Objective To investigate the effects of sodium ferulate on lung mRNA expression of TGF-β1 signal transduction molecule in rats with pulmonary fibrosis，and explore the mechanism of sodium ferulate on pulmonary fibrosis.Methods A rat model of pulmonary fibrosis was induced by intratracheal injection of bleomycin (5 mg/kg).Thirty SD rats were randomly divided into three groups (n=10 in each group)，ie.a control group，a pulmonary fibrosis model group，and a sodium ferulate group.The lung histopathology and the expression of collagen was examined by HE staining and collagen fibril staining respectively.The expressions of TGF-βRII and Smad4 mRNA in the lung tissue were detected by situ hybridization.And the expression of TGF-β1 mRNA was detected by real-time fluorescence-quantification RT-PCR.Results Collagen fibril staining indicated that the expression of pulmonary collagen in the model group was significantly higher than that in the control group and sodium ferulate group (Plt;0.001).The mRNA expressions of pulmonary TGF-β1，TGF-βRII and Smad4 were significantly higher in the model group than those in the control group (all Plt;0.01)，and were significantly lower in the sodium ferulate group than those in the model group (all Plt;0.05).Conclusions Sodium ferulate can effectively reduce pulmonary fibrosis through inhibition of the mRNA expression of TGF-β1，TGF-βRII and Smad4 in the lung tissue，thus influence the TGF-β1/Smad4 signal transduction way and inhibit the target gene activation.
Objective To investigate the role of IFN-γ in suppressing bleomycin-induced pulmonary fibrosis in rats.Methods Seventy-five SD rats were randomly divided into five groups (15 rats in each group)，ie.a normal group，a bleomycin-induced pulmonary fibrosis model group，a dexamethasone-treated group，a high-dose IFN-γ-treated group (150 000 U/kg) and a low-dose IFN-γ-treated group (50 000 U/kg).Five rats in each group were randomly killed in 7th day，14th day and 28th day after relative treatment respectively，and lung tissue samples were harvested for histopathology study.HE and Masson staining were used to determine the extent of alveolus inflammation and pulmonary fibrosis respectively.Histoimmunochemical method were adapted to determine protein levels of TGF-β1，CTGF，type Ⅰcollagen and type Ⅲ collagen in pulmonary tissues.Results Histopathological study showed that treatment with either dexamethasone or IFN-γ (both high dose and low dose) remarkably meliorated the extent of alveolus inflammation and suppressed pulmonary fibrosis (compared with model group，all Plt;0.05).Histoimmunochemical study suggested that both dexamethasone and IFN-γ could inhibit the expression of TGF-β1，CTGF，type Ⅰand type Ⅲ collagen (compared with model group，all Plt;0.05)，and the suppression of TGF-β1，type Ⅰand type Ⅲ collagen expression was more obvious in high-dose IFN-γ-treated group than those in low-dose group (Plt;0.05).Conclusions INF-γ possesses apparent anti-fibrosis effect that is similar to dexamethasone but with less side effect.Such effect may resulted from reduced production of type Ⅰand type Ⅲ collagen through expression inhibition of cytokines such as TGF-β1 and CTGF.
Objective To study the inhibitory effects of curcumin on bleomycin-induced pulmonary fibrosis in rats at the fibrosing stage and explore its possible mechanism.Methods 96 male SD rats were randomly divided into a normal control group，a fibrosis model group，a fibrosis model treated with prednisone group and a fibrosis model treated with curcumin group.Pulmonary fibrosis were induced by instilled bleomycin through tracheal.From day 15 after bleomycin administration，the curcumin group and prednisone group were given curcumin（300 mg/kg） or prednisone（5 mg/kg） per day by intragastric administration，respectively.The normal control group and fibrosis model group were given 1% sodium carboxymethyl cellulose（10 mL/kg） as control.Six rats of each group were randomly sacrificed on day 21，28，42 and 56 after bleomycin administration，respectively.The histological changes of the lung were evaluated by HE and Masson’s trichrome staining.Lung expressions of transforming growth factor-β1（TGF-β1） and hydroxyproline were assessed by immuno-histochemistry and digestion method，respectively.Results Pulmonary fibrosis and hydroxyproline level in the curcumin group were significantly reduced as compared with those in the model group on day 42 and 56.The expession of TGF-β1 in the curcumin group was significantly lower than that in the model group on day 28，42 and 56，and was not significantly different from the normal group on day 56.Conclusion Curcumin could alleviate bleomycin-induced pulmonary fibrosis in rats at the fibrosing stage by inhibiting the expressions of TGF-β1.
