Effect Of Baicalin On Influenza Virus H1N1 Infection Of A549 Cells And Its Immunomodulatory Mechanism

ObjectiveInfluenza, caused by influenza virus, is the cute respiratory infectious diseases usually occured in the winiter and spring. It is difficult to find an effective vaccine for the virus of influenza A 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 or has become a very meaningful research in Chinese medieine.To investigate the regulatory effects of baicalin which is active components of scutellaria baicalensis of the traditional Chinese medicine on immune function and the antiviral effect, the model was established by human pulmonary carcinoma cell A549 infected with type A virus H1N1. Base on the pattern recognition receptors in innate immune TLR (toll-like receptors) toll-like receptors, the reaserach explore the baicalin antiviral effect and molecular mechanism to regulated-immune in vitro, and the regulation on inflammatory factors and apoptosis related factors in infected cell. Gene chip used to find out the key molecules on the immune cells influence by drugs, which is advanced technology in studying the regulation of the immune system. The total RNA from A549 cell of each groups were isolated. Furthermore, the total RNA were screened with the gene chip technique to detected the differently expressed genes, and analyzed key molecules of baicalin played the effect of anti-influenza virus activity.Methods1. The research on method of baicalin against influenza virus proliferation 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 baicalin 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 baicailin, A549 cell infected with H1N1 were detected with MTT method,48 hours after calculating drugs suppress the virus effectively.2. The influence of baicailin to 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), high-dose-baicalin (3.96μg·ml-1) and low-dose-baicalin (0.99μg·ml-1). The model model.was established by added 100TCID50 influenza virus 100μl H1N1 diluent with medium into cell plate, while the control group was added in normal medium. After the above infectious treatment for 2 hours, cell were treated with high-dose-baicalin and low-dose-baicalin 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 Baicalin on inflammation related cytokines in vitro and its mechanismExperiment cell were divided into five groups randomly:control group, model group, Oseltamivir group, high-dose-baicalin and low-dose-baicalin. The model model was established by added 100TCIDso influenza virus 100μl H1N1 diluent with medium into cell plate, while the control group was added in normal medium. After the above infectious treatment for 2 hours, cell were treated with high-dose-baicalin and low-dose-baicalin for 48 hours. The mRNA and protein expression of inflammation related cytokines 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 baicalin on TLR3/7 signal pathway in A549 cell and its mechanismTo investigate the regulatory effects of baicalin on TLR3/7 signal pathway induced by virus H1N1 in A549 cell. Divided cell into five groups:control group, model group, Oseltamivir group, high-dose-baicalin and low-dose-baicalin, 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, TLR7 and MyD88, were verified by Real-Time PCR, and those protein expression were verified by Western-Blot.5. Effect of baicalin on NF-κand MAPK-JNK signal pathway in A549 cell and its mechanismTo investigate the regulatory effects of baicalin on inflammation related signal pathway, NF-κand MAPK-JNK signal pathway were observed by gene chip technology. Divided cell into five groups:control group, model group, Oseltamivir group, high-dose-baicalin and low-dose-baicalin, H1N1 Virus-infected A549 cells were cultured, except the control group. These RNA samples in virus-infected A549 were screened by gene chips, differentially expressed genes of NF-κB and MAPK-JNK signal pathway were selected out. The mRNA of NF-κB and JNK were verified by Real-Time PCR, and those protein expression were verified by Western-Blot.6. Effect of baicalin on JAK-STAT signal pathway in A549 cell and its mechanismTo investigate