Background: Inflammatory osteolysis after total joint replacement (TJR) may cause implant failure, periprosthetic fractures, and be a severe threat to global public health. Our previous studies demonstrated that melatonin had a therapeutic effect on wear-particles induced osteolysis. Gut microbiota is closely related to bone homeostasis, and has been proven to be affected by melatonin. However, whether melatonin could play its anti-osteolysis effects through reprogramming gut microbiota remains elusive. Results: Here, we demonstrated that melatonin could alleviate Ti-particles induced osteolysis, while this therapeutic effect was blocked by antibiotic cocktail treatment. Interestingly, transplantation of fecal microbiota from mice treated with melatonin reappeared the same beneficial effect. Analysis of the 16S rRNA revealed that melatonin could reverse dysbacteriosis triggered by osteolysis, and elevate the relative abundance of some short chain fatty acid (SCFA) producing bacteria. Moreover, butyrate was enriched by exogenous melatonin administration, while acetate and propionate did not show an evident difference. This was consistent with the results of the metagenomic approach (PICRUSt2) analysis, which revealed a general increase in the synthetic enzymes of butyrate. More importantly, direct supplementation of butyrate could also recapitulate the anti-osteolysis effect of melatonin. Further analysis identified that butyrate alleviated osteolysis via activating its receptor GPR109A, and thus to suppress the activation of NLRP3 inflammasome triggered by Ti-particles. Conclusions: Taken together, our results suggested that the benefits of melatonin mainly depend on the ability of modulating gut microbiota and regulating butyrate production. Graphic

Previous studies have indicated edema may be involved in the pathophysiology following hypoxic-ischemic encephalopathy (HIE), and melatonin may exhibit neuro-protection against brain insults. However, little is known regarding the mechanisms that involve the protective effects of melatonin in the brain and peripheral tissues after HIE. The present study aimed to examine the effects of melatonin on multiple organs, and the expression of edema related proteins in a neonatal rat model of hypoxic-ischemic brain damage (HIBD). One hundred ninety-two neonatal rats were randomly divided into three subgroups that underwent a sham surgery or HIBD. After the HIBD or sham-injury, the rats received an intraperitoneal injection of melatonin or an equal volume vehicle, respectively. We investigated the effects of melatonin on brain, kidney, and colon edema via histological examination and the expression of edema related proteins, including AQP-4, ZO-1 and occludin, via qPCR and western blot. Our data indicated (1) Melatonin reduced the histological injury in the brain and peripheral organs induced by HIBD as assessed via H-E staining and transmission electron microscopy. (2) Melatonin alleviated the HIBD-induced cerebral edema characterized by increased brain water content. (3) HIBD induced significant changes of edema related proteins, such as AQP-4, ZO-1 and occludin, and these changes were partially reversed by melatonin treatment. These findings provide substantial evidence that melatonin treatment has protective effects on the brain and peripheral organs after HIBD, and the edema related proteins, AQP4, ZO-1, and occludin, may indirectly contribute tothe mechanism of the edema protection by melatonin.

Background: A prompt diagnosis of HIE remains a challenge clinically. This study aimed to identify potential biomarkers of neonatal hypoxic-ischemic encephalopathy (HIE) via a novel proteomic approach, the isobaric tags for absolute and relative quantification (iTRAQ) method. Methods: Blood samples were collected from neonates with mild (n = 4), moderate (n = 4), or severe (n = 4) HIE who were admitted to the neonatal intensive care unit of Children’s Hospital of Soochow University between Oct 2015 and Oct 2017. iTRAQ was performed in HIE patients and healthy controls (n = 4). Bioinformatics analyses including Gene Ontology and KEGG pathway enrichment analysis were performed to evaluate the potential features and capabilities of the identified differentially expressed proteins. Results: A total of 51 commonly differentially expressed proteins were identified among the comparisons between mild, moderate, and severe HIE as well as healthy controls. Haptoglobin (HP) and S100A8 were most significantly up-regulated in patients with HIE and further validated via real-time PCR and western blotting. The differentially expressed proteins represented multiple biological processes, cellular components and molecular functions and were markedly enriched in complement and coagulation cascades. Conclusions: HP and S100A8 may serve as a potential biomarker for neonatal HIE and reflects the severity of HIE. The complement and coagulation cascades play crucial roles in the development of neonatal HIE.

Autophagy has been suggested to participate in the pathology of hypoxic-ischemic brain damage (HIBD). However, its regulatory role in HIBD remains unclear and was thus examined here using a rat model. To induce HIBD, the left common carotid artery was ligated in neonatal rats, and the rats were subjected to hypoxia for 2 hours. Some of these rats were intraperitoneally pretreated with the autophagy inhibitor 3-methyladenine (10 mM in 10 μL) or the autophagy stimulator rapamycin (1 g/kg) 1 hour before artery ligation. Our findings demonstrated that hypoxia-ischemia-induced hippocampal injury in neonatal rats was accompanied by increased expression levels of the autophagy-related proteins light chain 3 and Beclin-1 as well as of the AMPA receptor subunit GluR1, but by reduced expression of GluR2. Pretreatment with the autophagy inhibitor 3-methyladenine blocked hypoxia-ischemia-induced hippocampal injury, whereas pretreatment with the autophagy stimulator rapamycin significantly augmented hippocampal injury. Additionally, 3-methyladenine pretreatment blocked the hypoxia-ischemia-induced upregulation of GluR1 and downregulation of GluR2 in the hippocampus. By contrast, rapamycin further elevated hippocampal GluR1 levels and exacerbated decreased GluR2 expression levels in neonates with HIBD. Our results indicate that autophagy inhibition favors the prevention of HIBD in neonatal rats, at least in part, through normalizing GluR1 and GluR2 expression.