DY98 Improves Cognitive Dysfunction In Mice With Vascular Dementia By Inhibiting The Inflammatory Response Of Astrocytes

ObjectiveTo evaluate the effect of calmodulin inhibitor DY98 on cognitive dysfunction induced by permanent ligation of the right common carotid artery in Vascular Dementia(Va D)model mice,and to elucidate the mechanism of DY98 on cognitive dysfunction in vascular dementia model mice based on the inflammatory response of astrocytes.Methods1.To assess the ameliorative effect of DY98 on r UCCAO-induced cognitive dysfunction in mice suffering from Va D model and to explore the mechanism of ameliorative effect of DY98 based on astrocytesAfter seven days of adaptive rearing of purchased SPF-level male C57BL/6J mice,they were randomized into 5 groups of 10 mice each.By ligating the right common carotid artery(r UCCAO)of C57BL/6J mice,chronic cerebral blood flow hypoperfusion was caused in mice,simulating Va D lesions.0.5% CMC-Na was given to the sham and r UCCAO groups,donepezil hydrochloride(Donepezil)was given to the positive control group,and DY98(5mg/kg)and DY98(1 mg/kg)were given to the dosing group for gavage.The right common carotid artery of blunt isolated mice,except that carotid artery ligation was not performed,other operations were the same as those in the surgical group,and normal saline gavage was given as a sham surgical group.After 72 days of continuous administration,the relevant indicators were detected by materials.(1)The learning and memory functions of each mice were assessed using Y-maze experiment and Morris water-maze experiment behavioral experiments.(2)Immunofluorescence was used to colocate astrocytes in the brains of Va D model mice with inflammatory proteins such as IL-1β and GSDMD,and Quantitative Real-time Polymerase Chain Reaction was used,q PCR)detects m RNA levels of Caspase-1 and GSDMD pyrozotic proteins in the right hippocampus of mice.2.To investigate the effect and mechanism of DY98 on LPS/ATP-induced pyroptosis in astrocytesLPS/ATP was given to induce to construct a pyropyrosis inflammatory model of C8-D1 A astrocytes,and then sterile water,minocline 10 μM and DY98 1μM were administered respectively,and the control group was given the same amount of sterile water as the model group.(1)MTT assay was performed to detect the effect of different concentrations of DY98 on cell survival after the administration of LPS/ATP to induce astrocytes to undergo pyroptosis.(2)Cell morphological analysis of the changes of cell morphology after LPS/ATP induced pyroptosis in astrocytes by DY98.(3)Western Blot and Immunofluorescence were used to detect the expression of IL-1β,Caspase-1,GSDMD,NLRP3 proteins,and Quantitative Real-time Polymerase Chain Reaction was used to detect their m RNA levels.Results1.Under the gavage administration of DY98,the spontaneous alternation accuracy rate of C57BL/6J mice after r UCCAO was significantly improved in the Y maze experiment,while the spontaneous alternation accuracy rate of Va D model mice without any treatment decreased significantly,all of which were statistical significance.At the same time,in the Morris water maze experiment,it was found that when the mice in the r UCCAO group performed space exploration without the platform removed on the fifth day,the mice could not find the position of the platform within 45 s,and the mice in other groups could find the position of the platform.In addition,compared with the Sham group mice,the escape latency of the r UCCAO group was significantly extended,while the escape latency of the mice was shortened after giving DY98 and Donepezil gavage.At the same time,compared with the mice in the Sham group,the number of crossings of the platform in the r UCCAO model group was significantly reduced,which was statistically different.Compared with the mice in the r UCCAO model group,the number of platform crossings in the DY98 and Donepezil groups was significantly increased,with statistical significance The results of Y-maze and water maze experiments suggest that DY98 can improve learning and memory dysfunction in mice after r UCCAO.2.The preliminary mechanism of action of DY98 was explored by immunofluorescence experiments and q PCR experiments,and the colocalization and staining of astrocytes and inflammatory proteins in the brain tissues of each group of mice showed that the fluorescence intensity of GFAP and IL-1β and other inflammatory proteins in r UCCAO mice was enhanced compared with the Scham group of mice,and the results suggested that the cognitive dysfunction caused by r UCCAO after surgery may be related to the inflammatory response of astrocytes.The results of q PCR experiments showed that the m RNA levels of inflammatory proteins such as IL-1β,GSDMD and Caspase-1 in the hippocampus of mice in the r UCCAO group were upregulated and statistically different compared with mice in the Sham group.Compared with mice in the r UCCAO group,mice given DY98 and Donepezil for gavage respectively had m RNA levels of the above inflammatory proteins downregulated and statistically significant.Combined with the results of immunofluorescence colocalization experiment and q PCR experiment,it was suggested that DY98 improved learning and memory dysfunction in r UCCAO mice and was associated with improved inflammatory response of astrocytes and corponication pathway..3.The MTT test results showed that the survival rate of astrocytes decreased after LPS/ATP modeling,which was statistically significant compared with normal group cells.When DY98 was administered 3 h in advance,the survival rate of astrocytes was improved at DY98 concentrations of 1 μM and 500 n M,which was statistically different compared with the LPS/ATP model group.At the same time,the results of cell morphological analysis showed that the C8-D1 A astrocytes in the normal state were star-shaped,and the protrusions between the cells were stretched and tightly connected.After LPS/ATP molding,the C8-D1 A cells are swollen,rounded,the protrusions are broken,and most of the cells are scattered individually.However,the cells given MINO and DY98 groups did not show obvious swelling circles,and the protruding branches were relatively tightly connected.The results suggested that DY98 could alleviate LPS/ATP-induced pyroptosis in astrocytes.4.WB,IF and q PCR experiments were used to verify the mechanism of action,and it was found that the experimental results of the three were consistent.Compared with the normal group,the expression of inflammatory proteins such as IL-1β,Caspase-1,GSDMD,NLRP3 and other inflammatory proteins and the corresponding m RNA levels in the LPS/ATP model group were significantly upregulated,and there were statistical differences.Compared with the LPS/ATP model group,the expression and corresponding m RNA levels of inflammatory proteins such as IL-1β,Caspase-1,GSDMD,and NLRP3 were significantly downregulated in the group given DY98 and Minocycline,and there were significant differences.The results showed that DY98 could inhibit the inflammatory response caused by cell pyrozosis induced by astrocytes induced by LPS/ATP.ConclusionThe results found that both DY98 and Donepezil could improve learning and memory dysfunction in Va D model mice,which may be related to inhibition of pyroptosis in astrocytes and alleviating inflammation.Through LPS/ATP-induced pyroptosis inflammation model of astrocytes,DY98 was found to have similar pharmacological efficacy as MINO,with strong anti-inflammatory effect,inhibition of pyroptosis in astrocytes,alleviation of neuroinflammation,and may play a role by blocking NLRP3-mediated classical pyrosis pathway.Therefore,DY98 is expected to become a potential therapeutic drug for Va D disease,and the NLRP3-mediated classical pyrozoosis pathway may be a new therapeutic target.