Use of mere-5 in the preparation of a medicament and / or a pharmaceutical composition for preventing and / or treating ischemic cerebrovascular disease

Abstract

The present application relates to the technical field of biological medicine, and more particularly to application of Mere-5 in preparation of a medicine and / or a pharmaceutical composition for preventing and / or treating ischemic cerebrovascular disease, wherein the present application shows for the first time that Mere-5 can significantly improve neurological function impairment of a mouse caused by a thread blocking method and reduce the percentage of cerebral infarction volume of the mouse, and therefore, the Mere-5 provided by the present application has a good application prospect in preparation of a medicine and / or a pharmaceutical composition for preventing and / or treating ischemic cerebrovascular disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of Mere-5 in the preparation of drugs and / or pharmaceutical compositions for the prevention and / or treatment of ischemic cerebrovascular diseases. Background Technology

[0002] Ischemic cerebrovascular disease, especially ischemic stroke, which accounts for about 80% of all strokes, has become one of the diseases with the highest morbidity, mortality, and disability rates globally. This clinical emergency caused by narrowing or occlusion of the arteries supplying blood to the brain relentlessly attacks more than 12 million new patients every year. According to the Global Burden of Disease Study, stroke continues to rank as the second leading cause of death and the third leading cause of disability worldwide. Its heavy disease burden profoundly threatens the health of all humanity, with the elderly bearing the brunt. With increasing age, the natural aging of blood vessels and the accumulation of risk factors such as hypertension and atrial fibrillation cause the risk of ischemic stroke to rise exponentially. However, it is alarming that, influenced by unhealthy lifestyles, the disease is also increasingly occurring in young and middle-aged adults, showing a trend towards affecting younger people.

[0003] The devastating impact of this disease on health and quality of life is multidimensional and long-lasting. Survivors often suffer from hemiplegia, aphasia, dysphagia, sensory abnormalities, and even vascular cognitive impairment, instantly robbing a person of the basic abilities to walk independently, communicate freely, and eat on their own. Patients not only suffer from the loss of physical function but also experience a collapse in their psychological and social roles, leading to a sharp decline in their quality of life.

[0004] In recent years, thanks to the widespread adoption of primary prevention strategies such as hypertension control, smoking cessation, exercise, anticoagulation, and lipid-lowering, the age-standardized incidence of stroke in many regions worldwide has generally decreased compared to before. This is undoubtedly the result of the unremitting efforts of public health. However, this progress is not optimistic. The surging tide of population aging and the prevalence of metabolic diseases and other risk factors mean that the absolute number of stroke cases remains large, and is even rising in many countries. Therefore, stroke remains one of the greatest health challenges facing the population.

[0005] Among various neurological diseases, ischemic stroke stands out for its exceptionally high risk. Unlike the relatively slow-progressing Alzheimer's or Parkinson's diseases, it has a rapid onset, causing irreversible brain tissue necrosis within minutes to hours. Its short-term mortality and long-term disability rates consistently rank among the highest of neurological diseases. Unfortunately, correspondingly, the number of effective drugs currently available for the treatment of ischemic cerebrovascular diseases, both domestically and internationally, is extremely limited.

[0006] Therefore, in the face of this serious health challenge, the search for new and effective treatments remains an urgent matter for the scientific community and clinical medicine. Summary of the Invention

[0007] To address the aforementioned problems, the object of this invention is to provide the use of Mere-5 in the preparation of medicaments and / or pharmaceutical compositions for the prevention and / or treatment of ischemic cerebrovascular diseases.

[0008] Mere-5 is a pentapeptide extracted from clams and possesses antioxidant properties. Existing techniques have isolated a small molecule enzymatically hydrolyzed fraction (LMEC) from clam soft tissue. This fraction has a protein / peptide content of 79%, an average molecular weight of 1.05 kDa, and exhibits excellent performance in multiple antioxidant indicators, including total reducing power, hydroxyl radical scavenging, DPPH scavenging, and superoxide anion scavenging. DEAE ion exchange separation and UPLC-MS analysis of the LMEC revealed six possible peptide sequences. One of these is Val-Glu-Leu-Thr-Lys (molecular weight 588 Da, i.e., Mere-5), with its N-terminal Val being a hydrophobic amino acid, consistent with the common structural characteristics of antioxidant peptides; it also contains glutamate (Glu) and Lys, imparting certain charge properties that may influence its interaction with biological membranes or receptors. This invention is the first to discover that Mere-5 has a significant protective effect against cerebral ischemia, and it holds promise for use in the preparation of drugs for ischemic cerebrovascular diseases.

