Biochemical Mechanism Underlying Lipid Metabolism Disorder Induced By Acute Schisandrin B Treatment In Normal-and High Fat/Fructose Diet-fed Mice

Lipids are biological macromolecules that maintain normal physiological function of cells and body’s balance.There are two sources of lipids:exogenous and endogenous.The lipid metabolism disorder will caused hyperlipidemia which is referred to as hypertriglyceridemia,hypercholestermia and both in combined hyperlipidemia.In modern society,the morbidity of obesity,diabetes,cardiovascular diseases and non-alcoholic fatty liver disease induced by hyperlipidemia was significantly increased due to the changes of lifestyle.Therefore,the searching for therapeutic intervention of hyperlipidemia caused by the disorder of lipid metabolism has been an area of intensive research.The animal models of hyperlipidemia are more and more important,especially hypertriglyceridemia,which is hard to be established.Schisandrin B(Sch B)is the most abundant dibenzocyclooctadiene derivative isolated from Schisandrae fructus.Previous studies in our lab have demonstrated that high-dose Sch B increased serum TG,and the peak effects were observed at 24 h after dosing.Sch B treatment significantly increased hepatic TG level and decreased the fatty index at 48 h posttreatment.In this study,the alterations in lipid metabolism were measured after high dose of Sch B treatment in normal and high fat/fructose diet(HFFD)-fed mice,including its molecular mechanisms revealed by lipidomics and gene chip technology.In addition,the effects of Sch B on proliferation and differentiation of 3T3-L1 preadipocytes were also investigated.Part 1 Influence of Sch B on lipid metabolism in normolipidemic miceObjective:To evaluate the effect of Sch B on lipid metabolism in mice fed with normal diet.Method:Male ICR mice were intragastrically treated with either Sch B(0.25,0.5,1,2 g/kg)or FF(0.15 g/kg)suspended in olive oil or CMC.Serum/hepatic total cholesterol(TC),triglyceride(TG)and Glucose(GLU)levels,as well as serum apolipoproteins such as highdensity lipoprotein(HDL),low density lipoprotein(LDL),very low density lipoprotein(VLDL),apolipoprotein A1(Apo Al),apolipoprotein B48(Apo B48),apolipoprotein B100(Apo B100),apolipoprotein C Ⅱ(ApoC Ⅱ),apolipoprotein C Ⅲ(Apo C Ⅲ),apolipoprotein(Apo E),were measured at 48 h after Sch B or FF treatment.At the same time,the biomarkers and enzymes of lipid metabolism,including free fatty acid(FFA),leptin,adiponectin,resistin,visfatin,IL-6,lecithin cholesterol acyltransferase(LCAT),lipoprotein lipase(LPL),hepatic lipase(HL),and hormone sensitive lipase(HSL)and alanine aminotransferase(ALT,an indicator of potential hepatocyte damage)activity and hepatic growth factor(HGF)were determined with ELISA or chemical methods.Additionally,epididymal adipose tissues/liver mass,index,lipocytes size,and serum were recorded.Liver tissue section and lipid accumulation were measured using HE staining and oil red O staining,respectively.During the process of the experiment,we kept a record of food/water intake.Result:1.(1)Serum TG/TC levels were significantly increased by 111%/41%and 10%/18%after a single oral dose of Sch B(1 g/kg)suspended in olive oil or CMC,respectively,but decreased by 23%after FF(0.15g/kg,suspended in both vehicles)treatment.However,serum GLU level was not markedly changed in all groups.(2)The serum TG level was significantly increased by 32%-266%in a dose-dependent manner after Sch B treatment in olive oil.2.(1)Hepatic TG and TC levels were significantly increased by 164%and 38%after a single oral dose of Sch B(1 g/kg,suspended in olive oil),respectively.However,the same dose of Sch B suspended in CMC did not alter the hepatic TG and TC contains.FF suspended in both olive oil and CMC decreased hepatic TG and TC(up to 65%).However,hepatic GLU level was decreased in all treatment groups,compared with the control.