【作者】 马平；

【导师】 程远；

【作者基本信息】 重庆医科大学 ， 外科学， 2023， 硕士

【摘要】 背景与目的:创伤性脑损伤(Traumatic brain injury,TBI)是一种致残、致死率较高的疾病,其发病率逐年上升。TBI的病理生理包括原发性损伤和继发性损伤。然而,目前基于原发性损伤和继发性损伤的治疗仍未有突破性进展,导致重症TBI的预后不佳。究其原因除了原发性机械打击造成的脑组织损伤外,TBI后继发性损伤引起脑水肿、挫伤出血进展、神经炎症及神经凋亡等病理生理改变,将导致颅内压升高、脑血流量降低,脑疝发生以及细胞死亡。而这些改变被认为是TBI治疗的关键环节,成为继发性脑损伤药物治疗的焦点。血脑屏障(Blood-Brain barrier,BBB)和脑微血管内皮细胞(Brain microvascular endothelial cell,BMVEC)的损伤是继发性损伤的重点,与血管源性脑水肿、挫伤出血进展、细胞炎症免疫浸入等病理生理变化密切相关,加重脑组织损伤,严重影响TBI患者神经功能及预后。瞬时受体电位离子通路4(Transient receptor potential melastatin 4,TRPM4)是近年发现的一个新的与BBB损伤密切相关的蛋白,它的激活与TBI后的BBB和BMVEC的损伤有着密切的相关性。目前发现TBI后,TRPM4大量表达于血管内皮细胞、胶质细胞、神经元、周细胞等,不仅促使离子平衡失调和流动紊乱,还严重影响BBB和BMVEC的完整性,导致水流出异常,引起细胞肿胀和死亡。9-菲酚(9-phenanthrol)是新近发现针对TRPM4通道的抑制剂。大量体外研究证实9-菲酚对TRPM4的抑制具有较高的特异性。在一些体外实验中,9-菲酚可以抑制90%的TRPM4介导的离子跨膜流动。据此,本研究提出9-菲酚可能是一种极具潜力的TBI治疗药物,探究9-菲酚在TBI继发性损伤中的作用,有望成为治疗TBI的一种新策略与途径。方法:采用自由落体法建立大鼠TBI模型。将实验动物分为假手术组(Sham组),脑外伤组(TBI组),脑外伤+溶剂组(Veh组),脑外伤+9-菲酚组(9-PH组),脑外伤+PI3K抑制剂组(LY294002组)。应用行为学量化评分表、脑组织含水量、伊文思蓝渗透实验和H&E染色、尼氏染色评估损伤程度;蛋白免疫印迹(Western blot,WB)检测TRPM4、基质金属蛋白酶-9(Matrix metallopeptidase 9,MMP-9)、PI3K/AKT/NF-k B信号通路蛋白,紧密连接相关蛋白(ZO-1、Occludin、Claudin-5),凋亡相关指标(Bax和Bcl2)和炎症相关介质(TNF-α和IL-6)的表达;明胶酶谱实验检测MMP-9活性;免疫荧光(Immunofluorescence,IF)染色观察BMVEC损伤程度和神经元凋亡程度、胶质细胞活化和中性粒细胞浸润情况;酶联免疫吸附实验(Enzyme linked immunosorbent assay,ELISA)方法检测血清磺酰脲受体—1(Sulfonylurea receptor 1,SUR1)和TRPM4含量水平。结果:(1)9-菲酚治疗显著改善大鼠TBI后的神经功能评分。(2)在病理层面,9-菲酚治疗显著减少BMVEC损伤和脑微血管的碎裂,改善BBB的通透性,并且降低脑组织含水量和减少挫伤出血进展;另外,9-菲酚还可以抑制炎症介质释放,减少胶质细胞的活化和中性粒细胞的浸润,抑制细胞凋亡,促进神经元存活。(3)在分子层面,9-菲酚治疗降低损伤周围组织TRPM4和MMP-9表达,抑制MMP-9酶活性,减少BBB紧密连接蛋白(ZO-1、Occludin、Claudin-5)丢失,并上调Claudin-1表达;9-菲酚治疗还明显降低PI3K P85、AKT P55、NF-k B P65磷酸化程度,同时在使用PI3K抑制剂LY294002治疗后,MMP-9表达也显著降低。结论:本研究表明,9-菲酚具有良好的BBB保护和修复作用,从而起到减轻脑水肿,减轻BMVEC损伤,改善神经功能缺损和减轻炎症反应的作用,其部分机制如下:(1)9-菲酚抑制TBI后TRPM4表达,可减轻BMVEC损伤;(2)9-菲酚通过抑制PI3K/AKT/NF-k B信号通路抑制MMP-9蛋白表达,减少MMP-9对BBB的基底膜的破坏,从而抑制血管源性脑水肿、挫伤出血进展等。更多还原

