10-9mol·L-1) stimulated the cell proliferation and 10-7mol·L-1 PAF reached the maximal effect. The cell percentage of the ASMCs of 107 mol·L-1 PAF subgroup at G0/1 phase (68.67%) was much lower than that of control group (85.57%, P<0.01), in this subgroup, the percentage of expression of PCNA at 48 h (71.05%±1.22%) was significantly increased compared with the control group (53.27%±2.56%, P<0.05).CONCLUSION: PAF can stimulate the proliferation of cultured ASMC in a time-dependent, but not dose-dependent manner.     

Down-regulation of glutamyl cysteine cystathionine-lyase/hydrogen sulfide system in rat cardiovascular calcification

AIM：To observe the changes of cystathionine-lyase/hydrogen sulfide (CSE/H2S) in vivo in vascular calcification and to explore the role of CSE/H2S in vascular calcification.METHODS：Vascular calcification model in rats was induced by administration of vitamin D3 plus nicotine.The extent of calcification was estimated by assaying calcium content.［45Ca2+］ deposition and alkaline phosphatase (ALP) activity were detected.CSE mRNA amount was determined by using competitive quantitative RT-PCR.The content of H2S and activity of CSE in the plasma and cardiovascular tissues were also determined with biochemical methods.RESULTS：Calcium content in myocardium increased by 3.8 folds in a calcification model.Compared to control,calcium content,［45Ca2+］ accumulation and ALP activity in calcified arteries increased by 6.8,1.4,and 1.9 folds,respectively (P＜0.01).H2S contents in plasma,myocardium and aorta were 39%,39% and 31% lower than those in control group (P＜0.01).The gene expression of CSE was down-regulated in myocardium and aorta.Compared to control group,the amount of CSE mRNA in myocardium and calcified aorta were decreased by 28% and 36%,respectively (P＜0.01).The activity of CSE was 56% and 53% lower than that in control (P<0.01).CONCLUSION：The production of H2S,the gene expression and activity of CSE are down-regulated in the cardiovascular calcification,suggesting that the decrease in H2S production plays a role in the pathogenesis of vascular calcification.     

Protective role of endogenous hydrogen sulfide in cholecystokinin octapeptid against acute lung injury induced by lipopolysaccharide

AIM: To explore the role of endogenous hydrogen sulfide (H₂S) in the mechanism of cholecystokinin octapeptide (CCK-8) to alleviate acute lung injury (ALI) induced by lipopolysaccharide (LPS). METHODS: Eighty-four Sprague-Dawley rats were randomly divided into seven groups: control, LPS (instilled intratracheally to reproduce the model of ALI), NaHS (H₂S donor) +LPS, propargylglycine ［inhibitor of cysathionine-γ-lyase (CSE), PPG］+LPS, CCK-8+LPS, PPG+CCK-8+LPS and CCK-8 group. Animals were sacrificed at 4 h and 8 h after agent instillation. The wet and dry ratio (W/D) of the lung weight was measured and calculated. Morphological changes of lung tissues were observed. H₂S concentration in plasma, malondialdehyde (MDA) content, myeloperoxidase (MPO) and CSE activities in the lung were determined. Furthermore, the level of P-selectin of lung tissue was measured by radioimmunoassay, the CSE mRNA expression in the lung was detected by RT-PCR, and the protein content in bronchoalveolar lavage fluid (BALF) was detected. RESULTS: Compared with control, severe injury of lung tissues and increase in W/D, protein content in BALF, MDA content, MPO activity and P-selectin level in the lung were observed in rats treated with LPS. LPS also lead to a drop in plasma H₂S concentration, lung CSE activity and CSE mRNA expression. Administration of NaHS before LPS could attenuate the changes induced by LPS, while H₂S concentration, CSE activity and CSE mRNA expression were higher than those in LPS group. However, pre-treatment with PPG exacerbated the lung injury induced by LPS, H₂S concentration, CSE activity and CSE mRNA expression were lower than those in LPS and CCK-8 +LPS group, respectively. CONCLUSION: CCK-8 attenuates LPS-induced acute lung injury by means of anti-oxidation and inhibition of PMN adhesion and aggregation, both of which are mediated by endogenous H₂S.     

