Mechanistic Study Of The Weight Loss And Lipid-lowering Effects Of Wheat Bran Polyphenols And Wheat Bran Oil On Diet-induced Obese Rats

Objective: A large number of studies have shown that whole grain has the effect of weight loss and fat reduction,which may be related to the dietary fiber and phytochemical components contained in bran,but the exact mechanism is still unclear.Previous studies have shown that whole wheat naan has the effect of weight loss and lipid reduction.This study further systematically analyzed the effects of wheat bran extract polyphenol(TCJ)and wheat bran oil(WJB)on fat loss and lipid reduction in obese rats from multiple perspectives through pharmacodynamics,non-target metabolomics combined with network pharmacology,and metabolic omics combined with intestinal microbiome.Methods: This study was divided into four parts:(1)The main nutrients(protein,fat,carbohydrate,dietary fiber,10 minerals,17 amino acids,37 fatty acids,etc.)and phytochemicals(polyphenols,betaine,alkyl-hydroquinone,etc.)of 30 batches of wheat bran from 24 different counties and cities in Xinjiang and 6 different provinces and cities outside Xinjiang were quantitatively determined by national standard or reference method.Principal component analysis(PCA)was used to screen samples for follow-up intervention experiments.Through plant broad-target metabolomics detection,the compounds with the highest response in wheat bran extract were qualitatively identified as positive controls for intervention experiments.(2)To observe the effects of wheat bran extracts TCJ and WJB on weight and fat reduction in high-fat fed obese rats.A total of140 SPF-grade SD rats were selected,aged 6-7 weeks and weighing 80-90 g,half male and half female.After one week of adaptive feeding,10 rats were randomly selected as blank control group(B)and fed with basic diet,and the remaining 130 rats were fed with high-fat diet as obese rat model group(M).The modeling was considered successful if the body weight growth rate of rats was more than 20%.The rats were randomly divided into9 groups according to their body weight: Model control group(M),betaine positive control group(TMG,0.10 g/kg),linoleic acid positive control group(YYS,0.07 g/kg),wheat bran polyphenols extract low(TCJ-L,1.5 g/kg),medium(TCJ-M,3.00 g/kg),the high dose group(TCJ-H,6.00 g /kg)three dose groups,wheat bran oil low(WJB-L,1.25ml/kg),medium(WJB-M,2.5ml/kg),high dose(WJB-H,5.00ml/kg)three dose groups,10 in each group,were given each subject every day.Polyphenol extract was intragastric at the rate of 5m L/kg(dissolved in distilled water),while groups M and B were intragastric with distilled water(5.0 m L/kg)for 9 weeks.At the end of the intervention period,rats were treated with ether anesthesia after 12 hours of water deprivation,and related specimens were collected.The weight change,organ coefficient,obesity index and Lee’s index were calculated before and after the intervention.Blood lipid(TC,TG,HDL-C,LDL-C),blood glucose(GLU),liver function related indicators(ALP,ALT,AST and LDH),gallbladder function related indicators(TP,ALB and TBA),renal function related indicators(CREA,UA and UREA)were detected.Anti-inflammatory(IL-6,IL-1β and TNF-α),antioxidant(MDA,GSH-Px,CAT,SOD and T-AOC),other related indicators(ADPN,LEP,LPL and HL)levels and liver morphological changes.(3)Based on non-target metabolomics and network pharmacology to study the weight loss and lipid lowering substances of wheat bran extract and their mechanism of action,UHPLC-QE-MS non-target metabolomics technology was used,combined with univariate statistical analysis and multivariate statistical analysis methods,to screen wheat bran extract and the differential metabolites in rat liver tissue after intervention;The caloric value criteria for screening differential metabolites were: P <0.05,VIP > 1,FOLD CHANGE < 0.5 or > 2;The potential targets and metabolic pathways of major metabolites of wheat bran extract were further studied with network pharmacology,and the weight loss and lipid lowering substances and their mechanisms were analyzed.The main metabolite chemical components were docked with the core target,and the binding energy was used to evaluate the binding ability of the components and the target,and predict the effect.Finally,Western blot analysis was further used to verify the corresponding core targets.Finally,the mechanism of weight loss and lipid reduction of two wheat bran extracts(TCJ and WJB)was analyzed based on non-target metabolomics and network pharmacology.