| ObjectiveInfluenza is the cute respiratory infectious diseases which was caused by influenza virus usually occurred in the winter 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-effect 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 medicine.Toinvestigate the regulatory effects of Chinese medicine. Tetramethylpyrazine 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 recognized by TLR7, activate the TLR7 cell signal pathway. Investigated differences mechanism on anti-influenza-virus function in molecule and gene levels. To research regulatory effects of Tetramethylpyrazine on apoptosis and inflammation of A549 cells induced by virus HIN1.In order to find out the key molecules which tetramethylpyrazine plays the role of anti-influenza virus activity.Methods1. Antiviral action research of tetramethylpyrazine on Influenza Virus A H1N1 in Vitro.Choose A549 lung cancer cell, add broth by 3-4 times to extend after growth status back to normal cells used in the experiment, after doubling dilution join into the 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 Probitregression method to calculate TC0andTC50. 100TCID50 Virus vaccination add in has grown normal cells, 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 tetramethylpyrazine. Set oseltamivir phosphate positive for drug control group the negative control group and model group to observe the cell pathological changes, after 48h 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. Effect of tetramethylpyrazine on inflammation-related cytokines in vitro and its mechanism.The mRNA and protein expression of TNF-a, IL-1, INF-y, IL-10, were inspected by DNA microarray, Real-Time PCR and western-blot. Calculated the probe signal intensity ratio in each group vs model group.3. Effect of tetramethylpyrazine on TLR7 signal pathway in human pulmonary carcinoma cell A549 and its mechanism.To investigate the regulatory effects of Chinese medicine tetramethylpyrazine. Tetramethylpyrazine 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, H1N1 Virus-infected A549 cells were cultured except the control group. The mRNA of TLR3/7, MyD88, NF-kB. TNF-α、IL-1 were verified by Real-Time PCR, and those protein expression were verified by Westem-Blot.4. Effect of tetramethylpyrazine on apoptosis induced by H1N1 virus in vitroThe mRNA expressions of Caspase-3,-8, and-9, were verified by Real-Time PCR.Results1. Antiviral action research of tetramethylpyrazine on Influenza Virus A H1N1 in Vitro.The maximum drug concentration (TCo) of tetramethylpyrazine is as followed: 100.40μg·ml-1 half of drug toxicity concentration (TC50) is as followed:562.17 μg·ml-1. The drug concentration is 6.25μg· ml-1 on H1N1 cells induced by CPE has the best protection of ER85.3%, the positive control drug oseltamivir was 0.75μg ml-1 of cell concentrations of CPE has the biggest effect. The half of drugs inhibiting concentration (IC50) value is 51.13μg. The rapeutic index TI value is 10.99. Hemagglutinin titer results show titration end in these prescriptions are clear ahead of time.2. Effect of tetramethylpyrazine on inflammation-related cytokines in vitroRT-PCR showed that, as compared with cell control group, the mRNA expressions of IL-1, IL-10, INF-γ, TNF-a were significantly increased in HIN1-infected group (P <0.01). Compared with H1N1-infected group, mRNA expressions of IL-1, IL-10, INF-γ, TNF-a in low-dose tetramethylpyrazine and high-dose tetramethylpyrazine decreased (P<0.01, P<0.05). Western blotting showed that, the levels of IL-1, IL-10, INF-γ, TNF-a in H1N1-infected group were obviously increased as compared with those in cell control group (P<0.05). The levels of IL-1, IL-10, INF-y, TNF-a in low-dose tetramethylpyrazine and high-dose tetramethylpyrazine were decreased as compared with those in H1N1-infected group (P<0.05, P<0.01).3. Effect of tetramethylpyrazine on TLR7 signal pathway in human pulmonary carcinoma cell A549 and its mechanism.Real-Time PCR experiments showed that two formulae could significantly decrease mRNA expression of TLR3, MyD88, NF-κB, TNF-a, IL-1(P< 0.01), compared with the virus infected group. Western blot experiment showed that two formulae could significantly decrease mRNA expression of TLR3/7, MyD88, NF-κB, TNF-a and IL-1(P<0.01), compared with the virus infected group. The results also figured that the interference efficacy of low-dose tetramethylpyrazine was better than that of high-dose tetramethylpyrazine.4. Effect of on tetramethylpyrazine apoptosis induced by H1N1 virus in vitro and its mechanism.The results of Real-Time PCR experiments investigated that can significantly decrease mRNA expression of Caspase-3,-8,-9. Compared with control group, the mRNA expressions of TLR7, MyD88, NF-kB in model group were significantly up-regulated(P<0.01). Compared with model group, tetramethylpyrazine were shown statistically significant down-regulated.Conclusions1. Antiviral action research of tetramethylpyrazine on Influenza Virus A H1N1 in Vitro.Tetramethylpyrazine 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. Effect of tetramethylpyrazine on inflammation-related cytokines in vitroEffect of tetramethylpyrazine on inflammation-related cytokines in vitro and its: mechanism tetramethylpyrazine can down-regulate the over-expressions of IL-1, IL-10, INF-y, TNF-a mRNA and protein, reducing inflammation, restoring stability and balance of Body’s immune function.3. Effect of tetramethylpyrazine on TLR7 signal pathway in human pulmonary carcinoma cell A549 and its mechanism.Effect of tetramethylpyrazine on TLR7 signal pathway in human pulmonary carcinoma cell A549 and its mechanism.Tetramethylpyrazine could down-regulate the activity of NF-kB by regulating MyD88 in TLR3/7signal pathway, thus fighting against influenza virus in vitro.4. Effect of tetramethylpyrazine on apoptosis induced by H1N1 virus in vitro and its mechanism.Tetramethylpyrazine can be detected their suppression effect of Caspase-3,-8, and-9 mRNA expression, so it resists against the apoptosis to fight against influenza virus H1N1 in vitro. |
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