She-Ti-Zhi-Qiu Decoction Ameliorated Allergic Rhinitis By Modulating Microbiotath-Th17/Treg Axis

Background:The average incidence of allergic rhinitis（AR）is 10%-50%,and at least 600million people worldwide suffer from allergic rhinitis.With the development of economy,the prevalence of AR also shows a significant upward trend.Its treatment lies in correcting the disease state of yin and yang imbalance from the whole body of the patient.It can act on the body with multiple systems and multiple targets and has the advantage of relatively stable long-term efficacy.In recent years,with the extension of the"hygiene hypothesis"to the"old friend"hypothesis and the"microbiome"hypothesis,the key role of the gut microbiota in the pathogenesis and prevention of AR is gradually being revealed.Further studies have found that the gut microbiota can participate in the regulation of the host immune system in a variety of ways.Therefore,some scholars have begun to try to target the intestinal microbiota,such as maintaining intestinal homeostasis by supplementing probiotics and exploring its preventive and therapeutic effects on allergic diseases.Therefore,modulating the microbiota may be a promising approach for the treatment of AR.She-Ti-Zhi-Qiu decoction is the clinical experience prescription of Professor Peng from Chengdu University of Traditional Chinese Medicine in the treatment of AR patients with lung qi and cold deficiency.As a traditional Chinese medicine compound,She-Ti-Zhi-Qiu decoction has complex chemical components,and a considerable part of these complex traditional Chinese medicine components have low oral bioavailability.Difficulty being absorbed into the blood,which results in a limited concentration level in the blood.On the surface,these components are not absorbed by the small intestine.In fact,the components that are not absorbed by the small intestine will come into direct contact with the intestinal microbiota after entering the colon,so the interaction between the intestinal microbiota and traditional Chinese medicine is inevitable.The previous study showed that She-Ti-Zhi-Qiu decoction could correct the imbalance of Th17/Treg cytokine network in AR patients.Modern medicine has put forward the concept of the"gut-lung"axis,which emphasizes the interconnection between gut microbiota and respiratory diseases.Based on this,we put forward a hypothesis:She-Ti-Zhi-Qiu decoction can regulate the intestinal microbiota,further affect the balance of Th17/Treg cells and play a role in the treatment of AR.Objective:Clinical and experimental studies have shown that She-Ti-Zhi-Qiu decoction（STZQ）is effective in treating AR with lung-qi deficiency and cold.Recent studies have found that the pathogenesis of AR is closely related to the gut microbiota and Th17/Treg balance,and targeting the gut microbiota can effectively prevent AR.This study mainly explores STZQ in the treatment of AR by regulating the intestinal flora and further affecting the balance of Th17/Treg.Method:Twenty-eight 6-8 week old female SD rats were reared for 1 week and then induced and challenged by OVA+Al（OH）₃ to construct AR rat model.Twenty-eight rats were randomly divided into 4 groups,namely Control group,AR Model group,STZQ group,and Probiotics group.The Control group and Model group were intragastrically administered with normal saline,the STZQ group was intragastrically administered with She-Ti-Zhi-Qiu decoction,and the Probiotics group was intragastrically administered with probiotics.The rats in each group were given intragastric administration for 14 days,and then the nasal mucosa,serum,peripheral blood and colon contents were collected.Nasal mucosa histopathology was assessed using Hematoxylin-eosin staining（HE）.The structure and content of bacteria in colon contents were detected and analyzed by 16SrDNA sequencing.The relative proportions of regulatory T cells（Treg）and T helper cell 17（Th17）in peripheral blood mononuclear cells were detected by flow cytometer（FCM）.Enzyme Linked Immunoadsorption Assay（ELISA）was used to detect the levels of Th17 cells and Treg cell-related cytokines in serum.The expression of Foxp3mRNA and RORγt mRNA was detected by real-time PCR.The protein expressions of Foxp3 and RORγt