Antifibrotic action of pirfenidone and prednisolone: different effects on pulmonary cytokines and growth factors in bleomycin-induced murine pulmonary fibrosis

Pirfenidone, a broad-spectrum antifibrotic agent, is known to have efficacy in certain fibrotic disease models, and is under clinical trials in patients with idiopathic pulmonary fibrosis. We investigated the antifibrotic effect of pirfenidone, and its regulatory effect on various pulmonary cytokines, in bleomycin-induced lung fibrosis in mice at the protein level, using prednisolone as a reference agent. Pirfenidone attenuated the bleomycin-induced pulmonary fibrosis at a minimum effective dose of 30 mg/kg/day t.i.d. from the analysis of lung hydroxyproline content. Both pirfenidone (30, 100 mg/kg/day t.i.d) and prednisolone (3, 15 mg/kg/day q.d.) suppressed lung inflammatory edema; however, prednisolone failed to suppress pulmonary fibrosis, which was significantly suppressed only by pirfenidone. Both pirfenidone and prednisolone suppressed the increase in lung interleukin (IL)-1beta, IL-6, IL-12p40 and monocyte chemoattractant protein (MCP)-1 levels induced by bleomycin. On the other hand, pirfenidone prevented the bleomycin-induced decrease in lung interferon (IFN)-gamma levels, while prednisolone had no such effect. Furthermore, pirfenidone suppressed elevation of lung basic-fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta1 levels, but prednisolone had no such effect. The increases in lung stroma cell derived factor (SDF)-1alpha and IL-18 were also suppressed. These findings suggest that pirfenidone exerts its antifibrotic effect through regulation of lung IFN-gamma, bFGF and TGF-beta1 levels during the development of bleomycin-induced pulmonary fibrosis in mice. The effect on SDF-1alpha and IL-18 levels may also be related to the antifibrotic effects of pirfenidone.     

Effects of dimethyl sulfoxide (DMSO) on pulmonary fibrosis in rats and mice

Dimethyl sulfoxide (DMSO), a putative anti-inflammatory agent and free radical scavenger, was shown to protect against acute bleomycin-induced pulmonary fibrosis in the rat (Pepin and Langner, Biochem. Pharmacol., 34 (1985) 2386). We examined the effect of DMSO on bleomycin-induced pulmonary toxicity in Swiss outbred mice and Sprague-Dawley rats, and on butylated hydroxytoluene (BHT)-induced pulmonary toxicity in Swiss outbred mice. Bleomycin (BL)-induced mortality in mice (20% at 0.1 units BL) and rats (50% at 1.5 units BL) was increased to 100% by daily DMSO (5 g/kg 50% in saline). Similar DMSO treatment after lower doses of bleomycin (1 unit BL in rats and 0.075 or 0.050 units in mice) increased lung hydroxyproline content in the rat but had no effect in the mouse. Lung hydroxyproline content in mice 14 days after 400 mg/kg BHT in corn oil was also slightly increased by daily DMSO at 5 g/kg, but not at 1 or 2 g/kg. Daily DMSO (5 g/kg) did not alter cellular proliferation [( 14C]thymidine incorporation into pulmonary DNA) in the lung at 2 or 5 days after BHT. Thus, we found that DMSO potentiated the lethality of bleomycin, and potentiated or had no effect on bleomycin or BHT-induced pulmonary fibrosis in the rat and mouse.     

Ginkgo biloba inhibits bleomycin-induced lung fibrosis in rats.

