VPA alleviates bleomycin-induced pulmonary fibrosis in rats

AIM: To investigate the effect of sodium valproate (VPA) on bleomycin-induced pulmonary fibrosis (PF) and its mechanism. METHODS: SD rats (n=42) were randomly divided into model group and treatment group. Bleomycin at dose of 5 mg/kg was intratracheally injected to establish a rat PF model. The rats in treatment group were given normal saline (NS, 0.5 mL/d), VPA (300 mg·kg^-1·d^-1) or dexamethasone (DEX, 0.6 mg·kg^-1·d^-1) via intraperitoneal injection from the 14th day to the 28th day after modeling. The rats in model group were sacrificed on 0 d, 14 day and 28 d after modeling . The rats in treatment group were killed at 14th day after treatment. The effects of VPA on PF were evaluated by HE and Masson staining. The hydroxyproline (HYP) content in the rat lung tissues was detected, and the expression of α-smooth muscle actin (α-SMA) and E-cadherin was determined by Western blotting. RESULTS: HE staining showed that the alveolar structure and interstitial morphology in VPA group were better than those in NS group and DEX group. The level of collagen in VPA group was significantly lower than that in DEX group and NS group by determining the HYP content and Masson staining. VPA reduced the expression of α-SMA, a mesenchymal marker protein of PF, while increased the expression of epithelial marker protein E-cadherin. CONCLUSION: VPA reduces collagen content and distribution, and up-regulates the expression of the epithelial marker protein E-cadherin, while down-regulates mesenchymal marker protein α-SMA, thereby preventing the rat lung tissues from bleomycin-induced PF.     

Etanercept attenuates bleomycin-induced lung fibrosis in mice

AIM: To evaluate the effect of tumor necrosis factor α (TNF-α) antagonist etanercept on bleomycin-induced lung fibrosis in mice.METHODS: Forty-five Kunming female mice were randomly divided into 3 groups: the mice in control group were intraperitoneally injected with vehicle and intratracheally administered with saline aerosol, the mice in bleomycin group were intraperitoneally injected with vehicle and intratracheally administered with bleomycin (3 mg/kg) aerosol, and the mice in bleomycin+etanercept group were intraperitoneally injected with etanercept (4 mg/kg) every 3 d and intratracheally administered with bleomycin aerosol. All animals were sacrificed 28 d after treatments. The left lung was fixed in 10% neutral formalin and then stained with hematoxylin-eosin or Masson’s trichrome for the pathological examination. The tissues of right lung were sampled for measuring the content of hydroxyproline (HYP) by the method of alkaline hydrolysis. The serum concentrations of TNF-α and TGF-β were detected by ELISA. Total tissue protein was extracted for examination of ERK1/2, JNK and p38 by Western blotting.RESULTS: Etanercept prevented the collagen accumulation under the airway epithelium and decreased the scores of lung inflammation and fibrosis induced by bleomycin with significantly reduced the levels of tissue HYP, serum TNF-α and serum TGF-β. The protein phosphorylations of ERK/JNK/p38 in the lung tissues were remarkably decreased compared with BLM group.CONCLUSION: Etanercept decreases the phosphorylations of ERK1/2/JNK/p38 via inhibiting the expression of TNF-α and TGF-β. Etanercept might be useful in the treatment of pulmonary fibrosis.     

Astragalus membranaceus inhibits bleomycin-induced pulmonary fibrosis in mice by antioxidation

