| Objective:The epithelial barrier is an emerging research field in allergic rhinitis(AR),which serves as the first line of defense against allergens.Current research is focused on the physical barrier,with less emphasis on other barriers,such as chemical barrier and microbiological barrier.Several Traditional Chinese Medicine(TCM)treatments have been shown to repair epithelial barriers,such as the Yu-Ping-Feng San.Based on unique insights into the pathogenesis of "Qi deficiency of lung leading to weakened defensive,nasal obstruction caused by stagnant blood".Professor Tian proposed an external treatment method that invigorating qi and promoting blood circulation,and developed the Modified Yu-Ping-Feng Nasal Spray.Its efficacy and mechanism in AR have been preliminarily confirmed.This study aimed to explore the clinical and mechanistic aspects based on epithelial-derived cytokines in repairing the epithelial barrier in AR.This study involved the physical,microbiological,and chemical barriers of the nasal epithelium.It provides a new perspective for the treatment of AR,and promotes the application of the TCM external treatment method.Method:1.Clinical observation 1:A total of 160 outpatients with AR were enrolled as subjects,and allocated to either the treatment group or the control group through randomization.The treatment group was administered Modified Yu-Ping-Feng Nasal Spray(YPF),while the control group was administered Mometasone Furoate Nasal Spray(MMS).The study was based on observations made before and after treatment.We Chat mini-program was employed as a means of monitoring patients’ medical conditions.The Rhinitis Control Assessment Test(RCAT)was used as the clinical control standard.The observation indicators included:(1)Clinical data: TCM constitution,RCAT score,Visual Analogue Score(VAS),Rhinocou-junctivitis Quality of Life Questionnaire(RQLQ),and Qi-Deficiency score.(2)Cytokines: Serum and nasal lavage fluid(NALF)were collected.ELISA was used to detect IL-4,IL-13,IFN-γ,TSLP,and IL-25.(3)Chemical barrier: MUC5 AC protein expression and gene transcription were detected using Western blotting(WB)and reverse transcription-polymerase chain reaction(rt-PCR),respectively.The study aimed to explore the effects of Modified Yu-Ping-Feng Nasal Spray in AR from the perspective of clinical efficacy,epithelial-derived cytokines,and chemical barrier.2.Clinical study 2:24 subjects were recruited and divided into four groups,including the control check(CK)group,AR group,YPF group and MMS group.Samples were collected using nasal swabs and sequenced.This study involved sequence denoising and clustering to generate Amplicon Sequence Variant(ASV)and Operational Taxonomic Unit(OTU)data tables.Observation measures include:(1)Species composition analysis: the study analyzed species composition at different taxonomic levels.(2)Alpha(α)-diversity metrics such as Chao1,Observed species,Shannon,Simpson,Faith’s PD,Pielou’s evenness,and Good’s coverage index.(3)Beta(β)-diversity metrics include distance matrix and principal coordinate analysis,as well as nonmetric multidimensional scaling analysis.(4)Differential species can be identified through Venn diagrams and species composition heatmaps,as well as through LEf Se and Random Forest analyses.This study investigated the differences in epithelial microbiota between AR patients and healthy individuals from the perspective of microbiological barriers.Additionally,it examined the regulatory effect of Modified Yu-Ping-Feng Nasal Spray on the nasal epithelial microbiota of AR patients.3.Animal Experimentation:Forty SD rats were randomly divided into four groups: the control check(CK)group,the AR group,the MMS group,and the YPF group.Except for the CK group,all groups were induced with a model of allergic rhinitis.Observation indicators:(1)Pathological observation:the study collected the mucosa of both sides of the nasal septum,conducted HE staining,and carried out pathological evaluation.(2)Serum: the study detected levels of TSLP,IL-25 and TGF-β1 by ELISA.