To study the effect of cholesterol and 25-OH-cholesterol on cholesterol metabolism in HepG2 cells and the effect of coptisine (Cop) extracted from Coptidis Rhizoma (CR) in reducing and regulating cholesterol. In this study, TC, TG, LDL-c and HDL-c were measured by biochemical analysis; mRNA and protein expressions of LDLR, HMGCR and CYP7A1 were detected by qRT-PCR and Western blot. According to the results, cholesterol and 25-OH-cholesterol inducing could decrease in mRNA and protein expressions of LDLR and CYP7A1, so as to increase TC and LDL-c contents. However, Cop could up-regulate mRNA and protein expressions of LDLR and CYP7A1 and down-regulate that of HMGCR, so as to reduce TC and LDL-c levels. These findings suggested that Cop has potential pharmacological activity for reducing cholesterol, and may reduce cholesterol by regulating mRNA and protein expressions of key genes involved in cholesterol metabolism, such as LDLR, CYP7A1 and HMGCR. This study laid a firm theoretical foundation for developing new natural drugs with the cholesterol-lowering activity.
Berberine ; analogs & derivatives ; pharmacology ; Cholesterol ; metabolism ; Cholesterol 7-alpha-Hydroxylase ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Hep G2 Cells ; Humans ; Hydroxymethylglutaryl CoA Reductases ; genetics ; metabolism ; Receptors, LDL ; genetics ; metabolism ; Triglycerides ; metabolism

Animals ; Cholelithiasis ; genetics ; Cholesterol ; metabolism ; Cholesterol 7-alpha-Hydroxylase ; genetics ; Genetic Predisposition to Disease ; Hydroxymethylglutaryl CoA Reductases ; genetics ; Liver ; enzymology ; Male ; Mice ; Mice, Inbred AKR ; Mice, Inbred C57BL ; Receptors, LDL ; genetics ; Sterol O-Acyltransferase ; genetics

Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. As a metabolic regulator, FXR plays key roles in bile acid and cholesterol metabolism and lipid and glucose homeostasis. Therefore, FXR is a potential drug target for several metabolic syndromes, especially those related to lipidemia disorders. In the present study, we identified small molecule SIPI-7623, a derivative of an extract from Oriental wormwood (Artemisia capillaris), and found that it specifically upregulated the expression of cholesterol-7-alpha-hydroxylase (CYP7A1), downregulated the expression of sterol-regulatory element-binding protein 1c (SREBP-1c) in the liver, and inhibited the expression of ileal bile acid binding-protein (IBABP) in the ileum of rats. We found that inhibition of FXR by SIPI-7623 decreased the level of cholesterol and triglyceride. SIPI-7623 reduced the levels of cholesterol and triglyceride in in vitro HepG2 cell models, ameliorated diet-induced atherosclerosis, and decreased the serum lipid content on rats and rabbits model of atherosclerosis in vivo. Furthermore, SIPI-7623 decreased the extent of atherosclerotic lesions. Our resutls demonstrated that antagonism of the FXR pathway can be employed as a therapeutic strategy to treat metabolic diseases such as hyperlipidemia and atherosclerosis. In conclusion, SIPI-7623 could be a promising lead compound for development of drugs to treat hyperlipidemia and atherosclerosis.
Animals ; Artemisia ; chemistry ; Atherosclerosis ; drug therapy ; genetics ; metabolism ; Cholesterol ; metabolism ; Cholesterol 7-alpha-Hydroxylase ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Hyperlipidemias ; drug therapy ; genetics ; metabolism ; Hypolipidemic Agents ; administration & dosage ; Liver ; drug effects ; metabolism ; Male ; Rabbits ; Rats ; Rats, Sprague-Dawley ; Receptors, Cytoplasmic and Nuclear ; antagonists & inhibitors ; genetics ; metabolism ; Sterol Regulatory Element Binding Protein 1 ; genetics ; metabolism ; Triglycerides ; metabolism

