甘草甜素减轻博莱霉素致大鼠肺纤维化

目的探讨甘草甜素减轻博莱霉素致大鼠肺纤维化的机制。方法将大鼠随机分成对照组、博莱霉素致大鼠肺纤维化模型组、地塞米松(DXM)、甘草甜素低、中及高剂量治疗组。注博莱霉素后第28天,连续14 d同一时间段腹腔注射。第15天取左肺下叶供免疫组化检测TGF-β1、IFN-γ表达;右肺下叶供病理组织观察。用ELISA双抗体夹心法测定各组大鼠血清IL-4、IFN-γ含量。结果 (1)模型组大鼠肺组织成纤维细胞反应增强,TGF-β1染色及定量表达增强;而甘草甜素各组、DXM组减轻明显(P<0.01);(2)模型组大鼠血清IL-4含量较高,IFN-γ含量较低;甘草甜素各组及DXM组则相反(P<0.05)。(3)甘草甜素各组、DXM组肺组织IFN-γ染色及定量表达增强,甘草甜素低剂量组较其他组增强(P<0.01)。结论甘草甜素通过降低肺组织TGF-β1表达和血清IL-4含量,上调IFN-γ表达,提高血清IFN-γ含量,从而减轻肺纤维化程度。     

PTX3 genetic variations affect the risk of Pseudomonas aeruginosa airway colonization in cystic fibrosis patients

Cystic fibrosis (CF) is a common life-threatening autosomal recessive disorder in the Caucasian population, and the gene responsible is the CF transmembrane conductance regulator (CFTR). Patients with CF have repeated bacterial infection of the airways caused by Pseudomonas aeruginosa (PA), which is one of the predominant pathogen, and endobronchial chronic infection represents a major cause of morbidity and mortality. Pentraxin 3 (PTX3) is a gene that encodes the antimicrobial protein, PTX3, which is believed to have an important role in innate immunity of lung. To address the role of PTX3 in the risk of PA lung colonization, we investigated five single nucleotide polymorphisms of PTX3 gene in 172 Caucasian CF patients who were homozygous for the F508del mutation. We observed that PTX3 haplotype frequencies were significantly different between patients with PA colonization, as compared with noncolonized patients. Moreover, a protective effect was found in association with a specific haplotype (odds ratio 0.524). Our data suggest that variations within PTX3 affect lung colonization of Pseudomonas in patients with CF.     

Dasatinib attenuated bleomycin-induced pulmonary fibrosis in mice

Abstract

Anti-fibrotic effect of dasatinib, a platelet-derived growth factor receptor (PDGFR) and Src-kinase inhibitor, was tested on pulmonary fibrosis (PF). Adult mice were divided into four groups: mice dissected 21?d after the bleomycin (BLM) instillation (0.08?mg/kg in 200?µl) (I) and their controls (II), and mice treated with dasatinib (8?mg/kg in 100?µl, gavage) for one week 14?d after BLM instillation and dissected 21?d after instillation (III) and their controls (IV). The fibrosis score and the levels of fibrotic markers were analyzed in lungs. BLM treatment-induced cell proliferation and increased the levels of collagen-1, alpha smooth muscle actin, phospho (p)-PDGFR-alpha, p-Src, p-extracellular signal-regulated kinases1/2 and p-cytoplasmic-Abelson-kinase (c-Abl) in lungs, and down-regulated PTEN expression. Dasatinib reversed these alterations in the fibrotic lung. Dasatinib limited myofibroblast activation and collagen-1 accumulation by the inhibition of PDGFR-alpha, and Src and c-Abl activations. In conclusion, dasatinib may be a novel tyrosine and Src-kinase inhibitor for PF regression in mice.     

