Abstracts Background Many neurological diseases involve neuroinflammation, during which overproduction of cytokines by immune cells, especially microglia, can aggregate neuronal death. Ferroptosis is a recently discovered cell metabolism-related form of cell death and RSL3 is a well-known inducer of cell ferroptosis. Here, we aimed to investigate the effects of RSL3 in neuroinflammation and sensitivity of different type of microglia and macrophage to ferroptosis. Methods Here, we used quantitative RT-PCR analysis and ELISA analysis to analyze the production of proinflammatory cytokine production of microglia and macrophages after lipopolysaccharides (LPS) stimulation. We used CCK8, LDH, and flow cytometry analysis to evaluate the sensitivity of different microglia and macrophages to RSL3-induced ferroptosis. Western blot was used to test the activation of inflammatory signaling pathway and knockdown efficiency. SiRNA-mediated interference was conducted to knockdown GPX4 or Nrf2 in BV2 microglia. Intraperitoneal injection of LPS was performed to evaluate systemic inflammation and neuroinflammation severity in in vivo conditions. Results We found that ferroptosis inducer RSL3 inhibited lipopolysaccharides (LPS)-induced inflammation of microglia and peritoneal macrophages (PMs) in a cell ferroptosis-independent manner, whereas cell ferroptosis-conditioned medium significantly triggered inflammation of microglia and PMs. Different type of microglia and macrophages showed varied sensitivity to RSL3-induced ferroptosis. Mechanistically, RSL3 induced Nrf2 protein expression to inhibit RNA Polymerase II recruitment to transcription start site of proinflammatory cytokine genes to repress cytokine transcription, and protect cells from ferroptosis. Furthermore, simultaneously injection of RSL3 and Fer-1 ameliorated LPS-induced neuroinflammation in in vivo conditions. Conclusions These data revealed the proinflammatory role of ferroptosis in microglia and macrophages, identified RSL3 as a novel inhibitor of LPS-induced inflammation, and uncovered the molecular regulation of microglia and macrophage sensitivity to ferroptosis. Thus, targeting ferroptosis in diseases by using RSL3 should consider both the pro-ferroptosis effect and the anti-inflammation effect to achieve optimal outcome.

Background: Wound healing is a complex pathophysiological process that involves a variety of cells and cytokines. In this study, we found that local injection of human amnion mesenchymal stem cells into wounds in rats could promote wound healing. Therefore, we hypothesized that the exosomes of human amnion mesenchymal stem cells contain substances that regulate the migration of epidermal cells. It has been reported that miR-135a is involved in cell migration and transformation. However, there have been no reports of its function in skin wound healing. Methods: To test this hypothesis, we injected exosomes overexpressing miR-135a directly into the wound margin. In addition, we tested the migration of BJ cells with overexpression or knockdown of miR-135a in vitro. Additionally, Western blot analysis was used to detect the expression of fibroblast migration-associated proteins after treatment with miR-135a overexpression or knockdown. Results: MiR-135a significantly promoted wound healing compared to the control treatment. Western blot analysis showed a significant downregulation of LATS2 after overexpression of miR-135a. In addition, knockdown of miR-135a effectively attenuated the promoting effect of exosomes on cell migration. Conclusions: Our results indicated that miR-135a promotes wound healing, which may be mediated by downregulating LATS2 levels to increase cell migration. This study provides a rationale for the therapeutic effect on wound healing of miR-135a in exosomes derived from human amnion mesenchymal stem cells.

