| Pulmonary fibrosis is a common outcome of various causes of interstitial lung diseases. As the final outcome of many lung diseases are accompanied with varying degrees of fibrosis, the pulmonary fibrosis is also the main cause of further deterioration of the original disease. Currently, corticosteroids and other immunosuppressive drugs are used for treatment with pulmonary fibrosis. However, it is found to have little effect on patients with idiopathic pulmonary fibrosis. In recent years, the morbidity and mortality of pulmonary fibrosis rise gradually. Therefore, it is to be imminent to find new therapeutic strategies.Antiflammin-1(AF-1), a peptide with the sequence methionine glutamine-methionine-lysine-lysine-valine-leucine-aspartic acid-serine (MQMKKVLDS), is equivalent to the carboxyl-terminal part of the third a-helix of uteroglobin (UG) and has biological characteristics similar to those of its parent protein. AF-1has a wide range of functions. For example, in vitro, AF-1can inhibit leukocyte adhesion and function, suppress macrophage activation, reduce platelet aggregation, prevent mast cell degranulation, block lymphatic vessel contraction and decrease inflammatory mediator production and release. In vivo, AF-1has highly potent anti-inflammatory activity in different animal models, tissues and organs. Compared with UG, AF-1has smaller molecular weight and lower immunogenicity, therefore, it has a number of potential applications. Despite progress in recent decades, our understanding of the biological properties of AF-1remains incomplete.Although AF-1has been explored by numerous groups, there is no research on this molecule in the field of fibrosis. Because AF-1can inhibit the transforming growth factor-beta1(TGF-β1) induced proliferation of NIH3T3and UG plays an important role in inhibiting pulmonary fibrosis, we sought to determine whether AF-1could also protect against pulmonary fibrosis. In our previous experiments, we found that AF-1specifically binds to a saturable membrane receptor, the UG receptor, and activates the mitogen-activated protein kinase (MAPK) signaling pathway. Since AF-1can inhibit the TGF-β1induced proliferation of NIH3T3, we speculated that AF-1may also play a biological function by interacting with the UG receptor on lung epithelial cells. In our present study, we try to observe:①Whether AF-1is able to inhibit TGF-β1induced epithelial-mesenchymal transition (EMT)?②If so, whether the effect of AF-1is mediated by the UG receptor? Chapter1Antiflammin-1prevents bleomycin induced acute lung injuryMethod:Adult male C57BL/6mice were obtained from the laboratory animal unit of Central South University. After being anesthetized, mice were intratracheally injected with50μL of bleomycin (5mg/kg) on Day0. To determine whether continuous treatment with AF-1(2mg/kg/day) could inhibit the inflammatory response in the bleomycin mouse model, AF-1was injected from Day0to Day6, and mice were sacrificed on Day7. The wet/dry (W/D) method was used to measure pulmonary edema. HE staining was used to observe pathology changes in the lung tissue. The protein content in bronchoalveolar lavage fluid (BALF) was measured by lowry assay. The myeloperoxidase (MPO) activity in lung homogenate and lactic dehydrogenase (LDH) activity in BALF were detected with the corresponding kits. ELISA was used to quantify the level of interleukin-1β(IL-1β), tumor necrosis factor-alpha (TNF-α) and TGF-β1. Results:(1) The degree of acute lung injury was assessed by W/D ratio and pulmonary index. The result showed that compared with the bleomycin-treated mice without AF-1,the mice treated with AF-1showed a significant reduction in the lung W/D ratio and pulmonary index.(2) The degree of acute lung injury was assessed by pathological analysis. As shown in figure1-2, a large increase in inflammatory cells was observed in the bleomycin-treated lungs on Day7. In contrast, a decreased number of infiltrated inflammatory cells and reduced destruction of lung architecture in the AF-1treated group was observed(3) The degree of acute lung injury was assessed by MPO activity in lung homogenate. The result showed that administration of AF-1inhibited the MPO activity in bleomycin treated lung.