Identification of independent risk loci for Graves' disease within the MHC in the Japanese population

To identify genetic variants that confer the risk of Graves' disease (GD) in the Japanese population, we conducted a two-stage genome-wide association study (GWAS) using 1119 Japanese individuals with GD and 2718 unrelated controls, and a subsequent replication study using independent 432 GD cases and 1157 controls. We identified 34 single nucleotide polymorphisms (SNPs) to be significantly associated with GD in the GWAS phase. Twenty-two out of 34 SNPs remained positive in the replication study. All 22 SNPs were located within the major histocompatibility complex (MHC) locus on chromosome 6p21. No strong long-range linkage disequilibrium (LD) was observed among the 22 SNPs, indicating independent involvement of multiple loci within the MHC with the risk of GD. Multivariate stepwise logistic regression analysis selected rs3893464, rs4313034, rs3132613, rs4248154, rs2273017, rs9394159 and rs4713693, as markers for independent risk loci for GD. The analysis of LD between these seven SNPs and tagging SNPs for GD-associated human leukocyte antigen (HLA) alleles in the Japanese population (HLA-DPB1(*)0501 and HLA-A(*)0206) demonstrated that all of and five of seven SNPs were not in strong LD with HLA-DPB1(*)0501 and HLA-A(*)0206, respectively. Although causal variants remain to be identified, our results demonstrate the existence of multiple GD susceptibility loci within the MHC region.     

Genetic variation in GREM1 is a risk factor for fibrosis in pulmonary sarcoidosis

Sarcoidosis is a granulomatous systemic disorder most often affecting the lung. Pulmonary fibrosis develops in approximately 10%-15% of patients with sarcoidosis. The human gene GREM1 encodes gremlin, a member of the bone morphogenetic protein antagonist family. Bone morphogenetic proteins are essential for the maintenance of tissue homeostasis and regeneration after injury. We examined associations between genetic variation in GREM1 and pulmonary disease outcome in patients with pulmonary sarcoidosis. Four common tag single nucleotide polymorphisms spanning GREM1 were genotyped in 483 controls and in 237 sarcoidosis patients with radiographic data at pulmonary disease outcome, defined by chest X-ray after a minimum of 4 years follow-up. Highly significant differences were found between GREM1 genotype frequencies in sarcoidosis patients without chest X-ray abnormalities (stage 0) (n = 116) versus patients who had fibrosis on chest X-ray (stage IV) (n = 59) at pulmonary disease outcome. The most significant association was with GREM1 rs1919364. The recessive model resulted in an increased risk of fibrosis development for homozygous carriers of the C allele at GREM1 rs1919364 versus carriers of the G allele [P = 9.3 × 10??, χ2 = 24.1, odds ratio (OR) = 6.37 (2.89-14.1)]. This study is the first to suggest that genetic variation of GREM1 predisposes to pulmonary fibrosis in sarcoidosis patients. Carriers of the GREM1 CC genotype at position rs1919364 were at 6.4 times greater risk for developing fibrosis.     

Didecyldimethylammonium chloride induces pulmonary inflammation and fibrosis in mice

Didecyldimethylammonium chloride (DDAC) is used worldwide as a germicide, in antiseptics, and as a wood preservative, and can cause adverse pulmonary disease in humans. However, the pulmonary toxicity of DDAC has not yet been thoroughly investigated. Mice were intratracheally instilled with DDAC to the lung and the bronchoalveolar lavage (BAL) fluid and lung tissues were collected to assess dose- and time-related pulmonary injury. Exposure to 1500 μg/kg of DDAC caused severe morbidity with pulmonary congestive oedema. When the BAL fluid from survivors was examined on day 3 after treatment, exposure to 150 μg/kg of DDAC caused weakly induced inflammation, and exposure to 15 μg/kg did not cause any visible effects. Next, we observed pulmonary changes that occurred up to day 20 after 150 μg/kg of DDAC exposure. Pulmonary inflammation peaked on day 7 and was confirmed by expression of interleukin-6, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, and regulated upon activation, normal T-cell expressed and secreted in the BAL fluid; these changes were accompanied by altered gene expression of their chemokine (C–C motif) receptor (Ccr) 1, Ccr2, Ccr3, and Ccr5. Cytotoxicity evoked by DDAC was related to the inflammatory changes and was confirmed by an in vitro study using isolated mouse lung fibroblasts. The inflammatory phase was accompanied or followed by pulmonary remodeling, i.e., fibrosis, which was evident in the mRNA expression of type I procollagen. These results suggest that administering DDAC by intratracheal instillation causes pulmonary injury in mice, and occupational exposure to DDAC might be a potential hazard to human health.     

