4 apos; application of-O-methyl bavachalcone in medicine for treating vascular cognitive impairment

Abstract

The invention belongs to the field of new application of 4 '-O-methyl bavachalcone, and particularly relates to application of 4'-O-methyl bavachalcone in a medicine for treating vascular cognitive impairment. The invention discloses a new application of 4 '-O-methyl bavachalcone in development of drugs for treating vascular cognitive impairment for the first time, and the 4'-O-methyl bavachalcone is mainly used for effectively inhibiting inflammatory response of glial cells so as to improve symptoms of the vascular cognitive impairment. The preparation process of the medicine is simple and easy to implement, and the prepared medicine product is safe, stable and remarkable in curative effect and particularly has the pharmacological effects of repairing white matter damage, relieving neuroinflammation and the like.

Description

Technical Field

[0001] The invention belongs to the field of new uses of 4'-O-methyl psoralea chalcone, and in particular relates to the use of 4'-O-methyl psoralea chalcone in drugs for treating vascular cognitive impairment. Background Art

[0002] Vascular cognitive impairment (VCI) is an acquired syndrome of advanced cognitive dysfunction caused by various cerebrovascular diseases and their related risk factors. In China, the number of patients with cerebrovascular diseases is huge, exceeding 8 million, of which about 3.5 million patients have cognitive dysfunction. What is more worrying is that with the prevalence of unhealthy lifestyles and the trend of younger people suffering from cerebrovascular diseases, the incidence of VCI is rising year by year. Reduced cerebral blood flow and vascular risk factors are not only the leading factors of cognitive impairment, but also closely related to the occurrence of other neurodegenerative diseases. Aging, as an important risk factor for VCI, causes cells to produce senescence-associated secretory phenotype (SASP), in which the long-term effects of inflammatory factors (such as TNF-α, IL-1α / β, IL-6, IL-8) will induce chronic low-grade inflammation in the body, which in turn aggravates to inflammatory aging (inflammaging). This process promotes vascular embolism, vascular inflammation and vascular spasm, causing the brain tissue to be in a state of chronic hypoperfusion for a long time, which may eventually deteriorate to VCI. Chronic cerebral hypoperfusion (CCH) is a common pathological condition in VCI patients and an important part of the pathogenesis of VCI. CCH can lead to ischemia and hypoxia in the brain, which in turn causes a series of brain tissue damage, including cortical atrophy, leukoaraiosis, myelin sheath loss, oligodendrocyte loss, microglial activation and proliferation, astrocyte reactive proliferation, and neuronal degeneration and necrosis in the CA1 region of the hippocampus. These pathological changes can lead to axonal damage and interruption of dendrite-synaptic transmission, ultimately inducing cognitive dysfunction.

[0003] Although the medical community has made many attempts in the field of VCI treatment, no specific drugs have been developed so far. At present, the commonly used cholinesterase inhibitors (such as donepezil hydrochloride, galantamine), calcium channel blockers (such as nimodipine, flunarizine), NMDA receptor antagonists (such as memantine, piracetam), r-aminobutyric acid (GABA) receptor modulators and antioxidant drugs (such as vitamin E) can relieve symptoms but cannot effectively block the progression of the disease.

[0004] In traditional Chinese medicine theory, vascular cognitive impairment (VCI) is classified as "dementia", "forgetfulness" and "literary idiocy". This disease originates from "insufficient kidney essence", "decline of blood and qi", "depression of liver qi" and "extreme phlegm qi", which lead to blockage of brain collaterals, making it difficult for qi, blood, essence and fluid to flow smoothly to the brain. As a result, the brain orifices lose their fullness, and the clear orifices become unclear, which eventually leads to the loss of nourishment of the primordial spirit and then develops into dementia (i.e. cognitive impairment). Although the location of VCI is in the brain, its root cause lies in the kidney, which belongs to the category of deficiency in the root and excess in the superficial. The kidney, as the innate foundation, is responsible for bone production and marrow production. The marrow can transform essence into blood to nourish the brain, and the brain is regarded as the sea of ​​marrow. When the kidney essence is deficient, the sea of ​​marrow cannot be fully nourished, the brain will lose nourishment, and the spirit machine cannot function normally, which will cause a series of cognitive impairment symptoms such as decreased language ability, forgetfulness, and decreased logical judgment ability. Therefore, the main pathogenesis of VCI lies in kidney deficiency and brain malnutrition in old age. The treatment principle should be to tonify the kidney, replenish essence, resist aging and strengthen the brain.