Role of ERK on proliferation of airway smooth muscle cells in chronic asthmatic rats

AIM: To investigate the roles of extracellular signal-regulated kinase(ERK) signaling pathway on regulating proliferation of airway smooth muscle by observing the expression of ERK in airway smooth muscle(ASM) in chronic asthmatic rats.METHODS: Airway remodeling was detected in chronic asthmatic rats by using image analysis system. The expressions of ERK and proliferating cell nuclear antigen(PCNA) in lung tissue from chronic asthmatic rats were observed by immuocytochemistry staining. The expressions of ERK1/2, p ERK1/2 and PCNA were detected in airway smooth muscle (ASM) by immunofluorescence double staining with confocal microscopy, and the expressions of protein or mRNA of ERK and PCNA in ASM were also detected by immunoblotting and hybridization in situ,respectively.RESULTS: The thickening of smooth muscle and structural remodeling in airway were observed in chronic asthmatic rats by image analysis. The enhanced expressions of ERK and PCNA appeared obviously increased in same lung tissue and the expressions of protein or mRNA of ERK and PCNA were significantly increased in ASM.CONCLUSION: ERK signal pathway might be an important pathway on regulating cell proliferation of ASM resulting in asthmatic airway remodeling.     

Depressant effect and mechanism of atorvastatin on the chronic rejection of aortic allograft in rats

AIM: To investigate the depressant effect and mechanism of atorvastatin on the chronic rejection of aortic allograft in rats.METHODS: The models of abdominal aorta transplantation were made with micro-surgery in rats.The recipients were divided into three groups: allograft control group,atorvastatin-treated group and isograft control group.Vascular intimal thickness in all of the groups was observed by histological examination.The expression of proliferation cell nuclear antigenl(PCNA) and α-smooth muscle actin(α-SMA) were determined by immunohistochemistry.The content of nitric oxide was measured by nitrate reductase chromatometry.RESULTS: The vascular intimal thickness in atorvastatin-treated group (11.60%±2.40%) was lower than that in allograft control group (34.60%±6.40%,P<0.05) and higher than that in isograft control group (1.15%±0.65%,P<0.05).The expression level of PCNA was decreased in atorvastatin-treated group (4.80%±0.80%) than that in allograft control group (18.40%±1.80%,P<0.05) and higher than that in isograft group (1.20%±0.40%,P<0.05).CONCLUSION: The expression of PCNA in the transplanted aorta is suppressed by atorvastatin,which results in relief of chronic rejection of aortic allograft.     

Effects of endogenous nitric oxide on potassium channels of bronchial smooth muscle cells from asthmatic rats

AIM: To investigate the effects of in vivo application of L-arginine on potassium channels in bronchial smooth muscle cells (BSMC) isolated from asthmatic model rats. METHODS: Male SD rats were randomly divided into control group, asthmatic group and asthmatic rats treated with L-arginine (L-Arg group). Single BSMCs were obtained by acute enzyme separation method. The resting membrane potential (Em), Ca2+-activated K+ channels (BKCa) currents and voltage-dependent K+ channel (Kv) currents in BSMCs were recorded under whole-cell patch clamp technique. RESULTS: (1) The Em of asthmatic group was significantly lower than that in control group (P<0.05). In vivo application of L-Arg significantly hyperpolarized BSMCs near to control group (P>0.05). (2) The peak current density at +50 mV of KCa: IKca in asthmatic group ［(43.8±16.5) pA/pF］ was significantly lower than that in normal group ［(72.5±19.9) pA/pF］ (P<0.01). Treatment with 300 mg/kg L-Arg significantly increased IKca in asthmatic group to (58.7±12.4) pA/pF (P<0.05). (3) The peak currents density at +50 mV of Kv: IKv in asthmatic group ［(32.4±8.7) pA/pF］ was significantly lower than that in control group ［(57.7±9.8) pA/pF］ (P<0.01). Treatment with L-Arg also significantly increased IKv in asthmatic group to (43.6±7.9) pA/pF, (P<0.05). CONCLUSION: Endogenous NO improves Em in asthmatic BSMCs, increases the activities of BKCa channels and Kv channel. These findings implicate that NO may have a potential therapeutically role in airway hypersensitivity.     