Oxidative stress has been implicated in the pathogenesis of bleomycin-induced lung fibrosis and many antioxidant agents have been studied for prevention and treatment of the disease in animals and humans. We therefore examined whether Ginkgo biloba (Gb), a flavonoid-rich antioxidant, inhibits bleomycin-induced lung fibrosis in rats. Male Sprague-Dawley rats were given a single dose of bleomycin (2.5 mg/kg, intratracheally) in pulmonary fibrosis groups and saline in controls. First dose of Gb was given a day before the bleomycin injection and continued until sacrifice. At day 14, fibrotic changes in lung were estimated to occur by Aschoft's criteria and lung hydroxyproline content. Bleomycin challenge provoked severe pulmonary fibrosis with marked increase in hydroxyproline content of lung tissue and typical histological findings, which is prevented by Gb. Hydroxyproline level was significantly higher (13.51+/-0.87 mg/g dried tissue) in bleomycin treated rats than controls (9.2+/-1.33), and its level was remained to the control levels (7.38+/-0.76) in rats treated with prophylactic Gb. On the other hand, bleomycin injection significantly reduced activities of glutathione peroxidase, superoxide dismutase and catalase in lung tissue which is prevented by Gb. Also, bleomycin injection resulted in a marked increase of malondialdehyde and nitrite level which is attenuated by Gb. The data suggest that Gb has a potent antioxidant activity in the model of bleomycin-induced lung fibrosis in rats, and therefore has a potent antifibrotic activity against bleomycin-induced lung fibrosis model in rats.     

（5R）-5-hydroxytriptolide （LLDT-8） protects against bleomycin-induced lung fibrosis in mice

AIM: To study the protective effects of a triptolide-derived, novel compound, (5R)-5-hydroxytriptolide (LLDT-8), on bleomycin-induced lung fibrosis. METHODS: C57BL/6 mice received an intratracheal injection of bleomycin and were then treated with LLDT-8 (0.5, 1, 2 mg/kg, ip) once daily for 7 or 14 consecutive days. The body weight loss and lung index augmentation was observed; the inflammatory response including differential cells counts of neutrophils, macrophages, and lymphocytes in the bronchoalveolar lavage fluid (BALF), superoxide dismutase (SOD), and malondialdehyde (MDA) level in the lung homogenates was detected, and the fibrosis extent was evaluated by hydroxyproline content and histopathological changes in the lungs. In addition, the pro-inflammatory and pro-fibrotic cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-4 (IL-4), and transforming growth factor-alpha (TGF-alpha) production in the lungs were measured. RESULTS: LLDT-8 alleviated the body weight loss and lung index increase caused by bleomycin, reduced neutrophils and lymphocytes in the BALF, promoted SOD activity, decreased MDA production, and inhibited the hydroxyproline level and the amelioration of lung tissue histological damage. Moreover, LLDT-8 suppressed TNF-alpha, IL-4, and TGF-beta production in the lung homogenates. CONCLUSION: LLDT-8 showed protective effects against bleomycin-induced lung fibrosis, and the results suggested the potential role of LLDT-8 in the treatment of this disease.     

Oral chymase inhibitor SUN13834 ameliorates skin inflammation as well as pruritus in mouse model for atopic dermatitis

Chymase is a chymotrypsin-like serine protease exclusively stored in secretory granules of mast cells and has been thought to participate in allergic diseases. It has already been shown that chymase inhibitor SUN13834 improves dermatitis in NC/Nga mice that spontaneously develop dermatitis resembling atopic dermatitis. In the present study, effect of chymase inhibitor SUN13834 on itch, the major feature of atopic dermatitis, was examined using a mouse dermatitis model induced by repeated topical application of 2,4-dinitrofluorobenzene (DNFB). Oral administration of SUN13834 once a day for 5 weeks inhibited not only skin swelling but accumulation of inflammatory cells including mast cells and eosinophils in the skin of the mice. In addition, SUN13834 also decreased significantly at 10 and 50 mg/kg the amount of scratching behavior induced by the DNFB challenge. This result indicates for the first time that mast cell chymase may be involved in itch induction. In conclusion, SUN13834 is thought to be useful as therapeutic agent for atopic dermatitis.     

