Effect Of Endogenous Melatonin Deficiency On Physiological Function Of Mice

Melatonin is synthesized by the pineal gland and released into the internal circulation system.In human and animal bodies,it not only participates in regulating the circadian rhythm of peripheral organs,but also participates in regulating aging,tissue and organ development and other physiological processes.In addition to the pineal gland,which can synthesize melatonin,melatonin related enzymes and their receptors have been found in liver,kidney,skin,gut,platelets,lymphocytes and other tissues and cells.The function of melatonin has been widely studied,and it has been used as a potential therapeutic drug for a variety of diseases due to its anti-inflammatory,antioxidant,anti-cancer and other characteristics.However,the systematic effects of endogenous melatonin deficiency and its relationship with diseases have not been reported.Therefore,this study used CRISPR-Cas9 technology to knock out the encoding gene of aryl alkylamine N-acetyltransferase（AANAT）,a rate limiting enzyme for melatonin synthesis,and establish an endogenous melatonin deficiency（EMD）mouse model.First,the important physiological characteristics of EMD mice were observed.Then,the transcriptome and serum metabolome characteristics of important tissues and organs in mice were revealed.The relationship and mechanism between EMD and Alzheimer’s disease（AD）and obesity were studied.Finally,fecal bacteria transplantation（FMT）was used to further verify the role of gut microbiota in AD and obesity.The main research contents and results are as follows:1.Construction of EMD mouse model and observation of important physiological characteristicsIn this study,the serum melatonin level,body weight,reproductive performance,glucose tolerance,insulin tolerance and histological characteristics of main tissues and organs of EMD mice were first detected.The results showed that the serum melatonin level of Aanat^-/-mice was significantly reduced（P<0.001）,indicating that the establishment of EMD animal model was successful.Compared with WT group,there was no significant difference in body weight from birth to 9 weeks of age in EMD group.The results of testis weight detection and section showed that there was no difference in testis weight between the two groups,and there was no pathological feature in the testes of EMD mice.The results of cage closing test showed that EMD mice had fertility.The results of glucose tolerance（GTT）and insulin tolerance（ITT）tests showed that EMD mice showed glucose intolerance and insulin resistance.Histological observation showed that EMD did not cause obvious pathological features of brain,heart,liver,kidney,lung,skin,stomach,spleen,skeletal muscle and e WAT in 10 week old adult mice.The above results showed that EMD did not affect the weight and reproductive performance of mice.EMD did not cause obvious pathological characteristics of important tissues and organs in 10 week old mice.However,EMD caused metabolic disorder in mice,which manifested glucose intolerance and insulin resistance.2.Characteristics of transcriptome and serum metabolome in important tissues and organs of EMD miceThis study investigated the effects of EMD on the transcriptome and serum metabolome of 11 tissues and organs in mice.Brain,heart,liver,kidney,lung,skin,stomach,spleen,testis,skeletal muscle and e WAT were selected as research targets.The results showed that there were significant differences in transcriptiome of 11 tissues and organs.A total of 8321 differentially expressed genes（DEGs）were found（Log2Fold Change>2 or<0.5 and P<0.05）.Among them,the relative expression levels of 3209 genes were significantly up-regulated and 5112 genes were significantly down regulated.The results of GO and KEGG enrichment analysis showed that part of the pathway functions significantly enriched in the brain,liver,skin,lung and testis of EMD mice were related to tissue specific diseases.Further analysis showed that the changes in the relative expression of DEGs in some tissues were consistent with their changes in the pathogenesis and poor prognosis of tissue specific diseases.The results of serum metabolome showed that EMD changed 30 kinds of metabolites in mouse serum,and significantly enriched in 16 metabolic pathways.These metabolic pathways are mainly related to amino acid metabolism and lipid metabolism.The