Absence of proteinase-activated receptor-1 signaling affords protection from bleomycin-induced lung inflammation and fibrosis

Activation of the coagulation cascade is commonly observed in the lungs of patients with both acute and chronic inflammatory and fibrotic lung disorders, as well as in animal models of these disorders. The aim of this study was to examine the contribution of the major thrombin receptor, proteinase-activated receptor-1 (PAR-1), during the acute inflammatory and chronic fibrotic phases of lung injury induced by intratracheal instillation of bleomycin in mice. Inflammatory cell recruitment and increases in bronchoalveolar lavage fluid (BALF) protein were attenuated by 56 +/- 10% (P < 0.05) and 53 +/- 12% (P < 0.05), respectively, in PAR-1-deficient (PAR-1-/-) mice compared with wild-type (WT) mice. PAR-1-/- mice were also protected from bleomycin-induced pulmonary fibrosis with total lung collagen accumulation reduced by 59 +/- 5% (P < 0.05). The protection afforded by PAR-1 deficiency was accompanied by significant reductions in pulmonary levels of the potent PAR-1-inducible proinflammatory and profibrotic mediators, monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-beta-1 (TGF-beta1), and connective tissue growth factor/fibroblast-inducible secreted protein-12 (CTGF/FISP12). In addition, PAR-1 was highly expressed in inflammatory and fibroproliferative lesions in lung sections obtained from patients with fibrotic lung disease. These data show for the first time that PAR-1 signaling plays a key role in experimentally induced lung injury, and they further identify PAR-1 as one of the critical receptors involved in orchestrating the interplay between coagulation, inflammation, and remodeling in response to tissue injury.     

结缔组织生长因子在博莱霉素诱导的小鼠肺纤维化中的作用探讨

目的:检测结缔组织生长因子(CTGF)在小鼠肺内的表达,并探讨其在肺纤维化中的意义。方法:通过向气管内滴入博莱霉素建立小鼠肺纤维化模型。用免疫组化染色方法检测不同时期肺组织中CTGF蛋白的表达;用RT PCR检测CTGFmRNA的表达;用酸水解法测定肺羟脯氨酸的含量。结果:在正常小鼠肺组织中,CTGF蛋白和其mRNA几乎不表达;而在模型组的肺组织中表达明显升高(P<0.01)。CTGF蛋白表达于支气管/细支气管上皮细胞、肺泡Ⅱ型上皮细胞和肺成纤维细胞,表达量与肺组织中羟脯氨酸的含量呈显著的正相关(r=0.92,P<0.001)。结论:CTGF与肺纤维化的形成关系密切,CTGF检测可作为评价肺纤维化发生、发展的一种灵敏的指标。     

Caveolin-1 deficiency protects from pulmonary fibrosis by modulating epithelial cell senescence in mice

Idiopathic pulmonary fibrosis is associated with a decreased expression of caveolin-1 (cav-1), yet its role remains unclear. To investigate the role of cav-1, we induced pulmonary fibrosis in wild-type (WT) and cav-1-deficient (cav-1(-/-)) mice using intratracheal instillation of bleomycin. Contrary to expectations, significantly less collagen deposition was measured in tissue from cav-1(-/-) mice than in their WT counterparts, consistent with reduced mRNA expression of procollagen1a2 and procollagen3a1. Moreover, cav-1(-/-) mice demonstrated 77% less α-smooth muscle actin staining, suggesting reduced mesenchymal cell activation. Levels of pulmonary injury, assessed by tenascin-C mRNA expression and CD44v10 detection, were significantly increased at Day 21 after injury in WT mice, an effect significantly attenuated in cav-1(-/-) mice. The apparent protective effect against bleomycin-induced fibrosis in cav-1(-/-) mice was attributed to reduce cellular senescence and apoptosis in cav-1(-/-) epithelial cells during the early phase of lung injury. Reduced matrix metalloproteinase (MMP)-2 and MMP-9 expressions indicated a low profile of senescence-associated secretory phenotype (SASP) in the bleomycin-injured cav-1(-/-) mice. However, IL-6 and macrophage inflammatory protein 2 were increased in WT and cav-1(-/-) mice after bleomycin challenge, suggesting that bleomycin-induced inflammatory response substantiated the SASP pool. Thus, loss of cav-1 attenuates early injury response to bleomycin by limiting stress-induced cellular senescence/apoptosis in epithelial cells. In contrast, decreased cav-1 expression promotes fibroblast activation and collagen deposition, effects that may be relevant in later stages of reparative response. Hence, therapeutic strategies to modulate the expression of cav-1 should take into account cell-specific effects in the regenerative responses of the lung epithelium to injury.     

Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-beta in human fibrotic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice

Prostaglandin E(2) (PGE(2)) inhibits fibroblast proliferation and collagen production. Its synthesis by fibroblasts is induced by profibrotic mediators including transforming growth factor (TGF)-beta(1). However, in patients with pulmonary fibrosis, PGE(2) levels are decreased. In this study we examined the effect of TGF-beta(1) on PGE(2) synthesis, proliferation, collagen production, and cyclooxygenase (COX) mRNA levels in fibroblasts derived from fibrotic and nonfibrotic human lung. In addition, we examined the effect of bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. We demonstrate that basal and TGF-beta(1)-induced PGE(2) synthesis is limited in fibroblasts from fibrotic lung. Functionally, this correlates with a loss of the anti-proliferative response to TGF-beta(1). This failure to induce PGE(2) synthesis is because of an inability to up-regulate COX-2 mRNA levels in these fibroblasts. Furthermore, mice deficient in COX-2 exhibit an enhanced response to bleomycin. We conclude that a decreased capacity to up-regulate COX-2 expression and COX-2-derived PGE(2) synthesis in the presence of increasing levels of profibrotic mediators such as TGF-beta(1) may lead to unopposed fibroblast proliferation and collagen synthesis and contribute to the pathogenesis of pulmonary fibrosis.     

Thrombin enhances lung fibroblast proliferation in bleomycin-induced pulmonary fibrosis.

To clarify the role of thrombin in fibroblast growth and the development of pulmonary fibrosis in bleomycin-induced interstitial lung disease, we examined the relationship of thrombin activity to fibroblast growth-stimulating activity (FGA) in bronchoalveolar lavage (BAL) fluid from bleomycin-treated rats. Male Wistar rats were given a single intratracheal injection of bleomycin, BAL was performed 2, 6, and 15 days later, and the BAL fluid was assayed for thrombin activity and FGA. Higher FGA than the control value was detected in the BAL fluid from rats on day 6 after bleomycin administration. In bleomycin-treated rats, thrombin activity in the BAL fluid was significantly elevated on day 2 and maximal on day 6. The FGA of the BAL fluid from bleomycin-treated rats on day 6 was significantly decreased by its treatment with various thrombin inhibitors, such as alpha 1-protease inhibitor, antithrombin III, hirudin, and MD-805. In our assay, purified rat thrombin also showed FGA in vitro, and its FGA was inhibited by the same concentrations of these thrombin inhibitors as those inhibiting the activity in the BAL fluid. On ammonium sulfate fractionation, most of the thrombin activity was recovered in the fraction of 35 to 50% saturation in which most of the FGA was detected. These results suggest that the FGA of the BAL fluid from bleomycin-treated rats was at least partly due to thrombin is responsible, at least in part, for fibroblast growth and pulmonary fibrosis in bleomycin-induced interstitial lung disease.     