Chymase mediates paraquat-induced collagen production in human lung fibroblasts

Survivors of paraquat poisoning may be left with pulmonary fibrosis and a restrictive type of pulmonary dysfunction. Chymase converts angiotensin (Ang) I to Ang II, which is closely involved with lung fibrosis. The role played by chymase in paraquat-induced lung fibrosis is unclear. We examined the effects of paraquat on chymase, renin–angiotensin system components, and collagen expression in murine and human lung fibroblasts (MRC-5). Lung chymase and collagen type I mRNA and protein expression were significantly increased and angiotensin-converting enzyme (ACE) mRNA and protein expression were comparable between the control and paraquat-treated mice 1 and 3 weeks after administration. Paraquat significantly upregulated angiotensinogen mRNA expression in a dose-dependent manner while ACE activity and protein expression were similar in MRC-5 cells. Furthermore, paraquat enhanced Ang II and collagen type I mRNA and protein expression, α-smooth muscle actin, and chymase protein and chymase small interfering RNA inhibited these effects. The cDNA sequence of chymase in MRC-5 cells is identical to that in human mast cells. This study found increased chymase expression in paraquat-treated human lung fibroblasts and confirmed in vitro and in an in vivo paraquat model of lung fibrosis that chymase generates Ang II and enhances collagen expression. These data suggest a role for chymase in the pathogenesis of paraquat-induced lung fibrosis.     

1-(5-异喹啉磺酰基)-2-甲基哌嗪(H-7)对肺纤维化大鼠肺单核细胞趋化蛋白-1 mRNA表达的影响

研究蛋白激酶C(PKC)抑制剂 1 (5 异喹啉磺酰基 ) 2 甲基哌嗪 (H 7)对肺纤维化大鼠单核细胞趋化蛋白 1(MCP 1)mRNA表达的影响 ,为临床治疗肺纤维化提供理论基础 .博来霉素致大鼠肺纤维化动物模型 ,腹腔注射H 7后 ,定量RT PCR方法检测其对肺组织MCP 1mRNA表达的影响 ;测定实验组和对照组肺组织匀浆内羟脯氨酸的含量 ,观察肺组织病理学变化并计数单核细胞 .结果发现 ,H 7能显著抑制大鼠肺纤维化组织MCP 1mRNA表达和单核细胞的聚集以及羟脯氨酸的含量 .结果表明 ,H 7通过抑制肺组织MCP 1mRNA的表达 ,对博来霉素致肺纤维化有明显的防治作用 .     

Vildagliptin,a CD26/DPP4 inhibitor,ameliorates bleomycin-induced pulmonary fibrosis via regulating the extracellular matrix

BackgroundIdiopathic pulmonary fibrosis is a debilitating lung disease. CD26/DPP4 plays promotive roles in pulmonary damage and fibrosis. This study aimed to explore the roles of vildagliptin in bleomycin-induced pulmonary fibrosis, and to address its ameliorative effect on the extracellular matrix (ECM).MethodsIdiopathic pulmonary fibrosis mice models were induced by intratracheal injection of bleomycin. DPP4 activity was evaluated, and the fibrosis was investigated by Hematoxylin-eosin, Masson's trichrome staining and hydroxyproline assay. Expression of extracellular matrix proteins including α-SMA, collagen IV, collagen I, FN and TGF-β were analyzed by immunochemistry and western blot. Percentages of the numbers of monocytes, leukocytes, basophils and lymphocytes were classified, and inflammatory factors in plasma as well as lung tissues were examined by enzyme-linked immunosorbent assay and western blot. The influences of vildagliptin on TGF‐β1-induced cell proliferation, differentiation and inflammatory factors in MRC-5 cells were detected.ResultsVildagliptin effectively attenuated inflammation and fibrosis in bleomycin-induced pulmonary tissue via inhibiting the activity of CD26/DPP4. extracellular matrix proteins were suppressed by vildagliptin. Thus, lung tissue fibrosis was efficiently alleviated by vildagliptin.ConclusionAs an inhibitor of CD26/DPP4, Vildagliptin could be a promising therapeutic candidate for idiopathic pulmonary fibrosis.     

