| Part I: Changes of SMAC,cIAP1,TRAF3 and MAPK signaling pathway in RAW264.7 cells stimulated by LPSObjective: To investigate the changes of SMAC,c IAP1,TRAF3 and MAPK signaling pathway in RAW264.7 cells stimulated by LPS.Methods: The RAW264.7 cells were treated with 100 ug/ml LPS for 0 h,1 h,2 h and 4 h,respectively.Western blot was used to detect the protein level of SMAC,TRAF3,c IAP1,p-JNK and p-p38 in RAW264.7 cells.The protein expression of SMAC was further detected by cellular immunofluorescence technique.q RT-PCR detected the nucleic acid levels of TNF-? and IL-1 in RAW264.7 cell.Results:(1)With the extension of LPS treatment time,SMAC and TRAF3 protein expressions decreased gradually,and c IAP1 expression increased gradually.Besides,the protein expression of SMAC,c IAP1 and TRAF3 in RAW264.7 cells treated with LPS for 1 h,2 h and 4 h was significantly different from that treated with LPS for 0 h(P<0.05).(2)With the extension of LPS treatment time,p-JNK and p-p38 protein expression increased gradually,and the protein expression of p-JNK and p-p38 in RAW264.7 cells treated with LPS for 2 h and 4 h was significantly different from that treated with LPS for 0 h(P<0.05).(3)The m RNA relative expression of TNF-? and IL-1 increased significantly after treated with LPS(P<0.05).Conclusion: LPS inhibit the expression of SMAC,TRAF3 and promoted the expression of c IAP1 in RAW264.7 cells,and activated MAPK signaling pathway.Then,further activated RAW264.7 macrophages and induced the release of inflammatory factors.Part II: Effects of Birinapant on LPS-induced activation of RAW264.7 cells and its molecular mechanismObjective: To investigate the effects of SMAC mimetic Birinapant on LPS-induced activation of RAW264.7 macrophages and its molecular mechanism.Methods: After pretreatment of RAW264.7 macrophages with 300nmol/ml of Birinapant,the model was established by stimulation of macrophages with 100ug/ml LPS.The experiment was divided into four groups:(1)Blank control group without any treatment(Blank group);(2)Birinapant pretreatment + LPS group(Birinapant group);(3)LPS alone group(Vehicle group);(4)Only Birinapant and without LPS(Control group).Birinapant treat with 24 h and LPS treat with 4 h.The expression of c IAP1,TRAF3 and MAPK pathway proteins were detected by western blot.K48-linked polyubiquitination(K48-Ub)of TRAF3 in RAW264.7 were detected by immunoprecipitation(IP)and immunoblotting(IB).q RT-PCR detected the expression of TNF-? and IL-1.Results:(1)TRAF3 protein expression was low in Vehicle group and c IAP1 protein expression was high,which was consistent with the results of the part I.Compared with Vehicle group,the expression of c IAP1 was significantly low and TRAF3 in Birinapant group was significantly high,and the difference were statistically significant(P<0.05);The expression of c IAP1 and TRAF3 in Control group were not significantly different(P>0.05);(2)The K48-linked ubiquitination of TRAF3 was significantly decreased in the Birinapant group compared to the Vehicle group;(3)The protein expression of p-JNK and p-p38 was high in Vehicle group,and the protein expression of p-JNK and p-p38 in Birinapant group was significantly lower than that in Vehicle group(P<0.05);The phosphorylation of JNK and p38 was not promoted or inhibited by the use of Birinapant(Control group)only(P>0.05);(4)TNF-? and IL-1 m RNA expression was high in Vehicle group,which was consistent with the experimental results of part I,and the LPS-mediated expression of inflammatory factors was significantly inhibited by the addition pretreatment of Birinapant(Birinapant group)(P<0.05).Conclusion: Birinapant effectively inhibited LPS-mediated activation of RAW264.7 macrophages in vitro by inhibiting c IAP1 expression.Then inhibit TRAF3 degradation by inhibiting K48-linked ubiquitination of TRAF3,which futher inhibiting MAPK pathway activation.Thus reduce cellular inflammatory response.Part III: Changes of SMAC,c IAP1,TRAF3 and MAPK signaling pathway in KCs of mice stimulated by LPSObjective: To investigate the changes of SMAC,c IAP1 and TRAF3 levels in kupffer cells(KCs)and the effects of LPS on MAPK signaling pathway,and to observe the effects of LPS on liver injury,liver function and survival rate in mice.Methods:(1)Mice liver injury model was established by intraperitoneal injection of LPS(10 mg/kg).Four mice in each group were treated for 0 h,6 h,12 h and 24 h respectively,then the liver tissue of each group was removed and KCs was extracted from liver tissues.The purity of KCs was identified by F4/80 immunofluorescence staining;The activity and phagocytic ability of KCs were analyzed by Trypan Blue staining and ink swallowing experiments;Western blot