Effects Of Aconitum Alkaloids On CYP450 Enzymes And HERG Channel

FUZI is one of the famous traditional Chinese medicine,which mainly treats the deficiency of vital energy,the burst of yang qi and the exhaustion of pulse.The clinical application of FUZI in traditional Chinese medicine has a history of thousands of years,and it is known as"the primary medicine to restore yang and save reverse".Because of its outstanding effect,Shenfu compatibility（Shenfu decoction）is widely used in the treatment of heart failure and other diseases.However,the adverse reactions of FUZI have limited its clinical application,and frequently reported of poisoning caused by FUZI.Scholars all over the world have studied the toxicity of FUZI.For example,Professor Sun found that the water extract of FUZI at the prescribed dose of pharmacopoeia can obviously enhance the function of isolated heart,showing a certain"dose-effect"relationship.And at high doses,the function of isolated heart showed the toxic and side effects of insufficient myocardial contractility and decreased cardiac output with the increase of dose,showing a certain"dose-toxicity"relationship.Professor Peng Cheng discussed the significant effects of different decoction time on the toxic components and toxic components of FUZI.In the process of decoction of FUZI,aconitum diester diterpenoid alkaloids can be hydrolyzed into monoester diterpenoid alkaloids,and then hydrolysis to form amine alcohols,which is almost non-toxic.However,all these works have not been integrated analysis and in-depth exploration from the perspective of drug interaction in vivo.Our research group established a technical platform for detecting the induction or inhibition of Cytochrome P450（CYP）production by drugs,systematically studied the effect of Shenfu decoction on drug metabolic enzymes and found that aconitine was the substrate of CYP3A and had obvious induction effect.Is this effect mediated by PXR?In this research,we will take FUZI as the research object,put forward the scientific hypothesis that"PXR-CYP3A plays an important role in the transformation of toxic effect relationship of Shenfu decoction",and reveal the role and position of PXR-CYP3A in the transformation of toxic effect relationship of Shenfu decoction.It provides a new target for the integrated analysis of"toxic-effective"of traditional Chinese medicine and establishes a new model for the integrated analysis of integrated nuclear receptor-drug metabolic enzyme expression compound"toxic-effective".CYP,a drug metabolizing enzyme mediated by nuclear receptor pregnane X receptor（PXR）,constitutive androstane receptor（AhR）and constitutive androstane receptor（CAR）,is mainly distributed in the liver.It is the most important drug metabolizing enzyme in vivo and is involved in 70%drug metabolism.In recent years,more and more attention has been paid to the research of CYP.However,as the most ideal cell model in the study of CYP in vitro,the isolation and culture of primary human hepatocytes is time-consuming and costly.The passage hepatocytes are not only easy to culture,low cost,but also similar to the primary cells in characteristics.However,there are many kinds of cells in the liver,and the selection of suitable cell models suitable for different metabolic enzyme isoforms has been neglected in the traditional metabolic enzyme experiments.We selected three typical human subcultured hepatocytes representing different liver states,and determined the cell types suitable for the follow-up study on PXR-CYP3A4 through the comparison of the three hepatocyte lines.At the same time,we showed the gene and protein expression profiles of several CYP isoforms in LX-2 of human passage hepatocytes and provided suitable cell types for the study of different CYP isoforms.The main clinical manifestations of FUZI and its main components are cardiotoxicity,but the specific components and related mechanisms of action are not reported.Ion channel is a passive transport pathway of various inorganic ions across the membrane,which is closely related to cardiac toxicity.The human Ether-à-go-go-Related Gene（hERG）channel detection is considered to be the gold standard for the evaluation of drug cardiotoxicity.The effects of aconitine,the main toxic monomer of FUZI water extract,on hERG channel were detected by molecular biology combined with patch clamp technique.At the same time,the pathway of aconitine regulating hERG channel was explored,and the effect of aconitine on miRNA was preliminarily determined so as to regulate hERG channel,which laid a solid foundation for further study of the mechanism.The main research contents are summarized as follows:1.Comparison of induced CYP expression in three permanent human hepatocytes.First of all,we selected three different sources of human passage