Effects Of Alkaloids From Dendrobium Nobile Lindl. On Lipid Homeostasis In Mice Fed By High-Fat Diet Via Bile Acids Metabolism

Objective:To investigate the effects of DNLA on bile acid metabolism in mice fed by high-fat diet and the related mechanism.Methods:C57BL/6J mice were divided into six groups randomly,10 mice in each.Including control diet group（CD group）,high fat diet group（HFD group）,DNLA-Low treatment group（DNLA-L）,DNLA-Mid treatment group（DNLA-M）,DNLA-High treatment group（DNLA-H）and positive control（simvastatin）group.Mice in the control group were fed with control diet and the other groups were fed with high-fat diet.Lipid metabolism in mice was determined by serum LDL-C and TC.The treatment via oral was started after lipid metabolism was disrupted,once a day,lasted for 18weeks.CD and HFD groups were given 0.45%CMC-Na and DNLA-L group was given DNLA 15 mg/kg,DNLA-M group was given DNLA 30 mg/kg,DNLA-H group was given DNLA 60 mg/kg and simvastatin group was given simvastatin 20 mg/kg.After the treatment ended 12 hours,the liver was collected.Cell morphology and lipid were observed by Oil-Red-O staining and HE staining.Detected TG and TC in liver.Bile acids profile were analysed by UPLC-MS.The gradient elution chromatography separation was performed on a Thermo Hypersil Gold C₁₈ column（150 mm×2.1 mm,1.9μm）.The mobile phases were consisted of 20%acetonitrile with 5 mmol/L ammonium acetate（A）and 80%acetonitrile with 5 mmol/L ammonium acetate（B）.The injection volume was 2μL,the flow rate was 0.3 mL/min,and the temperature of column was 40℃.HESI ionization method.negative mode;spray voltage,3.5 kV;probe heater and capillary temp,300 and 350℃,aux gas flow 10 arb.sheath gas flow35 arb.The scan mode was negative full scan and ddMS scan.The expression of Cyp7a1,Cyp7b1,Cyp8b1,Cyp27a1,Fxr,Shp,Bsep,Mrp2,Mdr2,Fgfr4,Lrh-1,Cyp3a11,Baat,Bacs related to bile acids metabolism of liver tissues were measured by RT-PCR.Results:After 18 weeks of high-fat diet feeding,the body weight of mice was significantly increased compared with the normal control group（P<0.05）,and the serum LDL-C and TC were also significantly increased（P<0.05）.These results showed that after feeding high fat diet,abnormal lipid metabolism appeared in HFD mice and model was accomplished.Then entered treatment phase.After drug administration,the liver was collected and histomorphological changes were observed.HE staining showed that fat vacuoles could be seen in liver and lipid droplets in cells in the HFD group mice.Compared with the HFD group,the lipid accumulation and hepatocyte steatosis were decreased in DNLA groups（P<0.05）,DNLA-L had the most normal cells and the least lipid accumulation.Oil-Red-O staining showed that compared to CD group,the liver of mice in the HFD group was stained more significantly.Compared with the HFD group,the DNLA groups had lighter staining variously.The DNLA-L group had the least lipid in liver.The liver TG and TC in HFD group were significantly higher than CD control group（P<0.05）.Compared with the HFD group,liver TG and TC in DNLA-L group was significantly decreased（P<0.05）,and they also were much lower than positive control group.The DNLA-L group had exhibited the best treat effect among several groups in reducing hepatic lipid accumulation from high-fat diet,so we use DNLA-L group to take further study in metabolism mechanism.Principal component analysis（PCA）was used to detect the changes of liver bile acid profiles in each group.It was found that the profile of bile acid in CD group and HFD group was significantly different,while the profile of bile acid in DNLA-L group was similar to that in CD group and could be clearly distinguished from that in HFD group.The loading graphic showed that CA,CDCA,LCA,DCA,TMCAs,TDCA,TUDCA,TCDCA and THDCA contributed to the difference in bile acid profile from each group mice.One way ANOVA revealed that CA,DCA and CDCA in the liver of mice in the HFD group increased（P<0.05）,TCDCA decreased（P<0.05）.Compared with the HFD group,the bile acids CA,DCA,CDCA and LCA of DNLA-L group were decreased significantly（P<0.05）,while the conjugated bile acids TDCA,THDCA,TCDCA,TMCAs and TUDCA were significantly increased（P<0.05）.In addition,comparison of CA/CDCA between HFD group and DNLA-L group showed that DNLA obviously inhibited CA/CDCA ratio（P<0.05）.The expression of genes related to bile acid metabolism indicated that compared with CD group,the expression level of Cyp27a1 in liver was significantly increased in HFD group（P<0.05）,while the expression levels of Fxr and Bacs were significantly decreased（P<0.05）.Compared with CD group,the expression of Cyp27a1 and Shp were significantly increased in the DNLA-L group（P<0.05）.The expression of Fxr and Cyp3a11 were significantly increased in the DNLA-L group compared with the HFD group（P<0.05）.Conclusions:DNLA may improve the lipid metabolism of high fat diet fed mice,changed lipid accumulation in the liver significantly,this might related to influence bile acids metabolic pathways.It is mainly in enhancing bile acids biotransformation to the taurine-conjugated bile acids which are highly hydrophilic and contribute to the solubility and excretion of cholesterol,and decrease the CA/CDCA ratio which is positively related to cholesterol absorption.