[0005] Traditional Chinese medicine is known for its multi-target, multi-pathway mode of action and synergistic effects between its ingredients. Common single Chinese medicines for regulating VCI in clinical practice include: active ingredient extracts (such as tanshinone, ginsenosides, ginkgo leaf preparations, etc.), Chinese medicine compound decoctions (such as Tongqiao Huoxue Decoction), capsules (such as Naotong Capsules), granules (such as Tianzhi Granules), and other types (such as Danhong Injection). Statistics show that the common prescriptions used by modern doctors to treat VCI also have the highest frequency of use of tonic drugs, with the largest proportion (accounting for 45.68%, of which more than half are kidney-tonifying drugs), followed by blood-activating and blood-stasis-removing drugs and phlegm-removing and dampness-removing drugs; this is consistent with the understanding of traditional Chinese medicine that VaD is mainly "insufficient kidney essence, marrow sea is not full" and mixed with phlegm and blood stasis to form a mixed syndrome of deficiency and excess. Based on this, we consider using kidney-tonifying Chinese medicine to replenish kidney essence, resist aging, and nourish the brain in order to improve cognitive impairment.

[0006] Psoralea corylifolia Linn., as a traditional Chinese medicine for kidney tonification, its dried and mature fruits are often used in TCM clinical practice. 4′-O-methylbavachalcone (MeBavaC) in Psoralea corylifolia is a prenylated chalcone component with potential biological activity. Related studies have shown that MeBavaC can activate the activity of erythropoietin EPO, inhibit the senescence of human endothelial cells, and downregulate the gene expression of p16INK4a (a marker of replicative senescence) and IL-1α (a marker of inflammation). In animal models, MeBavaC can also promote blood flow recovery, increase the number of circulating endothelial progenitor cells and promote capillary angiogenesis, and at the same time show potential neuroprotective effects in a cerebral ischemia-reperfusion injury model. However, there are no detailed reports on the therapeutic effect of MeBavaC on vascular cognitive impairment and its molecular mechanism.

[0007] Patent document CN116036058A discloses the use of 4-O-methylpsoralen chalcone in the preparation of drugs for treating or preventing thrombosis. 4-O-methylpsoralen chalcone inhibits the formation of thrombus by blocking the interaction between 14-3-3ζ and integrin β3. However, there is no prior art report on the use of 4′-O-methylpsoralen chalcone in drugs for treating vascular cognitive impairment. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides an application of 4′-O-methylpsoralea chalcone (MeBavaC) in a drug for treating vascular cognitive impairment.

[0009] As an implementable case, the application of 4′-O-methylpsoralen chalcone (MeBavaC) in the treatment of vascular cognitive impairment includes: dissolving 4′-O-methylpsoralen chalcone with a solvent, and then ultrasonically treating it to obtain a 4′-O-methylpsoralen chalcone solution.

[0010] As an implementable example, the solvent includes a polysaccharide solvent.

[0011] As an implementable example, the raw materials for preparing the polysaccharide solvent include water and polysaccharides.

[0012] As an implementable example, the polysaccharide includes: sodium carboxymethyl cellulose (CMC-Na).

[0013] As an implementable example, the mass concentration of polysaccharide in the polysaccharide solution is 1-2wt%.

[0014] Furthermore, the mass concentration of the polysaccharide in the polysaccharide solution is 1.5wt%, and the polysaccharide solution is in a paste-like state.

[0015] As an implementable example, the injection dosage of the 4′-O-methylpsoralea chalcone solution includes a low-dose injection or a high-dose injection.

[0016] As an implementable case, the injection volume of the low-dose injection is 5-10 mg / kg.

[0017] Furthermore, the injection volume of the low-dose injection is 7 mg / kg.

[0018] As an practicable case, the injection volume of the high-dose injection is 11-20 mg / kg.

[0019] Furthermore, the injection volume of the high-dose injection is 14 mg / kg.

[0020] As an implementable case, the mass concentration of 4′-O-methylpsoralen chalcone in the 4′-O-methylpsoralen chalcone solution is 1-2 mg / mL.

[0021] As an implementable case, the dosage form of the 4′-O-methylpsoralea chalcone solution includes an oral dosage form.

[0022] As an implementable case, the administration method of the 4′-O-methylpsoralen chalcone solution includes: intragastric administration.

[0023] As an implementable case, the 4′-O-methylpsoralen chalcone improves vascular cognitive impairment by inhibiting glial cell inflammation.

