Use of matrix metalloproteinase 8 mutant mmp8-p326v

Abstract

The application belongs to the technical field of biological medicine, and relates to application of a matrix metalloproteinase 8 mutant MMP8-P326V, in particular to application of the matrix metalloproteinase 8 mutant MMP8-P326V in treatment of vascular dementia and senile dementia. The matrix metalloproteinase 8 mutant MMP8-P326V is applied to treatment of vascular dementia and senile dementia for the first time, and experiments prove that the matrix metalloproteinase 8 mutant MMP8-P326V can improve and treat vascular dementia and senile dementia, effectively improve pathological phenotypes of vascular dementia and senile dementia, improve neuron function of vascular dementia or senile dementia, improve cognitive impairment and cognitive function, and meanwhile, in a vascular dementia animal model, MMP8-P326V exhibits a double regulation effect of inhibiting nerve inflammation and improving cognitive function. The application is expected to provide a strategy for precise treatment of vascular dementia and senile dementia, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and to the application of matrix metalloproteinase 8 mutant MMP8-P326V, particularly the application of matrix metalloproteinase 8 mutant MMP8-P326V in the preparation of drugs for treating vascular dementia and Alzheimer's disease. Background Technology

[0002] Dementia is a clinical syndrome caused by a combination of acquired and progressive brain injuries, characterized by a persistent decline in cognitive function that significantly interferes with daily life and social functioning. According to the latest data from the Global Burden of Disease Study (GBD2021), dementia is now the second leading cause of disability in people aged 65 and older. Vascular dementia (VaD) and Alzheimer's disease (AD) account for 15-25% and 50-60% of all dementia cases, respectively, totaling over 75%, constituting the main disease entities of cognitive impairment in old age. VaD and AD not only have high prevalence but are also the leading causes of cognitive impairment and dementia in the elderly. With the increasing aging of the global population, the prevalence of these two diseases continues to rise, placing a heavy economic and care burden on patients' families and society. VaD is mainly caused by cerebrovascular diseases such as stroke and small vessel disease, which lead to ischemic and hypoxic damage to brain tissue; AD is a neurodegenerative disease characterized by progressive cognitive decline, with typical pathological features including senile plaques formed by the deposition of β-amyloid protein (Aβ) in the brain and neurofibrillary tangles composed of hyperphosphorylated Tau protein.

[0003] Although the initiating factors and some pathological mechanisms of VaD and AD differ, pure VaD or AD is relatively rare in clinical practice. More patients present with mixed dementia, meaning that vascular factors and Alzheimer's disease pathology coexist and exacerbate each other. This increases the complexity of diagnosis and treatment, and the harms have a cumulative effect: ① The average survival time of patients is shortened by 6-10 years; ② The risk of complications such as falls, fractures, pneumonia, and stroke increases by 2-4 times; ③ The proportion of moderate to severe patients requiring long-term care is >70%, and the average annual direct medical expenditure is 3-5 times higher than that of non-dementia people of the same age, placing a heavy economic burden on the public health system.

[0004] From a pathophysiological perspective, cerebral microcirculatory dysfunction and the resulting chronic hypoperfusion and hypoxia are the core links in the development and progression of VaD. Large and small vessel lesions such as arteriosclerosis, lipid hyaline degeneration, and amyloid angiopathy can lead to blood-brain barrier disruption, activation of local inflammatory cascades, and disintegration of neurovascular units, ultimately resulting in neuronal energy metabolism exhaustion and axonal loss. Notably, similar ischemia-hypoxia mechanisms also play a crucial role in the pathological process of AD: decreased cerebral blood flow can exacerbate β-amyloid (Aβ) deposition and tau protein hyperphosphorylation, while Aβ deposition, in turn, further constricts capillaries and reduces cerebral perfusion, forming a positive feedback loop of deteriorating vascular-metabolic-neural interactions. Therefore, improving cerebral vascular function and restoring oxygen supply to brain tissue have become intervention strategies for VaD and even AD.

[0005] Furthermore, chronic hypoxia can exacerbate cognitive decline through epigenetic mechanisms: persistently elevated HIF-1α induced by hypoperfusion leads to upregulation of DNA methyltransferase 1 (DNMT1), resulting in hypermethylation of the promoter regions of memory-related immediate early genes (such as Arc and Egr1), and suppression of transcription; simultaneously, hypoxia triggers overexpression of miR-210-3p, which targets and regulates mitochondrial iron-sulfur cluster assembly enzyme (ISCU), further inhibiting cytochrome C oxidase activity, causing a further 15-20% decrease in ATP production. Imaging-pathology studies have confirmed that for every 1 mL increase in periventricular white matter hypersignal (WMH), the whole-brain metabolic rate (CMRglc) decreases by 0.8%, and this is linearly correlated with a 0.12 standard unit decrease in executive function z-score. It is worth noting that the accumulation of vascular risk factors can advance the onset of dementia by 7-10 years; and a combined intervention of intensive blood pressure lowering (SBP <120 mmHg) and lipid target achievement (LDL-C <1.8 mmol / L) for 5 years can slow the progression rate of vascular malformation (WMH) by 32% and reduce the risk of cognitive decline by 18%. Therefore, early identification and intervention of the cerebrovascular-hypoxia axis may not only block the progression of VaD, but may also become an effective means of delaying the pathological spread of Alzheimer's disease (AD).

[0006] Currently, first-line clinical treatments for Alzheimer's disease (AD) primarily consist of cholinesterase inhibitors such as donepezil and rivastigmine, and NMDA receptor antagonists such as memantine and piracetam. These drugs can only alleviate symptoms to some extent but cannot block or reverse disease progression. For Vascular Depression (VED), there are currently no specific treatments widely approved by global regulatory agencies. Clinical management mainly focuses on controlling vascular risk factors such as hypertension, diabetes, hyperlipidemia, and antiplatelet therapy to prevent recurrence of cerebrovascular events. However, this preventative strategy has very limited repair effects on existing cognitive impairment. Therefore, developing therapies that can fundamentally intervene in disease progression, protect neurons, and promote neural repair is a critical scientific problem and a significant clinical need that urgently needs to be addressed in the field of neuroscience.

[0007] Cerebrovascular lesions or embolisms leading to persistent brain hypoperfusion and ischemic-hypoxic injury are the core pathological mechanisms of Vascular Dysplasia (VaD) and one of the important pathogenesis mechanisms of Alzheimer's disease (AD). Notably, dolphins (Delphinidae), marine mammals, face extreme hypoxic environments during long-term diving but maintain brain function integrity and avoid hypoxic brain injury through unique physiological adaptation mechanisms. This evolutionary advantage suggests that dolphins may possess special neuroprotective mechanisms, providing a valuable biological model for intervention research on human cerebrovascular diseases. Through cross-species comparative genomics analysis, the inventors' team has for the first time identified a specific Pro→Val mutation (P326V) at amino acid 326 of dolphin matrix metalloproteinase-8 (MMP8). The MMP8 gene has undergone strong positive selection in dolphins, and the ProVal mutation at amino acid 326 is unique to dolphins, significantly altering the 3D structure of MMP8 at this site.