(4)Based on the discovery of metabolomics and intestinal microbiome,the mechanism of weight loss and lipid reduction of wheat bran extract was analyzed.UHPLC-MS/MS and 16 Sr DNA sequencing were used to analyze the liver metabolites and intestinal flora of obese rats before and after the intervention of two wheat bran extracts(TCJ and WJB)by discovery metabolomics and intestinal microbiome techniques,and the correlation between different metabolites and microflora structure was analyzed.Combining the pharmacodynamic results and the pathways and important target proteins predicted by network pharmacology,the mechanism of regulating the weight loss and lipid reduction of two extracts of wheat bran(TCJ and WJB)based on metabolites and intestinal flora was explored.Results:(1)Among the three major nutrients in 30 batches of wheat bran,carbohydrate content ranged from 61.7 to 71.0 g/100g(dietary fiber was 12.9 to 40.6 g/100g),protein content ranged from 12.2 to 19.7g/100 g,and fat content ranged from 1.3 to 4.5 g/100g;Among the minerals,the content of potassium(K)was the highest(0.404～1.614 g/100g),and the content of K in 15# sample was the highest(1.614 g/100g),which was higher than the reference value(the content of nutrients in wheat bran in the Standard Edition of China Food Composition Table).The content of glutamic acid(Glu)was the highest among the17 amino acids detected,the content range was 1.08～5.42 g/100 g,the content of cystine(Cys)was lower than the reference value,and the rest content was within the reference value range.In the analysis of fatty acids,saturated fatty acids(SFA)accounted for16.8-21.0% of total fatty acids,monounsaturated fatty acids(MUFA)accounted for15.6-24.7%,and polyunsaturated fatty acids(PUFA)accounted for 57.9-66.5%.Among them,the content of free linoleic acid is 0.00057～0.00236 g/100 g,the highest content is29# sample,the content of α-linolenic acid is 0.000011～0.000148 g/100 g,the highest content is 12# sample.The content of polyphenols in wheat bran from different producing areas ranged from 0.0316 to 0.0729 g/100 g,and the highest content was 5# sample.The content of betaine was 0.00108～0.00273 g/100 g,the highest content was 15# sample;The content of total alkyl-pyrocinol(ARs)was 0.0511～0.2186 g/100 g,and the highest content was 5# sample.Based on the qualitative analysis of broad-target metabolomics scanning of plants,it is concluded that the extracts prepared by screening 5# samples can be used for subsequent animal experiments.(2)Both TCJ and WJB can reduce the food response ratio of obese rats fed high-fat diets,significantly reduce weight and fat,reduce liver weight and liver coefficient;The levels of serum lipids(TG,TC,LDL-C)and inflammation(IL-6,IL-1β and TNF-α)were decreased,the antioxidant capacity was increased(MDA,GSH-Px,CAT,SOD and T-AOC),and the liver and bile function was improved(ALP,ALT,AST,TP,ALB,TBA and CREA).Liver morphological examination: TCJ and WJB can alleviate liver injury caused by high fat feeding.(3)Metabolomic examination,compared with the M group,26 different metabolites were screened in the TCJ group and 13 different metabolites were screened in the WJB group;Compared with group B,a total of 190 differential metabolites were screened in group TCJ and 150 in group WJB.A total of 21 different metabolites were identified in TCJ group and WJB group.The possible metabolic pathways are vitamin B6 metabolic pathway and steroid hormone biosynthesis pathway.Through network pharmacological analysis,60 potential targets of 8 major metabolites were enriched in 18 pathways,among which c AMP signaling pathway and c GMP-PKG signaling pathway mainly reflected anti-inflammatory effects.The molecular docking analysis of metabolites and key targets showed that betaine-TNF(-6.85 k J·mol-1)was the most closely bound,followed by betaine-Pparg(-6.16 k J·mol-1),indicating that Betaine played an important role in weight loss and lipid reduction.Further analysis by western blot confirmed that the corresponding core targets were TNF-α,PPARG and AKT1,among which PPARG was closely related to the lipid and atherosclerosis signaling pathway and played a role in lipid lowering.At the same time,the increased expression levels of AKT and TNF-α m RNA also indicate that they play a certain anti-inflammatory role.