the regulatory effects of baicalin on JAK-STAT signal pathway, cell were divided into five groups:control group, model group, Oseltamivir group, high-dose-baicalin and low-dose-baicalin, H1N1 Virus-infected A549 cells were cultured, except the control group. These RNA samples in virus-infected A549 were screened by gene chips, differentially expressed genes of JAK-STAT signal pathway were selected out. The mRNA of IFN-y and IL-10 were verified by Real-Time PCR, and those protein expression were verified by Western-Blot.7. Effect of baicalin 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 Caspase3, Caspase8 and Fas were verified by Real-Time PCR. And protein expressions of Fas and Caspase3 were verified by Western-Blot.Results1. The research on method of baicalin against influenza virus proliferation in VitroThe 50% tissue culture infective dose of influenza virus H1N1 (TCID50) was 10-3778per decimus milliliter. Maximal atoxic concentration (TCo) and half of the toxic concentration (TC50) of baicalin was 7.92μg·ml-1 and 31.62μg·ml-1 in human lung adenocarcinoma cell A549.In addition, the best method of baicalin against viral infection were added 2 hours after cells infected. The high survival rate was 89.11%. In addition, the effect of baicalin has the effect of hemagglutination inhibition.2. The influence of baicailin to gene expression of A549 cell infected with H1N1 Based on the design of experiment, the GO analysis for differentially expressed genes of two doses of baicalin 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. Baicalin involves the immunology molecular function:enzyme binding, transcription activator activity, RNA binding, protein dimerization activity, protein kinase activity, nucleotide binding, protein serine/threonine kinase activity, phosphatase regulator activity, small GTPase binding, purine nucleoside binding and identical protein binding, etc. Baicalin regulates of metabolic pathways:DNA metabolic process, RNA biosynthetic process, response to DNA damage stimulus, chromosome organization, protein localization, intracellular transport, cellular response to stress, organelle fission, DNA repair, phosphate metabolic process, cell cycle, mitotic cell cycle, etc.Baicalin involves the components of cells:nuclear lumen, intracellular organelle lumen, nucleoplasm, membrane-enclosed lumen, envelope, organelle lumen, chromosomal part, intracellular non-membrane-bounded organelle, centrosome, endomembrane system, cytosol, etc.In KEGG database, the pathway analysis for differentially expressed genes of baicalin is shown. To identify differentially expressed genes, standard selection criteria are established at the number of genes involved no less than 3 and The P value was less than 0.05.Based on KEGG database, some signal pathways were noted to have been significantly affected. The genes in the pathways of MAPK signaling pathway, Pancreatic cancer, Toll-like receptor signaling pathway, Colorectal cancer, Adherens junction, NF-κB signaling pathway, Fc gamma R-mediated phagocytosis, Pathways in cancer, Apoptosis, mTOR signaling pathway, Jak-STAT signaling pathway, Endocytosis, and Phosphatidylinositol signaling system was significantly different compared with virus infection group. The P value of pathway analysis was little and difference striking.3. Effect of Baicalin on inflammation related cytokines in vitro and its mechanismIn DNA microarray and Real-Time PCR experiment, IL1B, TNFA, RANTES, IL10 and IL6 were up-regulated in virus-infected group. Oseltamivir group, high-dose-baicalin and low-dose-baicalin could down-regulate gene expressions of IL1B, TNFA, RANTES, IL10 and IL6. The result of qRT-PCR experiment showed that baicalin could significantly decrease protein expressions of IL1β, IL6, TNF-a and RANTES (P<0.01) compared with the virus-infected group. The result of Western blotting was coincide with qRT-PCR and gene chips experiment.4. Effect of baicalin on TLR signal pathway in A549 cell and its mechanismBased on KEGG database analyzed, TLR3, TLR7, MYD88, IRAK4, FOS, IL6 and IRF7 were up-regulated in virus-infected group. High-dose-baicalin could