Severity of lung injury in cyclooxygenase-2-deficient mice is dependent on reduced prostaglandin E(2) production

Levels of prostaglandin E(2) (PGE(2)), a potent inhibitor of fibroblast function, are decreased in the lungs of patients with pulmonary fibrosis, which has been shown to be because of limited expression of cyclooxygenase-2 (COX-2). To further investigate the relative importance of COX-2 and PGE(2) in the development of fibrosis we have used a selective COX-2 inhibitor and COX-2-deficient ((-/-) and (+/-)) mice in studies of bleomycin-induced lung fibrosis. We demonstrate in wild-type mice that bleomycin-induced lung PGE(2) production is predominantly COX-2 mediated. Furthermore, COX-2(+/-) mice show limited induction of PGE(2) and an enhanced fibrotic response with increased lung collagen content compared with wild-type mice after bleomycin injury (P < 0.001). In contrast, COX-2(-/-) mice show increased levels of lung PGE(2), compared with wild-type mice after injury (P < 0.05), because of compensatory up-regulation of COX-1, which appears to be associated with macrophage/monocytes but not fibroblasts derived from these mice. COX-2(-/-) mice show an enhanced and persistent inflammatory response to bleomycin, however the fibrotic response to injury was unaltered compared with wild-type animals. These data provide further direct evidence for the importance of up-regulating COX-2 and PGE(2) expression in protecting against the development of fibrosis after lung injury.     

Extracellular matrix powder protects against bleomycin-induced pulmonary fibrosis

Pulmonary fibrosis refers to a group of lung diseases characterized by inflammation, fibroblast proliferation, and excessive collagen deposition. Although the mechanisms underlying pulmonary fibrosis are poorly understood, current evidence suggests that epithelial injury contributes to the development of fibrosis. Regenerative medicine approaches using extracellular matrix (ECM) scaffolds have been shown to promote site-specific tissue remodeling. This led to the hypothesis that particulate ECM would promote normal tissue repair and attenuate bleomycin-induced pulmonary fibrosis. C57BL/6 mice were treated intratracheally with bleomycin or saline with or without a particulate form of ECM scaffold from porcine urinary bladder matrix (UBM-ECM) or enzymatically digested UBM-ECM. Mice were sacrificed 5 and 14 days after exposure. Compared to control mice, bleomycin-exposed mice had similar increases in inflammation in the bronchoalveolar lavage fluid regardless of UBM-ECM treatment. However, 14 days after exposure, lung histology and collagen levels revealed that mice treated with bleomycin and the particulate or digested UBM-ECM had negligible fibrosis, whereas mice given only bleomycin had marked fibrosis. Administration of the particulate UBM-ECM 24 h after bleomycin exposure also significantly protected against pulmonary injury. In vitro epithelial cell migration and wound healing assays revealed that particulate UBM-ECM promoted epithelial cell chemotaxis and migration. This suggests that promotion of epithelial wound repair may be one mechanism in which UBM-ECM limits pulmonary fibrosis.     

Complex regulation of pulmonary inflammation and fibrosis by CCL18

Elevated pulmonary levels of CCL18 have been associated with influx of T lymphocytes, collagen accumulation, and a decline in lung function in pulmonary fibrosis patients. We previously reported that overexpression of CCL18 in mouse lungs triggers selective infiltration of T lymphocytes and moderate lymphocyte-dependent collagen accumulation. We hypothesized that in combination with bleomycin injury, overexpression of CCL18 will worsen the severity of lung inflammation and fibrosis. Mice were infected with a replication-deficient adenovirus encoding CCL18 and then instilled with bleomycin; control mice were challenged with either CCL18 overexpression or bleomycin. Additive effects of CCL18 overexpression and bleomycin injury were observed on pulmonary inflammation, particularly on T-cell infiltration, and increased levels of tumor necrosis factor-alpha, interferon-gamma, matrix metalloproteinase (MMP)-2, and MMP-9. Despite the additive effect on inflammation, CCL18 overexpression unexpectedly attenuated the bleomycin-induced collagen accumulation. Pulmonary levels of active transforming growth factor-beta1 mirrored the changes in collagen levels. Depletion of T cells with antilymphocyte serum or pharmacological inhibition of MMPs with GM6001 abrogated accumulation of collagen and increases in the levels of tumor necrosis factor-alpha, interferon-gamma, and active transforming growth factor-beta1. Thus, CCL18-stimulated T-lymphocytic infiltration is by itself mildly profibrotic to a healthy lung, whereas it partially protects against lung fibrosis in an inflammatory profibrotic pulmonary milieu.     

