Study On The Therapeutic Effect And Immunoregulatory Mechanism Of The Spleen - Lungs In The Mice Infected With Influenza Virus

Influenza is a kind of a cute respiratory infectious diseases caused by influenza virus,which usually oecurs in the winiter and spring.Preparing an effective vaeeine for the influenza A virus is diffieult beeause of its mutable feature.The anti-influenza western medicines have strong side effeets and easy induetion of drug resistanee.Under the status quo, to find effeetiv e anti-influenza drugs and new preparations has become a very meaningful research in Chinese medieine.Established the model of Pneumonia by nasal dropping influenza virus A in mice. The TLRs play an important role of anti-infectious immunity in hunman body.the virus ssRNA can be recogniazed by TLR7,activate the TLR7cell signalpathway. Compare the two drugs,Shu feng Xuan fei Formula (the main ingredients including honeysuckle, forsythia, burdock, periostraum cicada, schizonepeta, et al.) of Jiebiao Qingli Formula [mainly ephedra, gypsum, scutellaria baicalensis, apricot (almond), raw glycyrrhiza, et al.] and study the differences mechanism on anti-influenza-virus function in molecule and gene levels. To investigate regulatory effects of Shufeng Xuanfei and Jiebiao Qingli Formulae on CD4+helper lymphocyte T1/2(Th1/2) cytokines of mice with influenza virus FM1pneumonia.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 lung tissue 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. Experiment one Eight-one male mice, ICR grade, were randomly divided into nine groups:control group, model group, Oseltamivir group, the high, middle and low dose groups of Shufeng Xuanfei Formula and the high, middle and low dose groups of Jiebiao Qingli Formula (each, n=9). The pneumonia model was established by dropping influenza virus strain FM1into the nose, while the control group was given normal saline0.05ml into the nose. After the above infectious treatment of mice for2hours, they were treated with Oseltamivir2.5g·ml-1·d-1for4days in Oseltamivir group, high, middle and low doses (3.762、1.881.0.941g·kg-1·d-1) of Shufeng Xuanfei Formula (the main ingredients including honeysuckle, forsythia, burdock, periostraum cicada, schizonepeta, et al.) and high, middle and low dose (4.368.2.184,1.092g·kg-1·d-1) of Jiebiao Qingli Formula [mainly ephedra, gypsum, scutellaria baicalensis, apricot (almond), raw glycyrrhiza, et al.] were administered intragastrically in the corresponding groups,0.2ml once a day for4days; in the control and model groups, the mice were treated by intragastrical administration of the same amount of normal saline for4days. Observing the pathological change of lung injury.2.Experiment two One hundred and eight male mice, ICR grade, were randomly divided into nine groups:control group, model group, Oseltamivir group, the high, middle and low dose groups of Shufeng Xuanfei Formula and the high, middle and low dose groups of Jiebiao Qingli Formula (each, n=12). The pneumonia model was established by dropping influenza virus strain FM1into the nose, while the control group was given normal saline0.05ml into the nose. After the above infectious treatment of mice for2hours, they were treated with Oseltamivir2.5g·ml-1·d-1for4days in Oseltamivir group, high, middle and low doses (3.762,1.881,0.941g·kg-1·d-1) of Shufeng Xuanfei Formula and high, middle and low dose (4.368.2.184,1.092g·kg-l·d-1) of Jiebiao Qingli Formula were administered intragastrically in the corresponding groups,0.2ml once a day for4days; in the control and model groups, the mice were treated by intragastrical administration of the same amount of normal saline for4days. Total RNA was extracted in each groups.we used the cutting edge technology, Microarray, to genome-widely compare the gene expression changes between different treatments in mice, following which we filtered out significantly changed genes.3. Experiment three Established the model of Pneumonia by nasal dropping influenza virus A in mice. Divided randomly into nine groups:normal group,model group,control group,low-dose,medium-dose and high-dose groups of Shufengxuanfei Fomula,low-dose,medium-dose and high-dose groups of Jiebiaoqingli Fomula. Total RNA was extracted in each groups.Then gene chips were used to screen these RNA samples. Some genes that were involved in TLR signal pathways were selected. Calculated the probe signal intensity ratio in each group vs model group.4. Experiment four Real-time PCR was adopted to determine the mRNA expression of TLR7、MyD88and NF-κB.Western blot was used to determine the protein expression of TLR7、MyD88and NF-κB.5. Experiment five The mRNA and protein expressions of interferon-γ (IFN-γ) and interleukin-4(IL-4) were determined by real-time fluorescent quantitative polymerase chain reaction (RT-PCR) and Western blotting.Results1.The pathological changes of lung:in normal control group, the lung tissue of mice was in good condition and the alveolar wall was normal, no inflammatory cell infiltration. In model group, there were marked with inflammatory cell infiltration, neutrophil infiltration, a large number of lymphocyte and thickening of alveolar walls, congestion in the blood vessels. Chinese medicine groups each have different degrees of improvement the inflammatory pathological changes of