down-regulate gene expressions of TLR3, TLR7, MYD88, IRAK4, FOS and IL6. Low-dose-baicalin could down-regulate gene expressions of TLR3, TLR7, MYD88 and IL6. The results of Real-Time PCR and Western-Blot experiments showed that two doses of baicalin can significantly decrease mRNA and protein expression of TLR3/7 and MyD88 (P<0.01), compared with the virus-infected group. As expected, real-time PCR and western-blot data were in good agreement with the microarray assay.5. Effect of baicalin on NF-κB and MAPK-JNK signal pathway in A549 cell and its mechanismBased on KEGG database analyzed the NF-κB singal pathway, IRAK1, TAB2, NFKB, IL1β, IL8, TNFA and CD40 were up-regulated and BIRC3 and TNFAIP3 down-regulated in virus-infected group. High-dose-baicalin could down-regulate gene expressions of IRAKI, TAB2, NFKB, IL1B, TNFA and IL1RAP, and up-regulate gene expressions of BIRC3, NFKBIA and TNFAIP3. Low-dose-baicalin could down-regulate gene expressions of IRAKI, TAB2, NFKB, IL10, IL8, CD40, TNFA and IL1RAP.Based on KEGG database analyzed the MAPK-JNK signal pathway, ILIA and MAP3K2 were up-regulated, but MAPK8IP2 was down-regulated in virus-infected group compared with the normal group. High-dose-baicalin could down-regulate gene expressions of ILIA, PPP3CA, MAP3K2 and JNK, and up-regulate gene expressions of MAPK8IP2 compared with the virus-infected group. Low-dose-baicalin could down-regulate gene expressions of MAP4K3, MAP3K2, JUN and JNK, and up-regulate gene expressions of MAPK8IP2.The results of Real-Time PCR and Western-Blot experiments showed that two doses of baicalin can significantly decrease mRNA and protein expression of NF-κB and JNK (P<0.01), compared with the virus-infected group. As expected, real-time PCR and western-blot data were in good agreement with the microarray assay.6. Effect of baicalin on JAK-STAT signal pathway in A549 cell and its mechanismBased on KEGG database analyzed the JAK-STAT signal pathway, IL10, IL20RB, PIK3CD and STAT1 were down-regulated in virus-infected group. High-dose-baicalin could up-regulate gene expressions of IL10, IFNA1, IFNB1, IL10RB, IL6R, IL20RB, IFNAR2, STAT3 and PIK3CD, and down-regulate gene expressions of PTPN2 compared with the virus-infected group. Low-dose-baicalin could up-regulate gene expressions of IL10, IFNA1, IFNB1, IL4R, IL17RA, IL10RB, IL6R, IFNAR2, STAT1, STAT3, STAT5Aand PIK3CD, and down-regulate gene expressions of PTPN2.The results of Real-Time PCR and Western-Blot experiments showed that two doses of baicalin can significantly decrease mRNA and protein expression of IL10 and IFN-y (P<0.01), compared with the virus-infected group. As expected, real-time PCR and western-blot data were in good agreement with the microarray assay.7. Effect of baicalin 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,-7,-8,-10, Fas and TRAIL were increased in virus-infected group. High-dose-baicalin could down-regulate gene expressions of FAS, Casp3, Casp4, Casp6, Casp8 and FADD, and up-regulate gene expressions of BCL2L1 and CFLAR. Low dose-baicalin could down-regulate gene expressions of FAS, Casp3, Casp4, Casp6, Casp8, IL1RAP, Cn and FADD, and up-regulate gene expressions of BCL2L1 and CFLAR.The results of Real-Time PCR experiments investigated that two doses of baicalin can significantly decrease mRNA expression of Caspase-3,-8 and Fas (P<0.01), and the results of Western-Blot investigated that two-doses of baicalin can significantly decrease protein expression of Caspase-3,-8 and Fas.Conclusions1. Baicalin had a significant inhibitory effect on influenza A HlN1 virus infection in epithelial cell line A549. Baicalin has inhibitive effect on influenza A H1N1 virus infection via antiviral biosynthesis activity. Furthermore, antiviral efficiency of baicalin low dose group was higher than that of baicalin high dose group.2. The influence of baicalin to gene expression of A549 cell infected with H1N1. Baicalin involves the immunology molecular function:enzyme binding, transcription activator activity, RNA binding, protein dimerization activity, etc. Baicalin regulates of metabolic pathways: DNA metabolic process, RNA biosynthetic