Cyclooxygenase-2 deficiency exacerbates bleomycin-induced lung dysfunction but not fibrosis

Cyclooxygenase (COX)-derived eicosanoids have been implicated in the pathogenesis of pulmonary fibrosis. Uncertainty regarding the influence of COX-2 on experimental pulmonary fibrosis prompted us to clarify the fibrotic and functional effects of intratracheal bleomycin administration in mice genetically deficient in COX-2. Further, the effects of airway-specific COX-1 overexpression on fibrotic and functional outcomes in wild-type and COX-2 knockout mice were assessed. Equivalent increases in airway cell influx, lung collagen content, and histopathologic evidence of fibrosis were observed in wild-type and COX-2 knockout mice 21 d after bleomycin treatment, suggesting that COX-2 deficiency did not alter the extent or severity of fibrosis in this model. However, bleomycin-induced alterations in respiratory mechanics were more severe in COX-2 knockout mice than in wild-type mice, as illustrated by a greater decrease in static compliance compared with genotype-matched, saline-treated control mice (26 +/- 3% versus 11 +/- 4% decreases for COX-2 knockout and wild-type mice, respectively; P < 0.05). The influence of COX-1 overexpression in airway Clara cells was also examined. Whereas the fibrotic effects of bleomycin were not altered in wild-type or COX-2 knockout mice overexpressing COX-1, the exaggerated lung function decrement in bleomycin-treated COX-2 knockout mice was prevented by COX-1 overexpression and coincided with decreased airway cysteinyl leukotriene levels. Collectively, these data suggest an important regulatory role for COX-2 in the maintenance of lung function in the setting of lung fibrosis, but not in the progression of the fibrotic process per se.     

Immunomodulation of experimental pulmonary fibrosis by intravenous immunoglobulin (IVIG)

OBJECTIVES: To assess the immunomodulatory effect of intravenous immunoglobulin (IVIG) using an experimental model of bleomycin-induced pulmonary fibrosis. METHODS: Pulmonary fibrosis was induced in C57BL/6 mice by direct intratracheal injection of bleomycin. Mice were treated with IVIG 1 week prior to (prevention protocol), or 10 days following bleomycin injection, when the disease was in progress. The controls used in the study included mice given phosphate buffered saline (PBS) and mice subjected to a commercial individual-IgG. Collagen-I deposits in the affected lungs were detected by Sirius red staining of paraffin embedded lung sections. The collagen-I content was measured by employing the hydroxyproline assay. RESULTS: Prevention of bleomycin-induced pulmonary fibrosis by IVIG has been demonstrated by reduced expression of collagen-I protein in the affected lungs. The hydroxyproline levels in the lungs of the IVIG-treated mice were 214.33 +/- 13.56 microg/1 g tissue, compared to the higher levels in lungs of IgG treated mice (342.44 +/- 35.60 microg/1 g tissue) or untreated controls 328.00 +/- 45.55 microg/1 g tissue, (p < 0.0001). Effective treatment of bleomycin-induced pulmonary fibrosis by IVIG has been demonstrated by the reduced expression of collagen-I protein in the affected lungs, detected by sirius red histological staining. The hydroxyproline levels in the lungs of the IVIG-treated mice were 261.00 +/- 18.81 microg/1 g tissue, in comparison to the higher levels in the lungs of the IgG treated mice (342.43 +/- 32.89 microg/1 g tissue) and of untreated controls (344.33 +/- 49.85 microg/1 g tissue), (p < 0.001). CONCLUSIONS: Based on these preliminary studies, we conclude that IVIG may have a beneficial effect in the down regulation of collagen-I levels in the lungs of mice with bleomycin-induced pulmonary fibrosis.     

