| Objective:Firstly,this study was conducted to observe the clinical effect of Xiao-qing-long Tang(XQLT)in the therapy of allergic rhinitis(AR),then we used network pharmacology to explore the active ingredients and potential targets of XQLT in treating AR,and to verified the active ingredient Catechin in which comes from in XQLT in the therapy of AR mice finally.Through conducting animal experiments,we aimed to explore the mechanism of XQLT in the treatment of AR,especially its active components whether by regulating specific pathways so that achieve immunomodulatory.Methods:1.Clinical observation:65 cases of moderate or severe persistent AR(PAR)patients who were diagnosed as lung qi deficiency-cold syndrome were recruited and randomly divided into XQLT group(treatment group)with 33 cases and loratadine group(control group)with 32 cases.The treatment group patients were treated with one prescription of XQLT mainly per day,2 weeks totally and herbals in prescription would be adjusted according to the patient’ s different symptoms.Patients of control group were given loratadine tablets orally,10 mg/time and once a day for 2 weeks.Before and after treatment,patient’ s symptoms and physical signs were recorded,individual nasal symptom score(INSS)and total nasal symptom score(TNSS)were also recorded to determine the subjective nasal sensitivity symptoms and degree of disease.Rhinoconjunctivitis Quality of Life Questionnaire(RQLQ)were reported and analysis before and after treatment to evaluate the clinical efficacy of XQLT in the treatment of moderate or severe PAR and its impact on quality of life.2.Network pharmacology study:In order to clarify the effective active ingredients of XQLT in the treatment of AR,and to integrate the target gene of XQLT and AR pathogenic gene,the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Plat form(TCMSP)was used to retrieve the bioactive components and their corresponding targets of Ephedra Herba(Mahuang),Cinnamomi Ramulus(Guizhi),Zingiberis Rhizoma(Ganjiang),Asari Radix Et Rhizoma(Xixin),Schisandrae Sphenantherae Fructus(Wuweizi),Paeoniae Radix Alba(Baishao),Arum Ternatum Thunb(Banxia),and licorice(Gancao)in XQLT.In addition,the AR related genes were searched through the NCBI Gene database,and the protein interaction network of drugs and disease targets was constructed by software to explore its target.3.Animal experiment research:SPF healthy female BALB/C mice with 6-8 weeks old,body weight 18-22g were randomly divided into model group,positive drug group,control group and low,medium and high doses of catechin groups.In addition to the control group,the AR mouse models of all groups were established by intraperitoneal injection combine nasal drop of OVA.After that,mouse of model group and control group were given phosphate buffer solution intragastrically,positive drug group was intragastrically administered with loratadine suspension,mice in the low,medium and high doses of catechin were intragastrically administered with the corresponding dose of catechin.Recorded and reported the behavioral changes of mice including scratching,sneezing and rhinorrhea.After mercy killing,the peripheral blood was used to detect the changes of inflammatory cell content.The thymus and spleen were taken to detect the changes of immune organ index,the nasal mucosa was taken for tissue section,and HE staining was used to observe nasal mucosal inflammation.The expression levels of OVA-sIgE,IFN-γ,IL-5 and IL-13 in serum were detected by ELISA.The protein expression of TSLP,OX40L,JAK2,P-JAK2,STAT5 and P-STAT5 in nasal mucosa was detected by Western Blot.Aim to clarify the material basis and possible immunoregulatory targets of XQLT in the therapy of AR,and to explore the specific pharmacological mechanism of XQLT in the therapy of ARResults:1.Result of clinical study:This study recruited 65 moderate or severe PAR patients who were classified as lung deficiency syndromes and were randomly divided into XQLT group(treatment group)and loratadine group(control group).The symptom score and RQLQ score of patient were recorded before and after 2 weeks of treatment.Among them,the comprehensive score of nasal symptoms in the treatment group decreased from 9.55±1.23 to 4.58±1.28.The comprehensive score of nasal symptoms in the control group decreased from 9.31 ± 1.03 to 4.72±0.96.After 2 weeks of treatment,the comprehensive score of nasal symptoms in the control group was higher than that in the treatment group,but the difference was not statistically significant(P>0.05).In the individual nasal symptom score,the nasal itching score of the treatment group decreased from 2.36±0.49 to 1.12±0.70,the sneezing score decreased from 2.36±0.65 to 1.09±0.72,and the rhinorrhea score decreased from 2.33±0.60 to 1.18 ± 0.64.The nasal congestion score decreased from 2.48±0.62 to 1.18±0.73,the control group’s nasal itching score decreased from 2.16±0.45 to 1.00±0.51,the sneezing score decreased from 2.38±0.55 to 1.31±0.54,and the rhinorrhea score decreased from 2.28±0.58 to 0.97±0.65,the nasal congestion score decreased from 2.50±0.62 to 1.44±0.62.There was no clinical difference in the symptoms of nasal symptoms,nasal itching,sneezing,rhinorrhea and nasal congestion(P>0.05).The RQLQ score of the treatment group decreased by 41.03±14.27 from 71.88±24.21,and the RQLQ score of the control group decreased from 72.16±22.54 to 41.16 ±14.63.There was no significant difference in the RQLQ score between the two groups(P>0.05).2.Results