| PART1:Effects of Fasudil on Bleomycin-Induced Pulmonary Fibrosis in MiceObjectiveTo determine the beneficial effects and mechanisms of fasudil, a selective ROCK inhibitor, on bleomycin-induced pulmonary fibrosis in mice.Materials and Methods1. The BPF model was induced by a single dosage of2.5mg/kg bleomycin intratracheal injection in mice.10mg/kg/d,30mg/kg/d, or100mg/kg/d fasudil intraperitoneal injection was given to the mice.2. The fibrosis degree was determined pathologically by using the Ashcroft scoring method and biochemically by hydroxyproline assay in lung tissue.3. To collect bronchoalveolar lavage fluid, the differential inflammatory cells were counted in BALF and the concentrations of PAI-1in BALF were determined by ELISA.4. The expression of TGF-β1, CTGF, a-SMA and PAI-1mRNA in lung tissue was detected by RT-PCR.5. The expression of TGF-β1, CTGF and a-SMA protein and the level of MYPT1phosphorylation in lung tissue was detected by Western blot.Results1. Compare to the mice administrated by bleomycin, the Aschcroft score and hydroxyproline content were significantly decreased in the mice administered10and100mg/kg fasudil.2. Compare to the mice administrated by bleomycin, treatment with10and100mg/kg fasudil significantly reduced the total number of cells and macrophages from day7to day21, and neutrophils by day7.3. Compared to the bleomycin alone group, treatment with10and100mg/kg fasudil significantly decreased the levels of TGF-01, CTGF,α-SMA, and PAI-1mRNA and protein.4. Treatment with100mg/kg fasudil significantly decreased the level of MYPT1phosphorylation in the lungs of bleomycin-treated mice. ConclusionAdministration of fasudil can attenuate bleomycin-induced pulmonary fibrosis in mice. The beneficial effect of fasudil was possibly related to the inhibition of inflammatory cell (predominantly macrophages and neutrophils) recruitment and decrease in the production of TGF-β1,CTGF,α-SMA, and PAI-1. These findings suggest that fasudil may be a potential therapeutic candidate for the treatment of pulmonary fibrosis. PART2:Effects of Fasudil on the Biological Behaviors in NIH3T3Mouse Fibroblast CelllineObjectiveTo determine the effects and mechanisms of fasudil on the biological behaviors in NIH3T3mouse fibroblast cellline.Materials and Methods1. NIH3T3mouse fibroblast cellline was cultured in vitro.1μg/ml or10μg/ml fasudil was given to the cell.2. The proliferation activity in NIH3T3cells were detected by MTT assay and flat colony forming experiment.3. The migration activity in NIH3T3cells were detected by scratch test and transwell chamber experiment.4. The expression of CyclinDl, MMP2and TIMP1mRNA in NIH3T3cells was detected by RT-PCR.5. The expression of CyclinDl, MMP2and TIMP1protein and the level of MYPT1phosphorylation in NIH3T3cells was detected by Western blot.Results1. MTT results showed that fasudil can decrease the OD value of NIH3T3cells in a time-concentration-dependent manner. Plate colony forming experiment results showed that fasudil can reduce the number of NIH3T3cell clones in a dose-dependent manner.2. Scratch test results showed that fasudil can shorten the migration distance of NIH3T3cells in a dose-dependent manner. Transwell chamber experiment results showed that fasudil can reduce the number of NIH3T3cells which migrated through the basement membrane in a dose-dependent manner.3.10μg/ml fasudil significantly decreased the levels of CyclinDl mRNA and protein, while increased the levels of TIMP1mRNA and protein. Both1μg/ml and10μg/ml fasudil can significantly decrease the levels of MMP2mRNA and protein in NIH3T3cells.4. Fasudil can decrease the level of MYPT1phosphorylation in a dose-dependent manner in NIH3T3cells.ConclusionAdministration of fasudil can reduce the ability of proliferation and migration in a dose-dependent manner in NIH3T3cells. The effect of fasudil was possibly related to increase the production of TIMP1and decrease the production of CyclinDl and MMP2. PART3:Adverse events of pirfenidone for the treatment of pulmonary fibrosis:a meta-analysis of randomized controlled trialsObjectivePirfenidone (PFD) is a novel antifibrotic agent approved for patients with pulmonary fibrosis. However, there are concerns regarding toxicity of the drug. In this meta-analysis, we analyzed the adverse events (AEs) of PFD for the treatment of pulmonary fibrosis.Materials and MethodsWe performed a systematic search of PubMed, Embase, ClinicalTrials.gov, and Cochrane Central Register of Controlled Trials for trials published between January1999and October2011. Data extracted from literature were analyzed with Review manager5.0.24.ResultsThe results of six randomized controlled trials (1073participants) revealed that the number of individuals who discontinued PFD therapy was significantly higher than patients receiving placebo. The PFD group had a significantly higher rate of gastrointestinal (nausea, dyspepsia, diarrhea, and anorexia), neurological (dizziness and fatigue), and dermatological (photosensitivity and rash) AEs compared to the placebo group.ConclusionPFD used for the treatment of pulmonary fibrosis is not so safe or well-tolerated. Notably, gastrointestinal, neurological and dermatological adverse effects were more common in patients receiving PFD therapy, and therefore appropriate precaution is needed. |
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