Expression of endoplasmic reticulum-associated apoptosis protein CHOP in lung tissues of COPD model rats / 中国病理生理杂志

AIM:To investigate whether cigarette smoke(CS)promotes the expression of endoplasmic reticu-lum-associated apoptosis protein CCAAT/enhancer-binding protein homologous protein(CHOP)in rat lung tissues. METHODS:Adult male Wistar rats(n=40)were randomly divided into 4 groups with 10 rats in each group: control group,CS-2 group(exposed to CS for 2 months),CS-4 group(exposed to CS for 4 months)and ex-smoking(Ex-S)group (exposed to CS for 4 months and then quit smoking for 1 month).The percentage of forced expiratory volume in 0.3 second to forced vital capacity(FEV0.3/FVC)and peak expiratory flow(PEF)were measured.TUNEL assay was used to detect the apoptotic cells.In situ hybridization and RT-PCR were used to determine the mRNA expression of CHOP.The methods of immunohistochemistry and Western blot were used to determine the protein expression of CHOP.Western blot was also used to determine the protein levels of protein kinase R-like endoplasmic reticulum kinase(PERK),p-PERK,eukaryotic initiation factor(eIF)2αand p-eIF2α.RESULTS:The pulmonary function greatly decreased in the rats exposed to CS for 2 months in comparison with control group(P<0.05),markedly decreased in the rats exposed to CS for 4 months as com-pared with the rats after exposure to CS for 2 months(P<0.05),and was improved little in ex-smoking rats(P>0.05). The structural destruction of the lung was observed in the rats exposed to CS for 2 months,and more obvious changes were found in the rats exposed to CS for 4 months.However,the structural destruction of the lung remained obvious in ex-smok-ing rats.The apoptotic cells were markedly increased in the rats exposed to CS for 2 months and were even more in the rats exposed to CS for 4 months.The apoptotic cells were alveolar epithelial cell I(ACE I),ACE II,vascular endothelial cells and bronchial epithelial cells.The protein levels of p-PERK,p-eIF2αand CHOP were remarkably increased in the rats af-ter exposure to CS for 2 months compared with the control rats(P<0.05),significantly elevated in the rats exposed to CS for 4 months compared with the rats exposed to CS for 2 months(P<0.05),and slightly decreased in ex-smoking rats in comparison with the rats after exposure to CS for 4 months(P>0.05).The total protein levels of PERK and eIF2αdid not change between the control rats and those exposed to CS.CONCLUSION: CS promotes the development of chronic ob-structive pulmonary disease(COPD)by inducing the expression of endoplasmic reticulum-associated apoptosis protein CHOP via PERK/eIF2α/CHOP signaling pathway.

The expression and possible role of SENP1 in the pulmonary vascular wall of rat during the development of hypoxic pulmonary hypertension / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the dynamic expression and role of SENP1 (SUMO-specific proteases-1) in the pulmonary vascular wall of rat during the development of hypoxic pulmonary hypertension (HPH).</p><p><b>METHODS</b>Forty adult male Wistar rats were randomly divided into 5 groups (n = 8), and exposed to normoxia (Control group) or exposed to hypoxia for 3, 7, 14 or 21 d, respectively. The HPH models were established by normobaric intermittent hypoxia. Mean pulmonary arterial pressure (mPAP), right ventricle hypertrophy index (RVHI), and vessel morphometry were measured. Reverse transcriptase-polymerase chain reaction(RT-PCR) and in situ hybridization were used to determine the mRNA expression of SENP1. Immunohistochemistry and Western blot were used to determine the protein expression of SENP1.</p><p><b>RESULTS</b>The hypoxic rats developed pulmonary vascular remodeling in pulmonary arterioles after 7 d of hypoxia exposure. Pulmonary vascular remodeling in pulmonary arterioles significantly increased after 14 d of hypoxia. The level of mPAP in hypoxic rats increased significantly after 7 d of hypoxia, reached its peak after 14 d of hypoxic exposure. RVHI was markedly increased after 14 d of hypoxia. In situ hybridization and immunohistochemical analysis showed that SENP1 mRNA and protein were positively stained in control. SENP1 mRNA expression had little changes after exposure to hypoxia compared with the control, however, SENP1 protein expression was declined gradually after 7 d of hypoxia. The results of RT-PCR and Western blot showed that the same dynamic expression of SENP1 mRNA and protein in lung tissues of rats. Linear correlation analysis showed that SENP1 protein were negatively correlated with mPAP, pulmonary vascular remodeling index and RVHI.</p><p><b>CONCLUSION</b>Under chronic hypoxia, SENP1 protein can be degradated. The dynamic expression of SENP1 protein may play a role in implicating in the development of HPH.</p>

