| Objective:There have been many cases of liver injury caused by Psoraleae Fructus during clinical administration,and the female patients were higher than the male ones.Psoralen（Pso）and isopsoralen（Isp）are the main material basis of liver injury caused by Psoraleae Fructus,and also the commonly used marker components for optimizing and evaluating the toxicity reduction process of Psoraleae Fructus at present.At present,the toxicity of Pso and Isp-induced liver injury is not clear,and it is not clear whether there is gender difference in hepatotoxicity induced by Pso and Isp.Toxic and side effects caused by drugs are often closely related to the absorption and distribution of toxic components in vivo and the storage amount in various organs.So far,there has been no report on the distribution process of Pso and Isp in the tissues of male and female rats.In this study,the toxicity of Pso and Isp-induced liver injury and the characteristics of gender differences were systematically studied from the perspective of gender differences,further,the effects of Pso and Isp on bile acid homeostasis and energy metabolism in male and female rats were investigated by targeted metabolomics.Finally,equal doses of Pso and Isp were given to male and female rats by intragastric administration to study the distribution of the main organs in rats and the characteristics of gender differences,so as to provide data support for the follow-up study.Methods:1.144 Wistar rats,half male and half female,were randomly divided into male control group（NC-Male）,female control group（NC-Female）,Male psoralen group（Pso-Male）,Female psoralen group（Pso-Female）,Male isopsoralen group（Isp-Male）and Female isopsoralen group（Isp-Female）,with 24 rats in each group.The rats were given 80mg/kg intragastric administration once a day for 4 weeks,and 6 rats were randomly selected from each group every week for biological sample collection,Samples were taken for 4 consecutive weeks.After the last administration,the rats were weighed,and blood and liver samples were collected.ALT,AST,TG and other biochemical indexes were determined in the serum of the rats.The liver tissues in the same position were stained with hematoxylin-eosin（HE）,and pathological sections were observed.2.The contents of 17 major bile acids and 12 organic acids related to energy metabolism in serum of rats in each group were determined by UPLC-MS after 4w of administration,and multivariate statistical analysis such as OPLS-DA was conducted,in order to investigate the effects of Pso and Isp on bile acid homeostasis and energy metabolism in male and female rats.3.After intragastric administration of 80 mg/kg Pso and Isp solution in male and female rats,the main organs of liver,kidney,heart,spleen and lung were collected at different time points.The drug concentration in biological samples was determined by HPLC,and pharmacokinetic parameters were calculated by DAS 2.0.Results:1.Compared with the NC group,the liver coefficient of rats in the Pso group and the Isp group increased after 1w,2w,3w and 4w of administration,and the liver function indexes and liver sections showed pathological changes.Compared with the Pso group,the increase of liver coefficient and the change degree of serum ALT,TBIL,GSH-Px and other liver function indexes in the Isp group were higher than those in the Pso group,and a large area of liver cell necrosis was also observed in the histomathological sections.Histopathological observation showed that the degree of liver lesions in the Isp group and the Pso group increased with the prolongation of administration week.Histopathological observation showed that the degree of liver lesions in the Isp group and the Pso group increased with the prolongation of administration week.The pathological sections of liver of female mice in the administration group showed more serious inflammatory cell infiltration,vacuolar degeneration of liver cells and cataclysis of nucleus.2.Serum metabolomics results showed that among the 17 kinds of main bile acids targeted,9 kinds of bile acid metabolites were significantly different between the female Pso group and the NC group,and 3 kinds were found in the male group.There were 10 kinds of bile acid metabolites with significant difference between female Isp and NC group,and 8 kinds were found in male.Compared with the NC group,the bile acid content in the Pso and Isp groups showed an overall increasing trend.The changes of the types and contents of bile acids in Isp group were higher than those in Pso group,and the disorder degree of bile acids in female mice was more serious than that in male mice under the same administration conditions.Through the determination and analysis of organic acids related to energy metabolism,it was found that the contents of TCA cycle intermediates such as citric acid,succinic acid and fumarinic acid in serum of rats in Pso and Isp groups were significantly increased.3.The results of tissue distribution showed that the AUC0-tof Pso in male and female rats was（1078.5±60.6）and（960.62±111.77）mg·h/L（P<0.05）,AUC0-tin kidney was（972.46±70.67）and（861.8±43.86）mg·h/L（P<0.01）,AUC0-tof Isp in liver tissue of female rats was（1439.08±84.27）mg·h/L,which was significantly higher than that of male rats（1239.55±26.25）mg·h/L（P<0.01）.Conclusions:1.Both Pso and Isp administration for 1 to 4 weeks can cause substantial liver injury in rats,and the effect of liver injury may be related to oxidative stress dysfunction and lipid metabolism disorder.The hepatotoxicity of rats induced by Isp was higher than that of Pso,and both of them were more likely to cause liver injury in female rats,and the degree of liver injury was correlated with the time of administration.2.Pso and Isp can destroy the balance of bile acid homeostasis in male and female rats,and there are differences in the degree of bile acid disorder between male and female rats.According to the changes of the type and content of different bile acids as the evaluation standard of the degree of liver injury,isopsoralen was more toxic to the liver of rats,and the liver injury of female rats was more serious.In addition,Pso and Isp can also cause the blockage of TCA circulation and the disorder of energy metabolism in rats.3.The content distribution of Pso and Isp in liver and kidney tissues was higher than that in other organs.There were differences in the process of tissue distribution between male and female,mainly reflected in the difference in the content distribution of liver and kidney,and the higher drug exposure in the liver tissue of female mice,which might be the reason why the two kinds of drugs could easily cause liver injury in female mice. |
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