| Objective:1.To analyze the correlation between ROS and NEK7 expression levels in liver ischemia-reperfusion（I/R）models in vivo;2.To observe the effect of NEK7 on expression of NLRP3 inflammasome related molecules,secretion of inflammatory factors,liver histological changes and degree of liver function injury in the liver I/R model in vivo;3.To investigate the regulatory effect of NEK7-mediated ROS on NLRP3 in hepatic Kupffer cells and its molecular mechanism affecting hepatic ischemia-reperfusion injury（IRI）.Method:An in vivo model of hepatic I/R in C57BL/6J mice and an in vitro model of I/R were established by treating RAW264.7 with hypoxia and reoxygenation（H/R）.Related protein expression in liver was tested by western-blot;ELISA was conducted to detect inflammatory cytokines IL-1βand IL-18 of serum;Microplate assay was prepared to measure the expression of AST and ALT of serum;The expression of ROS in liver tissue was labelled by DCFH-CA assay and evaluated by flow cytometry;HE was conducted to assess the liver tissue injury;liver tissue apoptosis was assessed by TUNEL;Double immunofluorescence staining of NEK7and F4/80 or CK18 was presented to observe the expression of NEK7 in hepatocytes and Kupffer cells of liver.Results:1.Compared with Sham group,the expressions of ROS,NEK7,NLRP3,Cleaved-caspase-1,AST,ALT,IL-1βand IL-18 in I1R3,I1R6 and I1R9 groups were up-regulated,and their expressions were gradually increased with the extension of reperfusion time.2.Pearson correlation analysis showed a positive correlation between ROS expression and NEK7 expression in liver（p<0.0001,r=0.8501）.3.Immunofluorescence double labeling showed that NEK7 in liver tissue overlapped with F4/80,but not with CK18.4.Compared with the I/R group,the expressions of NEK7,Cleaved-caspase-1,AST,ALT,IL-1βand IL-18 in the I/R+siNEK7 group were down-regulated（p<0.01;p<0.01;p<0.05;p<0.05;p<0.05;p<0.05）,and attenuated the liver tissue injury,while there was no significant difference between I/R and I/R+siRNA control groups（p>0.05）.5.Compared with the control group,the expressions of intracellular ROS,NEK7,NLRP3 and Cleaved-caspase-1 in the H/R group were significantly increased,and the release of inflammatory factors IL-1β and IL-18 in the cell supernatant was increased,with statistically significant differences（p<0.05;p<0.01;p<0.001;p<0.001;p<0.001;p<0.001）.Compared with the H/R group,the expressions of NEK7 and Cleaved-caspase-1 in cells of the H/R+siNEK7 group were significantly reduced,and the release of inflammatory factors IL-1β and IL-18 in the cell supernatant was reduced,with statistically significant differences（p<0.001;p<0.001;p<0.001;p<0.001）;However,there was no significant difference in the expression of ROS and NLRP3,and the difference was not statistically significant（p>0.05）.6.Compared with the H/R group,the expressions of NLRP3 and Cleaved-caspase-1 in cells of the H/R+siNLRP3 group were significantly reduced,and the release of inflammatory factors IL-1βand IL-18 in the cell supernatant was reduced,with statistically significant differences（p<0.001;p<0.001;p<0.001;p<0.001）;However,there was no significant difference in the expression of NEK7,and the difference was not statistically significant（p>0.05）.7.Compared with the H/R group,the expression of Cleaved-caspase-1in cells in the H/R+Ac-YVAD-CMK group was significantly reduced,and the release of inflammatory factors IL-1βand IL-18 in the cell supernatant was reduced,with statistically significant differences（p<0.001;p<0.001;p<0.001）;The expression of NLRP3 and NEK7 showed no significant difference,and the difference was not statistically significant（p>0.05）.8.Compared with the allopurinol group,the expression of NEK7 protein was increased in the allopurinol+rNEK7 group（p<0.001）,while the expression of NLRP3 and Cleaved-caspase-1 protein and the release of inflammatory factors IL-1βand IL-18 in the cell supernatant showed no difference（p>0.05）.Compared with the H2O2 group,the protein expressions of NEK7 and Cleaved-caspase-1 in cells of the H2O2+siNEK7 group were decreased,and the release of inflammatory factors IL-1βand IL-18 in the cell supernatant was reduced（p<0.001）,while the protein expression of NLRP3 in cells remained unchanged（p>0.05）.Conclusion:1.ROS is positively correlated with NEK7 expression in liver.2.NEK7 is involved in the positive regulation of hepatic IRI in mice,and this effect is mainly mediated by NEK7 in hepatic Kupffer cells rather than NEK7 in hepatocytes.3.Inhibiting the expression of NEK7 can reduce the release of inflammatory cytokines by inhibiting the activation of inflammasome NLRP3,leading to the alleviation of liver function,the alleviation of hepatic tissue injury and the reduction of hepatic cell apoptosis after hepatic IRI.4.NEK7 is a key molecule of the liver IRI induced by ROS induced NLRP3 inflammasome activation. |
