The Intervention Of Procyanidins On Calcium Metabolism Disorder In High-fat Diet Fed Mice Based On Multi-omics Analysis

As the standard of living has improved,people’s dietary structure and lifestyle have changed dramatically,and the percentage of high-fat foods in their daily meals has continuously increased.There have been reported that dyslipidemia and oxidative stress status generation by high-fat diet increased the prevalence of osteoporosis.Procyanidin B₂,a dimeric form of procyanidins,has been widely used as a dietary supplement to resist oxidative stress,cardiovascular diseases and their metabolic syndrome,and regulate gut microbiota.However,the mechanism of its intervention on calcium metabolism disorder is rarely reported.In this paper,we aimed to explore the underlying molecular mechanism of procyanidin B₂intervention in dyslipidemia model mice by comprehensive analysis of fecal gut microbiota,bone gene expression,and plasma metabolites in C57BL/6 mice.The specific conclusions obtained are as follows:（1）Twenty four C57BL/6 mice（male）were randomly set up into control（CON）group,high-fat（HF）group,and procyanidin B₂（PB₂）group,and continuously fed for 8 weeks.The results showed that high-fat diet led to abnormal body weight and organ indexes,dyslipidemia and oxidative stress,and decreased intestinal calcium absorption and bone calcium content.However,intervention with procyanidin B₂restored body weight and partial organ indexes to the levels of the control group in the high-fat diet fed mice,significantly reduced plasma total cholesterol,low-density lipoprotein,and atherosclerotic index levels.The activities of lipid metabolism-related enzymes and tissue antioxidant enzymes were increased,and the liver and duodenum malondialdehyde levels were decreased.In addition,intestinal calcium absorption and bone calcium content were significantly increased in the PB₂group（P<0.05）.The above results suggested that procyanidin B₂is able to ameliorate high-fat diet induced dyslipidemia and oxidative stress,thereby improving the calcium metabolism disorder.（2）The eukaryotic parametric transcriptomic analysis was performed using Illumina next-generation high-throughput sequencing.A total of 49 differentially co-expressed genes were selected,high-fat diet intake resulted in a significant up-regulation of 38 genes and a significant down-regulation of 11 genes,and 48 of these genes were significantly down-regulated by proanthocyanidin B₂supplement（P<0.05）.The Go enrichment analysis showed that the differentially co-expressed genes were assigned to body immunity,body stress response,composition of cell phagocytosis,etc.The KEGG enrichment analysis showed that a total of 13 pathways were significantly enriched（P<0.05）,thus the Cdkn2a,Adrb1,Fcgr4,Aldh1a3,and Calml3 genes were finally selected as potential regulatory genes.The above results indicated that procyanidin B₂could effectively regulate femoral gene expression in high-fat diet fed mice.（3）The metagenomics analysis was performed using Illumina next-generation high-throughput sequencing.The Venn diagram results based on OTU levels showed that there were 69 OTUs unique to the CON group,14 OTUs unique to the HF group,and 17OTUs unique to the PB₂group.The diversity analysis showed the compositions of fecal gut microbiota were differed significantly in the CON group,HF,and PB₂group.The student’s t-test data indicated that procyanidin B₂decrease the relative abundance of Firmicutes and increase the relative abundance of Bacteroidetes and Proteobacteria.And a total of nine genera were significantly different at the genus level,procyanidin B₂supplement significantly increased the relative abundances of the Escherichia-Shigella and Blautia genera,and decreased the relative abundances of Lachnoclostridium,Bilophila,Terrisporobacter and Proteus genera.In addition,compared with HF group,the contents of short chain fatty acids indicated that the butyrate content were significantly increased in the PB₂group（P<0.05）.The above results indicated that procyanidin B₂could increase the abundances of short chain fatty acid producing microbes in the intestine and improve the disorder of gut microbiota caused by high-fat diet.（4）Metabolomics analysis was performed using LC-MS/MS.The plasma metabolites of mice were analyzed by Met PA pathway,the lipid metabolism and calcium metabolism indexes and the potential biomarkers of key pathways were selected to analyze the relationship between gut microbes and metabolites.And the correlation analysis between metabolites and potential regulatory gene expression was performed.The data indicated that procyanidin B₂supplement increased purine,unsaturated fatty acids,glycerophospholipid and bile acid contents,and down regulated phenylglucuronide content in plasma of high-fat diet fed mice.The key metabolic pathways including glycerol phospholipid metabolism,bile acid synthesis,taurine metabolism,etc.The results of correlation analysis showed that the abundances of gut microbiota could regulate the levels of lipid metabolites（such as TC,TG,LDL-C,LPL and HL）,the enzyme activities,and calcium metabolism related indexes.However,the related gut microbiota,especially the Bilophila and Proteus genera are closely associated with the regulation of phospholipid,purine,carboxylic acid,unsaturated fatty acid,and bile acid metabolism.Aldh1a3 and Calml3 genes,which expression was significantly up-regulated were significantly correlated with the contents of metabolites such as phospholipids,linoleic acid,and acetylcholine.In conclusion,procyanidin B₂can intervene in the production of dyslipidemia and oxidative stress caused by high-fat diet.It can also regulate the metabolism of phospholipids,purines,carboxylic acids,unsaturated fatty acids,and bile acids through gut microbiota.Thereby regulating calcium metabolism-related gene expression and intervening in high-fat dietary calcium metabolism disorder.