(4)Analysis of the differential metabolite pathways identified by metabolomics showed that: compared with group M,the main metabolic pathways of group B were glyoxylic acid and dicarboxylic acid metabolism,and mutual conversion of pentose and glucuronic acid;The main metabolic pathways of TCJ group are niacin and niacinamide metabolism and purine metabolism.The main metabolic pathways in TMG group were α-linolenic acid metabolism and ether lipid metabolism.The main metabolic pathway of WJB group is taurine and taurine metabolism.The main metabolic pathways of YYS group were TCA cycle/citric acid cycle and propionic acid metabolism.From the analysis of intestinal flora,Bacteroidota had a great effect on LDA Score,and propionic acid,as a short-chain fatty acid(SCFA),significantly reduced the proportion of Bacteroides.Compared with WJB-M,TCJ-M increased various metabolism(including carbohydrate metabolism,amino acid metabolism and energy metabolism),as well as cell transformation and signal transduction,indicating that TCJ and WJB can increase metabolism,regulate cell signaling pathways and improve intestinal microbiota.Spearman method was used to analyze the correlation between 20 different intestinal flora and 20 different liver metabolites in TCJ and WJB.WJB had a stronger regulatory effect on intestinal flora than TCJ in obese rats,and was closely related to short-chain fatty acid metabolism.Conclusion:(1)By analyzing the content of nutrients and main phytochemicals in wheat bran in Xinjiang,the 5# samples with the highest content of ARs and polyphenols were selected for subsequent animal intervention experiments;1127 compounds in wheat bran extract were qualitatively identified by plant MRM broad-target metabolomics.Betaine,the compound with the highest response,was selected as a positive control in subsequent animal intervention experiments.(2)The two extracts of wheat bran(TCJ and WJB)can inhibit excessive weight gain,reduce blood lipid level,reduce liver injury degree and liver pathological injury caused by high-fat feeding in rats,and have anti-inflammatory and antioxidant activities;It is better than the positive control TMG and YYS in lipid reduction and liver protection,suggesting that it is beneficial to cardiovascular health.(3)Differential metabolites screened by non-target metabolomics combined with network pharmacological analysis showed that 60 potential targets of 8 major metabolites were enriched in 18 pathways,among which c AMP signaling pathway and c GMP-PKG signaling pathway mainly reflected anti-inflammatory effects,and were verified by western blot analysis.The results of the network pharmacological study by molecular docking technology showed that betaine was the effective substance of wheat bran for weight loss and lipid reduction,and TNF and PPARG were the effective targets of wheat bran for weight loss and lipid reduction.(4)Based on the analysis of the pathways of differential metabolites screened by metabolomics,it can be seen that the main metabolic pathways of TCJ group are niacin and niacinamide metabolism and purine metabolism;The main metabolic pathway of WJB group is taurine and taurine metabolism.Bacteroides had a significant effect on LDA Score,and propionic acid,as a short-chain fatty acid(SCFA)in intestinal tract,significantly reduced the proportion of bacteroides.It is suggested that TCJ and WJB can increase metabolism,regulate cell signaling pathways and improve intestinal microbiota.WJB has a stronger regulatory effect on intestinal flora than TCJ in obese rats and is closely related to short-chain fatty acid metabolism.Wheat bran contains a large number of bioactive ingredients,mainly including polyphenols,unsaturated fatty acids,etc.These ingredients exert pharmacological activities of weight loss and lipid reduction through various ways,such as anti-inflammatory,antioxidant,regulation(c AMP,c GMP-PKG and lipid and atherosclerosis)signaling pathways,and improvement of intestinal flora.