(3)Physical and chemical barriers: the study detected protein levels of tight junction,Claudin(CLDN)-1 and ZO-1,and transcript levels of MUC5 AC gene via WB and PCR.Investigate the therapeutic effect of Yu-Ping-Feng modified nasal spray on AR from the perspectives of physical and chemical barriers.This study investigated the therapeutic effects of Modified Yu-Ping-Feng Nasal Spray on AR from the perspectives of physical and chemical barriers.Results:1.Clinical observation 1:(1)Completion status of the study: A total of 160 patients were enrolled in this study,with80 patients in both the treatment and control groups.The number of completed cases in the treatment and control groups were 76 and 75,respectively.(2)Baseline data: This study analyzed the age,gender,education level,BMI,and family history of allergies.The results indicated no significant difference in baseline data between the two groups.(3)TCM constitution: In AR,common TCM constitutions included the specific endowment constitution,yang deficiency constitution and qi deficiency constitution.In the analysis of the risk factors of TCM constitution,sex and family history of allergies were found to have different effects on TCM constitution(p<0.05).When using mild physical constitution as a reference,the qi stagnation constitution tended to be more common in females,with the same trend for the yang deficiency constitution.In addition,patients with a family history of allergies tended to have a specific endowment constitution.At the same time,it was found that patients with qi deficiency constitution had the highest score for qi deficiency syndrome(p<0.05).(4)Clinical efficacy: When comparing the scores of various scales before treatment,both groups showed no difference.(1)RCAT: After treatment,both groups showed improvement in RCAT scores(p<0.05).The treatment group had a better improvement in nasal congestion than the control group,whereas the control group had a better improvement in sneezing and activity than the treatment group.(2)VAS: The treatment group was found to be more effective in improving nasal obstruction(p<0.05)than the control group.(3)RQLQ: After treatment,both groups improved in RQLQ scores(p<0.05);the treatment group was lower than the control group in sleep and non-ocular/nasal symptoms(p<0.05).(4)Qi deficiency Syndrome score:After treatment,the control group demonstrated improvement in shortness of breath,mental fatigue,weakness,and spontaneous sweating(p<0.05).In contrast,the treatment group exhibited favorable improvement in all manifestations of qi deficiency syndrome(p<0.05).Further,the treatment group was more effective in improving symptoms of mental fatigue,weakness,and laziness(p<0.05)than the control group.(5)Cytokines:(1)Serology: Both groups showed decreased levels of IL-4 and IL-13 and increased levels of IFN-γ(p<0.05),with no significant difference between the groups(p>0.05).(2)Nasal lavage fluid: Both groups demonstrated reduced levels of IL-25 and TSLP(p<0.05),with the treatment group showing superior downregulation of TSLP compared to the control group(p<0.05).Additionally,both groups showed decreased levels of IL-4,IL-13,and increased levels of IFN-γ(p<0.05).Significant differences were observed in the groups when downregulating IL-4 and IL-13(p<0.05).(3)Both groups were able to down-regulate TLR2 m RNA levels(p < 0.05).The treatment group showed a better effect than the control group(p< 0.05).(6)Chemical barrier: Both the treatment group and the control group inhibited MUC5 AC protein levels and gene transcription levels(p<0.05),with the treatment group showing better inhibition of gene transcription levels than the control group(p<0.05).2.Clinical observation 2:(1)Species composition analysis:(1)Comparison of the species abundance between AR patients and healthy controls: The abundance of microbiome in the AR group decreased compared to the CK group(p<0.05),while it increased in the MMS and YPF groups(p<0.05).Specifically,in the AR group,the abundance of Bacilli,Moraxellaceae,and Finegoldia decreased compared to the CK group(p<0.05).The abundance of Clostridia,Bacillales,[Tissierellaceae],Brucellaceae,Peptoniphilus,Anaerococcus,and Ochrobactrum increased(p<0.05).(2)Comparison between YPF group and MMS group: At the phylum level,Alphaproteobacteria,Bacteroidia,Anaerolineae,Coriobacteriia,and Verrucomicrobiae in the YPF group increased in abundance(p<0.05).At the order level,Acidimicrobiales increased in abundance in the MMS group,while Rhizobiales and Bacteroidales both increased in abundance in the YPF group(p<0.05).At the family level,the abundance of [Tissierellaceae]decreased in the YPF group,while the abundance of Moraxellaceae increased in the MMS group(p<0.05).At the genus level,the abundance of Peptoniphilus and Anaerococcus decreased in the YPF group(p<0.05),and the abundance of Peptoniphilus was lower in the YPF group than in the MMS group(p<0.05).