Study On Action Mechanism Of IGF ? In Inflammatory Pulmonary Injury Induced By Influenza Virus

To study the role and its molecular mechanism of IGF I in the acute inflammatory pulmonary injury induced by influenza virus, A/Porto Rico/8/1934(H1N1) influenza virus was used as the experimental material, and the influenza infected cells model and mouse model were established as study objects. At the cellular level: real-time quantitative PCR and Western blot were used to detect expression of IGF I after influenza infection, cells model A549 of IGF I over-expression and knowdown were used to detect the effects of IGF I on various inflammatory cytokines expression including IL-8, IL-6, TNF-?, IL-1? and INF-?. At the animal level: mouse model infected by PR8 was established to detected expression of IGF I mRNA and protein through qRT-PCR, Western blot and ELISA. And then the mice were divided into PBS group, PBS+ IGF I group, PBS+PPP group,PR8+PBS group, PR8+ IGF I group (0.25mg/kg/24h), PR8+PPP group(20mg/kg/12h). The role of IGF I in inflammatory pulmonary injury induced by influenza virus wasevaluated were evaluated according to mice body weight, survival rate and clinical symptoms, histopathology, viral load and protein expression of PI3K/AKT and MAPK pathways. The resulted showed that:(1) At the cellular level: the expression IGF I mRNA and protein in A549 cells increased after influenza infection. After PR8 infection, the expression of IL-8, IL-6,TNF-a, IL-1? and INF-y increased in pcDNA3.1 + IGF I group compared with control group. Interestingly, the expression of opposite phenomenon decreased in Lenti+sh IGF1 group (P<0.01).(2) At the Animal level: the expression of mRNA and protein of IGF I increased after PR8 infection in mice lung tissues. ELISA results showed that IGF I protein in mice serum increased significantly compared with PBS control after PR8 infection and the content of IGF I protein reached the highest which was (253.229 ±9.501)ng/mL(P<0.01) in mice serum on the seventh day.(3) Mice treated with PBS were fur smooth, flexible and good mental state.While mice of PR8+PBS group appeared symptoms, hair micro vertical, persistent cough, slow response on the third day after infection. And mice were extremely thin,eyes closed, piloerection arched, huddled together on the fifth day. The first death was occurred on sixth day and thesurvival rate was 25%. At the same time, miceclinical symptoms in PR8+ IGF I group was severer than that in PR8+PBS group, the first death occurred on the fifth day, by the eighth day all mice were dead. In the meanwhile, miceclinical symptoms in PR8+PPP group was less serious than that in PR8+PBS group and the mice survival rate was 75% . (P<0.001)(4) Lung tissuein flammatory infiltration in PR8+ IGF ? group was more serious than that in PR8+PBS group. Mice lung index of PR8+ IGF ? group increased significantly from (1.953 + 0.074)% to (2.515 + 0.121)% (P<0.01), and the area of lung injury increased from (0.38 + 0.042) to (0.78 + 0.069) (P<0.01),compared with PR8+PBS mice. On the other hand, inflammatory infiltration in PR8+PPP group was less serious than that in PR8+PBS group. Mice lung index of PR8+ PPP group decreased to (1.393 + 0.032)% (P<0.01), the area of lung injury decreased to (0.25 + 0.062) (P>0.05). ELISA results demonstrated that expression of inflammatory factors including IL-6,TNF-?,IL-1? and INF-? in PR8+ IGF ? group mouse serum were significantly higher than that in PR8+ PBS group, while the expression of IL-6,TNF-?,IL-1? and INF-? in PR8+ PPP group mouse serum were significantly lower than that in PR8+ PBS group.(5) Compared with PR8+PBS group, viral load of PR8+ IGF ? group increased significantly reaching value of 1.598×10-2±5.42±10-4 ng/?L. Viral load of the mice lung increased very significantly reaching value of 3.256×10-2±1.19×10-4ng/?L after injecting IGF ? . However no significant difference was found in the viral load levels between PR8+PBS group and PR8+PPP group.(6) Western Blot results showed that PR8 infection increased the expression of p-IGF ? R, p-AKT p-p38 and p-JNK compared with control. The expression of p-AKT, p-p38 and p-JNK in group PR8+ IGF ? was higher than that of PR8+PBS group while the expression of p-AKT, p-p38 and p-JNK in PR8+PPPgroup waslowerthan that of PR8+PBS group.Conclusions:(1) Cells model of IGF ? overexpression and knowdown A549 were established.(2) Inhibition of IGF ? could effectively relieve the lung inflammation induced by influenza, on the contrary, overexpression of IGF ? could enhance the lung inflammation induced by influenza.(3) IGF ? increased influenza virus replication, which could be one of the mechanisms inducing excess death rate.(4)Influenza virus infection increased the expression of IGF1 and the phosphorylation of IGF ? receptor, then activated downstream PI3K/AKT and MAPK pathways,induced inflammation response, so, inhibiting the expression of IGF ? or IGF ? R, blocking the downstream signaling pathway would be a new target for treating influenza.