Study On Toxicity And Mechanism Of "Fuzi" Under Different Extraction Methods Based On PXR-CYP3A Pathway

Background: The secondary roots ofAconitum carmichaeli(Aconitum carmichaeli Debx.),a famous Chinese traditional herbal known as“Fuzi”(FZ),is a commonly found herbal remedy in Jiangyou City,Sichuan Province in China.According to practitioners of traditional Chinese medicine,the roots act upon Yang Yang to save inverses,reinforce fire and help Yang,as well as dispersing colds and relieving pain,and is praised as "the first medicine for recovering Yang and rescuing inverse".Although readily used,the active ingredients in the FZ extracts are known to cause cardiac toxicity,and there are numerous reports of poisoning with aconitum caused by ingestion of raw or incorrectly processed FZ.The toxicity of FZ can be reduced by foaming,bleaching,cooking and processing.Therefore,we studied the metabolism,toxicity and mechanism of FZ under different extraction methods.Purpose: This experiment is based on the toxicity of PXR-CYP3 A pathway to FZ under different extraction methods,and to explore the toxicity mechanism of aconite.This study is divided into three parts,the first part is to explore the relationship between different extracts of FZ on metabolic enzymes;The second part is based on the metabolic enzyme to explore the toxicity of the extract of FZ;The third part is based on the method of metabonomics to explore the cardiac toxicity of FZ extract to rats and its mechanism.Materials and Methods: In this study,traditional decoction and alcohol precipitation with 80% ethanol were used to extract FZ,and the components were identified by Ultra Performance Liquid Chromatography/Quad Time Of Flight Mass Spectrometry(UPLC/Q-TOF-MS).Three diester-diterpene alkaloids(DDAs)type were quantified.The expression of PXR and CYP3A4 in HepG2 cells treated with FZ extracts was detected by real-time Real-time PCR(RT-qPCR),Western blot analysis and reportergene method.The effects of DDAs on PXR and CYP3A4 in HepG2 cells were detected by RT-qPCR and reporter gene method.The effect of monoester-diterpene alkaloids(MDAs)on the expression of PXR and CYP3A4 in HepG2 cells was analyzed by reporter gene method.After 7 days,the protein was extracted from rat liver and the expressions of PXR,CYP3A4,and caspase-3 were detected.Effects of FZ extract on HepG2 and HepG2 cells with stable PXR-CYP3A4.Annexin V-FITC and propidium iodide staining assays were used to measure the cell cytotoxicity of these extracts.The protein was extracted from two kinds of cells treated with FZ extracts,and the protein expression of caspase-3 was detected.Pathological section staining of heart tissue.ELISA was used to measure serum cardiac troponin(CTnI),serum creatine kinase MB isoenzyme and catecholamin.Colorimetric analysis was used to determine serum lactate dehydrogenase.The metabolic expression of small molecules in rats was measured by a metabolomics method.Western blotting was used to detect the expression of phosphoinositide 3-kinase(PI3K),protein kinase B(Akt),mammalian target of rapamycin(mTOR),transforming growth factor-?1(TGF-?1),and caspase-3;Immunohistochemistry was used to detect the expression of CTnI,mTOR,and TGF-?1.Results: The results showed that the extracts of FZ contained more alkaloids,including DDAs and MDAs.Two kinds of FZ extracts and three DDAs could induce the activation of PXR and mediate the expression of CYP3A4,but there were individual differences in dose-dependent manner.In the process of inducing high expression of PXR and CYP3A4,the ethanol extract,mesaconitine and hypaconitine were positively correlated with the dose.The results also showed that MDAs could significantly inhibit the expression of PXR and CYP3A4.And the results showed that the extracts of FZ could activate the overexpression of PXR,CYP3A4 and caspase-3in rat liver.And the ethanol extraction of FZ was higher than that of water extraction.The two extracts of FZ had toxic effects on the two kinds of cells,and the cytotoxicity of the two extracts to HepG2 cells with stable transfer of PXR-CYP3A4 was higher than that of ordinary HepG2 cells.Cardiac index,pathological section and stainingshowed that the toxicity of different extracts of FZ on rat heart was different,and the toxicity of ethanol extract was higher than that of water extract.There were significant differences in metabolites between the groups of rats.The FZ extracts activated PI3 K,Akt,mTOR,TGF-?1 and caspase-3 in rat cardiomyocytes.Conclusion: Because the contents of DDAs and MDAs are different,and the effects and degrees of different alkaloids on PXR and CYP3A4 are also different.Therefore,the difference of activation of PXR and CYP3A4 between the two extracts may be caused by the drug-drug interactions between the different substances in the extract of FZ.In the rat experiment,the extracts of FZ has cytotoxicity,and the ethanol extract activates metabolic enzymes,and the more toxic effect.The results suggest that FZ may be caused by PXR drug transporter after entering the blood circulation of the toxic substances in the FZ.The water and ethanol FZ extracts exert cardiotoxic effects via activating the PI3K/Akt/mTOR signaling pathway to induce cardiomyocyte apoptosis.Follow-up can explore the use of different extraction methods and different concentrations of FZ extracts,detection of pharmacokinetic,mainly focus on the serum concentration of toxic alkaloids,and observe the toxic reaction.The activation effects of PXR and CYP3A4 in liver were detected to explore the internal relationship between them and to guide the clinical use of FZ.