A Multicenter Clinical Study Of Modified Yupingfeng Nasal Spray In Controlling The Recurrence Of Allergic Rhinitis And Its Mechanism Verification

Objective:Allergic Rhinitis(AR)is one of the most common chronic and Allergic diseases,with a prevalence of up to 50% in some countries.Since the 1960 s,the prevalence of AR has been on the rise worldwide,and even a steep increase in recent years.Recurrent episode of AR is one of the leading illnesses that affects patients.A study of 7-year-olds in South Korea found that the two-year recurrence rate of AR was 72%.Although drug therapy for AR can quickly control immune inflammation and relieve immediate symptoms.Few pharmacologic treatments for AR have been studied and evaluated for their effectiveness in controlling relapse.In the long-term clinical experiences,Professor Tian summarized the clinical evidences of AR patients.She proposed the syndrome of Qi deficiency and blood stasis(QDBS)as the core TCM syndrome of AR recurrence,and created the modified Yupingfeng nasal Spray(MYN).Intranasal administration not only has the advantages of overall regulation of multi-components and multi-targets of traditional Chinese medicine,but also a superior way of administration for nasal diseases,which is an innovative use of classic formulations.In the previous clinical application and research,it was found that MYN can effectively control the immediate symptoms of AR and has obvious advantages in controlling the recurrence.However,the efficacy of MYN in controlling the recurrence has not been studied specifically,and its mechanism of action is still unclear.Therefore,the 3-month recurrence rate was used as the primary outcome in this research to study the effecacy of MYN on controlling AR recurrence.Then the possible mechanism of MYN in the treatment of AR was screened by network pharmacology.Finally,the mechanism of MYN screened by network pharmacology was verified in clinical subjects’ sample and animal models.Methods:Chapter 1:A total of 126 patients diagnosed as perennial and moderate to severe AR(QDBS)by Western medicine from September 2021 to February 2022 in the Teaching Hospital of Chengdu University of Traditional Chinese Medicine,the Sichuan Provincial Hospital of Integrated Traditional Chinese and Western Medicine,and the West China Hospital of Sichuan University were selected for this study.This study has been approved by ethics committees of all medical centers.The random sequences were generated using SAS9.4 software from a given seed and appropriate block length and sealed in opaque envelopes by statisticians who were not involved in the actual conduct of the trial.After the beginning of the study,the envelopes were opened in order of grouping,and recruited subjects were assigned to treatment groups according to the information in the envelope.Subjects were randomly assigned to receive the modified Yupingfeng nasal spray or mometasone furoate aqueous nasal spray(NSN).When subjects’ Rhinitis Control Assessment Test(RCAT)score was >21 for two weeks,they stoped taking the medication and entered the follow-up(The shortest treatment time was not less than four weeks,and the longest treatment time was not more than three months.).Once a relapse occurs,the time point will be recorded,and the follow-up stops.The primary outcome was the three-month recurrence rate of AR.The secondary outcomes were the RCAT score,the duration of follow-up,and questionnaires to evaluate symptoms,signs,and quality of life.Chapter 2:Drug targets datasets were obtained using the ETCM database by inputting herbs in MYN prescription(Astragalus membranaceus var.mongholicus(Bunge)P.K.Hsiao,Atractylodes macrocephala Koidz.,Saposhnikovia divaricata(Turcz.)Schischk.,Ligusticum sinense ’Chuanxiong’,etc.).Then Dis Ge NET database and Gene Cards database were used to screen out the disease genes datasets of AR.Cytoscape software was used to capture the disease gene network in the HINT background network and perform network topology analysis on the disease gene network.The direct and indirect targets of MYN in the treatment of AR were captured in the disease gene network,and the core targets of MYN formulation on AR were screened out.GO and KEGG enrichment analysis were performed on the core targets to predict the possible key mechanism of MYN controlling AR.Chapter 3:In the clinical study,a subgroup of 18 subjects were asked to participate in an optional study,in which the epithelial cells of the nasal mucosa was extracted with a nasal brush,and the serum and nasal lavage fluid were collected to measure the mechanism indicators.These samples were collected at baseline and after drug withdrawal.(1)The protein expression levels of TLR4,My D88,and TRIF in the nasal mucosa were measured by western blotting.(2)The m RNA expression levels of TLR4,My D88,TRIF,TRAF6,TRAF3,claudin1 and ZO-1 were measured by Real-time PCR.(3)The levels of TNF-α,NF-κβ,IL-6,IRF3 and IRF7 in nasal lavage fluid and serum were measured by ELISA.To explore the mechanism of MYN regulating claudin1 and ZO-1 through TLR4-MYD88 /TRIF pathway.Results:Chapter 1:1.Baseline: There were no statistically significant differences in age,height,weight,BMI,gender,ethnicity,native place,residence place,education level and occupation between group A and B(P >0.05).The baseline was consistent and comparable between group A and B.2.Assessment at drug withdrawal:(1)The effective rate was 77.8% in group A and 87.3% in group B.There was no significant difference in the effective rate between the two group by Chi-square test /Fisher exact tests(P>0.05).There were no significant differences in the total scores of RCAT,RQLQ,TNSS and TOSS scales between group A and B at the time of drug withdrawal by t-test /Wilcoxon rank sum test(P>0.05).There was no significant difference in TNSS symptoms(sneezing,runny nose,nasal congestion,and nasal itching)and TOSS symptoms(eye itching,tears,redness and swelling)between group A and B at the time of drug withdrawal(P>0.05).The AR control status was consistent in the two group at the time of drug withdrawal,and the subsequent recurrence assessment was comparable between group A and B.3.Assessment at recurrence:(1)The 3-month recurrence rate of AR in group A and group B was 55.1% and 76.4%,respectively.The 3-month recurrence rate of AR in group A and B was significantly different measured by Chi-square test /Fisher exact tests(P<0.05).