| Objective:Previous studies showed that the brain impairment of patients with cerebral ischemia injury associated with comorbid diabetes is related to non-specific immunity.This study aimed at exploring the relationship between the activation of complement C3 in the nonspecific immune system and the aggravation of ischemic brain damage by diabetes,and discovering the mechanism of brain impairment of patients with cerebral ischemia injury associated with comorbid diabetes.Methods:A transient middle cerebral artery occlusion(tMCAO)model was used for cerebral ischemia/reperfusion(I/R)injury in streptozotocin-induced diabetic mice.Cerebral infarct volume and neurological function were measured at different times of reperfusion.Complement C3 was measured by ELISA and western blotting.Results:(1)Increased complement C3 activity was associated with cerebral I/R injury in diabetes.The expression of complement C3 in diabetic group was significantly higher than that in non-diabetic group(45.0±2.4 vs.29.7±3.1,p<0.01).After reperfusion for 0 hours(2.1±0.3 vs.1.0±0.2,p<0.01),6 hours(3.8±0.5 vs.3.1±0.3,P<0.01),24 hours(5.6±0.5 vs.4.3±0.5,p<0,01),the expression of complement C3 in the ischemic penumbra was increased significantly,and the expression of complement C3 in diabetic neurons was increased.(2)The deficiency of complement C3 could reduce the ischemic brain injury in diabetes.After cerebral ischemia reperfusion injury,the volume of cerebral infarction in C3-/-mice decreased significantly(43.4±7.8 vs.63.6±6.8,p<0.01).And 2 hours after cerebral ischemia and reperfusion(1.2±0.1 vs.1.2±0.1,p>0.05),6 hours(1.3±0.1 vs.1.5±0.2,p>0.05),12 hours(1.4±0.2 vs.2.5±0.3,p>0.05),and 24 hours(1.9±0.2 vs.3.9±0.4,p<0.01)were also improved compared with the wild type mice.There was a significant difference in the number of cell deaths between the wild type and the complement C3 knockout mice(4.2±5.7 vs.13.4±1.9,p<0.01).The activation of complement C3 enhanced the cell death of oxygen glucose deprivation/reperfusion(32.8±4.3 vs.57.5±9.3,p<0.01).After 3 months of diabetes,the complement C3 knockout mouse TU was compared with the wild type mice.NEL staining showed that the number of nerve cells was significantly different(8.5±1.2 vs.18.6±2.76,p<0.01).(3)The complement C3 participates in the diabetes combined with ischemic brain injury through Toll like receptor.The expression of TLR2 protein in mice after cerebral ischemia is significantly increased(0.73±0.07 vs.0.95±0.08,p<0.05),and the expression of TLR2 mRNA is also significantly increased(0.58±0.06vs.0.98±0.07,p<0.05).In primary C3 knockout mice,the expression of TLR2 decreased significantly after reperfusion for 24 hours(0.51 ±0.07vs.0.93±0.08,P<0.05).The transcriptional activity of NF-kappa B increased significantly(2.47±0.25 vs.0.98±0.08,p<0.05),and the expression of complement C3 promoted TLR2(0.43±0.06 vs.1.00±0.10,p<0.05)through TLR2 silencing also decreased significantly.After TLR2 silencing,P50 protein(0.46±0.05 vs.0.99±0.10,p<0.05)and mRNA level(0.53±0.06 vs.,1.00±0.12,p<0.05)also decreased significantly.(4)Complement C3 inhibitors protect diabetes and ischemic brain injury.Compstatin treatment can reduce the volume of cerebral infarction(47.14±7.62 vs.62.98±6.39 p<0.01)and nerve function score at the beginning of reperfusion(2 hours after reperfusion,1.27±0.12 vs.1.291±0.16,p>0.05;6 hours after reperfusion,1.38±0.19 vs.1.65±0.23,P>0.05;after reperfusion for 12 hours,1.45±0.21 vs.2.54±0.28,p>0.05;after reperfusion for 24 hours,2.03±0.24 vs.3.98±0.40,P<0.01).Conclusion:Thus,complement C3 promotes cerebral I/R injury by TLR2/NF-?B pathway in diabetic mice,and regulating complement C3 may be a novel target for therapeutic intervention in diabetic stroke. |
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