[0024] The chemical structural formula of the MeBavaC is:

[0025]

[0026] Beneficial Effects

[0027] (I) The present invention discloses for the first time a specific compound 4′-O-methylpsoralea chalcone (MeBavaC) derived from the traditional Chinese medicine psoralea corylifolia, and its application in improving vascular cognitive impairment. MeBavaC mainly achieves the purpose of improving cognitive function by effectively inhibiting neuroinflammation and thereby reducing white matter damage. In addition, the drug preparation process of the present invention is simple and clear, and the prepared drug is not only safe and stable, but also has a definite therapeutic effect, and is particularly suitable for alleviating the symptoms of cognitive impairment caused by neuroinflammation and cerebral ischemia.

[0028] (II) The present invention verifies that MeBavaC can significantly improve the problem of decreased learning and memory ability caused by CCH after oral administration, thereby improving the learning and memory ability of rats.

[0029] (III) The present invention verifies that MeBavaC, after oral administration, can improve the activation of glial cells in the white matter of CCH rats by inhibiting astrocyte proliferation and microglial activation.

[0030] (IV) The present invention demonstrates that MeBavaC can significantly improve the ultrastructure of myelin axons in the corpus callosum of model rats and promote the increase in the number of axons.

[0031] (V) The present invention discloses that the preparation process of MeBavaC drug is simple, the toxicity is low, the product is safe, stable and effective, and it has the functions of improving brain white matter damage, neuroinflammation, etc.; it fills the technical gap of MeBavaC drug treatment of VCI. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a graph showing the protective effect of 4′-O-methylpsoralen chalcone (MeBavaC) on learning and memory in rats with vascular cognitive impairment (VCI) induced by chronic cerebral hypoperfusion. Figure 1A The chemical structure formula is 4′-O-methylpsoralea corylifolia chalcone; Figure 1B Flow chart for animal experiments; Figure 1C The trajectory diagram of swimming distance and number of crossing the platform of rats in each group; Figure 1D This is a statistical chart of the swimming distance of animals four weeks after modeling (n=10-12); Figure 1E This is a statistical chart of the number of times rats in each group crossed the platform in four weeks (n=10-12); Figure 1F The figure is a statistical chart of the incubation period within four weeks after animal modeling (n=10-12); *P<0.05 vs. sham (control group); #P<0.05 vs. model (model group).

[0033] FIG2 is a graph showing the effect of 4′-O-methylpsoralen chalcone (MeBavaC) on reducing white matter ischemic injury and hippocampal neuron injury in rats; Figure 2A ~C is a schematic diagram of the corpus callosum (paramedian, medial) fast blue staining and quantitative analysis results (arrows indicate myelin fiber vacuolization), white matter damage grade: 0 = no damage; 1 = nerve fiber disorder; 2 = obvious vacuoles; 3 = myelinated fibers disappear, scale = 50μm and 5000μm; Figure 2D ~G is a schematic diagram of hippocampal Nissl staining and quantitative analysis results, scale bars = 50 μm and 500 μm; Figure 2H and Fig.2I Schematic diagram and quantitative analysis results of immunofluorescence staining for the detection of corpus callosum myelin (MBP) and myelinated neurons (NF200), scale bar = 100 μm and 200 μm; Figure 2J and Figure 2K Schematic diagram of the ultrastructure of myelinated axons in the corpus callosum (red arrows indicate myelinated axons, blue arrows indicate unmyelinated axons), scale bars = 10 μm and 20 μm; n = 3, **P≤0.01, ***P≤0.001, ****P≤0.0001VS.Sham; #P≤0.05, ##P≤0.01####P≤0.0001vs.2-VO.

[0034] FIG3 is a graph showing the results of 4′-O-methylpsoralen chalcone (MeBavaC) drug inhibiting corpus callosum neuroinflammation in rats; wherein Figure 3A and Figure 3B Schematic diagram of immunofluorescence staining for corpus callosum microglia (IBA1) and quantitative analysis results, scale bar = 20 μm; Figure 3C and Figure 2DSchematic diagram of immunofluorescence staining for detection of corpus callosum astrocytes (GFAP) and quantitative analysis results, scale bar = 20 μm; Figure 3E This is a picture of the relative expression level of AIM2 mRNA in rat corpus callosum; Figure 3F and Figure 3G Typical images of NLRP3, AIM2, GSDMD and Caspase-1-p20 protein expression; n=3-6, *P≤0.05, **P≤0.01 vs. Sham; #P≤0.05, ##P≤0.01 vs. 2-VO.

