Combined medicine for preventing and treating neurodegenerative diseases and application thereof

Abstract

The invention discloses a combined medicine for preventing and treating neurodegenerative diseases and application of the combined medicine, and belongs to the field of biological medicine. The combined medicine for preventing and / or treating neurodegenerative diseases comprises the following active pharmaceutical ingredients: bone-derived polypeptide; or, any one or more of pyrroloquinoline quinine and pyrroloquinoline quinine derivatives, and bone-derived polypeptide. According to the present invention, the bone-derived polypeptide is adopted as the drug active component for preventing and / or treating the neurodegenerative disease, such that the neurodegenerative disease can be effectively prevented and / or treated, and when any one or more of the pyrroloquinoline quinine and the pyrroloquinoline quinine derivative are combined with the bone-derived polypeptide, the neurodegenerative disease can be effectively prevented and / or treated. According to the present invention, the prevention and / or treatment effect on neurodegenerative diseases can be significantly improved, and particularly, the treatment effect on neurodegenerative diseases such as Alzheimer's disease with cognitive impairment can be further improved;

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a combination drug for preventing and / or treating neurodegenerative diseases and an application thereof. Background Art

[0002] Neurodegenerative diseases are a class of diseases that affect the brain, spinal cord, or peripheral nervous system, characterized by the gradual loss of function or death of neurons or their myelin sheaths over time, leading to a progressive degeneration of physical or mental abilities. These diseases are usually chronic and progressive, and there is currently no cure, only treatment to relieve symptoms and slow the progression of the disease.

[0003] Alzheimer's disease (AD) is a progressive neurodegenerative disease that mainly affects the cognitive function of the brain. As the disease progresses, symptoms such as language disorders, disorientation, impaired judgment, and mood swings may occur, especially in the elderly. At present, the exact cause of Alzheimer's disease is not yet fully understood, and there is no drug or method that can cure Alzheimer's disease.

[0004] Pyrroloquinoline quinone (PQQ) is a strong antioxidant. However, most studies on the use of PQQ alone focus on its antioxidant properties, and there are no reports on how to specifically apply it to complex neurodegenerative disease models.

[0005] Bone-derived polypeptides refer to a class of active polypeptide substances extracted from animal bone tissue. They have multiple biological activities, such as promoting bone cell growth, enhancing bone density, accelerating fracture healing, and improving osteoporosis. However, there have been no reports on their use in the treatment of neurodegenerative diseases. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a combination drug for preventing and / or treating neurodegenerative diseases and its application, so as to overcome the problem that the prior art has poor prevention and treatment effects on neurodegenerative diseases such as Alzheimer's disease.

[0007] In a first aspect, the present invention provides a combination drug for preventing and / or treating neurodegenerative diseases, wherein the combination drug comprises the following active pharmaceutical ingredients: a bone-derived polypeptide; or

[0008] Any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives, and bone-derived polypeptide.

[0009] Furthermore, the combined drug is a single drug preparation containing the bone-derived polypeptide; or,

[0010] A single-drug preparation comprising any one of pyrroloquinoline quinone and a pyrroloquinoline quinone derivative and a single-drug preparation comprising a bone-derived polypeptide; or,

[0011] The combined drug is a pharmaceutical composition containing any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives, and a bone-derived polypeptide.

[0012] Furthermore, when the active pharmaceutical ingredients in the combination drug include component A and a bone-derived polypeptide, component A is any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives, and the mass ratio of component A to bone-derived polypeptide is (1-5):(75-150).

[0013] Furthermore, the pyrroloquinoline quinone derivative includes one or more pharmaceutically acceptable salts or esters of pyrroloquinoline quinone.

[0014] Further, the pharmaceutically acceptable salt of pyrroloquinoline quinone includes one or more of the disodium salt, dipotassium salt, dicalcium salt, dimagnesium salt, hydrochloride, sulfate, tartrate, oxalate, maleate, fumarate, and citrate of pyrroloquinoline quinone;

[0015] Furthermore, the bone-derived polypeptide is one or more of bovine bone marrow polypeptide, bovine collagen polypeptide, sheep bone marrow polypeptide or horse bone marrow polypeptide.

