| Background and ObjectivesCerebral Small Vessel Disease(CSVD)is a disease caused by cerebral arterioles,capillaries and venules.CSVD is positively correlated with the risk of stroke and dementia,which seriously harms the health of middle-aged and elderly people and imposes a heavy burden on society.However,due to the lack of reliable research models,the pathogenesis of CSVD is still unclear and effective therapy are lacking.At present,blood brain barrier(BBB)injury is considered to be an important factor affecting the occurrence and development of CSVD,and how to prevent and control BBB injury has also become the focus of treatment.CADASIL caused by NOTCH3 gene mutation is the most common single-gene inherited CSVD with BBB damage.It’s helpful to do advance the overall understanding of CSVD.In the earlier stage,our team constructed the NOTCH3 gene R544 C point mutation model mice and found that BBB structure was damaged and matrix metalloproteinase-9(MMP-9)expression was increased.According to literature review,MMP-9 can degrade the extracellular matrix,thus damaging the integrity of the blood-brain barrier.In addition,MMP-9 can also promote the apoptosis of neurons,which is closely related to cognitive impairment.However,the mechanism of the increase of MMP-9 has been rarely reported.In order to explore the role and mechanism of increased MMP-9 in CADASIL,this study used mice with R544 C point mutation to determine whether this point mutation regulates the expression of MMP-9 through NF-κB and/or Akt/c-Jun signaling pathway,thereby damaging BBB and promoting neuron apoptosis.To provide a new theoretical basis for the pathogenesis of CSVD,and provide a new target for treatment.Methods1.Through CRISPR/Cas9 technology,model mice corresponding to the R544 C point mutation of human NOTCH3 gene were constructed.2.Activation of caspase-3 and caspase-9 was detected by Western blot to reflect apoptosis of R544 C point mutant mice;Evans blue leakage test was used to detect the amount and location of Evans blue leakage in the brain and cerebellum of R544 C point mutant mice.Meanwhile,Western blot and immunofluorescence staining were used to observe the albumin leakage to reflect the permeability of blood-brain barrier.The permeability of the blood-retinal barrier was measured by fundus fluorescein contrast.3.The expression of MMP-9 and TIMP-1 and their cell localization were observed by immunofluorescence staining and Western blot.In order to investigate the brain injury of mice with R544 C point mutation,the signaling pathways of NF-κB,c-Jun and Akt were detected by q-PCR,Western blot and immunofluorescence staining.Results1.Compared with WT mice,Evans blue leakage in brain and cerebellum of R544 C point mutant mice was increased by 96.11 ± 33.83%(P<0.05)and 61.91 ± 13.95%(P<0.001),respectively,and albumin content was increased by 30.13±9.26%(P<0.001).2.Fundus fluorescence imaging of 9-month-old WT mice and R544 C point mutant mice showed no contrast agent leakage.3.The content of MMP-9 in cortex and hippocampus of mice with R544 C point mutation was higher than that of WT mice.The increased MMP-9 was co-localized with neurons,but not with astrocytes and microglia.4.Compared with WT mice,the TIMP-1 level of R544 C mutant mice was decreased by 11.22 ± 4.90%(P<0.05),and the MMP-9/TIMP-1 ratio was increased by 50.45 ± 22.25%(P<0.05).5.Compared with WT mice,NF-κB(P65)m RNA was increased by 52.72 ± 19.29%(P<0.05)and c-Jun m RNA was increased by 42.3 ± 17.94%(P<0.05)in R544 C mutant mice,but immunofluorescence did not show NF-κB(P65)entering the nucleus,and the protein contents of NF-κB,c-Jun and AKT were decreased,and there was no statistical difference.Conclusion1.BBB permeability of NOTCH3 gene R544 C model mice was increased.2.The imbalance of MMP-9/TIMP-1 expression in R544 C Notch3 mutant mice may be the reason for the increased BBB permeability.3.The increase of MMP-9 in R544 C model of NOTCH3 gene is not regulated by the Notch3/NF-κB and Notch3/AKT/c-Jun signaling pathways. |
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