| Objective:The main research purpose of this thesis is to reveal the metabolic behavior of the toxic component arsenic trioxide in arsenic-containing preparations by studying the metabolic fate of the medicinal agent in the arsenic-containing preparations.According to the metabolic behavior of arsenic trioxide in the body,a new mechanism for the toxicity of arsenic-containing preparations was found,which provided clues for exploring the detoxification mechanism in the next step.At the same time,through the study of the compatibility of single drug and realgar in Realgar-Indigo naturalis formula,the possible clues of compatibility and attenuation are preliminarily explained,and it is hoped to provide useful experimental basis for the research and development and clinical safe use of other arsenic-containing compound preparations.Methods:The research in this thesis is divided into three parts.In the first part,in order to study the toxicity of inorganic arsenic,this experiment used CCK8 method to detect the inhibitory effect of arsenic trioxide on the growth of cells of different cell lines.In order to confirm the membrane toxicity and induce apoptosis of arsenic trioxide in leukemia cells,the apoptosis and reactive oxygen species of leukemia cells were detected by flow cytometry.The detection of apoptosis-related indicators was performed by real-time PCR and immunoblotting.Western blot technique was used to detect membrane damage related indicators such as lipid peroxide(LPO)and glutathione(GSH)by ELISA.In the second part,in order to further understand the metabolic pathway of inorganic arsenic,this experiment determined the appropriate cell target and the concentration of the drug,using real-time fluorescent quantitative PCR,Western blot and related techniques to detect arsenic trioxide,tanshinone IIA and indirubin to CYP450 enzyme.The effects of system and As3 MT expression were further explained.The effect of arsenic trioxide,tanshinone IIA and indirubin on the expression of As3 MT was further explained by the method of transfecting cells with small RNA.Meanwhile,the enzyme activity of As3 MT was detected by ELISA.In the third part,in order to grasp more evidence of the metabolic pathway of arsenic in the body,high-performance liquid-phase coupled inductively coupled plasma mass spectrometry(HPLC-ICP/MS)was used to detect the content of soluble arsenic in realgar.And different valence states to study arsenic metabolism behavior.Results:The results show that arsenic trioxide is generally toxic to different cell lines.Although the specific mechanism of cytotoxicity is still unclear,membrane toxicity and apoptosis-induced hidden dangers will always exist during its use.Then,in the study of arsenic trioxide metabolism,it was found that phase I metabolic enzyme is not a key enzyme affecting arsenic metabolism,and phase II metabolic enzyme(As3MT)is an important and key enzyme for arsenic metabolism.Finally,experiments at the animal level further confirmed that realgar can significantly induce As3 MT enzyme activity.The compatibility of realgar with salvia miltiorrhiza and medlar enhances this induction.Highly active As3 MT can accelerate the metabolic clearance of toxic trivalent arsenic.Conclusion:Throughout the full text,this experimental study is the first study of eight kinds of cells,starting from the cell viability test,determining the appropriate cell target and concentration,and using phase II metabolic enzyme(As3MT)as the key point,based on the metabolic point of view.To explore the metabolic behavior of arsenic in the body.The study found two important points.One is that the phase II metabolic enzyme(As3MT)is an important and key enzyme for arsenic metabolism,and As3 MT may be a clue to the detoxification of arsenic.Second,the experimental results of compatibility suggest that when the combination of arsenic-containing preparations is used in combination,how to achieve synergy and attenuation is not only the dose consideration,but also the metabolic regulation in the body should always pay attention.Based on the above,this experimental study expects that arsenic-containing preparations can effectively avoid or reduce the risk of toxicity in clinically safe use,and in the development of other arsenic-containing traditional Chinese medicines,rational compatibility can be achieved based on the perspective of metabolism to achieve synergy and attenuation. |
