| Purpose : Using network pharmacological method,the key targets and main pathways of effective components of Ganlu Xiaodu Micropill in the treatment of influenza were screened,and the mechanism of action was explored.Through the establishment of BALB/c mice model infected by influenza virus in haze environment,the effects of Ganlu Xiaodu Micropill Simplify Decoction on NLRP3 inflammasome,related inflammatory factors,upstream regulatory factors and viral load of IV infected mice in haze environment were studied from the levels of histology and molecular biology.To explore the mechanism of Ganlu Xiaodu Micropill Simplify Decoction on IV virus infection in haze environment,and to provide experimental basis for the clinical treatment of influenza with traditional Chinese medicine;Through the effect of Ganlu Xiaodu Micropill Simplify Decoction to clear heat and detoxify and aromatic turbidification on NLRP3 inflammasome pathway of IV infection in haze environment,the correlation between the activation of NLRP3 inflammasome pathway by influenza virus and the "external toxicosis leads to internal toxicosis" of traditional Chinese medicine was explored,so as to enrich and enrich the theoretical connotation of toxic evil of traditional Chinese medicine.Materials and methods:(1)TCMSP database was used to screen the active ingredients and potential targets of Ganlu Xiaodu Micropill,Drugbank and Disgenet database were used to obtain influenza disease targets,and R language was used to screen the intersection targets of drugs and diseases.Cytoscape3.6.1 software was used to construct and analyze the "active ingredient-key target" network and key target protein interaction network.The GO function enrichment analysis and KEGG pathway analysis of the key targets were performed using R language.Auto Dock Vina software was used to conduct molecular docking between the core target and the active ingredient,and Ligplus was used for visual processing.(2)Ninety SPF BALB/c mice were randomly divided into normal group,model group,Ganlu Xiaodu Micropill group,Ganlu Xiaodu Micropill Simplify Decoction group and oseltamivir phosphate group,with 18 mice in each group.After 1 day of adaptation feeding,the model group,Ganlu Xiedan Group,Ganlu Xiedan Compound,and oseltamivir phosphate group began modeling on the second day.Mice in each group were placed in an incubator simulating the humid and hot environment for 2 hours.After that,PM2.5 suspension was taken out and injected into the tracheas of mice,once every other day,for a total of 3 times.On the 7th day after modeling,except the normal group,the mice in other groups were inoculated nasally with the suitable strain of influenza A virus A/FM1/47(H1N1)0.1ml per mouse after ether anesthesia.24 h after modeling,Ganlu Xiaodu Micropill group,Ganlu Xiaodu Micropill Simplify Decoction group and oseltamivir phosphate group were given the corresponding drugs by gavage,once a day,for 10 consecutive days.Mice in each group were sacrificed in batches on the 3rd,7th and 10 th day after modeling,6 mice at a time.Lung index of mice was calculated.The pathological sections of lung tissue of mice were prepared and observed under light microscope,and the severity of pathological inflammation of lung tissue was scored according to the observation.Westernblot was used to detect the expression of NLRP3 protein in lung tissues of mice.The expression of IV viral load and m RNA of NF-κB and NLRP3 in lung tissues were detected by q PCR.The expression of IL-1β in serum of mice was detected by ELISA.Result:1 The network pharmacological results showed that there were 22 key active components of Ganlu xiangdan in the treatment of influenza,including 62 targets,including 19 core targets,which may play a role in the treatment of influenza by participating in the cell receptor binding,antioxidant and other biological functions as well as the regulation of interleukin 17,tumor necrosis factor and other pathways.2 After HE staining,no abnormalities were found in the lung tissues of normal mice.A large number of inflammatory cells infiltrated in the model group,thickening of alveolar septum,absence of alveolar,alveolar sacs,alveolar septum,edema of surrounding mucosal epithelium were observed.A small number of inflammatory cells were observed in Ganlu Xiaodu Micropill Simplify Decoction group.Compared with the model group,the alveolar septum was thickened and the mucosal epithelial edema was improved.The number of inflammatory cells in Ganlu Xiaodu Micropill group and oseltamivir group was significantly lower than that in the model group,accompanied by mild vascular swelling.3 After PM2.5 and IV combined infection,the lung index and pathological score of mice in each group were increased at each time point compared with the normal group,and reached the peak value on the 7th day(P<0.05).Compared with model group,lung index and pathological score in treatment groups were significantly decreased(P<0.05),and there was no significant difference in lung index among treatment groups(P>0.05).The pathological score of lung tissue in Ganlu Xiaodu Micropill group and oseltamivir group from the 7th day was significantly lower than that in the simplified formula group(P < 0.05).Other differences were not statistically significant(P >0.05).4 After PM2.5 and IV combined infection,the expression of NLRP3 protein in lung tissue of mice in each group increased at each time point(P<0.05),and reached the peak on the 7th day.After drug intervention,compared with model group,the expression of NLRP3 protein in treatment groups was down-regulated(P<0.05).The oseltamivir group was significantly lower than the Ganlu Xiaodu Micropill Simplify Decoction group(P<0.05),but there was no significant difference between the Ganlu Xiaodu Micropill Simplify Decoction group and the Ganlu Xiaodu Micropill group(P<0.05).5 After PM2.5 and IV combined infection,the m RNA expression of NLRP3,NF-κB and IV load in lung tissues of mice in each group were increased(P<0.05).After drug intervention,compared with model group,m RNA expression of NLRP3,NF-κB and IV load were decreased in all treatment groups.Compared with other groups,The lowest in oseltamivir group(P < 0.05),while the expression of NF-κ B was significantly different between the Ganlu Xiaodu Micropill Simplify Decoction group and Ganlu Xiaodu Micropillgroup only on day 3 and day 10(P<0.05).6 After PM2.5 and IV co-infection,the serum IL-1β content in each group increased(P < 0.05),and reached the peak value on the 7th day;After drug intervention,compared with the model group,the expression of IL-1β in each treatment group was down-regulated,and the oseltamivir group was significantly lower than the Ganlu Xiaodu Micropill Simplify Decoction group(P<0.05).On the 10 th day,the content of Ganlu Xiaodu Micropill group was lower than that of Ganlu Xiaodu Micropill Simplify Decoction group(P<0.05),and there was no significant difference at other time points(P<0.05).Conclusion:1 Ganlu Xiaodu Micropil to treat influenza may contain with quercetin,mignonette element and crude drug,such as composition,by acting on RELA,TNF,JUN targets,such as participate in cell receptor,antioxidant and other biological function and regulation of interleukin 17,tumor necrosis factor and other related pathways.2 By down-regulating the expression of NF-κB and inhibiting the activation of NLRP3 inflammasome pathway,and reducing the release of IL-1β inflammatory factors,Ganlu Xiaodu Micropill Simplify Decoction has the effect of clearing away heat and detoxification,dehumidifying turbidities and regulating pulmonary inflammation caused by IV infection in haze environment,which is similar to the original prescription.3 In the haze environment,IV infection activates NLRP3 inflammasome and a large number of inflammatory factors are released,which is the embodiment of the traditional Chinese medicine toxicology theory "external toxicology leads to internal toxicology".Ganlu Xiaodu Micropill Simplify Decoction can clear away heat and detoxify,and clearing damp can remove turbidification. |
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