| Background: It is necessary to establish a stable animal model for the study of the pathogenesis of liver diseases and the selection of appropriate drugs for liver protection.However,the existing animal models have shortcomings.So it is of great practical significance to establish a more stable animal model of acute liver injury.Objective: 1.To establish a stable acute liver injury animal model by sodium cyclamate;2.To explore the possible mechanism of sodium cyclamate induced acute liver,heart and kidney injury.Methods: Mice were randomly divided into the control group and the low,middle and high-dose group of sodium cyclamate,24 mice in each group.The control group was injected intraperitoneally with 0.04ml/g/d 0.9%normal saline,while the low,middle and high-dose groups of sodium cyclamate were injected intraperitoneally with 3000mg/kg/d,6000mg/kg/d and 12000mg/kg/d sodium cyclamate respectively,once every 24 hours.At the end of the day1,day3,day5,day7,blood was collected and liver,heart,kidney tissues were taken.Serum levels of Alanine Aminotransferase(ALT),Aspartate Aminotransferase(AST),Total Bilirubin(TBIL),Direct Bilirubin(DBIL),Creatine Kinase(CK),Creatine Kinase MB(CK-MB),Cardiac troponin T(cTnT),Serum Creatinine(Scr)and Blood Urea Nitrogen(BUN)were used to evaluate the toxic effects of sodium cyclamate on liver,heart and kidney.The changes of liver,heart,kidney and their tissues HE staining were observed;the ultrastructural changes of liver were observed under transmission electron microscope;the expressions of Tumor necrosis factor-?(TNF-?)and Interleukin-1?(IL-1?)in liver,heart and kidney tissues were detected by Enzyme Linked Immunosorbent Assay(ELISA)and immunohistochemistry.Results: 1.The results of serum enzymes levels:(1)Compared with the parallel control group,the serum level of ALT of the low-dose group were increased at the day1 and day3(P<0.05),also in the middle-dose,high-dose groups at the day1,day3,all sodium cyclamate-treated groups at the day5 and the low-dose,middle-dose groups at the day7(P<0.01);the serum level of AST of the middle-dose,high-dose groups at the day1,all sodium cyclamate treated-groups at the day3,day5 and the low-dose,middle-dose groups at the day7 were increased significantly(P<0.01);compared with the day1,the serum level of ALT of the low-dose group at the day5,day7,the middle-dose group at the day3,day5,day7 and the high-dose group at the day3,day5 were increased significantly(P<0.01);the serum level of AST of the low-dose,middle-dose groups at the day3,day5,day7 and the high-dose group at the day3,day5 were increased significantly(P<0.01).The serum levels of ALT and AST in all sodium cyclamate-treated groups were highly positive correlated with the dosage and treated time of sodium cyclamate(P<0.01,r=0.83~0.97).There were no significant difference in the serum levels of TBIL and DBIL(P>0.05).(2)Compared with the parallel control group,the serum levels of CK,CK-MB in the high-dose group at the day1 and the low-dose group at the day7 were increased(P<0.05),also in the high-dose group at the day3,day5 and the middle-dose group at the day7(P<0.01);the serum level of cTnT in the high-dose group at the day1 was increased(P<0.05),also in the high-dose group at the day3,day5 and the low-dose,middle-dose groups at the day7(P<0.01);compared with the day1,the serum levels of CK,CK-MB,cTnT in the low-dose,middle-dose groups at the day7 and the high-dose group at the day3,day5 were increased significantly(P<0.01).(3)There were no significant difference in the serum levels of Scr and BUN(P>0.05).2.Observation under light microscope:(1)The liver tissues of each sodium cyclamate-treated group showed different degrees of disorder in arrangement of hepatocytes,ballooning degeneration,inflammatory cell infiltration,congestion and expansion of hepatic sinuses.The damage of hepatocytes increased with the increase of sodium cyclamate dosage and treated time.(2)At the day5 in the low-dose and middle-dose groups,a small amount of granular degeneration and watery degeneration appeared in individual muscle fibers.At the day7 in the low-dose,middle-dose groups and at the day1,day3,day5 in the high-dose groups,the whole muscle bundles showed granular degeneration and watery degeneration in varying degrees.(3)At the day5 in the low-dose,middle-dose groups and at the day1 in the high-dose group,the lumen of individual renal tubules were expanded.At the day7 in the low-dose,middle-dose groups and at the day3,day5 in the high-dose groups,the epithelial cells of renal tubules in different degrees of brush edge fell off and necrosis,some of them expanded,basement membrane exposed,inflammatory cells infiltrated.3.Under the transmission electron microscope,no obvious abnormality were found in the organelles of the liver cells of the control group,but the mitochondria,endoplasmic reticulum,ribosome and chromatin of the liver cells of the middle-dose group at the day5 were damaged obviously.4.ELISA results: At the day5,compared with the control group,the levels of TNF-? and IL-1? in the liver tissues of the low,middle and high-dose groups were significantly higher(P<0.01);the levels of TNF-? and IL-1? in the heart tissues of the high-dose group were significantly higher(P<0.01);the levels of IL-1? in the kidney tissue of the middle-dose group were higher(P<0.05),and the levels of TNF-? and IL-1? in the kidney tissue of the high-dose group were significantly higher(P<0.01).5.Immunohistochemistry results: At the day5,compared with the control group,the IOD/Area of TNF-? and IL-1? positive expression in the low-dose group were increased significantly(P<0.05),also in the middle and high-dose groups(P<0.01).In the heart tissues,compared with the control group,the IOD/Area of TNF-? and IL-1? positive expression in the high-dose group increased significantly(P<0.05).In the kidney tissues,compared with the control group,the IOD/Area of TNF-? positive expression in the high-dose group was increased(P<0.05);the IOD/Area of IL-1? positive expression in the middle-dose group(P<0.05)and the high-dose group increased significantly(P<0.01).Conclusion: 1.The acute liver injury mouse model can be established by intraperitoneal injection of sodium cyclamate 6000mg/kg/d for 5 days successfully.2.Sodium cyclamate shows a dose-response and time-response relationship in liver injury.3.The pathogenesis of acute liver,heart and kidney injury induced by sodium cyclamate may be related to the inflammatory response mediated by TNF-? and IL-1?. |
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