were detected by Western Blot（WB）.To explore the role of She-Ti-Zhi-Qiu decoction in the prevention and treatment of AR.Result:1.AR typical symptom score,nasal mucosa pathological tissue,serum OVA-sIgE:Compared with the control group,the AR typical symptom score of the model group was significantly increased（p<0.05）,the inflammatory changes in the nasal mucosa of the rats,and the serum OVA-sIgE was significantly increased（p<0.05）,indicating that the AR rat model was successfully established by OVA+Al（OH）₃ sensitization;The intervention of STZQ significantly reduced the AR typical symptom score（p<0.05）,relieved the severity of inflammation in nasal mucosa,and down-regulated the level of serum OVA-sIgE（p<0.05）.2.Related indicators of Th17/Treg balance:She-Ti-Zhi-Qiu decoction can reduce the levels of IL-17A and IL-17E in the serum of AR rat model,and reduce the number of CD3+CD8-IL17A+Th17 cells in the peripheral blood.The expression of RORγt mRNA in nasal mucosa and the expression of RORγt protein in nasal mucosa were down-regulated;the level of IL-10 in serum and the number of CD4+CD25+Foxp3+Treg cells in peripheral blood were increased,and the expression of Foxp3 mRNA in nasal mucosa and the expression of Foxp3 protein in nasal mucosa were up-regulated.3.Colonic microflora detection:3.1 Species annotation:Control group,Model group,STZQ group and Probiotics group had 2077,3245,1606,and 2310 OTU,respectively.The number of OTU shared by the four experimental groups was 228,the control group had 1147 unique OTU,the Model group had 2009 unique OTU,the STZQ group had 884 unique OTU,and the Probiotics group had 1284 unique OTU.The analysis results showed that the types of microflorae in the intestinal tract of the experimental rats changed significantly after modeling and administration.3.2 Species richness:Compared with the Control group,the intestinal flora richness of the Model group increased,and the species richness decreased after the intervention of STZQ and Probiotics,which tended to be in the Control group.3.3 Community diversity:There was no statistical difference in the diversity of intestinal flora among the four groups（Control group,Model group,STZQ group,Probiotics group）.3.4 Similarities and differences of intestinal flora between groups:at the phylum level:at the phylum level:STZQ can up-regulate Firmicutes,down-regulate the abundance of Spirochaetes（Spirochetes）and Bacteroidetes（Bacteroidetes）;at the order level:STZQ can down-regulate Enterobacteriaceae and up-regulate the abundance of Turicibacterales;at the family level:STZQ and Probiotics can down-regulate the abundance of g＿unidentified＿S24-7 and Enterobacteriaceae;at the genus level:STZQ and Probiotics can down-regulate Unidentified＿S24-7 and Oscillospira abundance.3.5 Correlation between intestinal flora and biological indicators:STZQ increased the abundance of the intestinal flora Firmicutes,down-regulated the abundance of Spirochaetes,Bacteroidetes,Enterobacteriales,g＿unidentified＿S24-7and Enterobacteriaceae（Enterobacteriaceae）Degree,Unidentified＿S24-7 and Oscillospira,the correlation analysis results showed that the above differential bacteria Genus Firmicutes was positively correlated with IL-10;Spirochaetes,Bacteroidetes,Enterobacteriales,S24-7,Unidentified＿S24-7 and Oscillospira were negatively correlated with IL-10;Ruminococcaceae,S24-7,Unidentified＿S24-7,Oscillospira were positively correlated with IL-17A,IL-17E.Conclusion:1.The AR rat model was successfully established by OVA+Al（OH）₃ sensitization.She-Ti-Zhi-Qiu decoction could relieve the typical symptoms of AR model rats,relieve the severity of inflammation in nasal mucosa,and down-regulate the level of serum OVA-sIgE.2.She-Ti-Zhi-Qiu decoction could down-regulate the richness of colonic flora in AR model rats,making it tend to the Control group.She-Ti-Zhi-Qiu decoction had no effect on the diversity of flora.3.She-Ti-Zhi-Qiu decoction modulated the composition of gut microbiota and affected the balance of Th17/Treg in peripheral blood and the expression of related cytokines,which showed was correlated with serum IL-17A,IL-17E and IL-10.It is suggested that She-Ti-Zhi-Qiu decoction ameliorated allergic rhinitis by modulating microbiota-Th17/Treg axis.