(2)Diversity:(1)Alpha-diversity: The Chao1,Observed Species,Shannon,Simpson,Faith’s PD,Pielou’s evenness indices,and Good’s coverage indices all had p-values > 0.05.(2)Beta-diversity: Adonism and Permanova tests revealed significant differences in species composition among the groups.(3)Differential species analysis revealed that the unique OTUs in each group were 928 for the CK group,778 for the AR group,985 for the MMS group,and 990 for the YPF group,as determined by Venn diagrams.A heatmap displaying the top 50 abundant species revealed that Corynebacterium,Alloiococcus,Haemophilus,Peptoniphilus,Dermabacter,Finegoldia,and Anaerococcus were significantly upregulated in the AR group compared to the CK group(p<0.05),whereas their abundances were downregulated in the MMS group and YPF group(p<0.05).The abundances of Propionibacterium,Enhydrobacte,and Lactobacillus were decreased in the AR group compared to the CK group,while the YPF group exhibited an increase in their abundances(p<0.05).Finally,LEf Se and random forest analyses were performed,which identified the top 10 differentially abundant species,namely Ochrobactrum,Finegoldia,Anaerococcus,Paracoocus,Microcoocus,Lactobacillus,Peptoniphilus,Akkermansia,Allobaculum,and Acinetobacter.3.Animal Experimentation:(1)Pathological observations: Compared with the CK group,the pathological score of the AR group exhibited a significant increase in the pathological score,with disruptions in the epithelial cell structures and cilia,an increase in the gap junction.Additionally,swelling of the epithelial layer was observed.Conversely,the MMS and YPF groups showed a reduction in the pathological score(p<0.05).(2)Serum analysis: The levels of TSLP,IL-25,and TGF-β1 in the AR group were significantly increased compared with those in the CK group(p < 0.05).Their levels in the MMS group and the YPF group decreased significantly(p < 0.05).The YPF group showed a better down-regulation effect on TSLP and TGF-β1 than the MMS group(p < 0.05).(3)Physical and chemical barriers: Results demonstrated that the levels of CLDN-1 and ZO-1 proteins were significantly decreased in the AR group compared to the CK group(p<0.05).Their levels were increased in the MMS and YPF groups(p<0.05),but thers was no significant difference between the two groups.Compared to the CK group,the AR group demonstrated a marked upregulation in the transcriptional expression of the MUC5 AC gene.Both the MMS and YPF groups exhibited a significant suppression in transcriptional expression(p<0.05).YPF group showed a better suppression effect on MUC5 AC than the MMS group(p < 0.05).Conclusion:(1)In patients with AR,the most commonly observed TCM constitutions were the specific endowment constitution,yang deficiency constitution and qi deficiency constitution.While patients with TCM Qi deficiency constitution exhibited the most severe manifestations of Qi deficiency syndrome.(2)Regarding therapeutic effectiveness,there was no significant difference observed between YPF and MMS.However,compared to MMS,YPF was superior in improving nasal congestion and non-ocular/nasal symptoms,as well as improving symptoms of patients with Qi deficiency.(3)Both YPF and MMS effectively suppressed TSLP,IL-25 and TGF-β1 levels.YPF had more advantages in down-regulating TSLP and TGF-β1.Both interventions reduced the levels of IL-4 and IL-13,while increasing the level of IFN-γ.There are differences between two interventions used in NALF.(4)Both YPF and MMS significantly improved the level of physical barrier proteins,CLDN-1 and ZO-1,as well as the level of chemical barrier proteins,MUC5 AC.YPF was more advantageous in suppressing MUC5 AC gene transcription.(5)In patients with AR,the species abundance of nasal epithelium microbiota is decreased compared to healthy individuals,with both YPF and MMS stimulating the species abundance.YPF significantly improved the abundance of Peptoniphilus,while MMS improved the abundance of Moraxella. |
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