[0009] The objective of this invention can be achieved through the following technical solutions: The first object of the present invention is to provide the use of Mere-5 in the preparation of medicaments and / or pharmaceutical compositions for the prevention and / or treatment of ischemic cerebrovascular diseases, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] In one embodiment of the present invention, the ischemic cerebrovascular disease is ischemic stroke.

[0011] Preferably, the ischemic stroke is an acute ischemic stroke.

[0012] A second object of the present invention is to provide the use of the Mere-5 promoter in the preparation of medicaments and / or pharmaceutical compositions for the prevention and / or treatment of ischemic cerebrovascular diseases, the amino acid sequence of which is shown in SEQ ID NO.1.

[0013] In one embodiment of the present invention, the ischemic cerebrovascular disease is ischemic stroke.

[0014] Preferably, the ischemic stroke is an acute ischemic stroke.

[0015] A third objective of this invention is to provide a drug for the prevention and / or treatment of ischemic cerebrovascular disease, the drug containing Mere-5 and / or a Mere-5 promoter; the amino acid sequence of Mere-5 is shown in SEQ ID NO.1.

[0016] In one embodiment of the present invention, the dosage form of the drug includes one or more of the following: suspension, granules, capsules, powders, tablets, emulsions, pills, injections, suppositories, enemas, aerosols, patches, or drops.

[0017] In one embodiment of the invention, the drug contains pharmaceutically acceptable excipients.

[0018] In one embodiment of the present invention, the excipients include one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0019] In one embodiment of the present invention, the drug is administered via one of the following methods: intravenous injection, subcutaneous injection, intramuscular injection, oral administration, or transdermal absorption.

[0020] A fourth object of the present invention is to provide a pharmaceutical composition for the prevention and / or treatment of long fibrosis, said pharmaceutical composition containing the above-mentioned drug.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of Mere-5 in the prevention and treatment of brain diseases. Pharmacological experiments on mice show that Mere-5 can significantly improve neurological deficits induced by suture occlusion and reduce the percentage of cerebral infarction volume in mice. Therefore, Mere-5 provided by this invention has good application prospects in the preparation of drugs for the prevention and treatment of ischemic cerebrovascular diseases. Attached Figure Description

[0022] Figure 1 This diagram illustrates the effects of different experimental groups of drugs on the neurological function of mice.

[0023] Figure 2 Comparison of TTC staining images of the brains of mice in different experimental groups and the results of cerebral infarction volume.

[0024] Figure 3 A schematic diagram illustrating the effect of HE staining on the pathological damage of Mere-5 on brain tissue in MCAO / R mice.

[0025] Figure 4 A schematic diagram illustrating the effect of Mere-5 on pathological damage in brain tissue of MCAO / R mice as observed by Nissl staining.

[0026] Figure 5 This diagram illustrates the effect of Mere-5 on the expression of inflammation-related factors mRNA in ischemic brain tissue of MCAO / R mice; where (A) Il1b(B) Il6 (C) Tnf (D) Ccl2 .

[0027] Figure 6 This is a schematic diagram illustrating the effect of Mere-5 on oxidative stress levels in the brain tissue of MCAO / R mice. Figure 7 This is a schematic diagram showing the effect of Mere-5 on SIRT1 protein expression in ischemic brain tissue of MCAO / R mice; (A) Western blot analysis of SIRT1 protein expression in brain tissue of each group of mice; (B) Statistical analysis of the relative expression of SIRT1 protein.

[0028] Figure 8 This is a schematic diagram showing the effect of Mere-5 on NOX2 protein expression in ischemic brain tissue of MCAO / R mice; (A) Western blot analysis of NOX2 protein expression in brain tissue of each group of mice; (B) Statistical analysis of the relative expression of NOX2 protein.

[0029] Figure 9 This is a schematic diagram showing the effect of Mere-5 on NOX2 protein expression in ischemic brain tissue of MCAO / R mice; (A) Western blot analysis of NOX2 protein expression in brain tissue of each group of mice; (B) Statistical analysis of the relative expression of NOX2 protein.

[0030] Figure 10 This diagram illustrates the effect of Mere-5 on the mRNA levels of inflammatory factors in BV2 cells after ODG / R; where (A) Il1b (B) Il6 (C) Tnf .

