The Effection Of Wu Hu Decoction To Young Rat Of Airway Remodeling Of Asthma Induced By RSV

Objective:In vivo,to study Wuhu Decoction on the expression of NGF in serum,TGF-β1and IL-13in the lung tissue and airway remodeling as well as correlation analysis in the rat model of asthma induced by RSV.In vitro,to study effect of Wuhu Decoction drug containing plasma on the expression of NGF and Sema4D receptor Plexin-B1and CD72,and the relationship between Sema4D and NGF.Through adjusting the neuroendocrine immune from disorder to improve airway remodeling, provide experimental basis for the treatment of asthma by Wuhu Decoction.Methods:Establish the young SD rats airway remodeling of asthma model through the sensitization and inspire of Respiratory syncytial virus joint egg white protein.After the success of the model is established,in vivo study,with Wuhu Decoction of high,medium and low dose and dexamethasone intervention,to observe each reaction in rats,the pathological morphology methods to analyze related indexes of airway remodeling,to detect the expression of a-SMA in the lung tissue by immunohistochemical method,to determinate the level of NGF in serum by ELISA, to detect lung tissue TGF-β1and IL-13proteins relative content by Western blot,to determinate the expression of NGFmRNA,TGF-β1mRNA,IL-13mRNA by Real-time PCR.In vitro,after separation of airway epithelial cells of normal group and the model group rats,then cultured and passaged the cells in2-3generation.To the intervent by Sema4D,anti Sema4D and the high,middle dose Wuhu Decoction.To detect the number of proliferation of airway epithelial cells were detected and the amount of migration by MTT and Transwell chamber, respectively,Sema4D receptor Plexin-Bl and CD72mRNA and protein expression level by RT-PCR and Western Blot,the expression of NGF in airway epithelial cells detection by ELISA.Results:Compared with normal group,model group rats appeared obvious breathing,be agitated,arrector shrug,oral cyanosis,and poor weight slow growth and spirit, breathing symptoms such as Wuhu Decoction high,middle dose group compared with model group significantly reduce,the symptoms in DEX group rats also ease, low dose group Wuhu Decoction rats breathing level is not obvious;Compared with normal group,model group pathological microscopically a large number of inflammatory cells, luminal stenosis,mucus secretion increased,markedly thickened and the disordered arrangement of airway smooth muscle,more visible glue under the same material deposition under mucous membrane.At the same time,some quantitative indicators of Airway remodeling,the integral optical density(IOD)of a-SMA in the lung tissue significantly increased(P<0.01).Compared with model group,Wuhu Decoction high,middle dose group and DEX group microscopically pathological changes was significantly reduced,and the indicators,positive airway morphology parameters expression IOD of a-SMA values significantly decreased(P<0.01),there was no statistically significant difference between the three groups(P>0.05),the difference in low dose group is not obvious(P>0.05); Compared with normal group,the NGF expression in serum in model group rats increased obviously,statistically significant difference(P<0.01),the treatment groups can reduce the level of NGF,with Wuhu Decoction of high,middle dose group obviously(P<0.01),low dose group,DEX group loss difference has significant statistical significance(P<0.01),NGF expression of Wuhu Decoction each dose group were superior to dexamethasone group(P<0.01,P<0.05);Compared with normal group TGF-βt,IL-13protein expression of lung tissue in Model group significantly increased(P<0.01),after drug intervention,TGF-β1and IL-13protein expression in Wuhu Decoction high,middle dose group and DEX group were significantly decreased(P <0.01),and no difference between the three groups(P>0.05),low dose group of TGF-β1and IL-13proteins decreased there was no statistically significant difference compared with model group (P>0.05);Compared with the normal group,the rest of the groups relative expressions of NGFmRNA in lung tissue were higher.Compared with the model group,NGFmRNA expression in all treatment groups were significantly decreased(P<0.01),but Wuhu Decoction high dose,middle dose decreased significantly statistical significance than in low dose group and DEX group (P<0.01),but no difference between the two groups(P>0.05).Compared with the normal group,model group rats TGF-β1and IL-13mRNA expression in creased significantly(P<0.01).Compared with model group, after the treatment of Wuhu Decoction of high,medium dose can significantly reduce the expression levels of TGF-β1mRNA(P<0.01),and no obvious difference between the two groups(P>0.05),but still higher than normal group (P<0.01).Compared with model group low dose group and DEX group there was no significant difference(P>0.05);the expression levels of IL-13mRNA in Wuhu Decoction high, middle dose group and DEX group were significantly lower in the model group (P<0.01),and there was no statistically significant difference between Wuhu Decoction high and middle dose group(P>0.05),and IL-13mRNA expression in the DEX group is significant(P<0.01);Compared with model group, Wuhu Decoction low dose group there was no statistically significant difference (P>0.05).In vitro,compared with A group, the MTT method for the determination of airway epithelial cell proliferation quantity display B group OD value increased significantly;C group with Sema4D function of24h,compared with the B group OD value increased significantly(P<0.01);OD value of El decreased compared with group D,F1,F2add anti Sema4D and Wuhu Decoction drug containing plasma;The OD value of E2group decreased after the addition of Wuhu decoction drug containing plasma its.Each group compared with the control group,there were a significant difference(P<0.01). Determination of A-F2groups cell migration number respectively are70,102,155,140,102,93,126,137by transwell chamber.Compared with groupA and B, Sema4D and its receptor Plexin-Bl expression and NGF content were increased,receptor CD72expression decreased,C group in the presence of Sema4D,the number of OD and migrating cells increased than in B group significantly(P<0.01),the expression of Sema4D receptor Plexin-B1mRNA and protein in C group,compared with the B group were significantly increased (P<0.01),the expression of CD72receptor was significantly decreased (P<0.01);the expression of NGF in airway epithelial cells also increased(P<0.01);compared with C group, the expression of Plexin-B1mRNA and protein also decreased (P<0.01),while the expression of NGF decreased (P<0.01),and the expression of CD72mRNA and protein correspondingly increased (P<0.01).Compared with group D,E2and El,PlexinBlmRNA protein and NGF expression levels also decreased significantly(P<0.01),the expression of CD72mRNA and protein levels increased to a certain extent(P<0.01).The expression of group F1,F2and D,are consistent,there is no significant difference.Compared with the C group, the decreased degree of group F1,F2on number of OD and migration are less than groupE1,E2.The increase degree of Plexin-B1mRNA and protein and NGF,and decrease degree of CD72mRNA and protein expression were less than E1, E2group.Conclusions:Application the method of combined RSV added OVA sensitization and excitation,can successfully establish young SD rats airway remodeling of asthma model.The high and medium dose of Wuhu Decoction may be through improved under the bronchial wall thickening and epithelium,smooth muscle collagen deposition,and cut the a-SMA expression to slow down the airway remodeling process;Wuhu Decoction high and medium dose may be by reducing the TGF-β1and IL-13to cut model NGF expression,to implementation of NEI network disorder in asthma adjustment and to delay the process of airway remodeling.Sema4D participate in the proliferation and migration of the asthmatic airway epithelial cells,and through mutual promotion and the role of NGF,enhance their ability to bind with the receptor PlexinBl and inhibited the binding to its receptor CD72,then accelerate the proliferation of airway epithelial cells and the migration process,so as to promote the occurrence and development of airway remodeling;the high and middle dose medicated plasma of Wuhu Decoction can play the role of anti Sema4D,through inhibition of Sema4D and NGF and decrease the binding ability of Sema4D and its receptor PlexinBl, to enhance the bonding force and its receptor CD72,to relieve the pathological process of airway remodeling in asthma.