Increased and prolonged pulmonary fibrosis in surfactant protein C-deficient mice following intratracheal bleomycin

Recent reports have linked mutations in the surfactant protein C gene (SFTPC) to familial forms of pulmonary fibrosis, but it is uncertain whether deficiency of mature SP-C contributes to disease pathogenesis. In this study, we evaluated bleomycin-induced lung fibrosis in mice with genetic deletion of SFTPC. Compared with wild-type (SFTPC+/+) controls, mice lacking surfactant protein C (SFTPC-/-) had greater lung neutrophil influx at 1 week after intratracheal bleomycin, greater weight loss during the first 2 weeks, and increased mortality. At 3 and 6 weeks after bleomycin, lungs from SFTPC-/- mice had increased fibroblast numbers, augmented collagen accumulation, and greater parenchymal distortion. Furthermore, resolution of fibrosis was delayed. Although remodeling was near complete in SFTPC+/+ mice by 6 weeks, SFTPC-/- mice did not return to baseline until 9 weeks after bleomycin. By terminal dUTP nick-end labeling staining, widespread cell injury was observed in SFTPC-/- and SFTPC+/+ mice 1 week after bleomycin; however, ongoing apoptosis of epithelial and interstitial cells occurred in lungs of SFTPC-/- mice, but not SFTPC+/+ mice, 6 weeks after bleomycin. Thus, SP-C functions to limit lung inflammation, inhibit collagen accumulation, and restore normal lung structure after bleomycin.     

Protease-activated receptor-2 induces myofibroblast differentiation and tissue factor up-regulation during bleomycin-induced lung injury: potential role in pulmonary fibrosis

Idiopathic pulmonary fibrosis constitutes the most devastating form of fibrotic lung disorders and remains refractory to current therapies. The coagulation cascade is frequently activated during pulmonary fibrosis, but this observation has so far resisted a mechanistic explanation. Recent data suggest that protease-activated receptor (PAR)-2, a receptor activated by (among others) coagulation factor (F)Xa, plays a key role in fibrotic disease; consequently, we assessed the role of PAR-2 in the development of pulmonary fibrosis in this study. We show that PAR-2 is up-regulated in the lungs of patients with idiopathic pulmonary fibrosis and that bronchoalveolar lavage fluid from these patients displays increased procoagulant activity that triggers fibroblast survival. Using a bleomycin model of pulmonary fibrosis, we show that bleomycin induces PAR-2 expression, as well as both myofibroblast differentiation and collagen synthesis. In PAR-2-/- mice, both the extent and severity of fibrotic lesions are reduced, whereas myofibroblast differentiation is diminished and collagen expression is decreased. Moreover, fibrin deposition in the lungs of fibrotic PAR-2-/- mice is reduced compared with wild-type mice due to differential tissue factor expression in response to bleomycin. Taken together, these results suggest an important role for PAR-2 in the development of pulmonary fibrosis, and the inhibition of the PAR-2-coagulation axis may provide a novel therapeutic approach to treat this devastating disease.     

Antifibrotic effects of crocetin in scleroderma fibroblasts and in bleomycin-induced sclerotic mice

OBJECTIVE:

To investigate the antifibrotic effects of crocetin in scleroderma fibroblasts and in sclerotic mice.

METHODS:

Skin fibroblasts that were isolated from three systemic scleroderma (SSc) patients and three healthy subjects were treated with crocetin (0.1, 1 or 10 μM). Cell proliferation was measured with an MTT assay. Alpha-smooth muscle actin was detected via an immunohistochemical method. Alpha 1 (I) procollagen (COL1A1), alpha 1 (III) procollagen (COL3A1), matrix metalloproteinase (MMP)-1 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA levels were measured using real-time PCR. SSc mice were established by the subcutaneous injection of bleomycin. Crocetin (50 mg/kg/d) was injected intraperitoneally for 14 days. Dermal thickness and lung fibrosis were assessed with Masson''s trichrome staining. Plasma ET-1 was detected with an enzyme-linked immunosorbent assay (ELISA). Skin and lung ET-1 and COL1A1 mRNA levels were measured via real-time PCR.

