阻断肝脏Notch1/Jagged1通路对大鼠肝移植急性排斥反应的影响

Effects of blocking liver Notch1/Jagged1 pathway on acute rejection in rat liver transplantation

目的探讨阻断肝脏Notch1/Jagged1通路对大鼠肝移植急性排斥反应的影响。方法建立大鼠肝移植急性排斥反应模型,分为4组,每组15对。SHAM组:不做任何处理建立肝缺血再灌注模型;NS组:肝缺血再灌注术前14 d尾静脉注射100μL的生理盐水作为对照;shRNA-Jagged1组:肝缺血再灌注术前14 d尾静脉注射100μL 1×1012 PFU慢病毒;shRNA-GFP组:肝缺血再灌注术前14 d尾静脉注射100μL 1×1012 PFU空载慢病毒。检测各组丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、总胆红素(TBiL)水平,ELISA检测血清中炎性因子水平,PCR、Western blot检测肝组织Jagged1、Notch1表达,Western blot检测白细胞介素(IL)-6、IL-1、p-JNK、cleaved-caspase3、PTEN、p-P65等表达,HE染色评估大鼠肝移植情况。结果肝移植后Notch1、Jagged1表达在24 h最高。shRNA-Jagged1组AST、ALT、TBiL水平明显高于NS组和shRNA-GFP组(P<0.05),排斥反应评分明显高于NS组(P<0.05)。shRNA-Jagged1组IL-1、IL-6水平明显高于NS组,IL-10、TGF-β水平明显低于NS组,差异有统计学意义(P<0.05)。与NS组比较,shRNA-Jagged1组p-JNK、cleaved-caspase3、PTEN、p-P65表达明显升高,而p-AKT表达明显降低;NS组及shRNA-GFP组比较差异无统计学意义(P>0.05)。结论阻断肝脏Notch1/Jagged1通路可能是通过激活PTEN/AKT/TLR4/NF-κB通路加重肝移植炎性反应损伤,以及激活JNK通路从而加重肝细胞凋亡导致肝移植后发生急性排斥反应。

Objective To investigate the effect of blocking Notch1/Jagged1 pathway on acute rejection in rat liver transplantation.Methods The rat liver transplantation acute rejection models were established and divided into 4 groups,15 pairs in each group.The SHAM group had no any treatment and established the liver ischemic reperfusion model;the NS group was injected 100μL of normal saline by the caudal vein on 14 d before liver ischemic reperfusion as the control;the shRNA-Jagged1 group was injected by 100μL of 1×1012 PFU lentivirus by the caudal vein on 14 d before liver ischemic reperfusion;the shRNA-GFP group was injected by 100μL of 1×1012 PFU no-load lentivirus.The levels of ALT,AST and TBiL were detected in each group.ELISA was used to detect the levels of serum inflammatory factors.The expressions of liver tissues Jagged1 and Notch1 were detected by PCR and Western blot.The expressions of IL-6,IL-1 p-JNK cleaved-caspase3,PTEN and p-P65 were detected by Western blot.The HE staining was used to evaluate the rat liver graft situation.Results After liver transplantation,the expression levels of Notch1 and Jagged1 were the highest at 24 h.The levels of ALT,AST and TBiL in the shRNA-Jagged1 group were significantly higher than those in the NS group and shRNA-GFP group(P<0.05);the rejection reaction score of the shRNA-Jagged1 group was significantly higher than that of the NS group(P<0.05).The levels of IL-1 and IL-6 in the shRNA-Jagged1 group were significantly higher than those in the NS group,while the levels of IL-10 and TGF-βwere significantly lower than those in the NS group,and the differences were statistically significant(P<0.05).Compared with the NS group,the expression levels of p-JNK,cleaved-caspase3,PTEN and p-P65 in the shRNA-Jagged1 group were significantly increased,while the expression level of p-AKT was significantly reduced.There was no statistically significant difference between the NS group and shRNA-GFP group(P>0.05).Conclusion Blocking the liver Notch1/Jagged1 pathway aggravates the inflammatory response damage of liver transplantation possibly by activating the PTEN/AKT/TLR4 pathway and activating the JNK pathway to aggravate liver cell apoptosis and cause acute rejection after liver transplantation.