Research Of Lipid-lowering Effect Of Octacosanol And Related Potential Mechanism

Metabolic syndromes such as obesity,hyperlipidemia and non-alcoholic fatty liver disease have become the most prevalent diseases in modern times.It is of great significance to find safe and effective natural products and develop supplementary lipid-lowering functional foods.As the main component of rice bran fat,octacosanol has a broad application prospect.In this paper,the lipid-lowering efficacy of octacosanol was evaluated by establishing a high-fat diet（HFD）mouse model and a hepatic steatosis cell model,and the mechanism involved was also explored to provide scientific theoretical basis for the development of functional food and health food,and make positive contributions to promoting the high value use of rice by-products and alleviating the burden of related diseases.1.Evaluation of lipid-lowering effect of Octacosanol on high-fat diet mice and its mechanismThe high-fat diet-induced C57BL/6J mice were used to establish an obese animal model,after 11 weeks of feeding,the average body weight of the high-fat diet（HFD）group mice was significantly increased,compared with the control（Con）group mice.The average body weight of octacosanol（HFD+Oct）group mice was significantly reduced,which was significantly different from that of HFD group mice（p<0.05）.Compared with the HFD group mice,the total triglyceride（TG）,total cholesterol（TC）,and low Low-density lipoprotein cholesterol（LDL-C）in the serum of the mice in the HFD+Oct group were significantly decreased（p<0.01）,and the total triglyceride content was only 51%of that in HFD group mice,but the high-density lipoprotein cholesterol（HDL-C）content was not significantly changed（p>0.05）.Compared with the Con group mice,the volume and weight of the liver,fat pad and spleen of the mice in the HFD group was significantly increased.After the intervention of octacosanol,the weight of liver,fat pad and spleen were decreased by 7.8%,10.3%and 14.5%compared with the HFD group,respectively.Pathological analysis showed that the liver adipose tissue and the fat droplets of the mice in the HFD group was significantly increased,compared with the mice in the Con group;compared with HFD group,the adipose tissue（white space）of liver tissue slices in HFD+Oct group was significantly reduced,and the number of fat drops was also reduced.At the same time,the adipose tissue cells in the HFD group were significantly increased,while octacosanol could significantly reduce the volume of adipose cells.It is shown that octacosanol can inhibit the obesity of mice induced by high-fat diet,and has the effects of reducing weight and fat.2.Study on the molecular mechanism of lipid-lowering and antiinflammatory effect of OctacosanolUsing gene chip analysis,the results showed that octacosanol interfered with the transcription and expression of liver tissue genes in mice,and octacosanol interfered made 28 genes up-regulated and 44 genes down-regulated in the liver tissue of high-fat diet mice.The GO analysis of bioinformatics shows that octacosanol participates in the fatty acid metabolism process,long-chain fatty acid metabolism process,circadian rhythm,glucose metabolism process and other biochemical processes;can regulate the metabolism of fatty acids,muscle stretching,lipid metabolism,lipid homeostasis,insulin resistance and other effects of the body,and most of its regulatory effects are closely related to lipid metabolism.KEGG analysis showed that octacosanol can regulate the lipid metabolism,glucose metabolism and cholesterol metabolism related signaling pathways,PPAR signaling pathways,as well as insulin resistance,carbohydrate digestion and absorption,Toll like receptor pathways,etc.;IP A analysis showed that octacosanol was mainly involved in lipid metabolism,cell proliferation and differentiation,and other processes.and AMPK,ERK,JNK,MAPK14 and MAPK9 were at the center of its network,and participating in energy metabolism,inflammatory response and other biochemical metabolism.Through the above researches,this topic has determined for the first time that the changes in the transcriptional profile of mouse liver tissue caused by octacosanol,and has determined the potential signal pathway,important target genes and biological effects of its role.Using real-time fluorescence quantitative PCR analysis,Western blotting and other molecular biotechnology,it was found that octacosanol can significantly down-regulate the expression of lipid metabolism-related genes such as LXRα,FASN,ACC,and CD36 in the liver tissue of high-fat diet mice,and their mRNA expression levels was decreased by 55.58%,55.9%,52.02%and 43.7%,respectively（p<0.01）,and at the same time,their protein expression levels was decreased by 31.54%,33.97%,39.63%and 28.33%,respectively;octacosanol also significantly decreased the expression of SREBP-1c mRNA,decreased its expression by 27.86%,and down-regulated the protein expression level