Attenuation of bleomycin-induced lung fibrosis in rats by mesna

Lung fibrosis is a common side effect of the chemotherapeutic agent, bleomycin. Current evidence suggests that reactive oxygen species may play a key role in the development of lung fibrosis. The present study examined the effect of mesna on bleomycin-induced lung fibrosis in rats. Animals were divided into three groups: (1) saline control group; (2) Bleomycin group in which rats were injected with bleomycin (15 mg/kg, i.p.) three times a week for four weeks; (3) Bleomycin and mesna group, in which mesna was given to rats (180 mg/kg/day, i.p.) a week prior to bleomycin and daily during bleomycin injections for 4 weeks until the end of the treatment. Bleomycin treatment resulted in a pronounced fall in the average body weight of animals. Bleomycin-induced pulmonary injury and lung fibrosis was indicated by increased lung hydroxyproline content, and elevated nitric oxide synthase, myeoloperoxidase, platelet activating factor, and tumor necrosis factor-alpha in lung tissues. On the other hand, bleomycin induced a reduction in reduced glutathione concentration and angiotensin converting enzyme activity in lung tissues. Moreover, bleomycin-induced severe histological changes in lung tissues revealed as lymphocytes and neutrophils infiltration, increased collagen deposition and fibrosis. Co-administration of bleomycin and mesna reduced bleomycin-induced weight loss and attenuated lung injury as evaluated by the significant reduction in hydroxyproline content, nitric oxide synthase activity, and concentrations of myeoloperoxidase, platelet activating factor, and tumor necrosis factor-alpha in lung tissues. Furthermore, mesna ameliorated bleomycin-induced reduction in reduced glutathione concentration and angiotensin activity in lung tissues. Finally, histological evidence supported the ability of mesna to attenuate bleomycin-induced lung fibrosis and consolidation. Thus, the findings of the present study provide evidence that mesna may serve as a novel target for potential therapeutic treatment of lung fibrosis.     

Attenuation of bleomycin-induced lung fibrosis by oxymatrine is associated with regulation of fibroblast proliferation and collagen production in primary culture

There is no satisfactory treatment for pulmonary fibrosis, which is characterized by altered control of proliferation of mesenchymal fibroblasts and extracellular matrix production. Oxymatrine is an alkaloid extracted from the Chinese herb Sophora japonica (Sophora flavescens Ait.) with capacities of anti-inflammation, inhibition of immune reaction, antivirus, protection against acute lung injury and antihepatic fibrosis. In this study, the effect of oxymatrine on pulmonary fibrosis was investigated using a bleomycin-induced pulmonary fibrosis mouse model. The results showed that bleomycin challenge provoked severe pulmonary fibrosis with marked increase in hydroxyproline content of lung tissue and lung fibrosis fraction, which was prevented by oxymatrine in a dose-dependent manner. In addition, bleomycin injection resulted in a marked increase of myeloperoxidase activity and malondialdehyde level that was attenuated by oxymatrine. Administration of oxymatrine inhibited the proliferation of murine lung fibroblasts, arrested the cells at G(0)/G(1) phase and reduced the expression of cell cycle regulatory protein, cyclin D1 in vitro. Furthermore, the steady-state production of collagen and the expression of alpha1(I) pro-collagen and alpha2(I) pro-collagen mRNA in fibroblasts were inhibited by oxymatrine in a dose-dependent manner. These results suggested that oxymatrine may attenuate pulmonary fibrosis induced by bleomycin in mice, partly through inhibition of inflammatory response and lipid peroxidation in lung induced by bleomycin and reduction of fibroblast proliferation and collagen synthesis.     

VEGFR-2 antagonist SU5416 attenuates bleomycin-induced pulmonary fibrosis in mice

ObjectiveAbnormal angiogenesis is a central hallmark for the development and progression of idiopathic pulmonary fibrosis. It has been shown that vascular endothelial growth factor (VEGF) is one of the critical angiogenic factors in angiogenesis. The aim of the present study was to assess whether disruption of VEGF pathway would attenuate bleomycin-induced pulmonary fibrosis.MethodsBleomycin-induced pulmonary fibrosis mice were treated intraperitoneally with VEGF receptor tyrosine kinase inhibitor SU5416 at different phases after bleomycin infusion. We measured angiogenesis and inflammatory response in both bleomycin-treated and control mice, and correlated these levels with pulmonary fibrosis.ResultsThe increased expressions of VEGF/VEGFR (Flk-1) were correlated to a larger number of microvessels and a higher score of pulmonary fibrosis. Early administration of SU5416 inhibited pulmonary collagen deposition, histopathologic fibroplasias and the activation of TGF-β1/Smad3 signaling pathway in bleomycin-stimulated lung. These were also paralleled by a reduction of VEGF/VEGFR-2 (Flk-1) expression and microvessel numbers in lung. Furthermore, SU5416 inhibited inflammatory cell numbers and LDH activity in BALF and IL-13 expression in lung tissue at early inflammatory phase of bleomycin-induced pulmonary fibrosis.ConclusionThese results suggest that the VEGFR-2 inhibitor, SU5416, attenuates histopathologic fibroplasias and collagen deposition by regulating angiogenesis and inflammation in the lung.     

