Effect Of Shufeng Xuanfei Recipe On The Intervention Of Influenza Virus Infected Cells In

ObjectiveInfluenza is caused by influenza virus of acute respiratory infectious diseases, can cause a wide range of popular in a short time, due to the flu virus has a high degree of variability, thus the flu vaccine preparation and the treatment is very difficult, at present the clinical treatment of western medicine has significant limitations, and the unique theoretical system of traditional Chinese medicine and accumulated rich clinical experience and the role of multiple targets characteristics of traditional Chinese medicine compound, the prevention of influenza has huge advantage.Shufengxuanfei and jiebiaoqingli formula is made by professor of china-japan friendship hospital ChaoEnXiang’s experience, clinical research has proved that the two syndromes treatment for swine flu has obvious effect, this study through the in vitro experiment to observe the cytotoxicity of two formulas by acting on influenza a (H1N1) virus infection of A549 cells observation of CPE, calculate the drug to the virus inhibition rate and therapeutic index to evaluate the safety, through the drug-containing serum pharmacology experiment clear two antiviral effect, the formulas by PCR and Western blot method to detect the NF-kappaB and AKT protein and mRNA expression level, verify the two formulas for the NF-kappaB and PI3K/AKT regulation of cellular signal transduction pathways, thus further clear the mechanism of action of the two formulas, provide the experimental basis for its clinical application.Methods1、Choose A549 lung cancer cell, add broth by 3-4 times to extend after growth status back to normal cells used in the experiment, shufengxuanfei and jiebiaoqingli formula after doubling dilution join into zhe culture plate which Has grown into a single layer of cells,100μl/hole, each concentration after six holes, also set the negative control group. culture 48hAt 37 ℃ and 5% CO2 incubator, observe cell CPE, determined by MTT method to detect, Using SPSS 20.0 software Probit regression method to calculate TCOandTC50.Virus diluted 10 times by the sequence, inoculation on has grown monolayer cell culture plate, Cells lesions rate of 50% or more is the lesions hole, According to the Reed-Mμench method to calculateTCID50. 100TCID50Virus vaccination add in has grown normal cell’s plate including 100μl/ hole, put in 37 ℃ and 5% CO2 incubator, after 2h abandon the virus, Add the following five largest non-toxic concentration dilution degrees accordingly of shufengxuanfei and jiebiaoqingli formula.set oseltamivir phosphate positive for drug control group the negative control group and model group to observe the cell pathological changes, after 48 h the absorbance OD value determined by MTT method and calculation of antiviral drug efficient (ER) and drug therapeutic index (TI) and half inhibitory concentration (IC50). According to influenza virus experiment after 48 hours to determine the drop degree of coagulation, observe and record the results after 2 hours.2、shufengxuanfei and jiebiaoqingli formula medicated serum in vitro anti-influenzavirus activity.The male SD rats, weight 120-130g; animal level: SPF/VAF adaptability feeding 3 days, divided into negative control group take deionized water; positive control group take tamiflu liquid; shufengxuanfei high dose group, and low dose group; jiebiaoqingli high dose group, and low dose group, drug according to people and big rat body surface area conversion out to drug dose, multiplied by five times volume for high dose, twice a day, each time 0.2 ml, for 5 consecutive days, fasting 12h, blooding from the abdominal aorta, place 2 hours at room temperature and then placed inside the fridge for 2 hours, serum-4 OOOrpm after centrifugation. Shufeng xuanfei and jiebiaoqingli serum containing join the reconciliation table has grown intomonolayer cell culture plate, CPE was observed. Largest non-toxic concentration of the following five corresponding dilution degrees of Shufeng xuanfei and jiebiaoqingli serum containing joined 100TCID50Of virus infection of A549 cell culture plate cell lesion was observed.3、Take has grew up single layer cell of training board flu virus H1N1 adsorption 2h, respectively joined shufengxuanfei and jiebiaoqingli formula 2.50μg·ml-1 while set negative control group, model group, positive control group up Philippine 0.78μg·ml-1.after 24h with Tris-HCl cracking liquid collection cell cracking reset-80 ℃ refrigerator frozen save After extracting the total mRNA of cells recorded NF-κ b,Myd88,IL-1 and TNF-α amplified bandsof gray values,with each set of samples and reference gene GAPDH vale ratio of NF-κb,Myd88,IL-1 and TNF-α DNA, gene expression relative amount of statistics,2-Delta-Delta CT method is used to calculate.Collect cells extract total cellular protein, Western-blot determination of protein