| Qingke(Hordeum vulgare L.var.nudum Hook.f)belongs to the family of Poaceae and the genus of Hordeum.When lemma and palea are separated,the grain is exposed,and it is also called naked barley or hull-less barley.Qingke has been cultivated in China as early as 5000 years ago and is a very rich genetic resource of germplasm resources.Mainly distributes in the alpine and anoxic plateau region,with harsh environment.Qingke is the main staple food crop in Qinghai-Tibet Plateau,China and is being used as an essential food crop for human consumption.It has been studied that the ?-glucan content of hull-less barley,especially highland hull-less barley,is higher than normal barley.The health benefits of ?-glucans have been verified in terms of reducing serum cholesterol levels and attenuating postprandial blood glucose and insulin responses.Moreover,according to information from the health-epidemic prevention and medical institutions in the Tibet Autonomous Region,consumption of Qingke in daily life is associated with a reduced risk of cholesterol-related diseases.Qingke has high content of protein,fiber,and vitamin and low content of fat and sugar.Furthermore,it also has many other nutrition compositions,such as pentosan and phenolic compounds.The hypocholesterolemic effects of ?-glucan were previously reported.However,available information on the cholesterol-lowering effect or mechanism of whole-grain Qingke(WGQ)is limited.Liver and intestine play central roles in maintaining lipid homeostasis.Thus,the present study aims to investigate the effect and mechanism of WGQ on cholesterol metabolism in the liver and intestine.The main results are as follows.1.The nutrient composition and antioxidant activity of WGQ were investigated.Nutrient composition analysis showed that WGQ had relatively high contents of ash(1.95±0.08 g/100g),protein(17.00±0.26 g/100g),?-glucan(5.77±0.28 g/100g),total dietary fiber(19.01±0.54 g/100g),soluble dietary fiber(9.28±0.01 g/100g),total pentosans(10.74±0.47 g/100g),total phenolic(259.90±0.96 mg/100g)and free phenolic,low contents of fat(1.03±0.02 g/100g),insoluble dietary fiber(11.64±0.95 g/100g),and Water-extractable pentosans(0.44±0.03 g/100g).Antioxidants were subjected to gradient extraction using ethanol,NaOH,methanol,and water.Oxygen radical absorbance capacities(ORACs)of extracted antioxidants were investigated.Results showed that the ORAC values of total antioxidants were 418.05±5.65 ?mol/g.Therefore,WGQ has high nutrient composition and antioxidant activity.Finally,correlation analysis between nutrient composition and antioxidant activities were calculated.Positive and significant correlations(P < 0.01)were observed between the ?-glucan,phenolic,and pentosan compounds and ORAC values of antioxidants.These results suggested that the abovementioned compounds of WGQ may have antioxidant activities.2.To study the effect of WGQ on plasma lipid,a total of 72 male Sprague-Dawley rats were randomly assigned to 4 dietary groups and fed experimental diets for 4 weeks and 8 weeks.These feeding regimes included the normal control(NC)group fed with a normal AIN-93 G diet;the blank control(BC)group fed with a high-fat diet(HFD);the low dose(LD)group fed with HFD containing low dose(10%)of WGQ;and the high dose(HD)group fed with HFD containing high dose(48.95%)of WGQ.The effect of WGQ on body weight and Lee‘s index were investigated.Results showed that the body weight gain in both LD and HD groups were higher than BC group,while HD group being remarkably higher.Compared with other groups,the Lee‘s indexes in the HD group significantly(P < 0.05)decreased after fed for 8 weeks.These results suggested that WGQ diet,especially the high-dose diet,increased the body weight of HFD rats,whereas decreased the degree of obesity.Organ index tests showed that after administration of high-dose WGQ,the indexes of liver and abdominal fat of HFD rats were significantly(P < 0.05)decreased.These results suggested that WGQ can affect the liver metabolism and reduce the fat accumulation in HFD rats.Changes in plasma lipid levels were studied.Plasma lipid levels of LD group were slightly lower than BC group,while the difference was not significant.The