Objective To explore whether epithelial to mesenchymal transition ( EMT) occurs in bleomycin( BLM) induced pulmonary fibrosis, and the involvement of bronchial epithelial cells( BECs) in the EMT. Methods BLM-induced peribronchial fibrosis in an α-smooth muscle actin-Cre transgenic mouse( α-SMACre /R26R) was examined by pulmonary βgal staining and α-SMA immunofluorescence staining. Results BLMtreated mice showed significantly enhanced βgal staining in subepithelial areas in bronchi, terminal bronchioles and walls of pulmonary vessels. Some alveolar epithelial cells( AECs) in certain peribronchial areas or even a small subset of BECs were also positively stained, as confirmed by α-SMA immunostaining. Conclusions EMT occurs in BLM-induced peribronchial fibrosis mice. BECs, like AECs, have the capacity to undergo EMT and to contribute to mesenchymal expansion in pulmonary fibrosis.
Objective To study the pathology and possible mechanism of experimental hydrochloric acid（HCl） inhalation-indued pulmonary fibrosis in rats.Methods 120 male SD rats were randomly divided into a nomal control group，a bleomycin group，a high dose HCl group，a middle dose HCl group and a low dose HCl group.The bleomycin group was intratracheally injected with bleomycin once to induce pulmonary fibrosis.The three HCl groups were intratracheally injected with HCl once per week.The control group was given saline by the same way.Six rats of each group were randomly sacrificed on day 7，14，28 and 42 respectively.The histological changes of lung tissue were studied by HE and Masson’s trichrome staining.Hydroxyproline level in lung tissue was measured by digestion method.Protein and mRNA expression of transforming growth factor-β1（TGF-β1） were assayed by immunohistochemistry and RT-PCR respectively.Results Alveolitis in three HCl groups was significantl compared to control group，most severe at the second week，then remained at a high level which was equivalent to or exceeded the level of the bleomysin group after 28 days.Pulmonary fibrosis in three HCl groups was also significantly more severe than that in the control group，but milder than that in the bleomysin group.The high-dose and middle-dose HCl groups were not significantly different from the bleomysin group on day 42.There was no difference between three HCl groups in the earlier period，but the high-dose HCl group has a significantly difference from low-dose group on day 42.The content of hydroxyproline in high-dose and middle-dose HCl groups was also significantly higher than that in the control group.On day 42 hydroxyproline content in high-dose HCl dose rather middle –or low dose group was similiar with the level of bleomysin group.Content of TGF-β1 mRNA in three HCl groups was comparable to the level of bleomysin group on day 28 and exceeded on day 42.The expression of TGF-β1 in three HCl groups was not significantly different from the bleomysin group on day 42.Conclusion Experimental acid aspiration might contribute to pulmonary fibrosis in rats.Acid induced alveolar epithelial cell damage，abnormal proliferation and repair and fibrosis could be involved..
