| Objective:To collate the medical cases of bronchial asthma treated by masters of national medicine,to summarize the characteristics of the distribution of evidence elements in asthma patients and the rules and characteristic ideas of Chinese herbal medicine dispensing in the treatment of asthma by masters of national medicine.To explore the potential mechanism of action of the hemp peony and asthma pacifying formula for asthma treatment by means of network pharmacology and bioinformatics,and to verify the related mechanism by cellular experiments and setting up an MSPC drug-containing serum intervention lipopolysaccharide(LPS)-induced airway epithelial 16 HBE cell model.Methods:1.Medical cases of four national medical masters treating asthma were retrieved from China Knowledge Network(CNKI),Wanfang Data Knowledge Service Platform and Vipshop Journal Resource Integration Service Platform(VIP)databases,and evidence elements and Chinese herbal medicines were standardized and processed The cases were saved as xlsx and imported into SPSS Modeler 18.0 and SPSS 22.0software for analysis,and frequency analysis was performed on the evidence elements and traditional Chinese medicine,and association rules and systematic clustering were performed on high-frequency traditional Chinese medicine to summarize the rules of medication used by the masters in the treatment of asthma.2.Through GEO database,we obtained differentially expressed genes(DEGs)of asthma patients and normal groups;through TCMSP database,we obtained the main active ingredients and target genes of MSPC formula,and took the intersection of disease difference genes and herbal target genes as downstream key genes.The key genes were analyzed for protein interaction(PPI),GO,and KEGG enrichment to study the main pathways and targets of MSPC formula to prevent and treat asthma.Human bronchial epithelial cells 16 HBE were selected as the study target,and the cell proliferation and activity assay(CCK-8)method was used to detect the effect of LPSinduced 16 HBE activity and the effect of hemp peony asthma-containing serum on16 HBE cell activity;the LPS-induced 16 HBE cells with suitable conditions were screened by CCK-8 method,and the tumor necrosis factor-α(TNF-α)content was determined to identify The cells were divided into normal group,LPS group,LPS+MSPC group,LY294002+LPS group,LY294002+LPS+MSPC group,740YP+LPS group and 740Y-P+LPS+MSPC group.The changes of autophagic vesicles and ultrastructure of the cells were observed by transmission electron microscopy;protein expression levels of cellular PI3 K,phosphorylated PI3 K,p-PI3 K,Akt,p-Akt,mTOR,p-mTOR and microtubule-associated protein 1 light chain 3B(LC3B)were detected by protein immunoblotting assay(Western blot);enzyme-linked immunosorbent assay(ELISA)The expression levels of the inflammatory factors interleukin(IL)-5,IL-6,TNF-α and IL-10 were detected in each group.Results:1.A total of 176 prescriptions and 236 herbal drugs were included.The highfrequency drugs were ephedra,licorice,bitter almond,perilla,scutellaria,shegan.The drugs are most often warm,followed by cold,then slightly cold,great cold and slightly warm.The taste of medicine is most pungent,followed by bitter,and then sweet,slightly bitter,salty.The drug is mainly for the lung,followed by the spleen and stomach,and then the heart,bladder,liver and kidney.The highest pathogenic evidence is lung,followed by kidney and spleen,then table,liver and stomach;the highest pathogenic evidence is phlegm,followed by heat,wind,qi deficiency,yin deficiency,qi stagnation,yang deficiency,etc.Association rules showed that licorice-peony was a high confidence(95.83%)drug pair;ephedra,dry ginger,fine spice,wu wei zi,and gypsum were high frequency combinations.The clustering analysis was divided into five categories: the first category was Ephedra,Bitter almond,Gypsum,Cicada;the second category was Glycyrrhiza,Schisandra,Schisandra,Ginger,Cinnamomum,Paeonia;the third category was