Blocking Kupffer Cells Notch1/Jagged1 Pathway Aggravates Mouse Liver Transplantation Ischemia-reperfusion Injury

Object:Kupffer cells(KCs)play a dual role in promoting rejection and inducing immune tolerance in liver transplantation immunity,and the exact mechanism is still unclear.It has recently been found that the Notch1/Jagged1 pathway is involved in many immune regulatory aspects of the body,and its immune regulation mechanism may be stronger than the single factor regulatory factor,but the study of the Notch1/Jagged1 pathway has not yet involved the transplantation field.Therefore,this study aimed to select liver KCs as target cells,and use Notch1/Jagged1 pathway as the entry point to explore the specific molecular mechanism of Notch1/Jagged1 pathway through the activation of inflammatory response to KCs,and then induce the formation of liver immune tolerance.And use this effect to propose possible treatments for related diseases.Method:A modified Kamada's "two-sleeve cannula" method was used to establish an acute rejection model of orthotopic liver transplantation in C57BL/6?C3H mice.The experimental animals were randomly divided into 4 groups: sham group,NS group,AAV-GFP group,AAV-Jagged1 group.Each group was injected with adeno-associated virus again through the tail vein 2 weeks before surgery.The mice were given anesthetized blood for 0.5 mL at 3 h,6 h,and 24 h after transplantation.The mice were sacrificed and the middle lobe liver tissue was removed.The KCs of each group were extracted and the expression of KCs Notch1/Jagged1 was detected by immunofluorescence.The serum sAST and sALT of each group were detected by biochemical test.The apoptosis of liver tissue was detected by TUNEL method.The pathological changes of liver tissue of each group were detected by HE.The expression levels of p-JNK and cleaved-caspase3 in liver tissues of each group were detected by Western blot.KCs were extracted in vitro and the expression of Notch1/Jagged1 was verified.In vitro,they were divided into 4 groups,Control group,LPS+NS group,LPS+shRNA-GFP group,LPS+shRNA-Jagged1 group.In order to silence Jagged1 by lentiviral transfection,Western blot and qRT-PCR were used to verify how the Notch1-Jagged1 pathway regulates LPS-induced inflammatory response in vitro.Result:1.(1)The activation number of KCs in liver tissue after mouse transplantation gradually increased with time.The relative expression levels of KCs Notch1/Jagged1 protein and mRNA were significantly increased at 3h,6h and 24 h after operation,and higher than that of sham group(P<0.05).(2)Compared with the control group,the serum sAST and sALT levels of AAV-Jagged1 group were significantly increased(P<0.05).The liver injury score of AAV-Jagged1 group was significantly higher than that of the control group(P<0.05).The number of apoptotic cells in AAV-Jagged1 group was significantly higher than that in the control group(P<0.05).The expression levels of p-JNK and cleaved-caspase3 in AAV-Jagged1 group were significantly higher than those in control group(P<0.05).2.The KCs cultured in vitro under the stimulation of LPS(100ng/ml)had immunofluorescence.The expression of Notch1-Jagged1 was significantly higher than that of the control group by Western blot and qtPCR(P<0.05).The secretion of inflammatory factors was significantly increased in the shRNA-Jagged1 group under LPS stimulation(P<0.05).Using shRNA-Jagged1 to silence Jagged1 in KCs,we found that the expression of Notch and Hes1 was decreased in the shRNA-Jagged1 group 24 hours after LPS treatment,but not in the control group(P<0.05).After knocking out Jagged1 by shRNA-Jagged1 pretreatment,Hes1 and p-AKT in LPS-stimulated KCs were significantly inhibited(P<0.05).However,the expression of PTEN,TLR4 and NF-?B was up-regulated in the shRNA-Jagged1 group(P<0.05).Further downstream inflammatory factors,the expression of IL-1? and TNF-? in shRNA-Jagged1 group were significantly increased(P<0.05).Conclusion:1.Blocking KCs Notch1-Jagged1 may increase the release of inflammatory factors by affecting the PTEN/AKT/TLR4/NF-?B pathway,aggravating the damage of liver tissue rejection.2.After transplantation,blocking Notch1-Jagged1 in KCs aggravates apoptosis of liver cells by activating JNK signaling pathway.