The Protective Effect And Mechanism Of Luteolin On Carbon Tetrachloride-Induced Acute Liver Injury In Mice

Acute liver injury（ALI）is a process of pathological damage to the liver caused by multiple causative factors within a short period of time.The liver has a strong regenerative capacity,and minor acute liver injury can be repaired by hepatocyte proliferation,while sustained liver injury can cause liver fibrosis,which can lead to irreversible cirrhosis or even liver cancer.Currently,there are many kinds of liver-protective drugs for the treatment of acute liver injury,but long-term use will cause the body to develop drug resistance,thus affecting the efficacy.Therefore,it is important to find new therapeutic drugs to control the progression of liver disease.Lut is a natural flavonoid found in many plants,such as honeysuckle,with various pharmacological properties,including antioxidant and anti-inflammatory effects.However,whether it has a protective effect on carbon tetrachloride-induced acute liver injury in mice and its mechanism are unclear.Objective:In this study,the protective effect and mechanism of Lut on acute liver injury in mice were preliminarily explored by establishing a model of carbon tetrachloride-induced acute liver injury in mice.Methods:1.Male C57BL/6 mice were randomly divided into control,model and Lut（15,30,60 mg/kg）groups according to body weight.The control group and the model group were given 0.5%CMC-Na 20 m L/kg,and the Lut group（15,30,60 mg/kg）was given10 m L/kg for 7 days.10 m L/kg of ip olive oil solution in the control group and 0.25%CCl₄ in the remaining groups were used 1 h after the last dose to construct an acute liver injury model.After 24 h,blood and liver tissue were collected after anesthetized mice with 1%pentobarbital sodium.Biochemical kits were used to detect AST and ALT activities in serum and GSH and MDA contents in liver tissues.HE staining to observe the morphological changes of liver tissue;qRT-PCR detected TFR1 and FTH1 mRNA expression levels in liver tissues.Western Blot detects GPX4 and Nrf2 signaling pathway-related protein expression in liver tissue.2.Male C57BL/6 mice were randomly divided into control group,model group,ML385（30 mg/kg）group,Lut 60 mg/kg group and Lut+ML385 group according to body weight.The control group,model group and ML385 group were given 0.5%CMC-Na 20 m L/kg,and Lut 60 mg/kg group and Lut+ML385 group were given 10m L/kg of Lut 1 time/day for 7 days.1 h before dosing on days 6 and 7,10 m L/kg of ip ML385 solution in the ML385 group and Lut+ML385 group.10 m L/kg of ip olive oil solution in the control group and 0.25%CCl₄ in the remaining groups were used 1 h after the last dose to construct an acute liver injury model.After 24 h,blood and liver tissue were collected after anesthetized mice with 1%pentobarbital sodium.A biochemical kit was used to detect AST and ALT activities in mouse serum.HE staining observed the morphological changes of liver tissue;qRT-PCR detected changes in FTH1 and TFR1 mRNA expression in liver tissues.Western Blot detects GPX4 and Nrf2 signaling pathway-related protein expression in liver tissue.Results:1.Protective effect of luteolin on carbon tetrachloride-induced acute liver injury in mice（1）The liver of mice generally showed that the liver surface of mice in the control group was smooth and shiny;The surface of the liver of the mice in the model group had a grainy feeling;Lut 15,30,and 60 mg/kg all improved this morphological change,as a gradual normalization of liver color and texture.The results of HE staining showed that the liver tissue structure of the mice in the control group was normal and the size of hepatocytes was uniform.The liver tissue damage of the mice in the model group was serious,which was manifested as hepatocyte enlargement,impaired hepatic lobular integrity and cell necrosis.The liver tissue structure of each group of Lut gradually returned to normal and the hepatocytes were arranged in an orderly manner.