[0008] Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endopeptidases capable of degrading various components of the extracellular matrix. In the central nervous system, MMPs participate in neural development, synaptic plasticity, and tissue remodeling under physiological conditions. However, under pathological conditions, the overexpression and activation of MMPs have been shown to be closely related to the pathogenesis of various neurological diseases, including blood-brain barrier disruption, amplified inflammatory responses, neuronal damage, and white matter lesions. Among these, MMP8 has traditionally been thought to originate primarily from activated neutrophils and play a role in acute inflammation and tissue destruction. Recent studies have found that MMP8 also plays a complex and crucial role in chronic central nervous system diseases. For example, in models of cerebral ischemia and Alzheimer's disease (AD), MMP8 expression levels are upregulated, exacerbating neurological damage. Notably, in human and mouse studies, MMP8 has consistently been considered a destructive force: ① promoting microglia activation and TNF-α release, exacerbating neuroinflammation; ② degrading the tight junction protein claudin-5, disrupting the blood-brain barrier (BBB), and mediating peripheral immune cell infiltration; ③ breaking down plaque fibrous caps, making atherosclerotic arteries more prone to rupture, indirectly inducing cerebral embolism. In the fields of VaD and AD, existing research on the specific functions of MMP8 and its potential as a therapeutic target is insufficient and contradictory. Some studies even suggest that inhibiting certain MMPs may have adverse effects, highlighting the complexity of MMP function in the nervous system. However, a specific mutation has occurred in dolphins, which possess unique neuroprotective mechanisms, and this mutation undergoes strong positive selection. This suggests that the P326V mutation in the MMP8 gene may play a different role. However, whether MMP8-P326V can exert an anti-VaD effect remains unconfirmed.

[0009] According to the search, there are currently no reports of matrix metalloproteinase 8 mutant MMP8-P326V being used to treat vascular dementia and Alzheimer's disease. Summary of the Invention

[0010] To address the above problems, the present invention aims to provide a use of the matrix metalloproteinase 8 mutant MMP8-P326V, particularly the application of the matrix metalloproteinase 8 mutant MMP8-P326V in drugs for the treatment of dementia.

[0011] This invention protects the use of the matrix metalloproteinase 8 mutant MMP8-P326V in the preparation of drugs for treating vascular dementia or Alzheimer's disease.

[0012] Furthermore, the matrix metalloproteinase 8 mutant MMP8-P326V is used in the preparation of drugs that improve neuronal function in vascular dementia or Alzheimer's disease, and improve cognitive impairment and cognitive function.

[0013] Furthermore, the matrix metalloproteinase 8 mutant MMP8-P326V is injected via the common carotid artery, subcutaneous injection, or intracranial injection.

[0014] Furthermore, the vascular dementia mentioned refers to dementia caused by ischemic cerebrovascular disease or dementia caused by cerebrovascular injury.

[0015] Through in-depth research, the inventors unexpectedly discovered that a variant of the MMP8 protein (named MMP8-P326V) exhibits remarkable neuroprotective, cognitive function improvement, and pathological remission effects in animal models of VaD and AD. This discovery breaks through the traditional understanding of MMP8 as a "destructive" protease, revealing a novel application of its specific variant in the treatment of neurodegenerative diseases and vascular cognitive impairment, providing a new, efficient, and highly specific solution to the aforementioned long-standing technical challenges. The embodiments of this invention demonstrate that the matrix metalloproteinase 8 mutant MMP8-P326V can improve cognitive levels in VaD models, suggesting its potential use as a VaD prevention and treatment drug. According to an embodiment of the present invention, in order to demonstrate the therapeutic effect of MMP8-P326V on VaD, the inventors injected LV-MMP8-P326V (Group M) lentivirus and empty vector (Group V) lentivirus (viral titer: 1*10⁹ TU / mL, dose: 1 μL / g) subcutaneously into the scalp on the day of VaD model creation. Simultaneously, oxracetam (concentration 200 mg / mL) was administered intraperitoneally daily as a positive control (dose 1.8 μL / g). -1 ·d -1 After 14 and 30 days of treatment, the Y-maze neo-arm exploration experiment and the Barnes maze experiment were performed. The Y-maze experiment recorded the number of entries into the starting arm, neo-arm, and other arms; the Barnes maze experiment recorded the total distance to the target hole. Results showed that compared with the model group, the proportion of entries into the neo-arm in the sham surgery group, the positive drug group, and the MMP8-P326V group all showed a significant upward trend, with the MMP8-P326V group showing statistically significant (…). P <0.05, Figure 1 A); while the unloaded group and the model group showed similar performance. Similarly, compared with the model group, the walking distance for finding target holes in the Barnes maze was significantly reduced in the sham surgery group, positive drug group, MMP8-P326V group, and unloaded group. P <0.01, Figure 1B). Among them, the MMP8-P326V group reduced more than the positive drug group and was closer to the sham surgery group. Compared with the model group, the MMP8-P326V group reduced the most significantly. Although the positive drug group could effectively improve the cognitive level of the VaD model, the effect was still inferior to that of MMP8-P326V.

[0016] On the day of VaD model creation, the inventors injected LV-MMP8-P326V lentivirus (Group M), LV-MMP8 lentivirus (Group W), and empty vector (Group V) lentivirus via the common carotid artery (viral titer: 1*10⁹ TU / mL, dose: 0.13 μL / g). Simultaneously, piracetam (concentration 200 mg / mL) was administered intraperitoneally as a positive control (dose: 1.8 μL / g). -1 ·d -1 After 28 days, water maze training and testing were conducted. The results showed that on day 5 of training, the MMP8-P326V group rats had the shortest total distance required to find the target platform from other quadrants, significantly better than the model group. P <0.05, Figure 2 B), and the level was close to that of the sham-operated group rats; while compared with the wild-type MMP8 group and the empty group, the wild-type MMP8 group showed some improvement, but the platform-seeking trajectory was still messy. Figure 2 A). During the testing period, compared with the model group, the total distance from the wild-type MMP8 group, the unloaded group, and the MMP8-P326V group to the target platform was shortened, with the MMP8-P326V group showing the most significant statistical difference. P <0.01, Figure 2 D). Meanwhile, the MMP8-P326V group showed significantly better treatment efficacy than the positive control group ( P <0.05, Figure 2 (D) This indicates that MMP8-P326V is more effective than the positive control in improving cognitive impairment. Overall, MMP8-P326V significantly improved spatial learning and memory retention in VaD model rats. Literature indicates that current clinical practice for VaD treatment mainly focuses on controlling vascular risk factors and providing symptomatic support, lacking targeted drugs that can reverse cognitive impairment. This invention reveals for the first time that MMP8-P326V can be administered via subcutaneous injection and carotid artery injection, and only on the day of model establishment, with no subsequent treatment. The positive control group received intraperitoneal injections of piracetam (1.8 μL·g) daily after model establishment. -1 ·d -1 Subsequent results showed that the positive control group had a therapeutic effect on the VaD model, but the effect was not significant, while the MMP8-P326V group showed significant results and outstanding therapeutic effect. These results indicate that MMP8-P326V is significantly superior to piracetam and can significantly improve cognitive function in VaD model rats.

