Insulin-Attenuated Inflammatory Response of Dendritic Cells in Diabetes by Regulating RAGE-PKCβ1-IRS1-NF-κB Signal Pathway: A Study on the Anti-Inflammatory Mechanism of Insulin in Diabetes

Figure 3

Insulin dose dependently attenuating AGEs-BSA-stimulated BMDCs immune maturation and decreasing expressions of RAGE and phosphorylation of PKCβ1 and IRS1. (a) Flow cytometric analysis was performed for cell surface CD83, CD86, and MHC-II expression examinations. Insulin exerted an inhibitory effect on the expressions of CD83 and CD86 in a dose-dependent manner. (b) The concentrations of TNF-α, IFN-γ, IL-6, IL-1b, and CCL2 in the supernatants of the culture were measured by ELISA. (c, d) The expressions of cytokines and chemokines mRNA in BMDCs were analyzed by real-time quantitative RT-PCR. Results of (b–d) suggested insulin attenuated the expressions of AGEs-BSA-induced IL-1b, IL-10, IFN-γ, and TNF-α.; (e–i) The expressions of phosphorylated and total NF-κB (e), phosphorylated and total IκB (f), phosphorylation of PKC isoforms (g), serine phosphorylated IRS1 and RAGE (h), and serine phosphorylated IRS2 (i) were analyzed by western blot. IκB and NF-κB phosphorylation decreased significantly with the insulin treatment in a dose-dependent manner. Insulin treatment decreased significantly the upregulated expression of RAGE, phosphorylation of PKCβ1, and serine phosphorylation of IRS1, not the phosphorylation of PKCα and PKCβ2. AGEs significantly upregulated the expression of serine phosphorylated IRS2, while insulin had no significantly effect on phosphorylated IRS2. Data are expressed as ; ; vs. control group; ^# vs. AGEs-BSA group. AGEs-BSA: advanced glycosylation end-bovine serum albumin; BMDCs: bone marrow-derived dendritic cells; RAGE: receptor for advanced glycation end products; pPKC: phosphorylated protein kinase C; IRS1: insulin receptor subunit-1; IRS2: insulin receptor subunit-2; TNF-α: tumor necrosis factor-α; IFN-γ: interferon-γ; MHC-II: major histocompatibility complex-II; NF-κB: nuclear factor-κB.

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