[LIMR is involved in the inhibitory effect of antiflammin-1 on epithelial-mesenchymal transition in A549 cells].

Epithelial-mesenchymal transition (EMT) occurring in alveolar epithelial cells plays an important role in the development and progression of pulmonary fibrosis. Previous studies showed that antiflammin-1 (the active fragment of uteroglobin) effectively inhibited bleomycin-induced pulmonary fibrosis. However, its mechanism is still far from being clarified. In this study, we investigated the effects of antiflammin-1 on EMT in A549 cells induced by transforming growth factor-ß1 (TGF-ß1) and the underlying mechanism by using morphological observation and Western blot. The results showed that the expression of α-smooth muscle actin (α-SMA) increased significantly while the expression of E-cadherin decreased significantly in A549 cells following treatment with TGF-ß1 concomitant with morphological change of A549 cells from pebble-like shape epithelial cells to spindle-like mesenchymal shape. This process of EMT in A549 cells induced by TGF-ß1 was significantly inhibited when A549 cells were co-incubated with TGF-ß1 and antiflammin-1. Furthermore, the anti-lipocalin interacting membrane receptor (LIMR) antibody and PD98059 (an ERK signaling pathway blocker) attenuated the inhibitory effect of antiflammin-1 on TGF-ß1-induced EMT, respectively. Our findings indicate that antiflammin-1 can inhibit EMT in A549 cells induced by TGF-ß1, which is related to LIMR and its downstream ERK signaling pathway.

A Sustained Activation of Pancreatic NMDARs Is a Novel Factor of ß-Cell Apoptosis and Dysfunction.

Type 2 diabetes, which features ß-cell failure, is caused by the decrease of ß-cell mass and insulin secretory function. Current treatments fail to halt the decrease of functional ß-cell mass. Strategies to prevent ß-cell apoptosis and dysfunction are highly desirable. Recently, our group and others have reported that blockade of N-methyl-d-aspartate receptors (NMDARs) in the islets has been proposed to prevent the progress of type 2 diabetes through improving ß-cell function. It suggests that a sustained activation of the NMDARs may exhibit deleterious effect on ß-cells. However, the exact functional impact and mechanism of the sustained NMDAR stimulation on islet ß-cells remains unclear. Here, we identify a sustained activation of pancreatic NMDARs as a novel factor of apoptotic ß-cell death and function. The sustained treatment with NMDA results in an increase of intracellular [Ca2+] and reactive oxygen species, subsequently induces mitochondrial membrane potential depolarization and a decrease of oxidative phosphorylation expression, and then impairs the mitochondrial function of ß-cells. NMDA specifically induces the mitochondrial-dependent pathway of apoptosis in ß-cells through upregulation of the proapoptotic Bim and Bax, and downregulation of antiapoptotic Bcl-2. Furthermore, a sustained stimulation of NMDARs impairs ß-cell insulin secretion through decrease of pancreatic duodenal homeobox-1 (Pdx-1) and adenosine triphosphate synthesis. The activation of nuclear factor-κB partly contributes to the reduction of Pdx-1 expression induced by overstimulation of NMDARs. In conclusion, we show that the sustained stimulation of NMDARs is a novel mediator of apoptotic signaling and ß-cell dysfunction, providing a mechanistic insight into the pathological role of NMDARs activation in diabetes.

An excessive increase in glutamate contributes to glucose-toxicity in ß-cells via activation of pancreatic NMDA receptors in rodent diabetes.

In the nervous system, excessive activation of NMDA receptors causes neuronal injury. Although activation of NMDARs has been proposed to contribute to the progress of diabetes, little is known about the effect of excessive long-term activation of NMDARs on ß-cells, especially under the challenge of hyperglycemia. Here we thoroughly investigated whether endogenous glutamate aggravated ß-cell dysfunction under chronic exposure to high-glucose via activation of NMDARs. The glutamate level was increased in plasma of diabetic mice or patients and in the supernatant of ß-cell lines after treatment with high-glucose for 72 h. Decomposing the released glutamate improved GSIS of ß-cells under chronic high-glucose exposure. Long-term treatment of ß-cells with NMDA inhibited cell viability and decreased GSIS. These effects were eliminated by GluN1 knockout. The NMDAR antagonist MK-801 or GluN1 knockout prevented high-glucose-induced dysfunction in ß-cells. MK-801 also decreased the expression of pro-inflammatory cytokines, and inhibited I-κB degradation, ROS generation and NLRP3 inflammasome expression in ß-cells exposed to high-glucose. Furthermore, another NMDAR antagonist, Memantine, improved ß-cells function in diabetic mice. Taken together, these findings indicate that an increase of glutamate may contribute to the development of diabetes through excessive activation of NMDARs in ß-cells, accelerating ß-cells dysfunction and apoptosis induced by hyperglycemia.

Antiflammin-1 attenuates bleomycin-induced pulmonary fibrosis in mice.

BACKGROUND: Antiflammin-1 (AF-1), a derivative of uteroglobin (UG), is a synthetic nonapeptide with diverse biological functions. In the present study, we investigated whether AF-1 has a protective effect against bleomycin-induced pulmonary fibrosis. METHODS: C57BL/6 mice were injected with bleomycin intratracheally to create an animal model of bleomycin-induced pulmonary fibrosis. On Day 7 and Day 28, we examined the anti-inflammatory effect and antifibrotic effect, respectively, of AF-1 on the bleomycin-treated mice. The effects of AF-1 on the transforming growth factor-beta 1 (TGF-ß1)-induced proliferation of murine lung fibroblasts (NIH3T3) were examined by a bromodeoxycytidine (BrdU) incorporation assay and cell cycle analysis. RESULTS: Severe lung inflammation and fibrosis were observed in the bleomycin-treated mice on Day 7 and Day 28, respectively. Administration of AF-1 significantly reduced the number of neutrophils in the bronchoalveolar lavage fluid (BALF) and the levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) in the lung homogenates on Day 7. Histological examination revealed that AF-1 markedly reduced the number of infiltrating cells on Day 7 and attenuated the collagen deposition and destruction of lung architecture on Day 28. The hydroxyproline (HYP) content was significantly decreased in the AF-1-treated mice. In vitro, AF-1 inhibited the TGF-ß1-induced proliferation of NIH3T3 cells, which was mediated by the UG receptor. CONCLUSIONS: AF-1 has anti-inflammatory and antifibrotic actions in bleomycin-induced lung injury. We propose that the antifibrotic effect of AF-1 might be related to its suppression of fibroblast growth in bleomycin-treated lungs and that AF-1 has potential as a new therapeutic tool for pulmonary fibrosis.