A severe phenotype in mice with a duplication of exon 3 in the cystic fibrosis locus

To develop an animal model for cystic fibrosis (CF), targetedgene disruption in embryonic stem (ES) cells was used to generatea duplication of exon 3 (cftr^m1Bray allele) of the mouse CFgene. ES cells containing this mutation were used to generatechimeric animals that transmitted the mutant allele throughthe germline. Homozygous mutant animals display a severe phenotype,with approximately 40% dying within 1 week from intestinal obstruction.RNAase protection analysis of the cftr^m1Bray allele did notdetect any normal mRNA (<1–2% of wild-type) In mutantanimals. Pathologic changes in the intestines from mutant miceincluded mucus accumulation in the crypts and intestinal lumen,dilatation of the bases of the crypts, enlargement of gobletcells, and the presence of concretions in the crypts or betweenthe villi. Changes were also present in the mucosal glands ofthe pharynx and the minor sublingual glands, where dilatationof acini and accumulation of eosinophilic material were evident.Atrophy of acinar cells that may be secondary to nutritionaldeficiency and mild inflammation in the main pancreatic ductwere present in the pancreas of mutant animals. No changes werenoted in the lung, trachea, liver, or male reproductive tractof mutant animals, and mutant males were fertile. Homozygousmutant mice showed defects In cAMP-mediated ion transport bothin lleum and in cultured fetal tracheal explants. Thus, an additionalmouse model for CF has been generated that should prove usefulfor the understanding of the pathogenesis and the developmentof treatments for CF.     

姜黄素抑制小鼠矽肺纤维化

目的： 建立小鼠矽肺动物模型,探讨姜黄素在减轻小鼠矽肺纤维化中的作用。方法: 用手术方法暴露小鼠气管,定量注射二氧化硅（SiO2）悬液建立矽肺模型。建模2周后将小鼠随机分为模型组、低剂量组、中剂量组和高剂量组分别给药,给药2周以及6周后处死动物,对肺组织行病理组织学检查。结果:建立了矽肺的纤维化模型。与模型组相比,给药2周后,高剂量组矽结节大小及数量显著减小(P<0.05);给药6周后,模型组以成纤维细胞为主,同时夹杂少量的胶原纤维,而给药组仍以巨噬细胞和淋巴细胞为主的细胞性矽结节为主。结论：姜黄素能抑制矽肺纤维化进程。     

Bronchiolar and alveolar lesions in the pathogenesis of crocidolite-induced pulmonary fibrosis in mice

Asbestosis is generally considered to result in restrictive pulmonary disease associated with interstitial fibrosis. Recently, however, attention has focused upon bronchiolar lesions and concomitant obstruction to air flow. The responses of conducting airways and alveoli were studied over a 20 week period following instillation of crocidolite to mice. The location of the lesions and the sequential inflammatory changes were studied by bronchoalveolar lavage, light and electron microscopy; regenerative activity was monitored by autoradiographs. Within 48 h there was multifocal necrosis of bronchiolar epithelium, maximal at bifurcations where longer fibres tend to adhere. Subsequently, intralumenal exudates were overgrown by epithelium and incorporated into the bronchiolar connective tissue where active peribronchiolar granulomas persisted until 20 weeks. Alveolar lesions were located predominantly in peribronchiolar air sacs and at the junctions of bronchioles and alveolar ducts. Focal acute injury of type 1 cells and transepithelial passage facilitated transport of short asbestos fibres to the interstitium where they were phagocytozed by macrophages. Regenerative activity was prompt with active division of type 2 epithelial cells. Biochemically, collagen increased after 4 weeks, when fibrosis was predominantly located in the bronchiolar lumens and in peribronchiolar connective tissue with lesser amounts in the centrilobular alveolar interstitium. The results suggest that the longer fibres induce bronchiolar injury and a more severe fibrotic pattern similar to the recently described changes seen in human asbestosis.     

