Effects Of Pine Pollen Wall On Gut Microbiota And Biomarkers In Fatty Liver And Dyslipidemia Mice

In recent years,research on the physiological functions of Pinus yunnanensis pollen in our laboratory has focused on the polysaccharides and nutrient-rich contents of pine pollen,and found that it has prevented and inhibitory effects on a variety of diseases and cancers.The pine pollen wall accounts for nearly 40%of the pollen.There have been a lot of researches on dietary fiber to improve the metabolism of the body through the gut microbiota.However,the investigation of pine pollen wall mainly composed of cellulose and sporopollenin is still limited.This experiment used purified pine pollen wall to feed mice and explored its beneficial effects on non-alcoholic fatty liver and dyslipidemia induced by a high-fat diet.1.Optimized the extraction reagents and conditions.The pine pollen was treated with?-amylase,?-amylase and protease,and washed three times with ethanol and water respectively,finally obtained high purity pine pollen wall without nutrient content,such as protein,carbohydrates and fats.The yield of the pine pollen wall was about 43.75%,and the main components were cellulose and spore powder,of which cellulose accounts for about 41.80%,used in subsequent experiments.2.C57BL/6 male mice were used to establish a high-fat diet-induced model of non-alcoholic fatty liver and dyslipidemia(MC).A control group(HC),an experimental group containing 2.5%pine pollen wall(PW),and 2.5%soy dietary fiber positive control group(FC)were also set up.After 42 days of feeding,the liver,serum,perinephric and epididymal fats of mice were collected to determine body fat ratio,liver index,blood lipid level,inflammatory cytokines TNF-?and IL-6,and observe liver pathological sections.We found that high-fat diet induced liver fatty lesions in mice,body fat ratio and liver index slightly increased,serum TC,HDL-C,LDL-C levels were significantly increased,and liver cytokines TNF-?and IL-6 levels were significantly increased,while serum TG levels did not change.The intake of pine pollen wall can prevent high fat diet-induced increase in body fat and hepatic fat lesions in mice,reduce liver inflammatory cytokines TNF-?and IL-6 levels,and relieve the increase in serum TC and HDL-C levels,but the effects of pine pollen wall on liver index and serum LDL-C levels were limited.Compared with pine pollen wall,the intake of soy dietary fiber can prevent the increase of body fat induced by high-fat diet in mice,reduce the level of liver inflammatory cytokines TNF-?,alleviate the increase of serum HDL-C levels,and reduce liver fat lesions,but the effects of soy dietary fiber on liver index,IL-6levels and serum TC,LDL-C levels were limited.3.Metabolomics analysis of the cecum contents of mice using ~1H NMR spectroscopy.Through multivariate statistical analysis,it was found that the cecum contents of the four groups can be distinguished.The content changes of metabolites such as cholic acid,fatty acid,short chain fatty acid(SCFAs),sugar and amino acid were identified after matching with the database.Among them,biomarkers include bile acid,leucine,pantothenic acid,and long-chain fatty acids such as palmitic acid,stearic acid,and myristic acid.Their levels generally increased significantly after induction of high-fat diet,and decreased to varying degrees after the intake of pine pollen wall and soy dietary fiber.However,the content of SCFAs was generally decreased in the model group,and significantly recovered after the intake of pine pollen wall and soy dietary fiber.We analyzed the metabolic pathways involved by MetPA analysis and found that the intake of cellulose in the pine pollen wall can prevent the abnormal metabolism of bile acids and fats in mice caused by high-fat diets,and alleviate the reduction of short-chain fatty acid content.At the same time,the intake of cellulose in the pine pollen wall and soy dietary fiber caused an increase in carbohydrate metabolites.Combined with the molecular structure and metabolomics analysis results of sporopollenin,we speculate that sporopollenin may not be digested and utilized in the body.4.Used the Illumina MiSeq sequencing platform to analyze the gut microbiota in the contents of cecum and feces of mice,and to explore the changes in species diversity,community structure and composition at different classification levels.We found that the high-fat diet induced severe imbalance in the gut microbiota of mice,and the ratio of Firmicutes to Bacteroides was significantly increased.Among them,the abundance of beneficial bacteria such as Rikenellaceae,Ruminococcaceae,Marinifilaceae,Alloprevotella from Bacteroides and Alistipes,Lachnospiraceae,Lactobacillus decreased significantly,while the abundance of harmful bacteria such as Enterobacteriaceae,Staphylococcaceae and Helicobacter significantly increased.The intake of pine pollen wall can alleviate the gut microbiota imbalance in mouse feces caused by high-fat diet,It increased the abundance of beneficial bacteria,such as Rikenellaceae,Ruminococcaceae,Parabacteroides,Alloprevotella from Bacteroides,and effectively alleviated the increase in the abundance of various harmful bacteria like Staphylococcaceae,Desulfovibrio and Proteobacteria,thereby regulated the metabolism of the body,increased the production of SCFAs,and played an active role in alleviating non-alcoholic fatty liver and blood lipids.The intake of soy dietary fiber did not show an effective effect on gut microbiota imbalance.We only found that the abundance of erysipelotrichaceae decreased,and that of Faecalibaculum and Staphylococcaceae significantly increased.In the contents of the cecum,neither pine pollen wall nor soy dietary fiber showed an alleviating effect on gut microbiota imbalance.Soy dietary fiber significantly increased the abundance of Faecalibaculum and Akkermansia,indicating that pine pollen wall and soy dietary fiber,mainly play a role in the large intestine stage.In this study,we successfully extracted pine pollen wall with high purity,and confirmed that pine pollen wall can play a role in preventing fatty liver,dyslipidemia and inflammation by regulating bile acid,fat metabolism,SCFAs content,and composition and structure of gut microbiota.In addition,the component with the above effects may be mainly cellulose in the pine pollen wall,and sporopollenin cannot be digested and used in animals.Therefore,the pine pollen wall can be partially utilized by the body and play an active physiological role,which is of great significance for the more effective application of pine pollen in health products.