| Objective:Nasal epithelial barrier damage is the most fundamental cause of allergic rhinitis（AR）.At present,increasing studies have been confirmed that treating AR from the perspective of repairing the nasal epithelial barrier damage is feasible and effective.Nasal corticosteroids are still the first-line drugs recommended by domestic and foreign guidelines for treating AR.Also,studies have proved that it can effectively repair the nasal epithelial barrier damage of AR.Modified Yupingfeng nasal spray,a pure Chinese medicine nasal preparation developed by Professor.TIAN LI,previous studies have confirmed that it also can suppress TSLP-mediated inflammatory injury via regulating p38MAPK/PPARγsignaling.Study indicated that integrating and regulating of AMPK-NRF2/NF-κBp65 signaling also plays a very important role in speeding up the assembly of tight junction proteins as well as repairing the damage of AR nasal epithelial barrier.Based on that,this study will compare the repairing effects of following three treatment methods（intranasal glucocorticoids,Modified Yupingfeng nasal spray as well as oral administration of Chinese medicine,respectively）on nasal mucosal epithelial barrier damage in AR rats,and further explore the specific internal mechanism of Modified Yupingfeng nasal spray’s effect via integrating and regulating of AMPK-NRF2/NF-κBp65 signaling,in order to provide reference for its later clinical promotion and application.Methods:Fifty-two SD rats were randomly divided into five groups,blank control group（n=10）,AR model group（n=10）,positive control group（mometasone furoate nasal spray group,n=10）,Chinese medicine nasal spray group（Modified Yupingfeng nasal spray group,n=11）and Chinese medicine gavage group（same as the decoction and concentration made of the same drug composition of Modified Yupingfeng nasal spray,n=11）.The other four groups were established by intraperitoneal injection of ovalbumin（OVA）combined with aluminum hydroxide（Al（OH）3）to establish AR model,except for blank control group,administrating normal saline,mometasone furoate nasal spray,Modified Yupingfeng nasal spray,and traditional Chinese medicine（TCM）gavage for intervention respectively.After thirty minutes,5%ovalbumin solution was used for intranasal challenging.When twenty-four hours after the end of the last treatment finished,we would observe as follows:（1）Enzyme-linked immunosorbent assay（ELISA）was used to detect the levels of IL-13 and IL-17 in the serum of rats in each group.（2）Hematoxylin-eosin staining（HE staining）was used to observe the inflammatory infiltration of nasal mucosa of rats in each group.（3）Transmission electron microscope was used to observe the ultrastructure of the nasal mucosa epithelial cells of the rats in each group.（4）Western blotting was used to detect the expression level of proteins in nasal mucosa epithelial such as Claudin-4,Occludin,ZO-2,AMPK,NRF2,NF-κBp65.（5）Real-time PCR was used to detect the expression level of proteins’m RNA in nasal mucosa epithelial such as Claudin-4,Occludin,ZO-1.（6）Immunofluorescence was used to detect the expression level of proteins in nasal mucosa epithelial such as NRF2,NF-κBp65.Results:（1）General observation:The body weight of rats in each group almost maintained a similar speed rate.AR has little effect on the body weight of rats.meanwhile,there was no statistically significant difference in the body weight of the rats in the positive control group,the Chinese medicine nasal spray group,and the Chinese medicine gavage group（P>0.05）.AR has little effect on the body weight of rats.It can be seen that the above three treatments also had no significant effect on the body weight of rats.（2）Histopathological observation of nasal mucosa in rats:under light microscope,the nasal mucosa surface of rats in the blank control group was covered with pseudo-stratified columnar epithelium,the cells of each layer of the mucosal epithelium were arranged neatly,the glandular cells in the lamina propria were arranged neatly,and there was no edema and degeneration of the interstitium.In the AR model group,the mucosal epithelial cells degenerated and necrotic.There was interstitial edema in the lamina propria.A large number of eosinophils infiltrated in mucosa.The proliferating goblet cells were densely distributed in clusters and enlarged in size,some of which broke through the lamina propria.Large areas of glands were basophilic degeneration with necrosis,and severe edema was observed in the submucosa.The nasal mucosa of the positive control group,the Chinese medicine nasal spray group and the Chinese medicine gavage group were repaired,and the surface was covered with pseudo-stratified columnar epithelium.The mucosal epithelium and the glandular cells in the lamina propria were arranged relatively neatly,and the structure was basically complete.