AIM:To investigate the effect of Astragalus membranaceus on the balance of oxidation and antioxidation in bleomycin-induced pulmonary fibrosis in mice and the possible anti-fibrosis mechanism of Astragalus membranaceus.METHODS:Female KM mice (n=36) were randomly divided into 3 groups.The mice in control group were administered with saline aerosol intratracheally.The mice in fibrosis group were administered with bleomycin at dose of 3 mg/kg aerosol intratracheally.The mice in Astragalus membranaceus group were administered with bleomycin at dose of 3 mg/kg aerosol intratracheally and then intraperitoneal injected with Astragalus membranaceus parenteral solution at daily dose of 1.7 g/kg.All mice were sacrificed 14 d after the treatment,and the lungs and serum were collected for detection.Hematoxylin-eosin staining was performed in the lung tissue.The mRNA expression of superoxide dismutase 1/2/3(SOD1/2/3),catalase (CAT),NADPH oxidase 2/4(NOX2/4) and α-smooth muscle actin (α-SMA) was detected by RT-PCR,and the protein expression of α-SMA and NOX2/4 was determined by Western blot.The concentration of malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in the serum were measured by a colorimetric method.RESULTS:The pathological injury was obviously observed in bleomycin group compared with control group,but was attenuated in Astragalus membranaceus group.The α-SMA mRNA and protein expression,MDA/T-AOC,NOX2,NOX4 and SOD3 mRNA expression,and NOX2 protein expression in bleomycin group were significantly higher than those in control group,while those in Astragalus membranaceus group were significantly lower than those in bleomycin group.The protein expression of NOX4 in bleomycin group was significantly lower than that in control group,while that in Astragalus membranaceus group was higher than that in bleomycin group.The mRNA expression of SOD1 and CAT in Astragalus membranaceus group and bleomycin group were decreased compared with control group.No significant difference of SOD2 mRNA expression among the 3 groups was observed.CONCLUSION:Astragalus membranaceus inhibits bleomycin-induced pulmonary fibrosis in mice by maintaining the balance of oxidation and antioxidation.     

Matrine attenuates bleomycin-induced pulmonary injury partially via modulating mononuclear phagocyte phenotype switching in mice

AIM: To investigate the influence of matrine (MA) on the phenotype switching of mouse monocytes and alveolar macrophages induced by bleomycin (BLM).METHODS: All mice were randomly divided into normal saline (NS) group, BLM group, BLM+NS group and BLM+MA group. The mice were administered with BLM at 2.5 mg/kg via oropharyngeal instillation. The mice in BLM+MA group were treated with MA (15 mg·kg^-1·d^-1) by oral gavage following BLM administration. The mice were sacrificed on days 3, 7, 14, and 21. The lungs were removed for pathological analysis. The circulating monocyte subsets and polarization state of bronchoalveolar lavage fluid (BALF)-derived alveolar macrophages were analyzed by flow cytometry.RESULTS: The results of HE and Masson trichrome staining in BLM and BLM+NS groups exhibited classical pathological stages of lung fibrosis, including acute inflammation phase and later fibrosis phase. Compared with BLM+NS group, MA treatment alleviated the inflammatory response and the degree of fibrosis induced by BLM (P<0.05). There was a rapid change of circulating Ly6C^hi monocytes and its magnitude was positively associated with the pulmonary inflammatory response. An expansion of M2-like alveolar macrophages was positively correlated with the magnitude of lung fibrosis. Moreover, MA treatment partially normalized the phenotype switching of monocytes and alveolar macrophages.CONCLUSION: Matrine treatment attenuates BLM-induced pulmonary injury partially via modulating the phenotype switching of monocytes and alveolar mocrophages.     

Rhein attenuates bleomycin-induced rats pulmonary fibrosis through TGF-β1/Smad pathway by inhibiting miR-21 expression

AIM: To investigate the effect of rhein on bleomycin-induced pulmonary fibrosis and the expression of microRNA-21 (miR-21) and transforming growth factor-β1 (TGF-β1)/Smad signaling molecules in rats. METHODS: A single dose of bleomycin was intratracheal injected into the SD rats to induce pulmonary fibrosis. After injection of bleomycin, the rats were randomly divided into low-, medium-and high-dose rhein treatment groups and model group. The rats that were instilled with normal saline intratracheally served as control group. After the treatment for 28 d, the pulmonary pathologic changes were observed under microscope with hematoxylin-eosin staining. The lung coefficient and hydroxyproline content were also measured. The expression of miR-21 and the mRNA levels of TGF-β1 and Smad7 in the lung tissues were detected by real-time PCR. The protein levels of TGF-β1 and Smad7 were determined by Western blot. RESULTS: Rhein significantly attenuated the experimental alveolitis, pulmonary fibrosis, lung coefficient and hydroxyproline contents in the rats. Rhein obviously decreased the expression of miR-21,and the mRNA and protein levels of TGF-β1, but significantly increased the mRNA and protein levels of Smad7 in the lung tissues. CONCLUSION: Rhein effectively prevents bleomycin-induced pulmonary fibrosis by inhibiting the expression of miR-21 and promoting the expression of Smad7, thus regulating the TGF/Smad signaling pathway to decrease extracellular matrix deposition.     