total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),and non-high-density lipoprotein cholesterol(non-HDL-C)levels and atherogenic indexes(AI)of HD group was significantly(P < 0.05)lower,whereas high-density lipoprotein cholesterol(HDL-C)content were significantly higher(P < 0.05)than those in the BC group after 8-week feeding.These results indicate that high dose of WGQ has a hypocholesterolemic effect and can reduce the risk of atherosclerosis after 8-week feeding.3.To study the effect of WGQ on the liver cholesterol metabolism and the involved mechanisms,we investigated the changes of liver lipid and the expression of genes responsible for hepatic cholesterol metabolism in rats.TC,TG,and total lipid levels of the liver were investigated.Results showed that the TC,TG,and total lipid levels in the liver of all HFD groups,including BC,LD,and HD groups,were significantly(P < 0.05)higher than those in NC group after 4-and 8-week feeding.The TC content and lipid concentration in the liver of HD group was significantly(P < 0.05)lower than those in the BC group after 8-week feeding.Liver sections were further performed for hepatocyte morphologic observation.Compared with BC group,hepatic steatosis slightly decreased in LD group.In HD group,hepatic steatosis decreased remarkably,and the lipid droplets in the liver cells shrunk significantly after 8-week feeding.These results suggested that administration of high-dose WGQ for 8 weeks can significantly decrease liver lipid levels.Real-time PCR(RT-PCR)analysis was performed to investigate the mRNA levels of cellular biomarkers expressed in liver that involved in hepatic cholesterol synthesis,cholesterol accumulation in peripheral tissue,or cholesterol conversion into bile acids.About biomarkers involved in hepatic cholesterol synthesis,results showed that mRNA levels of AMP-activated protein kinase ?(AMPK?)in HD group were significantly(P < 0.05)higher,whereas mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMG-CoAr)were significantly(P < 0.05)lower compared with BC group.About biomarkers involved in cholesterol accumulation in peripheral tissue,the mRNA levels of low-density lipoprotein receptor(LDL-R),liver X receptor(LXR),and peroxisome proliferator-activated receptor ?(PPAR?)in HD group were significantly(P < 0.05)higher than those in BC group after 8-week feeding.About biomarkers involved in cholesterol conversion into bile acids,the mRNA levels of 7?-hydroxylase(CYP7A1)in HD group were significantly(P < 0.05)higher than those in BC group after 8-week feeding.Thus,WGQ exhibits cholesterol-lowering effects mainly by regulating hepatic cellular biomarkers(AMPK?,HMG-CoAr,LDL-R,LXR,PPAR?,and CYP7A1)that involved in cholesterol metabolism.4.To further study the lipid-regulating mechanism of WGQ,expressions of hepatic protein,CYP7A1,HMG-CoAr,and LDLR,were investigated after 8-week feeding.Western blot results show that the expression levels of CYP7A1 and LDLR in HD group were significantly higher,whereas levels of HMG-CoAr were significantly lower compared with BC group.Moreover,liver proteome analysis was conducted.Proteins were separated by two-dimensional gel electrophoresis,identified by mass spectrometry,and submitted for analysis in database.Seven spots were successfully identified.KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis showed that these differential proteins invovled in pathways of lipid,energy,and carbohydrate metabolism,etc.Four differential protein involved in lipid metabolism were further analyzed by RT-PCR.After WGQ diet,the expressions of heat shock protein 60(HSP60)and phosphatidylethanolamine binding protein 1(PEBP1)were down-regulated,and expressions of enoyl-coenzyme A hydratase(ECH)and peroxiredoxin 6(PRDX6)were up-regulated in rats with HFD.HSP60 and PEBP1 contribute to the pathophysiology of atherosclerosis;ECH catalyzes the beta-oxidation pathway of fatty acid metabolism;PRDX6 is a peroxiredoxin with peroxidase activities.Therefore,WGQ diet may accelerate fatty acid beta-oxidation,decrease oxidative stress levels,and reduce the risk of atherosclerosis.5.The effect of WGQ on intestinal cholesterol metabolism was investigated in this section.Multiple factors of the feces,small intestinal content and caecum of