Effects Of Macrophage Migration Inhibitory Factor SiRNA On Acute Lung Injury Induced By LPS Or Influenza A Virus And The Signal Pathways Involved In The Modulation

Objective:Macrophage migration inhibitory factor (MIF) plays an important role not only in the immune system but also in inflammation response. In this study, we used siRNA to knockdown MIF mRNA for evaluation of the role of MIF siRNA in acute lung injury (ALI) induced by LPS and influenza A virus (H1N1), and further examined the effect of MIF siRNA on the signal transduction system.Contents and methods:Part 1: Transfection of MIF siRNA in human alveolar epithial cells and its interference impact on targeting geneAfter MIF siRNA was transfected into human alveolar epithial cells (A549 cells) with INTERFERin, RT-PCR was carried out to assess the dose-response of MIF siRNA's effect on MIF mRNA expression, Cell Immumofluorescence Method was used to analyze the expression of MIF protein, MTT Cell Proliferation Assay was performed to detect the cytotoxicity of MIF siRNA to A549 cells, and the survival rate of A549 cells caused by LPS.Part 2: Effect of MIF siRNA on the expression of Toll-like receptors and down stream signaling components in A549 cellsIn vitro: we use following methods, including RT-PCR, Western-Blot, Cell Immunofluorescence Method and ELISA, and with three level, such as mRNA, protein and medium, in A549 cells stimulated by LPS or infected by influenza A virus, to assess the expression of TLR2, TLR3, TLR4 mRNA, to analyse the expression of MyD88 and IRF3 protein in down stream MyD88 -dependent and independent signaling pathways, to observed NF-κB (p65) nuclear translocation, to detect released level of TNF-α, IL-1β, IL-6 in the supernatant, and further evaluated the effect of MIF siRNA on above biological index.Part 3: Effects of MIF siRNA on acute lung injury induced by LPS or H1N1 and the signal pathways involved in the modulationIn vivo: Mouse models of ALI were made by injection of LPS into abdominal cavity, or inoculation of H1N1 into intranasally. We use following methods, including ELISA, EMSA, Western-Blot, Immunohistochemical staining and HE stained for histopathological examination, to detect the released level of TNF-α, IL-1β, IL-6 in lung homogenates, to observed the histopathological manifestation of the lung, to assess the expression of aquapn (AQP1, AQP4) in lung tissue and to correlate with inflammatory cells infiltration, lung edema, then to analyse the expression of NF-κB, IκB-α, p-p38/p38, p-ERK/ERK, p-JNK/JNK protein in signal transcription pathway of NF-κB and MAPK. In addition, we further examined the effect of MIF siRNA on acute lung injury induced by LPS or H1N1, and on these signal transduction system.Results:Part 1: Transfection of MIF siRNA in human alveolar epithial cells and its interference impact on targeting gene1. Once the MIF siRNA was transfected into the A549 cells, it could obviously reduce the MIF mRNA expression. But only the concentration over 50nM, it did.2. After MIF siRNA was transfected into A549 cells, the expression of MIF's green fluorescent protein reduced greatly, but could be reversed by MIF.3. Almost no toxicity to A549 cells when the concentration of MIF siRNA was low 50nM, but also it could reduce the cell mortality and survival rate caused by LPS. Part 2: Effect of MIF siRNA on the expression of Toll-like receptors and down stream signaling components in A549 cells1. The expression of MIF, TLR2, TLR4 mRNA increase greatly when the LPS stimulated A549 cells up to 6h, and the adding of MIF siRNA+DXM, MIF siRNA and DXM respectively could downregulate the expression of MIF, TLR2, TLR4 mRNA. But LPS did not work to TLR3 mRNA.2. MIF, TLR3 mRNA level increase greatly when the H1N1 infected A549 cells for 16h, and the adding of MIF siRNA+RBVR, MIF siRNA, RBVR, MIF siRNA+DXM, DXM respectively could also downregulate the expression of MIF, TLR3 mRNA. But H1N1 did not work to TLR2, TLR4 mRNA.It is known that the downregulation of MIF mRNA expression is the lowest in the group with the combination of MIF siRNA and DXM among all groups.3. The expression of MyD88 and IRF3 protein increase greatly when the LPS stimulated A549 cells up to 6h, and the adding of MIF siRNA+DXM, MIF siRNA and DXM respectively could downregulate the expression of MyD88. Only MIF siRNA+ DXM and DXM could downregulate the express of IRF3. But MIF siRNA did not work to express of IRF3.4. The expression of MyD88 and IRF3 protein also increase greatly after infection with H1N1 for 16h, and the adding of MIF siRNA+RBVR, MIF siRNA, RBVR, MIF siRNA+DXM, DXM respectively could downregulate the expression of MyD88. Except MIF siRNA+ DXM and DXM could downregulate the expression of IRF3, the other groups no effect on IRF3.5. When LPS stimulated A549 cells to 8h or H1N1 infected A549 