Gefitinib inhibits epithelial-mesenchymal transition by up-regulating Foxo3a expression in mice with bleomycin-induced lung fibrosis

AIM：To identify the effect of gefitinib on the expression of forkhead box protein O3a (Foxo3a), α-smooth muscle actin (α-SMA) and related signal pathway molecules in the mice with bleomycin-induced lung fibrosis and to investigate the inhibition mechanism of gefitinib on lung epithelial-mesenchymal transition. METHODS：Thirty Kunming female mice were randomly divided into 3 groups:control group (received normal saline intratracheally), bleomycin group (received bleomycin intratracheally, 3 mg/kg), and bleomycin plus gefitinib group (received bleomycin intratracheally and gefitinib orally, 20 mg/kg). All the mice were sacrificed 14 d after the treatments. Pulmonary histological changes were evaluated by hematoxylin-eosin staining and Masson trichrome staining. The mRNA levels of Foxo3a and α-SMA in the lung tissues were detected by RT-PCR. Nuclear Foxo3a, α-SMA, and phosphorylation of EGFR, Akt and Foxo3a in the lung tissues were determined by Western blotting. RESULTS：Gefitinib inhibited bleomycin-induced lung fibrosis and significantly decreased the scores of lung inflammation and fibrosis. Foxo3a mRNA expression and total Foxo3a protein expression were increased, while the phosphorylated Foxo3a was decreased. Nuclear Foxo3a was increased significantly. Meanwhile, phosphorylated EGFR and Akt were decreased. The level of α-SMA was observably increased. CONCLUSION:Gefitinib restores Foxo3a activity and reduces α-SMA expression by modulating EGFR/Akt activity, thus inhibiting bleomycin-induced lung fibrosis.     

Role of calcium-sensing receptor in hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells

AIM: To explore the role of calcium-sensing receptor (CaSR) in rat pulmonary artery smooth muscle cells (PASMCs) and its effect on hypoxia-induced proliferation of PASMCs. METHODS: The expression and distribution of CaSRs were detected by Western blotting and immunofluorescence observation. The intracellular concentration of free calcium ([Ca]_i) was determined by confocal laser scanning microscopy. The cell viability was analyzed by MTT assay. The expression of PCNA and CaSRs was determined by Western blotting. RESULTS: CaSR protein was expressed in rat PASMCs. Hypoxia significantly increased the expression of CaSR and PCNA,[Ca]_i and the cell viability. GdCl₃ (an agonist of CaSR) or NPS2390 (an antagonist of CaSR) amplified or weakened the effect of hypoxia, respectively.CONCLUSION: CaSR is expressed in rat PASMCs. The activation of CaSR is involved in the proliferation of PASMCs induced by hypoxia.     

Effect of mineralocorticoid receptor gene silencing by RNA interference on proliferation and apoptosis of murine macrophage cell line RAW 264.7

AIM: To establish stable knockdown of mineralocorticoid receptor (MR) expression through short hairpin RNA (shRNA)-mediated silencing in murine RAW 264.7 macrophages. METHODS: Stable MR silencing in RAW 264.7 cells was achieved by recombinant shRNA plasmid targeting murine MR gene via liposome-mediated transfection, followed by G418 selection. The efficacies of plasmid transfection and MR silencing in G418-resistant cells were verified by immunofluorescent microcopy and real-time PCR, respectively. Proliferative activity of MR-silencing cell line was analyzed by CCK-8 assay. Cell cycle and apoptosis were evaluated by flow cytometry. RESULTS: MR gene expression was down-regulated by 70% compared with the negative control (NC) plasmid transfection. In addition, MR-silencing cells exhibited lower proliferative activity compared with NC and wide type RAW 264.7 cells (P<0.05), along with reduced proliferation index of 31.0%±1.3% (P<0.05), compared with the wide type cells (37.2%±0.5%) and the NC cells (37.5%±1.6%). In resting state, the apoptotic rate in wide type, NC and MR-silencing cells were 2.18%±0.36%, 6.65%±0.81% and 7.70%±1.34%, respectively, and no statistical difference was observed between NC and MR-silencing cells (P>0.05). CONCLUSION: MR gene silencing inhibits the proliferation of RAW 264.7 macrophages, but has no obvious effect on the apoptosis of the resting state cells.     