(4) The degree of acute lung injury was assessed by LDH activity and protein content in BALF. The result showed that compared with control group, LDH activity and protein content in bleomycin induced lung were increased. After AF-1(2mg/kg) treatment, LDH activity and protein content were significantly reduced. (5) The degree of acute lung injury was assessed by cells count and cell classification in BALF. Administration of bleomycin elevated the number of inflammatory cells, including lymphocytes and neutrophils. Treatment with AF-1reduced the number of these inflammatory cells in BALF on Day7.(6) The degree of acute lung injury was assessed by the levels of TNF-α and IL-1β and TGF-β1in lung homogenate. The results showed that compared with bleomycin treated mice, AF-1resulted in a significant reduction in the level of these cytokines Conclusion:Bleomycin (5mg/kg) induced acute lung injury, and antiflammin-1could prevent bleomycin induced acute lung injury Chapter2Antiflammin-1attunates bleomycin induced pulmonary fibrosisMethod:(1) After being anesthetized, mice were intratracheally injected with50μL of bleomycin (5mg/kg) on Day0. For time course experiments, lung samples were collected on Days0,3,7,10,14,21and28for further analysis. ELISA was used to quantify the level of IL-1β, IL-6, TNF-α and TGF-(31. The collagen content in the lung homogenates was examined by a hydroxyproline (HYP) assay.(2) For the preventive antifibrotic treatment, AF-1(2mg/kg/day) was administered from Day0to Day27, and lung samples were collected on Day28. For the therapeutic antifibrotic treatment, AF-1(2or10mg/kg/day) was administered from Day11to Day27, and mice were sacrificed on Day28.The lung samples were fixed in4%buffered paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin-eosin and Masson’s trichrome. The Ashcroft score was used for the quantitative histologic analysis. RT-PCR was used to quantify the expression of EMT marker protein. We also examined the effect of AF-1on the survival rate each day and the weight loss on Day28in bleomycin treated mice. Results:(1) In time course experiments, the protein levels of TNF-α, IL-1β and IL-6exhibited similar changes over the entire experimental period. The expression of pro-inflammatory cytokines increased rapidly during the first3days and remained high until Day10. However, the expression of pro-inflammatory cytokines significantly decreased after Day10and continued to decrease until the end of the experiment.(2) In time course experiments, the expression of TGF-β1increased rapidly during the first3days and remained high until Day14. However, the expression of TGF-β1significantly decreased after Day14and continued to decrease until the end of the experiment.(3) In time course experiments, the expression profile of collagen was estimated by HYP assay at the indicated time points. The result showed that there was no significant change in collagen deposition in the first10days. After Day10, the collagen deposition increased rapidly and peaked on Day21(4) In preventative antifibrotic treatment, the effect of AF-1on bleomycin treated mice were assessed by the the survival rate and weight loss. The result showed that the bleomycin treated mice has a decrease in the survival rate and a significant increase in weight loss. The preventative treatment with AF-1(2mg/kg) improved the survival rate and weight loss of mice injected with bleomycin.(5) In preventative antifibrotic treatment, the degree of lung fibrosis was assessed by H&E staining and Masson staining. As shown in Figure2-5, administration of AF-1alone did not generate any changes in lung morphology. However, when AF-1was administered in bleomycin treated mice, a reduction of fibrotic lesions in the lung was observed.(6) In preventative antifibrotic treatment, we measured hydroxyproline content and Ashcroft score of the tissue to estimate degree of fibrosis in the lung. The results showed that HYP content and Ashcroft score were significantly increased in bleomycin treated mice. After AF-1treatment, HYP content and Ashcroft score were markedly decreased.