[0031] Figure 11 This is a schematic diagram illustrating the effect of Mere-5 on MDA levels in BV2 cells after OGD / R.

[0032] Figure 12 A schematic diagram showing the effect of Mere-5 on ROS levels in BV2 cells after OGD / R (quantitative detection by flow cytometry); where (A) is a typical flow cytometry diagram; and (B) is a statistical analysis of fluorescence intensity.

[0033] Figure 13 A schematic diagram showing the effect of Mere-5 on ROS levels in BV2 cells after OGD / R under fluorescence microscopy; (A) a representative image of ROS levels in BV2 cells detected by fluorescence microscopy (DCFH-DA staining, scale bar = 100 μm), with green fluorescence intensity reflecting ROS levels; (B) statistical analysis of fluorescence intensity.

[0034] Figure 14 This is a schematic diagram showing the effect of Mere-5 on SIRT1 protein expression in BV2 cells after OGD / R; (A) Western blot analysis of SIRT1 protein expression in BV2 cells; (B) Statistical analysis of the relative expression level of SIRT1 protein.

[0035] Figure 15 This is a schematic diagram showing the effect of Mere-5 on NOX2 protein expression in BV2 cells after OGD / R; (A) Western blot analysis of NOX2 protein expression in BV2 cells; (B) Statistical analysis of the relative expression level of NOX2 protein.

[0036] Figure 16 This is a schematic diagram showing the effect of Mere-5 on the expression of NF-κB-related proteins in BV2 cells after OGD / R. Among them, (A) Western blot analysis of p-p65 and p65 protein expression in the brain tissue of mice in each group; (B) Statistical analysis of p-p65 / p65 ratio; (C) Statistical analysis of p65 / β-actin ratio. Detailed Implementation

[0037] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0038] Unless otherwise specified, all reagents used in the following embodiments are commercially available reagents, and all detection methods and techniques used are conventional detection methods and techniques in the art.

[0039] Example 1 This embodiment provides a Mere-5, whose amino acid sequence is shown in SEQ ID NO.1, specifically VGLTL (Val-Glu-Leu-Thr-Lys), which was synthesized by Shanghai Taopu Biotechnology Co., Ltd. (purity 97.56%). Its chemical structural formula is shown below: .

[0040] Example 2 This embodiment provides an animal experiment to investigate the use of Mere-5 in the treatment of ischemic cerebrovascular disease, as detailed below: The pharmacological experiment of creating a local cerebral ischemia model by middle cerebral artery occlusion (MCAO) in mice is a commonly used animal experiment to verify the effect of drugs in preventing and treating ischemic cerebrovascular diseases. Therefore, it was used to verify the protective effect of Mere-5 on cerebral ischemia in experimental animals.

[0041] (1) Animals: Healthy male C57BL / 6J mice, weighing 20-25 g, were housed in groups of 6 per cage, for a total of 5 groups. They were purchased from Suzhou Sibefore Biotechnology Co., Ltd., Jiangsu Province, with quality certificate number (Animal Certificate No.: B202411280320), and housed in an SPF-grade animal facility.

[0042] (2) Experimental method: The left middle cerebral artery embolization / reperfusion (MCAO / R) surgery was performed on experimental mice using the suture occlusion method.

[0043] ① Mice were weighed and anesthetized with an intraperitoneal injection of 1% sodium pentobarbital at a dose of 100 mg / kg. The mice were then fixed in a supine position on the operating table, and the neck vessels were dissected and exposed. ② A silicone-coated suture was inserted into the internal carotid artery through the bifurcation of the common carotid artery, blocking the blood supply to the origin of the middle cerebral artery and all its collateral branches, causing focal ischemia in the middle cerebral artery area. After 2 hours of ischemia, the suture was gently pulled out. The patient was then returned to their cage for 24 hours of reperfusion. All of the above procedures were performed under constant room temperature conditions.

[0044] ③TTC staining: After neurological behavioral scoring, mice were anesthetized with sodium pentobarbital (100 mg / kg, IP) and euthanized. Brain tissue was collected and frozen at -20°C for 20 min to sclerosis for sectioning. After freezing, the brain tissue was placed on ice and cut into 5 coronal sections with a scalpel. The sections were placed in 6-well plates containing 1.5% TTC staining solution and stained at 37°C in the dark. During staining, the sections were rotated every 10 min, staining both sides for 10 min each. After staining, the TTC staining solution was discarded, and the brain slices were fixed with 4% paraformaldehyde. The next day, the infarct volume was quantified using Image J. The infarcted area appeared white; infarct volume (%) = white area volume / total brain slice volume × 100%.

