参知健脑方调控Notch1/RBP-Jκ/HES-1信号通路改善脑小血管病性认知障碍的机制研究OA

Objective To investigate the effects and mechanism of Shenzhi Jiannao Formula(SZJNF)in treating cerebral small vessel cognitive impairment(CSVCI).Methods Fifty-seven male SPF-grade SD rats were randomly assigned to the sham operation(n=15),modeling(n=15),SZJNF(n=15,2.21 g/kg),and memantine hydrochloride(n=12,2.1 mg/kg)groups using the random number table method.The sham operation group underwent sham surgery,whereas the other groups underwent bilateral common carotid artery ligation to establish a rat model of cerebral small vessel disease.All drug groups received oral administration of the corresponding drug doses.The sham operation and modeling groups received oral administration of an equal volume of sterile reverse osmosis water once daily for four consecutive weeks.Cognitive function was assessed using the Morris water maze test.Pathological morphology in the CA1 region of the rat hippocampus was observed via hematoxylin-eosin and Nissl staining.Immunofluorescence was used to detect fluorescent expression of cluster of differentiation 11b(CD11b)and ionized calcium-binding adapter molecule 1(IBA-1)in hippocampal tissue;enzyme-linked immunosorbent assays measured interleukin(IL)-6,IL-1β,and tumor necrosis factor-α(TNF-α)levels in hippocampal tissue and serum.Western blotting and quantitative fluorescent PCR were used to detect the protein and mRNA expression of neurogenic locus notch homolog protein 1(Notch1),J kappa-recombination signal-binding protein(RBP-Jκ),and hairy and enhancer of split 1(HES-1)in rat hippocampal tissue.A co-culture model of BV2 and Bend.3 cells was established using oxygen-glucose deprivation/reperfusion and conditioned medium co-culture method.Groups included BV2/Bend.3-normal group 1,BV2/Bend.3-model group 1,BV2/Bend.3-memantine hydrochloride(10 μmol/L),and BV2/Bend.3-SZJNF low-,medium-,high-dose groups(0.025,0.050,and 0.100 g/L).For the cellular recovery experiment,BV2 cells were transfected with Notch1-overexpression lentiviral transduction before modeling and co-culturing.Groups included BV2/Bend.3-normal group 2,BV2/Bend.3-model group 2,BV2/Bend.3-Notch1 overexpression,BV2/Bend.3-negative control,BV2/Bend.3-SZJNF(0.050 g/L),and BV2/Bend.3-Notch1 overexpression+SZJNF group(0.050 g/L).The normal and model groups received no intervention,whereas all drug groups were cultured with corresponding medications.Immunofluorescence was used to detect CD11b and IBA-1 expression in BV2 cells.Enzyme-linked immunosorbent assays measured IL-6,IL-1β,and TNF-α contents in Bend.3 cells.Western blotting and quantitative fluorescent PCR assessed Notch1,RBP-Jκ,and HES-1 protein and mRNA expressions in BV2 cells.Results In cell experiments,compared with the sham operation group,the modeling group showed prolonged escape latency,reduced number of platform crossings,decreased percentage of dwell time in the target quadrant,hippocampal CA1 neuronal damage,increased average fluorescence intensity of CD11b and IBA-1,elevated IL-6,IL-1β,and TNF-α levels,and upregulated Notch1,RBP-Jκ,and HES-1 protein and mRNA expressions(P<0.05).These pathological changes were improved after treatment with SZJNF(P<0.05).In cell experiments,compared with the BV2-normal group 1,the BV2-model group 1 showed increased CD11b and IBA-1 average fluorescence intensity,and upregulated Notch1,RBP-Jκ,and HES-1 protein and mRNA expressions(P<0.05).Compared with the Bend.3-normal group 1,the Bend.3-model group 1 showed elevated IL-6,IL-1β,and TNF-α levels(P<0.05).These pathological changes were improved after treatment with SZJNF medium-dose and memantine hydrochloride(P<0.05).In the cellular recovery experiment,compared with the BV2-normal group 2,the BV2-model group 2 and BV2-Notch1 overexpression group showed increased CD11b and IBA-1 average fluorescence intensity,and upregulated Notch1,RBP-Jκ,and HES-1 protein and mRNA expressions(P<0.05).Compared with the Bend.3-normal group 2,the Bend.3-model group 2 and Bend.3-Notch1 overexpression group showed elevated IL-6,IL-1β,and TNF-α levels(P<0.05).These pathological changes were improved after treatment with SZJNF(P<0.05).Conclusion SZJNF may improve CSVCI by downregulating the Notch1/RBP-Jκ/HES-1 signaling pathway,inhibiting microglial activation,alleviating inflammatory responses,and protecting neurovascular units.