Study On MRP-oxidative Stress-based Hepatotoxicity And Related Mechanisms Of Rhein

Rhein is an important active ingredient of many commonly used drugs containing anthraquinone compounds.Although special studies have been conducted on the toxicity of rhubarb and its main components,anthraquinones,the conclusions are not uniform.Many research reports believe that anthraquinones Class compounds can cause liver toxicity at certain doses.However,there are not many studies on the liver toxicity mechanism of rhein.This study is based on the toxicology of rhein in the past,through acute toxicity and 75d long-term toxicity experiments to determine the toxicity of rhein,and focus on the effect of rhein on the liver.The effects of oxidative stress,mitochondrial damage,cell inflammation,cell apoptosis,and MRP protein expression were discussed in terms of dose and age,and the possible toxic mechanism of rhein was discussed at the same time.Objective:To determine the LD₅₀ value of rhein through acute toxicity experiments,observe the liver toxicity of rhein at different doses in mice of different ages,and explore the possible effects of rhein through oxidative stress,mitochondrial damage,cell apoptosis,and the expression of MRP protein Mechanism of liver toxicity.Method:1.Acute toxicity test of rhein120 healthy male Kunming mice were randomly divided into 10 groups according to their body weight,with 12 mice in each group.The intragastric concentrations were 4000,2000,1000,500,250,125,62.5,31.2,15.6,and 0 mg/kg.Rhein.After 14 days,pathological examinations of possible toxic target organs were performed and the weighted probability unit method（Bliss method）was used to calculate the LD₅₀ and its 95%confidence interval.2.Long-term（75d）hepatotoxicity test of rhein on young and D-gal old miceThe experimental mice were five-week-old young mice and 60-day old mice modeled by D-gal.D-gal old mice are modeled by subcutaneously injecting 2.5%D-gal solution into the neck back of 30 healthy SPF mice with half male and female,and continuously administering them at a concentration of 0.125 mg/kg for 60 days.The aged mice were randomly divided into3 groups,namely D-gal blank group（DC）,D-gal low-dose group（DL）,and D-gal high-dose group（DH）.A group of 10 mice was divided into male and female groups.The young mice are another 30 five-week-old mice with half male and half males.They are randomly divided into three groups,namely the young blank group（IC）,the young low-dose group（DL）,and the young high-dose group（DH）10 Only one group,half male and half.Based on the LD50 value,it was determined that the doses of rhein were low dose 0.8 g/kg,high dose 1.6 g/kg,and the blank group was 0.5%CMC-Na solution,administered once in the morning and evening for 75consecutive days.After the administration,the activity state and morphological changes of the mice were observed,and the diet,water consumption,and body weight of the mice were recorded every week.After the administration,blood was taken from the abdominal aorta,and the blood was allowed to stand for 1 hour before the serum was separated for analysis of alanine aminotransferase（ALT）,aspartate aminotransferase（AST）and other indicators,mouse thymus,liver,heart,spleen,kidney And the brain is cleaned with saline and its weight is recorded with an electronic balance,and the liver is used for pathological examination.3.The mechanism of rhein’s hepatotoxicity in mice of different agesDetect relevant indicators of oxidative stress,including serum SOD,MDA,GSH/GSSG and GSH-Px.Detection of mitochondrial related indicators,including mitochondrial membrane potential and Ca²⁺swelling sensitivity FSC/SSC.Detect inflammation and apoptosis-related indicators,detect IL-6 and TNF-αthrough kits,and use immunohistochemical methods to detect caspase-3.Finally,Western Bolt is used to explore the expression of MRP1 and MRP2in the liver to find possible internal connections.Result:1.Research results of acute toxicity of rheinAccording to the Bliss method,the LD50 value of rhein is 2.9272±1.2144（2.1856-4.6143）g/kg.According to the prescribed classification standard for the LD50 value of mice one-time oral determination,rhein is judged to have low toxicity.The pathological results of HE showed that rhein can cause inflammation,swelling and necrosis of liver,kidney and colon cells to varying degrees.2.The 75-day long-term toxicity test results of rhein on young and D-gal old miceThe aging model mice were formed through D-gal modeling.The results of flow cytometry on thymic T cells showed that the ratio of CD₄⁺/CD₈⁺and the expression of CD⁺₂₈decreased,indicating that the aging mouse model was successfully established.After long-term administration of rhein,compared with the young blank group,serum AST increased significantly in the 0.6 mg/kg low-dose group in the youth group.The pathological results showed that the liver tissues of the young mice were swollen and fatty.Degeneration:Compared with the old blank group,the expression of serum AST and ALT increased in the old group of mice.The liver pathological results of the old group were consistent with the young group,but the degree of liver damage was more serious.In addition,the old group also had The phenomenon of reduced survival.3.Research results of the mechanism of rhein’s hepatotoxicity in young and D-gal old mice1)The effect of rhein on oxidative stress in mice:Compared with the control group,TNF-αand MDA positive cells in immature mice increased significantly（p<0.05）;in D-gal aging model mice and immature mice,SOD values were significantly reduced（p<0.05）.At the same time,GSH-Px and GSH/GSSG decreased significantly in each group（p<0.05）,but the overall D-gal aging model group was more than twice that of the immature group.2)The effect of rhein on mitochondrial damage and apoptosisThe overall MMP showed a downward trend.Compared with the control group,the MMP level of the immature low-dose group was significantly decreased（p<0.05）.The overall FSC/SSC showed an increasing trend.Compared with the elderly control group,the mitochondrial swelling（FSC/SSC）in the elderly group increased significantly（p<0.05）.The number of Caspase-3-positive cells in both the immature group and the old group increased significantly（p<0.05）3)The effect of rhein on the expression of multidrug resistance protein in miceWestern blotting showed that,compared with the control group,the relative expression of MRP1 and MRP2 protein in the rhein administration group of the immature group and the old group decreased,and the expression of the MRP2 protein appeared with the increase of the rhein administration concentration.The corresponding downward trend.However,the results of relative expression levels showed no statistically significant difference（p>0.05）.Conclusion:1.The LD₅₀ value of rhein is 2.9272±1.2144（2.1856-4.6143）g/kg,indicating that rhein has low toxicity.2.After long-term（75d）and high-dose（0.35 mg/kg）administration of rhein,the coefficients of heart,spleen and thymus of old mice have a significant downward trend（p<0.05 or p<0.01）,while the coefficient of liver of old mice has obvious The increase（p<0.05）.Pathological results and blood biochemical indicators of AST and ALT also showed that rhein has a certain toxicity to the liver of mice,and the liver toxicity to old mice is greater than that of young mice.3.The possible toxic mechanism of rhein is related to the competitive binding of MRP2 protein sites,reducing the expression of MRP2 protein,thereby blocking COSS efflux,resulting in increased oxidative stress,promoting cell apoptosis and mitochondrial function damage.