[0017] Collateral regeneration is key to reversing brain damage caused by cerebral ischemia in VaD patients. The inventors further employed laser speckle imaging to systematically and quantitatively evaluate the hemodynamic effects of MMP8-P326V on the brain tissue of each rat group, verifying whether MMP8-P326V achieves structural and functional recovery of cognitive function by specifically improving collateral circulation in the brain. The collateral circulation perfusion index (Region-of-Interest, ROI) was used for quantification. Results showed that the ROI of the MMP8-P326V group was significantly improved compared to the model group and the unloaded group. P <0.05, Figure 3 The results suggest that MMP8-P326V can rapidly open and stably maintain collateral circulation in ischemic brain tissue. Meanwhile, compared to the MMP8-P326V group, the ROI in the MMP8 group was not improved, and was even lower than that in the model group. This suggests that wild-type MMP8 may exacerbate ischemic brain injury under conditions of cerebral ischemia and hypoperfusion. P <0.01, Figure 3 The ROI of the empty-load group was comparable to that of the model group, indicating no therapeutic effect. The ROI of the positive drug group was higher than that of the sham-operated group, but not statistically significant, and lower than that of the MMP8-P326V group, suggesting that traditional nootropic drugs have a weak effect on promoting collateral circulation perfusion. Figure 3 The above in vivo hemodynamic evidence directly demonstrates that the MMP8-P326V protein significantly improves the ischemic environment by specifically upregulating collateral circulation perfusion in the brain, thereby providing a structural-functional basis for cognitive function recovery.

[0018] This invention further validates, through histological and cellular studies, the direct protective effect of the matrix metalloproteinase 8 mutant MMP8-P326V on the structural integrity and functional state of hippocampal neurons in a rat model of vascular dementia (VaD), thereby clarifying its medicinal use in improving hippocampal neuronal function and treating VaD. According to an embodiment of this invention, after the aforementioned behavioral and laser speckle blood flow imaging experiments, brain tissue samples from VaD model rats were collected, fixed with paraformaldehyde, and then processed and stained with hematoxylin and eosin (HE). Using surviving neurons with round nuclei, clear nuclear membranes, and prominent nucleoli in the hippocampus as the standard, changes in neuronal morphology were observed in each group of brain tissue. In the sham-operated group, hippocampal neurons were densely and neatly arranged, with cone-shaped cell bodies, uniform eosinophilic cytoplasm, and clearly stained nucleoli, showing no obvious degeneration or necrosis. In contrast, the model group showed a significant reduction in neuronal number, with obvious degeneration and necrosis, manifested as cell body swelling or shrinkage, deep nuclear pyknosis, and cell membrane rupture. This suggests that chronic hypoperfusion in the VaD model led to severe structural damage to hippocampal neurons. In the positive control group (Oxiracetam), neuronal density recovered, and the degree of damage was significantly reduced compared to the model group, although some cells still showed nuclear pyknosis and increased eosinophilic cytoplasm. In the MMP8-P326V group, hippocampal neurons were plump, neatly arranged, with uniform eosinophilic cytoplasm, clearly identifiable nucleoli, and restored neuronal density, with morphology similar to the sham-operated group. The MMP8 and Vector groups had similar neuronal density to the model group; although not completely necrotic, significant neuronal degeneration was observed, indicating that MMP8 had no significant salvage effect on neuronal morphological damage. Figure 4 Simultaneously, the neuronal marker NeuN was detected using immunofluorescence. Results showed that neurons in the hippocampus of the sham-operated group were densely packed, and NeuN-positive cell nuclei showed uniform staining. Figure 5 A); In the model group, the fluorescence intensity of NeuN decreased, and the nuclear condensation and fragmentation of NeuN-positive cells were obvious, indicating neuronal damage; In the MMP8-WT group, the fluorescence intensity of NeuN decreased ( Figure 5 B); The NeuN fluorescence intensity in the MMP8-P326V group was not significantly different from that in the sham-operated group and was higher than that in the model group. NeuN-positive cell nuclei were uniformly stained and their morphology was closer to that of the sham-operated group. Figure 5 A, Figure 5 (B) The above evidence indicates that MMP8-P326V protein can significantly inhibit neuronal degeneration, necrosis, and loss in the hippocampus of VaD model rats, restoring the number and normal morphology of neurons. Its protective effect is superior to that of the traditional positive control drug piracetam, while wild-type MMP8 does not have this activity. Therefore, it is confirmed that MMP8-P326V mediates cognitive function recovery by directly improving the structural integrity of hippocampal neurons, and thus possesses pharmaceutical value as an active ingredient in the preparation of drugs for the prevention and treatment of VaD.

[0019] Neuroinflammation is another major pathogenic factor in vascular dementia, and astrocytes are the most important neuroinflammation-related cells; therefore, immunofluorescence detection was performed. GFAP, short for glial fibrillary acidic protein, is mainly distributed in astrocytes of the central nervous system. Almost all CNS injuries (ischemia, hemorrhage, trauma, neurodegenerative diseases) are accompanied by upregulation of GFAP expression, which is a marker of astrocyte activation. GFAP immunofluorescence results showed that astrocytes in the sham-operated group were in a resting state. Compared with the sham-operated group, GFAP fluorescence was significantly enhanced in both the model group and the empty vector group, indicating severe central nervous system damage and a higher degree of astrocyte activation. Figure 5 A). Meanwhile, the GFAP fluorescence intensity in the positive drug and MMP8-P326V groups was significantly lower than that in the model group, and there was no statistically significant difference compared to the sham surgery group. P >0.05, Figure 5 C). Morphological analysis showed that the cell body area of ​​astrocytes in the MMP8-P326V group was restored to the resting state level, and the complexity of process branching was reduced, suggesting that it effectively inhibited VaD-induced excessive glial activation. Figure 5 A). Wild-type MMP8 did not show equivalent activity ( Figure 5 C). HE staining and immunofluorescence results jointly confirmed that MMP8-P326V significantly improves the central nervous system injury phenotype by maintaining the integrity of neuronal nuclei and inhibiting secondary glial inflammation, and has pharmaceutical use as an active ingredient in the preparation of drug compositions for the prevention and treatment of vascular dementia.

[0020] This invention also demonstrates the effect of the matrix metalloproteinase 8 mutant MMP8-P326V on the cognitive level of AD model mice, and its potential use as a dementia prevention and treatment drug. According to an embodiment of this invention, to demonstrate the therapeutic effect of MMP8-P326V on AD, the inventors used a 5XFAD transgenic mouse model (AD disease model), stereotactically located the right hippocampus of the mice, and injected LV-MMP8-P326V, LV-MMP8, and LV-Vector with a viral titer of 1*10⁹ TU / mL. To evaluate the intervention effect of MMP8-P326V on Alzheimer's disease (AD)-related cognitive impairment, a new object recognition (NOR) test was performed 14 days after treatment, recording absolute discrimination measure, recognition index, and preference index. The results showed that compared with the normal group, the three indices of the 5XFAD+Vector group, 5XFAD+MMP8 group, and 5XFAD group all showed a significant decreasing trend; among them, the three indices of the 5XFAD+MMP8-P326V group were significantly higher than those of the 5XFAD+Vector group. P <0.05, Figure 6 (BD), and is closest to the normal group, and even shows better results than the normal group.

