| Pulmonary fibrosis is a chronic,progressive,irreversible disease and final stage in the chronic interstitial lung disease.The average survival time is less than 5 years.The cost of pulmonary fibrosis is a huge burden to individuals,families,and society.Therefore,to delay the progression of pulmonary ibrosis and to reduce the mortality rate is a great challenge and great demand for the prevention and treatment of lung disease in China.Although the cause of pulmonary fibrosis is complicated,the common pathological features and final pathway of pulmonary interstitial fibrosis mainly manifested as infiltration of inflammatory cells,the accumulation of extracellular matrix,and eventually the extensive fibrosis in the lung.At present there is no effective methods to prevent its progress.Therefore,it is of great significance to reduce the mortality by analysising the mechanism of pulmonary fibrosis and seeking new effective intervention strategies.However,the mechanism of pulmonary fibrosis is yet unclear.Myofibroblasts present in the lung interstitium is an important pathological change of pulmonary fibrosis.At present,the source of myofibroblasts is contentious.The result showed EMT is the mainly viewpoint.An important effect of lung epithelium after activation is transformed into myofibroblasts,promoted the synthesis of extracellular matrix,activated various genes involved in the innate immune,inflammation,promote inflammation,oxidative stress injury and futherly aggravated pulmonary fibrosis.It is suggested that lung epithelium transition is an important reason of myofibroblasts accumulation.Futherly to detect the mechanism of lung epithelium transition and the important molecular biological effect is an important strategy for prevention intervention targets.The above suggests that pulmonary fibrosis is a complex and refractory disease.At present,the traditional hormone and single receptor blocker of signal pathway receptor(for example gefitinib tablets)may get some effect,but these can not prevent the progression of pulmonary fibrosis.In recent years,Chinese herbal formula and its monomer active components in the treatment of pulmonary fibrosis gradually show an important role.Our research group in the preliminary experiment has confirmed that blood-activating and stasis-dissolving prescriptioncan can restrain the pulmonary fibrosis.Glycyrrhizic acid is one of the most important active components in the blood-activating and stasis-dissolving prescriptioncan and the number of studies have showed that the glycyrrhizic acid has a significant improvement effect in organ fibrosis(liver).But at present,it is unclear whether the treatment of pulmonary interstitial fibrosis can be effectly ameliorated by glycyrrhizic acid.So we put forward the following scientific questions: A variety of inflammation,excessive activation of multiple signaling pathways is an important basis for pulmonary fibrosis and EMT is also an important way to cause pulmonary fibrosis,what is the regulation of these inflammatory cytokines and signaling pathway activity? Whether glycyrrhizic acid is able to find the common targets to control their activity?The great progress of proteomics has opened up a new way for us to answer the questions mentioned above.Core fucosylation(CF)is a crucial glycosylation modification that forms the core fucose by 1,6 alpha fucosyltransferase(FUT8)catalyzing fucosylated GDP-Fuc to transfer the Glc NAc core structure of glycoprotein N-linked oligosaccharides.Our research group has showed that the key proteins in the organ fibrosis were glycoproteins,so we thought: whether glycyrrhizic acid regulated these inflammatory factors and the signal pathway by CF?In order to solve the above scientific problems,this study was to establish the rat model of pulmonary fibrosis by BLM induced to clear the effect of glycyrrhizic acid on pulmonary interstitial fibrosis in rats;On the basis of the model of pulmonary interstitial fibrosis in rats,observed the effects of glycyrrhizic acid on the process of EMT;On the above basis,through the observation of the effect of glycyrrhizic acid on the CF,deeply analysis the specific mechanism of inhibiting EMT process by glycyrrhizic acid.This is the first time to find the important effect of glycyrrhizic acid on EMT process in pulmonary fibrosis;secondly from the perspective of the new glycobiology mechanism,deeply analysis of the mechanism of inhibiting EMT process by glycyrrhizic acid.This study will fill the blank of glycobiology field in pulmonary fibrosis and provides the new ideas for the clinical development of new drugs.Part ? Glycyrrhizic alleviate BLM induced pulmonary fibrosisObjective: To clarify the protective effect of glycyrrhizic acid on pulmonary interstitial fibrosis by investigating the effect of glycyrrhizic acid on the pathological changes of pulmonary fibrosis rat model induced by BLM.Method: Using BLM induced pulmonary fibrosis rat model and successfully establishing the lung tissues pathology,obesrved the effects of different doses of glycyrrhizic acid on lung tissues pathology and the protective effect of glycyrrhizic acid on pulmonary fibrosis.First of all,measured the wet / dry weight ratio of lung tissues in rats;HE,Masson