Study On The Intervention Effect And Immune Regulation Mechanism Of Influenza Virus H1N1 Infection On A549 Cells

ObjectiveInfluenza is the cute respiratory infectious diseases which was caused by influenza virus usually occured in the winiter and spring. An effective vaccine for the virus of influenza A is difficult because of its mutable feature. The western medicines which has anti-influenza effect has strong side-effeet and easy induced drug resistance. Therefore, to find effective anti-influenza herbs and new formulation or dosage form has become a very meaningful research in Chinese medieine.To investigate the regulatory effects of Shufengxuanfei and Jiebiaoqingli Chinese medicine formulaes on immune function and the antiviral effect, the model of infection used type A virus H1N1 in human pulmonary carcinoma cell A549 was established. TLRs were an important function of anti-infectious immunity in host body. The virus ssRNA can be recogniazed by TLR7, activate the TLR7 cell signalpathway.Shu feng Xuan fei Formula were mainly included honeysuckle, forsythia, burdock, periostraum cicada and schizonepeta. And Jiebiao Qingli Formula were mainly contained ephedra, gypsum, scutellaria baicalensis, apricot, raw and glycyrrhiza. Compared the two formulas and investigated differences mechanism on anti-influenza-virus function in molecule and gene levels. To reserach regulatory effects of Shufeng Xuanfei and Jiebiao Qingli Formulaes on apoptosis and inflammation of A549 cells induced by virus H1N1.Gene chip technology has advantages in studying the regulation of the immune system and finding out the key molecules on the immune cells influence by drugs. Isolated the total RNA from A549 cell of each groups, and then screened the differently expressed genes with the gene chip technique, in order to find out the key molecules which two formulas play the role of anti-influenza virus activity.Methods1. Inhibitory the best method of Shufeng Xuanfei and Jiebiao Qingli Formulaes on Influenza Virus A H1N1 in VitroThe morphologic study and cytopathogenic effect (CPE) of cell were observed used inverted microscope. To observing cytopathic effect (CPE) of cells were effected by Shufeng Xuanfei and Jiebiao Qingli Formulaes cause cytopathic effects. Recovery in the epithelial cells of the human lung adenocarcinoma cell A549, adjusted the concentration of cell for 1.5 x 105 per milliliter. According to the replication cycle of virus, design three antiviral drug targets:prevention group (Ⅰ), adsorption resistance (Ⅱ), the treatment group (III). Furthermore, to identify the best method of administration and action time of Shufeng Xuanfei and Jiebiao Qingli Formulaes, A549 cell infected with H1N1 were detected with MTT method,48 hours after calculating drugs suppress the virus effectively.2. The influence of Shufeng Xuanfei and Jiebiao Qingli Formulaes to all gene expression of A549 cell infected with H1N1Experiment cell were divided into five groups randomly:control group, model group, Oseltamivir group (0.75μg·ml-1), Shufeng Xuanfei Formula (2.50μg·ml-1) and Jiebiao Qingli Formula (2.50μg·ml-1). The model model was established by added influenza virus H1N1 into supernatant, while the control group was added in normal medium. After the above infectious treatment for 2 hours, cell were treated with Oseltamivir, Shufeng Xuanfei Formula and Jiebiao Qingli Formula for 48 hours. Total RNA of each groups was extracted. Used the cutting edge technology, Microarray, to genome-widely compare the gene expression changeed between each manner treatmented cell, following that we found out significantly changed genes.3. Effect of Shufengxuanfei formula and Jiebiaoqingli formula on inflammation-related cytokines in vitro and its mechanismThe mRNA and protein expression of TNF-a, IL-6, IL-1β, IL-10, MCP-1 and RANTES were inspected by DNA microarray, Real-Time PCR and western-blot. Calculated the probe signal intensity ratio in each group vs model group.4. Effect of Shufeng Xuanfei formula and Jiebiao Qingli formula on TLR7 signal pathway in human pulmonary carcinoma cell A549 and its mechanismTo investigate the regulatory effects of Chinese medicine formulae Shufeng Xuanfei formula and Jiebiao Qingli formula on inflammation-related cytokines induced by virus H1N1 in human pulmonary carcinoma cell A549. Divided cell into five groups:control group, model group, Oseltamivir group, Shufeng Xuanfei Formula and Jiebiao Qingli Formula, H1N1 Virus-infected A549 cells were cultured except the control group. Used gene chips