[0045] (3) Experimental grouping: Mere-5 low-dose group (Mere-5 L): The dose was 1.0 mg (Mere-5) / kg (mice); mice were administered the drug via tail vein injection once within 2 hours after surgery.

[0046] Mere-5 high-dose group (Mere-5 H): The dose was 5.0 mg (Mere-5) / kg (mice); mice were administered the drug via tail vein injection once within 2 hours after surgery.

[0047] Positive control group (Edaravone): mice were given a dose of 3 mg (edaravone) / kg (edaravone injection, produced by Nanjing Sinopharm Dongyuan Pharmaceutical Co., Ltd.); mice were given a single tail vein injection within 2 hours after surgery.

[0048] Sham surgery group: Blood vessels were separated but no ischemic brain surgery was performed.

[0049] Negative control group (also known as "model group", MCAO / R): Received the same amount of normal saline as the drug experimental group and underwent cerebral ischemia surgery.

[0050] Twenty-four hours after reperfusion, behavioral changes in mice were observed using the modified Garcia JH score. The degree of neurological impairment was assessed in six aspects: voluntary movement, body symmetry, forelimb extension function, climbing movement, bilateral tactile sensation, and bilateral whisker touch response. See Table 1 below for details.

[0051] Table 1. Neurobehavioral Scoring Rules (4) Conclusion Analysis 1) Effects of drugs on neurological function in a mouse model of cerebral ischemia.

[0052] like Figure 1 As shown, compared with the MCAO / R group, the neurological function scores of mice in the Mere-5 H group were significantly increased ( P <0.05).

[0053] 2) Effects of drugs on the percentage of cerebral infarction volume in a mouse model of cerebral ischemia like Figure 2 As shown, compared with the MCAO / R group, the percentage of cerebral infarction volume in the Mere-5 H group mice was significantly reduced ( P <0.05).

[0054] The above experimental results indicate that Mere-5 has a significant therapeutic effect on cerebral ischemia induced by suture occlusion in MCAO / R mice, improving cerebral blood flow and reducing the percentage of cerebral infarction volume. Therefore, Mere-5 can be used to prepare drugs for the prevention and treatment of ischemic cerebrovascular diseases.

[0055] Example 3 This embodiment provides further exploration of Mere-5 (based on Embodiment 2), as follows: (1) Mere-5 alleviates pathological damage to the ischemic cortex in MCAO / R mice To further clarify the protective effect of Mere-5 on ischemic brain tissue from a histomorphological perspective, HE staining and Nissl staining were performed on the brain tissue of each group of mice (based on the sham-operated group, negative control group (model group), and Mere-5 group (5.0 mg / kg) in Example 2), respectively, to observe the pathological changes in the ischemic cortical tissue of each group of mice. HE staining results showed (e.g.) Figure 3As shown in the figure, the sham-operated group exhibited clear cortical and hippocampal structures with uniform staining, neat and tightly arranged neurons, normal cell morphology, round or oval nuclei, clear nucleoli, and abundant cytoplasm, with no obvious pathological changes. In contrast, the model group showed severe pathological damage to the ischemic cortex, characterized by disordered neuronal arrangement, loose structure, extensive pyknosis or karyolysis of neurons, significantly widened intercellular spaces with edema, and extensive inflammatory cell infiltration and glial cell proliferation. Compared to the model group, the high-dose Mere-5 group showed significantly reduced pathological damage to the brain tissue, with more intact cell morphology, more regular arrangement, and reduced inflammatory infiltration.

[0056] Nissl staining specifically displays Nissl bodies within neuronal cell bodies and is an important indicator for assessing neuronal viability. Nissl staining shows (e.g.) Figure 4 As shown in the figure, the sham-operated group had abundant Nissl bodies in the cytoplasm of neurons, which stained dark blue and granular, and were evenly distributed, indicating good neuronal function. In the model group, Nissl bodies were significantly reduced or even disappeared, and the neuronal cell bodies were shrunken, showing obvious "dark neurons" changes, reflecting severe ischemic neuronal damage. The number of surviving neurons in the ischemic area of ​​mice in the high-dose Mere-5 group was significantly increased compared with that in the model group, and the number of Nissl bodies was somewhat restored.