Objective To investigate the proliferation inhibitory effect and to explore the molecular mechanism of curcumin on pulmonary fibroblasts. Methods Fibroblasts derived from lung tissue of patients with idiopathic pulmonary fibrosis ( IPF) was cultured in vitro and incubated with curcumin at different concentrations for different time. Fibroblasts were randomized into 5 groups, ie. a control group and 4 curcumin groups ( intervened by 5, 10, 20, 40 μmol / L curcumin, respectively) . MTT assay was used to determine the inhibitory rate of curcumin on the proliferation of pulmonary fibroblasts. Apoptosis and the Caspase-3 expression of pulmonary fibroblasts were identified by flow cytometry ( FCM) . Variables were compared with One-Way ANOVA. The correlations between variables were analyzed using Pearson’scorrelation coefficient. Results Curcumin inhibited pulmonary fibroblasts proliferation in a dose-dependent and time-dependent manner( r =0. 886, r = 0. 832, respectively, all P lt; 0. 01) . Apoptosis rate of pulmonary fibroblasts in 4 curcumin groups was ( 29. 58 ±2. 13) % , ( 64. 36 ±3. 92) %, ( 72. 98 ±4. 42) % , ( 83. 14 ±2. 51) % , respectively, which was significantly higher than that in the control group[ ( 3. 84 ±1. 88) % , P lt;0. 01] . The positive expression rate of apoptosis-regulating protein caspase-3 was ( 26. 24 ±3. 64) % ,( 44. 87 ±5. 31) % , ( 57. 44 ±4. 23) % , ( 73. 65 ±5. 01) % , respectively, which was significantly higher than that of the control group[ ( 4. 02 ±0. 62) % , P lt; 0. 01] . Conclusions In vitro, curcumin can significantly inhibit proliferation and induce apoptosis of pulmonary fibroblasts of patients with IPF. The mechanism maybe associated with up-regulating expression of Caspase-3.
Objective To investigate the expression of high mobility group protein-B1( HMGB1)and α-smooth muscle actin( α-SMA) in Bleomycin induced pulmonary fibrosis in mice. Methods Twenty C57BL/ 6 male mice were randomly divided into a Bleomycin group and a control group. The Bleomycin group was treated with Bleomycin( 3 mg/kg) by endotracheally injection to induce pulmonary fibrosis. The control group were treated with normal saline( NS) . Then they were sacrificed by abdominal aortic bleeding 10 days after the injection. The right lung was stained with hematoxylin-eosin and Masson trichrome respectively for pathological examination. Immunohistochemistry and RT-PCR were performed to identify the protein and mRNA levels of α-SMA and HMGB1 respectively. Results The mRNA( 0. 89 ±0. 12, 0. 61 ±0. 08) and protein( 13. 66 ±1. 01, 13. 12 ±1. 33) expressions of α-SMA and HMGB1 in the Bleomycin group were all significantly higher than those of the control group( mRNA: 0. 60 ±0. 07, 0. 15 ±0. 02; protein: 8. 18 ±1. 33,7. 92 ±1. 10; all P lt; 0. 01) . Conclusions The expressions of HMGB1 and α-SMA are increased in Bleomycin induced pulmonary fibrosis. HMGB1 participates in the pathological process of pulmonary fibrosis probably by activation of the α-SMA expression.
Objective To explore the inhibitory mechanism of Imatinib mesylate on pulmonary fibrosis induced by bleomycin inmice. Methods A total of 120 C57BL/6 mice were randomly divided into four groups, ie. a control group, a model group, a dexamethasone group, and an Imatinib group. The model of pulmonary fibrosis was established by a single intratracheal instillation of bleomycin in the rats. Then dexamethasone or Imatinib were given intraperitoneally respectively. On day 7, 14, 21 after the treatment, 10 mice of each group were sacrificed respectively. The expressions of TGF-β1 and α-SMA in lung tissue were analyzed by immunohistochemistry. The mRNA expression of TGF-β1 was measured by RT-PCR. Results The expressions of TGF-β1 and α-SMA in lung tissue at each time point were significantly increased in the model group compared with the control group. And the expressions were obviously decreased in the dexamethasone group and the Imatinib group compared with the model group, with no significant differences between the two treatment groups. The expression of TGF-β1 was positively correlated with the α-SMA expression ( r= 0. 251, P lt;0. 05) . Conclusion The inhibitory effect of Imatinib on pulmonary fibrosis may be related to the inhibition of TGF-β1 and α-SMA expressions.