[0016] Further, the dosage form of the single-drug preparation containing any one of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives is powder, pill or oral solution; the dosage form of the single-drug preparation containing bone-derived polypeptide is powder, pill or oral solution;

[0017] The combined drug is a pharmaceutical composition containing any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives and a bone-derived polypeptide, and its dosage form is aqueous solution injection, powder injection, pill, powder, tablet, patch, suppository, emulsion, cream, gel, granule, capsule, aerosol, spray or powder spray.

[0018] In a second aspect, the present invention provides use of any one of the above-mentioned combination drugs in the preparation of drugs for preventing and / or treating neurodegenerative diseases.

[0019] Further, the neurodegenerative disease is Alzheimer's disease, Lewy body dementia, or Parkinson's disease;

[0020] Prevention and / or treatment of Alzheimer's disease may include at least one or more of the following:

[0021] A) Improve spatial learning and memory abilities in patients with Alzheimer's disease;

[0022] B) Relieve anxiety in patients with Alzheimer's disease;

[0023] C) reduce the damage to neuronal cells in the brains of patients with Alzheimer's disease;

[0024] D) reduce Aβ deposition in the brains of patients with Alzheimer's disease and alleviate the inflammatory response caused by microglia;

[0025] E) Reduce the expression of inflammatory factors in the brain of patients with Alzheimer's disease and alleviate the inflammatory response.

[0026] The beneficial effects of the present invention are:

[0027] 1. Compared with the prior art, the present invention uses bone-derived polypeptide as an active ingredient of a drug for preventing and / or treating neurodegenerative diseases, which can effectively prevent and / or treat neurodegenerative diseases. When any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives are used in combination with bone-derived polypeptide, there is a synergistic effect at the molecular level, which significantly improves the prevention and / or treatment effect of neurodegenerative diseases, especially in preventing and treating Alzheimer's disease or other neurodegenerative diseases with cognitive impairment, and has a significant therapeutic effect.

[0028] 2. The present invention limits the mass ratio of any one of the active pharmaceutical ingredients pyrroloquinoline quinone and pyrroloquinoline quinone derivatives to the bone-derived polypeptide. Within this ratio range, the synergistic effect is optimal, and the efficacy of preventing and treating neurodegenerative diseases, especially neurodegenerative diseases with cognitive impairment such as Alzheimer's disease, is further enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In the following figures, **** indicates P < 0.0001, *** indicates P < 0.001, ** indicates P < 0.01, and * indicates P < 0.05;

[0030] Figure 1 Schematic diagram of the Morris water maze experiment in Test Example 1.

[0031] Figure 2 This is to test the spatial exploration ability of the mice in Example 1.

[0032] Figure 3 Schematic diagram of a 1 / 4 open field box in the open field experiment of Test Example 2.

[0033] Figure 4 The distance and time of the mice's activities in the mine in Test Example 2.

[0034] Figure 5 This is the Nissl staining image of the cerebral cortex and hippocampus of the mouse in Test Example 3.

[0035] Figure 6 This is a statistical analysis of the number of Nissl bodies in the cerebral cortex and hippocampus of mice in Test Example 3.

[0036] Figure 7 The results of Thioflavin S staining and Iba1 immunofluorescence staining of the mouse cerebral cortex in Test Example 4 are shown.

[0037] Figure 8 Statistics of Aβ plaque deposition and Iba1 immunofluorescence intensity in the mouse cerebral cortex in Test Example 4.

[0038] Fig. 9 The results of Thioflavin S staining and Iba1 immunofluorescence staining of the mouse hippocampus in Test Example 4 are shown.

[0039] Fig.10 Statistics of Aβ plaque deposition and Iba1 immunofluorescence intensity in the hippocampus of mice in Test Example 4.

[0040] Fig.11 This is the expression of BACE1 mRNA and ADAM10 mRNA in the cerebral cortex and hippocampus of the mice in Test Example 5.

[0041] Fig.12 This is the expression of BACE1 protein and ADAM10 protein in the cerebral cortex and hippocampus of mice in Test Example 6.