[0057] The combined HE and Nissl staining results show that Mere-5 can effectively alleviate brain tissue structural damage caused by MCAO / R at the histopathological level and protect the survival of neurons in ischemic areas, further confirming its protective activity against mouse brain from a morphological perspective.

[0058] (2) Mere-5 reduces the expression of inflammatory factor mRNA in the brain tissue of MCAO / R mice. To investigate whether Mere-5 can inhibit the neuroinflammatory response in the brain tissue of MCAO / R mice, qPCR was used to detect the neuroinflammatory response in the ischemic brain tissue of each group of mice (based on the sham-operated group, negative control group (model group), and Mere-5 group (5.0 mg / kg) in Example 2). Il1b , Il6 , Tnf and Ccl2 mRNA expression levels.

[0059] Experimental method: Mouse brain tissue was quickly removed after the experiment, and trizol was added to extract RNA, which was then reverse transcribed into cDNA.

[0060] Quantitative detection was performed using the SYBR Green fluorescent dye method. 10 μL of 2×SYBR Green Pro Taq HSPremix, 1 μL of cDNA template, 0.4 μL each of forward and reverse primers (10 μM), and RNase-free water were added to a reaction volume of 20 μL. The amplification program was set as follows: pre-denaturation (95℃ for 30 s), followed by denaturation and annealing extension (95℃ for 5 s, 60℃ for 30 s, for a total of 40 cycles), and finally melting curve acquisition (95℃ for 15 s, 60℃ for 1 min, 95℃ for 1 s). After amplification, melting curve analysis was performed to check if the melting curve showed a single peak, confirming the absence of primer dimers and non-specific amplification. Each sample was tested in triplicate, with Actb as an internal control gene.

[0061] qPCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0062] The primer sequences are shown in Table 2.

[0063] Table 2 Primer Sequences After the RT-qPCR experiment, the obtained fluorescence amplification data were processed and analyzed. This study used the 2^-ΔΔCt method to calculate the relative expression level of the target gene. First, ΔCt was calculated: ΔCt = Ct (target gene) - Ct (internal reference gene), then ΔΔCt was calculated: ΔΔCt = ΔCt (experimental group) - ΔCt (control group). The relative expression level of the target gene was then calculated: relative expression level = 2^-ΔΔCt.

[0064] qPCR test results show (e.g.) Figure 5 As shown in the figure, compared with the sham-operated group, the expression of pro-inflammatory cytokines in the model group was significantly upregulated. Tnf , Il1b , Il6 and Ccl2 The mRNA expression levels were significantly upregulated. P <0.05), indicating that MCAO / R induced a strong inflammatory response. After Mere-5 administration, the mRNA expression levels of the above-mentioned inflammatory factors all showed a downward trend to varying degrees ( P <0.05).

[0065] (3) Mere-5 reduces MDA levels in brain tissue of MCAO / R mice The content of MDA in the ischemic brain tissue of mice in each group (based on the sham-operated group, negative control group (model group), and Mere-5 group (5.0 mg / kg) in Example 2) was detected using the thiobarbituric acid reactive substances (TBARS) method. The results showed (e.g.) Figure 6 As shown in the figure, compared with the sham-operated group, the MDA content in the brain tissue of mice in the model group was significantly increased ( P <0.05) indicates that significant lipid peroxidation damage and a significantly enhanced level of oxidative stress occurred in the brain tissue after MCAO / R. Compared with the model group, the MDA content in the brain tissue of mice in the Mere-5 group was reduced ( P The value of <0.05 indicates that Mere-5 can effectively inhibit lipid peroxidation induced by cerebral ischemia-reperfusion and reduce oxidative stress damage.

[0066] (4) Mere-5 regulates the SIRT1 / NOX2 / NF-κB pathway to reduce oxidative stress and inflammatory response in MCAO / R mice. Western blot was used to detect the expression levels of SIRT1, NOX2, and key proteins (p-p65; total protein p65) in the ischemic brain tissue of mice in each group (based on the sham-operated group, negative control group (model group), and Mere-5 group (5.0 mg / kg) in Example 2) to systematically verify the regulatory mechanism of Mere-5 on the SIRT1 / NOX2 / NF-κB signaling axis at the protein level.

[0067] SIRT1 is a NAD-dependent... + The deacetylases play an important endogenous protective role in CIRI, negatively regulating inflammatory and oxidative stress responses by deacetylating various substrate proteins. Western blot results are as follows... Figure 7 As shown, compared with the sham-operated group, the expression level of SIRT1 protein in the ischemic side brain tissue of the model group mice was significantly decreased ( P <0.05). Compared with the model group, SIRT1 protein expression was significantly upregulated after Mere-5 administration ( P <0.05), indicating that Mere-5 can effectively restore SIRT1 expression in ischemic brain tissue and enhance endogenous protective mechanisms.

