The Protective Effect And Mechanism Of Total Flavonoids Of Murraya Paniculata Leaves On Acute Liver Injury Induced By CCl₄ In Mice

Acute liver injury（ALI）refers to the pathological phenomenon of sudden abnormal liver function caused by various factors in a short period of time.Virus infection,drug poisoning,immune response,and ischemia-reperfusion are common causes of acute liver injury.Acute liver injury can induce a series of complications.Some patients progress from acute liver injury to acute liver failure with a high fatality rate,but most patients with acute liver injury can recover with drug therapy.Therefore,it is of great practical significance to find a drug that has a therapeutic effect on acute liver injury.Total flavonoids of murraya paniculata leaves（TFMP）is an active ingredient mainly composed of flavonoids extracted and isolated from the leaves of murraya paniculata,and has pharmacological activities such as hypoglycemic,anti-inflammatory and antioxidant.However,there is no report on the protective effect and mechanism of TFMP on acute liver injury.Objective:In this study,we established a model of acute liver injury induced by carbon tetrachloride in mice,and observed the protective effect and mechanism of TFMP on acute liver injury.Methods:1.The protective effect of TFMP on carbon tetrachloride（CCl₄）-induced acute liver injury.40 C57BL/6 mice were randomly divided into Control,Model,TFMP low-dose（100 mg/kg）group and TFMP high-dose（200 mg/kg）group.The mice in the Control and Model were given 0.5%CMC-Na 20 m L/kg by ig,and the mice in the 100,200 mg/kg does groups were given the corresponding dose of 20 m L/kg by ig,once a day,for 7 consecutive days.One hour after the last administration,the mice in the Control were intraperitoneally injected（i.p）with olive oil 10 m L/kg,The mice in the Model and100,200 mg/kg does groups were i.p 0.25%CCl4 solution to establish model of acute liver injury.Fasting but not water.24 h later,mice were anesthetized with i.p1%sodium pentobarbital 20 m L/kg,blood was collected from the eyeball and take liver.Biochemical kits were used to determine the activities of AST,ALT and LDH in serum and the content of iron in serum and liver;the liver coefficient was calculated,the pathological changes of liver tissue were observed by HE staining;the activity of SOD and the contents of GSH and MDA in liver tissue were detected by spectrophotometry;the level of ROS in liver tissue;the expression of 4-HNE in liver tissue was detected by immunohistochemistry.2.The protective mechanism of TFMP on carbon tetrachloride-induced acute liver injury.40 C57BL/6 mice were randomly divided into Model,IKE,TFMP group and TFMP+IKE group according to body weight.The TFMP group and TFMP+IKE group were given TFMP 200 mg/kg,and the Model group and the IKE group were given an equal volume of 0.5%CMC-Na by ig,once a day,for 7 consecutive days.IKE group and TFMP+IKE group received i.p 0.008%IKE 5 m L/kg 3 hours before the last administration.One hour after the last administration,0.25%CCl₄was administered i.p to establish model of acute liver injury.After 24 h,the mice were anesthetized with i.p1%sodium pentobarbital 20 m L/kg,and blood was collected from the eyeball and take liver.Biochemical kits were used to determine the activities of AST,ALT and LDH in serum and the content of iron in serum and liver tissue;the liver coefficient was calculated,the pathological changes of liver tissue were observed by HE staining;the activity of SOD and the contents of GSH and MDA in liver tissue were detected by spectrophotometry;the level of ROS in liver tissue;the expression of 4-HNE in liver tissue was detected by immunohistochemistry;the expression levels of ferroptosis-related proteins YAP,p-YAP and ACSL4 in liver tissue were detected by Western Blot.Results:1.The protective effect of TFMP on carbon tetrachloride（CCl₄）-induced acute liver injury.（1）Compared with the Control,the activities of AST,ALT and LDH in the serum of the mice in the Model were increased（P<0.01）,and the liver index was significantly increased（P<0.01）;compared with the Model,TFMP 200 mg/kg could significantly reduce the activities of AST,ALT and LDH in serum of mice with acute liver injury（P<0.05 or P<0.01）,and the liver index was significantly reduce（P<0.05）.（2）The photographs of liver results showed that the liver surface of the mice in the Control was smooth;the liver of the mice in the Model had lipid degeneration;TFMP 100 and 200 mg/kg does group could improve the liver lipid degeneration.HE staining results showed that the liver structure of the mice in the Control was normal,with complete hepatocyte morphology and no necrosis or inflammatory cell infiltration;the Model had disordered liver structure,impaired hepatocyte integrity,and necrosis and inflammatory cell infiltration;the liver pathological damage of the mice in the 100mg/kg does group was not significantly improved;the liver tissue structure of the mice in the TFMP 200 mg/kg does group tended to be normal,the structure of liver cells tended to be complete,the area of necrosis of liver cells was reduced,and the infiltration of inflammatory cells was weakened.