Cholesterol 7alpha-hydroxylase (CYP7A1) regulates the balance between cholesterol supply and metabolism by catalyzing the rate-limiting step of bile acid biosynthesis. The transcriptional activity of CYP7A1 is tightly controlled by various nuclear receptors. A forkhead transcription factor O1 (FOXO1) plays a critical role in metabolism, and insulin inactivates FOXO1 through Akt-dependent phosphorylation and nuclear exclusion. We investigated the role of insulin-Akt-FOXO1 signaling pathway in CYP7A1 transcriptional regulation since we found putative insulin-response elements, FOXO1 binding sequences, in both rat and human CYP7A1 promoters. However, ectopic expression of FOXO1 increased the rat CYP7A1-, but mildly reduced human CYP7A1-promoter activities in a dose-dependent manner. Similarly to bile acids, insulin treatment increased small heterodimer partner (SHP) mRNA rapidly and transiently, leading to the suppression of CYP7A1 transcription in both human and rodents. Chromatin immunoprecipitation showed that FOXO1 directly bound to rat CYP1A1 promoter in the absence of insulin. FOXO1 binding to the rat promoter was diminished by insulin treatment as well as by expression of SHP. Our results suggest that the stimulation of insulin- signaling pathway of Akt-FOXO1 and SHP expression may regulate cholesterol/bile acid metabolisms in liver, linking carbohydrate and cholesterol metabolic pathways. A prolonged exposure of insulin in hyperinsulinemic insulin resistance or diabetic status represses CYP7A1 transcription and bile acid biosynthesis through SHP induction and FOXO1 inactivation, leading to impairment of the hepatic cholesterol/bile acid metabolisms.
Animals ; Bile Acids and Salts/metabolism ; Cholesterol/*metabolism ; Cholesterol 7-alpha-Hydroxylase/genetics/*metabolism ; Forkhead Transcription Factors/genetics/*metabolism ; Gene Expression Regulation/drug effects ; Glucose/*metabolism ; Hep G2 Cells ; Humans ; Insulin/pharmacology ; Lipid Metabolism/drug effects ; Liver/*metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Mutagenesis, Site-Directed ; Nerve Tissue Proteins/genetics/*metabolism ; Protein Binding/drug effects/genetics ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Receptors, Cytoplasmic and Nuclear/genetics/metabolism ; Sequence Deletion/genetics ; Signal Transduction/drug effects/genetics ; Transcriptional Activation/drug effects/genetics

Cholesterol 7alpha-hydroxylase (CYP7A1) regulates the balance between cholesterol supply and metabolism by catalyzing the rate-limiting step of bile acid biosynthesis. The transcriptional activity of CYP7A1 is tightly controlled by various nuclear receptors. A forkhead transcription factor O1 (FOXO1) plays a critical role in metabolism, and insulin inactivates FOXO1 through Akt-dependent phosphorylation and nuclear exclusion. We investigated the role of insulin-Akt-FOXO1 signaling pathway in CYP7A1 transcriptional regulation since we found putative insulin-response elements, FOXO1 binding sequences, in both rat and human CYP7A1 promoters. However, ectopic expression of FOXO1 increased the rat CYP7A1-, but mildly reduced human CYP7A1-promoter activities in a dose-dependent manner. Similarly to bile acids, insulin treatment increased small heterodimer partner (SHP) mRNA rapidly and transiently, leading to the suppression of CYP7A1 transcription in both human and rodents. Chromatin immunoprecipitation showed that FOXO1 directly bound to rat CYP1A1 promoter in the absence of insulin. FOXO1 binding to the rat promoter was diminished by insulin treatment as well as by expression of SHP. Our results suggest that the stimulation of insulin- signaling pathway of Akt-FOXO1 and SHP expression may regulate cholesterol/bile acid metabolisms in liver, linking carbohydrate and cholesterol metabolic pathways. A prolonged exposure of insulin in hyperinsulinemic insulin resistance or diabetic status represses CYP7A1 transcription and bile acid biosynthesis through SHP induction and FOXO1 inactivation, leading to impairment of the hepatic cholesterol/bile acid metabolisms.
Animals ; Bile Acids and Salts/metabolism ; Cholesterol/*metabolism ; Cholesterol 7-alpha-Hydroxylase/genetics/*metabolism ; Forkhead Transcription Factors/genetics/*metabolism ; Gene Expression Regulation/drug effects ; Glucose/*metabolism ; Hep G2 Cells ; Humans ; Insulin/pharmacology ; Lipid Metabolism/drug effects ; Liver/*metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Mutagenesis, Site-Directed ; Nerve Tissue Proteins/genetics/*metabolism ; Protein Binding/drug effects/genetics ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Receptors, Cytoplasmic and Nuclear/genetics/metabolism ; Sequence Deletion/genetics ; Signal Transduction/drug effects/genetics ; Transcriptional Activation/drug effects/genetics

OBJECTIVETo study the expressions of FXR, PPARa and Bile acid metabolism related genes in intrahepatic cholestasis of pregnant rats.