The effect of transcription factor KLF2 in expression of gamma-GCS depend on regulation by Nrf2 in lung of rats with chronic obstructive pulmonary disease / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To study the expression of lung Krüppel-like transcription factor (KLF2/LKLF) in lung tissues of rats with chronic obstructive pulmonary disease (COPD) and the relationship between KLF2 and NF-E2-related factor 2 (Nrf2), and make further explore the effects of KLF2 on the expression of gamma-glutamylcysteine synthetase (gamma-GCS).</p><p><b>METHODS</b>Twenty-two male SD rats were randomly divided into a COPD group (n = 10) and a normal control group (n = 11). The rat model of COPD established by cigarette smoking and intratracheal instillation of lipopolysaccharide (LPS), and lung tissues were obtained. The expressions of KLF2, Nrf2, gamma-GCS mRNA and protein in lung tissues were measured by immunohistochemistry (IHC), Western blot, in situ hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR). To explore the relationship between KLF2 and Nrf2 protein,we utilize the method of co-immunoprecipitation (CO-IP).</p><p><b>RESULTS</b>IHC and Western blot showed that protein expressions of KLF2, Nrf2, gamma-GCS were higher in the lung tissues from rats with COPD than those in the control groups (all P < 0.05). The levels of KLF2, gamma-GCS mRNA were markedly increased in the COPD group (all P < 0.01) while Nrf2 mRNA expression in COPD group had no significant difference with that in control group ( P > 0.05). CO-IP result showed that KLF2 were obviously present in immunoprecipitates of Nrf2 (P < 0.01) . Linear correlation analysis showed that the level of KLF2 protein was positively correlated with the level of Nrf2 protein (P < 0.05), and KLF2, Nrf2 proteins were positively correlated with gamma-GCS mRNA and protein (all P < 0.05).</p><p><b>CONCLUSION</b>The expression of KLF2 is significantly up-regulated in COPD, which maybe up-regulate gamma-GCS mRNA expression by increasing Nrf2 expression and nuclear translocation against oxidative stress.</p>

The expression of hypoxia-inducible factor-1alpha and its hydroxylases in pulmonary arteries of patient with chronic obstructive pulmonary disease / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To observe the expression of hypoxia-inducible factor-lalpha subunit (HIF-1alpha), HIF prolyl hydroxylase domain-containing protein(PHDs) and factor inhibiting HIF-1(FIH) in pulmonary arteries of patient with chronic obstructive pulmonary disease (COPD).</p><p><b>METHODS</b>Pulmonary specimens were obtained from patients undergoing lobectomy for lung cancer, 12 had concurrent COPD (COPD group) and 14 without COPD (control group). The ratio of vascular wall area to total vascular area (WA%) and pulmonary artery media thickness (PAMT) was observed, and HIF-1alpha and its hydroxylases(PHD1, PHD2, PHD3, FIH) mRNA and protein were detected by in situ hybridization and immunohistochemistry respectively.</p><p><b>RESULTS</b>WA% and PAMT of COPD patients(50 microm +/- 9 microm, 40% +/- 5%, were statistically different from those of the control subjects (39 microm +/- 6 microm, 31% +/- 4%, P < 0.01). Relative quantification of mRNA and protein levels (absorbance, A) showed that HIF-lalpha mRNA and protein levels in COPD group (0.230 +/- 0.036,0.275 +/- 0.039) were statistically higher than those of the control subjects (0.174 +/- 0.029, 0.102 +/- 0.015, P < 0.01 ), and that the protein level increased more markedly. PHD1 mRNA in COPD subjects (0.180 +/- 0.030) was comparable to that in control group (0.191 +/- 0.029, P > 0.05); PHD2 and PHD3 mRNA levels in COPD (0.245 +/- 0.044, 0.252 +/- 0.023) were significantly higher than those in control group(0.182 +/- 0.028, 0.127 +/- 0.017, P < 0.01). On the other hand, in COPD subjects PHD1 protein (0.104 +/- 0.015) was significantly lower(P < 0.01), whereas PHD2 protein (0.274 +/- 0.044) was significantly higher(P < 0.01) than those in control group(0.209 +/- 0.023, 0.219+/- 0.043). As for PHD3 protein, no significant changes were observed between the two groups (0.161+/- 0.023 in COPD, 0.146 +/- 0.021 in control, P > 0.05). FIH mRNA and protein both showed no differences between the two groups. Linear correlation analysis showed that HIF1alpha protein was positively correlated with WA%, PAMT, PHD2 mRNA and protein, PHD3 mRNA, and that HIF1alpha protein was negatively correlated with PHD1 protein.</p><p><b>CONCLUSION</b>PHDs may be involved in the process of hypoxic pulmonary vascular remodeling in COPD via regulation of HIF-1alpha gene expression</p>

