Mechanism Of Autophagy And Ang(1-7) In OVA Induced Airway Remodeling In Asthma

Bronchial asthma is a common chronic inflammatory disease in the upper respiratory tract.There are more than 300 million asthmatic patients in the world,and nearly 30 million in China.And the happen of asthma shows a continuous upward trend.in developing countries.The main pathological changes of bronchial asthma are airway inflammation and airway hyperresponsiveness.The repeated struggle and balance between long-term chronic airway inflammation and self-healing of the body will inevitably lead to airway wall thickening,the change of airway tissue structure and eventually irreversible airway remodeling.Therefore,it is a new hot spot to clarify the molecular mechanism of airway remodeling and explore new molecular targets that can reverse airway remodeling in the field of asthma research.And it is very important for the treatment of patients with severe asthma.Autophagy is a important physiological process.It can digest and decompose the dead cytoplasmic contents and organelles through catabolism pathway.And it acts an important role in cell growth and differentiation.At present,more and more studies have confirmed that autophagy is closely related to the occurrence and development of many diseases,such as tumor and metabolic diseases.However,its role in airway remodeling of asthma is rarely reported.Therefore,it has important theoretical significance to study the role and molecular mechanism of autophagy in asthma.Renin-angiotensin system(RAS)is one of the most important humoral regulation systems in the body,and it is a large family.It is composed of a variety of angiotensinogen by hydrolysis and enzymolysis.It has been proved that there are independent RAS systems in cardiovascular and respiratory system.And they play the important role in local pathological and physiological processes.However,it has not been reported whether Ang(1-7)has a reversal effect on OVA-induced airway remodeling.Based on this,we believe that exploring the role of Ang(1-7)in airway remodeling can provide a new molecular therapeutic target for the treatment of severe asthma patients with airway remodeling.Therefore,in this study,we constructed a classic OVA-induced asthma airway remodeling model.And we studied the effect and mechanism of autophagy and Ang(1-7)for airway inflammatory,fibrosis and airway remodeling in this model.Moreover,we hope our research is helpful to the treatment of severe asthma patients and to provide a new molecular target and new ideas of intervention.Part ? The role and mechanism of autophagy in airway remodeling induced by OVA in asthma ratsObjectiveTo establish a classical airway remodeling mouse model induced by OVA,to study the relationship between autophagy and airway remodeling after treatment with corresponding specific inhibitors,and to further explore the molecular mechanism of STAT3 and PKR signal molecules in this processMethodThe expression of a-SMA and CollI was detected by WB and IHC in the airway remodeling mouse model induced by OVA in order to confirm that the preparation of airway remodeling model was successful.And we explored the occurrence of autophagy in this model through detecting the expression of Beclinl,LC3 and p62 by WB,IHC and ELISA test.Furthermore,the effects of STAT3 and PKR signaling molecules were studied after treatment with specific inhibitors WP1066 and PKRI in this model.Result1.The mice showed obvious symptoms of shortness of breath and dyspnea after ova intervention.He,Masson and Sirius red staining showed there were obvious pathological changes,including smooth muscle layer thickening,collagen fiber deposition,airway wall thickening and inflammatory response in the OVA-induced model mice.WB showed the expression of ?-SMA and CollI protein also increased significantly.All these data indicated that the model of airway remodeling was successfully prepared in asthma mice.2.Western blot,IHC and ELISA were used to detect the expression of Beclinl,LC3 and p62.The results showed that the expression of Beclin1 and LC3 decreased significantly in the model group,while the expression of p62 increased significantly.These data indicated that autophagy was inhibited in the airway tissue in asthma mice with airway remodeling.3.Western blot,IHC and ELISA tests were used to detect the expression of Beclinl,LC3 and p62 after wp1066 inhibitor intervention.The results showed that the expression of Beclinl and LC3 was significantly increased,while the expression of p62 was significantly decreased in the model group.At the same time,HE,Masson and Sirius red staining also showed that WP1066 inhibitor can significantly reduce airway remodeling