concentration, assay fordetection of expression of NF-Kb, IL-1 and TNF-α proteins and analysis of the NF-Kb, IL-1, TNF-a and GAPDH absorbance ratio as the relative expression of target proteins.4、shfengxuanfei and jiebiaoqingli formula of H1N1 influenza virus infection invitro effect of PI3K/AKT pathway in A549 cells, Take has grew up single layer cell of training board flu virus H1N1 adsorption 2h, respectively joined shufengxuanfei and jiebiaoqingli 2.50μg· ml-1, while set negative control group model group, positive control group up Philippine 0.75 μg ml-1,24h with Tris-HCl cracking liquid collection cell cracking reset-80 ℃ refrigerator frozen save collection cell extraction total mRNA of cells Record AKT, Fas, FasL and caspase9 gene amplification bands of grey value, and calculate the internal gene GAPDH value and each sample AKT, the ratio of Fas and FasL and caspase9 gene, statistics the relative amounts of gene expression, using 2-△△CT method. Collecting cells extracted total protein, Western blot method-determination of protein concentration detection between groups AKT, of Fas and FasL protein expression, analysis each AKT, of Fas and FasL stripe and GAPDH absorbance ratio for the purpose of the protein expression of relative quantity.Results1、TCID50= 10-3.778 of the H1N1 influenza virus, shufeng xuanfei and jiebiaoqingli the maximum drug concentration (TCo) are as follows:10.20μg· ml-1,9.91μg ml-1, half of drug toxicity concentration (TC50) are as follows:.38.56 μg ml-1 · 38.88μg ml-1 Shufeng xuanfei and jiebiaoqingli the drug concentration is 2.50 μg ml-1 on H1N1 cells induced by CPE has the best protection of ER 74.69%and 72.25%respectively, the positive control drug oseltamivir was 0.75 μg ml-1 of cell concentrations of CPE has the biggest effect. Shufeng xuanfei and jiebiaoqingli the half of drugs inhibiting concentration (IC50) values are:2.36μg·ml-1 and 2.46μg·ml-1, two therapeutic index TI of formula values are as follows:16.31 and 15.78. Hemagglutinin titer results show titration end in these prescriptions are clear ahead of time.2、shufeng xuanfei and jiebiaoqingli containing drug serum on cell of maximum nontoxic diluted multiples for 2-1,while containing drug serum in 2-1-2-2 times diluted, on cell appeared minor of CPE, survival slightly good than virus infection group, but differences not significantly (p> 0.05),while containing drug serum in 2-3～2-6 times diluted, cell of survival and virus infection group basic no differences.3、Compared with the negative control group, model group’s NF-κB, Myd88, IL 1 and TNF-a mRNA expression has significant higher (P<0.01); and Compared with the model group the positive control group, shufengxuanfei group and jiebiaoqingli group’s NF-κB, Myd88, IL 1 and TNF-a mRNA expression were significantly lower (P<0.01). Model group’s NF-κB, IL-1 and TNF-a protein expression compared with the negative control group was significantly increased. Compared with the model group the positive control group, shufengxuanfei group and jiebiaoqingli group’s NF-κB, IL-1 and TNF-a protein expression were significantly lower.4、Compared with the negative control group, model group AKT, Fas, FasL and caspase9 mRNA expression was significantly increased (P<0.01); compared with model group positive control group, shufengxuanfei group and jiebiaoqingli group AKT, Fas, caspase9 and FasL mRNA expression were significantly lower (P<0.01). Model group’s AKT, Fas and FasL protein expression compared with the negative control group were significantly increased. Compared with the model group, positive control group, shufengxuanfei group and jiebiaoqingli group AKT, Fas and FasL protein expression were significantly reduced.Conclusion1、Shufeng xuanfei and jiebiaoqingli formula on the H1N1 influenza virus infection of A549 cells have a significant protective effect,the best rate with positive effects of the virus is as well as the positive drug.2、Two prescription drug-containing serum on the H1N1 influenza virus infection of A549 cells only a slight protective effect, no significant difference with the model group.3、Two prescriptions could significantly cut the influenza virus H1N1 infection in A549 cells NF-κB signaling pathway’s related factor mRNA and protein levels, suggesting that two herbs are available through inhibition of NF-κB signal transduction pathway, regulates the expression of IL-1 and TNF-a and other inflammatory cytokines, which reduces influenza virus together pathological damage to achieve antiviral activity.4、Two prescriptions can cut H1N1 influenza virus-infected A549 cells, PI3K/ AKT signaling pathway in the expression levels of mRNA and protein-related factors, Inhibition of PI3K/AKT signal transduction pathway to promote cell survival function, and can cut the expression of Fas and FasL and Caspase-9 antagonism against influenza virus caused by cell apoptosis, play a role of antivirus.