The cooperation of OS-9 and PHDs in hypoxia-induced pulmonary hypertension of rats / 中国应用生理学杂志

<p><b>AIM</b>To investigate the dynamic expression of hypoxia-inducible factor 1alpha, PHDs and OS-9 in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension.</p><p><b>METHODS</b>SD rats were randomly divided into 5 groups (n = 8) and exposed to hypoxia for 0, 3, 7, 14 or 21 d, respectively. RT-PCR and in situ hybridization were used to determine the expression of mRNA. Immunohistochemistry and Western blot were used to determine the expression of protein.</p><p><b>RESULTS</b>HIF-1alpha protein was poorly positive in control, markedly up-regulated after 3 d and 7 d of hypoxia (P < 0.05, vs group C), and then declined slightly after 14 d and 21 d of hypoxia. HIF-1alpha mRNA increased dramatically after 14 d of hypoxia (P < 0.05, vs group C). PHD1, PHD2 mRNA and protein was positive in group C. PHD2 mRNA and protein were up-regulated after 3 d of hypoxia (P < 0.05, vs group C), reaching its peak after 14 d of hypoxia while PHD1 protein declined after 14 d of hypoxia (P < 0.05, vs group C) without statistic mRNA changing. PHD3 mRNA and protein were detected at low level in control, markedly up-regulated after 3 d of hypoxia (P < 0.05, vs group C), and then PHD3 mRNA kept at high level while PHD3 protein declined after 14 d of hypoxia (P < 0.05, vs 7 d). OS-9 mRNA was positively in control, markedly decreased after 3 d of hypoxia (P < 0.05, vs group C), reaching its lowest lever after 14 d of hypoxia. Linear correlation analysis showed that OS-9 protein was positively correlated with OS-9 mRNA (r = 0.82, P < 0.01) and HIF-1alpha protein (r = 0.57, P < 0.01).</p><p><b>CONCLUSION</b>HIF-1alpha, PHDs and OS-9 are all involved in the pathogenesis of hypoxic pulmonary hypertension in rats. OS-9 may interact with both HIF-1alpha and PHDs to promote PHD-mediated hydroxylation of HIF-1alpha.</p>

The preventative effects of protein tyrosine kinase on the inflammation and airway remondeling in lung of guinea pigs with bronchial asthma / 中国应用生理学杂志

<p><b>AIM</b>To investigate the effects of protein tyrosine kinase on the inflammation and airway remodeling in lung of guinea pigs with bronchial asthma.</p><p><b>METHODS</b>30 adult male guinea pigs were randomly divided into 3 groups (n=3): control group (C group), asthmatic group(A group)and genistein group (B group). Asthmatic model was established by ovalbumin intraperitoneal injection and ovalbumin inhalation. The total cell and the proportion of inflammatory cell in bronchial alveolar lavage fluid(BALF), inflammatory cell infiltration and index of remodeling of bronchiole were measured, respectively. The expression of p-tyrosine in lung tissue was examined by immunohistochemistry.</p><p><b>RESULTS</b>The total cell and proportion of eosinophil in BALF of A group were significantly higher than that of C group (P < 0.01), but compared with A group, the total cell and proportion of eosinophil in BALF of B group were much lower (P < 0.01). The number of eosinophile and lymphocyte of bronchiole in A group were significantly higher than that of C group (P < 0.01), but compared with A group, the number of eosinophile and lymphocyte in bronchiole of B group were much lower (P < 0.01). Compared with A group, the remodeling of bronchiole of B group was significantly relieved (P <0.01), there was no difference between B and C group (P > 0.05). Immunohistochemistry indicated that in A group the p-tyrosine was more positively expressed at the bronchial smooth muscle, bronchial epithelium, smooth muscle of vessel and inflammatory cell, especially at smooth muscle of bronchi and vessel and inflammatory cell than that of C group (P <0.01), there was no difference between B group and C group (P > 0.05).</p><p><b>CONCLUSION</b>PTK played a key role in inflammation and bronchial remodeling in lung of guinea pigs with bronchial asthma. The Protein tyrosine kinase inhibitor genistein could prevent and inhibit the inflammation and bronchial remodeling in lung of guinea pigs with bronchial asthma.</p>

Roles of NRF2 regulating gamma-glutamylcysteine synthetase in lung of rats with chronic obstructive pulmonary disease / 中国应用生理学杂志