[0035] FIG4 shows the effect of 4′-O-methylpsoralen chalcone (MeBavaC) on oligodendrocytes (MO3.13) and microglia (BV2); Figure 4A This is a statistical diagram of the effect of 4′-O-methylpsoralen chalcone on the cell viability of oligodendrocytes (MO3.13); Figure 4B Statistical graph showing the effect of 4′-O-methylpsoralen chalcone on microglial (BV2) cell viability; n=(4-6), *P≤0.05, ****P≤0.0001 vs. Control. DETAILED DESCRIPTION

[0036] Example 1

[0037] The invention provides an application of 4'-O-methylpsoralea chalcone (MeBavaC) in a drug for treating vascular cognitive impairment.

[0038] The application of the 4′-O-methylpsoralen chalcone in the treatment of vascular cognitive impairment is as follows: 4′-O-methylpsoralen chalcone is dissolved in a sodium carboxymethyl cellulose aqueous solution with a mass concentration of 1.5wt%, and then ultrasonically treated to prepare 4′-O-methylpsoralen chalcone solutions with mass concentrations of 1 mg / mL and 2 mg / mL, respectively; the dosage form of the 4′-O-methylpsoralen chalcone solution is an oral dosage form, and the administration method is: intragastric administration; MeBavaC improves vascular cognitive impairment by inhibiting glial cell inflammation; the structural formula of MeBavaC is shown in Figure 1.

[0039] The following tests were performed to establish the rat cerebral chronic hypoperfusion model (CCH) by bilateral common carotid artery ligation (2-VO) and to evaluate the effect of 4′-O-methylpsoralen chalcone on improving vascular cognitive impairment. All data are presented as mean ± SD One-way analysis of variance (ANOVA) was used for comparison among multiple groups, and LSD test for multiple comparisons was performed using Graph Pad Prism 6.0 (GraphPad Software, La Jolla, CA, USA); P < 0.05 was considered statistically significant.

[0040] 1. Selection of experimental subjects

[0041] Seven-week-old SPF (Specific Pathogen Free) grade male SD rats were selected and provided by Shanghai Slake Experimental Animal Co., Ltd. (SCXK (Shanghai) 2017-0005).

[0042] The animals were housed in cages (5 / cage) at the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine in a breeding room maintained at a constant humidity (65% ± 5% RH) and temperature (24°C ± 1°C), with free access to food and water and 12-hour lighting cycles.

[0043] The experimental animals were handled uniformly in accordance with “The Guide for Care and Use of Laboratory Animals” (USA National Institutes of Health) throughout the process and were kept in the SPF-level breeding room of the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine.

[0044] 2. Establishment of chronic cerebral hypoperfusion model in rats

[0045] like Figure 1B As shown, SD rats were adaptively raised in the animal room until they were 8 weeks old and then the model was established. Fasting for 12 hours before the experiment, free access to water. The animals were given an intraperitoneal injection of 1wt% sodium pentobarbital, combined with isoflurane gas inhalation anesthesia. The SD rats were fixed on a mouse board and the head and neck were prepared. The center of the neck was cut open with surgical scissors, and each layer of tissue was bluntly peeled off to expose the bilateral common carotid arteries and vagus nerves, and the vagus nerves accompanying the common carotid arteries were separated. The distal and proximal ends of the common carotid arteries were ligated and cut in the middle to ensure that the blood supply to the bilateral common carotid arteries of the brain was blocked. When the wound was sutured after surgery, the wound was rinsed with sodium penicillin (400,000 units / mL) and placed on a heating pad to restore body temperature. Rats in the sham operation group only underwent the same vascular separation surgery.

[0046] After the 2-VO model was established, the rats were randomly divided into 4 groups: sham group, model group, low-dose group of 4′-O-methylpsoralen chalcone (7 mg / kg), and high-dose group of 4′-O-methylpsoralen chalcone (14 mg / kg).

[0047] The rats were given an intragastric administration method, 30 minutes after modeling, and then once a day at a fixed time for five consecutive weeks. The rats were weighed once a week and the dosage of the drug was adjusted according to their body weight. Rats in the Sham group and the Model group were given an equal volume of CMC-Na solvent.