Effect of mast cell chymase inhibitor on the development of scleroderma in tight-skin mice

1 Although the pathogenesis of scleroderma is not fully understood, activation of connective-tissue-type mast cells (CTMCs) has been implicated in various fibrotic diseases. 2 Our previous study showed that the number of CTMCs was markedly increased during fibrous proliferation in the skin of a scleroderma model, namely tight-skin (Tsk) mice. Because mast cells express numerous bioactive factors, such as cytokines, growth factors, proteases, and others, it is crucial to identify the primary factors that may be involved in the pathogenesis of scleroderma. Our previous study also showed that a CTMC-specific protease, chymase-4, was selectively upregulated in accordance with the development of skin fibrosis in Tsk mice. 3 To further elucidate the role of chymase secreted from CTMCs, we evaluated the therapeutic effects of a synthetic chymase-specific inhibitor, SUN-C8257, on the development of skin fibrosis in Tsk mice. SUN-C8257 (50 mg kg-1 day-1) was administered via intraperitoneal injection in 13-week-old Tsk mice for a period of 2 weeks. 4 Treatment with SUN-C8257 significantly reduced chymase activity by 43% and the chymase-4 mRNA level by 47%, and also decreased the thickness of the subcutaneous fibrous layer of Tsk mice by 42% compared with that of Tsk mice injected with vehicle. 5 Furthermore, immunohistochemical analysis revealed that transforming growth factor (TGF)-beta1 staining in the fibrous layer of Tsk skin was markedly reduced by the treatment with SUN-C8257. This chymase inhibitor may prevent the chymase-dependent pathway that activates the latent TGF-beta1 in fibrous tissue, and may exhibit beneficial effects that inhibit the development of fibrosis. 6 In conclusion, our results strongly support the assumption that CTMC-derived chymase may play a key role in the pathogenesis of scleroderma.     

糜酶抑制剂对实验大鼠肺纤维化的干预作用

目的观察用糜酶抑制剂对博莱霉素致大鼠肺纤维化模型干预作用。方法健康SD大鼠60只随机均分为正常对照组(生理盐水)、博莱霉素模型组和干预组(博莱霉素+糜酶抑制剂)。分别在第7、14、21和28天处死。HE染色观察支气管肺组织病理改变,免疫组化观察肺组织中Ⅰ型、Ⅲ型胶原的表达。结果干预组肺泡炎程度明显轻于模型组和对照组(P<0.05)。干预组肺纤维化程度比模型组明显减轻(P<0.05)。与对照组比较,干预组和模型组肺组织中Ⅰ型、Ⅲ型胶原均表达持续增强,但干预组明显减少(P<0.05)。结论糜酶抑制剂能减轻博莱霉素诱导的大鼠肺纤维化,可能是通过下调Ⅰ型、Ⅲ型胶原的表达。     

灯盏花素对博来霉素诱导小鼠肺纤维化的保护作用

目的观察灯盏花素对肺纤维化的保护作用,探讨其可能的作用机制。方法观察灯盏花素对体外培养小鼠胚肺成纤维细胞L929增殖、激活及细胞外基质分泌的影响;应用博来霉素诱导小鼠肺纤维化模型,观察灯盏花素对肺纤维化的保护作用。结果灯盏花素体外对小鼠胚肺成纤维细胞L929无直接细胞毒作用,但能抑制TGF-β1促进的增殖、激活及层粘连蛋白(LN)、Ⅰ型胶原(ColⅠ)分泌。体内灯盏花素能抑制博来霉素诱导的小鼠血清TGF-β1升高,阻止博来霉素诱导的小鼠肺脏SOD、POD、CAT降低,并降低肺脏羟脯氨酸、胶原、MDA及TGF-β1含量。结论灯盏花素有肺纤维化保护作用,其机制可能是通过增加抗氧化防御系统和阻止TGF-β信号实现的。     