of Network pharmacology analysis:By inquiring the TCMSP database,156 active ingredients in XQLT were screened out,and 299 human target proteins were collected(228 target proteins from Mahuang,40 target proteins from Guizhi,44 target proteins from Ganjiang,106 target proteins from Xixin,4 target proteins from Wuweizi,90 target proteins from Baishao,97 target proteins from Banxia,229 target proteins from Gancao).411 related human genes related to AR were searched by querying NCBI Gene database,Via integrating XQLT target gene and AR target genes,the protein interaction network of drugs and disease targets was constructed by software to explore the target and bioconcentration information,and 48 core targets are selected,and performs GO biological function analysis on core targets.It is found that XQLT can regulate interleukins-related signals,glucose metabolism,inflammatory response,fluid shear stress,cytokine synthesis,immune regulation,Thl7 cell differentiation,substance synthesis and metabolism,oxidative stress response,regulation of cell activation,MAPK cascade regulation,leukocytes involved in immune response biological processes such as activation and production of chemokines regulate AR.3.Out of animal experiments:(1)Catechin relieved nasal sensitive symptoms in AR mice.After intraperitoneal injection of OVA and nasal drop sensitization,the numbers of scratching the nose and sneezing in AR mice increased significantly,however,after treating by catechin,the events of scratching the nose and sneezing were reduced(P<0.05),and the nasal sensitivity symptoms in AR mice were relieved.(2)Catechin reduced the levels of inflammatory cells in peripheral blood of AR mice.The levels of leukocytes,monocytes,granulocytes and lymphocytes in the peripheral blood of AR mice were significantly higher than those in the control group(P<0.05).High doses of catechin reduced the levels of leukocytes,monocytes,granulocytes and lymphocytes in the peripheral blood and alleviated inflammation.(3)Catechin could improve the immune function of AR mice.The spleen and thymus index of AR mice are lower than those of the control group(P<0.05),and catechin could increase the spleen index of AR mice(P<0.05).The thymus index was improved,but it was not statistically significant(P>0.05).(4)Catechin could alleviate local nasal mucosal inflammation in AR mice.HE staining results showed that a large number of inflammatory cells infiltrated in the model group,and the integrity of the mucosa was destroyed,cilia rupture,and local tissue edema.After catechin intervention,the inflammatory cell infiltration was significantly reduced compared with the model group,the mucosa was recovered,and the cilia density was slightly improved,but it was difficult to return to the nasal mucosa of the normal control group.(5)Change of serum inflammatory factors OVA-sIgE,IFN-y,IL-5 and IL-13:Serum OVA-sIgE and Thl/Th2 cytokines were detected by ELISA,OVA-sIgE,IL-5,IL-13 in the serum of AR model mice was found increase significantly(P<0.05),IFN-y in the serum of AR model mice decreased significantly(P<0.05),and results showed that high dose of catechin could reduce serum OVA-sIgE Levels(P<0.05);catechin increased IFN-y and decreased IL-13 in serum(P<0.05);medium dose catechins decreased expressions of IL-5 in serum(P<0.05).(6)Catechin inhibits TSLP-OX40L pathway in nasal mucosa.Western blotting was used to detect the expression of TSLP and OX4OL proteins in TSLP-OX4OL pathway in nasal mucosa.The results showed that the TSLP-OX4OL pathway was activated in the nasal mucosa of the AR model group,at the same time,the expression of TSLP and OX4OL protein was increased.The activation status of cytokines could be changed after catechin intervention with dose dependent.(7)Catechin inhibits the expression of JAK2,P-JAK2,STAT5 and P-STAT5 proteins in nasal mucosa.Western blot analysis of JAK2/STAT5 and its phosphorylated protein expression in JAK2/STAT5 pathway in nasal mucosa.The results showed that the expression levels of JAK2 and STAT5 in the nasal mucosa of AR model group were significantly higher than those in the control group.The catechin could not only inhibit the expression of total protein,but also inhibit its phosphorylation protein.Conclusion:1.XQLT is efficacious and safe for moderate or severe PAR patients who were diagnosed as lung qi deficiency-cold syndromes.2.Through the network pharmacology method,the mechanism and material basis of XQLT in the therapy of AR were explored.It was found that XQLT can regulate various biological processes.Among them,the highest level of enrichment is the interleukin-related signaling pathway.We are concerned with interleukin-related signaling pathways.It is found that component of XQLT,Ma huang and Gui zhi are rich in catechin,which is also rich in the blood components of XQLT by detecting liquid-mass spectrometry(HPLC-MS).Catechin can inhibit the release of inflammatory factors by inhibiting the NF-κB/TSLP pathway,however,the specific regulation mechanism is unknown,which inhibits the release of inflammatory factors by TSLP,may this provides an ideas for subsequent animal experiments in future.3.Catechin may inhibit the phosphorylation of JAK2/STAT5,block the TSLP-OX4OL signaling pathway,reduce the level of Th2 cytokines,and restore the immune balance of AR,exerting therapeutic effects on AR.4.In the material basis of XQLT for the treatment of AR,catechin may play a certain role. |
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