[0042] Fig.13 This is the expression of inflammatory factors TNF, IL-1β, and IL-6 mRNA in the cerebral cortex and hippocampus of mice in Test Example 7. DETAILED DESCRIPTION

[0043] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] In a first aspect, an embodiment of the present invention provides a combination drug for preventing and / or treating a neurodegenerative disease, wherein the combination drug comprises the following active pharmaceutical ingredients: a bone-derived polypeptide; or

[0045] Any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives, and bone-derived polypeptide.

[0046] Bone-derived polypeptides, as active pharmaceutical ingredients, have excellent preventive and therapeutic effects on neurodegenerative diseases. Any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives, when used in combination with bone-derived polypeptides, can further enhance the preventive and therapeutic effects on Alzheimer's disease or other neurodegenerative diseases with cognitive impairment, and improve the quality of life of patients.

[0047] Further, based on the above embodiments, the combination drug in the embodiments of the present invention is a single drug preparation containing bone-derived polypeptide; or,

[0048] A single-drug preparation comprising any one of pyrroloquinoline quinone and a pyrroloquinoline quinone derivative and a single-drug preparation comprising a bone-derived polypeptide; or,

[0049] The combined drug is a drug composition containing any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives, and a bone-derived polypeptide.

[0050] Further, on the basis of the above embodiments, when the active pharmaceutical ingredients in the combination drug of the embodiment of the present invention include component A and bone-derived polypeptide, component A is any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives, and the mass ratio of component A to bone-derived polypeptide is (1-5): (75-150). For example, the mass ratio can be 1:75, 1:100, 1:150, 5:75 or 5:150, preferably 1:75.

[0051] The above technical scheme defines the mass ratio of component A, i.e., any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives, and bone-derived polypeptide in the combination drug. Within the range of this mass ratio, the synergistic effect is better, and the preventive and therapeutic effect on neurodegenerative diseases with cognitive impairment is further enhanced.

[0052] Further, based on the above embodiments, the pyrroloquinoline quinone derivatives in the embodiments of the present invention include one or more pharmaceutically acceptable salts or esters of pyrroloquinoline quinone.

[0053] In some embodiments, the pharmaceutically acceptable salt of pyrroloquinoline quinone can be one or more of the disodium salt, dipotassium salt, dicalcium salt, dimagnesium salt, hydrochloride, sulfate, tartrate, oxalate, maleate, fumarate, and citrate of pyrroloquinoline quinone.

[0054] In some embodiments, the pharmaceutically acceptable ester of pyrroloquinoline quinone may be one or more of methyl ester, ethyl ester, propyl ester or butyl ester of pyrroloquinoline quinone.

[0055] The above technical scheme defines the specific components of the active ingredient pyrroloquinoline quinone derivatives. The pharmaceutically acceptable salts or esters of pyrroloquinoline quinone can reduce the oxidative damage of nerve cells in neurodegenerative diseases, enhance the adaptability of cells to long-term oxidative stress, and prevent and treat neurodegenerative diseases.

[0056] Further, based on the above embodiments, the bone-derived polypeptide in the embodiments of the present invention is one or more of bovine bone marrow polypeptide, bovine collagen polypeptide, sheep bone marrow polypeptide or horse bone marrow polypeptide.

[0057] The source of the bone-derived polypeptide in the above technical solution can be directly purchased commodities. For example, the bovine bone marrow peptide can be yak bone marrow polypeptide powder / 20221105-1 / produced by Liaoning Taiaipeptide Bioengineering Technology Co., Ltd.

[0058] The above technical solution defines the specific components of the active ingredient bone-derived polypeptide, which, when used in combination with pyrroloquinoline quinone or its derivatives, will produce a synergistic effect and show a better therapeutic effect than the existing technology in improving the patient's cognitive function.

[0059] Further, based on the above embodiments, the dosage form of the single-drug preparation containing any one of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives in the embodiments of the present invention is powder, pill or oral solution; the dosage form of the single-drug preparation containing bone-derived polypeptide is powder, pill or oral solution;

[0060] The combined drug is a pharmaceutical composition containing any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives and bone-derived polypeptide, and its dosage form is aqueous solution injection, powder injection, pill, powder, tablet, patch, suppository, emulsion, cream, gel, granule, capsule, aerosol, spray or powder spray.

[0061] It should be understood that the combination drug in the above technical solution may also contain various pharmaceutical excipients and be prepared into a dosage form that is convenient for administration by conventional methods.

