| Chapter One Development of the model about concanavalin A-induced experimental liver injury in miceObjective:To establish the model about concanavalin A-induced experimental liver injury in mice.Methods:Forty Balb/C mice were randomly divided into 4 groups based on the dose of Con A(ten for each group),A,B,C and D.Con A 10mg/kg,20mg/kg or 30mg/kg was intravenous(tail vein) injected into the mouse group A,B or C respectively while NS was used for the group D(control group).Survival rate of each group at 8 hours after via was calculated.The survived mice were killed in order to get blood,heart, liver,lung,kidney and brain samples.Activity of aminnotransferase(ALT) was tested.Histopathological examination for heart,liver,lung,kidney and brain were also performed.The optimal Con A dose(20mg/kg) and treated different times were selected.The ALT level,death rate and histopathological results were observed under the optimal Con A dose.Results:The activity of ALT in both group A and group B was significantly higher than that in group D(P＜0.01).No dead mouse was found in group A and D,but 3 and 10 mice were dead in group B and C respectively.Hepatic histopathology showed only degeneration of liver cells was present in group A,while degeneration,necrosis and inflammatory cells were found infiltration in group B and C.There were no pathological changes in heart and brain,while congestion was found in lung and little vacuolar degeneration in renal cells respectively.Within 24 hours of injection of 20mg/kg of Con A,the death rate,activity of ALT, liver inflammatory cells infiltration,degeneration,hepatocyte degeneration and necrosis exacerbated with the prolongation of time.Conclusion:The Con A-induced liver injury in mice was established successfully.The optimal dose for Con A-induced experimental liver injury in mice is 20mg/kg. Chapter Two The protective effect of suppressive oligonucleotides on Con A-induced liver injury in miceSection one:The protection effect of suppressive oligonucleotides injected at 12 hours before Con A-induced liver injury in miceObjective:To investigate the protective effect of suppressive oligonucleotides(Sup ODN) injected at 12 hours before concanavalin A (Con A)-induced liver injury in mice.Methods:Eighty Balb/C mice were randomly divided into 4 groups, normal saline(NS) group,control oligonucleotides(Con ODN) group, cyclosporin A(CsA) group and Sup ODN group.Mice in each group were injected with Con A(20mg/kg) via the tail vein.NS(0.3mL per mouse),Con ODN(20mg/kg),CsA(130 mg/kg) or Sup ODN(20mg/kg) was injected intraperitoneally at 12 hours before Con A injection.Death rate of each group at 8 and 24 hours after intraperitoneal injection was calculated and survived mice were killed in order to get blood and liver samples.Activity of aminnotransferase(ALT) was tested and levels of TNF-α,IFN-γand IL-4 were detected by ELISA.Expressions of TNF-αand IFN-γmRNA in liver tissue were detected by RT-PCR. Histopathological examination for liver was also Performed.Results:The ALT activity in Sup ODN group was found significantly lower than that in NS and Con ODN group respectively(P＜0.01).Level of IFN-γ,and its mRNA expression were significantly decreased,while the level of IL-4 was significantly increased in Sup ODN group compared with NS,Con ODN and CsA group(P＜0.01). Compared to NS and Con ODN group,the liver necrosis and infiltration of inflammatory cells significantly reduced.Section two:The protective effect of Sup ODN injected simulativsly with the start of Con A-induced liver injury in miceObjective:To investigate the protective effect of Sup ODN injected simulativsly with the start of Con A-induced liver injury in mice.Methods:Eighty Balb/C mice were randomly divided into 4 groups, NS group,Con ODN group,CsA group and Sup ODN group.Mice in each group were injected with Con A(20mg/kg) via the tail vein. NS(0.3mL per mouse),Con ODN(20mg/kg),CsA(130 mg/kg) or Sup ODN(20mg/kg) was injected intraperitoneally at the same time point Con A injection.Death rate of each group at 8 and 24 hours after intraperitoneal injection was calculated and survived mice were killed in order to get blood and liver samples.Activity of ALT was tested and levels of TNF-α,IFN-γ,and IL-4 were detected by ELISA.Expressions of TNF-αand IFN-γ,mRNA in liver tissue were detected by RT-PCR. Histopathological examination for liver was also performed.Results:The ALT activity in Sup ODN group was found significantly lower than that in NS and Con ODN group respectively(P＜0.01).Level of IFN-γ,and its mRNA expression were significantly decreased,while the level of IL-4 was significantly increased in Sup ODN group compared with NS,Con ODN and CsA group(P＜0.01).Compared to NS and Con ODN group,the liver necrosis and infiltration of inflammatory cells significantly reduced.Section Three:The protective effect of Sup ODN injected at 2 hours after Con A-induced liver injury in miceObjective:To investigate the protective effect of Sup ODN injected at 2 hours after Con A-induced liver injury in mice.Methods:Eighty Balb/C mice were randomly divided into 4 groups, NS group,control Con ODN group,CsA group and Sup ODN group. Mice in each group were injected with Con A(20mg/kg) via the tail vein. NS(0.3 mL per mouse),Con ODN(20mg/kg),CsA(130 mg/kg) or Sup ODN(20mg/kg) was injected intraperitoneally at 2 hours after Con A injection.Death rate of each group at 8 and 24 hours after intraperitoneal injection was calculated and survived mice were killed in order to get blood and liver samples.Activity of ALT was tested and levels of TNF-α, IFN-γ,and IL-4 were detected by ELISA.Expressions of TNF-αand IFN-γ,mRNA in liver tissue were detected by RT-PCR.Histopathological examination for liver was also performed.Results:The death rate in group Sup ODN significantly lower than that in group CsA.The ALT activity in Sup ODN group was found significantlylower than that in NS and Con ODN group respectively(P＜0.01).Level of IFN-γ,and its mRNA expression were significantly decreased,while the level of IL-4 was significantly increased in Sup-ODN group compared with NS,Con ODN and CsA group(P＜0.01).Compared to NS and Con ODN group,the liver necrosis and infiltration of inflammatory cells significantly reduced.Conclusion:The research indicated Sup ODN had a obviously protective effect on Con A-induced mice liver injury. Chapter Three The preiminaly study of mechanism and the protective effect of suppressive oligonucleotidesObjective:To investigate mechanism of suppressive oligonucleotides (Sup ODN) on concanavalin A(Con A )-induced liver injury in mice.Methods:The levels of TNF-α,IFN-γ,and IL-4 were detected by ELISA and the other methods is the same to chapter two.Expression of pSTAT4(phosphorylated pSTAT4),pSTAT6,T-bet in spleen tissue were detected by Western blot.Results:Sup ODN obviously suppressed the expression of pSTAT4 and T-bet in spleen tissue,but had no influence on pSTAT6,cyclosporin A(CsA) had no influence on pSTAT6,pSTAT4 and T-bet.The other results were the same to chapter two.Conclusion:The protective effect of Sup ODN may suppressed the expressions of IFN-γ,via down regulatory the expressions of pSTAT4 and T-bet in spleen tissue,but had no influence on pSTAT6.The mechanism of Sup ODN differently from CsA on Con A-induced liver injury in mice. |
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