Role of endogenous nitric oxide in apoptosis of alveolar epithelial cells in the development of pulmonary fibrosis in rats

AIM: To study the role of high level of endogenous nitric oxide (NO) in apoptosis of alveolar epithelial cells in the development of pulmonary fibrosis in rats. METHODS: The content of nitrite/nitrate (NO₂^-/NO₃^-) in out-flowing pulmonary blood (OPB) was assayed by nitric acid reduction method. The apoptosis of alveolar epithelial cells was observed by TdT-mediated dUTP nick-end labeling (TUNEL) and electron microscopy, respectively. The above indices were observed on the day 14 and the day 30 after intratracheal administration of BLMA₅ alone or along with blockade of iNOS by aminoguanidine (AG) in rats. RESULTS: (1) Both the content of NO₂^-/NO₃^- in OPB and the number of apoptotic alveolar epithelial cells in lung were increased in BLMA₅ 14 d group, compared with normal control group and BLMA₅ 30 d group, respectively (P<0.05). The high level of NO₂^-/NO₃^- in OPB and the apoptosis of alveolar epithelial cells were ameliorated by AG. CONCLUSION: The apoptosis of alveolar epithelial cell is induced by high level of endogenous NO in the development of pulmonary fibrosis.     

Comparison of in vivo anti-fibrotic effects of pirfenidone and nintedanib in bleomycin-induced pulmonary fibrosis model

AIM: To compare the effects of pirfenidone and nintedanib on bleomycin-induced mice pulmonary fibrosis model in different periods. METHODS: Five mice models were established according to the schedule of drug administration:a inflammatory phase model and 4 fibrotic phase models including the early prevention study group, early therapy study group, late therapy study group and full-course therapy study group. The indicators of lung inflammatory and lung fibrosis were detected respectively. RESULTS: (1) The level of anti-inflammatory and antioxidant indicators:both pirfenidone and nintedanib reduced the number of inflammatory cells and inhibited the secretion of inflammatory factors. Pirfenidone had a better effect on inhibition of interleukin (IL)-1β and IL-4 (P < 0.01), while nintedanib had a better effect on inhibition of IL-6 and IFN-γ (P < 0.05). Pirfenidone significantly increased superoxide dismutase (SOD) activity (P < 0.01), and nintedanib significantly reduced malondialdehyde (MDA) and myeloperoxidase (MPO) levels (P < 0.01). (2) Collagen content in lung tissue:the inhibitory effect of nintedanib on hydroxyproline content in mouse lung was better than that of pirfenidone in the early therapy study group, late therapy study group and full-course therapy study group of the fibrotic phase models (P < 0.05). Pirfenidone had a better inhibitory effect on hydroxyproline content than nintedanib in the early prevention study group (P < 0.01). (3) Pathological evaluation of lung tissue:both pirfenidone and nintedanib reduced the inflammatory infiltration and fibrotic area of the lung tissues. The inhibition trend was consistent with that of collagen content. CONCLUSION: Both pirfenidone and nintedanib have anti-inflammatory, anti-oxidative and anti-fibrosis effects in bleomycin-induced pulmonary fibrosis in mice. Pirfenidone is more effective in the early prevention study group, and nintedanib has a better effect in early therapy study group, late therapy study group and full-course therapy study group.     