[0062] In some embodiments, the pharmaceutical excipients may be those conventionally used in various preparations, such as isotonicity agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, lubricants, and the like.

[0063] In other embodiments, the pharmaceutical excipients may also be emulsifiers, solubilizers or antibacterial agents that are compatible with the pharmaceutically active substances pyrroloquinoline quinone, pyrroloquinoline quinone derivatives or bone-derived polypeptides, so as to improve the stability, solubility or release rate of the combined drugs, thereby enhancing the administration effect of the combined drugs.

[0064] In a second aspect, an embodiment of the present invention provides use of any one of the above-mentioned combination drugs in the preparation of a drug for preventing and / or treating neurodegenerative diseases.

[0065] Compared with the prior art, the use of any one of the above-mentioned combination drugs provided in the embodiments of the present invention in the preparation of drugs for preventing and / or treating neurodegenerative diseases has the same beneficial effects as the above-mentioned combination drug for preventing and / or treating neurodegenerative diseases, which will not be repeated here.

[0066] Furthermore, the neurodegenerative disease in the above embodiment may be Alzheimer's disease, Lewy body dementia or Parkinson's disease, preferably Alzheimer's disease.

[0067] The above-mentioned prevention and / or treatment of Alzheimer's disease may be embodied in at least one or more of the following:

[0068] A) Improve spatial learning and memory abilities in patients with Alzheimer's disease;

[0069] B) Relieve anxiety in patients with Alzheimer's disease;

[0070] C) reduce the damage to neuronal cells in the brains of patients with Alzheimer's disease;

[0071] D) reduce Aβ deposition in the brains of patients with Alzheimer's disease and alleviate the inflammatory response caused by microglia;

[0072] E) Reduce the expression of inflammatory factors in the brain of patients with Alzheimer's disease and alleviate the inflammatory response.

[0073] In order to better illustrate the technical solution of the present invention, the present invention also provides the following specific examples. It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0074] Test Case

[0075] C57 mice were used as a healthy control group, and genetically modified APP / PS1 double transgenic mice were used as an Alzheimer's disease model group (no medication) and a medication group, with 8 mice in each group.

[0076] The settings of the drug administration groups are as follows:

[0077] APP / PS1 double transgenic mice were divided into three drug administration groups: pyrroloquinoline quinone administration group, bovine bone marrow peptide administration group, and pyrroloquinoline quinone and bovine bone marrow peptide combined administration group. The pyrroloquinoline quinone is CAS No. 72909-34-3 produced by Zhucheng Haotian Pharmaceutical Co., Ltd.

[0078] PQQ group: The dosage of mice in the pyrroloquinoline quinone administration group was 20 mg / kg, that is, 20 mg of pyrroloquinoline quinone per kilogram of body weight per day.

[0079] Group B: The dosage of the mice in the bovine bone marrow peptide administration group was 1.5 g / kg, that is, 1.5 g of bovine bone marrow peptide was administered per kilogram of body weight per day.

[0080] PB group: The dosage of mice in the pyrroloquinoline quinone and bovine bone marrow peptide combined administration group was (20 mg + 1.5 g) / kg, that is, 20 mg of pyrroloquinoline quinone and 1.5 g of bovine bone marrow peptide were administered per kilogram of body weight per day.

[0081] The mice in the above-mentioned drug-administered groups were all given drugs by oral gavage for six months.

[0082] The healthy control group and the model group were given the same volume of normal saline.

[0083] Test Example 1

[0084] Morris water maze test

[0085] A platform with a diameter of 5 cm is set in a barrel with a diameter of 120 cm and a depth of 50 cm. Clean water is poured into the barrel to cover the platform, and the water surface is 1 cm above the platform.

[0086] The drum is surrounded by dark curtains to prevent interference from external factors. The water surface area of ​​the drum is divided into four quadrants. The platform is located in the third quadrant and marked in the supporting software system (such as Figure 1 shown).

[0087] In order to enhance the black and white contrast in the field of vision and make it easier for the software to track mice, select experimental mice with black hair, add an appropriate amount of pure milk to the water and stir evenly to make the water milky white, and keep the water temperature at 22-24°C.