[0021] To investigate the impact of MMP8-P326V on Alzheimer's disease, an ELISA assay was performed to quantitatively analyze the Aβ42 / Aβ40 ratio, total tau (T-tau), and phosphorylated tau (p-tau) levels. In AD patients, the Aβ42 / 40 ratio was inversely associated with intracranial plaque burden. Figure 7 The results showed that the ratio significantly increased after the introduction of the MMP8-P326V mutation, indicating a reduction in Aβ deposition. The Aβ42 / 40 ratio was chosen instead of the total Aβ amount because this ratio more sensitively reflects the tendency for fibrous plaque formation: Aβ42 has strong aggregation, and an increased proportion often predicts an increase in toxic plaques; therefore, reversing this ratio is considered a key indicator of successful early intervention. Simultaneously, the levels of the core AD biomarkers T-tau and P-tau also changed synchronously: ELISA results showed that the total T-tau amount in the MMP8-P326V group was significantly lower than that in the Vector and MMP8 groups. Figure 7 B, P <0.01%, its phosphorylated form P-tau also decreased synchronously ( Figure 7 C, P <0.01). Detection of total tau (T-tau) can comprehensively assess the degree of neuronal damage, while P-tau, especially AD-specific epitopes such as phosphorylation sites 396 / 404, directly reflects the formation rate of neurofibrillary tangles (NFTs). The simultaneous decrease of both not only suggests that the pathological expansion of tau is limited, but also indirectly indicates that the process of synaptic degeneration and neuronal death is inhibited, further supporting the potential therapeutic value of MMP8-P326V in blocking Aβ-tau biaxial toxicity.

[0022] Compared with the prior art, the present invention has the following beneficial technical effects:

[0023] The matrix metalloproteinase 8 mutant MMP8-P326V provided by this invention not only significantly improves the structural integrity of neurons in brain tissue and the phenotype of central nervous system damage, but also improves the cognitive level of rats with VaD. This demonstrates that the matrix metalloproteinase 8 mutant MMP8-P326V can be used to treat dementia. This invention is the first to use the matrix metalloproteinase 8 mutant MMP8-P326V to treat VaD and AD, achieving good technical results and providing a new therapeutic approach for dementia. Attached Figure Description

[0024] Figure 1 The images show the Y-maze and Barnes maze results after subcutaneous injection of LV-MMP8-P326V into the scalp of a VaD rat model. Figure 1 A represents the trajectory diagram and data statistics of the Y maze novel arm exploration experiment. Figure 1B represents the trajectory diagram and data statistics of the Barnes maze experiment;

[0025] Figure 2 Trajectory and statistical graphs of the Morris water maze experiment after injecting LV-MMP8-P326V into the common carotid artery of a VaD animal model. Figure 2 A represents the trajectory diagram of the Morris water maze experiment. Figure 2 B represents the statistical graph showing the total distance required for rats to find the target platform from other quadrants during the Morris water maze test training period. Figure 2 C represents the statistical graph of the average speed of rat movement during the Morris water maze test period. Figure 2 D represents a statistical graph of the total distance the rats traveled from the first quadrant to the target platform during the Morris water maze test period.

[0026] Figure 3 A diagram showing the collateral circulation in the brain detected by a laser speckle blood flow analyzer and the results of perfusion data processing.

[0027] Figure 4 Figure 1 shows the HE staining changes in the hippocampus of rat brain tissue caused by the matrix metalloproteinase 8 mutant MMP8-P326V.

[0028] Figure 5 The changes in immunofluorescence staining of neurons and astrocytes in the brain tissue of VaD model rats after NeuN / GFAP double labeling were observed. Figure 5 A represents an image of a NeuN / GFAP double-labeled immunofluorescence staining section of brain tissue from a VaD model rat. Figure 5 B represents a statistical graph of the fluorescence intensity data of NeuN, a neuronal marker, in brain tissue. Figure 5 C represents a statistical graph of fluorescence intensity data of GFAP, a marker of astrocytes, in brain tissue;

[0029] Figure 6 Trajectory graphs and statistical graphs of the new object recognition experiment after unilateral hippocampal injection of drug for stereotactic localization in 5XFAD model mice. Figure 6 A represents the trajectory diagram of the new object recognition experiment in the 5XFAD model mouse. Figure 6 B represents a statistical chart of the absolute discrimination measurement index in the new object recognition experiment. Figure 6 C represents a statistical chart of the recognition index in the new object recognition experiment. Figure 6 D represents a statistical chart of the preference index in the new object recognition experiment;

[0030] Figure 7A statistical graph showing the Aβ42 / Aβ40 ratio, total tau (T-tau), and phosphorylated tau (p-tau) content in the brain tissue of 5XFAD model mice was used for quantitative analysis by ELISA. Figure 7 A represents a statistical graph of the Aβ42 / Aβ40 ratio data from the quantitative analysis of ELISA experiments. Figure 7 B represents a statistical chart of total tau (T-tau) data from the quantitative analysis of ELISA experiments. Figure 7 C represents a statistical graph of data from the quantitative analysis of phosphorylated tau (p-tau) using ELISA. Detailed Implementation

[0031] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention. It should be noted that, unless specific techniques or conditions are specified in the embodiments, experimental results can be obtained by following the descriptions in relevant literature or product instructions in the field. Reagents, instruments, cells, and animal models used, unless otherwise specified, are all conventional products that can be purchased from the market. The recombinant expression plasmids encoding wild-type matrix metalloproteinase 8 (MMP8) and the MMP8 mutant (MMP8-P326V) were prepared in-house by the project team, and their preparation method is described in patent number CN202310449149.7. The construction method is as disclosed in Examples 1 and 2 of patent number CN202310449149.7.

[0032] Based on this, the lentivirus preparation method is as follows:

[0033] The constructed MMP8 or MMP8-P326V recombinant lentiviral expression plasmids and packaging plasmids (psPAX2 and pMD2.G) were transfected into HEK293T cells in logarithmic growth phase at a molar ratio of 4:3:1. Transfection was performed using the PEI (polyethylenimine) method: the plasmids were mixed in serum-free medium according to the specified ratio, and an appropriate amount of PEI (1 mg / mL, calculated at a DNA:PEI mass ratio of 1:3) was added. The mixture was gently mixed and incubated at room temperature for 20 min. The complex was then added dropwise to HEK293T cells and gently shaken. 6–8 h after transfection, the medium was replaced with fresh complete medium, and the cells were cultured for another 48–72 h. The cell supernatant was collected, filtered through a 0.45 μm filter to remove cell debris, and then centrifuged (4000 rpm, 4°C for 40 min) using an ultrafiltration tube. The concentrated virus solution was collected, aliquoted, and stored at -80°C for later use.