effect.These data showed that WP1066 can reduce or reverse the airway remodeling effect induced by OVA through inhibiting STAT3 signaling molecules and activating autophagy.4.Western blot,IHC and ELISA were used to detect the expression of Beclin1,LC3 and p62 after PKRI intervention.The changes of Beclinl,LC3 and p62 expression are similar with the model group.At the same time,HE,Masson and Sirius red staining also showed that the change of airway remodeling in mice was similar to that in model group.These data showed that PKRI can inhibit autophagy and induce airway remodeling by inhibiting PKR expression.5.Western blot was used to detect the activity of p-STAT3 and p-PKR.The results showed that the activity of p-STAT3 and p-PKR increased in the model group.But the activity of p-STAT3 decreased significantly after WP1066 inhibitor intervention,and the activity of p-PKR increased at the same time.And the activity of p-PKR decreased significantly,but the activity of p-STAT3 had no obvious change significantly after PKRI intervention.It is suggested that autophagy plays an important role by influencing the activity of p-STAT3 and p-PKR and mediating autophagy happen in airway remodeling.ConclusionOur data suggested there was obvious airway remodeling change in OVA-induced mice.And autophagy was inhibited,which was related to stat3-pkr signal pathway.Part ?:The role and mechanism of Ang(1-7)in airway remodeling and airway inflammatory induced by OVA in asthma ratsObjectiveTo study the role of Ang(1-7)in airway inflammatory response and airway remodeling induced by OVA,and to reveal the internal signaling pathway.MethodThe BALF,serum and airway tissues were collected and eosinophils,lymphocytes and neutrophils were isolated after Ang(1-7)and A779 treatment in OVA-induced asthma rice.The effect of Ang(1-7)on airway inflammation and airway remodeling were determined through ELISA,HE,Masson,Giemsa staining and Western blot test.Result1.Giemsa staining showed that the number of eosinophils,neutrophils and lymphocytes were significantly increased and the number of inflammatory cells was significantly decreased after Ang(1-7)intervention in BALB/c mice,however the protective effect of Ang(1-7)was reversed by A779 inhibitor.The levels of pro-inflammatory factors in BALF was detected by ELISA Kit.The results are similar with Giemsa staining.At the same time,the results of airway reactivity tests showed that the airway reactivity of the model group was improved,and the airway reactivity of the model group was significantly reduced after the intervention of Ang(1-7).These data suggested that Ang(1-7)could alleviate OVA-induced airway inflammatory response in mice.2.The results of airway staining showed that the smooth muscle layer of the model group was thickened obviously,and the pathological changes of airway remodeling occurred in BALB/c mice treated with different treatment factors.The pathological changes could be alleviated obviously after Ang(1-7)intervention,and the protective effect of Ang(1-7)could be reversed by A779 inhibitor.The expression of?-SMA and CollI protein,as biomarkers of smooth muscle proliferation,also showed the same changes with the staining results.These data suggested that Ang(1-7)could alleviate the pathological changes of airway remodeling induced by OVA.3.Western blot test showed that ova could enhance the expression of a-SMA and CollI protein by increasing the activity of p-JNK,while Ang(1-7)could decrease the activity of p-JNK and down regulated the expression of ?-SMA and CollI protein.But A779 inhibitor can inhibit the effect of Ang(1-7).These data suggested that Ang(1-7)could improve the activity of p-JNK and affected the effect of airway remodeling by OVA-mediated in asthmatic mice.4.The expression of a-SMA and CollI protein in epithelial cells was detected after the intervention of JNK inhibitor sp600125 by Western blot.At the same time,the contents of pro-inflammatory factors IL-4,IL-5 and IL-13 were detected by ELISA.The results showed that the activity of p-JNK was inhibited,the expression of a-SMA and CollI protein was down-regulated,and the contents of IL-4,IL-5 and IL-13 were down-regulated after sp600125 intervention.These data indicated that Ang(1-7)could reduce or reverse the inflammatory response and airway remodeling induced by OVA through JNK signaling pathway.ConclusionOVA induced obviously the changes of airway inflammation and remodeling in mice.Ang(1-7)can reduce the expression of a-SMA and CollI protein,reduce or reverse the inflammatory response and airway remodeling induced by OVA through increasing the activity of p-JNK protein and reducing the contents of pro-inflammatory factors IL-4,IL-5 and IL-13.