cells for 24h, the expression of MyD88 green fluorescent protein was more and strong, but could be reversed by MIF siRNA. If A549 cells stimulated by LPS for 45min or infected by H1N1 for 6h, translocation of NF-κB (P65) took place from cytosol to nucleus, the adding of MIF siRNA could partially knockdown NF-κB translocation.6. The released levels of TNF-α, IL-1β, IL-6 and IFN-βin supernatant increase greatly when the LPS stimulated up to 6h, and the adding of MIF siRNA+DXM,MIF siRNA and DXM respectively could downregulate the released levels of TNF-α, IL-1β, IL-6.7. TNF-α, IL-1β, IL-6 and IFN-βlevel in supernatant were very similar to stimulation with LPS. All of them were markedly increasing by H1N1 infection. The adding of MIF siRNA+RBVR, MIF siRNA, RBVR, MIF siRNA+DXM, DXM respectively could downregulate the released levels of TNF-α, IL-1β, IL-6.Except the siRNA+DXM and DXM2 could downregulate IFN-βlevel, the other groups no effect on IFN-βrelease. Part 3: Effects of MIF siRNA on acute lung injury induced by LPS or H1N1 and the signal pathways involved in the modulation1. Stimulation with LPS for 2h, or infection with N1H1 for 24h, it could increase greatly the expression of NF-κB in nucleus, and decrease markedly the expression of IκB-αin cytosol. Adding of MIF siRNA, DXM, MIF siRNA +DXM during stimulation with LPS, and adding of MIF siRNA, RBVR, MIF siRNA+RBVR, DXM and MIF siRNA+DXM in H1N1 infection could downregulate the expression of NF-κB and upregulate the expression of IκB-α. These data indicate that the upregulation of IκB-αexpression is the highest in the group with the combination of MIF siRNA and DXM among all groups.2. Phosphorylation of p-p38, p-ERK, p-JNK were rapidly increasing by stimulation with LPS for 2h. But no effect on the expression of p38, ERK, JNK, steady-state expression levels were found in them. Adding of MIF siRNA, DXM and MIF siRNA+DXM could downregulate the expression of p-p38 and p-JNK, but did not work to the expression of p-ERK.3. In contrast, there was no significant activation phosphorylation of p-p38, p-JNK in H1N1 infection throughout the time course. Only phosphorylation of p-ERK was rapidly increasing by infection with H1N1 for 24h. Adding MIF siRNA, RBVR, MIF siRNA+RBVR, DXM and MIF siRNA+DXM, the expression of p-ERK was downregulated only by RBVR, MIF siRNA+RBVR.4. Stimulation with LPS or infection with N1H1, It showed that increased lung wet weight, obvious alveolar destruction, mesenchyma infiltration with inflammation cells, epithelial and vascular endothelial swelling and shedding were demonstrated. Adding of MIF siRNA, DXM, MIF siRNA +DXM during stimulation with LPS, and adding of MIF siRNA, RBVR, MIF siRNA+RBVR, DXM and MIF siRNA+DXM in H1N1 infection could attenuate the severity of lung injury and decrease rate of lung wet/dry weight.5. Compare with control, immunostaining examination showed weaker staining against AQP1 in capillary endothelial cells, and against AQP4 in bronchial sub- epithelial basement membrane in mice's lung by stimulation with LPS or infection with H1N1. After treatment, AQP1 and AQP4 staining were increased to some extent.6. The released levels of TNF-α, IL-1β, IL-6 were increased greatly by stimulation with LPS or infection with H1N1. After treatment, all of them were downregulated in a certain extent.Clonclusion:1. MIF siRNA could be transfected into the target cell by linear cationic polymers jetPEI, and it was special silence the express of target gene MIF mRNA and protein in the A549 cell. Therefore MIF siRNA might exert its safeguard to the death of cell induced by LPS.2. The study provide the first evidence that in A549 cell, MIF siRNA could block the expression both of TLR4 induced by LPS and of TLR3 infected by H1N1, further it could block the expression of MyD88 protein in down stream signaling pathways, and inhibit the secretion of the inflammation cytokine such as TNF-α, IL-1β,IL-6. There were different effect on the down stream signaling pathways between MIF siRNA and Dexamethasone(DXM), DXM could block the expression both of MyD88 and IRF3, and inhibit the reaction of inflammation and immune, but MIF siRNA only blocked the expression of MyD88, and no effect to the expression of IRF3.3. In mouse of acute lung injure induced by LPS/H1N1, MIF siRNA could inhibit the expression of NF-κB. In the MAPK signaling pathways, it was also the first evidence that MIF siRNA was able to downregulate the expression of p-p38 and p-JNK, and lead to inhibit the releasing of inflammation cytokine of TNF-α,IL-1β,IL-6, attenuate the severity of lung injure.MIF siRNA can ease the symptoms of acute lung injure induced by LPS or influenza A virus, such function maybe ground on their special silence gene MIF to inhibit the triggers of mutiplex signal transcription pathway during acute lung injure.