The Mechanism Of Kupffer Cells’ Regulation In Liver Failure Induced By Cona

Background and objectsLiver failure (LF), known as a rapid and severe clinical syndrome, can lead to numerous hepatocytes death, ultimately induce multiple organ dysfunction and failure. The pathogenesis of liver failure is complicated, it includes direct and immune mediated liver injury. Innate immune mediated injury occurred at the early stage of liver failure and it might be an important factor in the pathogenesis of liver failure. Kupffer cell, a special macrophage resided in the liver, is known as the crucial element in liver innate immune system and it is also involved in the development of liver inflammation during various liver diseases. However, detailed regulation mechanism of Kupffer cells during liver failure is still obscured. Present study aimed to realize the effect from dynamic change of Kupffer cells activation at the early stage of liver failure, find the reason of IL-1β production, and discover the regulation mechanism of certain microRNA in Kupffer cells at the early stage of liver failure, which would be benefit to our understanding in the detailed regulation mechanism of innate immune mediated injury during liver failure and provide the experimental data for valid therapy. Methods1. The effect from dynamic change of Kupffer cells’immunologic status and the reason of IL-1p production at the early stage of liver failure1.1Dynamic change of Kupffer cells’immunologic status:The mice model of liver failure was established by ConA treatment. Kupffer cells were isolated after1,3,6hours treated by ConA. Relative mRNA levels of IL-1β (Ml marker), Argl and Mrc2(M2marker) were measured by qRT-PCR.1.2Reason of IL-1p production:Relative mRNA levels of AIM2in Kupffer cells from mice treated with Con A for1h,3h and6h were measured by qRT-PCR. Protein levels of AIM2in Kupffer cells from mice stimulated with Con A for1h,3h and6h were detected by Western blotting. Kupffer cells from normal mice were isolated and then transfected with AIM2-siRNA or siRNA control. After transfection, cells were exposed to Con A (5u.g/ml) for1h,3h and6h. Quantified protein level of IL-1β was measured by ELISA.2. The regulation mechanism of certain microRNA in Kupffer cells at the early stage of liver failure.2.1MicroRNA screening:Relative levels of microRNA-155and microRNA-223in Kupffer cells from mice treated with Con A for lh,3h and6h were measured by qRT-PCR.2.2Relationship between microRNA-223and IL-1p:Kupffer cells from normal mice were isolated and then transfected with microRNA-223mimics/microRNA-223inhibitor and relevant negative control. After transfection, cells were exposed to Con A (5μg/ml) for1h,3h and6h. Quantified protein level of IL-1β was measured by ELISA.2.3Relationship between microRNA-223and AIM2:Kupffer cells from normal mice were isolated and then transfected with microRNA-223mimics/microRNA-223inhibitor and relevant negative control. After transfection, cells were exposed to Con A (5μg/ml) for1h,3h and6h. Relative mRNA levels of AIM2were measured by qRT-PCR. Protein levels of AIM2were detected by Western blotting. Kupffer cells from normal mice were isolated and then transfected with AIM2-siRNA or siRNA control. After transfection, cells were exposed to Con A (5μ,g/ml) for1h,3h and6h. Relative levels of microRNA-223were measured by qRT-PCR. Kupffer cells from normal mice were isolated and then co-transfected with luciferase reporter plasmid or empty vector, and miR-223mimics or relevant negative control. Firefly luciferase activities were measuredResults1. Dynamic change of Kupffer cells’ immunologic statusKupffer cells’ immunologic status were dynamicly changed at the early stage of liver failure. Activated Kupffer cells switched from pro-to anti-inflammatory status, ultimately it was back to pro-inflammatory status again.2. Reason of IL-1β productionIL-1β production was related to the expression levels of inflammasome AIM2at the early stage of liver failure. The level of IL-1β production was decreased when AIM2silenced by siRNA.3. Relationship between microRNA-223and IL-1pIL-1β production was inhibited by microRNA-223in Kupffer cells at the early stage of liver failure. The level of IL-1β production was decreased by microRNA-223overexpression. In contrast, the level of IL-1β production was increased after microRNA-223inhibition.4. Relationship between microRNA-223and AIM2MicroRNA-223down regulated the expression of AIM2in Kupffer cells at the early stage of liver failure. The level of AIM2was decreased by microRNA-223overexpression. In contrast, the level of AIM2was increased after microRNA-223inhibition. No change in the level of microRNA-223expression occurred when AIM2 silenced by siRNA. The result of Luciferase assay system suggested that AIM2was not the direct target of microRNA-223.Conclusion1. Kupffer cells’immunologic status were dynamicly changed at the early stage of liver failure. The high level of IL-1β produced from activated Kuppfer cells might influence the pathogenesis of liver failure.2. IL-1β production was related to inflammasome AIM2at the early stage of liver failure.3. MicroRNA-223could lead to the change in activation pattern of Kupffer cells and affecting the production of IL-1β via AIM2pathway in the early stage of liver failure. However, AIM2was not the direct target of microRNA-223. MicroRNA-223would be a potential target for liver failure treatment.