Experimental Study On The Toxic Effects And Attenution Mechanism Of Saponins Aryl Hydrocarbon Receptor Agonists

This paper focuses on the two part of the research work of AhR. The one is preliminary study on hepatotoxicity induced by dioscin and the association with AhR/CYP1 A, the another is further study on the reversal effect of ginsenoside Rb1 on the toxicity of AhR agonists and the pharmacological mechanism based on our laboratory found that ginsenosides are partical AhR ligands.Dioscin has a wide range of biological effects and broad application prospects. However the studies concerning the toxicology and mechanism of dioscin is small. This article is to study the hepatotoxicity of dioscin and the effect of dioscin treatment on expression of aryl hydrocarbon receptor(AhR) mRNA and CYP1 A mRNA and protein in HepG2 cells in vitro.Dioscin 0.5~32μM exposed to HepG2 cells for 12 h,cell viability was examined by CCK-8 assay and the release rate of lactate dehydrogenase(LDH) was to evaluate cell membrane damage. HepG2 cells morphologic changes were quantified by inverted Microscope,and the effect on production of reactive oxygen species(ROS) was detected by flow cytometry. The mRNA expression of CYP1 A and AhR was evaluated by RT-RCR. The protein expression of CYP1A1 was detected by western blot.The cell viability was significantly inhibited after HepG2 cells were exposed to dioscin 4~32μM. Compared with the control,the LDH release rate and ROS were significantly increased. The expression of CYP1 A and AhR m RNA was increased. The expression of CYP1 A 1 protein was increased after dioscin treatment,and resveratrol,an AhR antagonist,could downregulate the expression of CYP1A1. It follows that large doses dioscin has potential hepatotoxicity. The possible mechanism may be dioscin can active aryl hydrocarbon receptor(AhR) and induce the expression of CYP1 A.In order to further investigate the cytotoxicity of AhR agonists, especially on the heart. We study using doxorubicin, an AhR agonists, for cardiac toxicity and to prove part AhR agonists such as ginsenosides have intervention effect on the cardiac toxicity. Doxorubicin(DOX) is a highly effective chemotherapeutic agent, however, the dose-dependent cardiotoxicity significantly limited its’ clinical application.Due to the polycyclic aromatic structure of DOX, it has been demonstrated that DOX can activate the aryl hydrocarbon receptor(AhR) and induce the expression of CYP1A1 in ARVM and H9C2 cells. Thus, the cardiotoxicity of DOX may be associated with its activation of AhR. The recent studies further demonstrated that ginsenoside Rb1 is one of the AhR ligand. Therefore, ginsenoside Rb1 may be a agonist or(partial) antagonists of the aryl hydrocarbon receptor, which can compete with other AhR agonist such as DOX for binding to the receptor.The present study was performed to investigate whether Rb1 can prevent DOX-induced cardiac toxicity by the change of cell apoptosis index and the role of AhR. H9C2 cells were treated with Rb1 at concentration of 50,100, 200, 400μ M or vehicle for 6 h, and then exposed to 1μ M DOX for indicated time. CCK-8 assay was to examine the effects of Rb1 on cells viability after DOX treatmen. ELISA assay was to determine the production of DNA fragmentation. Hoechst 33258 staining was used to identify the morphological features of apoptosis. And the expression of apoptosis related genes was determined on enzyme activity and protein levels. AhR, CYP1A1 protein and mRNA expression were quantified with western blot and real-time PCR analyses.The results showed that ginsenoside Rb1 significantly reduced cell apoptosis in cells exposed to DOX in a dose-dependent manner as evidenced by cytotoxic activity assay. The DNA fragmentation assay also proved the effect of Rb1 against DOX-induced apoptosis and Rb1 at 200 μM have the best effect. Rb1 attenuated DOX-induced cardiomyocytes apoptosis and reduced caspase-3 and caspase-8, but not caspase-9 activity in DOX-treated H9C2 cells. Meanwhile, pretreatment with Rb1 can decrease the expression of caspase-3 and PARP in the protein levels and no effects on cytochrome c, Bax and Bcl-2 in DOX-stimulated cells. Rb1 markedly decreased the CYP1A1 and CYP1A2 expression induced by DOX. Furthermore, pretreatment with AhR siRNA and AhR antagonist CH-223191, can significantly inhibit the ability of Rb1 to decrease the induction of CYP1 A as well as the caspase-3 protein level in the condition of DOX. These findings indicate that AhR plays an important role in the protection of ginsenoside Rb1 against DOX-triggered H9C2 cells apoptosis. This experiment provides a new possibility to expand the scope of clinical application of DOX and expounds the cardiovascular protective effect of ginsenoside Rb1 from a new angle.In summary, AhR as the target and dioscin and ginsenoside Rb1 as the main object of this paper, which by the research of dioscin hepatotoxicity and its possible mechanism and the myocardial protective effect of ginsenoside Rb1 on DOX, points out that evaluating whether a compound is a ligand or agonist of AhR helps to understand its toxicological properties and inhibition of AhR is also a potential intervention, opens up a new way of thinking for us to study the toxicity mechanism of toxic components in other traditional Chinese medicine and compatibility attenuation mechanisms.