| Objective:This paper mainly studied the clinical observation and mechanism of traditional Chinese medicine Gubiao Jiedu Ling granule in the treatment of children with allergic rhinitis(AR).The first part is the theoretical study of traditional Chinese medicine Gubiao Jiedu ling;The second part is to study the mechanism of Gubiao Jiedu Ling in the treatment of children with allergic rhinitis by network pharmacology.The third part is the clinical study of Gubiao Jiedu ling granule in the treatment of children with allergic rhinitis;In the fourth part,an OVA rat model was used to study the mechanism of Gubiejiedu ling in the treatment of allergic rhinitis in children.To preliminarily explore the possible new targets of traditional Chinese medicine Gubiao Jiedu Ling in the treatment of children with allergic rhinitis,and to provide clinical and experimental basis for further promoting the clinical innovative application development and promotion of the hospital classic preparation.Method:1.Theoretical research: Referring to the four classics and other ancient Chinese medical books,inheriting and excavating the traditional Chinese medicine theory and clinical application experience,summarizing the history of TCM rhinopathy,the etiology and pathogenesis of TCM,the treatment rules and prescriptions.Chinese Biomedical Literature database,Wanfang Data platform,VIP journal,CNKI,Nature,Cochrane Library,Pubmed and Science Direct platform databases were searched from November 30,2020 to December31,2021.To comprehensively collect and summarize the western history of allergic rhinitis,the etiology mechanism and treatment of western medicine,the related theories of traditional Chinese medicine group prescription and single drug,and the research progress of modern pharmacy,so as to provide a basis for the theoretical research of traditional Chinese medicine and western medicine in children with AR.2.Network Pharmacology Research:The active ingredients with OB≥30% and DL≥0.18 of the eight traditional Chinese medicines(wild chrysanthemum,Astragalus membranaceus,Isatidis root,Paratracurium,forsytha,Angelica dahurica,Atractylodes atractylodes,and Magnolia)were searched through TCMSP,and the corresponding target information of each active ingredient was found.gene symbol corresponding gene names were subsequently obtained after Uni Prot standardization of potential targets of active ingredients.Ar-related targets were searched in Drug Bank,Pharm Gkb,Genecards,OMIM and TTD databases.R 4.2.1 language and Venn were used to obtain the intersection genes between the traditional Chinese medicine targets of Gubiao Jiedu Ling and AR.The "Gubiao Jieduling-active ingredients-disease targets" network diagram can be obtained by using the intersection genes as a bridge and Cytoscape3.9.1 to construct a visual network.The PPI protein interaction network was constructed by String,and the core PPI network was constructed by running the plug-in Cyto NCA to obtain the core targets of Gubiao Jiedu ling in the treatment of AR.GO function and KEGG pathway enrichment analyses were performed using Bioc Manager,and their results were visualized to obtain bar and bubble plots.The signal pathway map was obtained by manual screening according to the correlation.The core gene ligand 2D was downloaded from Pub Chem for ligand 3D structure by Chem3 D transformation.The ID of the core gene was found in Uni Prot database,the protein 3D structure was searched in PDB database,and the protein receptor 3D structure was obtained by deleting water molecules and small molecule ligands with Py MOL2.5.4.Auto Docks Tools1.5.7 software was used to hydrogenate the active pocket.Auto Dock Vina software was used to complete the molecular docking of the receptor and ligand.3.Clinical controlled study: This study was a prospective and open controlled study.The children with mild intermittent allergic rhinitis diagnosed as lung meridian occult heat syndrome in traditional Chinese medicine were selected as the research objects.A total of 60 children aged 3-10 years who met the diagnostic criteria of AR in traditional Chinese medicine and western medicine were recruited.The treatment group(n = 30)was treated with traditional Chinese medicine Gubiao Jiedu ling granules twice a day after meals,and the control group(n = 30)was treated with cetirizine hydrochloride drops once