| Backgrounds:Idiopathic pulmonary fibrosis(IPF) is the most common and severe form of idiopathic interstitial pneumonia and is characterized by an over abundant accumulation of extracellular matrix(ECM) collagen deposition in the distal lung interstitial tissue in association with an injured overlying epithelium and activated myofibroblasts,gradually irreversible losing the ability to exchange with the oxygen. The predominant symptom is shortness of breath, initially only during activity but in advanced disease also at rest. IPF incidence was higher among older male adults, on average 60–70 years old,and increased with age, usually with a history of smoking. The overall range of incidence of IPF varies from 0.22 to 93.7 per 100,000 per year worldwide, but excluding old studies and different populations(Asia and South America) is likely to be 2.8–9.3 per100,000 per year in Europe and North America.The prevalence of IPF is estimated to be between 200 and 500 cases per 100,000 people,that is equal to 2-5 million people getting it every year. The prognosis of IPF is poor, with an estimated 5-year survival of approximately 20% and median survival time of 3–5 years from the time of diagnosis.The therapeutic approach for IPF integrates both nonpharmacologic and pharmacologic strategies, however, they don't work well and effectively. Although many of the cellular and molecular mechanisms underlying the pathophysiology of lung fibrosis have emerged from numerous studies over the past several decades, the precise pathways involved, their regulation, and the role of crosstalk between cells are not fully understood.The current model regarding the pathogenesis of IPF implies aberrant fibrosis as a consequence of recurrent injury to alveolar epithelial cells in a susceptible host,eg, virus,tobacco, microaspireation, mechanical stresss as well as genetic and epigenetic hallmarks of aging. In an attempt to restore functional integrity, injured Type II alveolar epithelial cells aberrantly release pleonastic cytokines and growth factors, matrix metalloproteinases(MMPs), and procoagulant mediators, which promote the recruitment and activation of apoptosis-resistant fibroblasts, the key players in fibrotic tissue remodeling.Caveolin-1(cav-1), a principal component of caveolae, has been implicated in the regulation of numerous signaling pathways and diverse biological processes, including cellular metabolism and fibrosis pathogenesis. Indeed, the initial phenotype described inmice with targeted deletion of the Cav1 gene included both systemic metabolic derangements and the development of tissue fibrosis. The co-existence of these pathological phenotypes in Cav1 deficient mice is currently thought to be explained by unrelated mechanisms, which result primarily from loss of Cav1 expression in a wide range of cell types including adipocytes, fibroblasts and epithelium. However, whether metabolic alterations contribute to the development of fibrosis in the absence of Cav1 mice is unclear.Methods:First, we extracted the lung tissue and brochoalveolar lavage fluid from the B6/129 and Cav1 deficiency mice(male, 8-10 weeks old) which were purchased from the Jackson Laboratory(Bar Harbor, ME) and housed in a pathogen-free animal facility. The harvested lung and BALF was kept at-80 ? for collagen determination and TGF-?detection with the Sircol collagen assay and Enzyme-linked immunosorbent assay to prove the concept that Cav1 was involved in the development of lung fibrosis.Meanwhile, the ER stress marker, the activity of Caspase-3 and the expression level of cleaved Caspase-3 in the lung, ATII and A549 cells were quantified and the statistical analysis was performed. Furthermore, the method of terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL) was used to assertain the role of ER stress in the apoptosis of the epithelial cells in the loss of Cav1.Secondly, we used the Electron Microscope to observe the shape and number of the mitochondrial, and Mitotracker to trace the allocation of them in the cell.ROS/Superoxide measurement and mitochondrial membrane potential in A549 cells was quantified by ROS/Superoxide and MMP detection kit. At last we performed western blot to analyse the expression of PGC1? in the tissure and cells to make sure the role of Cav1 in the function of mitochondrial for the energy metabolism. Meanwhile, we tried to use Colorimetry kits to test the extracellular ATP and the expression of the phosphorylation of AMPK in the lung and A549 cells to verify the Cav1 deficiency induced fibrosis was associated to the metabolism change.Thirdly, to clarify whether the influence of ATP deletion would exacerbate Cav1-/-induced lung fibrosis, we admistrated 12.5mg of 2-deoxyglucose by daily intraperitoneal(ip) injection, an arobic glycolysis inhibitor. The lung tissue was stained by masson trichrom staining, AMPK and all ER stress marker were measured by western blot andthe production of collagen and TGF-? were assayed by ELISA. All the tools we were taking