[0048] 3. Morris water maze (MWM) experiment

[0049] Experimental preparation:

[0050] The MWM experiment was conducted in a specific room throughout the experiment. The environment was quiet, the light was balanced, the reference objects were stable, and the experimental water temperature was controlled at 25±1℃. The pool movement area, platform movement area and binarization domain value were constant. The MWM pool was black and divided into four quadrants. The animal entry point was marked on the wall of each quadrant. The animal entry platform was located in the middle of the southeast (SE) quadrant, with a diameter of 10 cm, transparent color, and close to the water. The pool was attached with a curtain, and the reference objects on the inside of the curtain were stable (star / square / triangle / circular markers, fixed in position). During the operation, the experimenter's coordination was fixed and the spatial position was kept as unchanged as possible. All of the above were to minimize the interference of the environment on the experiment.

[0051] ①Visual platform experiment:

[0052] During the experiment, the platform was placed in the middle of the southeast (SE) quadrant, with a color similar to that of water and about 1 cm above the water surface. Each rat entered the water at the entry points of the SW, W, N, and NE quadrants in turn, and the training was conducted 4 times, each time for 60 seconds. If the rat cannot swim to the platform within 60 seconds, it will be guided to the platform and stand for 5 seconds. If the rat swims onto the platform within 60 seconds, it will also be made to stand for 5 seconds. After the training, wipe the water stains on the animals with a soft towel and return them to the cage, keep them dry and warm, and prevent them from getting sick. After the whole batch of animals completes the water entry training in one quadrant, they will proceed to the training in the next quadrant.

[0053] ② Positioning navigation experiment

[0054] During the experiment, the platform was placed in the middle of the southeast (SE) quadrant, with a color similar to water and about 1 cm below the water surface. The experiment lasted for 5 days, and training was carried out in the water entry quadrants SW (southwest), W (west), N (north), and NE (northeast), for a total of four times, each training lasting 60 seconds. If the animal could not swim to the platform within 60 seconds, it was guided to stand on the platform for 5 seconds; if the animal swam onto the platform within 60 seconds, it also stood for 5 seconds. Track its swimming trajectory and record the time it took to get on the platform.

[0055] ③Space exploration experiment

[0056] The experiment started after 5 consecutive days of positioning navigation, and a space exploration experiment was conducted on the 6th day. The platform was removed, and the other parts remained unchanged. The animal entered the water from the new quadrant (NW) entry point, and the number of times it crossed the original position of the platform within 60 seconds was observed.

[0057] The above experiments were repeated for four weeks.

[0058] In the five-day positioning navigation experiment, the swimming path (recorded as the swimming distance) and swimming time (recorded as the latency) of the animals to find the platform within a fixed time were observed. The shorter the swimming distance and latency, the stronger the animal's learning ability. In the sixth day of the spatial exploration experiment, the number of times the animal crossed the platform within a fixed time was observed. The more times the animal crossed the platform, the stronger the animal's memory ability.

[0059] Results from Figure 1C , 1D , 1F, we can see that in the MWM experiment, the swimming distance and latency of the sham group were shorter, while the time of the model group was significantly prolonged, with statistically significant differences (P≤0.05), indicating that CCH reduced the learning ability of animals, and animals needed a longer path and more time to find the target platform. Compared with the model group, the drug group significantly shortened the swimming distance and time, indicating that MeBavaC can improve the learning ability of VCI rats, shorten their swimming distance and latency, and enable animals to find the target platform in a shorter time through a shorter path.

[0060] Results from Figure 1C and 1E It can be seen that in the four weeks, the sham group had more platform crossings, while the model group had significantly fewer platform crossings, indicating that CCH damaged the animals' memory ability. In the fourth week, the drug-treated group significantly increased the number of platform crossings, which was significantly different from the model group (P≤0.05), indicating that MeBavaC can restore the memory ability of VCI rats and enable animals to find the target platform repeatedly. In addition, the low-dose group of MeBavaC had a better improvement effect, so the subsequent mechanism exploration was mainly based on the low-dose group of 7 mg / kg.

[0061] The results of the MWM experiment showed that CCH caused a decline in the learning and memory abilities of animals, and intragastric administration of MeBavaC could significantly improve the learning and memory abilities of VCI rats.

[0062] After the behavioral experiment, the rats were anesthetized, and the brains were removed after systemic perfusion with 4 wt% paraformaldehyde through the heart. The whole brains were carefully removed and fixed in 4 wt% paraformaldehyde at 4°C for 24 h, then dehydrated with sucrose and gradient ethanol, embedded in paraffin, and coronal sections were obtained by microsectioning.

