Effects Of Inhaled Eugenol On The Energy Metabolism Of The Body

Food flavor is one of the directions of food science research,and various flavor substances in food can produce different responses through olfactory and gustatory feedback to the organism.The delivery of small molecule functional compounds directly to the brain through the nasal cavity to achieve the improvement of the nervous system as well as peripheral system functions has become a new field of nutrition and functional food research in recent years.The pathways and mechanisms by which flavors in foods exert their functional effects on the body through the nasal cavity are still unclear and need to be studied in depth.Eugenol is a functional compound found in cinnamon,clove and other food flavors,which has anti-inflammatory,antioxidant and other biological functions.Its good lipid solubility and easy to cross the blood-brain barrier make it one of the potential compounds for functional studies via the nasal route.Therefore,in this study,we investigated the dose effect of eugenol on energy metabolism by intranasal pathway through in vivo and ex vivo experiments,and determined the metabolic regulation mechanism of hypothalamic-pituitary-thyroid（HPT）axis by intranasal inhalation of eugenol through transient receptor potential vanilloid channel 1（TRPV1）-mediated energy sensing signal.Details of the study and results are as follows.1.The differences in the use of intranasal drops versus transoral gavage of eugenol on disorders of glucolipid metabolism in the organism were evaluated by HFD/STZ-induced T2DM model in mice.At the same dose,intranasal eugenol compared with oral gavage significantly improved the weight loss caused by HFD/STZ,alleviated the systemic inflammation and redox imbalance caused by HFD/STZ by decreasing the levels of pro-inflammatory factors TNF-αand IL-6 and increasing the levels of antioxidant enzyme GSH-PX,improved mice to reduce pancreatic damage,increased insulin secretion,alleviated mice insulin resistance level,reduce plasma triglyceride and low-density lipoprotein levels.2.The metabolism and distribution of eugenol in normal mice by intranasal drip and oral gavage were examined by UPLC-DAD system.The UPLC-DAD assay was developed and validated.The assay ranged from 0.1 g/ml-100 g/ml,with an average recovery of 101.41%,an average stability of 1.046%and a precision of 1.45%in various tissues in plasma,brain,liver,muscle and pancreas,which can be used for in vivo assay of eugenol.The concentration of eugenol in mice returned to pre-experimental levels 16 h after intranasal drip and oral gavage of eugenol;the peak blood eugenol concentration reached 4.5 g/ml after intranasal drip and was significantly higher than that in the oral gavage of eugenol group within 2 h.There was no significant difference between the concentrations of eugenol administered by the two ways in mouse liver,pancreas and muscle tissues at various time points,but in The concentrations of eugenol in various parts of brain tissue（hypothalamus,hippocampus,olfactory bulb,cerebral cortex,and cerebellum）were significantly higher in the intranasal drip than in the oral gavage group,and eugenol accumulated in the hypothalamus and hippocampus.3.The genetic changes in the hypothalamus of T2DM model mice after different modes of eugenol intake were investigated by transcriptome sequencing.A total of 10723 genes were detected,of which 8183 were non-differential genes;1288 and 1175 genes including long-strand non-coding RNAs related to neural signaling were significantly different between oral and intranasal eugenol dripping and T2DM groups,respectively,of which 429 were differential genes specific to intranasal dripping.Enrichment analysis showed that intranasal drip of eugenol significantly decreased the expression levels of genes related to glucose metabolism such as Slc2a1,Hk1,Aldob,and Idh1,and increased the expression levels of Slc27a1,Abca1,Srebf1 genes for lipid catabolism metabolism and Acbd6,Acaa2,Eci1 fatty acid mitochondrial oxidation and Agpat3 phospholipid synthesis in hypothalamus gene expression levels to reduce glucose uptake,enhance lipid metabolism,and improve cell membrane phospholipid synthesis;on the