METHODS60 clean SD pregnant rats were selected and divided randomly into three groups. Since the 13th day of pregnancy rats in control group were injected subcutaneously with refined vegetable oil 2.0 mg/kg/d Rats in no-treated group were injected subcutaneously with the 17-a-ethynylestradiol (EE) 1.25 mg/kg/d until the 17th day. Those rat ih treated group were injected subcutaneously with the 17-a-ethynylestradiol (EE) 1.25 mg/kg/d until the 17th day and then were treated with fenofibrate for another four days until the 21th day. All rats were killed at the 21th day and livers were collected for study. The levels of serum TBA were examined by ELISA. The mRNA expressions of PPARa, FXR, CYP7A1, CYP27A1 and CYP8B1 were examined by real-time PCR. (1)

RESULTSThe levels of TBA were significantly higher in no-treated group (68.7+/-4.2)mumol/L and treated group (69.5+/-3.8)mumol/L compared with that of control group (26.6+/-2.3)mumol/L at the 17th day (P value is less than 0.05) and no difference found between treated and no-treated groups (P value is more than 0.05). The levels of TBA were higher in no-treated group (69.4+/-3.7)mumol/L and treated group (48.5+/-4.8)mumol/L as compared to control group (27.1+/-3.2)mumol/L at the 21th day (P value is less than 0.05). The lever of TBA was significantly lower in Treated group compared with No-treated group (P value is less than 0.05). (2) The mRNA expressions of CYP7A1, FXR, CYP27A1 and CYP8B1 increased in No-treated group (1.55+/-0.03, 1.75+/-0.02, 2.45+/-0.01, 2.15+/-0.01, respectively) and were all higher as compared to control group (0.75+/-0.02, 1.25+/-0.03, 0.65+/-0.03, 1.50+/-0.02, respectively) (P value is less than 0.05). However, the mRNA expression of PPARa decreased in No-treated group (0.85+/-0.02) compared with control group (1.45+/-0.02) (P value is less than 0.05). The mRNA expressions of CYP27A1, PPARa and CYP8B1 increased in treated group (1.25+/-0.01, 1.65+/-0.05, 1.65+/-0.02, respectively) and were all higher than that of control group (P value is less than 0.05).

CONCLUSIONAbnormal expressions of CYP7A1, FXR, CYP27A1, CYP8B1 and PPARa may play a role in pathogenesis of estrogen-induced intrahepatic cholestasis. Activator of PPARa may be used as therapeutical drug for ICP.

Animals ; Bile Acids and Salts ; metabolism ; Cholestasis, Intrahepatic ; chemically induced ; metabolism ; pathology ; Cholesterol 7-alpha-Hydroxylase ; metabolism ; Ethinyl Estradiol ; administration & dosage ; Female ; PPAR alpha ; metabolism ; Pregnancy ; Pregnancy Complications ; chemically induced ; metabolism ; pathology ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, Cytoplasmic and Nuclear ; genetics

Cajanus cajan L. is a natural plant, which contains a lot of potential active components. In the present study, we identified the effects of the stilbene extract from Cajanus cajan L. (sECC) on hepatic cholesterol metabolism in diet-induced (for 4 weeks) hyperlipidemic Kunming mice. All experimental mice were divided into 5 groups: control group, high lipid model group, sECC-treated with 200 or 100 mg kg(-1), and simvastatin (Sim, 12 mg kg(-1)) treated group. The mice were fed with fat and cholesterol-enriched chow except control mice that were fed with standard diet. The effects of sECC were investigated by monitoring serum and liver lipid profile (i. e. cholesterol homeostasis) in mice. To further explore the mechanism of sECC, hepatic cholesterol 7alpha-hydroxylase (CYP7A1) and low density lipoprotein (LDL) receptor expressions in cholesterol homeostasis were analyzed by reverse transcription PCR. After 4 weeks pretreatment, the mice in the high lipid model group showed markedly higher serum and hepatic lipid contents than control group (P< 0.01). Compared with high lipid model group, the increased serum and hepatic lipid contents were markedly attenuated by sECC (200 mg kg(-1)), the serum and hepatic total cholesterol were reduced by 31.5% and 22.7% (P<0.05), respectively. The triglyceride contents of serum and liver were also lowered by 23.0% and 14.4%, respectively. At the same times, serum LDL cholesterol decreased by 53.0% (P<0.01). The mRNA expressions of hepatic CYP7A1 and LDL-receptor were significantly enhanced in the mice administered with sECC (200 mg kg(-1)), whereas those expressions were suppressed by the fat and cholesterol-enriched diet. These data indicate that sECC reduces the atherogenic properties of dietary cholesterol in mice. It is indicated that expression enhancement of hepatic LDL-receptor and cholesterol 7alpha-hydroxylase may be responsible for the hypercholesterolemic effect.
Animals ; Anticholesteremic Agents ; isolation & purification ; pharmacology ; Body Weight ; drug effects ; Cajanus ; chemistry ; Cholesterol ; blood ; metabolism ; Cholesterol 7-alpha-Hydroxylase ; biosynthesis ; genetics ; Cholesterol, LDL ; blood ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Gene Expression Regulation ; Hypercholesterolemia ; blood ; genetics ; metabolism ; pathology ; Liver ; metabolism ; pathology ; Male ; Mice ; Organ Size ; drug effects ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; metabolism ; Receptors, LDL ; biosynthesis ; genetics ; Stilbenes ; isolation & purification ; pharmacology ; Triglycerides ; blood ; metabolism