Mechanism Of Xiaoqinglong Decoction In The Treatment Of Allergic Rhinitis By Regulating ILC2s

ObjectiveILC2s are a kind of newly discovered lymphocytes,which play an important role in the pathogenesis of AR.Activation of ILC2s secretes type 2 cytokines that aggravate and maintain Type 2 inflammation.ILC2s were found to be regulated by activating and costimulatory cytokines.In our previous study,we found that Xiaoqinglong Decoction has a therapeutic effect on allergic rhinitis mice.To further confirm the mechanism of xiaoqinglong Decoction in treating AR.This experiment intends to establish a mouse model of AR and take ILC2s as the key point to explore the effects of Xiaoqinglong Decoction on ILC2s and related signal pathways in AR model mice,so as to provide scientific basis for clinical application of Xiaoqinglong Decoction in the treatment of AR.MethodsThe mouse model of allergic rhinitis was constructed by ovalbumin(OVA)combined with Aluminum hydroxide adjuvant(Alum).Except for control group,200μl PBS containing 25μl OVA and 2mg Alum were intraperitoneally injected to cause basic sensitization.During the stimulation phase,except for the control group,mice were given 40μ L PBS(20μL/side)containing 1000μg OVA from day 22 to day 28,and then AR model was established.Then the medicine was given for a week,loratadine group was given loratadine solution at 2.05mg/kg,and Xiaoqinglong decoction low-dose,medium-dose and high-dose groups were given concentrated decoction of xiaoqinglong at 5.46,10.92 and 21.84 g/kg,respectively.Gavage was given once a day for 7 days.The frequency of sneezing and nasal rubbing of mice was observed within 10 min after the last nasal instillation.The levels of OVA-sIgE,interleukin 4,5,13(IL-4,IL-5,IL-13)in peripheral blood were measured.Hematoxylin-eosin staining(H&E),Periodic acid-Schiff stain(PAS),and Giemsa staining were used to observe the histopathological changes of nasal mucosa in mice.Immunofluorescence staining was used to observe the numbers of ILC2 cells in nasal mucosa.The expression of related genes and proteins was detected by reverse transcription polymerase chain PCR(RT-PCR)and Western Blot(WB).Results1.Effects of Xiaoqinglong Decoction on clinical symptoms,type 2 cytokines and histopathology in AR mouse:Compared with the control group,the number of sneezing and nasal rubbing of mice in the model group increased(all P<0.001).After administration,compared with the model group,the sneezing frequency and nasal rubbing frequency of loratadine group,xiaoqinglong decoction low-dose,medium-dose and high-dose groups decreased(all P<0.05).Compared with control group,the levels of OVA-sIgE,IL-4,IL-5,and IL-13 in each group were significantly increased in model group(all P<0.001).After administration,compared with model group,the levels of OVA-sIgE,IL-4,IL-5 and IL-13 in loratadine group and xiaoqinglong decoction low-dose,medium-dose and high-dose groups decreased(all P<0.01).In the model group,compared with the control group,nasal mucosa was thickened,interstitial edema,eosinophil infiltration and goblet cell proliferation.After administration,compared with the model group,the nasal mucosa edema thickened,eosinophil infiltration,goblet cell proliferation and other conditions were alleviated in each group.After the cell counting,compared with the model group,the number of nasal mucosal goblet cells and eosinophils in the Xiaoqinglong decoction medium and high-dose group was significantly reduced(both P<0.01).2.Effects of Xiaoqinglong Decoction on ILC2s in nasal mucosa of AR mice:In the model group,compared with the normal group,we can see ILC2s in the nasal mucosa.However,in the loratadine group and xiaoqinglong decoction medium-dose and high-dose groups,the number of ILC2s was decrease.After cell counting,compared with model group,the number of ILC2s in nasal mucosa of mice in medium-dose and high-dose Xiaoqinglong Decoction groups was significantly decreased(P<0.01).At the same time,the expression of GATA3 and ROR-α was detected.Compared with normal group,the expression of GATA3 and ROR-α in model group was significantly increased(all P<0.01).After administration,compared with model group,GATA3 and ROR-α gene expression in loratadine group,xiaoqinglong decoction low-dose,medium-dose and high-dose groups were decreased(all P<0.05).Among them,the decrease degree was most obvious in xiaoqinglong decoction highdose group.3.Effect of Xiaoqinglong Decoction on IL-33/ST2 signaling pathway in AR mouse:The mRNA expression levels of IL-33,ST2,MYD88,IRAK4 and NF-κBP65 in nasal mucosa tissues of model group were significantly increased compared with that of control group(All P<0.05).After administration,xiaoqinglong decoction high-dose group had the best effect.Compared with model group,the expressions of IL-33,ST2,MYD88,IRAK4 and NF-κBP65 in nasal mucosa of xiaoqinglong Decoction high-dose group were significantly decreased(All P<0.05).Western Blot analysis showed that the protein expressions of IL-33,ST2,MYD88,IRAK4 and NF-κBP65 in the model group were significantly increased compared with the normal group(all P<0.05).After administration,compared with model group,the protein expression of IL-33,ST2,MYD88 and NF-κBP65 in Xiaoqinglong Decoction highdose group was significantly decreased(All P<0.05).4.Effect of Xiaoqinglong Decoction on JAK/STAT signaling pathway in AR mouse:Compared with control group,the mRNA expression levels of JAK2、STAT3、STAT5a in nasal mucosa tissues of model group were significantly increased(All P<0.05).After administration,xiaoqinglong decoction high-dose group had the best pesticide effect.Compared with model group,the protein expressions of JAK2、STAT3、STAT5a in nasal mucosa of xiaoqinglong Decoction high-dose group were significantly decreased(All P<0.05).Then Western Blot analysis showed that the protein expressions of P-JAK2、P-STAT3、p-STAT5a in the model group were significantly increased compared with the normal group(All P<0.05).After administration,compared with model group,the protein expression of P-JAK2、P-STAT3、p-STAT5a in Xiaoqinglong Decoction high-dose group was significantly decreased(All P<0.05).ConclusionsBased on the above experimental results,we can draw the conclusions.1.Xiaoqinglong Decoction could significantly improve nasal allergy symptoms,decrease the level of type 2 cytokines,and improve nasal mucosal histopathological changes of AR mice.2.Xiaoqinglong Decoction inhibited the proliferation and maturation of ILC2s by regulating the activation cytokine related IL-33/ST2 pathway and the costimulatory cytokine related JAK/STAT pathway in the ILC2s regulatory network.