A specific chymase inhibitor, NK3201, suppresses bleomycin-induced pulmonary fibrosis in hamsters

We evaluated whether a chymase inhibitor, 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[(3,4-dioxo-1-phenyl-7-(2-pyridyloxy))-2-heptyl]acetamide (NK3201), suppressed bleomycin-induced pulmonary fibrosis. Hamsters were orally administered NK3201 (30 mg/kg per day) or placebo, beginning 5 days before intratracheal instillation of bleomycin (10 mg/kg). Four weeks after the instillation of bleomycin, pulmonary chymase activity in placebo-treated hamsters was significantly higher than in control hamsters, whereas the activity in NK3201-treated hamsters was significantly lower than in placebo-treated hamsters. The ratio of fibrotic area to total area in NK3201-treated hamsters was significantly decreased to 54.0% of the ratio in placebo-treated hamsters. Therefore, NK3201 may be useful in the prevention of pulmonary fibrosis.     

Short courses of low dose dexamethasone delay bleomycin-induced lung fibrosis in rats

After comparing mortality and clinical signs in rats receiving different dexamethasone treatments, we investigated whether 0.5 mg/kg/d dexamethasone could delay pulmonary fibrosis induced by bleomycin and its time course (1, 3, 7, 14, 21 and 28 days). Tissue injury was assessed by apoptosis, lactate dehydrogenase (LDH) release, malondialdehyde content, and protein content; and inflammation was measured in terms of myeloperoxidase (MPO) activity, inflammatory cell count, and the mRNA expression of pro/inflammatory cytokines. Fibrogenic activity was analyzed by measuring the mRNA expression of fibrotic cytokines in tissue, and the promotion of fibroproliferation and synthesis of collagen type I by bronchoalveolar lavage fluids in vitro; and fibrosis was assessed by measuring the hydroxyproline content and collagen-I mRNA expression, and by histology. Bleomycin treatment induced tissue injury, inflammation and fibrogenic activity in lung, and led to fibrosis. Treatment with dexamethasone diminished the extent of fibrosis by strongly reducing inflammation, lung damage, and fibrogenic activity. These results demonstrate that the progression of bleomycin-induced pulmonary fibrosis in rats can be delayed by dexamethasone treatment, which appeared to alleviate not only inflammation but also lung damage and fibrogenic activity, indicating a possible new role for dexamethasone in the treatment of fibrosis.     

The effect of serine esterase inhibitors on ionophore-induced histamine release from human pulmonary mast cells

The serine proteases tryptase and chymase are present in human pulmonary mast cells. About 10-100 times more tryptase than chymase is found in these cells. However, a clear physiological role for both enzymes remains to be elucidated; angiotensin processing has been proposed as one possible function of chymase. A dose-dependent inhibition of A23187-induced histamine release from dispersed human lung mast cells was observed after pretreatment with the serine protease inhibitor diisopropylfluorophosphate (DFP) or the chymotrypsin-like enzyme inhibitor N-tosyl-L-phenylalanine chloromethylketone (TPCK) but not with the trypsin-like enzyme inhibitor N-tosyl-L-lysine chloromethylketone (TLCK). These results indicate that a chymase is probably an important factor in a late phase of human lung mast cell activation.     

Inhibitory effects of isoliensinine on bleomycin-induced pulmonary fibrosis in mice