<p><b>AIM</b>To investigate the expression and relationship of gamma-glutamylcysteine synthetase (gamma-GCS) and NF-E2-related factor2 (NRR2) in lung of rat with chronic obstructive pulmonary disease (COPD)in order to elucidate the possible important role of gamma-GCS and NRF2 in pathogenesis of COPD.</p><p><b>METHODS</b>The rat COPD model was established by intratracheal instillation of lipopolysaccharide twice and exposed to cigarette smoke daily. The gamma-GCS activity was measured, the expression of gamma-GCS mRNA in lung was examined by in situ hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR), the protein expressions of NRF2, gamma-GCS in lung were detected by immunohistochemical (IH) and Western blot respectively.</p><p><b>RESULTS</b>The gamma-GCS activity was higher in COPD group than that in control group. The expressions of gamma-GCS mRNA in COPD group was stronger than those in control group. ISH showed that the gamma-GCS mRNA was expressed in alveolar epithelium and bronchial smooth muscle cell in COPD. The protein expressions of NRF2, gamma-GCS were significantly higher than the control group. IH showed that NRF2, gamma-GCS proteins were expressed in alveolar and bronchial epithelium in the COPD group. There was a positive correlation between NRF2 and gamma-GCS and gamma-GCS mRNA.</p><p><b>CONCLUSION</b>NRF2 may play an important role in the mechanism of COPD oxidative stress vis up-regulation of gamma-GCS.</p>

Effects of Nrf2 on gamma-glutamylcysteine synthase in lung of guinea pigs with bronchial asthma / 中国应用生理学杂志

<p><b>AIM</b>To investigate the effects of Nrf2 (Nuclear-E2 related factor) on gamma-glutamylcysteine synthase (gamma-GCS) in lung of guinea pigs with bronchial asthma.</p><p><b>METHODS</b>20 adult male guinea pigs were randomly divided into two groups (n = 10): control group (C group) and asthmatic group (A group), asthmatic model was established by ovalbumin intraperitoneal and ovalbumin inhalation. The reactive oxygen piece (ROS), reduced glutathione (GSH), oxidant glutathione (GSSG) and total GSH in lung tissue were examined respectively. Inflammatory cell infiltration and index of remodeling of bronchiole were detected. In situ hybridization detected the gamma-GCS heavy subunit (gamma-GCS h) mRNA in lung tissue. Immunohistochemistry detected the expression of Nrf2 protein and gamma-GCS protein in lung tissue. RT-PCR measured the expression of Nrf2 mRNA in lung tissue. The activity of gamma-GCS was measured by coupled enzyme assay.</p><p><b>RESULTS</b>(1) The number of eosinophils and lymphocytes in bronchiole of A group were significantly higher than that of C group (P < 0.05), the remodeling of bronchiole in A group was definite. (2) ROS (U/mg pro), GSSG (micromol/g pro) and total GSH in lung tissue of A group were significantly higher than that of C group (P < 0.01). The GSH/GSSG in lung tissue of A group was much lower than that of C group (P < 0.01), GSH in lung tissue showed no difference between A group and C group. (3) Immunohistochemistry indicated that Nrf2 protein and gamma-GCS protein were more positively expressed in A group than that in C group (P < 0.01). In situ hybridization discovered that the expression of gamma-GCS-h mRNA in lung tissue of A group was more positive than that of C group. (4) RT-PCR showed that the expression of Nrf2 mRNA was no difference between A group and C group (P > 0.05). (5) The activity of gamma-GCS of A group was (28 +/- 8)U which was significantly higher than that of C group (9 +/- 2)U (P < 0.01). (6) Linear correlation analysis indicated that in lung tissue of guinea pig with asthma there existed strongly positive relationship among ROS, GSSG and the expression of Nrf2, gamma-GCS mRNA, gamma-GCS protein, the activity of gamma-GCS, there existed strongly negative relationship among GSH/GSSG and the expression of Nrf2, gamma-GCS mRNA, gamma-GCS protein, the activity of gamma-GCS.</p><p><b>CONCLUSION</b>There existed oxidative stress in lung of guinea pigs with bronchial asthma, which possibly positively regulated gamma-GCS via up regulating transcription factor Nrf2.</p>