[0088] Before the formal Morris water maze experiment, all mice received 4 days of training and testing. During the training and testing phase, the platform was visible, and each mouse was trained once in quadrants 1, 2, 3, and 4 respectively. This process was to familiarize the mice with the spatial layout of the water maze. Each time, the mouse was gently placed in the water along the edge of the quadrant, so that it could recognize the location of the platform within 60 seconds, and the time to find the platform was recorded. If it stayed on the platform for ≥5 seconds, it was considered to have successfully found the platform. After the end, the mouse was taken out, dried, and put into the mouse cage. If the mouse did not find the platform within the specified time, the experimental operator guided the mouse to the platform and made it stay on the platform for 5 seconds.

[0089] On the fifth day, the table was removed and the spatial exploration ability of the mice was tested. The number of times the mice crossed the platform within 60 seconds, as well as the distance and residence time in the quadrant where the platform was located, were recorded synchronously by an automatic tracking camera. The observation indicators included: the distance of activity in the quadrant where the original platform was located, the activity time in the quadrant where the original platform was located, and the number of times the platform was crossed (referring to the number of times the experimental mice crossed the original platform position within 60 seconds after the platform was removed).

[0090] The results are as follows Figure 2 As shown: Figure 2 The WT group was the healthy control group, the MOD group was the Alzheimer's disease model group, the PQQ group was the pyrroloquinoline quinone administration group, the B group was the bovine bone marrow peptide administration group, and the PB group was the pyrroloquinoline quinone and bovine bone marrow peptide combined administration group.

[0091] Depend on Figure 2 It can be seen that: Figure A shows the activity distance of mice in the quadrant where the original platform was located. The model group was lower than the control group, the PQQ group and the PB group were significantly higher than the model group, there was no significant difference between the B group and the model group, and the PB group was significantly higher than the PQQ group and the B group.

[0092] Figure B shows the activity time of mice in the quadrant where the original platform was located. The activity time of the model group was lower than that of the control group, and that of the PQQ group and the PB group were significantly higher than that of the model group. There was no significant difference between group B and the model group, and that of the PB group was significantly higher than that of the PQQ group.

[0093] Figure C shows the number of times the mice crossed the original platform position. The number in the model group was 0, while that in the PQQ group, the B group, and the PB group were all higher than that in the model group, and that in the PB group was significantly higher than that in the PQQ group and the B group.

[0094] The longer the distance and time of the mouse's activities in the quadrant where the original platform was located, the deeper the mouse's memory of the original platform was, and after the platform was removed, the mouse continued to look for the original platform. The more times the mouse crossed the original platform, the more accurate the mouse's memory of the original platform was. The above results show that the combined use of pyrroloquinoline quinone and bovine bone marrow peptide can produce a synergistic effect and further improve the spatial learning and memory ability of APP / PS1 mice.

[0095] Test Example 2

[0096] Open field test

[0097] The open-field box is a roofless square field divided into four equal-sized areas (each area is 50 cm long, 50 cm wide, and 35 cm high). The inner walls of the square area are painted black and the bottom is white. A camera is placed directly above the open-field box so that it can observe the entire area of ​​the open-field box (such as Figure 3 shown).

[0098] Each time, 4 mice were tested at the same time. During the test, the mouse was held by hand at the upper 1 / 3 of its tail and placed in four equal-sized areas. The autonomous activities of the mice were observed within 5 minutes, and recorded by the camera directly above the open field box. The environment was kept quiet and the temperature was appropriate. After each mouse test, the feces in the box was cleaned and 75% alcohol was sprayed to remove the smell left by the previous mouse. After the alcohol evaporated, the next group of experiments was carried out. The observation indicators included: the total distance and average speed of the mouse movement.

[0099] The results are as follows Figure 4 As shown: Figure 4 The WT group was the healthy control group, the MOD group was the Alzheimer's disease model group, the PQQ group was the pyrroloquinoline quinone administration group, the B group was the bovine bone marrow peptide administration group, and the PB group was the pyrroloquinoline quinone and bovine bone marrow peptide combined administration group.

