The Effects Of Traditional Chinese Medicine On The Antidiabetic Pathway Of Constitutive Androstane Recentor (CAR)and Its Mechanism

Diseases caused by metabolic disorder lack perfect therapeutic targets yet. Looking for therapeutic targets containing both anti-obesity and anti-diabetes effects, was extremely important for the development of prominent effect and low toxicity drugs for diseases induced by metabolic disorder. However, an increasing body of evidence suggests that nuclear receptor CAR has functions that influence glucose and lipid metabolism, and the pathogenesis of metabolic diseases, make CAR a potential therapeutic target in treating metabolic diseases. It has been long trying to find drugs that simultaneous hypolipidemic and hypoglycemic from traditional Chinese medicines (TCMs) and natural products. Indeed, some have been found that contain these effects, such as cinnamon, berberine, purslane, senecio and so on. However, most studies tend to focus on the level of efficacy studies only, and lack in-depth study for its down-regulation effects. This greatly limited the further development of these drugs. Hence, screening CAR agonists that mediate glucose/lipid metabolism from TCMs, and further study on the down-regulation effects are of great significance. This study firstly screening CAR agonists from TCMs by reporter gene assay that hCAR3 mediated CYP2B6 induction. Results showed that evodiamine and rutaecarpine markedly increase the CYP2B6 level via activating hCAR. The next experiments measured the mRNA level and DNA level of chromatin, in line with the luciferase report gene results, CYP2B6 expression could be greatly up-regulated by evodiamine and rutaecarpine. Furthermore, we studied the down-regulation effects of evodiamine and rutaecarpine on gluconeogenesis and make sure whether hCAR activation is incorporated in these changes.1. Screening CAR agonists from active constituents in TCM by reporter gene assayFirstly, we selected 14 kinds of hypoglycemic traditional Chinese medicines and obtained the alcohol and water extracts by crude extraction. Toxicity of the TCM extracts to HepG2 cells were confirmed by MTT assay to guarantee the appropriate concentrations in follow-up experiments. The hCAR mediated CYP2B6 and hPXR mediated CYP3A4 reporter gene assay was utilized to screen hCAR/hPXR agonists from the TCM extracts. Luciferase activity was in excess of 30% when compared to the positive group treated with CITCO, or rifampicin indicating that may be activator of hCAR or hPXR. Most kinds of TCM extracts were found to activate hPXR, while only the alcohol extracts, Euonymus alatus, Apocynum venetum, Oroxylum indicum, and the water extracts, Artemisia capillaris Thunb, Bupleurum chinense DC were found to activate hCAR. Then we investigated part of active constituents of these five TCM that may activate hCAR and other active constituents. Luciferase reporter gene results showed that rutin, wilfordine, apigenin, bupleurum saponins A, daidzein, oridonin, bergenin, rutaecarpine and evodiamine can increase the CYP2B6 level by activating hCAR, indicating that they may be activators of hCAR. And the activation of hCAR were dose-dependent.2. Confirmation of the TCM constituents activated CARThe LS174T cells were treated with TCM constituents and the CYP2B6 gene expression was detected with real-time PCR. In keeping with the luciferase report gene results, the expression of CYP2B6 can be up-regulated by evodiamine and rutaecarpine than the other active constituents. And experiments measured the mRNAand protein level of CYP2B6 on HepG2 cells indicated that CYP2B6 expression also be up-regulated, and this induction can be reversed by hCAR antagonists. In addition, the mCAR mediated reporter gene results showed that rutaecarpine may be the activator of mCAR. While, no change was observed that treated with evodiamine. Also, the results of chromatin immunoprecipitation indicated that activated hCAR bound to the gene promoter of CYP2B6 in response to rutaecarpine and evodiamine compared to the negative control which hCAR shows very weak binding to the promoter. All the above results revealed that rutaecarpine and evodiamine could induce CYP2B6 gene expression through activating hCAR.3. CAR involved in the hypoglycemic effect of rutaecarpine and evodiamine and the relevant mechanism were studiedVarious studies have indicated that rutaecarpine and evodiamine have advantageous pharmacological properties such as anti-inflammatory, antithrombotic and analgesic activities, however, whether there are better pharmacodynamic properties have no relevant information. And research related to the mechanism has been reported rarely. This study mainly focused on gluconeogenesis and explored the down-regulation effects of evodiamine and rutaecarpine on anti-diabetes effect, making it clear whether these changes are induced by hCAR activation. The human FOXO1, PGClα, HNF4a code sequences, amplified from human liver or kidney cDNA, were inserted into pcDNA3.1 (+) empty vector to construct expression plasmid pcDNA3.1(+)-FOXO1、pcDNA3.1(+)-PGClα and pcDNA3.1(+)-HNF4α. The promoters containing IRS and DR1 binding sites, amplified from human genomic DNA, were inserted into pGL3-promoter empty vector to construct the reporter gene plasmid pGL3-hPCK-Luc and pGL3-G6Pase-Luc. The expression plasmids and reporter gene plasmids were co-transfected into HepG2 cells. The empty vectors were used to investigate the interference of transcriptional factors in blank cells. The hCAR3-FOXO1/hCAR3-PGCla-HNF4α mediated PEPCK/G6Pase reporter gene assay was established successfully and could be utilized to explore the influence of FOXO1 and HNF4a binding to the promoter of PEPCK/G6Pase after hCAR3 expression plasmid transfected. When treated the HepG2 cells with rutaecarpine and evodiamine, the promoter activation of PEPCK/G6Pase were declined after transfecting FOXO1 or PGCla-HNF4a expression plasmid. And the suppression effects were more significant with hCAR3 expression plasmid co-transfected. Real-time PCR further measured the mRNA levels of gluconeogenesis genes (PEPCK and G6Pase), on the basis of the results, PEPCK and G6Pase can be down-regulated in different degrees when treated with evodiamine and rutaecarpine. The results of chromatin immunoprecipitation indicated that activated hCAR by rutaecarpine and evodiamine suppress HNF4a binding to the DR1 binding site on PEPCK and G6Pase, also suppress FOXO1 binding to the IRS binding site on PEPCK and G6Pase. As a result, the gene expression of PEPCK and G6Pase were decreased. The above results suggested that the influence of HNF4a/FOXO1 binding to DRl/IRS were tightly related to the activation of hCAR when treated with rutaecarpine and evodiamine. This conclusion provide a theoretical basis for the in-depth development of rutaecarpine and evodiamine, uncovering the possibility of hCAR be the potential therapeutic target in treating metabolic sydromes.