[0068] NOX2 is a key enzyme in the production of reactive oxygen species (ROS) in the brain, and its overactivation is closely related to oxidative stress damage after cerebral ischemia-reperfusion. Studies have shown that SIRT1 can inhibit the transcriptional activation and protein expression of NOX2 through mechanisms such as deacetylation modification, thereby reducing ROS production. Western blot results are as follows... Figure 8 As shown, compared with the sham-operated group, the expression level of NOX2 protein in the brain tissue of mice in the model group was significantly increased ( P <0.05%. After Mere-5 administration, NOX2 protein expression was significantly different from that in the model group ( PThe result was <0.05, indicating that Mere-5 exerts its antioxidant protective effect by upregulating SIRT1 and thus inhibiting the overexpression of NOX2.

[0069] Western blot results of p-p65 / p65 protein expression are as follows: Figure 9 As shown, compared with the sham-operated group, the p-p65 (phosphorylated p65) protein level in the brain tissue of mice in the model group was significantly increased, and the p-p65 / p65 ratio was significantly increased. P <0.05), indicating that the NF-κB pathway is strongly activated after MCAO / R. After Mere-5 intervention, both p-p65 protein levels and the p-p65 / p65 ratio were significantly reduced ( P <0.05), while there was no significant difference in total p65 protein expression among the groups (p65 / β-actin ratio). P The result was >0.05, indicating that Mere-5 mainly inhibits the NF-κB signaling pathway by suppressing the phosphorylation activation of p65, rather than affecting the overall expression level of p65.

[0070] In summary, Mere-5 upregulates SIRT1 expression, inhibits NOX2 protein levels, and suppresses NF-κB (p65) phosphorylation activation in the brain tissue of MCAO / R mice.

[0071] (5) Mere-5 inhibits OGD / R-induced expression of inflammatory factors in BV2 cells. The effect of Mere-5 intervention on the expression level of pro-inflammatory factor mRNA in OGD / R-damaged BV2 cells was detected by RT-qPCR to evaluate the anti-neuroinflammatory effect of Mere-5 at the cellular level.

[0072] 1) Cells: Mouse microglia (BV2) were purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0073] 2) Experimental methods An OGD / R model was constructed using BV2 cells to simulate the hypoxic, hypoglycemic, and reoxygenated microenvironment of microglia during cerebral ischemia-reperfusion, providing an experimental basis for evaluating the protective effect of Mere-5 on cell damage and its possible mechanisms.

[0074] Cells were cultured in 6-well plates. When they reached approximately 70% confluence, the original culture medium was aspirated, and the cells were washed twice with sugar-free PBS to thoroughly remove residual glucose. Sugar-free DMEM medium was then added, and the culture plates were quickly transferred to a hypoxic chamber (5% CO2, 95% N2, 37℃). After aeration for 15 min, the nitrogen valve and the hypoxia device valve were closed. The plates were then placed in a 5% CO2, 37℃ incubator for 2 h of timed hypoxia to perform OGD treatment. After OGD treatment, the sugar-free hypoxic medium was quickly aspirated and replaced with normal high-glucose DMEM complete medium containing 10% FBS. The culture plates were then transferred back to a conventional CO2 incubator (37℃, 5% CO2, 21% O2) for 24 h of reoxygenation / reglucose treatment to simulate the reperfusion process.

[0075] After OGD / R, discard the old culture medium and gently wash the cells twice with sterile PBS. Add 1 mL of trizol to each well to lyse the cells and extract total RNA. Add reverse transcription reagent to obtain cDNA, which is then used for RT-qPCR experiments.

[0076] Quantitative detection was performed using the SYBR Green fluorescent dye method. 10 μL of 2×SYBR Green Pro Taq HSPremix, 1 μL of cDNA template, 0.4 μL each of forward and reverse primers (10 μM), and RNase-free water were added to a reaction volume of 20 μL. The amplification program was set as follows: pre-denaturation (95℃ for 30 s), followed by denaturation and annealing extension (95℃ for 5 s, 60℃ for 30 s, for a total of 40 cycles), and finally melting curve acquisition (95℃ for 15 s, 60℃ for 1 min, 95℃ for 1 s). After amplification, melting curve analysis was performed to check if the melting curve showed a single peak, confirming the absence of primer dimers and non-specific amplification. Each sample was tested in triplicate, with Actb as the internal control gene.

