| Part Ⅰ Effects of tissue kallikrein on cerebral ischemia/reperfusion in diabetic ratsObjective:Ischemic stroke has been a life-threatening subtype of neurovascular disease.Diabetes mellitus(DM)reduces the tolerance to ischemic insults.Stroke survivors with diabetes have a poorer prognosis compared with non-diabetics.This part aimed to assess whether tissue kallikrein(TK)could ameliorate cerebral ischemia/reperfusion(I/R)injury in diabetic ratsMethods:Healthy male Sprague-Dawley rats were made to be diabetes by injecting of streptozotocin intraperitoneally combined with high glucose and fat diets.The model of focal cerebral ischemia reperfusion was made with an intraluminal vascular occlusion method.All the rats were randomly divided into six groups:sham operation;saline control,tissue kallikrein group,pretreatment of B1R antagonist combined with TK,pretreatment of B2R antagonist combined with TK,pretreatment of B1R antagonist and B2R antagonist combined with TK.Neurological deficit scores,infarct sizes and brain water content were assessed at 24 hours after reperfusion.The Iba1-positive cells and MPO-positive cells were detected by immunohistochemistry.RT-qPCR was performed to observe the expression of TNF-α and IL-1β.Fluoro-Jade C,TUNEL and Cleaved caspase-3 staining was used to detect the cell apoptosis and degeneration.Results:The neuroprotective effects of TK on I/R injury in diabetic rats involved the reduction of brain edema and infarct volume,suppression of inflammation and apoptosis,and amelioration of ischemia-induced behavioral deficits.Additionally,the neuroprotective effects of TK was promoted by a B1R antagonist but were abrogated by treatment with a B2R antagonist.Conclusion:Early administration of TK provides robust resistance to I/R damage in the diabetic brain,which was attributed to the anti-apoptotic and anti-inflammatory effects mediated primarily through B2RPart Ⅱ The protection mechanism of tissue kallikrein against cerebral ischemia/reperfusion in diabetic ratsObjective:To explore the underlying signaling mechanism of TK against cerebral I/R injury in diabetic rats.Methods:1.Western blot was used to observe the expressions of phosphor-ERKl/2(p-ERK1/2),total-ERK1/2(t-ERKl/2),phosphor-CREB(p-CREB)and total-CREB(t-CREB)at 0h,1h,3h,6h and 24h of reperfusion in brain tissues of diabetic rats.Additionally,the levels of ERK1/2,CREB and apoptosis-related molecules such as Bcl-2,Bax of rats in the six group of part I were studied by western blot.2.Diabetic rats were randomly divided into five groups,including saline-treated group,TK-treated group,DMSO-treated group,U0126-treated group and U0126 combined with TK-treated group.Neurological deficit scores,infarct sizes,brain water content,cell apoptosis and degeneration,inflammatory reaction were assessed at 24 hours after reperfusion.The expressions of ERK1/2,CREB,Bcl-2 and Bax were analyzed by western blot.Results:1.TK led to up-regulated expression of p-ERK1/2,p-CREB and Bcl-2 proteins and down-regulated expression of Bax protein in the cerebral I/R injury of diabetic rats.Pre-treatment with the B1R antagonist strengthened the actions of TK on the abovementioned signaling pathways,whereas the B2R antagonist produced the opposite effects on these signaling proteins.2.Compared with the saline group,blockade of ERK1/2 signaling pathway in DM rats not only increased infarct volume,neurological severity scores,brain edema,cell apoptosis and neuron degeneration,but also promoted post-ischemic inflammation.Additionally,pretreatment with U0126 could partially but significantly counteract the efficacy of TK against cerebral I/R injury in diabetic rats.ERK1/2 inhibitor completely suppressed the activation of p-ERK1/2,p-CREB and Bcl-2 and promoted the activation of Bax in TK-treated group,indicating that ERK1/2 may be the main upstream protein in this signaling pathway activated by TK.Conclusion:TK exhibited neuro-protective effects after cerebral I/R in diabetic rats through activating of ERK1/2 signaling pathway. |
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