| Acute liver injury is caused by various factors and is the common features of chronic liver injury, characterized by hepatocyte death and massive inflammatory cytokines secretion. If the acute liver injury can not get effective treatment, it will eventually lead to liver cirrhosis and hepatoma. Therefore, to further study the pathogenesis of acute liver injury and characteristics of its molecular mechanism, will provide a new way for understanding the formation of chronic liver disease and have a profound significance in liver treatment.Kupffer cells (KCs) is the resident macrophage of liver,and the polarization macrophage is an important contributor to many diseases, such as liver diseases, tumor, obesity and so on.Previous studies showed that mouse bone marrow-derived macrophages (BMDM) and Raw-264.7 macrophages both expressed voltage-gated K+ channels and Kvl.3 is the major K+ channels in these cells. Several lines of evidence have indicated that Kv1.3 plays a vital role in the cell proliferation, cycle, apoptosis, migration, and differentiation.However, the knowledge of Kvl.3 in the regulation of macrophage polarization is still largely undefined. And there is little known about Kvl.3 in liver disease, and the relationship between Kvl.3 and KCs has not been found, we further study the effect and potential mechanism of Kvl.3 in macrophage polarization and the partial role of Kvl.3 in liver disease, which is used to develop novel targets for the prevention and treatment of acute liver injury.so this research mainly divided into the following parts:1. To build the model of acute liver injury induced by LPS.We chose C57BL/6Jmice as the object of our research. The model group mice was was induced by intraperitoneal injection ofLPS for 24 h. Serology, HE, qRT-PCR, western blot were used to demonstrate that the model is successfully built.2. The expression of Kv1.3 in liver tissues and KCs Immunohistochemical, qRT-PCR, western blot were used to detect Kvl.3 expression in liver tissues. The expression of Kvl.3 in KCs was examed by qRT-PCR. The results indicated that Kvl.3 was low expression both in liver tissues and KCs of acute liver injury model.3.The role of Kvl.3 on KCs released cytokines. We chose RAW264.7 as the object of our research. Pretreatment with MgTx(10 nM), the RAW264.7 cells was then stimulated with LPS (1 mg/ml) for 24 h after 2 h. qRT-PCR andwestern blot results revealed Kvl.3 may played a role in regulating KCs cytokines secretion.4. Introduction of M1/M2 phenotypic modulation of RAW264.7 cellsWe chose RAW264.7 as the object of our research, Ml-like macrophage induced by LPS 1000 ng/ml and IFN-y 20 ng/ml 24 h and the activation of M2-like macrophage is stimulated by IL-420 ng/ml 24 h.5. Expression level of Kv1.3 in M1-and M2-type macrophageThe mRNA and protein expressions of Kv1.3 in M1 and M2 were measured by qRT-PCR and Western Blotting. These results suggest that the expression of Kv1.3 decreased in M1-like macrophage and increased in M2-like macrophage.6. The role of Kvl.3 in macrophage phenotypic modulationTreatmentof M2-type macrophage with Kvl.3 special inhibitor MgTx and LipofectamineTM 2000 liposome transfected Kv1.3 RNAi, then the expression of M2-associated genes were detected. The results suggested Kvl.3 promoted M2 macrophage polarization.7. The regulation of Kvl.3downstream in macrophage phenotypic modulation It was reported that PI3K/AKT signaling pathway play an important role in M1 macrophage polarization and Kv1.3 channel is tightly associated with PI3K/AKT pathway. The expression of P-AKT was decreased,whenM2-type macrophage was pretreated with MgTx or LipofectamineTM 2000 liposome transfected Kvl.3 RNAi. The PI3K inhibitor LY294002 did not affected Kvl.3 expression. These results indicated Kvl.3 promoted M2 macrophage polarization via AKT pathway. |
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