| Objective:According to the report on nutrition and chronic diseases in China(2020),the prevalence of dyslipidemia in China has been increasing over the years due to the dramatic change in lifestyle,and the consequent cardiovascular events have also increased the medical burden of related diseases in China.However,healthy dietary pattern,one of the most economical and effective ways to improve dyslipidemia,is often ignored.The present study was designed to recruit Chinese people with mild dyslipidemia to conduct a randomized,single-blind,placebo-controlled trial to investigate whether oats supplementation could improve serum lipid profiles.Its effects on gut microbiota and serum metabolites were also initially explored by multi-omics techniques such as metabolomics,16S rDNA sequencing,and metagenomics.Finally,the cholesterol-lowering related signaling pathways of oats was further investigated through fecal microbiota transplantation and omics technology in a model rat.Methods:(1)In this multicenter,randomized,single-blind,placebo-controlled trial,a total of 210 eligible volunteers recruited in Beijing,Nanjing and Shanghai are randomly assigned to receive 80g oats or rice daily for 45 days.Blood samples were collected at baseline,day 30 and day 45,respectively.General physical information and 3d×24h dietary recalls were performed twice at the start and the end of the study.Difference in clinical parameters between groups after intervention was compared using variance for repeated measures,in which the principle of intention to treat was followed.(2)The samples are from the volunteers recruited in Nanjing and Shanghai in Part 1.Blood samples were collected three times(day 0,day 30,and day 45)to detect clinical biochemical parameters(based on samples collected from Nanjing);the detection of non-target metabolomics analysis and shortchain fatty acid were performed based on the blood samples collected before and after the trial(based on samples collected from Nanjing).In addition,16S rDNA sequencing(based on samples collected from Nanjing)and metagenomics(based on samples collected from Nanjing and Shanghai)were employed to assess the change in gut microbiota.Differences between groups were assessed by using variance for repeated measures,multivariate statistical methods,and Spearman correlation analysis(3)Animal experiment NO.1:a total of 48 SD rats were randomly divided into normal control group,normal control+10%Oat group,model control group,high-fat diet(HFD)+5%Oat group,HFD+10%Oat group and HFD+20%Oat group,with 8 rats in each group.Changing their bedding and water regularly and recorded their body weight and food intake during 12-week intervention.At the end of the experiment,blood samples,fecal and cecum contents,small intestine and liver were taken from each group of rats after sacrificing,and the level of serum lipid,fecal short-chain fatty acid,gut microbiota,cecum bile acids profiles and the relative expression of small intestine FXR,ABCG5/8,LXR,CYP7A1,CYP8B1,CYP27A1,CYP7B1 proteins were detected and compared.Animal experiment NO.2:Another 32 SD rats were purchased and give 2 weeks of antibiotic treatment(metronidazole 1 g/L+ vancomycin 0.5 g/L+neomycin sulfate 1 g/L+ampicillin 1 g/L dissolved in drinking water combined with gavage daily)after 12-week high-fat feeding to establish a germ-free rat model.Then all the rats were divided into eight groups that received the fecal bacteria from the model control and the three oat intervention groups in animal experiment NO.1 with a dosage of 400μL and 800μL respectively.The rats were fed with high-fat diet for another 3 weeks after fecal microbiota transplantation,then the serum lipid profiles was measured in each group.Results:(1)A total of 187 volunteers completed the whole trial(93 in rice group,94 in oat group).There is no significant difference in general demographic characteristics and clinical parameters at baseline(P>0.05).After 45-day oat intervention,the volunteers in oat group had significantly lower serum TC,LDL-C and NON-HDL-C than rice group(P for interaction<0.05 and P for time<0.05).Compared to baseline,the reductions in TC,LDL-C and NON-HDL-C in oat group could be as much as-7.89%,-9.06%and-10.22%,respectively.However,no significant between-groups difference in TG,Apo B and GA were found after intervention.(2)Blood samples were collected from 58 volunteers recruited from Nanjing(28 in rice group,30 in oat group).There is no significant difference in general demographic characteristics,blood lipids,oxidative stress and inflammatory factors at baseline(P>0.05).After 45-day oat intervention,the volunteers in oat group had significant improvements in oxidative stress and lipids level than rice group(P for interaction<0.05 and P for time<0.05).Compared to baseline,the net changes in T-AOC,SOD and MDA in oat group could be as much as 21.98%,14.98%and-18.61%,respectively.However,no significant between-groups differences in TNF-α and IL-6 were found after intervention.A total of 758 metabolites were identified according to the MS2 database by non-targeted metabolomics,most of which belong to the superclass of lipids and lipids-like molecules,organic acids and derivatives.OPLS-DA analysis showed a clear separation of clustering trends in metabolite profiles between groups after 45-day oat intervention,in which the discrimination and prediction power in this model is satisfying.Based on the thresholds of VIP values of the OPLS-DA>1 and P<0.05 of the Student’s t-tests,a significantly different metabolite profiles were screened to identify essential changes in serum metabolites after oat consumption.Briefly,a total of 21 differential metabolites were screened between groups.Some metabolites(e.g.varenicline,sphinganine,PC(20:5(5Z,8Z,11Z,14Z,17Z)/20:1(11Z)),myristicin,retinoic acid)may be closely correlated to serum lipid improvements.Finally,pathway analysis based on KEGG database suggested that oat consumption significantly changed 4 pathways:glycerophospholipid metabolism,alanine,aspartate,and glutamate metabolism,sphingolipids metabolism and retinol