The current study investigated the effects of nesfatin-1 in the hypothalamic paraventricular nucleus (PVN) on gastric motility and the regulation of the lateral hypothalamic area (LHA). Using single unit recordings in the PVN, we show that nesfatin-1 inhibited the majority of the gastric distention (GD)-excitatory neurons and excited more than half of the GD-inhibitory (GD-I) neurons in the PVN, which were weakened by oxytocin receptor antagonist H4928. Gastric motility experiments showed that administration of nesfatin-1 in the PVN decreased gastric motility, which was also partly prevented by H4928. The nesfatin-1 concentration producing a half-maximal response (EC50) in the PVN was lower than the value in the dorsomedial hypothalamic nucleus, while nesfatin-1 in the reuniens thalamic nucleus had no effect on gastric motility. Retrograde tracing and immunofluorescent staining showed that nucleobindin-2/nesfatin-1 and fluorogold double-labeled neurons were observed in the LHA. Electrical LHA stimulation changed the firing rate of GD-responsive neurons in the PVN. Pre-administration of an anti-nucleobindin-2/nesfatin-1 antibody in the PVN strengthened gastric motility and decreased the discharging of the GD-I neurons induced by electrical stimulation of the LHA. These results demonstrate that nesfatin-1 in the PVN could serve as an inhibitory factor to inhibit gastric motility, which might be regulated by the LHA.

Orexin-A is a circulating neuropeptide and neurotransmitter that regulates food intake and gastric motility. The central nucleus of the amygdala (CeA), which regulates feeding behavior and gastric function, expresses the orexin-1 receptor. The aim of this study was to evaluate the effects of microinjection of exogenous orexin-A into the CeA, on food intake and gastric motility, and to explore the mechanisms of these effects. Normal chow and high fat food (HFF) intake were measured, gastric motility and gastric emptying were evaluated, extracellular single unit firing was recorded, and c-fos expression was determined. The results showed that microinjection of orexin-A into the CeA resulted in increased HFF intake but did not affect normal chow intake. This effect was blocked by an orexin-1 receptor antagonist-SB-334867 and was partially blocked by a dopamine D1 receptor antagonist-SCH-23390. Gastric motility and gastric emptying were enhanced by orexin-A, and the former effect was abolished by subdiaphragmatic vagotomy. The firing frequency of gastric distention-related neurons was regulated by orexin-A via the orexin-1 receptor. Furthermore, c-fos expression was increased in the ventral tegmental area (VTA) and the nucleus accumbens (NAc), the lateral hypothalamus (LHA), and the dorsal motor nucleus of the vagus (DMV) in response to microinjection of orexin-A into the CeA. These findings showed that orexin-A regulated palatable food intake and gastric motility via the CeA. The LHA, the VTA, and the NAc may participate in palatable food intake and the CeA-DMV-vagus-stomach pathway may be involved in regulating gastric motility through the regulation of neuronal activity in the CeA.

Although the novel satiety peptide nesfatin-1 has been shown to regulate gastric motility, the underlying mechanisms have yet to be elucidated. The study aimed to explore the effects of nesfatin-1 on ghrelin-responsive gastric distension (GD) neurons in the arcuate nucleus (Arc), and potential regulation mechanisms of gastric motility by the paraventricular nucleus (PVN). Single-unit discharges in the Arc were recorded extracellularly, and gastric motility in conscious rats was monitored during the administration of nesfatin-1 to the Arc or electrical stimulation of the PVN. Retrograde tracing and fluo-immunohistochemistry staining were used to determine NUCB2/nesfatin-1 neuronal projections. Nesfatin-1 inhibited most of the ghrelin-responsive GD-excitatory neurons, but excited ghrelin-responsive GD-inhibitory neurons in the Arc. Gastric motility was significantly reduced by nesfatin-1 administration to the Arc in a dose-dependent manner. The firing activity in the Arc and changes to gastric motility were partly reduced by SHU9119, an antagonist of melanocortin 3/4 receptors. Electrical stimulation of PVN excited most of the ghrelin-responsive GD neurons in the Arc and promoted gastric motility. Nonetheless, pretreatment with an anti-NUCB2/nesfatin-1 antibody in the Arc further increased the firing rate of most of the ghrelin-responsive GD-excitatory neurons and decreased the ghrelin-responsive GD-inhibitory neurons following electrical stimulation of the PVN. Gastric motility was enhanced by pretreatment with an anti-NUCB2/nesfatin-1 antibody in the Arc following PVN stimulation. Furthermore, NUCB2/nesfatin-1/fluorogold double-labeled neurons were detected in the PVN. These results suggest that nesfatin-1 could serve as an inhibitory factor in the Arc to regulate gastric motility via the melanocortin pathway. The PVN could be involved in the regulation of the Arc in gastric activity.