RESULTS:

Crocetin inhibited the proliferation of SSc and normal fibroblasts, an effect that increased with crocetin concentration and incubation time. Crocetin decreased the expression of α-SMA and the levels of mRNA for COL1A1, COL3A1 and matrix metalloproteinase-1, while crocetin increased TIMP-1 mRNA levels in both SSc and normal fibroblasts. Skin and lung fibrosis was induced, and the levels of ET-1 in the plasma, skin and lungs were elevated in bleomycin-injected mice. Crocetin alleviated the thickening of the dermis and lung fibrosis; decreased COL1A1 mRNA levels in the skin and lung; and simultaneously decreased ET-1 concentrations in the plasma and ET-1 mRNA levels in the skin and lungs of the bleomycin-induced sclerotic mice, especially during the early phase (weeks 1-3).

CONCLUSION:

Crocetin inhibits cell proliferation, differentiation and collagen production in SSc fibroblasts. Crocetin alleviates skin and lung fibrosis in a bleomycin-induced SSc mouse model, in part due to a reduction in ET-1.     

Intercellular adhesion molecule-1 and L-selectin regulate bleomycin-induced lung fibrosis

The development of bleomycin-induced lung injury, a model of pulmonary fibrosis, results from inflammatory cell infiltration, a process highly regulated by the expression of multiple adhesion molecules. At present, the identity and role of the adhesion molecules involved in the fibrotic process are unknown. Therefore, bleomycin-induced pulmonary fibrosis was examined in mice lacking L-selectin (L-selectin(-/-)) expression, intercellular adhesion molecule-1 (ICAM-1) expression, or both. After 16 days of intratracheal bleomycin challenge, collagen deposition was inhibited in both L-selectin(-/-) and ICAM-1(-/-) mice when compared with wild-type littermates. Interestingly, collagen deposition was virtually eliminated in L-selectin/ICAM-1(-/-) mice relative to either the L-selectin(-/-) or ICAM-1(-/-) mice. Decreased pulmonary fibrosis was associated with reduced accumulation of leukocytes, including neutrophils and lymphocytes. Decreased mRNA expression of proinflammatory cytokines and transforming growth factor (TGF)-beta1 paralleled the inhibition of collagen deposition. The present study indicates that L-selectin and ICAM-1 play a critical role in pulmonary fibrosis by mediating the accumulation of leukocytes, which regulate the production of proinflammatory cytokines and TGF-beta1. This suggests that these adhesion molecules are potential therapeutic targets for inhibiting human pulmonary fibrosis.     

Effects of a calmodulin inhibitor on bleomycin-induced lung inflammation in hamsters. Biochemical, morphometric, and bronchoalveolar lavage data.

Previous studies have shown that bleomycin-induced pulmonary fibrosis is accompanied by elevated levels of calcium and calmodulin, which are important in the regulation of many biologic processes. The authors have further extended these observations and assessed the effect of a calmodulin inhibitor, trifluoperazine, on bleomycin-induced lung damage with biochemical, morphometric, and bronchoalveolar lavage techniques. The cumulative mortality due to bleomycin was not significantly reduced in animals receiving trifluoperazine. Trifluoperazine had no apparent effect on lung levels of collagen and DNA elevated by bleomycin. However, morphometric studies showed that the volume density of the lesion, the volume of amorphous material and interstitial inflammation, and the number of monocytes within lesions were less in the lungs of bleomycin-treated hamsters receiving trifluoperazine daily. When compared with hamsters treated with bleomycin alone, animals treated with both bleomycin and trifluoperazine had significantly fewer lymphocytes in their bronchoalveolar lavage fluid. The data suggest that trifluoperazine reduced the acute inflammation which accompanies bleomycin pneumotoxicity but did not affect the subsequent development of pulmonary fibrosis. It has been postulated that the observed antiinflammatory action of trifluoperazine may be due to inhibition of calmodulin-dependent leukocyte functions.     