Effect of aldosterone on rat peritoneal fibrosis induced by peritoneal dia-lysis

AIM: To investigate the pathologic role of aldosterone and protective effect of aldosterone receptor antagonist on peritoneal fibrosis in peritoneal dialysis rats. METHODS: A peritoneal fibrosis rat model was established by intraperitoneal injection of lipopolysaccharide(at 1 d, 3 d, 5 d and 7 d, 0.6 mg/kg) and dialysate(daily intraperitoneal injection of 4.25% dialysate, 100 mL/kg). At the same time, spironolactone(an aldosterone receptor antagonist, 100 mg·kg^-1·d^-1) was given to the model rats. After 4 weeks, the expression of aldosterone synthase CYP11B2, 11β-hydroxysteroid dehydrogenase type 2(11β-HSD2), mineralocorticoid receptor(MCR), and inflammatory factors were detected by immunohistochemistry, real-time PCR and Western blotting. RESULTS: The rat model of peritoneal fibrosis was successfully established. At the same time, the injury of mesothelial cells, deposition of collagen fibers and thickness of peritoneal were increased. Moreover, the infiltration of macrophages in the peritoneum/dialysate was increased. The level of aldosterone and the expression of MCR, 11β-HSD2 and CYP11B2 in fibrotic peritoneum were obviously up-regulated as compared with normal rats. The expression of NF-κB/MCP-1 was also increased. However, treatment with spironolactone alleviated peritoneal fibrosis and reduced the expression of NF-κB/MCP-1. CONCLUSION: Local aldosterone is involved in the process of peritoneal fibrosis via NF-κB/MCP-1 pathway. Spironolactone alleviates peritoneal fibrosis of peritoneal dialysis.     

The kinetic alteration of nitric oxide formation in the lungs in the development of pulmonary fibrosis of rats

AIM: To observe the kinetic alteration of nitric oxide formation in the lungs in the development of pulmonary fibrosis in the rat. METHODS: The contents of hydroxyproline in the lungs, NO₂^-/NO₃^- (nitrite/nitrate) in out-flowing and in-flowing pulmonary blood (OPB, IPB) were assayed on the day 7, 14, 21, 30 and 70 after intratracheal administration of bleomycin A₅ . The content of NO₂^-/NO₃^- in supernatants of culture of the alveolar macrophages (AMs) and the amount of iNOS positive stain cells in lung tissue section were also observed in the rat on 14th day after-bleomycin A₅ administration. RESULTS: The content of lung hydroxyproline had no change on the 7th day, increased on the 14th day (P＜0.05), increased significantly on the 21th day, 30th day and 70th day post-bleomycin A₅ compared with control rats. On the 7th day and 14th day, the content of NO^- ₂ /NO₃^- increased in OPB and decreased in IPB (P＜0.01). On the 21th day, the content of NO₂^-/NO₃^- abated in OPB (P＞0.05) but still decreased in IPB (P＜0.01). On the 30th day and the 70th day, the NO₂^-/NO₃^- level recovered both in OPB and IPB. AMs from rats on the 14th day post-bleomycin A₅ showed significant elevation (P＜0.01) in NO₂^-/NO₃^- level. The amount of iNOS positive stain cells increased in rats on the 14th day post-bleomycin A₅. CONCLUSION: The amount of NO in the lungs was high in the initial phase of fibroproliferative reaction induced by bleomycin A₅ ,and these might be associated with the enhanced ability of AMs to release NO and the increased amount of iNOS.     

The changes in proliferation and apoptosis of macrophages in the development of pulmonary fibrosis of rats

AIM: To observe the changes in proliferation and apoptosis of macrophages in the development of pulmonary fibrosis in rats. METHODS: The number of macrophages, apoptotic cells, the proliferation index (PI) and MTT activity of macrophages were assayed on the day 14 and the day 30 after intratracheal adminstration BLMA₅ in rats. RESULTS: (1) The number of alveolar macrophages was increased in BLMA₅ 14 d group and in BLMA₅ 30 d group, compared with sham 14 d group and sham 30 d group, respectively. The number of macrophages in BLMA₅ 14 d group was higher than that in BLMA₅ 30 d group. (2) The PI of macrophages increased in BLMA₅ 14 d group, and decreased in BLMA₅ 30 d group. (3) The number of apoptotic cells increased both in BLMA₅ 14 d group and BLMA₅ 30 d group. The number of apoptotic cells in BLMA₅ 14 d group was lower than that in BLMA₅ 30 d group. (4) The MTT activity of macrophages was higher in BLMA₅ 14 d group and in BLMA₅ 30 d group than that in sham 14 d group and sham 30 d group, respectively. CONCLUSIONS: The ability of proliferation increased at first, and then decreased, but the apoptosis of macrophages increased all the time, in the development of pulmonary fibrosis. This might be partly contributed to the changes of the number and function of macrophages in lung.     