[0034] Lentiviral titers were determined by detecting the proportion of GFP-positive cells. HEK293T cells were infected with serially diluted lentiviruses, and the percentage of GFP-positive cells was detected by flow cytometry after 48-72 hours. The number of functionally active viral particles per milliliter of viral solution (TU / mL) was calculated. Finally, MMP8 and MMP8-P326V lentiviruses with suitable titers were obtained and could be used for subsequent cell infection experiments.

[0035] Example 1: Effects of the matrix metalloproteinase 8 mutant MMP8-P326V on cognitive function in VaD model rats

[0036] The inventors employed the classic bilateral carotid artery ligation (2-Vessel Occlusion, 2-VO) procedure in rats, surgically and permanently ligating both sides of the common carotid artery to induce chronic cerebral hypoperfusion and construct a VaD disease model. Adult male Sprague-Dawley (SD) rats (8 weeks old, weighing 280–350 g) were anesthetized with 2% isoflurane, and the bilateral common carotid arteries were aseptically isolated and permanently ligated. Qualified model animals were randomly divided according to their cognitive baseline into a model group, an MMP8 wild-type group (MMP8-WT), an MMP8-P326V group, a sham operation group (Sham), a positive control group, and an empty vector group; n=10 in each group.

[0037] Dosing regimen 1: LV-MMP8-P326V lentivirus (Group M) and empty vector (Group V) lentivirus, viral titer: 1×10⁻⁶ 9 TU / mL, administered subcutaneously to the scalp on the day of modeling, at a dose of 1 μL / g; the positive control group received piracetam injection at a concentration of 200 mg / mL, administered intraperitoneally after modeling, at a dose of 1.8 μL / g. -1 ·d -1 The animals were administered the drug continuously for 14 and 30 days; the model group received an equal volume of PBS; the sham-operated group received no treatment. During the experiment, all animals had free access to food and water, a 12-hour light-dark cycle, a room temperature of 22 ± 2℃, and a relative humidity of 50–60% to eliminate environmental interference.

[0038] Cognitive function assessments were conducted 14 and 30 days after modeling, using two classic behavioral paradigms: the Y-maze novel arm exploration experiment and the Barnes maze experiment, spaced 4 hours apart. The subjects were double-blinded.

[0039] The Y-maze novel arm exploration experiment uses a device made of black acrylic, with three arms at a 120° angle, each arm measuring 40cm in length, 10cm in width, and 20cm in height. Removable partitions control the connection between the arms. The experiment consists of two phases:

[0040] (1) Training phase: Close one arm (the new arm) and allow the animal to explore freely in the starting arm and other arms for 5 minutes;

[0041] (2) Testing phase: Remove the partition, open the new arm, put the animal back to the starting point, and record the total number of times and time of entering the starting arm, the new arm and other arms within 5 minutes.

[0042] Cognitive indicators: Percentage of new alien arm entries (%) = Number of new alien arm entries / Total number of entries to all three arms × 100%; Percentage of new alien arm dwell time (%).

[0043] The results are as follows Figure 1 As shown in A: the proportion of new heteroarm entries in the MMP8-P326V group was significantly higher than that in the model group ( P <0.05, one-way ANOVA followed by Bonferroni post hoc test), and there was no statistically significant difference compared with the sham surgery group ( P >0.05), indicating that spatial working memory was significantly restored in VaD model rats treated with MMP8-P326V.

[0044] Barnes Maze Experiment: The platform is 122 cm in diameter with 20 evenly distributed 10 cm diameter holes. The target hole is connected to a black PVC safety box. 300 lx white light and 65 dB white noise are placed 50 cm above the platform as aversive stimuli.

[0045] Experimental procedure:

[0046] (1) Days 1–4 are for training, with 2 rounds per day and a maximum of 180 seconds per round. Record the latency, total path length and number of errors of the animal when it finds the target hole.

[0047] (2) On the 5th day, the exploration test was conducted. The safety box was removed, and the latency period of the first arrival at the target hole, the percentage of time spent in the target quadrant, and the total exploration distance were recorded within 90 seconds.

[0048] The results are as follows Figure 1 As shown in B: In the exploration test, the total exploration distance of the target hole in the MMP8-P326V group was reduced compared to the model group. P The value <0.05 indicates that spatial reference memory was significantly improved in VaD model rats after MMP8-P326V intervention.

[0049] Dosing regimen two: LV-MMP8-P326V lentivirus (Group M), LV-MMP8 lentivirus (Group W), and empty vector (Group V) lentivirus, with a viral titer of 1×10⁻⁶. 9 TU / mL, administered via bilateral common carotid artery injection on the day of modeling, at a dose of 0.13 μL / g; the positive control group received piracetam injection at a concentration of 200 mg / mL, administered intraperitoneally after modeling, at a dose of 1.8 μL / g. -1 ·d-1 The animals were administered the drug continuously for 28 days; the model group received an equal volume of PBS; the sham-operated group received no treatment. During the experiment, all animals had free access to food and water, a 12-hour light-dark cycle, a room temperature of 22 ± 2℃, and a relative humidity of 50–60% to eliminate environmental interference. Cognitive function was assessed 28 days after modeling using the Morris water maze test, and the experiment was double-blinded.

[0050] Morris water maze experiment

[0051] The experimental setup consisted of a circular stainless steel pool with a diameter of 120 cm and a height of 50 cm. The water temperature was kept constant at (24±1) ℃. The platform had a diameter of 10 cm and was positioned 1 cm below the water surface in quadrant IV. The experiment was divided into:

[0052] Acquisition (Days 1–5): Four training sessions per day, with random entry into the water from different quadrants, recording escape latency (EL) and swimming speed;

[0053] Space exploration (Probe test, Day 6): Remove the platform, enter the water in any non-platform quadrant, and record the number of times the original platform position is crossed within 60 seconds, the percentage of time spent in the target quadrant, and the latency period of the first crossing.

[0054] The experiment continued to use the aforementioned six groups (n=10 / group): ① Wild-type MMP8 group (WT-MMP8), ② MMP8-P326V group, ③ Empty vector group (Vector), ④ Positive drug group (Oxiracetam), ⑤ Sham operation group (Sham), and ⑥ Model group. All data acquisition was completed using the ANY-maze 3.0 automated tracking system, and the experimenters remained blinded.

[0055] There were no significant differences in swimming speed among the groups, thus ruling out the interference of motor dysfunction on the results.

[0056] The results of the one-way ANOVA and the trajectory plot are as follows: Figure 2 As shown, rats given MMP8-P326V were able to quickly locate the platform on the second day of training, and their swimming path tended to be straight. In the later stages of training, they showed clear goal orientation. Figure 2 A); After the platform was removed, the distance taken by MMP8-P326V rats to reach the target quadrant was reduced, and the dwell time was prolonged, both significantly better than the model group and close to the level of normal rats. Figure 2 B Figure 2 C Figure 2 D). In comparison, although the wild-type MMP8 group and the empty vector group showed some improvement, their station-seeking trajectories were still quite chaotic. Figure 2A); the positive drug group swam widely with no particular preference for the target quadrant; the empty carrier group mostly circled the pool wall, exhibiting a typical "peripheral" search, indicating significant spatial memory deficit. Figure 2 A). Therefore, this indicates that MMP8-P326V significantly enhances the spatial learning and memory retention abilities of VaD model rats.