to screen these RNA samples in virus-infected A549, differentially expressed genes of TLR3/7 signal pathway were selected out. The mRNA of TLR3/7, MyD88, NF-κB were verified by Real-Time PCR, and those protein expression were verified by Western-Blot.5. Effect of Shufengxuanfei formula and Jiebiaoqingli formula on apoptosis induced by H1N1 virus in vitro and its mechanismUsed the chips to screen the RNA samples of virus-infected A549 cells, differentially expressed genes were selected in the pathway of apoptosis. The mRNA expressions of Caspase-3,-8,-9, Fas and FasL were verified by Real-Time PCR. And protein expressions of Fas and FasL were verified by Western-Blot.Results1. Inhibitory the best method of Shufeng Xuanfei and Jiebiao Qingli Formulaes on Influenza Virus A H1N1 in VitroThe survival rate of A549 was significantly higher which added Shufengxuanfei formula and Jiebiaoqingli formula compared with the model without the drug. In addition, the best method of Shufengxuanfei formula and Jiebiaoqingli formula against viral infection were added 2 hours after cells infected. The best survival rate was 74.68% with 70.24μg·ml-1 of two formulas. In addition, the effect of Shufengxuanfei formula was superior to the effect of Jiebiaoqingli formula.2. The influence of Shufeng Xuanfei and Jiebiao Qingli Formulaes to all gene expression of A549 cell infected with H1N1Based on the design of experiment, the GO analysis for differentially expressed genes of Shu Feng Xuan Fei Formula and Jie Biao Qing Li Formula were shown below. To identify differentially expressed genes, standard selection criteria are established at the number of genes involved no less than 70 or 40 and P<0.05. Shu Feng Xuan Fei Formula involves the immunology molecular function:enzyme binding, RNA binding, protein kinase activity, protein serine/threonine kinase activity, small GTPase binding and identical protein binding, etc. Jie Biao Qing Li Formula involves the immunology molecular function:protein serine/threonine kinase activity, protein kinase activity, RNA binding, enzyme binding, nucleotide binding, transcription activator activity, etc.In KEGG database, the pathway analysis for differentially expressed genes of Shu Feng Xuan Fei Formula and Jie Biao Qing Li Formula is shown below. To identify differentially expressed genes, standard selection criteria are established at the number of genes involved no less than 3 and P<0.05. Shu Feng Xuan Fei Formula regulates of metabolic pathways:DNA metabolic process, response to DNA damage stimulus, protein localization, intracellular transport, organelle fission, phosphate metabolic process, etc. Jie Biao Qing Li Formula regulates of metabolic pathways:cellular response to stress, response to DNA damage stimulus, osteoblast differentiation, DNA metabolic process, organelle fission, DNA repair, mitotic cell cycle, etc.Shu Feng Xuan Fei Formula involves the components of cells:nuclear lumen, intracellular organelle lumen, membrane-enclosed lumen, organelle lumen, nucleoplasm and intracellular non-membrane-bounded organelle, etc. The components of cells handled with Jie Biao Qing Li Formula involves:nuclear envelope, nuclear lumen, endomembrane system, organelle envelope, membrane-enclosed lumen, intracellular non-membrane-bounded organelle and intracellular organelle lumen, etc. The P value of pathway analysis was little and difference striking.3. Effect of Shufengxuanfei formula and Jiebiaoqingli formula on inflammation-related cytokines in vitro and its mechanismIn DNA microarray and Real-Time PCR experiment, TNF-a, IL-6, IL-1β, IL-10, MCP-1 and RANTES were up-regulated in virus-infected group. Shufengxuanfei Formula could down-regulate gene expressions of TNF-a, L-10, IL-1β, IL-6 and MCP-1, however, Jiebiaoqingli Formula could down-regulate gene expressions of IL-la, TNF-β, IL-6 and MCP-1. The result of Western blot experiment showed that Shufeng Xuanfei and Jiebiao Qingli formulas could significantly decrease protein expressions of TNF-a, IL-6, IL-1β, IL-10, RANTES and MCP-1 (P<0.01), compared with the virus-infected group. The results also figured that the regulation efficacy of Shufeng Xuanfei formula was better than that of Jiebiao Qingli.4. Effect of Shufengxuanfei formula and Jiebiaoqingli formula on TLR7 signal pathway in human pulmonary carcinoma cell A549 and its mechanismTlr3, Tlr7, Myd88, Nfbkl, Mapk8, Mapk13, Ifnal and Ifnβ1 were up-regulated in virus-infected group. Shufengxuanfei and Jiebiaoqingli Formulae could down-regulate gene expressions of