The augmentative effect of inducible nitric oxide synthase on pulmonary fibrosis progression

AIM: To study the up-regulation of inducible nitric oxide synthase (iNOS) in lung of pulmonary fibrosis and its relationship with fibrosis. METHODS: The changes of amount of iNOS positive stain cells and type Ⅰ?Ⅲ collagen were examined on the day 7, 14 and 30 after intratracheal administration of bleomycin A₅. The contents of NO₂^-/NO₃^- (nitrite/nitrate) in out-flowing pulmonary blood (OPB), hydroxyproline in lung and the histological changes were detected after iNOS was blocked by aminoguanidine (AG). RESULTS: (1) The number of iNOS-positive stain cells increased significantly in BLMA₅ 7 d, 14 d and 30 d groups compared with that in control group (P<0.01). Furthermore, the increment of the number of iNOS-positive stain cells in BLMA₅ 7 d, 14 d groups was more than that in BLMA₅ 30 d group. There was an increment of collagen in BLMA₅ 14 d group and in BLMA₅ 30 d group , with an increase in type Ⅲ collagen in BLMA₅ 14 d group and an increase in type Ⅰcollagen in BLMA₅ 30 d group. (2) The high level of NO₂^-/NO₃^- in OPB and hydroxyproline level in lung could be reversed by AG, a selective inhibitor of iNOS. Large amount of fibroblasts and macrophages were also abated by AG. CONCLUSION: In the development of pulmonary fibrosis, the expression of iNOS is up-regulated, which induces nitric oxide (NO) production and promotes propagation of pulmonary fibrosis.     

Role of TGFβ1/Smad3 signaling pathway-mediated lysine hydroxylase 2 activity in collagen deposition of pulmonary fibrosis

AIM: To investigate the effect of TGFβ1/Smad3 signaling pathway on the changes of lysyl hydro-xylase2 (LH2) activity, and to study the role in the relationship between LH2 and collagen deposition of pulmonary fibrosis. METHODS: Human lung fibroblast cell line HFL1 was cultured in F12 medium with 10% fetal bovine serum. The cells were divided into control group, TGFβ1 (10 μg/L) stimulation group, and minoxidil (5 μmol/L) intervention group. The cells in control group were treated with the equivalent volume of medium. The RNA and protein were collected after 48 h. The mRNA levels of PLOD2, α-SMA and COLⅠ were detected by RT-qPCR. The protein levels of LH2, total Smad3, phosphorylated Smad3, α-SMA, COLⅠ and COL Ⅳ were determined by Western blot. Hydroxylysylpyridinoline (HP) content was detected by ELISA. RESULTS: After stimulation with TGFβ1, the mRNA expression of PLOD2, α-SMA and COLⅠ was increased (P<0.01), and the protein levels of LH2, p-Smad3, α-SMA, COLⅠ and COL Ⅳ were also up-regulated, but the total Smad3 protein did not change. Treatment with minoxidil decreased the levels of above indexes (P<0.01). Compared with control group, stimulation with TGFβ1 increased the content of HP. However, treatment with minoxidil decreased the synthesis of HP (P<0.05). CONCLUTION: Activation of TGFβ1/Samd3 signaling pathway enhances LH2 expression. Minoxidil inhibits the TGFβ1/Samd3 signaling transduction, thereby reducing the expression of LH2 and the synthesis of hydroxylysyl collagen pyridine chain, and reducing pulmonary fibrosis.     

