Protective Mechanism Of Magnesium Isoglycyrrhizinate On CCl₄-induced Liver Injury

Objectives:Magnesium isoglycyrrhizinate has been shown to possess prevention and treatment of liver diseases,and has a good pharmacological activity and is widely used in clinic,Which is a fourth generation glycyrrhizin preparation,and has good application prospect.magnesium isoglycyrrhizinate is a drug for clinical listed,However,the liver protective effects of magnesium isoglycyrrhizinate,especially the underlyingmechanisms,and remain less studied.This study investigated whether magnesium isoglycyrrhizinate to activate Nrf2/ARE signaling pathway,and to protect liver function.Nuclear factor-related factor-2（Nrf2）is one of the most important transcription factors in the process of oxidative stress,which is responsible for regulating the transcription and translation of downstream factors such as various antioxidant genes and phase II detoxification enzyme genes,thereby exerting the protective effect against oxidative stress.Basic research shows that there is a serious inflammatory reaction and oxidative stress response in CCl₄-induced rats and hepatocytes,which is may directly cause hepatocyte apoptosis or even necrosisthe.It is unclear whether Nrf2 involves this process or not.Therefore,this project intends to explore the protective effect of magnesium isoglycyrrhizinate on liver injury and its related mechanisms on the basis of previous studies in the further.To study the effect of magnesium isoglycyrrhizinate on the expression of related genes and proteins through the liver injury model induced by CCl₄ in rats and the injury model of L02cells in vitro.To explore the protective mechanism of magnesium isoglycyrrhizinate on CCl₄-induced hepatic injury was discussed from the angle of antioxidation,and to provide a wide range of experimental basis in the clinical application.Methods:1.Mechanism of liver protective effect of magnesium isoglycyrrhizinate on liver injury CCl₄-induced in rats.The SD rats were divided into five groups:1)Normal group,2)Model group,3)Low dose model group（15 mg·kg^-11 MgIG）,4)Medium dose model group(30 mg·kg^-1MgIG),5)High dose model group（45 mg·kg^-11 MgIG）.According to grouping,the following experiments were carried out:1)Detecting the change of biochemical index in serum;2)Detecting the content of GSH and MDA and the activity of SOD in liver tissue;3)Observing the histopathology of liver tissue;4)RT-PCR and Western Blot were applied to measure mRNA and protein expression of Nrf2 and its downstream target gene.2.The mechanism of magnesium isoglycyrrhizinate on on CCl₄-induced injured L02Cells.L02 cells were divided into five groups:1)Normal group,2)Model group,3)Low dose model group（15μM MgIG）,4)Medium dose model group（30μM MgIG）,5)High dose model group（60μM MgIG）.According to grouping,the following experiments were carried out:1)MTT assay was used to detect survival rate;2)LDH assay was used to detect cell leakage rate;3)The level of intracellular ROS was detected by the fluorescent probe substrate method;4)The content of GSH and MDA,and the activity of SOD were detected in L02 cells;5)Nrf2 nuclear translocation was detected by immunofluorescence;6)RT-PCR and Western Blot were applied to measure mRNA and protein expression of Nrf2 and its downstream target gene.Results:1.Magnesium isoglycyrrhizinate significantly improved CCl₄-induced liver injury in rats.Pathological examination of rat liver showed that administration of magnesium isoglycyrrhizinate restored the hepatic tissue structure and decreased the levels of serum ALT and AST in liver tissue.Compared with the model group,the administration of magnesium isoglycyrrhizinate significantly increased the activities of SOD and thecontent of GSH,and decreased the content of MDA in liver tissue.magnesium isoglycyrrhizinate may up-regulate the mRNA and nuclear protein expression levels of Nrf2,and up-regulate the mRNA and protein expression levels of downstream factors HO-1 and UGT1A1by activating the Nrf2/ARE signaling pathway in vivo.2.Magnesium isoglycyrrhizinate significantly improved CCl₄-induced injured L02cells.The time of CCl_4-induced model was determined by MTT assyed and the concentration of magnesium isoglycyrrhizinate was selected as 15μM,30μM and 60μM.Morphological study of L02 cells showed that the morphology of L02 cells tended to be normal after administration of magnesium isoglycyrrhizinate;The activity of SOD,the content of GSH were significantly increased in L02 cells,and the content of MDA and ROS were significantly decreased in L02 cells.The results of immunofluorescence assay showed that magnesium isoglycyrrhizinate could enhance the fluorescence of Nrf2,RT-PCR and Western Blot experimental results were shown that magnesium isoglycyrrhizinate up-regulate Nrf2 mRNA and nuclear protein expression levels,and up-regulate its downstream factors HO-1 and UGT1A1mRNA and protein expression levels.Conclusion:Magnesium isoglycyrrhizinate may up-regulate Nrf2 gene expression and nuclear protein expression in liver tissue and L02 injured cells through activation of Nrf2/ARE signaling pathway,and up-regulate the mRNA and protein levels of downstream regulatory factors HO-1 and UGT1A1.The results of this study may provide experimental evidence for the widespread use of magnesium isoglycyrrhizinate in clinical practice.