Study On The-mechanism Of The Regulation Of Immune Factors And Immune System Related Pathway In Asthma Rats By Baijiaozi Powder Acupoint Application

Objective:Based on the previous research,this experiment intends to establish a gene function expression profile database from the perspective of the regulation of Baijiazisan on immune factors in asthmatic rats,and to screen the pathways related to the immune system of asthmatic rats by whole transcriptome sequencing.The acupoint sticking of scattered points provides experimental basis for the treatment of asthma.Methods:First,pre-experiment was carried out to observe the pathological changes of lung tissue through pathology.Observation of lung tissue in the model group under the microscope revealed a large number of infiltration of inflammatory cells,and the presence of alveolar and interstitial hyperemia and edema,which proved the success of modeling.Then conduct a formal experiment.36 male clean SD rats,4-6 weeks old,body weight(150± 20),were divided into 5 groups according to the balanced random method(stratified by body weight),which were normal group(A group)and model group(B Group),the administration group is divided into three different dose groups: high-dose administration group(group C),intermediate-dose administration group(group D),low-dose administration group(group E),each group of experimental animals 6 Only 6 rats are left for use.On the first day,the model group and three different dosage groups were divided into four groups: freshly prepared OVA,100 mg supplemented with aluminum hydroxide gel 200 mg and physiological saline 1ml suspension in both inguinal and hindfoot plantar of rats A total of 4 points were injected subcutaneously,0.2ml at each point,and 0.2ml was injected intraperitoneally.The normal group did not do anything.On the 15 th to 24 th day,the three administration groups began to apply white mustard patch application.Before the point application,8% sodium sulfide was used to remove the hair at the acupuncture point.At Yu(double),Dingchuan,Fuzhong,and Jiaohuang(double),apply once a day for 2 to 4hours each time.After the application treatment,place the sensitized rats in the drug administration group in a closed container with ultrasound The nebulizer inhales the suspension of 1% OVA physiological saline for 30 minutes every day,and stimulates and induces asthma once for 10 consecutive days.The remaining 6 in the model group were applied blankly,and the treatment methods were the same as those in the administration group.The normal group had free access to regular feed and water.On the 25 th day,the ratswere sacrificed,lung tissue samples were collected for pathological detection,RNA extraction and identification,whole transcriptome sequencing detection,and screening of the immune system-related pathways in asthmatic rats.Results:1.Observing the pathological slice under the microscope,it can be found that the alveolar structure of the normal group is normal,and the thickness of the alveolar wall is normal;the model group shows that the alveolar wall structure is obviously damaged,a large number of mucosal epithelial cells are shed,and the mucus plug secretion is increased,which is consistent with asthma.Pathological manifestations,which proves the success of modeling.The model group was compared with the three administration groups: the bronchial lumen of the high-dose group was not significantly stenotic,and the inflammatory cell infiltration in the alveolar area was also significantly reduced;Alleviation occurred;the alveolar cavity of the low-dose administration group was slightly larger than that of the model group,and the degree of inflammatory cell infiltration was alleviated compared with the model group.By comparison,the symptoms of Baikangzisan's high-dose group were significantly improved after treatment;the low-and medium-dose groups also improved,but the effect was not as good as the high-dose group.From this,it can be seen that Baikuazizi acupoint application has obvious therapeutic effect on asthma,and the treatment effect is better in the high-dose group.2.Measure the gray value of MMP-9 in the lung tissue of each group of rats,and analyze the experimental results: compared with the normal group,the model The gray value of MMP-9 in the lung tissue of the type group rats was significantly reduced(P<0.001).Compared with the model group,the gray value of MMP-9 in the lung tissue of the high,medium and low dose groups of the drug administration group was significantly increased(P <0.01);the IL-4 gray value of the lung tissue of each group was determined Analysis of results: Compared with the normal group,the IL-4 gray value of the lung tissue of the model group was significantly reduced(P <0.001).Compared with the model group,the IL-4 gray value of the high,medium and low dose groups of the drug administration group was significantly increased(P <0.001);the positive expression analysis of TGF-?1 in the lung tissue of rats in each group: compared with the normal group,the model The gray value of TGF-?1 in the lung tissue of rats in the group decreased significantly(P <0.001).Compared with the model group,the gray value of TGF-?1 positive expression in the high-dose group of the drug administration group(P <0.001),the gray value of TGF-?1positive expression in the middle-dose group of the drug administration group(P <0.05),and the low dose of the drug administration group The gray value of TGF-?1 in the group was not statistically significant compared with the model group.3.Take RNA from rat lung tissue and perform full-transcription gene sequencing.Through full-transcriptome data,401,381,and 402 differentially expressed mRNAs were found in the high,middle,and low dose groups,respectively,and the resulting differential mRNA was further passed through trend analysis.Screening by trend analysis,we obtained206 genes whose expression changes continuously with the change of dose,and performed enrichment analysis of GO and KEGG.Through conditional screening,the functional items mainly involved in the treatment of asthma by Baiguanzi Powder mainly include cofactor metabolic processes,anatomical morphogenesis,immune system processes,and apoptosis,and Baiguanzi Powder mainly uses IL-17 signaling pathway and hematopoiesis in the immune system 18 related pathways,such as cell lineage,Th17 cell differentiation,Toll-like receptor signaling pathway,NOD-like receptor signaling pathway,Th1 and Th2 cell differentiation,are used to treat asthma.Conclusion:1.Asthmatic rats have significant changes in behavior and pathology,revealing that Bai Jie Zi San acupoint application has significant effects on asthma treatment.2.The gray value of positive expression of MMP-9,IL-4 and TGF-? in the lung tissue of rats was detected.The values of MMP-9,IL-4 and TGF-? in the lung tissue of asthmatic rats were all increased,and the points of white mustard seeds were scattered.After the application,all three indicators showed a downward trend,and the three indicators in the high-dose administration group had the largest declines,indicating that Baijiezi Powder was effective in the treatment of asthma,and the high-dose group had a better treatment effect.3.Enrichment analysis of GO function by analyzing the full-transcription gene sequencing data of rat lung tissue: The functional items mainly involved in the treatment of asthma by Baiguanzi Powder mainly include cofactor metabolism process,anatomical structure morphogenesis,immune system process,cell apoptosis,etc.;KEGG enrichmentanalysis: The white mustard powder is mainly related to 18 aspects of the immune system through IL-17 signaling pathway,hematopoietic cell lineage,Th17 cell differentiation,Toll-like receptor signaling pathway,NOD-like receptor signaling pathway,Th1 and Th2 cell differentiation Access to asthma.