The Study On Neuroprotective Effects Of Ulinastatin On Focal Cerebral Ischemia/reperfusion Injury By Regulating TLR4/NF-κB And Nrf2-ARE Signaling Pathway In Mice

Acute ischemic stroke is a leading cause of death and the mostfrequent cause of permanent disability in adult worldwide.Due to the lack of effective treatment and poor prognosis,it is still a critical medical and social problem.Acute cerebral infarction lesions are composed of ischemic core and penumbra.In penumbra restoring blood flow as early as possible and using neuroprotectant are key factors for improving neurologic outcome and decreasing the deformity rate.But restoring blood flow in penumbra will injury brain,and it is called ischemia reperfusion injury(IRI).The mechanisms of IRI are complex.The events that follow during I/R: calcium overload,free radical generation,oxidative stress,inflammation and neuron apoptosis may be involved.Inflammation is an attractive point in the research of IRI pathophysiological mechanism.Inflammation is a part of the innate immune response and it provides a rapid defense against microbes.Innate immune cells such as monocytes and macrophages produce inflammatory mediators like toll-like receptors(TLRs)in response to the presence of microbes following engagement of microbial components with pattern recognition receptors(PRR)expressed by the cells.TLRs family is widely expressed in various cells,including epithelial cells,endothelial cells,mesothelials,fibroblasts,neutrophils,T lymphocytes,natural killer cells,macrophages etc,and they play an important role in innate immunity and adaptive immunity.The first TLR to be described in humans was TLR4.It was reported that TLR4,the nuclear factor-kappa B(NF-κB),and the myeloid differentiation primary-response protein 88(MyD88)activated as mechanisms relating to the cerebral ischemia pathogenesis.MyD88-TLR4 complexes activate inflammatory cytokines and culminate in NF-κB,a transcription factor that regulates the expression of a wide array of genes involved in immune responses,such as mitogen-activated protein kinases(MAPKs),then they took part in IRI inflammatory mechanism.p38 MAPK signaling pathway was hot aspect in IRI pathogenesis.Among the mechanisms that lead to IRI,oxidative stress injury is the main mechanism and an important target for the clinical treatment of ischemic stroke.Nuclear factor erythroid 2-related factor 2(Nrf2)-antioxidant response element(ARE)signaling pathway,as one of the main mechanisms of cells against oxidative stress,has become the hot topics in the study of ischemic stroke.Nrf2 proteins combines with Keap1 protein in the cytoplasm and it can not play its transcriptional activity in the nucleus.In oxidative stress reaction Nrf2 protein is activated and dissociated from Keap-1.Then it combines with antioxidant response element after nuclear transition.This process promotes a variety of antioxidant enzymes’ expression,such as heme oxygenase-1(HO-1)and NAD(P)H quinone oxidoreductase(NQO-1),GSH-S-transferase(GST),superoxide dismutase 1(SOD1).The antioxidant enzymes expression plays a anti-oxidative effect.As well known blood-brain Barrier(BBB)is key factor for central nervous system homeostasis.The brain microvascular endothelial cells and tight junction between them are the important parts of BBB.The permeability of BBB is related with TJ’s structure and function.The tight junction protein ZO-1 and occluding are the important parts of TJ,and they can regulate the permeability of BBB.Matrix metalloproteinases(MMPs)is the crucial proteolytic enzymes mediating degradation of extracellular matrix(ECM).Particularly,elevated MMP-9 activity is associated with vascular permeability.It has been documented that elevated MMP-9 activity and loss of ZO-1 and occludin proteins cause brain edema and neuron death after ischemia reperfusion.Ulinastatin(UTI),a glycoprotein and multivalent enzyme inhibitor,purified from fresh healthy human urine.It had been found that UTI could be useful for treating acute inflammatory and autoimmune disorders,such as ulcerative colitis and crescentic glomerulonephritis.The neuroprotective mechanism of Ulinastatin on focal cerebral ischemia/reperfusion injury needs to be researched deeply.This study is to investigate possible neuroprotection of UTI against focal cerebral