staining was used to observe the effect of glycyrrhizic acid on the lung tissues pathology;using the method of immunohistochemistry and Western blotting to detect the changes of collagen I and III;Secondly,using ELISA method to detect the content of Hyp in rats lung tissue,clear the protective effect of glycyrrhizic acid on pulmonary fibrosis.Result: Compared with the normal group,BLM induced rat pulmonary fibrosis present significantly lower expression of lung wet / dry weight ratio;Masson,HE staining showed a significant aggravation in lung tissues pathology;The expression of collagen I and III was significantly increased by immunohistochemistry and Western blotting;The content of Hyp in ELISA was obviously increased.Through the glycyrrhizic acid treatment,the expression of collagen type I and III and Hyp were significantly lower than fibrosis group,the the expression of collagen type I and III and Hyp were significantly reduced with increasing dose of glycyrrhizic acid and high dose glycyrrhizic acid had significant inhibitory effect.Conclusion: glycyrrhizic acid plays a key role in the inhibition and protection of pulmonary fibrosis,which is dependent on dose.Part ? Glycyrrhizic have effects on EMTObjective: By investigating the effect of glycyrrhizic acid on the lung epithelial cells transition,clear the EMT of glycyrrhizic acid inhibition.Methods: Using the BLM induced rat pulmonary fibrosis model,fixed position of lung epithelial cells by using a unique marker E-cadherin and observed glycyrrhizic acid with different doses effect of lung epithelial cells transition into myofibroblast,clear glycyrrhizic acid inhibition of EMT in pulmonary fibrosis.Co-immunofluorescence staining method for detect the change of different dose of glycyrrhizic acid on the expression of E-cadherin,?-SMA in lung tissues by using co-immunofluorescence staining method and the quantitative analysis of the protein in lung tissues by western blotting method,clear the effect of different dose of glycyrrhizic acid on lung epithelial cell transition.The expression of p-Smad2/3??-catenin was detected in rats lung tissues by using immunofluorescence and quantitative analysis of the above protein was detected by Western blotting method,make sure the effects of different dose of glycyrrhizic acid effect on the key proteins in the pulmonary fibrosis.Results: The results of immunofluorescence and Western blotting showed that the expression of E-cadherin in normal lung tissue was equal and ?-SMA was low expression in the normal tissue.Compared with the normal group,the expression of ?-SMA was significantly increased and the expression of E-cadherin was significantly decreased in the BLM-induced rat pulmonary fibrosis model;After the treatment of glycyrrhizic acid,Immunofluorescence and Western blotting showed that the two proteins present a reverse change and the EMT phenomenon was significantly inhibited.The results of immunofluorescence and western blotting showed that the expression of p-Smad2/3,?-catenin was low in normal lung tissue.Compared with normal lung tissue,the expression of p-Smad2/3,?-catenin protein was significantly increased in BLM-induced rat pulmonary fibrosis model;After the glycyrrhizic acid treatment,the expression of the two proteins was inhibited by using immunofluorescence and western blotting.The above indexes were all shown to depend on the dose of glycyrrhizic acid.Conclusion: In BLM induced rat pulmonary fibrosis,the key proteins play an important role in EMT,glycyrrhizic acid can inhibit the EMT and the two protein activation.Part ? Glycyrrhizic regulate the mechanism of EMT by CF modifiedObjective: To investigate the core fucosylation modification effect on EMT key signaling pathways,deeply analyse that glycyrrhizic acid inhibit EMT by regulating FUT8.Methods: Using the BLM induced rat pulmonary fibrosis model,observed the core fucosylated modification levels and core fucosylations modification have effect on the TGF-?/Smad2/3,Wnt/?-catenin signaling pathway,analysis glycyrrhizic acid changes in the level of core fucosylation.First,the expression of CF and the change of CF in rat lung tissues were observed by immunofluorescence double staining method;Sencondly,detected the core fucosylation level of TGF-?R,WNT and glycyrrhizic acid have effect on the change of the above receptor protein and CF levels by using immunofluorescence and western blotting.Results: In normal lung tissue,there was an equal level of CF modification;Compared with the normal group,the level of CF modification was significantly increased in BLM induced rat pulmonary fibrosis model;TGF-?R,Wnt and their core fucosylation level in rats lung tissues increased significantly(P<0.001);Glycyrrhizic acid can significantly inhibit CF level of TGF-?R,Wnt receptor protein in pulmonary fibrosis,but did not affect the receptor protein theirself expression(P<0.01).Conclusion: Key receptor protein TGF-?R,WNT post-translational modification of CF plays an important role in the process of EMT,glycyrrhizic acid can block EMT process and alleviate the pulmonary fibrosis by inhibiting the TGF-?R,WNT core fucosylation modification in BLM-induced rats pulmonary fibrosis. |
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