metabolism.In terms of gut microbiota,16S rDNA sequencing showed that oat consumption resulted in a significant improvement in β-diversity but not α-diversity of gut microbiota in mildly hypercholesterolemic population.Besides,oat consumption led to significant increases in the relative abundance of probiotics such as Akkermansia,Dialister,and Lactobacillus.Correlation analysis suggested that these genera may play an important role in improving blood lipids and increasing antioxidant activity of the host.Although no between-group difference in short-chain fatty acid level was found,compared with baseline,oat consumption significantly increased the level of serum acetic,propionic and valeric acid;correlation analysis revealed that some short-chain fatty acid(e.g.acetic acid and propionic acid)negatively correlated with serum TC and LDL-C,indicating that short-chain fatty acid may be one of the pathways by which oat exert its hypolipidemic effect through gut microbiota.Finally,metagenomics showed that oat consumption significantly increased the abundance of retinol metabolism,fatty acid metabolism,fatty acid biosynthesis and sphingolipid signaling pathway,but no significant difference was found in the number of genes within and between groups.Additionally,the bile secretion pathway was also significantly affected by oat consumption,suggesting that bile metabolism is crucial to elucidate the mechanisms of cholesterol-lowering effect of oat.(3)Animal experiment NO.1:no significant between-group difference was observed in baseline body weight and serum lipids.In addition,the gained body weight and food intake were not significantly different between groups at each time point during 12-week intervention.Compared with normal control group,serum TC,TG and LDL-C in model control group were significantly increased,indicating a success of hyperlipidemia model.All oat intervention groups showed significant improvements in serum TC and LDL-C compared to model control group,suggesting that oats may exert a beneficial effect in modulating lipid in a dose-response manner.Results from 16S rDNA sequencing showed that high-fat diet can induce a notably dysbiosis of gut microbiota as evidenced by significantly decreased relative abundance of Lactobacillus,Butyricimonas,and Clostridiaceae_Clostridium and increased relative abundance of Lachnospirceae_Clostridium;while oat intervention can increase a-diversity and improve variety and composition structure of gut microbiota,as evidenced by significantly increased relative abundance of Allobaculum,Subdoligranulum and Bifidobacterium,and reduced relative abundance of Lachnospirceae_Clostridium,which reversed the dysbiosis of gut microbiota caused by high-fat diet to some extent.At the same time,oat intervention also significantly increased fecal propionic acid level,and there was a tendency to increase other short-chain fatty acid such as acetic acid and butyric acid,but the differences were not significant.In addition,high-fat diet also induced a disturbance in bile acids metabolism,as evidenced by a significant increase in the level of 31 bile acids and a decrease in the level of three LCA derivatives(all oLCA,6-ketoLCA and IALCA)in comparison with normal control group.In contrast,oat intervention can improve the disturbance induced by high-fat acid to some extent,as demonstrated by decreased level of 120H bile acid such as TCA,TDCA,23DCA,increased level of NON-120H bile acid such as GCDCA-3S,as well as a significant reduction in the 120H bile acid/NON-120H bile acid ratio.Correlation analysis between gut microbiota and short-chain fatty acid,bile acid suggested that some bacteria such as Bifidobacterium,Allobaculum、Subdoligranulum and Clostridium may play an important role in oat-mediated lipid improvement.Western Blot results showed that compared with model control group,oat intervention significantly increased the relative expression of key proteins in bile acid synthesis such as LXR,CPY7A1,CPY27A1,CPY8A1,inhibited the relative expression of small intestinal FXR and liver HMG-CoA(a limited enzyme for endogenous synthesis of hepatic cholesterol),and promoted the protein expression of ABCG5/8(an intestinal efflux transporter protein).Animal experiment NO.2:The antibiotic treatment used in current study was reliable to establish a germ-free animal model.Compared with high-fat fecal microbiota transplantation control group,the fecal bacteria from both 10%and 20%oat donor group could significantly reduce serum TC and LDL-C levels in rats,and the effect was independent of the amounts of fecal bacteria donated.Conclusion:(1)Supplementation with 80g/d of whole-grain oats for 45 days could significantly improve serum TC,LDL-C and NON-HDL-C in population with mild dyslipidemia,but no significant effect on serum TG and HDL-C level.(2)Supplementation with whole-grain oat for 45 days can significantly improve serum oxidative stress levels in individuals with mild dyslipidemia,and the effect might be related to the improved composition of gut microbiota and the changed serum metabolite profiles.The underlying mechanisms may involve metabolic pathways such as glycerophospholipid metabolism,alanine,aspartate and glutamate metabolism,sphingolipid metabolism,retinol metabolism,fatty acid metabolism and bile secretion.(3)The effect of oat on lipid improvement is gut microbiota-dependent.On the one hand,oat can increase the level of short-chain fatty acid such as propionic acid to inhibit endogenous synthesis of hepatic cholesterol and promote the efflux of intestinal cholesterol by regulating the structure and composition of gut microbiota;on the other hand,the improved gut microbiota may also inhibited intestinal FXR by inducing the conversion of bile acids to NON-120H bile acid and reducing the ratio of 120H bile acid/NON-120H bile acid,and activated the biological processes of the pathways(classical and alternative)in bile acid synthesis.The hypolipidemic effect of oat might be a synergistic consequence from the actions all above. |
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