lung tissue, the therapeutic effect of hydrophobic group was obviously, reduce inflammatory cells, the morphological structure of lung tissue basically recovered. the structure of alveolar, alveolar sacs, alveolar ducts, alveolar septum are intact in normal control group.in model group, mononuclear cells, lymphocytes and neutrophils extensively infiltrated into alveolar walls. Compared with model group,the scope and degree of pathological changes of all the treatment groups got alleviative.2. Based on the experimental design, the GO analysis of differentially expressed genes for Shu Feng Xuan Fei Formula and Jie Biao Qing Li Formula is shown below. Standard selection criteria to identify differentially expressed genes are established at the number of genes involved no less than70or40and P<0.05. Shu Feng Xuan Fei Formula involves the immunology molecular function:plasma membrane,intracellular signaling cascade,immune response,regulation of cell proliferation,regulation of apoptosis,defense response,etc.Jie Biao Qing Li Formula nvolves the immunology molecular function:plasma membrane,extracellular region, immune response,regulation of cell proliferation,intracellular signaling cascade,defense response,etc.In KEGG database,the pathway analysis of differentially expressed genes for Shu Feng Xuan Fei Formula and Jie Biao Qing Li Formula is shown below. Standard selection criteria to identify differentially expressed genes are established at the number of genes involved no less than3and P<0.05. Shu Feng Xuan Fei Formula regulates of metabolic pathways:Cytokine-cytokine receptor interaction,MAPK signaling pathway,Pathways in cancer,Chemokine signaling pathway,Toll-like receptor signaling pathway,Jak-STAT signaling pathway,Natural killer cell mediated cytotoxicity, etc.Jie Biao Qing Li Formula regulates of metabolic pathways:Cytokine-cytokine receptor interaction, MAPK signaling pathway, Pathways in cance, Chemokine signaling pathway, Regulation of actin cytoskeleton, Toll-like receptor signaling pathway, Jak-STAT signaling pathway, Focal adhesion, etc.The P value of pathway analysis was little and difference striking.3.TLR7、MYD88、CCL5、IFNB1、IL6、IL12a、NFKBIA、IKBKB were up-regulated in model group.Down-regulated genes in medium-dose、low-dose Shufengxuanfei and medium-dose Jiebiaoqingli included TLR3. TLR7、 MYD88、CCL5、IFNB1、IL6、IL12a. NFKBIA. IKBKB.The role. Shu Feng Xuan Fei Formula was more effective than Jie Biao Qing Li Formula. 4. Compared with control group, in model group, the expressions of TL7、NF-κB、 MyD88mRNA and protein were significantly up-regulated(P<0.01). Compared with model group, high-dose,medium-dose and low-dose Shufengxuanfei and medium-dose Jiebiaoqingli were shown statistically significant down-regulated (P<0.05,P<0.01).5.Compared with control group, in model group, the expressions of IFN-γ mRNA and protein were significantly down-regulated, while those of IL-4mRNA and protein, obviously up-regulated (all P<0.05).Compared with model group, in medium-dose and low-dose Shufengxuanfei and medium-dose Jiebiaoqingli groups, the expressions of IFN-γ mRNA and protein were significantly up-regulated (P<0.01), while those of IL-4mRNA and protein, obviously down-regulated, especially marked being in the medium-dose and low-dose groups of Shufeng Xuanfei Formula (P<0.05)Conclusions1. Based on the pathological changes of lung, medium-dose,low-dose of Shu Feng Xuan Fei Formula and Jie Biao Qing Li Formula have an obviously protective effect in the lungs of mice infected with influenza virus.2. Shu Feng Xuan Fei Formula involves the immunology molecular function:plasma membrane,intracellular signaling cascade,immune response,regulation of cell proliferation,regulation of apoptosis,defense response,etc. Shu Feng Xuan Fei Formula regulates of metabolic pathways:Cytokine-cytokine receptor interaction,MAPK signaling pathway,Pathways in cancer,Chemokine signaling pathway,Toll-like receptor signaling pathway,Jak-STAT signaling pathway,Natural killer cell mediated cytotoxicity, etc.Jie Biao Qing Li Formula involves the immunology molecular function:plasma membrane,extracellular region, immune response,regulation of cell proliferation,intracellular signaling cascade,defense response,etc. Jie Biao Qing Li Formula regulates of metabolic pathways:Cytokine-cytokine receptor interaction, MAPK signaling pathway, Pathways in cance, Chemokine signaling pathway, Regulation of actin cytoskeleton, Toll-like receptor signaling pathway, Jak-STAT signaling pathway, Focal adhesion, etc.3. Down-regulated genes in medium-dose、low-dose Shufengxuanfei and medium-dose Jiebiaoqingli included TLR3、TLR7、MYD88、CCL5、IFNB1、IL6、IL12a. NFKBIA、IKBKB.The role. Shu Feng Xuan Fei Formula was more effective than Jie Biao Qing Li Formula.4. medium-dose、low-dose Shu Feng Xuan Fei Formula and medium dose group of Jie Biao Qing Li Formula can inhibit the inflammatory induced by influenza virus by down-regulating the NF-κB through TLR7signal pathway dependent on MyD88.Shu Feng Xuan Fei Formula was more effective than Jie Biao Qing Li Formula.5. The middle and low dose of Shufeng Xuanfei Formula can ameliorate the immune-pathological injury of influenza viral pneumonia by regulating the balance of Th1/Th2through up-regulating the expression of IFN-γ and down-regulating the expression of IL-4.