(2)The hepatic TG levels were markedly increased by 57%-353%in a dose-dependent manner after Sch B(0.25,0.5,1,2 g/kg)suspended in olive oil,but Sch B suspended in CMC the hepatic TG was increased up to 243%.3.Sch B-treated mice the serum HDL and LDL levels were significantly increased by 25%30%,26%-31%,respectively,but VLDL、ApoB 100、ApoC Ⅱ、ApoC Ⅲ and Apo E levels were markedly decreased by 13%-16%,20%-21%,21%,18%and 11%-29%,respectively.However,ApoB48 level was decreased(up to 16%)at doses of 0.5 and 1 g/kg,but increased(up to 97%)at a dose of 2 g/kg.4.Sch B treatment decreased the serum FFA level by 16%-18%at doses of 0.5 and 1 g/kg,but increased the serum FFA level by 37%at a dose of 2 g/kg.5.Serum LCAT,LPL,HSL activity was significantly decreased by 48%,22%and 22%,respectively,after Sch B treatment.6.Sch B treatment decreased the fatty index(18%-49%),lipocytes volume(39%),serum leptin(23%-38%),adiponectin(19%)and visfatin(13%-29%)levels.7.Sch B treatment increased the liver index(20%-52%)and serum HGF level(4%-30%).But,ALT activity was no markedly changed in the current study.8.Histological examination and oil red O staining of liver tissue showed that hepatic lipid deposits was promoted by Sch B treatment.9.Sch B treatment inhibited the body weight gain by 54%-223%and the food/water intake by 2%-32%/10%-33%.Conclusion:Sch B suspended in olive oil augmented the hypertriglyceridemia caused by it,compared with suspended in CMC;Olive oil can improve the influences of Sch B on hepatic lipids and GLU levels in normal mice compared with CMC;It is reasonable speculate that the generation of exogenous TG mainly contributes to Sch B-induced hypertriglyceridemia in mice;Sch B could alter the serum FFA levels and showed double-acting.Small dose of Sch B decreased serum FFA levels,but high-dose Sch B increased serum FFA levels;Sch B treatment decreased serum LCAT,LPL,HSL activity.This may be one of the mechanisms of Sch B on lipid metabolism;Sch B treatment might result in lipolysis and then deceased serum leptin,adiponectin and visfatin levels.Sch B treatment decomposed the adipose tissue may be contribute to the elevation in serum TG level;Sch B increased HGF level,this may be,at least in part,contributes to increase in liver mass;Sch B promoted lipids accumulation in the liver tissue.Part 2 Effect of Sch B on lipid metabolism in dyslipidemic miceObjective:To evaluate the effect of Sch B on lipid metabolism in mice fed with high fat/fructose(10/10%,w/w)diet(HFFD).Method:Male ICR mice were fed with HFFD for successive 10 days and intragastrically administered with Sch B at doses of 0.5,1,or 2 g/kg on the eighth day.Serum/hepatic TC,TG,GLU levels,serum HDL,LDL,VLDL,Apo Al,Apo B48,Apo B100,ApoC Ⅱ,Apo C Ⅲ,Apo E,FFA,HGF,leptin,adiponectin,resistin,visfatin,and IL-6 levels,LCAT,LPL,HL,HSL and ALT activity were measured at 48 h after Sch B/FF treatment.Epididymal adipose tissues/liver mass,index,and lipocytes size were also measured at 48 h post-treatment.Liver tissue section and lipid accumulation were measured using HE staining and oil red O staining,respectively.During the process of the experiment,we kept a record of food/water intake.Result:1.Mice fed with HFFD for 10 days,the serum TC and GLU levels were prominently increased by 18%and 17%,respectively,compared with normal mice.Sch B markedly elevated serum TG level by 65%-317%in mice fed with HFFD compared with HFFD-fed mice without Sch B treatment;2.The hepatic TG and TC levels were increased by 107%and 49%in HFFD-fed mice,when compared with normal mice.Sch B increased hepatic TG(257%-466%)and TC(29%-102%)levels,but decreased hepatic GLU content by 37%-32%in mice fed with HFFD,when compared with that of HFFE-fed mice without Sch B treatment.3.VLDL level was increased by 15%in HFFD-fed mice,compared with that of normal mice.Sch B treatment markedly decreased serum HDL(21-36%),LDL(27%),VLDL(21-34%),ApoB 100(21-32%),ApoC II(22-26%)and ApoE(17-19%)levels,but increased ApoB 48 level by 94%when compared with those fed with HFFD only.4.Sch B treatment markedly increased serum FFA level by 33%when compared with HFFD control group.5.Mice fed with HFFD significantly increased serum HSL activity by 94%,when compared with normal mice.Treatment with Sch B decreased serum LCAT(28%-39%)and LPL activity(28%),but decreased/increased serum HSL activity by 25%/43%at dose of 0.5/2 g/kg in HFFD-fed mice,when compared with that of mice fed with HFFD only.6.Mice fed with HFFD markedly increased fatty index(50%),serum resistin(24%),IL-6(89%)levels.Sch B treatment decreased the fatty weight/index(52%/50%),lipocytes volume(36%),serum leptin(51%-5 6%),adiponectin(39%),resistin(28%-31%),and visfatin(19%-24%)levels,but significantly increased serum IL-6 level by 67%in mice fed with HFFD.7.HFFD significantly increased the serum HGF level by 48%,but the liver weight and serum ALT activity were not