[0063] 4. Axonal fiber staining experiment

[0064] Luxol fast blue (LFB) staining was performed on the axonal fiber density to detect the severity of white-matter lesions (WMLs). After the paraffin sections were dewaxed to water, the fast blue staining solution was preheated and the sections were stained for 4 hours. After cooling, the sections were washed with water. Next, the sections were alternately differentiated in 70% mass concentration ethanol and lithium carbonate solution, the degree of differentiation was controlled under a microscope, and the washing was terminated. Then, the sections were dehydrated and made transparent with anhydrous ethanol (5min×3), n-butanol, and xylene (5min×2), and sealed with neutral gum after drying slightly. Finally, the staining effect was observed under a microscope, and image acquisition and analysis were performed. Figure 2B and 2C Fast blue staining showed that the white matter of the corpus callosum of the rats in the model group showed vacuolation and disorder of myelin fibers, sparse white matter, and damaged white matter integrity, while MeBavaC had a significant improvement effect.

[0065] 5. Nissl staining of hippocampal neurons to observe neuronal damage

[0066] Nissl staining was performed on hippocampal neurons to observe neuronal damage. The animal tissue slices were immersed in the dye solution for 5 minutes, stained, washed with water, and then placed in 1wt% mass concentration of glacial acetic acid for differentiation. The degree of differentiation was controlled under a microscope, and the slices were baked dry; transparentized in xylene for 5 minutes, taken out, dried, and sealed with neutral gum; microscopic examination, collection and analysis of images. Figures 2D to 2G Nissl staining showed that compared with the sham group, the number of Nissl-positive neurons in the hippocampus of the model group (Model) rats was reduced and their arrangement was loose, indicating neuronal damage and degeneration. The drug group significantly improved the above-mentioned neuropathy.

[0067] 6. Immunohistochemical observation of the expression of myelin protein MBP in the corpus callosum and the degree of axonal damage marked by NF-200

[0068] The tissue sections were microwave-repaired in EDTA antigen retrieval buffer (pH=8.0), cooled naturally and washed with PBS buffer solution; the sections were slightly dried and circled with a histochemical pen, and a quencher was added into the circle for 5 min (to quench autofluorescence), and then rinsed with running water for 10 min; BSA was added into the circle and blocked for 30 min; then the sections were incubated with the corresponding glial fibrillary acidic protein (GFAP, diluted volume ratio 1:500, Proteintech), ionized calcium binding adapter molecule 1 (IBA1, diluted volume ratio 1:500, Proteintech), neurofilament protein 200 (NF200, diluted volume ratio The sections were incubated with primary antibody solution of 1:160, Sigma) and myelin basic protein (MBP, diluted volume ratio 1:500, Affinity) at 4°C overnight; after washing with PBS, the sections were incubated with secondary antibodies of the corresponding species of the primary antibody at room temperature at 25°C in the dark for 50 min; then, PBS was washed (5 min × 3 times), DAPI was added to the circle to re-stain the cell nucleus, and incubated at room temperature at 25°C in the dark for 10 min; the slides were placed in PBS and washed on a decolorizing shaker for 3 times, 5 min each time; the sections were dried, sealed with anti-fluorescence quenching sealing agent, and stored at 4°C in the dark; finally, the images were observed and collected under a fluorescence microscope.

[0069] like Figure 2H and 2I As shown, the expression of MBP and NF200 in the corpus callosum of rats in the model group was significantly reduced, indicating a decrease in oligodendrocytes, neuronal loss, and myelin density in the corpus callosum, which in turn indicated axonal damage, while the MeBavaC group had a significant improvement effect.

[0070] 7. Transmission electron microscopy observation of the ultrastructure of myelinated axons in the corpus callosum of rats

[0071] The corpus callosum (CC) was microdissected into 1 mm 3 The cube was placed in a 2.5wt% glutaraldehyde fixative; then rinsed with phosphate buffer and fixed with 1wt% osmium acid; rinsed again with phosphate buffer and dehydrated with gradient ethanol, then dehydrated with a mixture of 90wt% ethanol and 90wt% acetone in a volume ratio of 1:1 for 10 min; soaked in 100wt% acetone for 3 times, each time for 10 min, and then soaked in 100wt% acetone in a volume ratio of 1:1 for 10 min. The tissue was immersed in 0wt% acetone and embedding agent (812 embedding agent, CAS: 90529-77-4, SPI) for 1 hour, and then immersed in embedding solution for 1.5 hours; the embedded tissue was polymerized in a 37°C incubator for 12 hours after adding the embedding agent, and then polymerized in the embedding solution at 60°C for 48 hours, and then semi-thinly positioned and ultra-thinly sectioned (thickness 50-80nm), double-stained with uranyl acetate-lead citrate, and after the sample was dried, its ultrastructure was observed under a transmission electron microscope.