a day for 4 weeks.Firstly,the age,gender and symptom score of the two groups were compared.Then,the clinical symptoms and secondary symptoms of children with AR were observed and recorded before and after the treatment of traditional Chinese medicine Gubiao Jieduling and cetirizine hydrochloride drops,respectively.The VAS score was recorded,and the influence of AR on the quality of life of children before and after the treatment was observed and recorded,and the RQLQ questionnaire was filled out.Then the eosinophil count and percentage,IL-6,IL-8 and TNF-ɑ of AR children before and after4 weeks of treatment were compared.Then the total effective rate and the recurrence after 4 weeks of drug withdrawal were compared between the two groups.Finally,the adverse reactions of the two groups were observed.To evaluate the clinical efficacy and safety of traditional Chinese medicine Gubiejiedu ling in the treatment of children with allergic rhinitis and its correlation with eosinophils and inflammatory cytokines.4.Animal experiments: 40 SPF grade rats were selected and randomly divided into blank control group(group B,10 rats)and modeling group(group M,30 rats).AR rat models were subsequently replicated by intrabitoneal sensitization with ovalbumin OVA and stimulated by nasal drip.After successful modeling,30 rats in the modeling group were divided into 3 groups according to random number method: model control group(MC group,10 rats);Gubiao Jiedu Ling TCM observation group(MZ group,10 PCS);Cetirizine hydrochloride drops Western medicine control group(MX group,10 PCS).The blank control group was fed normally until the end of the experiment.Model control group was given normal saline 0.9% Nacl intragastric administration.Traditional Chinese medicine observation group and western medicine control group were given Gubiao Jiedu Ling granules and cetirizine hydrochloride drops,respectively.The changes of symptoms of rats in each group were observed and recorded.Nasal mucosa sections of rats were taken for microscopic examination,and the inflammatory manifestations and statistical results of eosinophils in each group were observed and compared by HE staining.Serum levels of IL-4,IL-13,IL-37,CCL-11 and Ig E were determined and compared by ELISA.Result:1.Theoretical research results:Allergic rhinitis in children belongs to allergic rhinitis of traditional Chinese medicine.The pathogenesis of allergic rhinitis in children is congenital deficiency of lung qi,which leads to spleen dysfunction caused by excessive fullness and excessive warmth,easy to external influence heat,leading to deficiency of lung qi,latent heat of lung meridians,and stagnation of pathogenic toxin in nasal cavity.Western medicine believes that it is closely related to genetics,environment and diet.After children with specific constitution are exposed to allergens,it is mainly caused by Ig E mediated,a variety of vasoactive mediators and inflammatory cytokines.Although western medicine in the treatment of children with allergic rhinitis is immediate,there are many complications and side effects,which are easy to be delayed and repeated.Traditional Chinese medicine Gubiao Jiedu Ling granule has good efficacy and safety in the treatment of children with AR,and it is not easy to relapse.Zhang Jiemei,a master of traditional Chinese medicine,added and modified the classic formula Yupingfeng powder to form Gubiao Jiedu Ling,which has the effect of beneficial qi,tonifying lung,clearing heat and promoting orifices.Modern pharmacological studies have shown that the traditional Chinese medicine Gubiao Jiedu Ling granule has anti-allergic,anti-inflammatory,immune regulation and anti-virus effects.2.Results of network pharmacology studies showed that there were 162 active ingredients in eight herbs of Gubiao Jiedulinggranule from TCMSP,including 22 forsythus forsythae,39 Isatidis root,12 wild chrysanthemum flower,20 astragalus membranaceus,21 paratractylodes,7 Atractylodes atractylodes,19 Astragalus membranaceus and 22 angelica dahurica.A total of 1163 target proteins were predicted by these components.207 target genes were obtained by Uni Prot normalization.A total of 2044AR-related targets were obtained through five databases.A total of 97 intersection genes between Gubiejiedu ling and AR were obtained by R and Venn.Cytoscape 3.9.1 software was used to construct