（2）The results of the biochemical kit showed that the activity of AST and ALT and the content of MDA in the serum of the model group were significantly increased（P<0.05 or P<0.01）,while the content of GSH was significantly decreased compared to the control group（P<0.01）.Compared with the model group,Lut 15 mg/kg could significantly increase the content of GSH in the liver of mice and reduce the content of MDA（P<0.01）.Lut 30 and 60 mg/kg could significantly increase GSH content,significantly reduce AST and ALT activity and MDA content in serum（P<0.01）.（3）The qRT-PCR results showed that TFR1 mRNA levels were significantly increased and FTH1 mRNA levels were significantly decreased in the liver tissues of mice in the model group compared with the control group（P<0.01）.Lut 15,30 and 60mg/kg significantly decreased the level of TFR1 mRNA and increased the level of FTH1 mRNA compared with the model group（P<0.01）.（4）Western Blot results showed that the expression levels of GPX4,Nrf2,HO-1 and SCL7A11 in liver tissues of mice in the model group were significantly lower compared with the control group（P<0.01 or P<0.05）,while the expression levels of GPX4,Nrf2,HO-1 and SCL7A11 in liver tissues of Lut 60 mg/kg compared with the model group expression levels were significantly higher（P<0.05）.2.Study on the mechanism of luteolin on carbon tetrachloride-induced acute liver injury in mice（1）The liver of mice generally showed that the liver color of mice in the control group was normal and the surface was smooth.The liver surface of the model group mice was grainy and dull.The surface of the liver of mice in the ML385 group had a grainy feeling;The liver surface of the mice in the Lut group was shiny;The liver surface of the Lut+ML385 group was dull and dark.HE staining showed that the liver tissue structure of the control group was normal,the structure of the liver lobule was intact,and there was no hepatocyte necrosis.The liver tissues of mice in the model group were arranged in disorder,and the structure of liver cells was incomplete and accompanied by cell necrosis.The liver tissues of mice in the ML385 group were arranged irregularly and a large number of cells were necrosis.The liver tissue structure of mice in the Lut group tended to be normal,and the arrangement of liver cells gradually became regular.The liver tissue structure of mice in the Lut+ML385 group was incomplete.（2）The results of the biochemical kit showed that the activity of serum AST and ALT was significantly increased in the model group compared to the control group（P<0.01）.Serum AST and ALT activities were significantly lower in the Lut group and significantly higher in the ML385 group compared to the model group（P<0.05）.Serum AST and ALT activities were significantly increased in the Lut+ML385 group compared to the Lut group（P<0.05）.（3）The qRT-PCR results showed that:compared with the control group,the liver tissue of mice in the model group had significantly higher TFR1 mRNA levels and significantly lower FTH1 mRNA levels（P<0.01）;compared with the model group,the liver tissue of mice in the Lut group had significantly lower TFR1 mRNA levels and significantly higher FTH1 mRNA levels,while the liver tissue of mice in the ML385 group had significantly higher TFR1 mRNA levels and significantly lower FTH1 mRNA levels（P<0.01）;compared with the Lut group,the Lut+ML385 group could significantly increase TFR1 mRNA levels and decrease FTH1 mRNA levels（P<0.01）.（4）Western Blot results showed that compared with the control group,GPX4,Nrf2,HO-1 and SCL7A11 protein expression in liver tissues of mice in the model group decreased significantly（P<0.01 or P<0.05）.Compared with the model group,Lut significantly increased the expression of GPX4,Nrf2 and SLC7A11 proteins（P<0.01or P<0.05）in mouse liver tissues,and significantly decreased the expression of GPX4and Nrf2 proteins in the ML385 group（P<0.01）.Compared with the Lut group,the Lut+ML385 group significantly reduced the expression of GPX4,Nrf2 and SLC7A11proteins（P<0.05 or P<0.01）in mouse liver tissue.Conclusion:Lut can significantly reduce the serum AST and ALT activities,reduce liver histopathological damage,alleviate iron metabolism disorders,and inhibit the process of iron diosis,indicating that luteolin has a protective effect on carbon tetrachloride-induced acute liver injury in mice,which may be related to the activation of Nrf2signaling pathway.