| Objective: To observe the role of Caspase-1-dependent pyroptosis in CPB global ischemia/reperfusion injury in T2 DM rats by establishing a rat model of type 2 diabetes mellitus(T2DM)and a model of global ischemia/reperfusion under CPB.Methods: To establish a T2 DM rat model,The successful establishment of the model was verified by the glucose tolerance test(IPGTT&OGTT).Twelve normal rats were randomly divided into 2 groups with 6 rats in each group: normal+sham operation group(Nor+S),normal+ischemia/reperfusion group(Nor+I/R);Twelve T2 DM rats were randomly divided into 2 groups with 6 rats in each group: T2DM+sham operation group(T2DM+S),T2DM+ischemia/reperfusion group(T2DM+I/R);Subsequently,24 T2 DM rats were randomly divided into 4 groups with 6 rats in each group:T2DM+ischemia/reperfusion+ROS scavenger group(T2DM+I/R+NAC),T2DM+ischemia/reperfusion+physiological saline control group(T2DM+I/R+NS),T2DM+ischemia/reperfusion+caspase-1 inhibitor group(T2DM+I/R+VX-765),T2DM+ischemia/reperfusion+organic solvent(dimethyl sulfoxide)control group(T2DM+I/R+DMSO).Sham operation group: continuous CPB for 150 minutes;I/R group: CPB for 10 minutes,global ischemia for 30 minutes,and reperfusion for 120 minutes.T2DM+I/R+NAC group and T2DM+I/R+NS group: NAC(150 mg/kg)or NS was continuously infused by tail vein 30 minutes before ischemia,and then I/R treatment was performed;T2DM+I/R+VX-765 group and T2DM+I/R+DMSO group: 30 min before ischemia by intraperitoneal injection of VX-765(16mg/kg)or DMSO(0.5ml/kg),followed by I/R treatment.record mean arterial pressure(MAP),heart rate(HR)and hematocrit(Hct).At the end of reperfusion,the morphological changes of myocardial cells,myocardial infarction area,mitochondrial ROS and caspase-1 contents were observed and compared in each group,as well as NLRP3,pro-caspase-1,caspase-1 p10 and GSDMD protein expression and plasma CK-MB,c Tn I,IL-1β and IL-18 content.Results:(1)OGTT and IPGTT results: The blood glucose increased immediately after gavage or intraperitoneal injection of 20% GS in normal rats and T2 DM rats,but decreased after 30 minutes.Compared with the blood glucose value of normal rats,the blood glucose value of T2 DM rats at each time point has been maintained at a higher level(>16.7mmol/L)(P<0.05).(2)Changes of MAP、HR and Hct: MAP of rats in each group showed a decreasing trend from T0 to T4,but remained above 60 mm Hg;I/R-treated rats achieved complete arrest at time T2,and successfully resumed jumping at time T3,HR close to the corresponding S group at time point T4;the Hct at T1 time point was significantly lower than that at T0 in each group,and there was no significant difference between T1 and T4,and the Hct at each time point was above 20%.(3)Comparison of mitochondrial score and myocardial infarction area:compared with the Nor+S group,the mitochondrial score of the T2DM+S group increased(P<0.05);Compared with the corresponding S group,the mitochondrial score of rats in Nor+I/R group and T2DM+I/R group increased,and the myocardial infarction area increased(P<0.05);Compared with the Nor+I/R group,the mitochondrial score in the T2DM+I/R group increased(P<0.05),and the myocardial infarction area increased(P<0.05);However,the mitochondrial score and myocardial infarction area in T2DM+I/R+NAC group and T2DM+I/R+VX-765 group were lower than those in control group(P<0.05).(4)Evaluation of ROS and caspase-1 content: Compared with the Nor+S group rats,the myocardial ROS and caspase-1 production of the T2DM+S group rats increased(P<0.05);Compared with the corresponding S group,the myocardial ROS and caspase-1 production in the Nor+I/R group and the T2DM+I/R group increased(P<0.05),and the T2DM+I/R group had more than the Nor+I/R group(P <0.05);However,the production of myocardial ROS and caspase-1 in the T2DM+I/R+NAC group were lower than those in the control group(P<0.05).(5)Expression of NLRP3,pro-caspase-1,caspase-1 p10 and GSDMD:Compared with normal rats,the expression of each protein in T2 DM rats increased(P<0.05);Compared with the corresponding S group,the protein expression of the rats in the Nor+I/R group and the T2DM+I/R group increased(P<0.05);among them,the T2DM+I/R group was more than the Nor+I/R group(P<0.05);The T2DM+I/R+NAC group had less protein expression than the control group(P<0.05);while the T2DM+I/R+VX-765 group had no effect on the upstream NLRP3 and pro-caspase-1expressions,The activation of caspase-1 was decreased,and the expression of caspase-1 p10 and GSDMD was decreased(P<0.05).(6)Release of CK-MB,c Tn I,IL-1β and IL-18: Compared with the corresponding S group,the release of Nor+I/R group and T2DM+I/R group was increased(P<0.05);Among them,T2DM+I/R group was more than Nor+I/R group(P<0.05).However,the T2DM+I/R+NAC group and T2DM+I/R+VX-765 group decreased compared with the control group(P<0.05).Conclusion:(1)Pyroptosis was activated during global ischemia/reperfusion under CPB in rats,and T2 DM rats were more activated than normal rats,resulting in more severe MI/RI;(2)Caspase-1-dependent pyroptosis plays a key role in inducing inflammation and injury in global ischemia/reperfusion under CPB in T2 DM rats;(3)The high expression of pyroptosis-related molecules NLRP3 、pro-caspase-1 、 caspase-1 p10 and GSDMD is related to excessive ROS generated by oxidative stress during reperfusion;... |
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