the "Gubiao Jiedu Lining-active ingredient-target" network consisting of 97 intersection genes,including 152 nodes and 423 interactions.The intersection genes were entered into String to construct PPI network map and PPI core network map,and7 core gene targets were obtained,namely TNF,MAPK14,MAPK1,RELA,TP53,HSP90AA1 and ESR1.The core active ingredients of Gubiaojiedu ling in the treatment of AR are wogonin baicalein,kaempferol,luteolin,and quercetin.GO functional enrichment analysis showed that Gubiao Jiedu Ling was mainly involved in the response to lipopolysaccharide and response to molecule of bacterial in the treatment of AR origin response to metal ion response to metal ion response to xenobiotic stimulus response to xenobiotic stimulus response to nutrient levels to nutrient levels,response to oxygen levels,and response to reactive oxygen species.KEGG pathway enrichment analysis obtained 162 signaling pathways,and the treatment of AR with Gubiao Jiduling was mainly involved in IL-17 signaling pathway,TNF signaling pathway,PI3K-Akt signaling pathway,NF-KB signaling pathway,AGE-RAGE signaling pathway,TLR signaling pathway and HIF-1 signaling pathway.Finally,Baicalein,the core active ingredient of Gubiejiedu Ling,was selected for molecular docking with the core target TNF,and the results showed that it had high binding activity.3.Clinical research results: A total of 67 children with AR were included in this study,and 7 cases(4 males and 3 females)were dropped out.Finally,60 cases(32 males and 28 females)were actually completed.There were 30 cases in the treatment group and30 cases in the control group.There were no significant differences in gender,age,and disease scores between the two groups before treatment(P>0.05).The total efficacy rates of the treatment group and the control group were 93.33% and 96.66%,respectively,and there was no statistically significant difference(P>0.05).The VAS scores of AR main symptoms(sneezing,nasal congestion,nasal itching,runny nose)secondary symptoms(spontaneous sweating,cough,dry throat,pharyngeal itching)in the two groups before and after treatment were significantly lower than those before treatment,P<0.05 was statistically significant.The RQLQ scores of AR children after treatment in the two groups were significantly lower than those before treatment,P<0.05 was statistically significant.The eosinophil count and percentage in peripheral blood of AR children after treatment were significantly lower than those before treatment in the two groups,P<0.05 was statistically significant.After treatment,the serum levels of IL-6,IL-8 and TNF-ɑ in the two groups were significantly lower than those before treatment,P<0.05 was statistically significant.The treatment group was better than the control group in the recurrence after 4weeks of drug withdrawal,P<0.05 was statistically significant.All the children with allergic rhinitis treated with Gubiao Jieduling granules did not show any obvious adverse reactions.Two patients in the control group had uncomfortable symptoms of enuresis,and the symptoms of enuresis disappeared after drug withdrawal for 1-2weeks.There were no abnormalities in blood and urine routine,electrocardiogram,liver and kidney function,and liver and kidney color Doppler ultrasound before and after treatment.4.Results of animal experiment: The symptom scores of 30 model rats were all above 5 points.Rats in the blank control group(0.174±0.038)had no obvious allergic symptoms and had good mental appetite activity,which had statistical significance compared with those in the modeling group(6.410±1.325)(P<0.01).The model control group showed the most obvious allergic symptoms,including irritability,frequent sneezing,scratching nose and face,runny nose,wheezing,etc.The score was(6.110±1.125),and the symptoms of the model control group were statistically different from those of the traditional Chinese medicine group and Western medicine group(P<0.05).The allergic symptoms of rats in Chinese medicine group(1.815±0.406)and western medicine group(1.663±0.264)were significantly reduced,and there was no significant difference between Chinese and Western groups(P>0.05).HE staining results:Nasal mucosa of blank control group was arranged neatly,and no inflammatory reaction was observed.In the model control group,nasal mucosa inflammation was the most severe,with mucosal