Objective To establish a mouse model of acute lung injury ( ALI) and pulmonary fibrosis by low dose lipopolysaccharide ( LPS) intermittent intraperitoneal injection, and to explore the pathogenesis of ALI and pulmonary fibrosis induced by endotoxin. Methods Forty C57BL/6 mice were randomly divided into a control group, a 3-days LPS group, a 2-weeks LPS group, and a 4-weeks LPS group,with 10 mice in each group. LPS was injected intraperitoneally at dose of 5 mg/ kg for three consecutive daysin the three LPS groups. Equivalent normal saline was injected by the same way in the control group. The mice lung tissues were obtained respectively 3 days ( the control group and 3-days LPS group) , 2 weeks ( the 2-weeks LPS group) , and 4 weeks ( the 4-weeks LPS group) after LPS or saline stimulation. HE staining,Van-Gieson collagen staining, and Ashcroft fibrosis score assessment were applied to evaluate the development of inflammation and fibrosis in lung tissue at various stages of ALI after LPS-stimulation. The mRNA expression of type Ⅰ procollagen and alpha smooth muscle actin ( α-SMA) were detected by realtime PCR. The deposition of collagen and fibrosis in lung tissue were detected by hydroxyproline assay. The survival condition of each group was also recorded. Results Acute inflammation occurred in mice lung tissue 3 days after intraperitoneal injection of LPS. Collagen deposited in pulmonary interstitium2 weeks afterLPS-stimulation and formed typical pulmonary interstitial fibrosis 4 weeks later accompanying with increase of Ashcroft fibrosis score. Real-time PCR and hydroxyproline assay showed that the expression of collagen and α-SMA increased 3 days after LPS-stimulation and reached the peak 4 weeks later. The animals were all survived up to the endpoint of experiment. Conclusions Accompanying with inflammation, pulmonary fibrosis initiated at early stage of ALI induced by LPS. Intraperitoneal injection of LPS at dose of 5 mg/kg for three consecutive days was able to establish the mouse model of ALI and pulmonary fibrosis with high successrate and low animal mortality, which provide an ideal experimental platform for further investigation.
Objective To observe the effects of different doses of atorvastatin on bleomycin-induced pulmonary fibrosis in rats. Methods Seventy-five healthy female SD rats were randomly divided into five groups ( 15 rats in each group) , ie. a normal group , a model group, a 10 mg/ kg atorvastatin-treated group, a 20 mg/ kg atorvastatin-treated group, and a 40 mg/ kg atorvastatin-treated group. The rats in the model group and treatment groups were instilled with bleomycin in trachea( 5 mg/kg) , and the normal group were instilled with equal volume of normal saline. The treatment groups were gastric gavaged with different doses of atorvastatin each day from2 nd day on after instillation, and the normal group and model group were gavaged with normal saline. Blood samples were obtained from abdominal aorta in five rats in each group and blood gas analysis was performed on1st week, 2nd week and 4th week respectively after BLM instillation. Then the animals were killed and lung tissue samples were harvested for histopathology study. HE and Masson staining were used to determine the extent of alveolus inflammation and pulmonary fibrosis respectively.Histoimmunochemical stain were used to determine the protein levels of transforming growth factor-β1 ( TGF-β1 ) and connective tissue growth factor( CTGF) in pulmonary tissues. Results The arterial partial pressure of oxygenate ( PaO2 ) in the treatment groups were increased gradually with the increasing of therapeutic dose at each time point and decreased with prolongation of time in the same group. The protein levels of TGF-β1 and CTGF in pulmonary tissues were decreased gradually with prolongation of time. TGF-β1 and CTGF expressed obviously less in the treatment groups than those in the model group at each time point .The higher therapeutic doses were, the less the expressions of TGF-β1 and CTGF were. Conclusion Atorvastatin has remarkable inhibitory effects on BLM-induced pulmonary fibrosis of rats in a dose- and timedependentmanner.