ABSTRACT
Objective To explore the role of the pyrin domain-containing 3 ( NLRP3) inflammasome in advanced glycation end products ( AGEs )-induced mice pancreatic β-cell damage. Methods AGEs were administered intraperitoneally for 6 weeks in NLRP3 knockout mice or C57BL/6J mice. Intraperitoneal glucose tolerance test and insulin releasing test were performed. Pancreatic sections were stained with haematoxylin and eosin, or with F4/80 and NLRP3 antibodies. Insulin and pancreatic tissue monocyte chemotactic protein 1 ( MCP-1) as well as interleukin-1β( IL-1β) levels were measured with ELISA kits. Expression of MCP-1 protein was determined by western blot. MIN6 cells and mouse peritoneal macrophages cells were treated with AGEs and different interventions (antioxidant NAC, adenovirus NLRP3 shRNA or NLRP3 knockout). Reactive oxygen species production, NLRP3 mRNA expression, IL-1β secretion, caspase 1 activity, apoptosis and glucose stimulated insulin release were determined. Results Injection of AGEs induced an abnormal response to glucose, enhanced the insulitis score, and increased the levels of pancreatic tissue MCP-1 and IL-1β, as well as raised the expression of NLRP3 and F4/80 in pancreatic islet. Remarkably, co-localization of NLRP3 and macrophage marker F4/80 was observed in islet. The damages were improved in NLRP3 knockout mice. After incubation with AGEs, reactive oxygen species production and cell apoptosis was enhanced, NLRP3 inflammasome activated, with glucose-stimulated insulin release impaired in MIN6 cells. NAC treatment alliviated the above damages, but NLRP3 gene silencing had no effect on ROS level, apoptosis, and insulin secretion. Finally NAC treatment and NLRP3 gene knockout inhibited activation of NLRP3 inflammasome induced by AGEs in mouse peritoneal macrophages cells. Conclusion NLRP3 knockout ameliorates the islet β-cell damage induced by AGEs. These effects were associated with AGEs-induced islets macrophage infiltrating by up-regulation of MCP-1 expression, and AGEs-induced activation of NLRP3 inflammasome in macrophage through ROS pathway, which results in the release of active IL-1βand leads to the lesions of β-cell.
ABSTRACT
Objective To observe the change of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression during 3T3-L1 adipocyte differentiation as well as other reference gene expressions.Methods The mRNA expressions of several common reference genes were detected by real time-PCR on day 0,1,3,5,and 7 of 3T3-L1 adipocyte differentiation.Western blot was used to confirm the protein expressions of three common reference genes.Results (1) GAPDH and transferrin receptor(TFRC) mRNA expressions were significantly increased during adipocyte differentiation.GAPDH mRNA level was increased by 5.7,7.6,22.0,and 24.5 folds on day 1,3,5,and 7 after induction of adipocyte differentiation,but no apparent changes of β-actin,α-tubulin,peptidylprolyl isomerase A (PIPA),and 18S mRNA expressions were detected.The expression changes of key transcript factors for adipocyte differentiation such as PPARγ2,C/EBPα,and C/EBPβ were under-estimated by real time-PCR if GAPDH was chosen as the reference gene.Western blotting results showed that the GAPDH protein level increased gradually during adipocyte differentiation,especially on day 5 and 7 after adipocyte differentiation.There were no obvious changes of β-actin and α-tubulin protein expressions.(2) Berberine significantly inhibited mRNA and protein expressions of GAPDH in the process of adipocyte differentiation.GAPDH mRNA levels were reduced by 68.1％ and 66.3％ on day 5 and 7 after induction of adipocyte differentiation,but with no significant change in other reference genes.Conclusion It is not suitable for GAPDH to be used as an endogenous reference gene during 3T3-L1 adipocyte differentiation.