| Background and ObjectiveCerebral ischemia-reperfusion injury（CIRI）refers to the restoration of blood perfusion after the brain tissue is temporarily ischemic,the damage to its biological function and tissue structure will further increase,and even permanent damage will occur.The pathophysiological mechanism of CIRI is related to multiple mechanisms,including excitotoxicity,inflammatory response,oxidative damage,Ca2+overload,and apoptosis.Among them,the mechanism of inflammatory response is closely related to CIRI,and it has received extensive attention from researchers in recent years.A large number of studies have shown that the inflammatory response caused by ischemic injury will further aggravate nerve cell damage and dysfunction,and anti-inflammatory treatment can improve nerve function.The inflammatory response is characterized by the recruitment and activation of innate immune and adaptive immune system related immune cells.After cerebral ischemia,the damaged neurons release purer（ATP/UTP）,high mobility group protein 1（HMGB1）,heat shock protein,peroxidase and mitochondrial-derived N-formyl peptide Danger-related molecular pattern（DAMP）,a pattern-related receptor（PRR）that activates microglia,astrocytes,and surrounding immune cells.The expression of these receptors is increased after activation by secreting MMP-9 and TNF-αAnd IL-1βand other inflammatory factors mediate systemic inflammatory response syndrome.Dectin-1/Syk/NF-κB signaling pathway is an important pathway for mediating inflammatory response in vivo.Studies have shown that this pathway plays an important media role in activities such as cell proliferation,differentiation,stress,inflammation and autoimmune regulation.Dectin-1 is a pattern recognition receptor on the cell,when it is activated,it recruits Syk to cause PLCγ2 phosphorylation,which induces the formation of CARD9-Bcl10-Malt1 complex;activated Syk can also directly activate NIK,Both can induce IKK complex to release NF-κB and regulate gene transcription.The activated Dectin-1 receptor mediates the secretion of pro-inflammatory factors TNF-α,IL-6 and other factors involved in the regulation of inflammation.Studies have found that in the mouse MCAO model,the expression of Dectin-1,Syk and p-Syk is significantly increased after ischemia-reperfusion,and the volume of cerebral infarction and nerve function are significantly improved after using Dectin-1 inhibitor Laminarin.Studies have also shown that Dectin-1 signaling channel blockers can significantly down-regulate the expression and secretion of pro-inflammatory cytokines,and are accompanied by a decrease in cardiomyocyte apoptosis.This indicates that there is a certain relationship between Dectin-1/Syk/NF-κB signal pathway and CIRI,and inhibiting the activation of this pathway will weaken the inflammation after ischemia.Ulinastatin（UTI）is a glycoprotein extracted and purified from the fresh urine and blood of adult men.It is an inherent serine protease inhibitor and is mainly synthesized in the liver.It has strong anti-inflammatory and anticoagulant activity and is widely used clinically for patients with acute inflammatory diseases,such as disseminated intravascular coagulation,shock,organ transplantation,severe burns,severe pancreatitis etc.Multiple studies have shown that ulinastatin can reduce the inflammatory response induced by cerebral ischemia-reperfusion injury,thereby improving the neurological function score,reducing brain tissue edema and reducing the volume of cerebral infarction.However,it has not been reported whether Ulinastatin can reduce brain function damage caused by ischemia-reperfusion by regulating Dectin-1/Syk/NF-κB signaling pathway when cerebral ischemia reperfusion injury occurs.In this study,adult SD rats were selected to establish a focal ischemia-reperfusion model（MCAO model）for left middle cerebral artery embolism,with 2 hours of ischemia and 24 hours of reperfusion.Immediately after ischemia,ulinastatin was pretreated intravenously to observe neuroethological changes,brain edema and cerebral infarction volume changes,serum TNF-αand IL-6 levels,and cerebral cortex Dectin-1,Syk and NF-κB protein expression,to explore its possible mechanism of action,to provide a new theoretical basis for clinical research and treatment of stroke.Methods120 adult healthy male SD rats,230～280g（6-8 weeks old）,were randomly divided into normal group（N group,n=15）;sham operation group（S group,n=15）;ischemia-reperfusion Group（I/R group,n=15）;I/R+DMSO group（Vehicle group,n=15）;I/R+Ulinastatin group（U group,n=15）;I/R+Laminarin（Dectin-1 inhibitor）group（LAM group,n=15）;I/R+R406（Syk inhibitor）group（R406 group,n=15）;I/R+PDTC（NF-κB inhibitor）group（PDTC group,n=15）.No operation was performed in group N,and only neck blood vessels were separated in group S,and no arterial embolization was performed.The remaining experimental