【Abstract】 Objective: Traumatic brain injury(TBI)is a serious disease with high morbidity and mortality,and its incidence rate is increasing year by year.The pathophysiology of TBI includes primary injury and secondary injury.However,there has been no breakthrough in the treatment of primary and secondary injuries,leading to poor prognosis in severe TBI.In addition to the tissue damage caused by primary mechanical impact,the secondary injury of TBI can cause pathological and physiological changes such as cerebarl edema,hemorrhagic progression of contusion,neuroinflammation,and neuronal apoptosis,leading to increased intracranial pressure,decreased cerebral blood flow,cerebral hernia,and cell death.These changes are considered a key link in the treatment of TBI and have become the focus of drug therapy for secondary brain injury.Disruption of blood-brain barrier(BBB)and brain microvascular endothelial cells(BMVEC)is the key process in secondary injury.It participates in pathological and physiological processes such as vascular cerebral edema,hemorrhagic progression of contusion,immune cells infiltration,and can exacerbate tissue injury,seriously affecting the neurological prognosis of TBI patients.Transient receptor potential melastatin 4(TRPM4)is a newly discovered protein closely related to BBB injury.Activation of TRPM4 plays an important role in BBB and BMVEC injuries.After TBI,TRPM4 is largely expressed in vascular endothelial cells,glial cells,neurons,pericyte,etc.,which not only promotes the imbalance of ion balance and flow disorder,but also seriously affects the integrity of BBB and BMVEC,leading to abnormal water outflow,causing cell swelling and death.9-phenanthrol(9-PH)is a newly discovered specific TRPM4 channel inhibitor.Previous in vitro studies have confirmed that9-phenanthrol has a high specificity and sensitivity in inhibiting TRPM4,and it can even block 90% of TRPM4-mediated ions flow in some cell types.Based on these studies,this study proposes that 9-phenanthrene is a highly promising drug for the treatment of TBI,and exploring its effects on TBI may become a new strategy and pathway for the treatment of TBI.Methods: In this study,rat model of TBI was induced using the modified Allen’s method.Group the experimental animals as five groups:sham group,TBI group,TBI + vehicle group,TBI + 9-PH group,TBI +LY294002 group.Evaluating the degree of injury using quantitative behavioral scoring scales,brain tissue water content test,Evans blue extravasation test,H&E staining,and Nissl staining;Western blot was used to detect the expression of TRPM4,Matrix metallopeptidase 9(MMP-9),PI3K/AKT/NF k B pathway protein,tight junction proteins(ZO-1 、Occludin 、 Claudin-5),apoptosis related indicators(Bax and Bcl2)and inflammatory mediators(TNF-α and IL-6);and gelatin zymography was used to detect the activity of MMP-9;Immunofluorescence staining was used to detect the degree of microvascular injury,neuronal apoptosis,glial cells activation,and neutrophils infiltration;Enzyme-linked immunosorbent assay is used to measure serum Sulfonylurea receptor-1(SUR1)and TRPM4 levels.Results:(1)9-phenanthrol treatment significantly improved the neurological function scores of rats after TBI.(2)At the pathological level,9-phenanthrol reduced brain microvascular fragmentation,improved the permeability of the BBB,reduced secondary bleeding and water content of brain tissue;In addition,9-phenanthrol treatment also inhibited inflammations,cells apoptosis,activation of microglia and astrocytes,and attenuated neutrophils infiltration,and promoted neuronal survival.(3)At the molecular level,9-phenanthrol treatment reduced the expression of TRPM4 and MMP-9,inhibited the enzyme activity of MMP-9,reduced the loss of tight junction proteins(ZO-1,Occludin,Claudin-5),and it upregulated the expression of Claudin-1 to promote the repair of the blood-brain barrier in the injured surrounding tissues.Treatment with 9-phenanthrol also reduced the phosphorylation of PI3 K P85,AKT P55,and NF-k B P65 proteins.Moreover,treatment with LY294002 significantly reduced the expression of MMP-9 at rats after TBI.Conclusion:This study indicates that 9-phenanthrol has a good protective and repairing effect on BBB,thereby reducing edema and BMVEC damage,alleviating inflammations,and improving neurological deficits.The possible mechanisms are as follows:(1)9-phenanthrol inhibits the expression of TRPM4 after TBI,which can alleviate BMVEC damage;(2)9-phenanthrol can inhibit the expression of MMP-9 protein by inhibiting the activation of the PI3K/AKT/NF-k B signaling pathway,and reducing the damage of MMP-9 to the BBB,thereby inhibiting vascular cerebral edema,hemorrhagic progression of contusion,and other secondary injuries.更多还原