rats were detected.Results showed that the pH of feces and small intestinal content in BC group were higher than those in other groups,while HD group had the lowest values during the experimental period.After 4-and 8-week feeding,the fecal dry weight,cecal weight,cecal surface area,and cecal content weight in HD group were significantly higher(P < 0.05)than those in other groups.The bile acid levels of small intestinal contents and feces,and fecal neutral sterol levels in LD groups were higher than those in BC group with no significant difference.However,the bile acid levels and fecal neutral sterol levels of HD groups were significantly(P < 0.05)higher than those of other groups.A statistically significant(P < 0.05)increase was found in the acetate,propionate,butyrate,and total SCFA concentrations in the HD group compared with those in the BC and LD groups after 4-and 8-week feeding.These results suggest that WGQ diet can lead to a large mass of indigestible residue and high fermentation activity.mRNA and protein expression levels of cellular biomarkers involved in ileal cholesterol metabolism were investigated.Results showed that the mRNA levels of ileal farnesoid X receptor(FXR)in HD group were significantly(P < 0.05)higher,whereas mRNA and protein levels of apical sodium-dependent bile acid transporter(ASBT)were significantly(P < 0.05)lower than those in BC group after 4-and 8-week feeding.These results suggested that WGQ may inhibite bile acid reabsorption by decreased level of ASBT,which may be caused by the increased level of ileal FXR.6.The effect of WGQ on the intestinal bacterial communities of HFD rats were determined to investigate the changes and functions of bacterial communities in the cholesterol-lowering effects of WGQ.Firstly,nested PCR-denaturing gradient gel electrophoresis(PCR-DGGE)technology was used to study the effect of WGQ on fecal and cecal bacterial communities in HFD rats after 4-and 8-week feeding.Results showed that the compositions of the bacterial communities in feces and cecal content of different groups were similarly.However,the dominant bacterial bands were distinctly different.The compositions of the bacterial communities in different groups after 4-week feeding were different with those after 8-week feeding.Moreover,the compositions in LD group were different with those in BC and HD group.A total 21 bands were excised from the gel,including 15 mutual bands,1 fecal specific band,and 5 cecal specific bands.After the double DGGE,gel extraction,DNA sequencing,and sequence alignment,19 bands were classified to Bacteroidetes and Firmicutes phyla,besides 2 bands identified as unclassified uncultured bacteria.Then,Illumina Miseq sequencing approach was used to analysis the changes of cecal microbial communities of rats after 8-week feeding.Result showed that HFD didn‘t affect the alpha diversities of rats,while high dose of WGQ significantly(P < 0.05)decrease the alpha diversities of HFD rats.A total of 23 bacteria phyla were identified in all the samples.Bacteroidetes and Firmicutes were the two most dominant phyla,accounting for >92.08% of the reads.At the genus level,all 204 detected genera were shared by all samples.Prevotella is the most dominant genus in all groups.The relative abundance of Prevotella and Anaerovibrio increased with the increase in WGQ dosage.Furthermore,WGQ diets decreased many bacterial genera.Since Prevotella and Anaerovibrio can produce SCFAs,these findings indicate that WGQ affect cholesterol metabolism potentially by selectively enriching the SCFA-producing intestinal microbiota.The present study innovatively investigated the effect of WGQ on cholesterol metabolism of rats under HFD and the involved mechanisms from multiple levels by using multiple technologies.We confirmed that WGQ affect the cholesterol metabolism of HFD rats and this effect exhibited dose and time effect.Several biomarkers in liver and intestine that involved in this effect were found.We further proposed that WGQ affect cholesterol metabolism potentially by selectively enriching the SCFA-producing intestinal microbiota.Our study provide basis for future functional food development of WGQ. |
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