The effects of isoliensinine (IL), a bisbenzylisoquinoline alkaloid extracted from the Chinese traditional medicine seed embryo of Nelumbo nucifera Gaertn., on bleomycin (BLM)-induced pulmonary fibrosis in mice were investigated. Seventy-two male Kungming mice were divided randomly into eight groups as BLM-IL10, BLM-IL20, BLM-IL40, BLM-Sal, Sal-IL10, Sal-IL20, Sal-IL40 and Sal-Sal groups. BLM (0.1 mg in 0.05 ml saline per animal, once) or saline (0.05 ml per animal, once) was applied intratracheally, and IL (10, 20, 40 mg/kg) or saline was administered orally 3 times per day in the appropriate groups. Animals were sacrificed 14 days after intratracheal treatment. Lung tissue and serum superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta 1 (TGF-beta (1)) were determined by biochemical measurements and immunohistochemistry. BLM treatment resulted in a significant increase of the hydroxyproline content and an obvious lung histological injury as compared to the Sal-Sal group. Administration of IL remarkably suppressed the increase in hydroxyproline content and abated the lung histological injury induced by BLM. There was a decrease in SOD activity and an increase in MDA level in lung tissue and serum in the BLM-Sal group (p < 0.01 , p < 0.01, vs. Sal-Sal group, respectively). And IL could obviously enhance the SOD activity and decrease the MDA level in a concentration-dependent manner. Moreover, IL also significantly inhibited the overexpression of TNF-alpha and TGF-beta (1) induced by BLM. These results indicated that IL possessed a significant inhibitory effect on BLM-induced pulmonary fibrosis, probably due to its antioxidant and/or anti-inflammatory activities and inhibitory overexpressing TNF-alpha and TGF-beta (1) induced by BLM.     

苦参碱对博来霉素所致肺间质纤维化大鼠的保护作用

目的:研究苦参碱对肺间质纤维化大鼠的保护作用。方法:采用气管内灌注博来霉素(BLM)的方法建立大鼠肺纤维化模型,设正常对照组、模型组、小剂量苦参碱组(50 mg/kg)、大剂量苦参碱组(100 mg/kg)和氢化可的松组(100 mg/kg),每组18只,再各分为d 7、14和d 28组。观察各组不同时期病理切片和肺组织羟脯氨酸(HYP)浓度,测定肺组织丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH- PX)含量。结果:苦参碱组和氢化可的松组的肺泡炎和肺纤维化程度及HYP含量显著地低于模型组(P<0.05);苦参碱和氢化可的松能明显降低肺间质纤维化大鼠肺组织MDA水平,提高GSH-PX含量(P<0.01)。结论:苦参碱能减轻BLM诱导的大鼠肺纤维化,这种作用有可能通过改善肺间质纤维化大鼠体内氧化应激状态实现。     

TIMP-1 is a key factor of fibrogenic response to bleomycin in mouse lung

Pulmonary fibrosis is characterized by the excessive deposition of extracellular matrix in the interstitium, resulting in respiratory failure. The role of remodeling mediators such as metalloproteinases (MMPs) and their inhibitors (TIMPs) in the fibrogenic process remains misunderstood. We investigated MMP-9, MMP-2, TIMP-1, TIMP-2 and TIMP-3 in the fibrotic response to bleomycin of fibrosis prone C57BL/6J and fibrosis resistant BALB/c mice. Mice were administered with 0.1 mg bleomycin by intranasal administration. Either 24 h or 14 days after, the mice were anesthetized and underwent either bronchoalveolear lavage (BAL) or lung removal. Collagen deposition in lung tissue was determined by hydroxyproline measurement, MMP activity was analyzed by zymography, and other mediators were analyzed by ELISA. TIMP-1 was localized in lung sections by immunohistochemistry and real time PCR was performed to gene expression in lung. Non parametric Mann-Whitney and Spearman tests were used for statistical analysis. Fourteen days after bleomycin administration, hydroxyproline assay and histological study revealed that BALB/c mice developed significantly less fibrosis compared to C57BL/6J mice. At day 1, bleomycin enhanced TIMP-1, MMP-2 and MMP-9 protein levels in BALF, and induced corresponding genes in lung tissue of both strains. The rise of Timp-1, Mmp-9 and Mmp-2 gene levels were significantly stronger in lungs of C57BL/6J, whereas gelatinase activities of MMP-2 and MMP-9 were similar. Immunohistochemistry revealed that TIMP-1 macrophages and epithelial cells were prominent TIMP-1 producers in both strains. At day 14, neither MMP-2 nor MMP-9 levels exhibited strain-dependent protein level or gene expression, although TIMP-1 was strongly associated with fibrosis. Interestingly, bleomycin induced neither Timp-2 nor Timp-3 in lung tissue at any time of the study. The present study shows that early altered regulation of TIMP-1 following bleomycin administration may be involved in bleomycin-induced pulmonary fibrosis.