[0100] Depend on Figure 4It can be seen that: Figure A shows the total distance of the mice in the open field area, Figure B shows the average speed of the mice in the open field, Figure C shows the distance of the mice in the central area of ​​the open field box, and Figure D shows the activity time of the mice in the central area of ​​the open field box. The four results all showed that the model group was lower than the control group, the PQQ group was higher than the model group, and there was no significant difference between the B group and the model group. In Figure C, the PB group was further higher than the PQQ group, and in Figures A, B, and D, the PB group was significantly higher than the PQQ group. The above results show that the motor activity and exploratory behavior of mice in the PQQ and PB groups increased significantly, and anxiety decreased. Pyrroloquinoline quinone and bovine bone marrow peptide have a synergistic effect, and the effect of the PB group is the most significant.

[0101] Test Example 3

[0102] Nissl staining experiment

[0103] (1) paraffin sections were prepared from the cerebral cortex and hippocampus of the five groups of mice, the thickness of the paraffin sections was 4 μm, and the sections were routinely dewaxed to water;

[0104] (2) Stain the sections in toluidine blue dye for 1-2 minutes, rinse with distilled water, differentiate the background with 0.5% glacial acetic acid, and wash with water while differentiating. The degree of differentiation can be monitored under a microscope.

[0105] (3) Dry the differentiated slices in a 65°C oven for 4 h;

[0106] (4) Place the dried slices in clean xylene I for 2 min and clean xylene II for 15 min;

[0107] (5) Finally, seal the slide with neutral gum;

[0108] (6) Observe and take photos under a microscope and use Image J software to count Nissler bodies.

[0109] The results are as follows Figure 5 As shown: Figure 5 The PQQ group was the pyrroloquinoline quinone administration group, the B group was the bovine bone marrow peptide administration group, and the PB group was the pyrroloquinoline quinone and bovine bone marrow peptide combined administration group.

[0110] Depend on Figure 5It can be seen that the neurons in the cerebral cortex and hippocampus of the control group mice were neatly and densely arranged, with clear cell hierarchy and complete morphology, and the Nissl bodies in the cytoplasm were clearly visible and abundant, and the cell nuclei were light blue. The neurons in the cerebral cortex and hippocampus of the model group mice were chaotically arranged, irregularly shaped, with unclear nuclear boundaries, and shrunken cells and reduced numbers. Compared with the model group, the cells in the PQQ group were relatively regular, with clear nuclear boundaries and clear Nissl bodies. The cells in the B group were compactly and regularly arranged, with relatively clear nuclear boundaries. The neuronal damage in the PB group was significantly improved, with regular arrangement, light blue nuclei, and clear boundaries, which was close to the control group.

[0111] The results are as follows Figure 6 As shown: Figure 6 The WT group was the healthy control group, the MOD group was the Alzheimer's disease model group, the PQQ group was the pyrroloquinoline quinone administration group, the B group was the bovine bone marrow peptide administration group, and the PB group was the pyrroloquinoline quinone and bovine bone marrow peptide combined administration group.

[0112] Depend on Figure 6 It can be seen that: Figure A shows that the number of Nissl bodies in the cerebral cortex of mice in the model group is lower than that in the control group, while the numbers in the PQQ group and the PB group are higher than the model group. There is no significant difference between group B and the model group, and the PB group is significantly higher than the PQQ group and the B group; Figures B and C counted the number of Nissl bodies in the CA1 and CA3 regions of the hippocampus of mice in the model group. The results showed that the number of Nissl bodies in the CA1 and CA3 regions of the hippocampus of mice in the model group were lower than those in the control group, the difference between the PQQ group and the B group and the model group was not significant, and the PB group was significantly higher than the model group, the PQQ group and the B group. Figure D counted the number of Nissl bodies in the DG region of the hippocampus of mice. The results showed that the number of Nissl bodies in the DG region of the hippocampus of mice in the model group was lower than that in the control group, the difference between the PQQ group and the B group and the model group was not significant, and the PB group was significantly higher than the model group, the PQQ group and the B group.

[0113] The number of Nissl bodies in the brain can indirectly reflect the activity and damage of neuronal cells. The above results show that the combined use of pyrroloquinoline quinone and bovine bone marrow peptide can reduce the damage of Alzheimer's disease to neuronal cells in the brain.

