RESUMO
Objective:To analyze the level of interleukin-36(IL-36) family cytokines in peripheral blood, and explore the regulatory role of recombinant human IL-36α in monocyte function in patients with diabetic kidney disease(DKD).Methods:A total of 41 type 2 diabetes mellitus(T2DM) patients, 36 DKD patients, and 20 controls were consecutively enrolled. Plasma and peripheral blood mononuclear cells(PBMCs) were isolated. Enzyme-linked immunosorbent assay(ELISA) was used to measure plasma levels of IL-36α, IL-36β, IL-36γ, and IL-36 receptor antagonist(IL-36Ra). PBMCs were sorted, and real-time quantitative PCR was performed to detect the mRNA expression of IL-36 receptor subunits in monocytes. Monocytes were stimulated with recombinant IL-36α, and levels of cytotoxic molecules and cytokines in the culture supernatant were measured. Flow cytometry was used to assess the expressions of programmed death receptor-1(PD-1) and cytotoxic T-lymphocyte-associated protein 4(CTLA-4). Co-culture of monocytes with Vero cells was performed to evaluate monocyte cytotoxicity.Results:Plasma levels of IL-36α and IL-36β in the T2DM and DKD groups were significantly higher than those in the control group. The DKD group also showed higher plasma levels of IL-36α compared to the T2DM group( P<0.05). There were no significant differences in IL-36γ and IL-36Ra levels among the three groups( P>0.05). The mRNA expression of IL-36 receptor subunits in monocytes was comparable among the three groups( P>0.05). The DKD group had higher level of tumor necrosis factor-alpha(TNF-α) compared to the control and T2DM groups( P<0.05). The levels of PD-1 and CTLA-4 were lower in the DKD group than those in the control and T2DM groups( P<0.05). The proportion of monocyte-induced Vero cell death was significantly higher in the DKD group compared to the control and T2DM groups( P<0.05). After stimulation with recombinant human IL-36α, monocytes from DKD patients showed a significant increase in the secretion of granzyme B and TNF-α( P<0.05), as well as an increased proportion of monocyte-induced Vero cell death( P=0.024). Conclusion:In DKD patients, elevated IL-36α and granzyme B levels in monocytes enhance monocyte function.
RESUMO
Objective:To explore the effect of recombinant human syntaxin-4(STX4) on lipopolysaccharide(LPS)-induced injury in islet β-cells(INS-1).Methods:Pancreatic islet β-cells(INS-1) were divided into Control (blank control), LPS (LPS treatment), LPS+ NC (transfection of negative control vector, LPS treatment), and LPS+ STX4 (transfection of pcDNA-STX4, LPS treatment) groups. RT-qPCR and Western blot were used to detect STX4 mRNA and protein expression, flow cytometry to detect apoptosis, DCFHDA method to detect reactive oxygen species(ROS) level, xanthine oxidation method to detect superoxide orgotein dismutase(SOD) level, colorimetric method to detect glutathione peroxidase(GSH-Px) level, ammonium molybdate colorimetric method to detect catalase(CAT) level, thiobarbituric acid method to detect malonaldehyde(MDA) level, ELISA method to detect the level of interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and insulin secretion levels under glucose conditions secreted by cells, Western blot method to detect Cleared Caspase-3, Bcl-2 Associated X Protein(Bax), p65 protein expression. After treatment with NF-κB signaling pathway activator, STX4 up-regulated islet β-cell INS-1 was given LPS stimulation, and the same method was used to measure apoptosis, ROS, SOD, GSH-Px, CAT, MDA levels and secreted IL-1β, TNF-α, insulin levels and Cleaved Caspase-3, Bax, p65 protein levels.Results:Compared with the Control group, the expression of STX4 mRNA and protein in islet β cells of the LPS group decreased, the apoptosis rate, ROS level, and MDA levels increased, and the levels of SOD, GSH-Px, and CAT decreased, the levels of IL-1β, TNF-α increased, the level of insulin secreted by the cells decreased, and the expression levels of Cleaved Caspase-3, Bax, and p65 also increased. NF-κB pathway activator treatment reversed the effect of up-regulated STX4 on islet β-cell apoptosis, ROS, SOD, GSH-Px, CAT, MDA levels and secreted IL-1β, TNF-α levels, and Cleaved Caspase-3 , Bax and p65 protein levels.Conclusion:Up-regulation of STX4 alleviated LPS-induced islet β cell oxidative damage, apoptosis and inflammatory factor release. The underlying mechanism might be related to the inhibition of activated NF-κB signaling pathway.