Study On The Mechanism Of The Regulation On Liver CYP7A1of Active Components Of Hypolipidemic TCM Huanglian

Background:Hyperlipidemia is a dangerous disease, and Huanglian is an important TCM that canlower lipid. Our previous study found that up-regulating cholesterol7-alpha–hydroxylase(CYP7A1)----a key rate-limit enzyme for cholesterol transformation into bile acid in theliver is related to the lipid-lowering effect of Huanglian, but the specific mechanism isunknown.Objective:The research is to clarify the mechanism and material basis of the regulation onCYP7A1of active components in lipid-lowering TCM Huanglian via LXRα/PPARα.Methods:1. In vivo study: To establish hyperlipidimic model, healthy male SD rats weighed180-200g, were fed with high lipid diet, blood was collected for the determination of TC,TG, LDL-C and HDL-C. Then50hyperlipidimic rats were divided into six groups ofcontrol, model group, low dose coptis alkaloid extract (CAE,50mg/kg) group, middle doseCAE(100mg/kg)group, high dose CAE (200mg/kg)group and simvastatin (10mg/kg) group.Except control group10rats were fed as uaual, all the hyperlipidimic rats were fed withhigh lipid diet, CAE or simvastatin was given once a day for4weeks. During the last threedays before the end of the experiment, samples of rat feses were collected. At the end of theexperiment, rats were sacritified and the samples of rat blood and liver were collected for the determination of TC, TG, HDL-C and LDL-C in serum, TC and TBA in liver and feces,the activity of CYP7A1in liver. Reverse transcription PCR (RT-PCR) was used to detectthe expression of CYP7A1, LXRα and PPARα mRNA in the rat livers.2. In vitro study:(1) Effects of berberine (BBR) on CYP7A1, LXRα and PPARαmRNA and protein expression in BRL-3A cells: BRL-3A cells were cultivated. Choosingthe safe dose range by MTT test, the cultivated confluent BRL-3A cells were exposured to0,0.3,3and10μM of BBR for12/24h. Then cells were collected to extract total RNA andprotein, RT-PCR and western blot were used to detect the expression of mRNA and proteinof CYP7A1, LXRα and PPARα in the cells.(2) LXRα and PPARα gene blocking test:BRL-3A cells were cultivated. Gene blocking agent(GGPP/MK-886,10μM) were added tothe cultivated confluent BRL-3A cells before BBR was given. Then the cells werestimulated for24h. Thereafter cells were collected to extract total RNA and protein,RT-PCR and western blot were used to detect the expression of mRNA and protein ofCYP7A1, LXRα and PPARα in the cells.(3) High concentration cholesterol induction: TheBRL-3A cells were cultivated and grouped into control, cholesterol (5μM), cholesterol+lowBBR (0.3μM), cholesterol+high BBR (10μM), cholesterol+low BBR (0.3μM)+GGPP(10μM), cholesterol+high BBR (10μM)+GGPP (10μM), cholesterol+lowBBR (0.3μM)+MK-886(10μM), cholesterol+highBBR (10μM)+MK-886(10μM). Then the cultivatedcells were exposued to the different composition drug for24h, Thereafter cells werecollected to extract total RNA and protein, RT-PCR was used to detect the expression ofmRNA of CYP7A1, LXRα and PPARα.Results:1. Effect of CAE on the liver related gene expression of hyperlipidemic ratsLevels of TC, TG and LDL-C were reduced by CAE in hyperlipidimia rats compared withmodel (P<0.05or P<0.01), at the same time, CYP7A1, LXRα, PPARα mRNA wasup-regulated in a dose-dependent way (P<0.05or P<0.01). The activity of CYP7A1in theliver was increased, the content of TC in the liver was also reduced, while TBA in liver andfeces, TC in the feces were increaced dose-dependently (P<0.05or P<0.01). 2. Effect of BBR on the related gene and protein expression of BRL-3A cells.(1)Effect of BBR on CYP7A1, LXRα and PPARα mRNA and protein expression inBRL-3Acells. BBR could promote mRNA and protein expression of CYP7A1after24h ofthe addition of BBR, while LXRα and PPARα were induced dose-dependently earlier at12h after the addition of BBR (P<0.05or P<0.01);(2) Effect of BBR on CYP7A1mRNA andprotein expression in BRL-3Acells after LXRα or PPARα were blocking After LXRα orPPARα gene expression was blocked, CYP7A1gene and protein expression were all alsoinhibited even BBR was given (P<0.05or P<0.01);(3) Effect of BBR on CYP7A1, LXRαand PPARα mRNA and protein expression in BRL-3Acells at high cholesterol condition. Inthe stimulation of high doses of cholesterol, CYP7A1mRNA expression was elevated, butLXRα and PPARα mRNA were reduced. When BBR was added, CYP7A1and PPARαmRNA were increased even more in a dose-dependent manner, while LXRα mRNA waspromoted at a low dose of BBR. After blocking gene expression of LXRα or PPARα,CYP7A1gene expression was inhibited (P<0.05or P<0.01).Conclusion:CAE, that an active components of lipid lowering herb Huanglian (a vital componentherb of famous hypolipidemic TCM composition FTZ) has a therapeutic action inhyperlipidemia, the possible mechanisms might be related to:(1) Increasing the expression of mRNA, protein and the activity of CYP7A1inhyperlipidemic rat liver, and to promote the cholesterol transformation into bile acid andexcretion of cholesterol in vivo, decreasing levels of seum cholesterol and improving thesymptom of hypercholesterolemia and hyperlipidimia;(2) Via inducing the expression of upstream regulation factors LXRα and PPARα,up-regulating CYP7A1gene and protein expression.