[0072] like Figure 2G and 2K As shown, the myelin sheath of the corpus callosum of the normal group rats surrounded the axons in concentric layers, and other organelles, microtubules, and neurofilaments were arranged regularly; in the model group rats, the normal structure of the corpus callosum was unclear, the myelin lamellar structure was loosened, local vacuoles were formed, and the number of axons decreased; MeBavaC can significantly improve the ultrastructure of myelin axons in the corpus callosum of the model rats and promote the increase in the number of axons.

[0073] 8. Glial cell activation experiment

[0074] Microglial activation and astrocyte proliferation are both manifestations of neuroinflammation, and CCH promotes the occurrence of neuroinflammation. Coronal sections of paraffin blocks of brain tissue were taken, and immunofluorescence was used to observe microglial activation (IBA-1) and astrocyte proliferation (GFAP) in the corpus callosum.

[0075] like Figure 3A and 3B As shown, the expression of IBA1 in the corpus callosum of rats in the model group was increased, indicating that CCH would cause microglial activation; after MeBavaC treatment, the positive expression of IBA1 in microglia was significantly reduced, indicating that MeBavaC could significantly improve the activation of microglia in rats with CCH.

[0076] like Figure 3C and 3D As shown in the data, the expression of GFAP in the corpus callosum of rats in the model group was increased, which provided a basis for the proliferation of astrocytes; while the positive expression of GFAP in astrocytes of rats in the MeBavaC group was significantly reduced, indicating that MeBavaC can significantly improve the activation of astrocytes in CCH rats.

[0077] In order to further determine the regulatory effect of MeBavaC on neuroinflammation, quantitative real-time polymerase chain reaction (qRT-PCR) and protein immunoblotting (Western blotting, WB) were used to detect the expression of corpus callosum inflammation-related genes and proteins, respectively.

[0078] 9. Corpus callosum inflammasome AIM2 mRNA inhibition test

[0079] Total RNA from rat brain corpus callosum was extracted using Trizol reagent, and cDNA was synthesized according to the instructions of Invitrogen cDNA reverse transcription kit (Applied Biosystems, UA). Fast SYBR Green Master mix (Life Technologies, US) kit was used to premix reagents and samples, and gene expression analysis was performed on ABI 7500Fast real-time fluorescence quantitative PCR system.

[0080] Gene expression was normalized using ACTIN as a reference gene.

[0081] The relative mRNA expression level was calculated according to the ΔΔCt method using primers with the following sequences (designed and synthesized by Shanghai Jierui Bioengineering Co., Ltd.).

[0082] The primer sequences are as follows: AIM2: forward primer (5'-GAGAGAAGCCTGCGGTTGAT-3'); rear primer (5'-CCCATATCCAACCTCCACCC-3'). ACTIN: forward primer (5'-CGCGAGTACAACCTTCTTGC-3'); rear primer (5'-CCTTCTGACCCATACCCACC-3').

[0083] like Figure 3E As shown, the AIM2 mRNA of inflammatory body in corpus callosum of rats in model group increased significantly, and the drug group had obvious inhibitory effect.

[0084] 10. Western Blot analysis

[0085] Rat brain corpus callosum tissue was lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors and used for Western Blot analysis. After protein lysis, centrifuge at 12000rpm for 15min at 4℃, collect the total protein in the supernatant, and determine the protein concentration of the supernatant using the BCA concentration determination kit. Dilute the total protein to 3μg / μL using loading buffer and then heat at 95℃ for 10min. 30μg of total protein per sample was loaded on SDS-PAGE for electrophoresis, and then the protein on the gel was transferred to a PVDF membrane. Then, the membrane was blocked with skim milk at a mass concentration of 5wt% for 1 hour, and incubated with primary antibodies against NLRP3 (dilution volume ratio 1:1000, Abcam), AIM2 (dilution volume ratio 1:1000, Abcam), GSDMD (dilution volume ratio 1:1000, Abcam), Caspase-1 (dilution volume ratio 1:1000, Proteintech), Tubulin (dilution volume ratio 1:10000, Proteintech) and HRP secondary antibody. The membrane was developed using an ECL chemiluminescence kit, and imaged and analyzed using a Tanon 5200 Multi chemiluminescence developer.

[0086] Compared with the Sham group, the expression levels of NLRP3, AIM2, GSDMD, and Caspase-1-p20 proteins in the Model group were significantly increased ( Figure 3F and 3G ), suggesting that CCH can cause neuroinflammation, while the MeBavaC group has a significant inhibitory effect. Therefore, MeBavaC intervention can alleviate neuroinflammation caused by CCH.