here is to see the effect of 2-DG on the Cav1-/- mice, and to understand well the hypothesis that metabolic change is related closely to the phathophysilogy process in Cav1-/- induced pulmonary fibrosis. Finally, we gave PQQ, an agonist of PGC1? to improve the generation of mitochondrial and try to supply part of the energy, to assure the important role of energy metabolism in the Cav1-/- induced lung fibrosis. We gave AICAR to the A549, an agonist of AMPK as well to see the recovery of mitochondrial either from the number or the function and the response capability of the ER stress.Through the above experiment, we could get an elementary idea that Caveolin-1deficiency might cause pulmonary fibrosis via altering energy metabolism and inducing endoplasmic reticulum stress in the alveolar epithelium.Results:1. The role of Cav1 deficiency and ER stress in the pulmonary fibrosis.First, we confirmed the fibrosis model in the Cav1 deficient mice by detecting the content of the collagen and TGF-? production with Sircol collagen assay, ELISA, and collagen deposition with massason trichrome straining in the 8 weeks. We also detected an ER stress marker and Cleaved-caspase 3 in the lung, AEC and A549 cells, and the apoptosis of AEC with TUNEL staining. We found that ER stress was existed in the tissue and cells without Cav expression.2. The role of Cav1 in the mitochaodrial and ER stress.Then we observed the expression of phosphorylation AMPK and its down target of ACC, which was 3 times and twice higher in the loss of Cav1 than in the normal group,perspectively. Moreover, ATP production analysis showed the the generation of ATP was half that in its wildtype counterpart. We could make a conclusion based on what we found that energy metabolism could be involved in the pathology of Cav-/- induced lung fibrosis. Apart from that, to ascertain the similar role of Cav1 in the AEC, we first proved the expression of Cav1 in the AEC and cell line A549 cells and then we down-regulated Cav1 in the A549 cells to see the phosphorylation of AMPK/ACC and the ATP production. The result showed the similar consequence with the lung tissue. Accouding to the result we got, we tried to see the change in the mitochondrial. We found that the expression of PGC1? decreased significantly in the lung tissue and A549 cells, which was half of that compaired to the wildtypes. Similar date was showed in the PCR. Exceptto the low level of PGC1? in the A549 cells, we observed an increased MMP. The Mitotracker and Mito SOX showed the high ROS production in the lack of Cav1. And no change was detected in the glycolysis pathway.3. Interruption of engey metabolism in the cav1 deficient induced fibrosisBy administrating the glycolysis inhibitor 2-DG for 7 days, we sacrificed them and extracted the lung tissue and BALF. We found the phosphorylated AMPK and ACC levels were incredibly higher then the uninjecting Cav1-/- mice. Meanwhile, the UPR pathway was activated with the outstanding IRE1 response and the activity of cleaved-caspase 3 was initiaved. The content of the collagen and TGF-? production was improved twices and more accumulation of the collagen deposition was found compared with the negative group. The same result could be seen in the A549 cells.After given the agonist of PGC1?—PQQ, the ability of mitochondria generation was repaired and the evergy supplement was recovered. We harvested the lung tissue and BALF after injection in the abdomen for 1 week and we found that the ATP production was higher than the PBS group, plus the marker of ER stress was lower, the lung fibrosis level was alleviated after PQQ treatment, which indicated that PQQ was helpful to relieve the progress of fibrosis. Apart from that, we also gave the AICAR to A549 cell,the agonist of AMPK. We ovserved the similar response, the marker of ER stress was lower and the cleaved-caspase 3 level was descended either. Meanwhile AICAR increased the ATP synthase and PGC1? expression, decreased the ROS production according to the Mito SOX staining, which might hint the protective role of AICAR in the improvement of mitochondrial.Conclusion:1. Cav1-/- mice could be good model of lung fibrosis after born 8 weeks. Moreover,ER stress initiated the apoptosis of alveolar epithelial cells in the absence of Cav1.2. The donw regulation of ATP in these mice showed energy metabolism could be involved in the pathology of this condition, and knockout of Cav1 resulted in a smaller number of mitochondrial and the malfunction. However, no change was observed in the glycolysis pathway.3. 2-DG could inhibit the glycolysis and stop the energy supplyment pathway,causing the umbanlance of the metabolism and aggregating the ER stress, which further exacerbate the progress of already-existed lung fibrosis. The treatment of PQQ andAICAR could improve the function of mitochondrial, recover the evergy supply and alleviate the ER stress response, relieveing the pulmonary fibrosis of Cav1-/- mice. |
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