Mice Model For Therapy On Asthma Airway Hyper-reactivity And Anti-influenza Drug Screening

As the increasing environmental population, the cases of asthma are rising year by year. Research found that the influenza virus-induced (non-allergy) asthma is corticosteroid-resistant, and it is urgent to find ways to cure such kind asthma. Similarly, Oseltamivir and Amantadine are most popular drugs to treat with influenza virus infection. However, these two kinds of chemicals were found with increasing virus resistance and side-effects. It is an important work to develop or find new kinds of anti-flu drugs for industrial and academic fields.This study comprises of two research projects. The first one is aim to establish influenza virus induced mouse Airway Hyper-reactivity (AHR) models, and to explore the relationship between influenza virus and AHR, and try to find potential therapy for this condition. C57BL/6 mice were infected with H1N1 influenza virus (PR8) The lung resistance (RL) also measured for confirming of Airway Hyper-reactivity. Infected with 1.5 ×103 EID50 PR8 viruses, mice were induced 325%RL value on 5dpi compared with base value. The rag2-/- mice were challenged similarly, and showed typical AHR too. These animal models were used to explore potential therapy for treating influenza-induced asthma. We found that the wild type C57BL/6 and Rag2-/- mice showed AHR with increasing IL-5 (86 pg/ml) and IL-13 (85 pg/ml) level. The rate of Innate Lymphote Cells 2 (ILC2) in the lung of Rag2-/- mice increase quickly post infection compared with the uninfected mice, with 13.4% and 5.8% respectively. The mRNA copy of ST-2 was 3-fold higher than uninfected mice and IL-33 level in mice lung rised significantly. To verify the role of IL-33 in flu induced AHR, we administrated recombinant mouse IL-33 protein intra-nasally to wild type C57BL/6 and Rag2-/- mice without influenza virus challenge, and mice showed typical AHR on 5dpi. Furthermore, we also treated infected animal model with mouse mAb IL-33, and found the severity of AHR, level of IL-5/IL-13 cytokines drop significantly. Our results indicated that the IL-33 mAb could be a potential therapy for influenza induced AHR therapy.The second part of this study is to establish in vitro and in vivo models for screening potential anti-flu chemicals. To establish in vitro screening model, influenza virus M2 gene was cloned and expressed in host cell-Eschericia coli. And the cell showed growth retardation with IPTG induction, with OD600 value less than 0.2. The retarded recombinated Eschericia coli resumed growth (OD600 value 0.47) with 70uM amantadine. To evaluate 7 chemicals, model bacterial were induced with 1mM IPTG and cultured with candidate drug, and OD 600nm value was detected. The results showed that 3 candidate A21, A189 and A193 display anti-flu potency. Then, the lethal dose (10 LD50) influenza virus infected animal model was used for further assessing the anti-influenza effect of drug A21, A189 and A193 in vivo. The lethal dose infected mice were rescued with 100mg/kg/d A193, but not A21 and A189. The dose of 10 and 50 mg/kg/d of A193 also effectively rescued animals from lethal dose influenza virus challenge. Our results showed that we established anti-flu drug screening model in vitro and in vivo, and found one candidate drug with anti-flu potency.To sum up, we established animal models for Air Hypersensitive and found that the IL-33 mAb could be a potential therapy for influenza induced AHR therapy. We established anti-flu drug screening model in vitro and in vivo, and found one candidate with anti-flu potency. These anima modes would be valuable for future research.