| Intracerebral hemorrhage（ICH）is a common and fatal subtype of stroke due to bleeding from small blood vessel within cerebral parenchyma and subsequent hematomas.Because of Brain injury after ICH results in the high rates of mortality and morbidity,ICH represents a serious threat to public health currently.Although considerable progress made in animal and preclinical studies,we still lack effective therapeutic strategies for ICH.Vascular disruption is the initial cause of ICH and blood–brain barrier（BBB）dysfunction is a secondary consequence.BBB disruption is a hallmark of ICH-induced brain injury and one of the most important pathophysiological changes in the acute injury phase following ICH.ICH-induced an increase in BBB permeability play an important role in perpetuating secondary injury after ICH.Such an increase may occur via changes in the paracellular pathway（alterations in tight junction function）and/or the transcellular route（vesicular transcytosis）across the cerebral endothelium.Numerous studies have demonstrated a variety of causes of BBB dysfunction after ICH,including thrombin,fibrin,erythrocyte components,and inflammatory injury.Therefore,to protect the BBB seems pressingly needed and was considered a promising disease control strategy for the treatment of brain injury at early period after ICH.Major facilitator superfamily domain containing protein 2a（Mfsd2a）,also known as sodium-dependent lysophosphatidylcholine symporter 1,is a protein that in humans is encoded by the Mfsd2a gene.It may also be responsible for uptake and transport of tunicamycin.A previous study show that Mfsd2a is a membrane transport protein that is selectively expressed in blood vessel endothelium constituting the blood–brain barrier and has an essential role in BBB formation and function.And recent studies indicate that Mfsd2a might be an important gene involved in BBB integrity modulation.However,it is unclear whether Mfsd2a participates in the pathophysiology process,especially in BBB disruption,of the early brain injury after ICH.In the present study,we aimed to investigate the role of Mfsd2a plays in ICH-induced BBB injury.We found that downregulation of Mfsd2a in brain blood vessel endothelium increased the cerebrovascular permeability of BBB,brain edema and neurologic deficit score,whereas in a manner that were reversible by Mfsd2a re-expression.Futhermore,our data suggest that endothelial Mfsd2a maybe has a protective role in ICH-induced BBB disruption via inhibition of vesicular transcytosis,which is critical for regulation of homeostasis in the brain.Methods:Part I.Changes of Mfsd2a expression after ICH1.Establishment of the ICH mice model.The mice were killed on 12h,1,3,5 and 7days respectively and the proteins extracted from the brain tissue of the hemorrhagic hemisphere.The expression of Mfsd2a in the tissues around hematoma was analyzed by Western Blot.2.To observe changes of Mfsd2a protein of cerebral hemorrhage in mice brain tissue around hematoma compared with the blank control and the sham operation group at24h/48h/72h time points by immunofluorescence,Western Blot.The trend of time variation was analyzed.3.Part II.Down-regulation of Mfsd2a by short interfering RNA（siRNA）increases BBB permeability and aggravated neurological dysfunction after ICH1.Mfsd2a siRNA was constructed by gene engineering technique and transfected into the mouse brain.The expression of Mfsd2a at different time points in wild-type（WT）mice was detected by WesternBlot.If after si-RNA interference of Mfsd2a expression decreased further,use siRNA to interfere the expression of Mfsd2a in mice after cerebral hemorrhage model,respectively 12h,1,3,5 and 7 days,the mice were sacrificed to take brain protein extraction.The expression of Mfsd2a in the tissues around hematoma was analyzed by Western Blot and immunofluorescence.2.Change the expression and regulation of Mfsd2a activity,the neurological deficit score,wet and dry weight method for the determination of brain water content,EB method to detect BBB permeability after ICH.Investigate the expression of Mfsd2a in cerebral hemorrhage group at 24h/48h/72h time point is increased BBB damage and aggravate brain edema than the control group.Analyze the time correlation and its molecular mechanism.Part III.Mfsd2a-/-mice exhibited significantly increased BBB injury and neurological deficits caused by ICH1.Mfsd2a gene knockout mice were prepared by CRISPR/Cas9 technique,and the expression of Mfsd2a in Mfsd2a knockout mice was verified by immunofluorescence and Western Blot.2.Using knockout mice construct autologous blood model of intracerebral hemorrhage,the expression of various signaling molecules associated with BBB pathways and mRNA were determined by neurological deficit score,wet and dry weight method for the determination of brain water content,EB method was used to detect the permeability of BBB.Part IV.Using gene engineering technology to construct Mfsd2a adeno associated virus（AAV）expression vector,increase the expression of Mfsd2a,reduce the permeability of BBB and neurological impairment after intracerebral hemorrhage.1.Using genetic engineering technique to construct Mfsd2a AAV,transfect the mouse brain,and detect the expression of Mfsd2a at different time points in WT mice by Western Blot.Autologous blood brain hemorrhage models were constructed using Mfsd2a AAV transfected mice.The mice were sacrificed at 12h,1,3,5 and 7 days,and the proteins extracted from the brain tissue of the hemorrhagic hemisphere were taken.The expression of Mfsd2a in the tissues around hematoma was analyzed by Western Blot and immunofluorescence.2.By upregulating the expression of Mfsd2a,the neurological deficit score,wet and dry weight method for the determination of brain water content,EB method was used to detect the permeability of BBB,to investigate the expression of Mfsd2a after cerebral hemorrhage compared with simple cerebral hemorrhage group whether to reduce BBB damage and relieve brain edema in 24h/48h/72h at different time points,and analysis the time correlation and its molecular mechanism.Part V.The mechanism of Mfsd2a expression in the BBB of mice was analyzed by electron microscopy,Western Blot and multiple reaction monitoring（MRM）.1.The expression of endothelial protein ZO-1,Claudin-5,Occludin,and and VE-Cadherin in brain tissue of Wild-type（WT）,Mfsd2a-/-,WT+control AAV and WT+Mfsd2a AAV mice After ICH was detected by Western Blot.2.The microstructure of vascular endothelium in brain tissue and brain hemorrhage of WT,Mfsd2a-/-,Mfsd2a and AAV mice were observed by electron microscope.3.MRM was used to analyze the protein changes in brain tissue after intracerebral hemorrhage in WT,Mfsd2a-/-mice.Conclusions:1.The expression of Mfsd2a protein decreased in WT mice after ICH.2.Mfsd2a protein further reduced by the use of siRNA interference decreased expression of Mfsd2a mice after the construction of autologous blood cerebral hemorrhage model,increase the permeability of the BBB,and aggravate the neurologic impairment after cerebral hemorrhage.3.In the knockout Mfsd2a mice,the BBB permeability and the neurological deficits increased significantly after ICH;4.Mfsd2a AAV expression vector transfected mice increased the expression of Mfsd2a,decreased the BBB permeability and impaired neurological function after ICH;5.Endothelial junction protein ZO-1,Claudin-5,Occludin,and VE-Cadherin expression did not show significantly differ in WT,Mfsd2a-/-,WT+control AAV,and WT+Mfsd2a AAV mice after cerebral hemorrhage.Electron microscopy showed that transport vesicles increased in Mfsd2a-/-and Mfsd2a-/-+ICH mice,and transport vesicles in Mfsd2a AAV mice after cerebral hemorrhage was not significantly increased.The analysis of WT,Mfsd2a-/-mice of vesicular transport protein in brain tissue using MRM,Srgap2,Stx7,and Sec22b from 31 vesicle trafficking-related proteins were markedly up-regulated.Our results indicate that selectively expressing endothelial Mfsd2a suppresses ICH-induced BBB disruptionmay via inhibiting vesicular transcytosis. |
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