epithelial detachment,lamina propria vascular congestion,tissue edema,and significant eosinophil infiltration.The nasal mucosal inflammation of rats in Chinese medicine group and western medicine group was significantly reduced,and there was no significant statistical difference between the two groups.The number of eosinophils in nasal mucosa of blank control group was the lowest(1.87±0.92),and there were statistical differences compared with the other three groups(P<0.01).The number of eosinophils in nasal mucosa of rats in model control group was the highest(12.07±6.25),which was statistically significant compared with that in Chinese medicine group and Western medicine group(P<0.05).The number of eosinophils in the Chinese medicine group(4.91±1.41)and western medicine group(5.16±0.37)was significantly reduced,and there was no significant statistical significance between the two groups(P>0.05).Reichjimsa staining: No eosinophils were found in the nasal lavage solution in the blank control group(5.1±1.2),and there was a statistical difference compared with the other three groups(P<0.01).The maximum number of eosinophils in nasal lavage solution of rats in model control group(91.3±1.6)was also statistically different from that in Chinese medicine group and Western medicine group(P<0.05).Eosinophils in nasal lavage of rats in Chinese medicine group(35.2±0.5)and western medicine group(30.7±1.1)were significantly reduced,and there was no statistical difference between the two groups(P>0.05).The results of serum inflammatory cytokines in each group were compared with those in the blank control group(Ig E: 10.61±1.76,IL-4:11.86 ±2.84,IL-13:8.82 ±1.23,CCL-11:21.36 ±6.55,IL-37: 47.21±8.76),Serum Ig E(31.04±5.49 in the model control group,17.71±4.11 in the traditional Chinese medicine group,15.41±5.16 in the western medicine group),IL-4(33.12±4.18 in the model control group,16.91±3.79 in the traditional Chinese medicine group,18.13±2.94 in the western medicine group),and IL-13(30 in the model control group).The levels of 25±3.51,23.21±1.38 in the Chinese medicine group,21.43±3.07 in the western medicine group,and CCL-11(96.48±16.17 in the model control group,44.33±6.63 in the Chinese medicine group,41.43±3.07 in the western medicine group)were significantly increased.The levels of IL-37(15.83±5.08 in model control group,35.94±5.27 in TCM group and 24.45±5.55 in western medicine group)were significantly decreased,with statistical differences(P<0.01).Compared with model control group,Ig E,IL-4,IL-13 and CCL-11 levels in serum of rats in both Chinese and western groups were decreased to varying degrees after treatment,while IL-37 content was significantly increased,with statistical significance(P<0.05).There were no significant differences in serum Ig E,IL-4,IL-13 and CCL-11 between TCM group and western medicine group(P>0.05).Compared with Western medicine control group,the serum content of IL-37 in TCM observation group was significantly increased,with statistical significance(P<0.05).Conclusions:1.Through the network pharmacological research,the mechanism of Gubiao Jiedu Ling to play its role in the treatment of AR is realized by multi-target and multi-pathway.It acts on AR through pharmaceutical active ingredients such as baicalein,kaempferol,luteolin,and quercetin.It can also regulate the molecular network around core target genes,such as TNF,MAPK14,MAPK1,RELA,TP53,HSP90AA1,and ESR1.The specific mechanism may be related to the interaction among IL-17,NF-KB,TNF,TLR and AGE-RAGE signaling pathways.2.Compared with western medicine,traditional Chinese medicine Gubiao Jiedu Ling granule has better long-term clinical efficacy and safety in the treatment of mild intermittent pediatric allergic rhinitis(lung meridian latent heat type).3.Gupyujidueling granules can promote the newly discovered inflammatory suppressor of IL-1 family(IL-37),inhibit Ig E,Th2cytokines(IL-4,IL-13)and chemokines(CCL-11),and thus reduce the immune response of Th2 cells in AR model rats.Inhibit eosinophil inflammatory infiltration,reduce the production of inflammatory transmitters,and ultimately inhibit allergic inflammatory response.Therefore,IL-37/ IL-4 /IL-13/CCL-11 signaling pathway can be used as a new target to study the mechanism of TCM treatment in children with allergic rhinitis. |
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