Mechanism Of Evodia Alkaloids On Glucose And Lipid Metabolism Through Activating Constitutive Androstane Receptor(CAR)

The traditional Chinese medicine evodia rutaecarpa (Juss. Benth), originally recorded in Sheng Nong’s Herbal Classic, has been widely studied in the past decades. The major active ingredients in evodia rutaecarpa are limonoids and alkaloids. Rutaecarpine, along with evodiamine and dehydroevodiamine, is the three major alkaloids in evodia rutaecarpine. Various studies have indicated that these three alkaloids have beneficial pharmacological properties such as anticancer, antithrombotic, anti-inflammatory and analgesic activities. A recent study has shown the anti-obesity effects of rutaecarpine and its two analogues were mediated by AMPK activation and UPR suppression, while there are still other pathways mediated the anti-obesity and anti-diabetes effects of evodiamine rutaecarpa alkaloids.Diabetes induced by metabolic disorder still lack perfect therapeutic targets, however, an increasing body of evidence suggests that CAR has endobiotic functions that impact glucose and lipid metabolism, as well as the pathogenesis of metabolic diseases, make it a potential therapeutic target in managing metabolic diseases. Hence, this study firstly focused on elucidating the relationship between evodia rutaecarpa alkaloids and human constitutive androstane receptor (hCAR) activation. On this basis, we further studied the downregulation effects of evodia rutaecarpa alkaloids on lipogenesis and gluconeogenesis and make it clear whether hCAR activation is involved in these changes.1. Crosstalk between evodia rutaecarpa and hCAR activationIn order to elucidate whether alkaloids from evodia rutaecarpa can activate hCAR, we conducted our experiments on HepG2with the gene report assay, real-time PCR, western blotting and other methods. Toxicity of the three alkoids on HepG2cells were measured by MTT assay to make sure the appropriate concentrations in later experiments. Luciferase reporter gene results showed that rutaecarpine and evodiamine can significantly increase the CYP2B6level by activating hCAR. Luciferase activity were both about or over70%when compared with the positive group treated with6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime (CITCO), indicating that the two alkaloids may be strong activators of hCAR. However, no change were observed in cells treated with dehydroevodiamine. The next experiments measured the mRNA and protein level of CYP2B6, in accordance with the luciferase report gene results, CYP2B6expression can be greatly up-regulated by rutaecarpine and evodiamine other than dehydroevodiamine, and this induction can be reversed by hCAR antagonists1-(2-Chlorophenylmethylpropyl)-3-isoquinoline-(PK11125) and3β-Hy-droxy-5α-androstane. All the above experiments revealed that rutaecarpine and evodiamine can induce CYP2B6through activating hCAR. Also, the results of chromatin Immunoprecipitation suggested that activated hCAR bound to the CYP2B6gene promoter in response to rutaecarpine compared with the negative control from which hCAR shows weak binding to the promoter.2. Hypoglycemic and hypolipidemic effects of rutaecarpine and evodiamineHepG2cells were treated with bovine serum albumin (BSA) and oleic acid (OA) to make the hyperlipidemia cell, then oil red staining and triglyceride assay were used to measure the lipid accumulation and triglyceride contents in cells. From the results, we can see that hyperlipidemia cells exhibited obvious lipid accumulation and increasing triglyceride contents. When treated the hyperlipidemia cells with rutaecarpine and evodiamine, lipid accumulation can be ameliorated and triglyceride content went down, the differences are statistically significant. Real-time PCR and western blotting further measured the mRNA and protein levels of gluconeogenesis genes (PEPCK and G6Pase) and lipogenesis genes (FAS, SREBP1and SCD1), According to the results, all these five genes can be down-regulated in different extents when treated with rutaecarpine and evodiamine. The above results suggested that rutaecarpine and evodiamine exhibit hypoglycemic and hypolipidemic effects on HepG2cells. 3. hCAR involved in the hypoglycemic and hypolipidemic effects of rutaecarpine and evodiamineTo make clear whether hCAR is involved in the hypoglycemic and hypolipidemic effects of rutaecarpine and evodiamine, we treated cells with two alkaloids together with two hCAR inhibtors PK11195and3β-Hydroxy-5a-androstane, then real-time PCR were used to examine the mRNA levels of PEPCK, G6Pase, FAS, SREBP1and SCD1. The results showed that mRNA level of the five genes were reversed into different extents with two inhibitors and the differences are statistically significant. Also, PK11195showed stronger reverse ability than3β-Hydroxy-5α-androstane, suggesting the PK11195has higher activity when inhibiting hCAR. In conclusion, the above experiments confirmed that down-regulation effects of rutaecarpine and evodiamine on lipogenesis and gluconeogenesis genes were tightly related to the activation of hCAR, revealing the possibility of hCAR to be potential therapeutic targets in managing metabolic sydromes.