Discovery And Mechanism Of Potential Hepatotoxic Components In Zuojin Pill

Purpose Zuojin Pill,composed of Coptis and Evodia in the ratio of 6:1,is one of the classic prescriptions from Zhu Danxi in the Ming Dynasty.Because of its medicinal effects such as regulating gastrointestinal motility,reducing gastric acid secretion,anti-inflammatory and inhibiting ulcers,Zuojin Pill is often used in modern clinics for diseases such as gastrointestinal inflammation,ulcerative gastroenteropathy and reflux esophagitis.In recent years,with the gradual deepening of the pharmacological research and the expanding scope of clinical use,Zuojin Pill plays an important role in anti-cancer,anti-ulcer and gastrointestinal functions,in addition to inflammatory diseases of the digestive system.With the deepening of the safety evaluation and supervision of Zuojin Pill,clinical cases of hemolytic jaundice,diarrhea and neurotoxicity caused by Coptidis have appeared one after another at local and overseas.For the high proportion of Coptisine in Zuojin Pill,the safety problems has attracted great attention from scholars.At present,the main toxic components of Zuojin Pill are concentrated in alkaloids,especially the isoquinoline alkaloids included in Coptis chinensis.In this project,the potential toxicity analysis method of the components was adopted to systematically evaluate the potential toxicity of the components of Zuojin Pill through cell models and animal models.We started from the material basis and network target screening technology,combined with the three levels of"cytotoxicity mechanism-traditional drug safety evaluation-metabolomic targets"to comprehensively analyze the potential toxicity mechanism of coptisine in Zuojin Pill.The research team selected coptisine,an effective component of Zuojin Pill,as the research object,and discussed the cytotoxicity and target organ toxicity in depth.We clarified the toxicity mechanism by combining the classic biological mechanism exploration model with the effective components of traditional Chinese medicine.Methods（1）Different doses of Rhizoma Coptidis water extract and Evodia japonicus water extract were used to treat L02 cells,and the cell viability was examined by CCK-8 assay to determine the main hepatotoxic components in Zuojin Pill.（2）Through flow cytometry and laser confocal microscopy,the effects of cell apoptosis and mitochondrial membrane potential after treatment of L02 cells with different concentrations of coptisine were observed,and the changes in the content of reactive oxygen species（ROS）and calciumions(Ca²⁺)in the cells were also tested.（3）Western-blot was applied to exam the protein expression related to cell apoptosis and endoplasmic reticulum stress.（4）Using traditional methods of evaluating liver toxicity of Chinese medicine（acute toxicity test and sub-acute toxicity test）,liver damage caused by coptisine was detected at the animal level,including body weight,organ coefficient,serum biochemical,and histopathological tests.（5）The non-targeted metabolomics method was used to identify the endogenous metabolites in mouse plasma,and then combined with the multivariate statistical method to screen out the variant metabolites between the different groups and analyze the metabolic pathways to explore the mechanism of liver toxicity caused by coptisine.Results（1）The water extract of Coptis chinensis,the water extract of Evodia and coptisine,the effective component of Zuojin Pill,have obvious cytotoxicity to L02 cells.The IC₅₀ of the water extract of Coptidis is 0.7872mg/ml,the IC₅₀ of the water extract of Evodia is 6.38mg/ml,and the IC₅₀ of Coptis is 26.81μmol/L.（2）Coptisine can induce apoptosis of L02 cells in a dose-dependent manner when L02 is treated at low dose（12.5μmol/L）,medium dose（25μmol/L）,and high dose（50μmol/L）,with significantly reducing of the mitochondrial membrane potential,causing a large amount of ROS accumulation and the broken calcium ion homeostasis in the cell.（3）The western-blot detection results showed that coptisine can dose-dependently induce the expression of proteins Bax,caspase-3,cytochrome c related to apoptosis,and proteins CHOP,ATF-4,and caspase-12related to endoplasmic reticulum stress.（4）The results of the acute toxicity test showed that coptisine did not have obvious acute toxicity.The results of the subacute toxicity test showed that the liver tissue showed different degrees of hepatocellular edema and scattered inflammatory cell foci after coptisine intervened in ICR mice for 28 days.It is preliminary judged that long-term use of coptisine can cause liver damage,which is related to the dose.（5）Metabonomics research results show that the toxic mechanism of coptisine on the liver is closely related to amino acid metabolism,fatty acid metabolism,purine metabolism,platelet activation.Conclusion In the process of screening the hepatotoxic components of Zuojin Pill,it was found for the first time that the hepatotoxicity of the water extract of Coptis was greater than that of the water extract of Evodia japonicas.Among its main components,coptisine has the most hepatotoxicity.In order to further explore the mechanism of coptisine hepatotoxicity,this study included apoptosis,flow cytometry detection（reactive oxygen species,mitochondrial membrane potential）,laser confocal detection（mitochondrial membrane potential,intracellular calcium ion）,and western blot.And then non-targeted metabonomics technology was used to investigate multiple indicators closely related to liver toxicity in vivo and in vitro experiments.The results show that the main manifestation of coptisine hepatotoxicity is that the mitochondrial membrane potential has changed significantly under low-dose conditions,and the endoplasmic reticulum emergency PERK pathway is activated under high-dose conditions;both are involved in the accumulation of intracellular reactive oxygen species（ROS）,which further leads to calciumions（Ca2+）disorder and eventually induces cells to go to apoptosis.It was found that taking coptisine for 4 weeks can cause liver damage in ICR mice through sub-acute toxicity experiments.It was found that coptisine can significantly affect amino acid metabolism and fatty acid metabolism by using non-targeted metabonomics.Meanwhile,whether liver toxicity caused by coptisine is related to its participation in two metabolic processes needs to be further studied.Therefore,we can infer that the disorder of amino acid metabolism and lipid metabolism may be the main reasons for coptisine hepatotoxicity.Coptisine may cause oxidative stress through lipid metabolism disorder,which in turn stimulates the mitochondrial pathway and the endoplasmic reticulum stress pathway to promote cell apoptosis and liver damage.And this suggests that long-term use of Chinese medicine preparations containing coptisine may have potential hepatotoxicity risks.