process, response to DNA damage stimulus, chromosome organization, etc. Baicalin involves the components of cells:nuclear lumen, intracellular organelle lumen, nucleoplasm, membrane-enclosed lumen, envelope, organelle lumen, endomembrane system, etc. Based on KEGG database, some signal pathways were noted to have been significantly affected. The genes in the pathways of MAPK signaling pathway, Toll-like receptor signaling pathway, NF-κB signaling pathway, Fc gamma R-mediated phagocytosis, Apoptosis, mTOR signaling pathway and Jak-STAT signaling pathway system was significantly different compared with virus infection group. The P value of pathway analysis was little and difference striking.3. Baicalin could down-regulate the over-expressions of inflammation-related cytokines in vitro. Two doses of baicalin could inhibit inflammation of host cell, and down-regulate the over-expressions of IL1β, TNF-a, IL6, IL8, IL10 and RANTES mRNA and protein, thus reducing inflammation and restoring stability and balance of body’s immune function in vitro. The results also figured that the interference efficacy of low-dose baicalin was better than that of high-doses.4. Baicalin have significant regulation to TLR signal pathway in human pulmonary carcinoma cell A549. Baicalin could downregulate TLR7 and MyD88 expression after virus infection and inhibit the secretion of inflammatory factors. It was demonstrated in the current study that TLR7/MyD88 in the Toll-like receptor pathway was specifically inhibited by baicalin, which downregulated the production of inflammatory factors induced by virus infection and alleviated the immune inflammatory injury.5. Baicalin could significantly down-regulated NF-κB and MAPK-JNK signal pathway in host cell A549. The low-dose baicalin could reduce the activation of NF-κthrough cytokine-cytokine receptor interaction; the high-dose baicalin (group D) could enhance negative feedback; and NF-κB pathway was inhibited from different aspects so that the host cell inflammation injury mediated by virus infection was suppressed. And, baicalin could inhibit over-active JNK to reduce the synthesis of virus RNA and protein antagonist influenza A virus amplification to antagonize inflammatory injury and cell apoptosis mediated by virus infection by inhibiting over-activation of JNK via the MAPK signal pathway.6. Baicalin could significantly regulated JAK-STAT signal pathway in host cell A549. Baicalin could increase membrane receptor expression including IL10RB, IL6R, and IFNAR2 and activate the JAK-STAT pathway; meanwhile, treatment with baicalin could decrease the expression of PTPN2 and reduce the inhibition mediated by JAK, upregulate the expression of STATs as well as their downstream factors MCL1 and PIM1, to play a role in anti-apoptosis and reduce HA protein-mediated immune injury. Besides, the expression of PIK3CD was upregulated, which was preferential to cell cycle and cell survival by increasing the expression of IFNA1 and IFNPyl to inhibit virus directly.7. Baicalin could significantly regulated Apoptosis signal pathway in host cell A549. Baicalin could down-regulate the expression of Caspase3,6,1,9 to resists against the apoptosis of host cell in vitro. And, baicalin could upregulate the anti-apoptotic protein expression of Bcl-xl, FLIP, IAP to inhibit the host cell apoptosis after virus infection. Therefore, baicalin could play anti-apoptotic function through multiple pathways in the infected cell.TLR7 was the antiviral targets of Baicalin in infected cells. Multiple downstream signaling pathways were activated while the virus infected cells such as NF-κB signaling pathway, Jak-STAT signaling pathway, and MAPK signaling pathway. These activated signaling pathways result in overexpression of inflammatory factors and apoptosis of host cells. Treatment with baicalin demonstrated the antivirus effect by regulating the tested signal pathways to modulate the expression of membrane receptor, reduce the inflammatory factors to decrease the inflammatory injury, and induce the anti-apoptotic factor activity and expression to suppress the cell apoptosis by virus infection.