| It has been thousands of years for the theory and application of traditional Chinese medicine(TCM) treating chronic and complex disease. The mechanisms of action of TCM are very complicated because the TCM contains a variety of chemical compositions which can target multiple targets and pathways. Currently, it is lack of systematic experimental methods to study the mechanisms of action of TCM. The development of network pharmacology provides us a new approach. There are lots of reports about experimental studies and clinical applications of YinQiaoSan(YQS) but few reports about its mechanisms of action. This paper is based on the theory and method of network pharmacology to investigate the mechanism of action of YQS to provide a reference for research of mechanism of TCM.1. The lungs of mice in normal control group, viral controlgroup, Oseltamivir and YQS treatment groups were obtained on day 1, 3, 5, 7 and 9 post-infection. Total RNA of the lungs were extracted with Trizol. The gene expression data was obtained through highthroughput sequencing and analyzed by bioinformatics methods. Then the network fingerprint of gene expression in lung tissue was obtained. The impact of influenza A virus on genes and pathways was predicted and verified by real-time PCR and Weastern-blotting. It was found that JAK-STAT, PI3K/Akt pathway in mice was infected by influenza A virus, antigen processing and presentation pathway and intestinal immune was suppressed, Adrengergic signal pathway, Salivary Secretion and Gastric acid Secretion was activated. Oseltamivir could recover the inhibition of JAK-STAT and antigen processing and presentation pathway while the effect of YQS was weaker. Oseltamivir could reduce the degree of apoptosis activation to normal while the effect of YQS was stronger. The effect of YQS on other pathways was similary to Oseltamivir but was generally weaker than Oseltamivir. The changes of relative levels of genes Saa1、Iigp1、Ms4a6d、Oasl2、Sectm1a、Gbp6、Plac8、Gbp11 were increased determined by real-time PCR. The results of Western-blotting found that the levels of proteins Iigp1 and Oasl2 were increased when the infection time was prolonged, while the level of protein Gbp6 were adverse. According to the results of the first part and the literature, Gbp6, Iipg1 and Oasl2 were selected as the targets and their antisense oligodeoxynucleotide were designed. The effects of the antisense oligodeoxynucleotide on the mice infected with influenza A virus were observed. The relationship between Oasl2 and replication of influenza A virus was investigated by gene silence and overexpression. The levels of gene and protein Oasl2 were decreased after treated with Si-Oasl2 through intratracheal administration. Compared with the Si-control, the levels of gene M, protein HA and virus titers in lung of mice treated with Si-Oasl2 were increased. When the level of Oasl2 were overexpressed in A549 cells, compared with the empty plasmid control, the levels of gene M, protein HA and virus titers in lung of mice were decreased.2. YQS is composed of nine Chinese herbs. Flos lonicerae and fructus forsythia are the monarch herbs; mint, schizonepetae, fructus arctii and Semen Sojae Praeparatum(SSP) are the ministerial herbs; bamboo leaf and platycodon grandiflorum are the adjuvant herbs; and Radix liquiritiae is the conductant herbs. JCZ(monarch, ministerial and adjuvant herbs), JCS(monarch, ministerial and conductant herbs), JZS(monarch, adjuvant and conductant herbs) and JC(monarch and ministerial herbs) combinations were obtained through recombining the nine herbs. The lungs of mice in normal control group, viral controlgroup, Oseltamivir and YQS treatment groups were obtained on day 3 and 5 post-infection. Total RNA of the lungs were extracted with Trizol. The gene expression data was obtained through highthroughput sequencing and analyzed by bioinformatics methods and the network fingerprint of gene expression in lung tissue was formed. it investigated that JCZ, JCS, JZS and JC have influence on the JAK-STAT and PI3K/Akt pathway Combination with the results in the first part, the impact of YQS, JCZ, JCS, JZS and JC on genes and pathways was verified by real-time PCR and Weastern-blotting.3. YQS is composed of nine Chinese herbs and each herb has many chemical components. The main chemical components in YQS were researched about their protection against influenza A virus infection in vivo. Phillyrin was found to be effective against influenza A virus infection in vivo. The survival rate, mean survival time, lung index, viral titers, the levels of influenza HA protein, gene M and serum cytokines and histopathological changes in lung tissue were examined. The Raman spectroscopy technology was used to detect the impact of Phillyrin on the chages of chemical components in the srum of mice. Compared with the viral group, administration of Phillyrin at a dose of 20 mg/kg/day improved the survival rate of mice infected with influenza A virus from 0% to 20%, significantly prolonged the mean survival time and slowed down the weight loss caused by infelunza A virus infection. Compared with the viral group on day 5 post-infection, administration of Phillyrin significantly reduced the lung index and the level of IL-6 and GM-CSF. Compared with the viral group on day 3 and 5 post-infection, administration of Phillyrin could attenuate lung tissue damage. Compared with the viral group on day 5 post-infection, administration of Phillyrin at a dose of 20 mg/kg/day significantly decreased the relative level of gene M and the virus titers, reduced expression of haemagglutinin(HA) in mice infected with influenza A virus. And this paper analyzed the influence of Phillyrin on the biomacromolecule by Raman spectroscopy.In conclusion, YQS could treat influenza A virus through multiple targets and pathways because of its varity of Chinese medicine and chemical components. |
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