[0057] The above results suggest that the matrix metalloproteinase 8 mutant MMP8-P326V can improve and enhance the cognitive level of VaD model rats, and MMP8-P326V can be used for the treatment of VaD.

[0058] Example 2: Effects of MMP8-P326V on hemodynamics in brain tissue of rats with vascular dementia

[0059] The experimental design strictly adhered to the principles of randomization, blinding, and control, and included the following six groups (n=10 / group): ① Wild-type MMP8 (W) group, ② MMP8-P326V (M) group, ③ Empty vector group, ④ Positive drug group (Oxiracetam), ⑤ Sham operation group, and ⑥ Model group. All groups received viral vector or drug intervention after bilateral common carotid artery ligation (2-VO) and underwent laser speckle imaging at week 4.

[0060] Hemodynamic quantification was performed using the collateral circulation perfusion index (ROI). The imaging conditions were standardized as follows: laser wavelength 785 nm, power 20 mW, exposure time 20 ms, and spatial resolution 5 µm pixel. -1 The room temperature was maintained at 24 ± 1℃, and the rectal temperature of the rats was 37.0 ± 0.2℃. The data were recorded continuously for 5 min, and the stable signal after the last 3 min was used for spatiotemporal frequency domain filtering (0.05–0.5 Hz band-pass) and artifact removal to finally generate a high signal-to-noise ratio cerebral blood flow distribution map.

[0061] The results are as follows Figure 3As shown: In the sham-operated group, because cerebral blood supply was not blocked, the cerebral perfusion pressure remained normal, and no hypoxia or shear stress signals were triggered. The laser speckle ROI value remained stable at the baseline, and no collateral vessel opening was observed. In the model group, after bilateral common carotid arteries were ligated and blocked, the perfusion of the cerebral core area dropped sharply. Due to the persistent blockage and insufficient distal pressure gradient, the anterior-posterior cerebral artery anastomoses failed to open effectively, and the ROI reading was not significantly different from that in the sham-operated group, presenting a "low perfusion - uncompensated" state. The positive control drug (piracetam) intervention only slightly increased the ROI of the ischemic periphery compared to the model group, suggesting that it can only partially relieve microvascular spasm and is insufficient to establish effective collateral circulation. After injection of empty vector (V group) virus, the ROI value was similar to that of the model group, indicating that the vector itself does not induce angiogenesis or collateral opening, and the interference of the viral vector on subsequent experiments can be ruled out. Wild-type MMP8 (W group) on the contrary significantly decreased the ROI value of the ischemic periphery compared to the MMP8-P326V (M group). P < 0.01), significantly lower than group V; indicating insufficient activation efficiency of wild-type protease, which exacerbates microvascular basement membrane degradation and collapse in the early stages, further worsening perfusion. Conversely, MMP8-P326V (group M) effectively improved the ischemic peri-ROI, significantly increasing it compared to the model group ( P <0.05%, which is also a significant improvement compared to group V ( P < 0.05); confirming that MMP8-P326V can specifically open and stably maintain collateral circulation, providing a structural and perfusion basis for cognitive function recovery. Data statistics and graphing: All data were statistically analyzed using SPSS 26.0. Significance differences were compared using analysis of variance, and the results were expressed as... P A difference of <0.05 indicates a statistically significant difference. A statistical graph was then plotted using GraphPad Prism 10 software.

[0062] Example 3: Effects of the matrix metalloproteinase 8 mutant MMP8-P326V on the structure and function of neurons and the central nervous system

[0063] 1. Sample Acquisition and Fixation

[0064] After the above behavioral and laser speckle blood flow imaging experiments, each group of rats (n=10 / group) was deeply anesthetized (3% isoflurane), and the blood was flushed by perfusion of 100 mL of pre-cooled physiological saline via the heart. Then, 200 mL of 4% paraformaldehyde (PFA, pH 7.4) at 4 ℃ was perfused for 30 min. The whole brain was completely dissected, post-fixed in 4% PFA for 24 h, and then transferred to 30% sucrose-PBS solution at 4 ℃ for dehydration for 48 h until the brain tissue settled to ensure that there was no ice crystal damage in subsequent sections.

[0065] Tissue processing and sectioning: Sections were made continuously along the sagittal plane using a cryostat (Leica CM3050S) with a section thickness of 4 μm. Sections were taken at intervals and attached to poly-L-lysine-treated slides and stored at -80°C for later use.

[0066] Hematoxylin-Eosin staining: Sections were warmed in PBS for 10 min → hematoxylin staining of nuclei for 5 min → differentiation with 1% hydrochloric acid and ethanol for 3 s → blueing with running water for 10 min → eosin staining of chromatin for 2 min → dehydration with graded ethanol (75%, 85%, 95%, 100%, 2 min each) → clearing with xylene for 2 × 5 min → mounting with neutral resin. Observation and image acquisition were performed under an optical microscope.

[0067] Morphological evaluation was conducted, using the following indicators:

[0068] (1) Neuron density: Count the number of surviving neurons in the hippocampus that have round nuclei, clear nuclear membranes, and prominent nucleoli;

[0069] (2) Degeneration score: A double-blind scoring method of 0–4 grade was used—Grade 0: normal morphology, abundant cytoplasm, clear nucleoli; Grade 1: slight eosinophilic enhancement of cytoplasm; Grade 2: shrunken cell body, nuclear pyknosis; Grade 3: cell membrane rupture, deep staining of cytoplasm; Grade 4: cell lysis and disappearance;

[0070] (3) Necrosis rate: the percentage of neurons with a degeneration score ≥2.

[0071] 2. Sample Acquisition and Preprocessing

[0072] After the behavioral and laser speckle experiments, rats in each group (n=10 / group) were fixed by cardiac perfusion with 4% paraformaldehyde. The whole brain was taken and fixed at 4℃ for 24 h, and then dehydrated with 30% sucrose until it sank to the bottom. Sagittal sections were cut continuously using a cryostat, and 20 interstitial sections were collected from each animal and stored at -80℃ for later use.