hepatocytes.The difference morphology of these cells suggests that there may be differences in the expression of metabolic enzymes.Select five kinds of CYP enzyme inducers to induce seven main CYP isoforms,and detect the changes of three kinds of cell activity after administration to verify the rationality of the range of cell induction concentration.Then human hepatic stellate cell LX-2 was used as the research object,and RT-qPCR technique was used to detect the changes of CYP isoforms gene expression after induction.In addition,Western blot technique was used to detect and compare the protein expression of CYP isoforms in three kinds of hepatocytes before and after induction.Finally,fluorescence staining was used to detect the changes of CYP3A4enzyme activity in the three kinds of cells after induction.The results showed that there were significant differences in the expression abundance of different CYP enzyme isoforms among the three passage hepatocytes.It is suggested that the optimal cell line should be considered in the experiment of CYP enzyme related in vitro.At the same time,this is the first time to report the expression of the main CYP enzyme isoforms in LX-2 cells.The enzyme activity test suggests that the passage cells may not be an ideal cell model for enzyme activity test.2.Cytotoxicity induced by water extracts from FUZI.L02 cells were selected for the highest protein expression of CYP3A4 and treated with different concentrations of FUZI water extracts for 24 hours.Compared with the control group,the change of cell activity was not high,but the expression of PXR and CYP3A4protein decreased significantly after treatment with FUZI water extracts.At the same time,the FUZI water extracts metabolized by L02 cells was collected and centrifuged to remove the precipitation.Another group of FUZI water extracts was prepared and collected after being placed at 37℃for 24 hours in 5%CO₂ environment.Human passage cardiomyocytes AC16 were selected and treated with FUZI water extracts for24 hours to compare the survival rate between the two groups.The results showed that the FUZI water extracts without metabolism in L02 cells showed more toxicity to AC16cells,also inhibited the expression of hERG.It is suggested that PXR-CYP3A4 plays an important role in the detoxification of FUZI.3.Aconitum aconitum alkaloids inhibited hERG channel.A hypothesis has be thrown up that the mechanism of cardiomyocyte toxicity of FUZI water extract may be due to the inhibition of hERG channel by the main toxic monomer of FUZI water extract.Then we selected three main bisfat aconitine alkaloids（hconitine,hypaconitine and hesaconitine）from the FUZI water extract to treat AC16 cells for 24hours.Detection of hERG channel protein gene expression by Western blot,RT-qPCR and laser confocal microscopy.The results showed that all three aconitine alkaloids could inhibit the expression of hERG protein and gene in AC16 cells,among which hypaconitine had the strongest inhibitory effect.At the same time,patch clamp test was used to detect the changes of hERG channel opening and closing before and after induction of those three alkaloids.The results showed that,corresponding to the results of protein gene experiment,the three alkaloids could inhibit the opening of hERG channel,among which hypaconitine had the strongest inhibitory effect.Then we detected the effects of different concentrations of hypoaconitine on hERG channels.Protein gene and patch clamp assay showed that the inhibitory effect of hypoaconitine on hERG channel enhanced with the increase of concentration.IC50 was 43.07μM.The strong inhibitory effect of hypoaconitine on hERG channel suggests that as the highest content of dilipid alkaloid in FUZI water extract,hypoaconitine should be paid close attention to in drug preparation and clinical use.4.Hypaconitine inhibits hERG channels by regulating the expression of related miRNA.The inhibitory mechanism of hypoaconitine on hERG channel may be related to miRNA.First of all,we summarized all the miRNA involved in regulating the hERG channel which eight kinds of related miRNA can inhibit hERG channel in the literature.We detected the expression of 8 kinds of miRNA in AC16 cells before and after hypoaconitine treatment.The results showed that the expression of 8 kinds of miRNA changed in varying degrees,and the increase of miR-134 expression was the most obvious.In further experiments,we used miRNA inhibitor to inhibit the expression of miR-134,and then continued to treat the cells with hypoaconitine.Western blot,RT-qPCR and laser confocal assay showed that hypoaconitine inhibited the expression of hERG protein gene,suggesting that hypoaconitine could inhibit the opening of hERG channel and the expression of protein gene by regulating the expression of related miRNA.