Role Of TAK1 Signaling Pathways In Bone Marrow Derived Macrophage Activation Induced By High Glucose

Background and Objective The incidence of diabetic nephropathy(DN) has been rising accompany with the increasing number of diabetes year by year. Now is generally accepted that diabetic kidney disease is a chronic low-grade inflammatory disease. Macrophages play an important role in the initiation and progression of diabetic nephropathy. Inactive macrophages do not cause kidney injury. Only activated macrophages play a critical role in the regulation of inflammation and contribute to fibrosis in diabetic nephropathy. However, the molecular mechanism of macrophage activation is unclear. Transforming growth factor beta activated kinase 1(TAK1) as a member of the MAP kinase kinase kinase(MAP3K) family, has been identified a pro-inflammatory pathway, which is located in the upstream of MAPKs and NF-κB signaling pathways. While, under high glucose conditions, whether or not TAK1 signaling pathway been involved in macrophage activation has not been reported. The present study designed to explore the likely mechanism of macrophage activation through detect the secretion levels of TNF-α, IL-1β, MCP-1 in each group cell media and the expression of p-TAK1, TAB1, p-JNK, NF-κBp65 proteins from bone marrow-derived macrophages(BMDM) stimulated by high glucose.Methods 1. Isolation and culture of mouse bone marrow-derived macrophages. 2. Bone marrow macrophage purity testing by flow cytometry.3. CCK-8 method was used to detect TAK1 inhibitor(5Z-7-oxozeaenol) cytotoxicity in different concentration(10-1000 nmol/L). 4. The model of macrophages and mesangial cells co-culture was building in Transwell chamber. 5. The BMDMs were cultured separately or co-cultured with mesangial cells and divided into seven groups: inhibitor control group(OZ300), mannitol control group(MC), normal control group(NC), high glucose group(HG) and inhibitor groups( HG + OZ30, HG + OZ100, HG + OZ300). 6. M1 macrophage marker i NOS was detected by cell immune fluorescence. 7. The concentration of TNF-α, IL-1β and MCP-1 in each group cell media was determined by enzyme-linked immune sorbent assay(ELISA). 8. The expression of p-TAK1, TAB1, p-JNK, NF-κB p65 proteins were analyzed by Western blotting. 9. The intracellular localization of NF-κB p65 was analyzed by cell immune fluorescence.Results 1. The purity of BMDM was about 99.36%. 2. Compared with control group, treatment with less than 300 nmol/L inhibitor has no negative effect on viability of BMDM stimulated by high glucose(P﹥0.05), while 1000nmol/L inhibitor was harmful to BMDM cell viability(P<0.05). 3. The fluorescence intensity of i NOS in high glucose group was significantly higher than those of control groups. While the fluorescence intensity of i NOS in inhibitor group was weaker than high glucose group. 4. In both cell culture models, the levels of TNF-α, IL-1β and MCP-1 were increased in high glucose, comparing with those in control groups(P<0.05).Meanwhile, exposed to high glucose, the levels of TNF-α, IL-1β and MCP-1 in co-culture were higher than those in monolayer BMDMs(P<0.05). 5Z-7-oxozeaenol can decrease those pro-inflammatory cytokines secretion with a concentration dependent manner, comparing with high glucose group(P<0.05). 5. Under the condition of BMDMs been cultured separately, the expression levels of p-TAK1, TAB1, p-JNK, NF-κB p65 proteins in high glucose group were higher than that in control groups(P<0.05). While 5Z-7-oxozeaenol down-regulates those proteins expression with a concentration dependent manner, comparing with high glucose group(P<0.05). 6. In high glucose stimulated BMDMs, NF-κB p65 nucleus translocation was significantly inhibited by 300nmol/L 5Z-7-oxozeaenol.Conclusions 1. High glucose can induce BMDM switch to the M1 phenotype, expressing higher i NOS. 2. High glucose stimulates BMDM activation via TAK1/JNK and TAK1/ NF-κB signaling pathways. 3. Exposed to high glucose, BMDMs and mesangial cells can interact with each other, which can promote BMDMs to secrete more pro-inflammatory cytokines, including TNF-α, IL-1β and MCP-1. 4. TAK1 inhibitor(5Z-7-oxozeaenol) prevents high glucose-induced macrophage activation to down-regulate pro-inflammatory cytokines via JNK and NF-κB pathways.