[0114] Test Example 4

[0115] Thioflavin S staining and Iba1 immunofluorescence staining

[0116] (1) Paraffin sections prepared from the cerebral cortex and hippocampus of the five groups of mice were taken, dewaxed to water, and then placed in environmentally friendly dewaxing liquid I for 10 min, environmentally friendly dewaxing liquid II for 10 min, environmentally friendly dewaxing liquid III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then washed with distilled water.

[0117] (2) Antigen retrieval: The retrieval conditions are shown in Table 1. During the retrieval process, the buffer should be prevented from evaporating excessively and the slides should not be dried. After retrieval, cool naturally. Place the slides in PBS (PH7.4) and wash them on a decolorizing shaker three times, 5 minutes each time.

[0118] (3) Circle serum blocking: After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue, add BSA, and block for 30 minutes.

[0119] (4) Add the mixed reagent of Iba1 and thioflavin primary antibodies: Mix the primary antibodies from two different sources, add the prepared primary antibodies dropwise, and incubate the sections flat in a humidified chamber at 4°C overnight.

[0120] (5) Adding secondary antibody: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 min each time. Add the corresponding secondary antibody and incubate at room temperature in the dark for 50 min.

[0121] (6) DAPI counterstaining of cell nuclei: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 min each time. Add DAPI staining solution and incubate at room temperature for 10 min in the dark.

[0122] (7) Quenching tissue autofluorescence: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 min each time. Add autofluorescence quencher B solution for 5 min and rinse with running water for 10 min.

[0123] (8) Sealing: Use anti-fluorescence quenching sealing medium to seal the slices.

[0124] (9) Image acquisition: DAPI excitation wavelength 330-380 nm, emission wavelength 420 nm; 488 excitation wavelength 465-495 nm, emission wavelength 515-555 nm; CY3 excitation wavelength 510-560 nm, emission wavelength 590 nm; CY5 excitation wavelength 608-648 nm, emission wavelength 672-712 nm.

[0125] Table 1 Immunofluorescence antibody table

[0126]

[0127] The results are as follows Figure 7 , Figure 8 , Fig. 9 , Fig.10 As shown: WT group is the healthy control group, MOD group is the Alzheimer's disease model group, PQQ group is the pyrroloquinoline quinone administration group, B group is the bovine bone marrow peptide administration group, and PB group is the pyrroloquinoline quinone and bovine bone marrow peptide combined administration group.

[0128] Depend on Figure 7It can be seen that: there is no Aβ deposition in the cerebral cortex of mice in the control group, obvious Aβ deposition can be seen in the model group, and the Aβ deposition in the PQQ group, group B and group PB is less than that in the model group. The Iba1 immunofluorescence intensity in the cerebral cortex of mice in the model group was significantly higher than that in the control group, and the cells were in an activated state. The Iba1 immunofluorescence intensity in the PQQ group, group B and group PB was lower than that in the model group, the number of activated cells was significantly reduced, and most of the cells were in a resting state.

[0129] Depend on Figure 8 It can be seen that: Figure B counts the percentage of Aβ plaques in the same area of ​​the mouse cerebral cortex. The Aβ deposition in the PQQ group, B group and PB group was significantly less than that in the model group, and the PB group was significantly less than that in the PQQ group and B group. Figure C counts the average fluorescence intensity of Iba1 in the same area of ​​the mouse cerebral cortex. The model group was significantly higher than the control group, and the difference was statistically significant (P<0.01). Compared with the model group, the PQQ group, B group and PB group were all reduced, and the PB group was significantly lower than the PQQ group and B group.

[0130] Depend on Fig. 9 It can be seen that there was no Aβ deposition in the hippocampus of mice in the control group, but significant Aβ deposition in the model group, while that in the PQQ group, B group and PB group was reduced compared with the model group; the Iba1 immunofluorescence intensity in the hippocampus of mice in the model group was significantly increased compared with the control group, while that in the PQQ group, B group and PB group was reduced compared with the model group.

[0131] Depend on Fig.10 It can be seen that Figures B and C respectively counted the percentage of the area occupied by Aβ plaques and the average fluorescence intensity of Iba1 in the same hippocampal region of mice. The results showed that the Aβ deposition in the model group was significantly higher than that in the control group, and the average fluorescence intensity of Iba1 in the model group was also significantly higher than that in the control group. Compared with the model group, the Aβ deposition in the PQQ group, group B and group PB was reduced, and the PB group was significantly lower than the PQQ group and group B. Compared with the model group, the average fluorescence intensity of Iba1 in the PQQ group, group B and group PB was also significantly reduced, and the PB group was significantly lower than the PQQ group and group B.

