Hepatotoxicity And Nephrotoxicity Mechanism Of Aloe-emodin Based On MRP-oxidative Stress In Aging Mice

Objective:In this experiment,aloe-emodin was used as the research object,and D-gal solution was used to establish aging model mice,and mechanisms of hepatotoxicity and nephrotoxicity in aged mice were investigated.From the"dose-age"level,hepatotoxicity and nephrotoxicity mechanism of aloe-emodin based on MRP,oxidative stress,mitochondrial function and apoptosis in aging mice.Provide scientific basis for the clinical safe use of traditional Chinese medicine containing terpenoids.Methods:There were 30 6-week-old mice.After 20 weeks,the mice were injected subcutaneously with 2.5%D-galactose solution,0.05 mL/10 g,for 60 days.Successfully established aging model mice were randomly divided into 3 groups according to body weight:old control group（D-gal）,old aloe-emodin low dose group（D-gal+AE-L）,old aloe-emodin high dose group（D-gal+AE-H）.Thirty SPF-class6-week-old mice were randomly divided into three groups:young control group（Control）,young aloe-emodin low dose group（AE-L）,and young aloe-emodin high dose group（AE-H）.Young and old low dose mice were given aloe-emodin 0.8 g/kg,young and old high dose mice were given aloe-emodin 1.6 g/kg.The sodium carboxymethy cellulose solution was administered once a day in the morning and evening for 75 days.1.Model identification:The flow cytometry method was used to detect the changes of CD₄⁺/CD₈⁺and CD⁺₂₈ proteins on the surface of thymic T cells.2.Hepatotoxicity and nephrotoxicity test:Biochemical method was used to detect the contents of alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,creatinine（SCr）and urea nitrogen（BUN）in the blood of mice,and HE staining was used to detect liver and kidney damage.3.The effect of aloe-emodin on the expression of MRP protein in aged mice:Western blot was used to detect the expression of MRP1 and MRP2 protein in liver tissue.4.Effect of aloe-emodin on oxidative stress in aged mice:biochemical method for detecting superoxide dismutase（SOD）activity and malondialdehyde（MDA）in blood,and reducing glutathione in liver and kidney The ratio of oxidized glutathione（GSH/GSSG）and the activity of glutathione peroxidase（GSH-Px）;5.Effects of aloe-emodin on mitochondrial function and apoptosis in aged mice:Flow cytometry was used to detect liver mitochondrial membrane potential（MMP）and liver mitochondrial Ca²⁺swelling,and immunohistochemistry was used to detect liver aspartic protease-expression of 3（caspase-3）and renal caspase-3 and TGF-β₁.Results:1.Compared with the young group,the expression of CD₄⁺and CD₈⁺CD₄⁺/CD₈⁺in the thymic T cells of the aged group was significantly decreased（P<0.05）,and the expression of CD⁺₂₈ was aslo decreased（P<0.05）.2.Compared with the young control group,the spleen,thymus,liver,heart and kidney index of the low dose and high dose aloe-emodin group were significantly lower（P<0.05）.Compared with the old control group,the aloe-emodin low dose and high dose group.The spleen,thymus,heart and kidney index of the mice were also significantly decreased（P<0.05）,and the liver index of the high dose group was increased（P<0.05）.1)Hepatotoxicity:Compared with the young and old control group,the levels of AST in the low dose and high dose groups of was significantly increased（P<0.05）;compared with the young aloe emodin low dose,the change in the old aloe emodin low dose ALT and AST level were increased by 0.95 and 1.2 times.Compared with the young people aloe-emodin high dose,the change in the old aloe-emodin high dose ALT and AST level were increased by 1.3 and 1.1 times,respectively.Liver pathological conditions:Compared with the young control group,the liver cells of the low and high dose group showed different degrees of steatosis and granular degeneration.The pathological scores of the high dose group were different（P<0.05）;compared with the old control group,the hepatocytes of the low dose group showed moderate steatosis,mild granular degeneration,and hyperkedosis in the nucleus;high dose mice had severe cytoplasmic fat lesions,nucleus staining,and severe particle degeneration.2)Nephrotoxicity:Compared with the young control group,the content of SCr in the low dose group of aloe-emodin increased（P<0.05）,and the content of BUN and SCr in the high-dose group increased（P<0.05）.Compared with the old control group,the level of BUN and SCr in old aloe-emodin low dose group were increased（P<0.05）,and the levels of BUN in high dose group was increased（P<0.05）.Compared with young aloe-emodin low dose group,the change of BUN and SCr levels in young aloe-emodin low dose group increased by 1.2 and 1.0 times;compared with the old aloe-emodin high dose group,the change of BUN and SCr in the high dose group in old aloe-emodin was increased by 1.1 and 0.7 times.kidney