Toxic Mechanism Of Emodin Based On Age-related Difference In The Expression Of MRP In Liver And Kidney Of Mouse

Objective:In this project,the mechanism of potential toxicity of emodin to animals was studied by using multidrug resistance protein（MRP）as the breakthrough point and MRP function,internal oxidative stress state and mitochondrial functional pathway as the main line.By comparing the toxicity of different doses of emodin to the liver and kidney of young and aged mice,the microscopic toxicological mechanism of the dose and age difference of emodin were further elucidated.Methods:1.Establishment of aging mouse model:125mg/kg/d D-galactose was injected into 25-week-old mice by subcutaneous for 60 d.Flow cytometry was used to detect the CD⁺₂₈ and CD₄⁺/CD₈⁺ratio of thymic T cell subsets in mice to test the success of establishment of the model.2.Effects of long-term administration of emodin on liver and kidney toxicity in mice:Five-week-old mice were selected as youth group and D-galactose model mice as old group.They were divided into blank group,low dose emodin group and high dose emodin group.The dosage of emodin in low dose group was 0.8g/kg,and that in high dose group was 1.6g/kg.The dose was given intragastrically for 75 days.1)Hepatotoxicity:In order to evaluate the toxicity of emodin in different doses to mice liver,serum AST,ALT activity was detected by kit,the morphology of mouse liver cells was evaluated by hematoxylin-eosin（HE）staining,and the expression of caspase3 in liver was detected by immunohistochemical method.2)Nephrotoxicity:In order to evaluate the toxicity of emodin in different doses to mice kidney,the serum Cr,BUN activity was detected by kit,the morphology of mouse kidney cells was evaluated by HE staining,and the expression of TGF-β1 and caspase3 in kidney was detected by immunohistochemical method.3)The effect of emodin on oxidative stress in mice:The relationship between emodin toxicity and oxidative stress was revealed by detecting serum MDA content and SOD activity,renal homogenate GSH-Px activity and GSH/GSSG ratio,liver mitochondrial membrane potential and mitochondrial sensitivity to Ca²⁺induced swelling.4)The effect of emodin on the expression of MRP protein in mouse liver:The expression of MRP1 and MRP2 proteins was detected by westernbolt,and the relationship between emodin and MRP family was revealed.Results:1.Establishment of aging mouse model:The ratio of CD₄⁺/CD₈⁺and CD⁺₂₈ in thymic T cells of D-galactose group was significantly lower than that of control group,indicating that the aging model was established successfully.2.The liver and kidney toxicity of long-term administration of emodin in mice1)Hepatotoxicity:HE staining showed that there were different degrees of cell swelling and steatosis in the liver of young and elderly mice,and the pathological score showed that the more serious the liver injury was with the increase of dose.Compared with the youth control group,the activity of AST in the youth emodin groups increased,and the increase was more significant in the high dose group.Compared with the old group induced by D-galactose,there was a significant increase in the activity of AST in the high dose group in the old groups.The number of caspase3 in the youth and old emodin groups was higher than that in the youth and old control groups.2)Nephrotoxicity:HE staining showed that the kidneys of young and elderly mice showed different degrees of cell swelling,protein tube type,hyperemia and proliferation of lymphocytes.The pathological score showed that the renal injury was more serious with the increase of dose.Compared with the youth control group,the contents of Cr and BUN increased significantly in the youth emodin groups;The optical density of TGF-β1 increased in the youth emodin groups,and there was statistical difference in the high dose group.The number of caspase3 positive cells in the youth emodin groups was significantly higher than that in the youth control group.Compared with the old group induced by D-galactose,the contents of Cr and BUN in the old emodin groups showed an increasing trend,and the high dose group had statistical significance.The optical density of TGF-β1 in the old emodin groups showed an increasing trend,and there was a statistical difference among these groups.The number of caspase3 positive cells increased significantly in the low dose group,but there was no significant change in the high dose group.3)The effect of emodin on oxidative stress in mice:Compared with the youth control group,the activity of SOD in the youth emodin groups were significantly decreased;the liver GSH-Px activity and GSH/GSSG ratio were decreased to some extent;The activity of GSH-Px in kidney showed a decreasing trend,and the ratio of GSH/GSSG in the high dose group was significantly lower than that in the youth control group.The mitochondrial membrane potential of liver tissue decreased significantly in the high dose group.Compared with the old control group,the activity of SOD in the old group showed a decreasing trend;The GSH-Px activity and GSH/GSSG ratio of the emodin high dose group increased.The activity of GSH-Px in kidney decreased,but the ratio of GSH/GSSG did not change significantly.The mitochondrial membrane potential of liver tissue showed a decreasing trend,and there was a significant decrease in the high dose group.The sensitivity of mitochondria to Ca²⁺induced swelling showed that there was no significant difference in the ratio of FSC/SSC among these groups.4)The effect of emodin on the expression of MRP protein in liver of mice:Emodin at different concentrations could down-regulate the expression of MRP1and MRP2 protein in the liver of youth emodin mice,And the high dose group was significantly lower than that of the youth control group;Emodin could up-regulate the expression of MRP1 and MRP2 protein in the liver of the old emodin mice,and the high dose group was significantly up-regulated compared with the old control group.Conclusion:The effect of emodin on the hepatorenal toxicity of young and old mice is different,as follows:1.For young mice:1)Emodin induced oxidative stress injury in mice,resulting in imbalance in the function of GSH antioxidant system.Through destroying the function of cell mitochondria,activating caspase apoptosis pathway leads to cell apoptosis,and finally shows the liver and kidney toxicity of emodin.2)Emodin may induce oxidative stress in mice by down-regulating the expression of MRP1 and MRP2,which is the promoter of emodin toxicity.2.For old mice:In the early stage of drug use,emodin induce the reduction of MRP expression,and cause imbalance of GSH antioxidant system in elderly patients with weak expression of MRP.This induces excessive oxidative stress and triggers the initiation of defense mechanisms after long-term high-dose administration.Finally,the expression of MRP was enhanced.