I/R injury inducedby transient middle cerebral artery occlusion(tMCAO)in mice.Neurological deficit scores,brain water content,infarct volume were measured at 24 h after reperfusion.Immuno-histochemistry staining,Western blot and RT-PCR were used to analyze the expressions of TLR4,NF-κB,p-p38 MAPK,Nrf2,NQO-1,HO-1,MMP-9,ZO-1,occluding in ischemic brain cortex.We detectits targeted and protectivepathway so as to offer important experimental basis for IRI neuroprotection.Part one Building cerebral ischemia reperfusion injury model in miceObjective: In this part,we builded cerebral ischemia reperfusion injury model in mice,and it is the basis of IRI research.Methods:60 adult male CD1 mice(25-30g)were divided into two groups randomly: Sham-operated group,I/R group.I/R group: in which mice were subjected to 60-min middle cerebral artery occlusion(MCAO)by modified Longa suture method.Reperfusion was achieved by withdrawing the suture after 60 min of occlusion to restore blood supply to the MCA territory.Sham group: sham-operated group,in which mice was underwent the same surgical procedure without inserting a filament.A modified five point scale was used to assess neurologicalimpairment.Infarct volume was measured at 24 h after reperfusion by TTC staining using image analysis software(Image-Pro Plus5.1).Brain water content was measured using the standard wet/dryweight method at 24 h after reperfusion.Results: Compared with sham group,mice in the I/R group had the higher neurological deficit score(P<0.001).At 24 h after reperfusion infarct volume was determined by 2,3,5-triphenyltetrazolium chloride(TTC).No infarction was observed in Sham group.Extensive lesion was found in both striatum and cortex in I/R group.There was significantly difference between I/R group and sham group(P<0.001).Compared with sham group,ipsilateral brain water content of I/R group was considerably increased(P<0.001).Conclusion: Themodified Longa suture method was efficient and easy method to master to make brain ischemia-reperfusion injury model in mice.And the model could be used in IRI research.Part two Experimental study on the effects of TLR4/NF-κB signaling pathway in neuroprotective effects of Ulinastatin on focal cerebral ischemia/reperfusion injury in miceObjective: This study is to investigate the potential neuroprotection of UTI on focal cerebral ischemia-reperfusion injury in mice,and explore whether the TLR4/NF-κB pathway mediated inflammation is involved in this protection in the acute phase of cerebral I/R in mice.Methods: Male CD1 mice were subjected to transient middle cerebral artery occlusion(tMCAO)and randomly assigned into four groups: Sham(sham-operated)group,I/R(tMCAO+0.9% saline)group,UTI-L(tMCAO+ UTI 1500 U/100g)and UTI-H(t MCAO+UTI 3000 U/100g)group.UTI was administered immediately after reperfusion both the UTI-L and UTI-H groups.I/R and Sham groups received equal volume 0.9 % normal saline.At 24 h after reperfusion,neurological deficit,brain water content and infarct volume were detected.Immunohistochemistry,western blot and quantitative reverse transcription polymerase chain reaction(RT-qPCR)were used to detect the expression of TLR4,NF-κB,p-p38 MAPK in ischemic cerebral cortex.Results: Compared with I/R group,both UTI-L and UTI-H groups dramatically ameliorated neurological deficit(P<0.001),relieved brain water content(P<0.001)and infarct volume(P<0.001).The number of positive cells in the ischemic cortex of I/R mice was significantly increased compared with Sham group(P<0.001).In UTI-L and UTI-H groups,the number of positive cells of TLR4,NF-κB and p-p38 MAPK was significantly decreased than the I/R group(P<0.001).Results from Western blot analysis and RT-qPCR analysis show that at protein level and the mRNA level of TLR4,NF-κB and p-p38 MAPK were significantly increased in the I/R group compared with sham group(P<0.001).Compared with I/R group,the levels of TLR4,NF-κB and p-p38 MAPK was significantly decreased in UTI-L and UTI-H groups(P<0.001).Conclusion:Systemicadministration ofUTI decreases neurologicimpairment and tissue injury after cerebral ischemia,and it may be via inhibiting the TLR4/ NF-κB signaling pathway thereby inhibitinginflammatory reaction.Part three Experimental study on the effects of Nuclear factor erythroid 2-related factor 