Study on the correlation of interleukin-13 polymorphism and susceptibility to chronic obstructive pulmonary disease in Chinese Han population / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the polymorphism of interleukin (IL)-13 gene in Chinese Han population, and to study the possible association of IL-13 polymorphism and the susceptibility to chronic obstructive pulmonary disease (COPD).</p><p><b>METHODS</b>Using genomic DNA extracted with phenol:chloroform:isoamyl alcohol from the blood of 94 healthy smokers and 88 COPD smokers, three single nucleotide polymorphism (SNP) sites in IL-13 gene marked as 1103C/T, 4257G/A, 4738G/A were determined by polymerase chain reaction/restriction fragment length polymorphism analysis or polymerase chain reaction/single strand conformation analysis. In addition, plasma IL-13 concentration was measured by ELISA on 24 healthy smokers and 24 smokers with chronic obstructive pulmonary disease.</p><p><b>RESULTS</b>(1) To 4257G/A and 4738G/A SNP sites, the genotype frequencies and allele frequencies were not significantly different between healthy smokers and COPD smokers (P > 0.1). (2) To 1103C/T SNP site, relative higher CC genotype frequency and C allele frequency were found in healthy smokers (P < 0.05), also C allele could prevent smokers from chronic obstructive pulmonary disease, with OR 0.56 and 95% confidence interval was 0.34 - 0.91. (3) Plasma IL-13 concentration was not affected by genotype of 4257G/A or 4738G/A SNP sites. However, 1103CC showed for significantly lower IL-13 concentration in plasma (P < 0.05).</p><p><b>CONCLUSION</b>The genotype of IL-13 1103C/T SNP site was associated with the susceptibility to COPD in Chinese Han population and 1103CC accounted for relative lower plasma IL-13 concentration and lower risk for COPD.</p>

Correlation of HDEFB1 polymorphism and susceptibility to chronic obstructive pulmonary disease in Chinese Han population / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Inherited factors are involved in the development of chronic obstructive pulmonary disease (COPD). This study was designed to investigate the relationship between polymorphisms of HDEFB1 668 C/G and 1654G/A loci and susceptibility to COPD in Chinese Han population.</p><p><b>METHODS</b>After the process of extracting genomic DNA from peripheral blood of COPD smokers and healthy smokers, the loci of genotypes 668C/G and 1654G/A were determined by polymerase chain reaction-restriction fragment length polymorphism analysis and polymerase chain reaction-single strand conformation polymorphism analysis.</p><p><b>RESULTS</b>With respect to HDEFB1 668 locus, the occurences of CC, CG, GG genotypes were 72.7%, 25.0%, 2.3% in COPD smokers and 53.2%, 38.3%, 8.5% in healthy smokers (P < 0.05, respectively). The allele frequencies of 668 C and 668G were 85.2% and 14.8% in COPD smokers and 72.3% and 27.7% in healthy smokers (P < 0.01, respectively, odds ratio was 2.32 with 95% confidence interval 1.37 to 3.72). As to HDEFB1 1654G/A locus, neither genotype distribution difference nor allele distribution difference was found when comparing COPD smokers with healthy smokers.</p><p><b>CONCLUSION</b>The polymorphism of HDEFB1 668C/G is associated with susceptibility to COPD in Chinese Han population; furthermore, the 668G allele represents relatively lower susceptibility to COPD.</p>

Polymorphism of interleukin-10 gene promoter and its association with susceptibility to chronic obstructive pulmonary disease in Chinese Han people / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the polymorphism of interleukin-10 (IL-10) gene promoters in Chinese Han people, and to disclose whether such polymorphism is associated with susceptibility to chronic obstructive pulmonary disease (COPD).</p><p><b>METHODS</b>After the process of extracting genomic DNA from blood of 94 health smokers and 88 COPD smokers by use of phenol-chloroform-isoamyl alcohol, three single nucleotide polymorphism (SNP) sites in IL-10 gene promoter marked as -1082G/A,-819C/T,-592C/A were determined by polymerase chain reaction/restriction fragment length polymorphism analysis.</p><p><b>RESULTS</b>Eleven different promoter genotypes were detected from all of the 182 smokers, and AA*TT*AA, AA*TC*AC, AA*TC*AA genotypes accounted for about 80% of genotypes in the research subjects. Two previously unreported haplotypes of IL-10 gene promoter (ATC and ACA) were found in Chinese Han people by analyzing the promoter genotypes. -1082G/A and -592C/A SNP sites polymorphisms were not associated with susceptibility to COPD, whereas the genotypes of -819C/T SNP site were associated with susceptibility to COPD in Chinese Han people. In respect to the alleles frequencies of the three SNP sites respectively, the Chinese Hans were similar to Japanese, but different from whites.</p><p><b>CONCLUSION</b>Polymorphism of IL-10 -819C/T SNP site is associated with susceptibility to COPD in Chinese Han people; at least five haplotypes of IL-10 gene promoter (ATA, ACC, GCC, ATC and ACA) exist in Chinese Han people.</p>