静脉注射博来霉素诱导肺纤维化模型小鼠的稳定性评价

背景：建立理想的肺间质纤维化动物模型对探究肺间质纤维化发病机制及筛选有效防治药物尤为重要。目的：建立一个反映人肺纤维化形成的小鼠模型改良方案。方法：雄性C57BL/6小鼠56只,随机等分为A,B两组。A组为单次剂量注射模型,一次性尾静脉注入博来霉素200 mg/kg;B组为多次小剂量注射模型,每周经尾静脉注射博来霉素50 mg/kg,连续6周。结果与结论：Micro-CT扫描显示A组在造模后第4周所形成的肺CT表现最具有代表性,可见周围肺野及肺底网格状阴影,不均匀的斑片状阴影,粗网状不透光影,6周时肺内病变范围有所减少;B组造模后肺CT病变类似A组,但随时间延长纤维化病变逐渐增多。A组肺泡炎、肺纤维化评分于造模后第4周达最高,6周有所下降,而B组于造模后逐渐增加,6周时较A组高。免疫组化显示异常的Ⅰ型胶原蛋白主要沉积在胸膜下、血管周围和肺泡间隔,与Masson染色所见的胶原纤维分布一致;转化生长因子β1和α平滑肌动蛋白在肺泡炎和肺纤维化部位表达明显增多;A组Ⅰ型胶原蛋白、转化生长因子β1、α平滑肌动蛋白的表达在造模后第4周达到峰值,并在第6周有所下降,而B组于造模后逐渐增加,6周时较A组高。A组小鼠肺组织羟脯氨酸含量于造模后第4周达到峰值,第6周有所下降,而B组于造模后逐渐增加,造模后第6周较A组高。说明尾静脉注射博来霉素可有效诱导肺间质纤维化,转化生长因子β1和α平滑肌动蛋白在肺纤维化发生发展中起着重要作用,且多次小剂量博来霉素诱导肺纤维化模型具有部分典型寻常型间质性肺炎特征,尤其在模型稳定性上明显优于单次大剂量博来霉素诱导肺纤维化模型,更加符合特发性肺间质纤维化演变过程。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Increased expression of epimorphin in bleomycin-induced pulmonary fibrosis in mice

Epimorphin was originally identified as a mesenchymal, cell surface-associated protein that modulates epithelial morphogenesis in embryonic organs, whereas pulmonary fibrosis is a process of wound healing, which in part mimics the process of fetal lung development. We investigated the temporal and spatial changes in the distribution of epimorphin protein and expression of its messenger RNA (mRNA) in bleomycin-induced pulmonary fibrosis in mice. Immunohistochemical analysis showed that low levels of epimorphin were present in the bronchiolar, alveolar, and vascular walls of normal adult lungs. However, from Day 7 until Day 28 after bleomycin treatment, increasing levels of epimorphin immunoreactivity were detected in the mesenchymal cells and in the extracellular matrix within intra-alveolar fibrotic lesions. Moreover, Northern blots showed corresponding increases in epimorphin mRNA expression. Re-epithelialization of epimorphin-rich intra-alveolar fibrosis was complete by Day 28 after bleomycin, and by Day 56, epimorphin immunoreactivity had declined. In situ hybridization and confocal microscopic studies confirmed expression of epimorphin mRNA by mesenchymal cells situated within early fibrotic lesions, whereas immunoelectron microscopy localized the epimorphin to the endoplasmic reticulum of the mesenchymal cells and to the basement membrane and collagen fibrils in the area. These results suggest that epimorphin may contribute to the remodeling of pulmonary fibrosis via epithelial-mesenchymal interactions.     

Crocidolite-induced pulmonary fibrosis in mice. Cytokinetic and biochemical studies.

The responses of pulmonary alveolar and bronchial cells to asbestos exposure were studied by relating the cytokinetic changes of injury and repair to the inflammatory process and subsequent fibroblastic activity. The lesions were induced by intratracheal instillation of 1 mg crocidolite asbestos in mice, which were killed up to 20 weeks thereafter; 3H-thymidine was injected 1 hour before death. A rapid inflammatory response with elevated polymorphonuclear leukocytes and lysosomal enzyme release was largely over by 2 weeks, but the increase in alveolar macrophages was maintained. Focal necrosis of bronchial epithelial cells was repaired by cell regeneration, whereby new epithelial cells overgrew luminal exudates to incorporate long asbestos fibers into the peribronchial interstitium, where macrophagic granulomas formed. Increased collagen levels were largely due to stimulation of peribronchial fibroblasts. A lesser reaction of epithelial damage, Type 2 cell proliferation, and fibroblast stimulation also occurred in the alveolar walls. The results suggest that macrophage-fibroblast interactions associated with enhanced fibrosis occur readily in the peribronchial interstitium following injury and repair of epithelial cells by long asbestos fibers.     