above results indicate that compared with WT group,EMD mice have significant changes in the transcriptiome and serum metabolome,and EMD may be related to the pathogenesis and poor prognosis of tissue specific diseases.3.Characteristics of gut microbiota in EMD miceEMD did not affect the alpha diversity of gut microbiota in mice.However,the beta diversity of gut microbiota changed significantly,and the gut microbiota composition of the two groups of mice changed at the level of phyla,class,order,family and genus.LEf Se（LDA Effect Size）analysis showed that there were significant differences in relative abundance between the two groups of mice in all species.Compared with WT group mice,the relative abundance of Bacteroides in the gut microbiota of EMD group mice was significantly reduced（P<0.05）,the relative abundance of Lactobacillus was significantly increased（P<0.05）,and the relative abundance ratio of Firmicutes and Bacteroides was significantly different（P<0.05）.Compared with WT group,the level of gut inflammatory marker LCN2 in EMD group was significantly increased（P<0.01）,and gut permeability was significantly increased（P<0.001）.At the same time,EMD causes serum proinflammatory cytokine TNF-αand MCP-1 were significantly up-regulated（P<0.05）.Pearson correlation analysis showed that proinflammatory cytokine TNF-αand MCP-1 levels were significantly positively correlated with the relative abundance of Ruminiclostridium5（P<0.01）and significantly positively correlated with the relative abundance of Ruminiclostridium5（P<0.05）,respectively.The above results indicate that EMD causes gut microbiota dysbiosis in mice,accompanied by gut inflammation and increased gut permeability.At the same time,EMD causes systemic inflammation,which may be related to the increase of the relative abundance of Ruminiclostridium 5.4.Relationship between EMD,AD and obesityThe detection results of AD serum markers showed that the serum uric acid level of 8-month old EMD group mice was significantly lower than that of 8-month old WT group mice（P<0.05）,and the serum T-tau protein level of mild AD precursor was significantly increased（P<0.05）.Compared with WT mice,EMD mice brain proinflammatory cytokine TNF-αand IL-6 levels were significantly increased（P<0.05）.Immunofluorescence analysis of hippocampus tissue showed that microglia in EMD group were activated,Aβprotein deposition.The results of T maze test showed that the spatial memory ability of EMD mice decreased significantly（P<0.05）.The two groups of 10-week old adult mice were fed with high fat（HF）diet for 10 weeks at the same time.The relationship between EMD and obesity was explored through body weight,GTT,ITT and liver oil red o staining.The results showed that there was no significant difference in body weight between the two groups fed with basic diet.From the fourth week,the weight of EMD+HF group mice was significantly higher than that of WT+HF group mice（P<0.05）,until the end of the tenth week.The weight of e WAT in EMD+HF group was significantly higher than that in WT+HF group（P<0.05）.The weight of perirenal adipose tissue and liver was significantly higher than that of WT+HF mice（P<0.01）,and the liver steatosis in EMD+HF group was significantly increased.The results of GTT and ITT tests showed that the mice in EMD+HF group showed more obvious glucose intolerance and insulin resistance than those in WT+HF group.The above results indicate that EMD causes the AD like phenotype of 8-month old mice,aggravates the obesity of high-fat induced mice,and reduces the resistance of mice to stress.5.fecal mictobiota transplantation alleviates AD and obesity symptoms in EMD miceFMT significantly improved the gut permeability（P<0.05）,significantly reduced gut inflammatory marker LCN2 level and significant reduced serum proinflammatory factor TNF-αand MCP-1（P<0.05）of 8-month-old EMD mice.At the same time,FMT reversed the activation of microglia and Aβprotein deposition.FMT test results of mice fed with high-fat diet showed that FMT significantly reduced the weight of EMD mice（P<0.05）,and alleviated the severity of liver steatosis,glucose tolerance and insulin resistance.The results showed that FMT could alleviate the increase of gut permeability,gut inflammation and systemic inflammation induced by EMD in mice.At the same time,FMT can alleviate the exacerbation of AD like phenotype and obesity caused by EMD.