| Pulmonary fibrosis is thought that a kind of lung inflammatory diseases. Theepidemiology researches find that the morbidity and mortality of pulmonary fibrosisare increased with aging in recently years. Now the pathogenesis of pulmonary fibrosisis also completely not clarified. It might been focused on epithelium cell damageinduced by oxidative stress, inflammatory cell infiltration, and cytokine networkinteraction imbalance, and reduced to extracellular matrix remodeled and broadcollagenic emerged. However, the current pharmacologic therapy for PF is limited andthere are no effective treatments. Searching for new agents to meet this unmet medicalneed is a priority.It was reported that endoplasmic reticulum stress（ERS） is one of apoptosispathways with the exception of mitochondria apoptosis pathway and death receptorpathway which have been two classics apoptosis pathways. It has been discussed thatmany diseases are in contact with the apoptosis of endoplasmic reticulum stress suchas angiocardiopathy, neurodegenerative diseases, liver diseases and so on. But it havebeen fewly reported whether this kind of apoptosis can be existed in pulmonaryfibrosis. So we observe changes of Glucose regulated protein（GRP78） and C/EBPhomologous protein（CHOP） which are landmark proteins of endoplasmic reticulumstress in lung tissue to discuss the relationship between endoplasmic reticulum stressand pulmonary fibrosis by establishing a model of bleomycin A5induced pulmonary fibrosis in rats and cell model.1. Changes of Endoplasmic retieulum stress relevant protein on bleomycin A5inducedpulmonary fibrosis in ratsAim To study changes of Endoplasmic retieulum stress relevant protein onbleomycin （BLM A5） induced pulmonary fibrosis in rats. Methods Forty-eighthealthy adult male Wistar rats were randomly divided into two groups: control group（Cgroup） and BLM A5group （M group）. M group was treated with a single intratrachealinstillation of BLM at a dose of5mg/kg, C group was treated with saline instead ofBLM A5. Eight rats in each group were randomly killed on7,14and28days afterinstillation. The rat lungs were harvested for HE and Masson trichrome stainrespectively to evaluate the histological change of the lung. Apoptosis was detected byTUNEL method. The expression of GRP78and GADD153/CHOP were examined byRT-PCR and Western blot. Conclusion The rat model of pulmonary fibrosis wassuccessfully established by the examination of HE and Masson trichromestain.Massive cell apoptosis took place in the lung of rats following BLM treatment. Tocontrast with the control group, the expression of GRP78decreased significantly from7to28day after BLM administration while the expression of CHOP increasedmarkedly at the same time. The elevated expression of relevant protein of ERSoccurred after BLM A5treatment, which indicates ERS participate in the process of thepulmonary fibrosis caused by BLM A5in rats.2. Thapsigargin induced apoptosis of MRC-5cells and its mechanismAim To study changes of endoplasmic retieulum stress relevant protein on theapoptosis in MRC-5cells and explore the apoptotic effect of thapsigargin （TG） onMRC-5cells through ER Stress activation. Methods Morphological changes ofapoptotic cells were observed by Annexin V-FITL/PI double fluorescent staining underfluorescent microscope; Apoptosis rate in MRC-5cells was observed using flowcytometric analysis. The expression levels of GRP78, CHOP mRNA were evaluated by RT-PCR. The protein levels of GRP, CHOP, Caspase-3were assessed by Western blot.Conclusions The results showed that MRC-5cells cultured in4mol/L thapsigargin（TG） for24hours exhibited typical morphological changes of apoptotic cells underfluorescent microscope, including shrinkage of cell condensation of chromatin,breakage of nuclear, formation of apoptotic bodies, fluorescence of green and pelletobserved in early apoptotic cells and red fluorescence of chromatin showed in lateapoptotic cells. The percentages of apoptosis induced by1,2,4,8,10μmol· L-1thapsigargin after24h intervention were respectively4.39%,6.66%,13.08%,22.58%,55.34%, and were statistically significant when compared with the control. RT-PCRand Western blot results indicated that when the cells were separately treated with TGfor24h, the relative content of GRP, CHOP mRNA and proteins were significantlyincreased compared to control group. Meanwhile, the expression of Caspase-3proteinin TG was upregulated. The elevated expression of relevant protein of ERS occurredindicated ERS participated in the process of the apoptosis in MRC-5Cells. |
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