Tlr3, Tlr7, Myd88, Nfbkl, Mapk8, Mapkl3, Ifnal and Ifnβ1. The results of Real-Time PCR and Western-Blot experiments showed that two Formulas can significantly decrease mRNA and protein expression of TLR3/7, MyD88 and NF-κB (P<0.01), compared with the virus-infected group. These results also figured that the regulation efficacy of Shufeng Xuanfei formula was better than that of Jiebiao Qingli. As expected, real-time PCR and western-blot data were in good agreement with the microarray assay.5. Effect of Shufengxuanfei formula and Jiebiaoqingli formula on apoptosis induced by H1N1 virus in vitro and its mechanismWith the DNA microarray, the functions of changed expressed genes involved in apoptosis biological pathways were analyzed with Pathway databases which named Kyoto Encyclopedia of Genes and Genomes (KEGG). Caspase-3,-8,-9, Fas and FasL were increased in virus-infected group. Shufengxuanfei and Jiebiaoqingli Formulae could down-regulate gene expressions of Caspase-3,-8,-9, Fas and FasL. The results of Real-Time PCR experiments investigated that two Formulas can significantly decrease mRNA expression of Caspase-3,-8,-9, Fas and FasL (P<0.01), and the results of Western-Blot investigated that two Formulas can significantly decrease protein expression of Fas and FasL, compared with the virus-infected group. The results also figured that the regulation efficacy of Shufeng Xuanfei formula was better than that of Jiebiao Qingli. As expected, real-time PCR and western-blot data were in good agreement with the microarray assay. Compared with control group, the mRNA expressions of TLR7, MyD88, NF-κB and protein in model group were significantly up-regulated (P<0.01). Compared with model group, Shufeng Xuanfei and Jiebiao Qingli were shown statistically significant down-regulated (P<0.05,P<0.01).Conclusions1. Inhibitory the best method of Shufeng Xuanfei and Jiebiao Qingli Formulaes on Influenza Virus A H1N1 in VitroBased on the CPE, Shufeng Xuanfei and Jiebiao Qingli Formulaes had a significant inhibitory effect on influenza A H1N1 virus infection in epithelial cell line A549 via antiviral biosynthesis activity.2. The influence of Shufeng Xuanfei and Jiebiao Qingli Formulaes to all gene expression of A549 cell infected with H1N1Shu Feng Xuan Fei Formula involves the immunology molecular function:enzyme binding, RNA binding, protein kinase activity, small GTPase binding and identical protein binding, protein serine/threonine kinase activity, etc; regulates of metabolic pathways:DNA metabolic process, response to DNA damage stimulus, protein localization, intracellular transport, organelle fission, phosphate metabolic process, etc; involves the components of cells:nuclear lumen, intracellular organelle lumen, membrane-enclosed lumen, organelle lumen, nucleoplasm and intracellular non-membrane-bounded organelle, etc.Jie Biao Qing Li Formula involves the immunology molecular function:protein serine/threonine kinase activity, RNA binding, protein kinase activity, enzyme binding, nucleotide binding, transcription activator activity, etc; regulates of metabolic pathways: cellular response to stress, response to DNA damage stimulus, osteoblast differentiation, DNA metabolic process, organelle fission, DNA repair, mitotic cell cycle, etc; involves the components of cells:nuclear envelope, nuclear lumen, endomembrane system, organelle envelope, membrane-enclosed lumen, intracellular non-membrane-bounded organelle and intracellular organelle lumen, etc. The P value of pathway analysis was little and difference striking.3. Effect of Shufengxuanfei formula and Jiebiaoqingli formula on inflammation-related cytokines in vitro and its mechanismShufengxuanfei and Jiebiaoqingli Formulae could down-regulate the mRNA and protein over-expressions of TNF-α, IL-6, IL-1β, MCP-1 and RANTES, thus reducing inflammation and restoring stability and balance of body’s immune function in vitro.4. Effect of Shufengxuanfei formula and Jiebiaoqingli formula on TLR7 signal pathway in human pulmonary carcinoma cell A549 and its mechanismShufengxuanfei and Jiebiaoqingli Formulae could down-regulate the activity of NF-κB by regulating MyD88 in TLR3/7 signal pathway, thus fighting against influenza virus in vitro.5. Effect of Shufengxuanfei formula and Jiebiaoqingli formula on apoptosis induced by H1N1 virus in vitro and its mechanismShufengxuanfei formula can be detected their suppression effect of Caspase-3,-8,-9, Fas and FasL mRNA expression, so it resists against the apoptosis to fight against influenza virus H1N1 in vitro.