other hand,intranasal drops of eugenol enhanced the expression levels of Ca2+channel-related genes such as Cacnala,Cacnalc,Canmk2,and neurotransmitter receptor（Gng2/Tubb6/Tubb5/Gabra1/2/4）genes by enhancing inter-synaptic excitability,which in turn upregulates the expression levels of peptide hormone secretion genes induced by melanocortin（POMC）.4.To investigate the possible mechanism of the effect of intranasal eugenol on energy metabolism through a short-term（120 min）intranasal eugenol intervention on blood glucose.Both intranasal and oral gavage of eugenol significantly reduced blood glucose in mice in the short term,but intranasal regulation of blood glucose and insulin secretion could be inhibited by parasympathetic depressant（atropine）;intranasal eugenol enhanced hypothalamic POMC-mediated secretion of adrenocorticotropic hormone（ACTH）and thyrotropin-secreting hormone（TRH）;intranasal eugenol increased intestinal-and Brain-derived glucagon-like peptide-1（GLP-1）secretion and enhanced Ca²⁺signaling in hypothalamic neurons via parasympathetic elicitation after GLP-1 sensing of glucose;it was verified by human-derived SH-SY5Y cell model that GLP-1 promotes neuronal cell Ca²⁺inward flow through TRPV1-mediated ion channels and upregulates melanocortin POMC expression,while Eugenol could facilitate this process by activating TRPV1.5.The effect of intranasal inhalation of eugenol on energy metabolism through the central nervous system was verified in a 12-week experiment in mice fed a high-energy diet.The results showed that transnasal inhalation of eugenol affected thyroid hormone secretion through the central nervous system,up-regulated the expression of thyroid hormone secretory genes（TPO,Tg,NIS and TSHR）,up-regulated deiodinase I（DIO1）activity in the liver,gene and protein expression of thyroid hormone receptor TRβ1 and transporter MCT-8,and promoted the conversion of thyroid hormone T4 to T3;up-regulated the expression of thyroid hormone receptor TRPV1 in the pancreas The expression of thyroid hormone receptor TRβ1 and transporter MCT-8 in the pancreas was upregulated,and the expression of thyroid hormone-regulated insulin secretion-related genes（Maf A,PDX-1 and GLUT2）and the gene and protein expression of anti-apoptotic factor Bcl2 in pancreaticβ-cells were upregulated,and the gene and protein expression of pro-apoptotic factor Bax was downregulated,which improved pancreatic function and enhanced insulin secretion.It alleviated hepatic insulin resistance,enhanced hepatic insulin signaling pathway gene expression,reduced fasting blood glucose levels in mice,and improved systemic insulin resistance;downregulated the expression of hepatic thyroid hormone-regulated lipid synthesis-related genes（SREBP-1C,SCD1,ACC1 and FAS）and upregulated lipolysis-related genes（CPT-1α,PGC-1α,PPARαand CYP7A1）expression,reduced liver index,liver fat vacuole area ratio and fat infiltration area ratio as well as liver lipid level in mice,and slowed down the fat deposition in the liver.Meanwhile,transnasal inhalation of eugenol inhibited apoptosis of hippocampal cells caused by high glucose,reduced hippocampal insulin resistance level and alleviated hippocampal oxidative stress by increasing glucose metabolism in hippocampal neuronal cells,which in turn improved the non-spatial and spatially dependent memory ability of HFFD mice.In summary,transnasal inhalation of eugenol can reduce insulin resistance in the liver by enhancing signaling（calcium ions）from energy substance receptors（GLP-1）to the central nervous system（hypothalamus）via the peripheral nervous system（parasympathetic）,mediating the hypothalamus to promote thyroid hormone release via the HPT axis,alleviating pancreatic injury,enhancing pancreatic insulin release,and promoting hepatic insulin signaling response.Promotes enhanced energy metabolism;meanwhile the improvement of energy metabolism relieves hippocampal insulin resistance,alleviates hippocampal neuronal damage,and improves cognitive impairment caused by energy metabolism disorders.The effect and mechanism of transnasal inhalation of eugenol on the body’s energy metabolism found in this study suggests a theoretical basis for the development of functional food flavor compounds.