Effect of arsenic trioxide on bleomycin-induced pulmonary fibrosis in rats

AIM: To observe the effect of arsenic trioxide (ATO) on bleomycin-induced pulmonary fibrosis in rats. METHODS: Pulmonary fibrosis was induced in Sprague-Dawley (SD) rats by intratracheal instillation of bleomycin (BLM). The rats in ATO treatment group, steroid treatment group and model group were intraperitoneally injected with ATO, dexamethasone or normal saline (NS), respectively, while the control rats received NS both intratracheally and intraperitoneally. The effects of ATO were evaluated by analyzing the median survival time, hydroxyproline level in the lung, semi-quantitative grading of alveolitis and pulmonary fibrosis, and quantitative analysis of the collagen in lung tissue (Masson’s trichrome staining). Apoptosis index (AI) of the lung was detected by using the terminal transferase dUTP-digoxygenin nick end-labeling (TUNEL) method. The results of immunohistochemical staining for some cytokines were quantitatively analyzed. RESULTS: ATO (1) prolonged the median survival time of rats with BLM-induced pulmonary fibrosis at some extent; (2) attenuated the alveolitis and pulmonary fibrosis, reduced hydroxyproline level and collagen deposition in the lung tissue; (3) increased the AI of lung tissue at a certain phase; and decreased the levels of transforming growth factor-β1 (TGF-β1) and tissue inhibitor of metalloproteinase-1 (TIMP-1), increased the content of interferon-γ (IFN-γ), but did not influence the concentration of matrix metalloproteinase-9 (MMP-9) significantly. CONCLUSION: ATO might attenuate BLM-induced pulmonary fibrosis in rats via increasing the AI in the lung tissue.     

Kinetics of pathologic changes in bleomycin-induced murine pulmonary fibrosis model

AIM: To Investigate the kinetics of pathologic changes in bleomycin-induced pulmonary fibrosis in rats. METHODS: Sixty male SD rats were randomized as a negative control group and pulmonary fibrosis model groups (B3, B7, B14, B28, B56 sub-groups). Except for control group, rats in the other groups were intratracheally administered with bleomycin. Animals in pulmonary fibrosis model groups were sacrificed on day 3, 7, 14, 28 and 56. The sections of the right lung were stained by HE, Masson and sirius red. The left lung was weighed and its hydroxyproline content was assayed. The mRNAs of TGF-β1, MMP-9 and TIMP-1 in the lung homogenate were measured by semi-quantitative RT-PCR. The expressions of TGF-β1, MMP-9 and TIMP-1 in lungs were observed by immunohistochemistry. RESULTS: (1) The content of lung hydroxyproline in pulmonary fibrosis model groups was significantly increased than that in control group (P<0.05). The pulmonary inflammation in pulmonary fibrosis model groups was significantly serious than that in control group, pulmonary fibrosis in B14, B28 and B56 groups was also significantly serious than that in control group. (2) A small quantity of TGF-β1, MMP-9 and TIMP-1mRNA were measured in normal lung, and the expression increased significantly after administration of bleomycin. Different expressions of TGF-β1, MMP-9 and TIMP-1 in different days after bleomycin administration were observed. CONCLUSION: The pathological changes in different days after bleomycin administration are different. TGF-β1, MMP-9 and TIMP-1 may play important roles in the pathogenesis of pulmonary fibrotic process.     

Inhibitory effect of metformin on alveolar epithelial-mesenchymal transition in lung tissue of rats with pulmonary fibrosis

AIM: To study the inhibitory effect of metformin on alveolar epithelial-mesenchymal transition (EMT) in rats with pulmonary fibrosis and the possible mechanism. METHODS: SD rats (n=48) were used, 12 of which were set up as normal control group, and 36 of which were induced by bleomycin (5 mg/kg) by tracheal instillation to establish pulmonary fibrosis. The pulmonary fibrosis rats were randomly divided into bleomycin group, low dose (100 mg/kg) of metformin group, and high dose (300 mg/kg) of metformin group. The rats in metformin groups were given the corresponding dose of metformin daily for 4 weeks. HE staining and Masson staining were used to observe the changes of lung histopathology and collagen deposition. Real-time PCR, Western blot and innunohistochemical staining were used to detect the mRNA and protein expression of α-smooth muscle actin (α-SMA), E-cadherin, vimentin, zonula occludens-1 (ZO-1), collagen I, collagen III and transforming growth factor-β₁ (TGF-β₁), and the protein phosphorylation levels of Smad2/3 and extracellular signal-regulated kinase 1/2 (ERK1/2) were also determined. RESULTS: Metformin up-regulated the expression of E-cadherin and ZO-1, down-regulated the expression of α-SMA, vimentin, collagen I and collagen III, and the protein phosphorylation levels of Smad2/3 and ERK1/2 were also decreased (P<0.05). CONCLUSION: Metformin inhibits alveolar EMT in the rats with pulmonary fibrosis, and its mechanism may be related to the inhibition of TGF-β₁ signal transduction pathway.