(2)Cox proportional hazards model analysis showed that MYN was a protective factor for AR recurrence(HR=0.57,95%CI: 0.34~0.97,P=0.037).Gender,age and BMI were not correlated with the recurrence of allergic rhinitis(P>0.05).(3)Univariate survival analysis of 3-month AR recurrence shows: I.The recurrence rate of group A and B increased over time,with a steeper trend in the first 45 days and a gentle trend after 45 days.II.The difference in recurrence rate between group A and B gradually widened after one month.III.The increasing trend of relapse rate over time in group A was more gradual than that in group B.(4)I.The t test /Wilcoxon rank sum test was used to compare the RCAT,RQLQ,TNSS,TOSS,TNSS symptoms(sneezing,runny nose,nasal congestion,and nasal itching)and TOSS symptoms(eye itching,tearing,redness and swelling)between group A and B when AR relapse.There was no significant difference in the score of each scale between group A and B(P>0.05).(5)The total scores of RQLQ scale at recurrence in group A and B were compared with those before treatment by t test /Wilcoxon rank sum test.The total score of RQLQ scale in recurrent group A and B was significantly lower than that before treatment(P<0.01).Chapter 2:(1)A total of 382 drug targets of MYN were screened by ETCM database.(2)885 AR genes were screened by Genecards database and Disgenet database.(3)The Cytoscape software was used to analyze the disease genes and drug targets,and 17 core targets of MYN for the treatment of AR were obtained.They were TLR4,MAPK14,STAT3,TGFBR1,ESR1,MYC,STAT1,MAPK1,EGFR,ERBB2,SYK,RELA,AKT1,HDAC1,JUN,SMAD3 and CASP8.(4)GO enrichment analysis showed that MYN may regulate the body’s immunity,inflammation and oxidative stress by regulating gene expression,transcription,protein synthesis and modification,so as to control allergic rhinitis.(5)KEGG pathway enrichment analysis showed that MYN may control AR by regulating Toll-like receptor signaling pathway.Chapter 3:(1)RT-PCR test results: There were significant differences in TRIF m RNA gene transcription levels between group A and B after treatment(P<0.05).After treatment,TRIF m RNA gene transcription in group B was significantly inhibited compared with that before treatment in group B(P<0.01),while there was no significant difference between group A before after treatment(P>0.05).After treatment,the m RNA levels of TLR4 and TRAF3 in group A were significantly lower than those before treatment(P<0.05).After treatment,TLR4 m RNA,My D88,TRAF6 and TRIF in group B were significantly decreased compared with those before treatment(P<0.01).ZO-1 m RNA in group A and B was significantly increased after treatment compared with that before treatment(group A: P<0.05;Group B(P<0.01).After treatment,Claudin1 m RNA in group A and B also increased compared with that before treatment,but there was no significant difference(P>0.05).(2)Western blotting test results: The protein expressions of TLR4,My D88 and TRIF in group A were significantly decreased after treatment compared with those before treatment(P<0.01).After treatment,the protein expressions of TLR4 and My D88 in group B were significantly decreased compared with those before treatment(P<0.01),and the protein expression of TRIF was significantly decreased compared with that before treatment(P<0.05).There were no significant differences in TLR4,My D88 and TRIF protein expressions between group A and B after treatment(P>0.05).(3)ELISA test results: Serum: After treatment,the expression of IL6 in group A was significantly decreased compared with that before treatment(P<0.01),and the expression of IRF3 and NF-κB were significantly decreased compared with that before treatment(P<0.05).There was no statistical difference in each index between group A and B after treatment.Nasal lavage fluid: After treatment,the levels of IL-6 in group A and B were significantly lower than those before treatment(P<0.01),and the levels of NF-κB and TNF-α were significantly lower than those before treatment(P<0.05).There was no statistical difference in each index between group A and B after treatment.Conclusion:Chapter 1:1.MYN can effectively control the 3-month recurrence rate of AR,which has obvious advantages compared with NSN.MYN is a protective factor for the recurrence of allergic rhinitis.2.MYN and NSN have similar efficacy in controlling the immediate symptoms of AR.3.After standardized use of MYN or NSN,even if AR relapse,the symptoms of relapse are milder than those before the administration of MYN or NSN.Chapter 2:1.MYN’s direct and indirect targets can cover the key disease genes of AR pathogenesis indicated that MYN is a treatment suitable for the pathogenesis of AR.2.TLR4-My D88/TRIF signaling pathway may be one of the key pathways to control AR recurrence.Chapter 3:1.MYN may control AR recurrence by regulating TLR4-MYD88/TRIF-epithelial barrier axis.2.The difference in inhibition of TRIF m RNA between MYN and NSN may be one of the reasons for the difference in recurrence rate between MYN and NSN.