The goal of this study was to interrogate the role of inducible NO synthase (iNOS) in the late phase of ischemic preconditioning (PC) in vivo. A total of 321 mice were used. Wild-type mice preconditioned 24 h earlier with six cycles of 4-min coronary occlusion͞4-min reperfusion exhibited a significant (P < 0.05) increase in myocardial iNOS protein content, iNOS activity (assessed as calciumindependent L-citrulline formation), and nitrite ؉ nitrate tissue levels. In contrast, endothelial NOS protein content and calcium-dependent NOS activity remained unchanged. No immunoreactive neuronal NOS was detected. When wild-type mice were preconditioned 24 h earlier with six 4-min occlusion͞4-min reperfusion cycles, the size of the infarcts produced by a 30-min coronary occlusion followed by 24 h of reperfusion was reduced markedly (by 67%; P < 0.05) compared with sham-preconditioned controls, indicating a late PC effect. In contrast, when mice homozygous for a null iNOS allele were preconditioned 24 h earlier with the same protocol, infarct size was not reduced. Disruption of the iNOS gene had no effect on early PC or on infarct size in the absence of PC. These results demonstrate that (i) the late phase of ischemic PC is associated with selective up-regulation of iNOS, and (ii) targeted disruption of the iNOS gene completely abrogates the infarct-sparing effect of late PC (but not of early PC), providing unequivocal molecular genetic evidence for an obligatory role of iNOS in the cardioprotection afforded by the late phase of ischemic PC. Thus, this study identifies a specific protein that mediates late PC in vivo.

Melatonin has been shown to improve lipid metabolism and gut microbiota communities in animals and humans; however, it remains to know whether melatonin prevents obesity through gut microbiota. Here, we found that high-fat diet promoted the lipid accumulation and intestinal microbiota dysbiosis in mice, while oral melatonin supplementation alleviated the lipid accumulation and reversed gut microbiota dysbiosis, including the diversity of intestinal microbiota, relative abundances of Bacteroides and Alistipes, and functional profiling of microbial communities, such as energy metabolism, lipid metabolism, and carbohydrate metabolism. Interestingly, melatonin failed to alleviate the high-fat-induced lipid accumulation in antibiotic-treated mice; however, microbiota transplantation from melatonin-treated mice alleviated high-fat diet-induced lipid metabolic disorders. Notably, short-chain fatty acids were decreased in high-fat diet-fed mice, while melatonin treatment improved the production of acetic acid. Correlation analysis found a marked correlation between production of acetic acid and relative abundances of Bacteroides and Alistipes. Importantly, sodium acetate treatment also alleviated high-fat diet-induced lipid metabolic disorders. Taken together, our results suggest that melatonin improves lipid metabolism in high-fat diet-fed mice, and the potential mechanisms may be associated with reprogramming gut microbiota, especially, Bacteroides and Alistipes-mediated acetic acid production. Future studies are needed for patients with metabolic syndrome to fully understand melatonin's effects on body weight and lipid profiles and the potential mechanism of gut microbiota.