of SREBP-1c,decreased its expression by 16.9%;octacosanol can increase the mRNA expression level of SIRT1 upstream of the lipid metabolism pathway,and up-regulate the protein level of SIRT1（p<0.01）.The above researches found that octacosanol can regulate the expression changes of key factors of lipid metabolism in liver tissue of mice caused by high-fat diet,promote fat oxidation and reduce fat accumulation.Using Western blotting and other molecular biotechnology,further research found that octacosanol can significantly up-regulate the phosphorylation level of AMPK in liver tissue and inhibit the phosphorylation of PPARα and PPARγ proteins,and the inhibition rates were 26.17%and 20.84%,respectively.The experiment showed that octacosanol activated the AMPK and PPAR signaling pathways in mouse liver tissue,and promoted fat metabolism,which further confirmed the results of gene chip bioinformatics analysis.High-fat diet will cause liver inflammation in mice.Molecular biology experiments shown that octacosanol can significantly inhibit the increased of the mRNA expressions of inflammatory factors IL-1β,TNF-α,IL-6 and iNOS in the liver tissue of high-fat diet mice（p<0.01）,which decreased by 58.76%,50.48%,45.04%and 58.95%,respectively,and also significantly inhibited their protein expression levels,which decreased by 31.86%,24.92%,43.59%and 28.02%,respectively.The results suggested that octacosanol can reduce the expression of inflammatory factors in liver tissue induced by high-fat diet,and has anti-inflammatory effect.3.The effect of Octacosanol on the fecal bacterial flora of mice fed with high-fat dietThe gut microbiota is closely related to the lipid metabolism of the body.The changes of octacosanol on the gut microbiota of mice with a high-fat diet were analyzed,the results showed that octacosanol can significantly change the gut microbiota,the relative abundance of Candidatus Saccharibacteria was decreased,and the relative abundance of Erysipelotrichia and Actinobacteria was significantly reduced at the class level;the relative abundance of Clostridiaceae₁,Streptococcaceae,Clostridiales_Incertae_Sedis_ⅩⅢ and Coriobacteriaceae was extremely significantly reduced,and the relative abundance of Unclassified_Candidatus_Saccharibacteri was significantly increased at the family level;the relative abundances of Streptococcus,Tannerella,Saccharibacteria_genera_incertae_sedis,Unclassified_Ruminococcaceae and Xylanibacter were significantly reduced,and the relative abundances of Clostridium_sensu_stricto,Clostridium_Ⅳ,Clostridium_XIVa,Eubacterium,Unclassified_Coriobacteriaceae,Turicibacter,Anaerovorax and Allobaculum were significantly increased at the genus level.Octacosanol exerts lipid-lowering and inhibiting inflammation effects by affecting the composition of gut microbiota,reducing the ratio of Firmicutes/Bacteroidetes,and reducing the relative abundance of obesity-related and inflammation-related microorganisms in Erysipelas,Actinomyces and Saccharomycetes.Based on the above research,this topic has determined that octacosanol can change the transcriptional profile of liver tissue and gut microbiota in high-fat diet,and promote the oxidation and decomposition of fat by regulating lipid metabolism-related signaling pathways and key target genes.At the same time,it also inhibits the synthesis of fat,and octacosanol has the effect of lowering lipid.Octacosanol can also inhibit the chronic inflammation of mouse liver induced by high-fat diet by inhibiting the expression of inflammatory factors in mouse liver tissue.4.Octacosanol inhibits linoleic acid-induced fat accumulation in HepG2 cells and its molecular mechanismHepG2 cells of liver were treated with oleic acid,and oil red staining showed that 12.5,25 and 50μg/mL of octacosanol intervention can significantly reduce the fat accumulation of cells in a dose-dependent manner.RT-qPCR and Western blotting analysis showed that octacosanol can significantly inhibit the expression levels of LXRα,SREBP-lc,CD36,ACC and FASN in cells,and up-regulate the expression level of SIRT1 gene.Octacosanol can promote AMPK phosphorylation,block AMPK activation with AMPK inhibitor,AMPK phosphorylation is significantly inhibited,by 34%,and affect the expression of SIRT1 and FASN.It shows that octacosanol depends on AMPK pathway to regulate lipid metabolism,thus inhibiting fat synthesis and reducing intracellular fat accumulation.Octacosanol inhibited p38 phosphorylation,PPARa phosphorylation and PPARy in high-fat cells.It reducing the ratio of p-p38/p38,p-PPAR α/PPARα and p-PPARγ/PPARγ by 85.16%,67.82%and 45.51%respectively.After intervention with the p38 activator Dehydrocorydaline,the downregulation of PPARγ was significantly inhibited,while the ratio of p-PPARγ/PPARγ was increased by 1.17-fold,showing that octacosanol reduced lipogenesis and promoted fatty acid metabolism by inhibiting phosphorylation of p38 and PPARy,and activating the PPAR signaling pathway.