（3）Transmission electron microscopy observation of nasal mucosa in rats:in the blank control group,the cilia on the surface of the nasal mucosa epithelium were arranged regularly.The cell morphology and structure were normal,and all kinds of organelles were arranged orderly.In the AR model group,the mucosal epithelial cilia were sparse,a large number of cilia were broken,shed,and irregularly arranged,the epithelium was loosely arranged,the cell volume became smaller,and the Golgi apparatus,mitochondria,and other organelles were reduced,swollen,and vacuolated.While the nasal mucosa cilia in the positive control group,the Chinese medicine nasal spray group and the Chinese medicine gavage group were repaired to varying degrees.（4）Detection by ELISA:compared with blank control group,the level of IL-13,IL-17 in AR model group were significantly increased（P<0.01）.Compared with AR model group,the level of IL-13,IL-17 in positive control group,the Chinese medicine nasal spray group and the Chinese medicine gavage group were significantly decreased（P<0.01）.There was no significant difference between the other groups（P>0.05）.（5）Detection by Western Blotting:compared with blank control group,the proteins’expression level of Claudin-4,Occludin,ZO-1,AMPK,NRF2 in AR model group were significantly decreased（P<0.01）,while the protein expression level of NF-κBp65 was significantly increased（P<0.01）.Compared with AR model group,the proteins’expression level of Claudin-4,Occludin,ZO-2,AMPK,NRF2 in positive control group,the Chinese medicine nasal spray group and the Chinese medicine gavage group were significantly increased（P<0.01）.While the protein expression level of NF-κBp65 was significantly decreased（P<0.01）.There was no significant difference between the other groups（P>0.05）.（6）Detection by Real-time PCR（RT-PCR）:compared with blank control group,the genes’transcription level of Claudin-4,Occludin,ZO-1 in AR model group were significantly decreased（P<0.01）,Compared with AR model group,the genes’transcription level of Claudin-4,Occludin,ZO-1 in positive control group,the Chinese medicine nasal spray group and the Chinese medicine gavage group were significantly increased（P<0.01）.There was no significant difference between the other groups（P>0.05）.（7）Detection by immunofluorescence staining microscopy:under the microscope of 200 times and 400 times,compared with the AR model group,the expression levels of NRF2 protein in the positive control group,the Chinese medicine nasal spray group and the Chinese medicine gavage group were higher,while the expression level of protein NF-κBp65 were lower in the positive control group,the Chinese medicine nasal spray group and the Chinese medicine gavage group.Conclusion:（1）In the state of AR,it can be seen that the proteins’expression level of Claudin-4,Occludin,ZO-2 and the genes’transcription level of Claudin-4,Occludin,ZO-1 in the nasal mucosa of rats were significantly decreased,and the epithelial barrier was damaged.While administrated mometasone furoate nasal spray,Modified Yupingfeng nasal spray and traditional Chinese medicine gavage,the proteins’expression level of Claudin-4,Occludin,ZO-2 protein and the genes’transcription level of Claudin-4,Occludin,ZO-1 can be improved,and the epithelial barrier damage can be repaired.（2）In the state of AR,the expression level of AMPK and NRF2 protein reduced obviously in nasal mucosa of rats.The expression level of NF-κBp65 protein enhanced significantly.While,when administrated mometasone furoate nasal spray,Modified Yupingfeng nasal spray and traditional Chinese medicine gavage,the expression level of AMPK and NRF2 protein can be obviously improved.The expression level of NF-κBp65 protein can be significantly inhibited.Therefore,we speculate that Modified Yupingfeng nasal spray may repair nasal mucosal epithelial barrier damage in AR rats by integrating and regulating AMPK-NRF2/NF-κBp65 signaling. |