pathology conditions:Compared with the young control group,the renal cells of the low and high dose groups showed different degrees of cell degeneration and interstitial capillary congestion,and the pathological scores were statistically significant（P<0.05）;In the low and high dose groups,the renal cells showed different degrees of granular degeneration,renal interstitial capillary congestion and lymphocytic infiltration.The pathological scores of the high dose group were statistically significant（P<0.05）.3.Compared with the young and old control group,the expression of MRP1 and MRP2 protein in the low and high dose groups decreased,but no statistical significance（P>0.05）.But the expression of MRP1 protein was increased in the elderly high-dose group（P<0.05）.4.Compared with the young control group,the SOD activity in the low and high dose groups was significantly decreased（P<0.05）,and the MDA content was significantly increased（P<0.01）.Compared with the old control group,the SOD activity was significantly lower in the low and high dose groups（P<0.05）,the MDA content in the low-dose group was significantly increased（P<0.05）.Compared with the young aloe-emodin low dose group,the low dose SOD activity of the old aloe-emodin was reduced by 1 time,the MDA content was increased by 0.9 times,and the young aloe rhubarb In the low dose group,the high dose SOD activity of the old aloe-emodin was reduced by 1 time,and the MDA content was increased by 0.8 times.1)Liver:Compared with the young control group,the GSH content decreased,the GSSG content increased,and the GSH/GSSG ratio decreased in the low and high dose groups,but there was no statistical significance（P>0.05）,and the high dose GSH-Px activity was significantly decreased（P<Compared with the old control group,the GSH content in the high dose group was significantly lower（P<0.05）,the GSSG content was significantly increased（P<0.01）,the GSH/GSSG ratio was significantly decreased（P<0.01）,and the GSH-Px content was significantly higher.Reduced（P<0.05）;2）Kidney:Compared with the young control group,the GSH/GSSG ratio in the high dose group of aloe-emodin was significantly lower,and the high dose GSH-Px activity was significantly lower（P<0.01）.Compared with the elderly control group,the low and high dose GSH/The proportion of GSSG decreased significantly（P<0.01）,and the activity of GSH-Px in high dose group decreased significantly（P<0.05）.5.1)Liver:Compared with the young and old control groups,the mitochondrial membrane potential of the mice in the low and high dose groups decreased,and the mitochondria induced Ca²⁺swelling was sensitive,but the data were not statistically significant（P>0.05）.Compared with the young and elderly control group,the expression of caspase-3was increased in the low and high dose groups（P<0.05）.Compared with the young aloe emodin low dose group,the expression of caspase-3 in the low dose group of aged aloe-emodin was increased by 0.9.Compared with the high dose group of young aloe-emodin,the expression of caspase-3 in the high dose group of aloe-emodin was increased by 1.7 times.2)Kidney:Compared with the young control group,the expression of caspase-3and TGF-β₁ was increased in the high dose group of aloe-emodin（P<0.05）.Compared with the old control group,the expression of caspase-3 was higher in the high dose group of aloe-emodin.<0.05)and TGF-β₁ expression increased（P>0.05）;the changes of caspase-3 and TGF-β₁ in the low dose group of aloe-emodin in the elderly were 1.8-fold and 0.9-fold higher than the low-dose group in the young aloe-emodin,respectively.The changes of caspase-3 and TGF-β₁ protein in the high dose group of old aloe-emodin were 1.5 times and 0.8 times higher than those in the high dose group of aloe-emodin.Conclusions:1.The continuous subcutaneous injection of 2.5%D-gal solution for 60 days in25-week old mice can decrease the expression of CD₄⁺/CD₈⁺and CD⁺₂₈ on the surface of thymic T cells,and the aging mouse model can be successfully established.2.Long-term high-dose administration of aloe-emodin in mice can cause a decrease in thymus and spleen index,an increase in liver index,and a toxic effect on liver and kidney of young and old mice,and older mice are younger than young adults.Rat liver and kidney are more toxic.3.Hepatotoxicity mechanism of aloe-emodin of aged mice:It may be that aloe-emodin competitively binds to MRP1 and MRP2 protein sites,which reduces the oxidative stress products of MRP efflux,resulting in increased oxidative stress and damage to mitochondrial function.Promotes the release of caspase-3,a downstream factor of apoptosis.4.Nephrotoxicity mechanism of aloe-emodin on the kidney of aged mice:aloe-emodin cause increased oxidative stress in the kidney,activates TGF-β₁ expression and release of caspase-3,a downstream factor of apoptosis.