Perilla,Aster,Radix et Rhizoma;the fourth category was Scrophularia,Pericarpium;the fifth category was Fructus Sempervirens,Fusarium,Morus alba,Cyperus,Salviae Miltiorrhizae,Salviae Miltiorrhizae;the sixth category was Scutellaria,Radix et Rhizoma,Radix et Rhizoma,Qian Hu.2.116 normal control microarray data and 405 microarray data of patients with different severity of asthma were finally included,with a total of 1111 up-regulated genes and 1619 down-regulated genes;106 effective drug components and 255 target genes of hemp peony and asthma formula were obtained;30 genes were crossed,and the enrichment results of these genes suggest that the key genes involved in the intervention of hemp peony and asthma formula in bronchial asthma The targets are mainly related to the effects of transcription,inflammatory response,cellular response to lipopolysaccharide,etc.The functional pathways mainly involve cancer pathway,metabolic pathway,PI3K-Akt signaling pathway,etc.The inhibition rate of LPS on 16 HBE cells was dose-dependent;compared with the normal group,the model group could significantly increase the expression of proinflammatory factor TNF-α for 24 h(P<0.05);the promotion effect of hemp peony and asthma formula containing serum on 16 HBE was concentration-dependent;compared with the normal group,the autophagosome formation increased in the model group,and hemp peony and asthma formula could inhibit autophagy and improve the cell status to a certain extent.Western blot results showed that compared with the normal group,the expression of p-PI3 K,p-Akt and p-mTOR proteins in the LPS group was significantly lower(P<0.05),and the expression of LC3 B protein was significantly higher(P<0.01);compared with the LPS group,the expression levels of p-PI3 K,p-Akt and p-mTOR proteins in the drug-containing serum group were significantly higher(P <0.05)and LC3 B expression levels were significantly lower(P<0.05)in the LY294002+LPS+MSCP group compared with the LPS+LY294002 group,and p-PI3 K,p-Akt,and p-mTOR protein expression levels were significantly higher(P<0.05)and LC3 B expression levels were significantly lower(P<0.05)in the LY294002+LPS+MSCP group compared with the 740Y-P+LPS group.Compared with the 740Y-P +LPS+MSCP group,the expression levels of p-PI3 K,p-Akt and p-mTOR were significantly higher(P<0.05)and LC3 B was significantly lower(P<0.05)in the740Y-P+LPS+MSCP group.ELISA results showed that cellular inflammation IL-5,IL-6,TNF-α,IL-10 levels were significantly higher in the LPS group compared with the normal group(P<0.01),and after treatment with the drug,TNF-α,IL-6,IL-8 levels were significantly decreased(P<0.01)and IL-10 was elevated(P<0.01)compared with the LPS group.Compared with LY295002 + LPS group,TNF-α,IL-5,IL-6 were significantly lower(P<0.01)and IL-10 expression level was significantly higher(P<0.01)in LY295002+LPS +MSCP group.Compared with 740Y-P +LPS group,TNF-α,IL-5 and IL-6 were significantly lower(P<0.01)and IL-10 expression level was significantly higher(P<0.05)in the740Y-P +LPS+MSPC group.Conclusions:1.National medical masters treat asthma mostly according to the nature of external evil,treating from cold and heat,wind and phlegm,and stasis,treating the lung position,taking into account the spleen,stomach and kidney,and flexibly combining antispasmodics,qi-activating drugs and blood-boosting drugs,etc.,which have some significance for clinical workers.2.The key genetic targets involved in the intervention of Ma Shao Ping Chuan formula for bronchial asthma created by National Medical Master Professor Han Mingxiang are mainly related to inflammatory response,cellular response to lipopolysaccharide,transcription and other effects,mainly involving cancer,metabolic pathways,PI3K-Akt signaling and other pathways.3.The Ma Shao Ping Chuan formula containing serum had a significant inhibitory effect on LPS-induced 16 HBE autophagy in human airway epithelial cells,which was mediated by PI3K/Akt/mTOR signaling pathway. |