（3）Compared with the Control,the SOD activity and GSH content in the liver of the Model were significantly decreased（P<0.01）,the MDA content was significantly increased（P<0.01）;compared with the Model,the content of GSH in the liver of TFMP 100 mg/kg does group was significantly increased（P<0.01）,the activity of SOD and the content of GSH in the liver of the mice in the TFMP 200 mg/kg does group were significantly increased（P<0.05 or P<0.01）,and the content of MDA was significantly decreased（P<0.01）.Compared with the Control,the level of ROS in the liver of the Model was significantly increased（P<0.01）;compared with the Model group,the level of ROS in the liver of the TFMP 100 and 200 mg/kg does groups was significantly decreased（P<0.05 or P<0.01）.（4）Compared with the Control,the contents of iron in serum and liver tissue in the Model were significantly increased（P<0.01）;compared with the Model,the contents of iron in serum and liver tissue in the TFMP 200 mg/kg does group were significantly decreased（P<0.01）,the contents of iron in serum and liver tissue in the TFMP 100 mg/kg does group were decreased,but there was no statistical significance（P>0.05）.The results of immunohistochemistry showed that there was a small amount of 4-HNE expression in the liver of the mice in the Control,the expression of 4-HNE in the liver of the mice in the Model was increased;the expression of 4-HNE in the liver of the mice in the TFMP 100 and 200 mg/kg does group was decreased.2.The protective mechanism of TFMP on carbon tetrachloride-induced acute liver injury.（1）Compared with the Model,the activities of AST,ALT and LDH in the serum of the mice in the TFMP 200 mg/kg does group were significantly decreased（P<0.05or P<0.01）,and the liver index was significantly reduce（P<0.01）;compared with the TFMP 200 mg/kg does group,the AST,ALT and LDH activities of the TFMP 200mg/kg+IKE group were significantly increased（P<0.05 or P<0.01）,and the liver index was significantly increased（P<0.01）.（2）The photograph of liver results showed that the liver lipid degeneration in the Model;the hepatic lipid degeneration in the TFMP 200 mg/kg does group was weakened;the liver lipid degeneration in the TFMP 200 mg/kg+IKE group was aggravated.The results of HE staining showed that the liver tissue structure of the Model was disordered,the integrity of hepatocytes was damaged,and necrosis and inflammatory cell infiltration occurred;the liver structure of the mice in the TFMP 200mg/kg does group tended to be normal,the structure of hepatocytes tended to be perfect,and the necrosis occurred.The area was reduced,and the inflammatory cell infiltration was alleviated;the liver structure of the mice in the TFMP 200 mg/kg+IKE group was disordered,the cell integrity was impaired,and the necrosis and inflammatory cell infiltration were aggravated.（3）Compared with the Model,the content of MDA in the liver of the mice in the TFMP 200 mg/kg does group was significantly decreased（P<0.01）,and activity of SOD and content of GSH were significantly increased（P<0.05 or P<0.01）;Compared with TFMP 200 mg/kg does group,the content of MDA in the liver of mice in the TFMP200 mg/kg+IKE group was significantly increased（P<0.05）,and the content of GSH was significantly decreased（P<0.05）.Compared with the Model,the level of ROS in the liver of the mice in the TFMP 200 mg/kg does group was decreased（P<0.01）.Compared with the TFMP 200 mg/kg does group,the level of ROS in the liver of the mice in the TFMP 200 mg/kg+IKE group was increased（P<0.01）.（4）Compared with the Model,the contents of iron in serum and liver tissue in the TFMP 200 mg/kg does group were significantly decreased（P<0.05 or P<0.01）;compared with the TFMP 200 mg/kg does group,the contents of iron in serum and liver tissue in TFMP 200 mg/kg+IKE group were significantly increased（P<0.05）.The results of immunohistochemistry showed that 4-HNE was significantly expressed in the liver of mice in the Model;the expression of 4-HNE in the liver of the mice in TFMP200 mg/kg does group was decreased;the expression of 4-HNE in the liver of the mice in TFMP 200 mg/kg+IKE group was increased.（5）Compared with the Model,the expressions of ferroptosis-related proteins YAP and ACSL4 in the liver of mice in the TFMP 200 mg/kg does group were significantly decreased（P<0.05 or P<0.01）,and the expression of p-YAP was significantly increased（P<0.05）.Compared with the 200 mg/kg does group,the expressions of YAP and ACSL4 in the liver of the TFMP 200 mg/kg+IKE group were significantly increased（P<0.01）,and the expression of p-YAP was significantly decreased（P<0.01）.Conclousion:The results of this study show that TFMP can significantly reduce the exudation of enzymes in the acute liver injury induced by CCl₄in mice,improve the pathological changes in the liver of mice,enhance the ability of liver to resist oxidative stress,and reduce ferroptosis.Its effect may be related to inhibition YAP/ACSL4 signaling pathway.