[0073] The results are as follows Figure 4 As shown:

[0074] In the sham-operated group (Sham), rats did not experience ischemic stimulation. HE staining of the hippocampus showed densely packed, well-formed neurons with uniform eosinophilic cytoplasm and clearly stained nucleoli, showing no degeneration or necrosis, indicating intact normal tissue structure. In the model group (VaD), due to chronic cerebral hypoperfusion injury, the number of neurons in the hippocampus was significantly reduced, with obvious cell degeneration and necrosis, deep nuclear pyknosis, and cell membrane rupture. In the positive control group, neuronal density recovered somewhat compared to the model group, and the degree of damage was significantly reduced. However, some cells still showed nuclear pyknosis and increased eosinophilic cytoplasm, and the neuronal morphology was slightly similar to that of the sham-operated group, suggesting that piracetam only partially alleviated neuronal damage but failed to completely restore normal neuronal structure. In the vector group, hippocampal neurons were similar to those in the model group, with obvious cell degeneration, sparse arrangement, and widespread nuclear pyknosis, confirming that the vector virus itself had no protective effect against neuronal damage. The degree of neuronal degeneration in the wild-type MMP8 group (WT-MMP8) was basically the same as that in the empty-load group, and no obvious neuroprotective effect was observed, indicating that wild-type MMP8 had no significant rescue effect on neuronal structural damage in the VaD model. The hippocampal neurons in the MMP8-P326V group (MMP8-P326V) had the closest morphology to the sham-operated group, with neatly arranged neurons, full cell bodies, uniform eosin staining of cytoplasm, clearly distinguishable nucleoli, significantly restored neuronal density, and less degeneration and necrosis, which was significantly better than the positive drug group.

[0075] The morphological results above suggest that MMP8-P326V can significantly inhibit neuronal degeneration, necrosis, and loss in the hippocampus of VaD model rats, restoring the number and normal morphology of neurons. Its neuroprotective effect is significantly better than that of the traditional positive control drug piracetam, while wild-type MMP8 does not have this activity. Therefore, MMP8-P326V mediates the recovery of cognitive function by specifically improving the structural integrity of hippocampal neurons, and thus has a clear pharmaceutical use as an active ingredient in the preparation of drug compositions for the prevention and treatment of vascular dementia (VaD).

[0076] Immunofluorescence staining procedure

[0077] (1) Rinse with 0.01 M PBS for 3 × 5 min;

[0078] (2) 0.3% Triton X-100 was used to break the membrane at room temperature for 15 min;

[0079] (3) Block with 10% normal goat serum at 37℃ for 30 min;

[0080] (4) Incubate with primary antibody at 4 ℃ overnight:

[0081] - Mouse anti-NeuN monoclonal antibody (Millipore MAB377, 1:500);

[0082] - Rabbit anti-GFAP polyclonal antibody (DAKO Z0334, 1:800);

[0083] (5) Incubate the corresponding fluorescent secondary antibody at 37°C in the dark for 1 hour:

[0084] - Alexa Fluor 488 goat anti-mouse IgG (Invitrogen A11001, 1:1000, green);

[0085] - Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen A11012, 1:1000, red);

[0086] (6) Counterstain the nucleus with DAPI (1 μg / mL) for 5 min;

[0087] (7) Mount the slide with anti-fluorescence quenching mounting medium, store at 4°C in the dark, and acquire images within 24 hours.

[0088] Image acquisition and quantitative analysis

[0089] A laser confocal microscope (Zeiss LSM 980, 20×40× oil immersion) was used to set a fixed field of view (400μm×400μm) for the CA1 region, with a Z-stack thickness of 1.0μm. Laser power, gain, and offset parameters were standardized. Double-blind quantitative analysis was performed using ImageJ 1.54c software.

[0090] NeuN: The mean fluorescence intensity (MFI) of NeuN fluorescent pixels in DAPI-positive nuclei was statistically analyzed, with the MFI of the sham-operated group set at 100%, and the relative fluorescence intensity was calculated.

[0091] GFAP: Calculate the percentage of GFAP-positive area to the total visual field (%Area), and correct for it with the average fluorescence intensity to obtain the "GFAP activation index";

[0092] Neuron morphology: The ratio of cell body major diameter to minor diameter and the total length of processes were measured to assess the degree of cell swelling or shrinkage. Statistical analysis: All data are expressed as mean ± standard deviation. One-way ANOVA combined with Bonferroni post-hoc test was used, with a significance level of α = 0.05.

[0093] The results are as follows Figure 5 As shown:

[0094] (1) NeuN signal

[0095] Sham surgery group: Hippocampal neurons were densely arranged, and NeuN-positive cell nuclei were uniformly stained. Figure 5 A); Model group: NeuN fluorescence intensity decreased, and NeuN-positive cell nuclei showed significant condensation and fragmentation, indicating neuronal damage ( Figure 5 B); MMP8-WT group: NeuN fluorescence intensity decreased ( Figure 5 B); MMP8-P326V group: NeuN fluorescence intensity was not significantly different from that of the sham-operated group and was higher than that of the model group. NeuN-positive cell nuclei were uniformly stained and their morphology was more similar to that of the sham-operated group. Figure 5 B).

[0096] (2) GFAP signal

[0097] Sham-operated group: Astrocytes were in a resting state; Model group: Cells were enlarged and had dense processes, indicating severe reactive gliosis. Figure 5 A); Morphological analysis showed that the cell body area of ​​astrocytes in the MMP8-P326V group recovered to the resting state level, and the complexity of process branching decreased ( Figure 5 A) and the fluorescence intensity of GFAP, a marker of astrocytes, in the MMP8-P326V group was significantly lower than that in the model group and the empty vector group. Figure 5 C), suggesting that it effectively inhibits VaD-induced excessive colloid activation.

[0098] Example 4: Effects of the matrix metalloproteinase 8 mutant MMP8-P326V on cognitive function in AD model mice

[0099] The inventors used a 5XFAD transgenic mouse model (AD disease model): Adult male 5XFAD mice (16 weeks old, weighing 20–25g) were randomly divided into four groups based on cognitive baseline: 5XFAD+Vector group, 5XFAD+MMP8 group, 5XFAD+MMP8-P326V group, 5XFAD group, and normal group; n=10 in each group. After anesthesia with 2% isoflurane, stereotactic brain localization was performed on the right hippocampus of the mice under sterile conditions, and LV-MMP8-P326V, LV-MMP8, and LV-Vector were injected to evaluate the intervention effect of MMP8-P326V on Alzheimer's disease (AD)-related cognitive impairment. Subsequently, a novel object recognition (NOR) test was performed to evaluate the cognitive performance of each group of mice.

[0100] Dosage regimen: LV-MMP8-P326V lentivirus (5XFAD+MMP8-P326V group), LV-MMP8 lentivirus (5XFAD+MMP8 group), and empty vector (5XFAD+Vector group) lentivirus were administered at a viral titer of 1×10⁻⁶. 9TU / mL, stereotactic injection into the brain on the day of treatment, dose 0.1 μL / g; the 5XFAD group received an equal volume of PBS; the normal group received no treatment. During the experiment, all animals had free access to food and water, a 12-hour light-dark cycle, a room temperature of 22 ± 2℃, and a relative humidity of 50–60% to eliminate environmental interference.

[0101] Cognitive function was assessed 14 days after drug administration using a novel object recognition experiment, a classic behavioral paradigm that assesses learning and memory abilities based on spontaneous exploratory behavior in mice without the need for reward or punishment stimuli. The experiment was double-blind.

[0102] Test box: 40 cm × 40 cm × 40 cm gray PVC square open box, with matte film on the bottom and four walls to eliminate reflections; a 4K camera is fixed on the top, operating at 30 fps, with uniform illumination under weak white light (50 lx). Object selection: ① Cylindrical LEGO bricks (5cm high, 3cm Φ); ② Cube LEGO bricks (3cm side length); all weighing > 50g to prevent movement; surfaces wiped with 75% ethanol and dried to eliminate residual odor.