[0077] qPCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0078] The primer sequences are shown in Table 2.

[0079] After the RT-qPCR experiment, the obtained fluorescence amplification data were processed and analyzed. The relative expression level of the target gene was calculated using the 2^-ΔΔCt method. First, ΔCt was calculated: ΔCt = Ct(target gene) - Ct(internal reference gene), then ΔΔCt was calculated: ΔΔCt = ΔCt(experimental group) - ΔCt(control group). The relative expression level of the target gene was then calculated: relative expression level = 2^-ΔΔCt.

[0080] 3) Experimental Grouping ① Control group: Routine culture with complete culture medium added; ②Mere-5 high-dose group: No OGD / R treatment was performed, and Mere-5 (10 μmol / L) was added only during the reoxygenation time. ③OGD / R Group (Model): OGD / R processing; ④ OGD / R + Mere-5 low-dose group: OGD / R treatment, Mere-5 (1 μmol / L) was administered during reoxygenation. ⑤ OGD / R + Mere-5 high-dose group: OGD / R treatment, Mere-5 (10 μmol / L) was administered during reoxygenation. After OGD / R-induced BV2 cell damage, Mere-5 was added, and the expression levels of pro-inflammatory factor mRNA were detected. Figure 10 As shown, compared with the control group, BV2 cells in the OGD / R model group... Il1b , Il6 , Tnf The mRNA expression levels were significantly upregulated. P <0.05). Compared with the OGD / R model group, the expression of inflammatory factor mRNA decreased in all Mere-5 dose groups, among which... Il1b ( P <0.05) Il6 ( P <0.05) Tnf ( P The difference was statistically significant (<0.05).

[0081] The above results indicate that Mere-5 can effectively inhibit OGD / R-induced transcription of pro-inflammatory cytokine genes in BV2 cells at the in vitro cellular level, and this inhibitory effect is dose-dependent, which is highly consistent with the anti-inflammatory results observed in animal experiments, further validating the anti-neuroinflammatory activity of Mere-5 at the cellular level.

[0082] (6) Mere-5 reduces OGD / R-induced oxidative stress levels in BV2 cells. Further, the oxidative stress status of BV2 cells in each group (the same as the control group, high-dose Mere-5 group, OGD / R group, low-dose OGD / R+Mere-5 group, and high-dose OGD / R+Mere-5 group in (5)) was systematically detected from two dimensions: lipid peroxidation end products (MDA) and total intracellular ROS levels.

[0083] The TBA method test results are as follows: Figure 11 As shown, compared with the control group, the MDA content in BV2 cells of the OGD / R group was significantly increased ( P<0.05), indicating that OGD / R treatment triggered a significant lipid peroxidation chain reaction, resulting in severe oxidative damage to the cell membrane; after Mere-5 intervention, the MDA content in cells of the OGD / R+Mere-5 low-dose group was lower than that of the model group ( P <0.05); the MDA content in the OGD / R+Mere-5 high-dose group was significantly lower than that in the model group ( P <0.05), indicating that high-dose Mere-5 can effectively antagonize OGD / R-induced lipid peroxidation damage.

[0084] DCFH-DA fluorescent probe combined with flow cytometry for quantitative detection, such as Figure 12 As shown, compared with the control group, the mean fluorescence intensity of DCF in BV2 cells of the OGD / R group was significantly enhanced ( P <0.05 indicates that intracellular ROS accumulates significantly after OGD / R treatment, and the level of oxidative stress is significantly increased. After low-dose Mere-5 intervention, the intracellular ROS fluorescence intensity showed a decreasing trend compared with the model group ( P <0.05); ROS levels were further reduced in the OGD / R+Mere-5 high-dose group ( P <0.05).

[0085] Fluorescence microscopy observation results as follows Figure 13 As shown, only weak green fluorescence was visible in the control group cells, indicating that the intracellular ROS was at a low level under basal conditions; the intensity of green fluorescence in the OGD / R group cells was significantly enhanced, and the fluorescence distribution was diffuse, covering the entire cytoplasm; the fluorescence intensity in the OGD / R+Mere-5 low-dose group was weaker than that in the model group; the fluorescence signal in the OGD / R+Mere-5 high-dose group was significantly weakened, approaching the level of the control group.