(7) In therapeutic antifibrotic treatment, the degree of lung fibrosis was assessed by H&E staining and Masson staining. The Figure2-7showed that the severe fibrosis was found using light microscopy in all bleomycin treated mice. In contrast, AF-1(10mg/kg but not2mg/kg) alleviated the bleomycin induced pulmonary fibrosis(8) In therapeutic antifibrotic treatment, we measured hydroxyproline content and Ashcroft score of the tissue to estimate degree of fibrosis in the lung. The quantitative histologic analysis demonstrated that the Ashcroft score and HYP content in mice treated with bleomycin and AF-1(10mg/kg but not2mg/kg) was significantly lower than that treated with bleomycin alone.(9) In therapeutic antifibrotic treatment, the effect of AF-1on bleomycin treated mice were assessed by the expression of EMT marker protein. We found that the therapeutic treatment with AF-1(10mg/kg) significantly reduced EMT marker in bleomycin treated lung. Conclusion:Bleomycin induced pulmonary fibrosis. Both the preventative treatment and therapeutic treatment attunates bleomycin induced pulmonary fibrosis, and the effective dose of therapeutic treatment is higher than that of preventative treatment. Chapter3Antiflammin-1inhibits TGF-β1induced EMT in A549cellsMethod:A549cells were seeded in culture dish and grown for24h. Before the experiments, the medium was changed to the incubation medium containing TGF-(31(5ng/mL) in the absence or presence of AF-1(100μM) and with or without anti-UG receptor antibody (50μg/mL). Then, cells were cultured for an additional48h. After that, cells were lysed in RIPA buffer and the expression of E-cadherin and a-smooth muscle actin (a-SMA) were analyzed by western blot. Results:(1) The degree of EMT was assessed by cellular morphology. After culture with TGF-β1for48h, untreated A549cells show a pebble-like shape and cell-cell adhesion is clearly observed. TGF-β1treated cells show a decrease in cell-cell contacts and adopt a more elongated morphological shape, however, treatment with AF-1(100μM) could significantly inhibit the TGF-β1induced morphological changes(2) AF-1(100μM) attenuated TGF-β1induced EMT in A549cells. The expression of E-cadherin and a-SMA were analyzed by western blot. The result showed that AF-1inhibited the effect of TGF-β1on the expression of a-SMA and E-cadherin.(3) AF-1(100μM) suppressed the TGF-β1induced EMT in A549cells by interacting with UG receptor. The expression of E-cadherin and a-SMA were analyzed by western blot. The result showed that AF-1attenuated TGF-β1-induced expression of a-SMA and restored cell border E-cadherin. However, The effect of AF-1(100μM) was reversed by pretreatment with anti-UG receptor antibody (50μg/mL).(4) AF-1induced c-Jun N-terminal kinase (JNK), p38MAPK (p38), and extracellular regulated protein kinases (ERK) activation, which was mediated by UG receptor. The results showed that the phosphorylation of JNK, p38and ERK were induced by AF-1. After pretreatment with anti-UG receptor antibody (50μg/mL), the phosphorylation of JNK, p38and ERK were reduced.(5) The effect of MAPK pathway on TGF-β1induced EMT in A549 cells was assessed by the expression of E-cadherin and a-SMA. The results showed that the specific inhibitors of p38attenuated TGF-β1induced mesenchymal markers in A549cells and JNK inhibitors had partial inhibitory effects on E-cadherin expression, whereas specific ERK inhibitor had no effect on TGF-β1induced EMT in A549. This results suggested that the ERK pathway should be not involved in TGF-β1induced A549cells EMT(6) AF-1suppressed the TGF-β1induced EMT in A549cells through ERK pathway. The result showed that AF-1inhibited the effect of TGF-β1on A549cells. However, The effect of AF-1(100μM) was reversed by pretreatment with ERK inhibitor (10μM). Conclusion:(1) AF-1suppressed the TGF-β1induced EMT in A549cells by interacting with UG receptor.(2) AF-1induced JNK, p38, and ERK activation, which was mediated by UG receptor, and AF-1suppressed the TGF-β1induced EMT in A549cells through ERK pathway Conclusions(1) AF-1could prevent bleomycin induced acute lung injury(2) Both the preventative treatment and therapeutic treatment attunates bleomycin induced pulmonary fibrosis, which suggested that AF-1has a antifibrotic activity in vivo(3) AF-1suppressed the TGF-β1induced EMT in A549cells by interacting with UG receptor.(4) AF-1induced JNK, p38, and ERK activation, which was mediated by UG receptor, and AF-1suppressed the TGF-β1induced EMT in A549cells through ERK pathway... |
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