Searching for a locus for schizophrenia within chromosome Xp11

Three gene-rich loci-HS212G6, HSU93305, and HS884M20-within the short arm of the X chromosome have been examined for allelic association with schizophrenia by the transmission disequilibrium test in 70 families of male individuals affected with schizophrenia. Neither the HS212G6 nor HS884M20 was found to be associated with schizophrenia. The HSU93305 locus, however, was significantly associated with schizophrenia (X(2)=17.92, df=3, P<0.001). The HSU93305 locus contains four distinct genes. They code, respectively, for A4 differentiation-dependent protein, triple LIM domain protein, synaptophysin, and calcium channel alpha-1 subunit. It is possible that one of these genes or some loci near to it may predispose a vulnerability to schizophrenia. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:4-7, 2000.     

ATP清除剂三磷酸腺苷双磷酸酶对实验性矽肺的干预作用

 目的:观察三磷酸腺苷双磷酸酶(apyrase,Apy)对实验性矽肺小鼠肺纤维化的干预作用及可能机制。方法:160只雄性C57BL/6小鼠,随机分为对照组、矽肺组、Apy组及溶剂组,采用经口咽吸入法给予SiO₂悬浊液(50 mg/kg)建立小鼠实验性矽肺模型,对照组给予等量生理盐水。Apy组在造模的同时及造模后4 h采用经口咽吸入法给予Apy(40 mg/kg),溶剂组给予等体积的无菌生理盐水。分别于术后3 h、7 d、14 d和28 d处死小鼠,计算各组小鼠肺指数,采用生物发光法检测小鼠肺组织的ATP含量,采用HE染色和苦味酸-天狼星红染色观察肺组织的病理学变化,采用real-time PCR法检测各组肺组织中Ⅰ型胶原(Col Ⅰ)、Ⅲ型胶原(Col Ⅲ)和转化生长因子β1(TGF-β1)的mRNA表达水平,采用ELISA法测定肺泡灌洗液TGF-β1的蛋白水平。结果:与对照组相比,模型组和溶剂对照组小鼠的肺指数和胶原含量明显升高,证明矽肺模型制备成功。Apy组与溶剂组相比,肺组织的ATP含量下降,肺组织炎症表现明显减轻,炎症评分显著下降;同时肺指数、胶原容积分数及Col Ⅰ和Col Ⅲ的 mRNA表达水平均显著下降。Apy可以明显下调肺组织中TGF-β1的mRNA表达水平和肺泡灌洗液中TGF-β1蛋白表达水平。结论:Apy可以明显减轻实验性矽肺小鼠肺组织的炎症反应和纤维化,该作用可能与Apy减少肺组织ATP的浓度、下调TGF-β1的表达有关。     

IL-10 inhibits inflammation but does not affect fibrosis in the pulmonary response to bleomycin

Bleomycin yields pulmonary injury characterized by inflammation that proceeds to fibrosis. The production of IL-10 by pulmonary macrophages is increased in the inflammation that accompanies bleomycin lung injury. In the present study, IL-10 deficient and wildtype mice received 0.075 units of bleomycin intratracheally at day 0 and were sacrificed at day 7 or day 14. At day 7, pulmonary inflammation was increased in IL-10-deficient mice as reflected by increased representation of CD3+ and CD4+ lymphocytes and GR-1+ pulmonary granulocytes in the bronchoalveolar lavage (BAL) fluid. Pulmonary interstitial CD80+ and CD86+ mononuclear cells were increased in situ. At day 14, mononuclear cell inflammation was comparable between groups but pulmonary eosinophils were increased in the wildtype. There was no difference in the degree of pulmonary fibrosis, as judged by histology or lung hydroxyproline content. Lung chemokine expression of MIP-1alpha/beta, MIP-2, and eotaxin was increased at days 7 and 14 with a trend towards increased MCP-1 expression at day 14. The findings suggest an immunomodulatory role for IL-10 in the inflammatory response but not in the pulmonary fibrosis yielded by bleomycin.     