detected the expression of SMAC,c IAP1,TRAF3,p-JNK and p-p38 protein in KCs;ELISA detected the levels of TNF-? and IL-1 in serum of mice;Blood biochemistry detected the levels of ALT,AST and TBil;HE staining detected liver injury;(2)Twenty-four mice were randomly divided into two groups.One group was intraperitoneally injected with lethal dose of LPS(45 mg/kg)to establish sepsis model,and the other group was intraperitoneally injected with the same amount of saline.Kaplan-Meier methods analysis the survival rate of mice.Results:(1)The purity of KCs was 78.57% and the activity of KCs was above 80%;(2)With the extension of LPS treatment time,TRAF3 and SMAC protein expressions decreased gradually,and c IAP1 expression increased gradually.Besides,the protein expression of SMAC,c IAP1 and TRAF3 in RAW264.7 cells treated with LPS for 6 h,12 h and 24 h was significantly different from that treated with LPS for 0 h(P<0.05)(P<0.05);(3)The protein expression of p-JNK and p-p38 increased gradually and the contents of TNF-?,IL-1,ALT,AST and TBil in serum of mice were significantly increased under LPS stimulation(P<0.05);(4)HE staining showed LPS-induced liver injury;(5)The median survival time was 36.0 h in the experimental group(LPS lethal dose group),and no death occurred in all mice in the Control group(injection of the same amount of saline).Conclusion:(1)LPS inhibit the expression of SMAC,TRAF3 and promote the expression of c IAP1 in KCs in vivo.Then,activation of MAPK signal pathway,which promote the production of inflammatory factors,and induced liver injury and abnormal of liver function;(2)Lethal doses of LPS could significantly shorten the survival time of mice.Part IV: Effects and mechanisms of Birinapant on LPS-induced KCs activation,liver injury and survival of miceObjective: To investigate the effects and mechanisms of SMAC mimetic Birinapant on LPS-induced KCs activation,liver injury and survival rate in mice and its molecular mechanism.Methods:(1)The experiment was divided into two groups,twenty mice in each group:(1)Mice in experimental group(Birinapant)were intraperitoneally injected Birinapant(30 mg/kg)in advance;(2)The control group(Vehicle)mice were intraperitoneally injected with the same amount of saline.24 h later,the two groups of mice were divided into five groups,four mice in each group,and the animal models were established by intraperitoneal injection of LPS(10 mg/kg).Western blot detected the expression of c IAP1,TRAF3 and MAPK pathway proteins;K48-linked polyubiquitination(K48-Ub)of TRAF3 in KCs were detected by immunoprecipitation(IP)and immunoblotting(IB);Immunohistochemistry staining detected the expression of TRAF3 and c IAP1;ELISA detected the levels of TNF-? and IL-1 in serum of mice;Blood biochemistry detected the levels of ALT,AST and TBil;HE staining detected liver injury;(2)Twenty-four mice were randomly divided into two groups.Twelve mice each group.One group was intraperitoneally injected with lethal dose of LPS(45 mg/kg)to establish sepsis model,and the other group was intraperitoneally injected with the same amount of saline.Kaplan-Meier methods analysis the survival rate of mice.Results:(1)The expression of c IAP1 was increased and TRAF3 was decreased in Vehicle group after intraperitoneal injection with LPS.There was no significant change of c IAP1 and TRAF3 expression in Birinapant group.The levels of c IAP1 and TRAF3 protein expression at the same time point were statistically different between Vehicle group and Birinapant group(P<0.05);(2)The K48-linked ubiquitination of TRAF3 was significantly decreased in the Birinapant group compared to the Vehicle group;(3)The immunohistochemical results of TRAF3 and c IAP1 were consistent with those of western blot;(4)Birinapant can significantly inhibit LPS-mediated phosphorylation of JNK and p-38;(5)The contents of TNF-?,IL-1,ALT,AST and TBil in serum of mice of Birinapant group were significantly lower than those of Vehicle group(P<0.05);(6)HE staining showed Birinapant can inhibit LPS-induced liver injury;(7)The median survival time was 58.0 h and the survival rate was 75.0% at 72 h in the Birinapant pretreatment group,and the median survival time was 36.0 h and the survival rate was 16.7.0% at 72 h in the Vehicle group(P<0.05).Conclusion:(1)Birinapant effectively inhibited LPS-mediated activation of KCs in vivo by inhibit c IAP1 expression.Then,inhibit TRAF3 degradation by inhibiting K48-linked ubiquitination of TRAF3,which futher inhibiting MAPK pathway activation.Thus effectively reduce that inflammatory reaction of the liver and the live injury;(2)Birinapant can prolong the survival time of sepsis mice. |
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