Inhibition of pulmonary fibrosis by the chemokine IP-10/CXCL10

Pulmonary fibrosis is an enigmatic and devastating disease with few treatment options, now thought to result from abnormal wound healing in the lung in response to injury. We have previously noted a role for the chemokine interferon gamma-inducible protein of 10 kD (IP-10)/CXC chemokine ligand 10 in the regulation of cutaneous wound healing, and consequently investigated whether IP-10 regulates pulmonary fibrosis. We found that IP-10 is highly expressed in a mouse model of pulmonary fibrosis induced by bleomycin. IP-10-deficient mice exhibited increased pulmonary fibrosis after administration of bleomycin, suggesting that IP-10 limits the development of fibrosis in this model. Substantial fibroblast chemoattractant and proliferative activities were generated in the lung after bleomycin exposure. IP-10 significantly inhibited fibroblast responses to the chemotactic, but not the proliferative activity generated, suggesting that IP-10 may attenuate fibroblast accumulation in bleomycin-induced pulmonary fibrosis by limiting fibroblast migration. Consistent with this inhibitory activity of IP-10 on fibroblast migration, fibroblast accumulation in the lung after bleomycin exposure was dramatically increased in IP-10-deficient mice compared with wild-type mice. Conversely, transgenic mice overexpressing IP-10 were protected from mortality after bleomycin exposure, and demonstrated decreased fibroblast accumulation in the lung after challenge compared with wild-type mice. Our findings suggest that interruption of fibroblast recruitment may represent a novel therapeutic strategy for pulmonary fibrosis, which could have applicability to a wide range of fibrotic illnesses.     

当归注射液及前列腺素E_2对结缔组织生长因子在大鼠肺纤维化模型中表达作用的比较研究

目的探讨结缔组织生长因子(CTGF)在大鼠肺间质纤维化中的表达及其在25%当归注射液、前列腺素E2(dmPGE2)的肺脏保护作用中的意义。方法SD大鼠随机分为对照组、模型组、当归组、PGE2组。按博莱霉素(BLM)5mg/kg体重复制大鼠肺纤维化模型,当归组、dmPGE2组术后分别给于25%当归注射液(腹腔内注射)、dmPGE2(肌肉注射),于第28天处死全部大鼠。制备大鼠肺组织切片,用原位杂交及免疫组化SABC法分别检测CTGF mRNA、纤维连结蛋白(FN)和Ⅲ型胶原(Col-Ⅲ)在肺内的表达,HE染色评价肺纤维化程度。结果模型组CTGF mRNA、FN和Col-Ⅲ的表达及肺纤维化程度明显高于对照组(P<0.01),而当归组及PGE2组明显低于模型组(P<0.01)。两治疗组间比较,除CTGF外,其余指标无显著性差异(P>0.05),各组指标的相关分析表明,CTGF表达水平与肺纤维化程度(r=0.886;P<0.01)、FN(r=0.836;P<0.01)和Col-Ⅲ(r=0.918;P<0.01)呈正相关关系。结论25%当归注射液可能通过下调CTGF而减轻肺间质纤维化。     

Male sex hormones exacerbate lung function impairment after bleomycin-induced pulmonary fibrosis

The roles of sex hormones as modulators of lung function and disease have received significant attention as differential sex responses to various lung insults have been recently reported. The present study used a bleomycin-induced pulmonary fibrosis model in C57BL/6 mice to examine potential sex differences in physiological and pathological outcomes. Endpoints measured included invasive lung function assessment, immunological response, lung collagen deposition, and a quantitative histological analysis of pulmonary fibrosis. Male mice had significantly higher basal static lung compliance than female mice (P < 0.05) and a more pronounced decline in static compliance after bleomycin administration when expressed as overall change or percentage of baseline change (P < 0.05). In contrast, there were no significant differences between the sexes in immune cell infiltration into the lung or in total lung collagen content after bleomycin. Total lung histopathology scores measured using the Ashcroft method did not differ between the sexes, while a quantitative histopathology scoring system designed to determine where within the lung the fibrosis occurred indicated a tendency toward more fibrosis immediately adjacent to airways in bleomycin-treated male versus female mice. Furthermore, castrated male mice exhibited a female-like response to bleomycin while female mice given exogenous androgen exhibited a male-like response. These data indicate that androgens play an exacerbating role in decreased lung function after bleomycin administration, and traditional measures of fibrosis may miss critical differences in lung function between the sexes. Sex differences should be carefully considered when designing and interpreting experimental models of pulmonary fibrosis in mice.