Transforming growth factor-alpha deficiency reduces pulmonary fibrosis in transgenic mice

Despite evidence that implicates transforming growth factor-alpha (TGF-alpha) in the pathogenesis of acute lung injury, the contribution of TGF-alpha to the fibroproliferative response is unknown. To determine whether the development of pulmonary fibrosis depends on TGF-alpha, we induced lung injury with bleomycin in TGF-alpha null-mutation transgenic mice and wild-type mice. Lung hydroxyproline content was 1.3, 1.2, and 1.6 times greater in wild-genotype mice than in TGF-alpha-deficient animals at Days 10, 21, and 28, respectively, after a single intratracheal injection of bleomycin. At Days 7 and 10 after bleomycin treatment, lung total RNA content was 1.5 times greater in wild-genotype mice than in TGF-alpha-deficient animals. There was no significant difference between mice of the two genotypes in lung total DNA content or nuclear labeling indices after bleomycin administration. Wild-genotype mice had significantly higher lung fibrosis scores at Days 7 and 14 after bleomycin treatment than did TGF-alpha-deficient animals. There was no significant difference between TGF-alpha-deficient mice and wild-genotype mice in lung inflammation scores after bleomycin administration. To determine whether expression of other members of the epidermal growth factor (EGF) family is increased after bleomycin-induced injury, we measured lung EGF and heparin-binding- epidermal growth factor (HB-EGF) mRNA levels. Steady-state HB-EGF mRNA levels were 321% and 478% of control values in bleomycin-treated lungs at Days 7 and 10, respectively, but were not significantly different in TGF-alpha-deficient and in wild-genotype mice. EGF mRNA was not detected in normal or bleomycin-treated lungs of mice of either genotype. These results show that TGF-alpha contributes significantly to the pathogenesis of pulmonary fibrosis after bleomycin-induced injury, and that compensatory increases in other EGF family members do not occur in TGF-alpha-deficient mice.     

黄芪通过抗氧化抑制博莱霉素诱导的小鼠肺纤维化

目的:研究黄芪对博莱霉素诱导的肺纤维化小鼠氧化/抗氧化水平的影响,探讨黄芪抗纤维化的可能机制。方法:将36只SPF级雌性昆明小鼠随机分为对照组(生理盐水气管内雾化)、博莱霉素组(博莱霉素3mg/kg气管内雾化)和黄芪治疗组(博莱霉素3 mg/kg气管内雾化后黄芪注射液1.7 g·kg~(-1)·d~(-1)腹腔内注射),实验第14天收集小鼠肺组织及血清标本,取小鼠肺组织行HE和Masson染色;RT-PCR法测小鼠肺组织超氧化物歧化酶(SOD)1/2/3、过氧化氢酶(CAT)、NADPH氧化酶2/4(NOX2/4)和α-平滑肌肌动蛋白(α-SMA)的mRNA水平;Western blot法测α-SMA和NOX2/4蛋白表达水平;比色法检测血清丙二醛(MDA)和总抗氧化能力(T-AOC)。结果:博莱霉素组小鼠肺组织病理损伤较正常组明显加重,α-SMA mRNA和蛋白表达,MDA/T-AOC,NOX2、NOX4和SOD3 mRNA表达,以及NOX2蛋白表达较正常组显著上升,黄芪治疗组则显著逆转上述改变;博莱霉素组小鼠NOX4蛋白表达较正常组显著下降,而黄芪治疗组较博莱霉素组显著上升;博莱霉素组和黄芪治疗组小鼠SOD1和CAT mRNA表达均较正常组显著下降;SOD2 mRNA在3个组中表达的差异无统计学显著性。结论:黄芪能够减缓博来霉素诱导的肺纤维化形成,其机制可能与调节氧化/抗氧化平衡有关。