obviously changed.Sch B treatment increased liver weight/index(up to 64%/68%)and serum HGF(29%)level,but decreased serum ALT activity by 23%in mice fed with HFFD.8.Histological examination and oil red O staining of liver tissue showed that Sch B treatment promoted hepatic lipid deposits in mice fed with HFFD.9.Sch B treatment decreased body weight gain by 118%249%,but has no effect on the food/water intake.Conclusion:Sch B significantly elevated TG level in HFFD-fed mice.Sch B combined with HFFD may build a novel model of combined hyperlipidemia;Sch B increased hepatic TG and TC contents,but decreased hepatic GLU contents in HFFD-fed mice;Sch B may alter the lipoprotein metabolism in mice fed with HFFD;High levels of serum FFA might be one of the reasons for Sch B-induced elevation in serum TG in HFFD-fed mice;Sch B affected the enzyme activity related to lipid metabolism,this may be,at least in part,contributes to the elevation in serum TG after Sch B treatment;Sch B treatment might cause lipolysis in HFFD-fed mice;Sch B-induced hepatomegaly might at least partly result from the HGF secretion;Sch B treatment promoted lipid accumulation in the liver tissue in HFFD-fed mice;Sch B treatment decreased body weight gain in HFFD-fed mice.Part 3 Serum lipidomics analysis in normal diet-and HFFD-fed mice treated with Sch BObjective:The aim of this study is to identify the potential biomarkers of lipid metabolism in normal-and HFFD-fed mice treated with Sch B.Method:Male ICR mice were fed on a normal diet or HFFD diet for 10 days,and were given a single dose of Sch B 2 g/kg on the eighth day.After 48 h treatment of Sch B,serum were obtained and subjected to lipidomics analysis.Result:Ten metabolites were identified as potential biomarkers in normal diet fed mice,including 19 TG,7 phosphatidylcholine(PC),and 2 phosphatidylethanolamine(PE).Twentyeight potential biomarkers were identified in HFFD-fed mice,including 15 TG,5 PC,4 PE,and 1 cholesteryl ester(CE).The level of PE was dramatically decreased in both mice fed with normal diet and HFFD,but others were increased.Conclusion:Sch B can increase serum biomarkers of lipid metabolism,especially TG metabolism.Part 4 Alternation of gene expression profiles in adipose tissue and liver of normal dietand HFFD-fed mice treated with Sch BObjective:To study the different genes expressed in the liver and fat tissue by gene chip,preliminary forecast the possible signaling pathway of Sch B on lipid metabolism.Method:Male ICR mice were fed with a normal diet or HFFD for 10 days.Sch B treatment groups were given a single dose of Sch B 2 g/kg on the eighth day.Detecting the different genes in the fat and liver with gene chip technology,then classify the different genes by GO and pathway analyses.Results:Compare the gene expression in normal diet fed mice with or without Sch B treatment,886 genes were significantly down regulated,1269 genes were markedly up regulated.Compare the gene expression in HFFD fed mice with or without Sch B treatment,861 genes were dramatically downregulated,1093 genes were significantly upregulated.Furthermore,the effects of Sch B on lipid metabolism associated with PPAR signaling pathway,Steroid biosynthesis,Insulin signaling pathway,Adipocytokine signaling pathway,and Metabolism of xenobiotics by cytochrome P450,et al.Conclusion:The present results indicated that Sch B regulated lipid metabolism via lipidrelated metabolism pathways.Part 5 Effect of Sch B on proliferation and differentiation of 3T3-L1 preadipocytesObjective:To study the effects of Sch B on preadipocyte proliferation,differentiation and adipocytokines secretory in the murine 3T3-L1 preadipocytes.Method:3T3-L1 cells were cultured with Sch B(2.5,5,10,20,or 40μM).Cells were stained with Oil Red O after differentiation to detect droplets in adipocytes.Exploring the mRNA expression of PPARγ,C/EBP α,FAS,aP-2,HSL,Leptin,Adiponectin,Resistin by RTPCR to study the possible mechanism.Results:Sch B markedly inhibited the lipid accumulation of 3T3-L1 adipocyte.PPARy,C/EBP α,FAS,aP-2,and HSL mRNA gene expression were significantly inhibited by Sch B,including Leptin,Adiponectin and Resistin mRNA expression.Conclusion:Sch B inhibited lipid accumulation in 3T3-L1 adipocyte through reducing PPARγ,C/EBP α,FAS and aP-2.The adipocytokines,including Leptin,Adiponectin and Resistin also showed markedly decreased by Sch B treatment.