Assessment Of Anti-Hyperlipidemic Effects Of Rice Bran Polysaccharides And Related Potential Mechanisms

China has a large yield of rice bran.It harbors varies kinds of active substances,which is a tremendous treasure-house for valuable resources.Recent studies found that rice bran polysaccharide(RBP)contribute to various physiological functions such as anti-obesity,lipid-lowering,anti-cardiovascular diseases,hypoglycemic effect,anti-cancer and immune enhancement.However,the utility research of rice bran has lagged behind,which has caused a huge waste of this valuable resource,leaving the majority of it only for feed so far.The study of lipid-lowering action of RBP stays at a primary stage,its molecular mechanism remains also far from recognized.In this study,RBP was extracted by ultrasonic-assisted extraction and the obtained RBP was further tested.Its lipid-lowering effect was then evaluated separately using the hyperlipidemic mouse model and HepG2 cells model.Moreover,its molecular mechanism was also explored to provide a scientific theoretical basis for the further study of the RBP and the development of functional foods and health products based on RBP.Extraction of rice bran polysaccharidesAfter removing the impurities,rice bran was pulverized;the powder was then added into hot water.After ultrasonic treatment,the mixture was centrifuged and the supernatant is mixed with amylase and protease.The mixture is centrifuged again,and the supernatant was added with ethanol.The obtained RBP was tested after concentration by rotary evaporation,which showed that the water-soluble dietary fiber is composed of carbohydrates without the presence of reducing sugars,starch,amino acids,proteins and uronic acids.The content of polysaccharide in the RBP sample was 81.40%and the extraction ratio was 1.66%.The RBP could be roughly divided into two different parts according to molecular weight:500 000 Da,count for 85%,and 25 000 Da,count for 15%.In addition,mannose,glucose,xylose and arabinose were included in the extracted RBP with the molar ratio of 1.35:3.32:1:1.25 without the presence of galactose,rhamnose and fucose.The absorption spectrum of the extracted RBP was determined finally by infrared spectroscopy,which showed that there exist a-glycosidic bond in the sample and the molecule was heteromonosaccharide without the presence of uronic acid.2.The lipid-lowering effect of RBP and molecular mechanism in high fat-diet miceIn this study,we investigated the anti-hyperlipidemic effect of the RBP and its mechanism in high-fat diet induced hyperlipidemic animal model.60 ICR male mice were randomly divided into 3 groups,which include Control,HFD(high fat diet)and HFD+RBP,and each group includes 20 mice.The control group was fed with standard diet while the other two groups were fed with HFD.In addition,the HFD+RBP group was fed with 500 mg/kg of RBP solution by intragastric administration while the other two groups were intragastrically administrated with equal amount of water.After the 11th week,the body weight,liver weight and fat pad weight of HFD+RBP mice were all significantly lower than that in the HFD group.The serum test showed that RBP could significantly decrease the levels of TC,TG and LDL-C in the plasma and increase HDL-C at a certain degree,however,there showed no significant difference.H&E staining of liver tissues showed that the hepatocytes of the HFD group were loosely arranged and enlarged with different sizes of fat droplets in the cytoplasm,while the RBP treatment could significantly decrease the size of the cell and number of fat droplets as well as arranged the cells more closely.The results of H&E staining of adipocytes showed that the size of fat cells in HFD group was significantly bigger than that in control group while RBP could significantly decrease their size.The mRNA and protein of each group were extracted from liver and analyzed further by microarray,RT-PCR and Western blotting.Microarray analysis revealed that RBP treatment resulted in the up-regulation of 93 genes and down-regulation of 72 genes in the hepatic tissues.GO analysis indicated that RBP caused differential expression of genes in the GO process of metabolism,respiratory chain and ATPase in mice fed HFD.KEGG pathway analysis suggested that RBP appeared to be involved in P450 cytochrome pathway,chemical carcinogenesis pathway,PPAR pathway and steroid hormone pathway in mice fed HFD.IPA software analysis indicated that lipid metabolism pathway was the second important pathway influenced by RBP.A total of 35 hepatic genes in lipid metabolism pathway were affected by RBP.It suggested further that NF-?B may play a vital role in the lipid-lowering effect of RBP.Real-time quantitative PCR confirmed that the mRNA levels of PPARa,PPAR?,PPAR?,SREBP-1C,FASN,ACC,SIRT and CD36,which were all related with lipid metabolism,were significantly regulated by RBP supplementation compared to HFD.The Western blot analysis further confirmed these altered expressions after RBP treatment.Taken together,these results suggested that RBP relieved the symptoms of hyperlipidmia in high-fat diet mice through regulating the gene expression profile and affecting some key genes of lipid metabolism in the liver.3.The effect of RBP on lipid-lowering and molecular mechanism in HepG2 cellsHepG2 model was built to observe the inhibitory effect of fat accumulation from RBP after oleic acid(OA)/BSA-treatment.First,the MTS assay and DAPI fluorescence microscopy found that 0-200 ?g/ml RBP had no significant effect on HepG2 cell viability.The Oil Red 0 stanning test showed that the addition of 25,50 and 100 ?g/mL of RBP could significantly reduce the fat accumulation in a dose dependent manner.RT-qPCR was consequently tested and showed that RBP could significantly up-regulate the relative mRNA expression of PPARa and PPAR? and down-regulate mRNA relative expression of PPAR?,SREBP-1C,FASN,ACC,SIRT and CD36 in HepG2 cells after OA/BSA treatment.Finally,the Western blot analysis further confirmed these altered expressions from protein aspect after RBP treatment,indicating that the regulatory effect of RBP on these genes played a key role in lipid metabolism,which leads a reduction of intracellular lipid accumulation.4.The effect of RBP on inflammation caused by HFD and its molecular mechanismMedical evidence shows that obesity is often accompanied by low levels of inflammation in the body.This study found that HFD could also stimulate the expression of inflammatory cytokines using the hyperlipidemic mice model induced by high fat fodder.The results showed that the spleen volume and weight of mice in HFD group were significantly higher than those in control group,while the spleen of mice in HFD+RBP group was relatively smaller and lighter.Moreover,high-fat diet increased the relative mRNA expressions of TNF-a,IL-6 and iNOS and RBP significantly decreased the relative mRNA expression levels of these factors by RT-qPCR analysis.Western blot analysis further confirmed that RBP could inhibit the protein expression levels of these inflammatory factors.These results suggested that RBP could inhibit the expression levels of inflammatory factors thus may reduce the inflammation accompanied with hyperlipidmia caused by high-fat diet.