[0087] 11. Drug safety test

[0088] The effect of MeBavaC on the cell viability of oligodendrocytes (MO3.13) and microglia (BV2) was investigated. MO3.13 cells and BV2 cells in the logarithmic growth phase were taken and inoculated in 96-well plates at 4000 cells / well, and 100 μL of culture medium was added to each well. After the cells adhered to the wall, different concentrations of MeBavaC (0.001 μm, 0.01 μm, 0.1 μm, 1 μm, 10 μm, 20 μm, 40 μm, 80 μm) were given, and the culture was continued for 24 hours. The old culture medium was discarded, and the CCK-8 solution was taken and diluted with serum-free culture medium to a final mass concentration of 10wt%. 100 μL / well was added to the 96-well plate, and it was placed in the incubator for further incubation for 1 hour, and then the absorbance was detected at 450nm using an ELISA reader.

[0089] The formula for calculating cell viability = (absorbance of experimental well - absorbance of blank well) / (absorbance of control well - absorbance of blank well).

[0090] like Figure 4A and 4B As shown, after stimulating cells with different concentrations of MeBavaC for 24 hours, MO3.13 cells had no obvious cytotoxicity when the drug was lower than 10 μM, and BV2 cells had no obvious cytotoxicity when the drug was lower than 0.01 μM, indicating that MeBavaC has higher safety at lower concentrations.

[0091] In summary, the present invention provides for the first time the use of MeBavaC as a drug for the treatment of vascular cognitive impairment, and proves that MeBavaC can effectively reduce brain white matter damage, which may be closely related to the inhibition of neuroinflammation.

[0092] At the same time, the present invention also found that the neuroprotective effect of MeBavaC may be related to the regulation of the NLRP3 / AIM2 / GSDMD signaling pathway. Therefore, MeBavaC is expected to become a potential therapeutic drug for vascular cognitive impairment.

Claims

1. A use of 4'-O-methylpsoralen chalcone in a drug for treating vascular cognitive impairment, characterized in that: The application of 4'-O-methylpsoralen chalcone in the treatment of vascular cognitive impairment includes: dissolving 4'-O-methylpsoralen chalcone with a solvent, and then ultrasonically treating it to obtain a 4'-O-methylpsoralen chalcone solution.

2. The use of 4'-O-methylpsoralea corylifolia chalcone in a drug for treating vascular cognitive impairment according to claim 1, characterized in that: The solvent includes a polysaccharide solvent.

3. The use of 4'-O-methylpsoralea corylifolia chalcone in a drug for treating vascular cognitive impairment according to claim 2, characterized in that: The raw materials for preparing the polysaccharide solvent include water and polysaccharide.

4. The use of 4'-O-methylpsoralea corylifolia chalcone in a drug for treating vascular cognitive impairment according to claim 3, characterized in that: The polysaccharide comprises sodium carboxymethyl cellulose.

5. The use of 4'-O-methylpsoralea corylifolia chalcone in a drug for treating vascular cognitive impairment according to claim 2, characterized in that: The mass concentration of polysaccharide in the polysaccharide solvent is 1-2wt%.

6. The use of 4'-O-methylpsoralea corylifolia chalcone according to any one of claims 1 to 5 in a drug for treating vascular cognitive impairment, characterized in that: The injection dosage of the 4'-O-methylpsoralea chalcone solution includes low-dose injection or high-dose injection.

7. The use of 4'-O-methylpsoralea corylifolia chalcone in a drug for treating vascular cognitive impairment according to claim 6, characterized in that: The injection amount of the low-dose injection is 5-10 mg / kg.

8. The use of 4'-O-methylpsoralea corylifolia chalcone in a drug for treating vascular cognitive impairment according to claim 6, characterized in that: The injection amount of the high-dose injection is 11-20 mg / kg.

9. The use of 4'-O-methylpsoralea corylifolia chalcone in a drug for treating vascular cognitive impairment according to claim 1, characterized in that: The mass concentration of 4'-O-methyl psoralea chalcone in the 4'-O-methyl psoralea chalcone solution is 1-2 mg / mL.

10. The use of 4'-O-methylpsoralea corylifolia chalcone in a drug for treating vascular cognitive impairment according to claim 1, characterized in that: The dosage form of the 4'-O-methylpsoralea chalcone solution includes an oral dosage form.

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