groups were prepared with a left middle cerebral artery embolization model（MCAO）of rats,which was ischemic for 2 hours and reperfused for 24 hours.The vehicle group was injected intraperitoneally with the same amount of each blocker solvent dimethyl sulfoxide（DMSO）1h before modeling,the LAM group was intraperitoneally injected with Dectin-1 blocker Laminarin 1h before modeling,and the R406 group was injected 1h before modeling Syk blocker R406 was injected intraperitoneally,and PDTC group was intraperitoneally injected with NF-κB blocker PDTC 1h before modeling.Immediately after the start of ischemia,U group was injected with 100,000 U/kg via tail vein,and I/R group,Vehicle group,LAM group,R406group,PDTC group were injected with equal volume of normal saline.At 24h of cerebral ischemia-reperfusion,the neurological deficit score（NDS）of each group of rats was evaluated,the volume ratio of cerebral infarction was determined by TTC staining,the brain moisture content was calculated by electronic balance weighing dry and wet weight of brain tissue,and the serum TNF-αand IL-6 were determined by ELISA,and the expression of Dectin-1,Syk and NF-κB protein in ischemic brain tissue was determined by Western Blot method.Results1、The neurological deficit scores of rats in each group:Compared with the S group,the NDS scores of rats in other experimental groups have increased to varying degrees（P<0.05）.Compared with the I/R group,there was no statistically significant difference in the NDS scores of the Vehicle group（P>0.05）;while the NDS scores of the U group,LAM group,R406 group and PDTC group all decreased significantly（P<0.05）.Compared with the U group,there was no statistically significant difference between the LAM group,R406 group,and PDTC group（P>0.05）.2、Cerebral infarction volume of rats in each group after ischemia-reperfusion for 24h:Group N and Sh had no cerebral infarction,and the remaining groups had cerebral infarction in varying degrees（P<0.05）.Compared with the I/R group,the cerebral infarction volume of the U group,LAM group,R406 group and PDTC group was significantly reduced（P<0.05）.Compared with group U,the cerebral infarction volume of rats in LAM group,R406 group and PDTC group increased（P<0.05）.3、The water content of the brain tissue of rats in each group during 24 hours of ischemia-reperfusion:Compared with the S group,the water content of the brain tissue of the other experimental groups increased significantly（P<0.05）.Compared with the I/R group,the water content of the brain tissue of the U group,LAM group,R406 group and PDTC group decreased（P<0.05）.Compared with the U group,the water content of brain tissue in the LAM group,R406 group and PDTC group increased（P<0.05）.4、Serum TNF-αand IL-6 levels in rats of each group after ischemia-reperfusion for 24h:Compared with group S,serum TNF-αin I/R group,Vehicle group,U group,LAM group,R406 group and PDTC group The content ofαand IL-6 increased significantly（P<0.05）.Compared with the I/R group,the levels of serum TNF-αand IL-6in the U group,LAM group,R406 group and PDTC group were significantly reduced（P<0.05）.Compared with group U,the levels of serum TNF-αand IL-6 in LAM group,R406 group and PDTC group increased（P<0.05）.5、The expression of Dectin-1,Syk and NF-κB protein in the cerebral cortex of rats in each group after ischemia and reperfusion for 24h:At 24h of ischemia-reperfusion,the cerebral cortex of each group expressed Dectin-1,Syk and NF-κB protein.Compared with the S group,the expression of Dectin-1,Syk,and NF-κB protein in the I/R group and Vehicle group were significantly increased（P<0.05）;the expression of Dectin-1 and Syk in the U group also increased（P<0.05）,but the expression of NF-κB protein increased but not obvious（P>0.05）.Compared with the I/R group,the expression of Dectin-1,Syk and NF-κB protein in the U group and LAM group were significantly reduced（P<0.05）;the expression of Dectin-1 protein in the R406 group was not significantly changed（P>0.05）,Syk and NF-κB protein were significantly reduced（P<0.05）;PDTC group Dectin-1 and Syk protein expression had no significant changes（P>0.05）,NF-κB protein expression was significantly reduced（P<0.05）.Conclusion1.Intravenous administration of ulinastatin 100,000U/kg during cerebral ischemia can reduce the volume of cerebral infarction and the degree of brain tissue edema in rats with cerebral ischemia for 2 hours and reperfusion for 24 hours,and improve the score of neurological deficit.2.Ulinastatin can down-regulate the expression of Dectin-1,Syk,NF-κB protein in ischemic brain tissue,reduce the serum IL-6 and TNF-αcontent,suggesting that Ulinastatin can inhibit Dectin-1/Syk/NF-κB signaling pathway reduces the inflammatory response induced by cerebral ischemia-reperfusion,thereby reducing cerebral ischemia-reperfusion injury. |
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