2(Nrf2)-antioxidant response element(ARE pathway in neuroprotective effects of Ulinastatin on focal cerebral ischemia/reperfusion injury in miceObjective: This study is to investigate the potential neuroprotection of UTI on focal cerebral ischemia-reperfusion injury in mice,and explore whether the Nrf2-ARE pathway mediated anti-oxidative effect is involved in this protection in the acute phase of cerebral I/R in mice.Methods: Male CD1 mice were subjected to transient middle cerebral artery occlusion(tMCAO)and randomly assigned into four groups: Sham(sham-operated)group,I/R(tMCAO+0.9% saline)group,UTI-L(tMCAO+ UTI 1500 U/100g)and UTI-H(t MCAO+UTI 3000 U/100g)group.UTI was administered immediately after reperfusion both the UTI-L and UTI-H groups.I/R and Sham groups received equal volume 0.9 % normal saline.At 24 h after reperfusion,neurological deficit,brain water content and infarct volume were detected.Immunohistochemistry,western blot and quantitative reverse transcription polymerase chain reaction(RT-qPCR)were used to detect the expression of Nrf2、NQO-1、HO-1 in ischemic cerebral cortex.Results: Compared with I/R group,both UTI-L and UTI-H groups dramatically ameliorated neurological deficit(P<0.001),relieved brain water content(P<0.001)and infarct volume(P<0.001).The number of positive cells in the ischemic cortex of I/R mice was significantly increased compared with Sham group(P<0.001).In UTI-L and UTI-H groups,the number of positive cells of Nrf2,HO-1 and NQO-1 was significantly increased than I/R group(P<0.001).Results from Western blot analysis and RT-qPCR analysis show that at protein levels and the mRNA level of Nrf2,NQO-1,HO-1were significantly increased in UTI-L and UTI-H groups than I/R group(P<0.05).Conclusion:Systemicadministration ofUTI decreases neurologicimpairment and tissue injury after cerebral ischemia,and it may be via the activation of the Nrf2-ARE pathway then playing its anti-oxidative stress effect.Part four Effect of Ulinastatin on permeability and integrity of BBB in neuroprotective effects on focal cerebral ischemia/reperfusion injury in miceObjective: This study is to detect the effect of Ulinastatin on permeability and integrity of BBB,and it may be involved in the potential neuroprotection of UTI on focal cerebral ischemia-reperfusion injury in mice.Methods: Male CD1 mice were subjected to transient middle cerebral artery occlusion(tMCAO)and randomly assigned into four groups: Sham(sham-operated)group,I/R(tMCAO+0.9% saline)group,UTI-L(tMCAO+ UTI 1500 U/100g)and UTI-H(t MCAO+UTI 3000 U/100g)group.UTI was administered immediately after reperfusion in both UTI-L and UTI-H groups.Sham and I/R groups received equal volume 0.9 % normal saline.At 24 h after reperfusion,brain water content and infarct volume were detected.Western blot and quantitative reverse transcription polymerase chain reaction(RT-qPCR)were used to detect the expression of MMP-9,ZO-1 and occludin in ischemic cerebral cortex.The loss of BBB integrity was assessed by leakage of Evans blue.Results: Compared with I/R group,Evans blue leaked of brain tissues was significantly decreased in UTI-L and UTI-H groups(P<0.001).Western blot and RT-qPCR analysis show that at protein and mRNA levels ZO-1 and occludin were significantly increased in both UTI-L and UTI-H groups(P<0.05)than I/R group.We observed significantly elevated protein and mRNA levels of MMP-9 in the ischemic cortex in I/R group compared with the sham group(P<0.001),and administration of UTI significantly(P<0.001)suppressed MMP-9 protein expressions triggered by I/R.Conclusion:Systemicadministration ofUTI decreases neurologicimpairment and tissue injury after cerebral ischemia by down-regulation of MMP-9 and alleviation of loss of ZO-1 and occludin proteins.This ameliorates BBB permeability.Conclusion:1.Systemic administration of UTI decreases Neurological deficit scores,brain water content,infarct volume and ameliorates BBB permeability after I/R in mice.2.The neuroprotection of UTI against focal cerebral I/R injury may be via inhibiting the TLR4/ NF-κB signaling pathway thereby inhibiting inflammatory reaction and activating of the Nrf2-ARE pathway then playing its anti-oxidative stress effect.It down-regulates of MMP-9 and alleviates the loss of ZO-1 and occludin proteins.This ameliorates BBB permeability.