| Rutaecarpine is one of the main active ingredients of evodia rutaecarpa, some reports have proved it may have some relationship with the deduction of glucose and triglycerides in mice serums. As a regulation factor to some drug metabolic enzymes and transporters, constitutive androstane receptor (CAR) also can affect the glucose and lipid metabolism. Our previous researches indicated that rutaecarpine could activate hCAR, and various studies have shown that rutaecarpine could induce the expression of CYP2B10, a target gene of mCAR, it suggests that rutaecarpine may be the activator of both hCAR and mCAR, the activation of CAR may be involved in the rutaecarpine-mediated gluconeogenesis and lipogenesis.1. The effect of rutaecarpine on gluconeogenesis and lipogenesis and the activation of mCAR by rutaecarpineIn order to elucidate the hypoglycemic and hypolipidemic of rutaecarpine on mice with diabetes, we conducted experiments on DB/DB, OB/OB mice and HFD-STZ induced diabetic mice, each model was divided into three or four groups randomly, then those mice were treated with blank solvent, phenobarbital and different concentrations of rutaecarpine. Weights and food intake were recorded during the treatment; the glucose tolerance and the serum content of glucose and triglyceride (TG) were determined after treating with drugs for a period of time; at last, we detected the expression of genes involved in gluconeogenesis and lipogenesis and CYP2B10 by RT-qPCR and the effect of rutaecarpine on the hepatic steatosis of DB/DB mice by H&E staining. The results showed that rutaecarpine could improve the glucose tolerance, reduce the serum glucose level and suppress the mRNA expression of G6Pase and PEPCK, but the concentration of serum TG and the mRNA expression of SREBP-1 and FAS were not affected by rutaecarpine, the hepatic steatosis was ameliorated by rutaecarpine to some extent. The above results suggested that rutaecarpine could regulate glucose metabolism but had slightly effect on lipid metabolism.The mRNA expression of CYP2B10 was up-regulated by rutaecarpine compared with control in all three kinds of mice, it suggested that rutaecarpine might induce the expression of mCAR. In order to confirm this speculation, first, the hepatocyte primary cells were separated from wild-type (WT) mice and CAR-null mice, and were treated with 0.1% DMSO,250 nmol/L TCPOBOP and different concentrations of rutaecarpine for 48 h respectively. The RT-qPCR was used to quantify the expression of CYP2B10; then, WT and CAR-null mice were divided into three groups respectively and treated with blank solvent, phenobarbital and rutaecarpine through gavage for 3 weeks, the expression of CYP2B10 in liver was determined by RT-qPCR. The results showed that the expression of CYP2B10 was increased in hepatocyte primary cells and liver of WT mice after treated with rutaecarpine, but the expression of CYP2B10 was undetermined in CAR-null mice. The results suggested that rutaecarpine could activate mCAR.2. the relationship between the effect of rutaecarpine on gluconeogenesis and lipogenesis and the activation of CARThe above results suggest that mCAR can be activated by rutaecarpine, and according to the previous researchs, CAR can regulate the of glucose and lipid metabolism. In order to clarify whether CAR plays a role in the hypoglycemic and hypolipidemic of rutaecarpine, the hepatocyte primary cells were separated from WT and CAR-null mice and incubated with hyperlipidemia medium. The cells were treated with 0.1% DMSO,250 nmol/L TCPOBOP and a series concentrations of rutaecarpine respectively for 48 h, then the expression of genes in gluconeogenesis and lipogenesis were determined. The results showed that the expression of G6Pase, PEPCK, SREBP-1 and FAS was decreased significantly after treated with rutaecarpine compared to the DMSO group. On the contrary, the expression of above genes did not exhibit obvious differences among different groups in CAR-null hepatocyte primary cells. Meanwhile, WT and CAR-null mice were divided into three groups respectively and treated with blank solvent, phenobarbital and rutaecarpine by gavage, the glucose tolerance and the mRNA expression of genes involved in gluconeogenesis and lipogenesis were determined after three weeks. After treated with rutaecarpine, the glucose tolerance was improved, the expression of PEPCK was decreased, but this influence was disappeared in CAR-null mice. The above experiments revealed that CAR could regulate the rutaecarpine-mediated glucose metabolism.3. Study on the pharmacokinetics of rutaecarpineAccording to the previous reports, rutaecarpine can induce the expression of CYPs, meanwhile, rutaecarpine also can be metabolized by those CYPs, so we investigated whether the elimination rate would be expedited after treated with repeated-dose rutaecarpine in mice. First, LC-MS/MS method was established to determine the concentration of rutaecarpine in plasma, the results showed a good specificity, the liner range was 0.5-1 ng/ml, the accuracy, the precision, the matrix effect and the extraction recovery were conform to the requirements of biological sample analysis.The plasma samples from mice treated with single-dose and repeated-dose rutaecarpine were determined, the pharmacokinetic parameters were calculated by DAS2.0 program. The AUC0-t was 68.189μg/L·h, Cmax was 16.902μg/L, CLz/F was 146.65 L/h/kg of mice treated with single-dose rutaecarpine, the AUCo-t was 155.462 μg/L·h, Cmax was 20.058μg/L, CLz/F was 64.324 L/h/kg of mice treated with repeated-dose rutaecarpine. From the results, it could be found that the AUCo-t and Cmax of repeated-dose group were more than single-dose group, the CLz/F was less than single-dose group, it suggested that when mice were treated with rutaecarpine for 7 days the plasma concentration of rutaecarpine was increased, the elimination rate was slowed down. |
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