[0103] Adaptation period: Each mouse was placed in an empty test chamber and allowed to explore freely for 10 minutes; after the 10 minutes, the mice were returned to their rearing cages, the chambers were thoroughly cleaned with 75% alcohol and allowed to air dry; the next mouse was placed after an interval of ≥ 5 minutes.

[0104] Acquaintance period: Fix two identical objects (A1, A2) diagonally opposite each other in the enclosure, 5cm from the enclosure wall. Place the mouse in the center with its back to the objects and record its exploration behavior towards the two objects for 10 minutes (nose tip ≤ 1cm from the object and front paw contact / scent meter is considered valid exploration; climbing or sitting is not counted). Clean the enclosure, and repeat the process with the second mouse in the same order after 1 hour, until all mice have been introduced.

[0105] Delay period: Mice were returned to their rearing cages and kept in a quiet environment for 2 hours.

[0106] Test period: One of the objects was randomly replaced with a new object B (B has a different shape, color, and material than A, but the same height and base area). Positional balancing was performed (random left / right) to eliminate spatial bias. The same mouse was placed back in the center, and video was recorded for 5 minutes. Exploration time was calculated: The effective exploration time for the familiar object (A) and the novel object (B) was recorded separately.

[0107] The results are as follows Figure 6 As shown: In the new object recognition experiment, normal C57 mice exhibited a stable exploration preference, while the 5XFAD group showed a more significant decrease compared to the 5XFAD+MMP8-P326V group. Figure 6D), indicating that the genetically modified background has caused significant cognitive deficits; the absolute discrimination measure, recognition index, and preference index of the 5XFAD+Vector group all showed a tendency to explore old objects, which were statistically significant compared with the 5XFAD+MMP8-P326V group. P <0.05, Figure 6 B Figure 6 C Figure 6 (D) This confirms that the viral vector itself has no restorative effect on cognitive function. While wild-type MMP8 (WT-MMP8) showed better exploration of new objects than the 5XFAD group, it was significantly lower than the 5XFAD+MMP8-P326V group. Figure 6 B Figure 6 C Figure 6 In contrast, MMP8-P326V significantly improved the cognitive level of AD model mice, with no statistically significant difference compared to the normal group (D). P >0.05), and significantly improved compared to the 5XFAD+Vector group ( P <0.05), almost completely reversing the loss of new object exploration preference caused by 5XFAD ( Figure 6 B Figure 6 C Figure 6 (D) demonstrates that the MMP8-P326V mutant protein can significantly improve memory recognition function in the pathological microenvironment of AD.

[0108] Example 5: Effects of the matrix metalloproteinase 8 mutant MMP8-P326V on the core pathology of AD model mice.

[0109] ELISA was used to detect the levels of Aβ42, Aβ40, total tau, and phosphorylated tau proteins.

[0110] 1. Sample preparation

[0111] Brain tissue from AD mice was collected, flash-frozen in liquid nitrogen, and stored at -80°C.

[0112] Add 1 mL of pre-chilled RIPA lysis buffer (containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor) to every 100 mg of tissue, homogenize, and incubate on ice for 30 minutes.

[0113] Centrifuge at 4℃, 12,000×g for 15 minutes, and collect the supernatant.

[0114] Total protein concentration was determined using the BCA method. The protein concentration of each sample was adjusted to be consistent (e.g., 1 mg / mL). After aliquoting, the samples were stored at -80℃ for later use.

[0115] 2. ELISA testing

[0116] The contents of Aβ42, Aβ40, total tau, and p-tau were detected using a commercial ELISA kit (Lunchangshuo Biotechnology Kit). The procedure was performed according to the instructions, and the brief steps are as follows:

[0117] Aβ42 and Aβ40 detection: Add standards and samples (diluted to the linear range, e.g., 1:10–1:100) to a 96-well plate pre-coated with anti-Aβ antibody and incubate at 37°C for 2 hours. After washing, add biotin-labeled detection antibody and incubate for 1 hour. After washing, add HRP-streptavidin and incubate for 30 minutes. Add TMB for color development, react at room temperature in the dark for 15 minutes, and measure absorbance at 450 nm. Calculate the concentrations of Aβ42 and Aβ40 based on the standard curve, and calculate the Aβ42 / Aβ40 ratio.

[0118] Total tau (T-tau) and phosphorylated tau (p-tau) detection: Add standards and samples (diluted to the linear range, e.g., 1:50–1:200) to a 96-well plate pre-coated with anti-tau antibody and incubate at 37°C for 2 hours. After washing, add specific detection antibodies (for p-tau, use anti-phosphorylation site antibodies, such as p-tau181) and incubate for 1 hour. After washing, add HRP-labeled secondary antibody and incubate for 30 minutes. Add TMB for color development and measure absorbance at 450 nm. Calculate the concentrations of T-tau and p-tau based on the standard curve.

[0119] 3. Data Analysis

[0120] All samples were tested in triplicate, and the average value was taken. Data are expressed as the target protein content per milligram of total protein (pg / mg). Statistical analysis was performed using GraphPad Prism. One-way ANOVA and Tukey's post-hoc test were used for comparisons among multiple groups. The significance level was set at 100%. P < 0.05.

[0121] The results are as follows Figure 7 As shown: ELISA was used to quantitatively analyze the Aβ42 / Aβ40 ratio, total tau (T-tau), and phosphorylated tau (p-tau) levels. Aβ42 / 40 was negatively correlated with Aβ plaque burden in the brain. It was found that the 5XFAD+MMP8-P326V group, compared with the 5XFAD+Vector group and the 5XFAD+MMP8 group, effectively increased the Aβ42 / Aβ40 ratio in the brains of 5XFAD mice. Figure 7 A), that is, it reduced the Aβ load in the mouse brain. However, brain tissue ELISA showed that the 5XFAD+MMP8-P326V group significantly reduced the T-tau content in 5XFAD mice compared with the normal group and the 5XFAD+MMP8 group. Figure 7 B), and the p-tau content in the 5XFAD+MMP8-P326V group decreased ( Figure 7 (C), which suggests that MMP8-P326V can have a positive effect on changes in p-tau protein in 5XFAD mice.

Claims

1. Application of matrix metalloproteinase 8 mutant MMP8-P326V in the preparation of drugs for the treatment of vascular dementia or Alzheimer's disease.

2. The application according to claim 1, characterized in that: The application of the matrix metalloproteinase 8 mutant MMP8-P326V in the preparation of drugs that improve neuronal function, cognitive impairment and cognitive function in vascular dementia or Alzheimer's disease.

3. The application according to claim 1, characterized in that: The matrix metalloproteinase 8 mutant MMP8-P326V is injected via the common carotid artery, subcutaneous injection, or intracranial injection.

4. The application according to claim 1, characterized in that: The vascular dementia mentioned refers to dementia caused by ischemic cerebrovascular disease or dementia caused by cerebrovascular injury.

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