[0086] In summary, the high degree of consistency between the trends of MDA and ROS indicates that Mere-5 can effectively alleviate OGD / R-induced oxidative stress damage in BV2 cells at multiple levels, with the high-dose group showing a more pronounced effect. This provides important cellular evidence for further exploration of its upstream molecular targets for antioxidant mechanisms.

[0087] (7) Mere-5 regulates the expression of SIRT1 / NOX2 / NF-κB pathway proteins in OGD / R-damaged BV2 cells. The expression levels of SIRT1, NOX2, p-p65 and total p65 proteins in BV2 cells of each group (the same as the control group, high-dose Mere-5 group, OGD / R group and OGD / R+Mere-5 high-dose group in (5)) were detected by Western blotting. The regulatory effect of Mere-5 on the SIRT1 / NOX2 / NF-κB signaling axis was systematically verified at the protein level.

[0088] SIRT1 protein detection results are as follows Figure 14 As shown, compared with the control group, the expression level of SIRT1 protein in BV2 cells of the OGD / R group was significantly reduced ( P <0.05 indicates that OGD / R injury can downregulate the protein expression of endogenous SIRT1, weakening its inhibitory function on downstream oxidative stress and inflammatory signaling. Compared with the OGD / R group, SIRT1 protein expression was significantly upregulated after Mere-5 intervention ( P <0.05).

[0089] NOX2 protein test results are as follows Figure 15 As shown, compared with the control group, the NOX2 protein expression level in the OGD / R group cells was significantly increased ( P <0.05). Compared with the OGD / R group, NOX2 protein expression level was significantly reduced after Mere-5 administration ( P <0.05).

[0090] NF-κB pathway protein detection results are as follows Figure 16 As shown, compared with the control group, the OGD / R group showed a significant increase in the level of p-p65 (phosphorylated p65, Ser536 site) protein and a significantly increased p-p65 / p65 ratio. P <0.05), and there was no significant difference in total p65 protein expression among the groups, indicating that OGD / R mainly activates the NF-κB signaling pathway by promoting p65 phosphorylation, rather than affecting the synthesis level of total p65 protein. Compared with the OGD / R group, after Mere-5 intervention, both p-p65 protein levels and the p-p65 / p65 ratio were reduced ( P <0.05), while the total p65 protein level remained largely unchanged ( P The value >0.05 indicates that Mere-5 selectively inhibits the phosphorylation activation process of p65.

[0091] In summary, Mere-5 can upregulate SIRT1, inhibit NOX2, and repress the activation of NF-κB (p-p65) at the cellular level.

[0092] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. The use of Mere-5 in the preparation of drugs and / or pharmaceutical compositions for the prevention and / or treatment of ischemic cerebrovascular diseases, characterized in that, The amino acid sequence of Mere-5 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The ischemic cerebrovascular disease mentioned is ischemic stroke.

3. The use of Mere-5 promoter in the preparation of drugs and / or pharmaceutical compositions for the prevention and / or treatment of ischemic cerebrovascular diseases, characterized in that, The amino acid sequence of Mere-5 is shown in SEQ ID NO.

1.

4. The application according to claim 3, characterized in that, The ischemic cerebrovascular disease mentioned is ischemic stroke.

5. A drug for the prevention and / or treatment of ischemic cerebrovascular disease, characterized in that, The drug contains Mere-5 and / or a Mere-5 promoter; the amino acid sequence of Mere-5 is shown in SEQ ID NO.

1.

6. A drug for the prevention and / or treatment of ischemic cerebrovascular disease according to claim 5, characterized in that, The dosage form of the drug includes one or more of the following: suspension, granules, capsules, powders, tablets, emulsions, pills, injections, suppositories, enemas, aerosols, patches, or drops.

7. A drug for the prevention and / or treatment of ischemic cerebrovascular disease according to claim 5, characterized in that, The drug contains pharmaceutically acceptable excipients.

8. A drug for the prevention and / or treatment of ischemic cerebrovascular disease according to claim 7, characterized in that, The excipients include one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

9. A drug for the prevention and / or treatment of ischemic cerebrovascular disease according to claim 5, characterized in that, The drug can be administered via one of the following methods: intravenous injection, subcutaneous injection, intramuscular injection, oral administration, or transdermal absorption.

10. A pharmaceutical composition for the prevention and / or treatment of long fibrosis, characterized in that, The pharmaceutical composition contains the drug as described in claims 5 to 9.

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