Effects of M. vaccae on eosinophil apoptosis and Bcl-2 protein expression in lung tissues of asthmatic guinea pigs

AIM: To study the effects of M. vaccae on eosinophil apoptosis and Bcl-2 protein expression in lung tissues of asthmatic guinea pigs. METHODS: 30 guinea pigs were divided into normal saline (NS) group, asthma group and M. vaccae treatment group at random, every group included 10 guinea pigs. Guinea pigs in M. vaccae treatment group were injected intramuscularly with 22.5 μg M. vaccae 10 days before OVA immunization. TdT-mediated dUTP nick end labeling (TUNEL) technique was used to investigate the apoptosis of eosinophils and immunohistochemistry method was used to study the expression of Bcl-2 protein in lung tissues. RESULTS: The apoptosis index (AI) of eosinophils in lung tissues in M. vaccae treatment group was significant higher than that in asthma group ［(23.78±5.42)% vs (4.56±0.68)%, P<0.01］. The mean optical density value of Bcl-2 protein in lung tissues of M. vaccae treatment group was significant lower than that of asthma group ［(1 556.3±492.4) vs (2 321.9±751.2), P<0.05］. CONCLUSION: The apoptosis of eosinophils induced by M. vaccae in lung tissues of asthmatic guinea pigs may be due to the inhibition of Bcl-2 protein expression.     

Effect of proliferating cell nuclear antigen specific antisense oligonucleotide on differentiation of cord blood CD34+ cells

AIM:To study the effect of proliferating cell nucler antigen antisense oligonucleotide on ex vivo expansion of cord blood CD34+ hematopoietic stem/progenitor cells. METHODS:CD34+ cells were purified from fresh cord blood by immunomagnetic beads. CD34+ cells were incubated in liquid culture system with different concentrations of PCNA-ASODN. Using flow cytometry, the number of different kinds of stem/progenitor cells and PCNA expression were measured after CD34+ cell incubation. RESULTS:PCNA was lowly expressed in low experiential group, with a positive rate of (27.2±3.6)% and (19.0±1.5)%, the positive rate of control group was (53.8±8.3)% (P<0.01), a high significant difference was observed. Just in low concentration of PCNA-ASODN, the percentage of CD34+cells were increased to (33.4±3.2)%, CD34+CD38-cells expanded (57.8±9.9) folds, and the percentage of CD34+cells were (25.2±2.6)% (P<0.01), the CD34+CD38-cells expanded (43.5±7.4) folds (P<0.05), it has significant difference compared with the control group. CONCLUSION:Low concentration of PCNA-ASODN decreases PCNA expression effectively, slows down differentiation of CD34+cells during ex vivo expansion procedure, and improves the expansion efficiency.     

Effects of PFOA exposure on inflammatory mediators IL-4, IFN-γ and glucocorticoid receptor in asthmatic mice

AIM: To investigate the effects of perfluorooctanoic acid (PFOA) exposure on the changes of asthmatic mouse airway inflammation, inflammatory mediators interleukin-4 (IL-4) and interferon-γ (IFN-γ) in serum, and glucocorticoid receptor (GR) expression in the lung tissue.METHODS: BALB/c mice (n=30) were randomly divided into 5 groups:normal control (C) group, asthma (A) group, asthma+low-dose PFOA (AP10) group, asthma+ mode-rate-dose PFOA (AP50) group and asthma+high-dose PFOA (AP100) group. Asthma model and PFOA exposure model of mice were established according to the grouping. The animals were sacrificed and their lungs were collected for HE staining, transmission electron microscopy, Western blot and immunohistochemical staining. ELISA was applied to detect the levels of IL-4 and IFN-γ in the serum.RESULTS: HE staining of the lungs showed that the asthmatic mice, compared with the normal control mice, had obvious mucus secretion around the airways and infiltration of inflammatory cells around airways and blood vessels, and the effects were much more marked in AP groups. Ultrastructural alteration of the lung tissues in the asthmatic mice were indicated by transmission electron microscopy. Compared with C group, the results of ELISA in A group and AP groups proved that IL-4 in the serum was increased and IFN-γ was decreased significantly (P<0.05). Compare with A group, IL-4 was significantly increased and IFN-γ was decreased in AP100 group (P<0.05), and no difference of those between AP10 group and AP50 group was found. The results of Western blot indicated that GR protein expression in the asthmatic mice were decreased compare with the normal mice (P<0.05), and no difference of that among A group and AP groups was observed. Immunohistochemical staining manifested that GR protein was mainly located in the cytoplasm of bronchial columnar epithelial cells, airway smooth muscle cells and vascular smooth muscle cells.CONCLUSION: Acute airway PFOA exposure in asthmatic mice dose-dependently exacebates lung inflammation by inducing Th2 type immune responses, promotes infiltration of inflammatory cells and mucus secretion around the airways and blood vessels, and destroys the ultrastructure of the lung tissues.