| China’s rice production ranks the first in the world,and rice bran,as the main byproduct of its processing,contains a variety of bioactive ingredients.Conducting research on the biological functions of rice bran active ingredients can help promote the development of the rice industry.In this thesis,octacosanol(OCT)is the research object.OCT is an effective active component in rice bran and other natural by-products which mainly exists in the form of waxiness.The study of health food with OCT as the main factor has practical and economic significance.OCT is a kind of monohydric higher linear fatty alcohol in nature,which has various physiological functions such as antifatigue,anti-inflammation,anti-oxidation,and hypolipidemic.However,the mechanism of anti-fatigue has not been studied,and the biological activity of anti-liver injury has not been reported.A fatigue model induced by forced exercise in mice and a liver injury model induced by alcoholic diet in mice were established in this study,evaluated the effects of OCT on anti-fatigue and anti-liver injury,and studied its potential molecular mechanisms.1.Functional assessment and mechanism study of octacosanol anti-fatigueThe fatigue model of C57BL/6J mice induced by forced exercise was established and after intervention with OCT,it was found that OCT had a significant effect on alleviating exercise fatigue in mice through experimental comparison,and it was confirmed that OCT could regulate the gene expression in gastrocnemius muscle tissue and improve the diversity of intestinal microbiota in fatigue mice.(1)The blank control group,the exercise fatigue model group,and the exercise fatigue-octacosanol protection group(200 mg/kg/day OCT continuous intervention)were compared and analyzed.After the experiment lasted for 4 weeks,no significant differences were found in body weight,food intake,and main organ weights of the mice.Exercise fatigue leads to a significant decrease in the autonomous activity ability,forelimb grip,and autonomous swimming ability of mice,and OCT intervention can effectively improve the exercise ability and endurance of mice.Fatigue could significantly reduce the contents of LG,MG,SOD,and GSH-Px in mice,while increase the contents of BLA,LDH,and MDA.OCT intervention can regulate the homeostasis of biochemical indicators in mice.The muscle tissue of fatigued mice showed striated muscle degeneration and local fiber structure looseness,and OCT intervention could alleviate muscle tissue damage in mice.(2)Through gene microarray analysis revealed that OCT intervention up-regulated 29 genes and down-regulated 38 genes in the gastrocnemius muscle tissue of fatigued mice.GO functional enrichment analysis indicated that these DEGs were mainly involved in processes such as myofibril,contractile fiber,and calcium-dependent ATP activity.Correlation analysis between gene microarray and RT-qPCR results revealed identical expression trends for 10 DEGs,with OCT intervention significantly upregulated the mRNA expression of Pmp22,Trim63,Ulk3,Arrdc2,and Prx,while significantly down-regulated the mRNA expression of Bcl3,Cga,Sv2a,Cacna1h,and Mybpc3.WB analysis showed that OCT intervention was involved in regulating the protein expression of PRX,TRIM63,CACNA1H,and MYBPC3.(3)By combining multi-database mining,327 common targets of OCT and fatigue were obtained,and 19 key targets were screened,which are mainly involved in cell irritability,signal transduction,cell communication,and other functions.KEGG analysis showed that they are mainly enriched in the HIF-1 and PI3K-Akt signaling pathways.The 4 core genes enriched in these two signaling pathways are Egfr,Nos3,Prkca,and Rela,which were validated by microarray dataset and molecular docking to show their potential role in the diagnosis of muscle diseases,and can be used as promising markers for monitoring and treating muscle fatigue.(4)Through 16S rRNA sequencing analysis,it was found that OCT intervention had a regulatory effect on the intestinal microbiome of mice with long-term exercise fatigue,and can partially restore the richness and diversity of the intestinal microbiota community.Among a total of 318 OTUs,there were some differentially expressed microbiota in the three groups of samples.OCT is involved in influencing the changes of metabolites in the organism by regulating the composition of the intestinal microbiota and the relative abundance of specific microbiota,thus exerting a potential anti-fatigue effect.2.Functional assessment and mechanism study of octacosanol anti-liver injuryThe liver injury model of C57BL/6J mice induced by alcoholic diet was established and after intervention with OCT,it was found that OCT had the effect of alleviating alcohol-induced liver injury in mice through experimental comparison,and it was revealed that OCT has the effect of regulating gene expression in liver tissue,improving the diversity of intestinal microbiota and its metabolite content in liverinjured mice.(1)The blank control group,the alcoholic liver injury model group,and the alcoholic liver injury-octacosanol protection group(200 mg/kg/day OCT continuous intervention)were compared and analyzed.After the experiment lasted for 7 weeks,it was found that there were differences in the body weight of the mice,and the rate of body weight gain of mice with liver injury was significantly reduced,while the OCT intervention could alleviate the body weight change of the mice.Long-term alcohol intake resulted in significant increases in tissue weight of the liver,epididymal fat pad,and spleen in mice,and OCT intervention was able to alleviate tissue edema in mice.The levels of ALT,AST,ALP,TG,TC,LDL-C,and MDA in the serum and liver tissue of of mice with liver injury were significantly increased,while the contents of SOD,GSH-Px,and HDL-C were significantly decreased.OCT intervention effectively regulated the abnormal biochemical indicators in mice.Pathological analysis revealed that the liver tissue of mice with liver injury showed significant steatosis and hepatocyte swelling,with more lipid accumulation and large vesicular lipid droplets.OCT intervention could effectively alleviate hepatocyte damage and reduce the symptoms of fat accumulation.OCT also has the effect of alleviating the structural damage of the colon and brain tissue caused by alcoholic diet.(2)Through RNA-seq transcriptome analysis revealed that OCT intervention upregulated 36 genes and down-regulated 42 genes in the liver tissues of mice with alcoholic liver injury.GO function enrichment analysis showed that these DEGs were mainly acted in the regulation of biological quality,small molecule metabolism,and multicellular organism processes,and they were mainly annotated to metabolic pathways,steroid hormone biosynthesis,and PPAR signaling pathways,etc.OCT alleviates the symptoms of alcohol-induced liver injury by regulating the expression and interaction function of DEGs in mouse liver tissue.(3)Through 16S rRNA sequencing analysis,it was found that OCT intervention could regulate the changes of intestinal microbiota in the mice with alcoholic liver injury,restore the species and evenness of intestinal microbiota composition,and improve the richness and diversity of the community.OCT alleviates alcohol-induced liver injury by regulating the composition of intestinal microbiota and the relative abundance of special microbiota,affecting the changes in metabolites of the organism.There is a potential regulatory effect between changes of these differential microbiota and the expression of DEGs in mouse liver tissue and the changes of biochemical indices in serum.(4)A total of 379 metabolites were collected by GC-MS analysis.There are some differential metabolites among these metabolites,and hierarchical cluster analysis and correlation analysis indicated that these differential metabolites and the correlation between metabolites may have a potentially contribute to the anti-alcoholic liver injury effect of OCT.10 major differential metabolites were obtained through differential screening,and correlation analysis showed that the generation of differential metabolites in the mouse intestine may be related to gene expression,intestinal microbiota,and their secreted products.(5)HepG2 cells were induced by 300 mmol/L alcohol,and the studies found that OCT had a protective effect on alcohol-damaged cells.OCT had no significant effect on the viability of HepG2 cells,but could restore the viability of HepG2 cells after alcohol injury.OCT can reduce the increase in cellular ROS and MDA levels induced by alcohol,increase SOD and GR activities,and enhance the oxidative defense ability of HepG2 cells. |
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