[0132] Thioflavin S staining was used to identify and visualize the deposited beta-amyloid protein (Aβ) in the mouse brain, which is considered to be one of the key factors in the pathogenesis of Alzheimer's disease. Iba1 immunofluorescence staining can be used to assess the degree of microglial activation. Iba1 is a calcium-binding protein specifically expressed in brain microglia. It is upregulated when microglia are activated, so it is widely used as a marker of microglial activation, which represents the presence of neuroinflammation.

[0133] The above results show that pyrroloquinoline quinone, bovine bone marrow peptide and the combination of the two can significantly reduce Aβ deposition in the brains of Alzheimer's disease model mice, alleviate inflammatory responses, and have a therapeutic effect on Alzheimer's disease. The combination of the two has a synergistic effect and a better therapeutic effect.

[0134] Test Example 5

[0135] RT-qPCR experiments

[0136] The cerebral cortex and hippocampus of mice in the above five groups were respectively obtained, and the total RNA was extracted using a kit and reverse transcribed into cDNA. The two-step PCR fluorescence quantitative detection was performed (the reaction conditions were pre-denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, and annealing & extension at 60°C for 30 s). β-actin was used as the internal reference gene, and the 2-ΔΔCt method was used to calculate the relative expression levels of the target genes BACE1 and ADAM10 (the primer sequences are shown in Table 2). The experiment was repeated three times.

[0137] Table 2 Primer sequence list

[0138]

[0139] The experimental results are as follows Fig.11 As shown: CON group is the healthy control group, MOD group is the Alzheimer's disease model group, PQQ group is the pyrroloquinoline quinone administration group, B group is the bovine bone marrow peptide administration group, and PB group is the pyrroloquinoline quinone and bovine bone marrow peptide combined administration group.

[0140] Depend on Fig.11 It can be seen that: Figure A and Figure B are the BACE1 mRNA expression in the cerebral cortex and hippocampus of mice, respectively. The results show that the model group is higher than the control group, the PQQ group, the B group and the PB group are lower than the model group, and the PB group is significantly lower than the PQQ group and the B group. Figure C and Figure D are the ADAM10 mRNA expression in the cerebral cortex and hippocampus of mice, respectively. The results show that the model group is lower than the control group, the PQQ group and the B group have no significant difference from the model group, and the PB group is significantly higher than the model group, the PQQ group and the B group. This shows that the combination of pyrroloquinoline quinone and bovine bone marrow peptide has a synergistic effect, which can reduce the relative expression level of the BACE1 gene in the cerebral cortex and hippocampus of mice and increase the relative expression level of the ADAM10 gene.

[0141] BACE1 is the β-site amyloid precursor protein cleaving enzyme-1, which plays an important role in the pathogenesis of AD. When the activity of neuronal BACE1 increases, it can promote the production of amyloid protein (Aβ) in the brains of Alzheimer's patients.

[0142] ADAM10 is an α-secretase that is involved in the hydrolytic metabolism of APP (amyloid precursor protein), a process that can prevent the production of Aβ and exert its effects by inducing the secretion of soluble APPα, which has a neuroprotective effect.

[0143] The above results indicate that the combination of pyrroloquinoline quinone and bovine bone marrow peptide can have a therapeutic effect on Alzheimer's disease by reducing the relative expression level of the BACE1 gene in the cerebral cortex and hippocampus of mice and increasing the relative expression level of the ADAM10 gene.

[0144] Test Example 6

[0145] Western Blot Experiment

[0146] The cerebral cortex and hippocampus of mice in five groups were taken respectively, and 1 mL of RIPA lysis buffer containing PMSF was added to extract proteins on ice. The protein concentration was determined by the BCA method, adjusted to the same protein concentration, mixed, boiled at 100°C for 5 minutes to denature the protein, and stored at -20°C. The separated protein blot was transferred to a PVDF membrane by SDS-PAGE gel electrophoresis, and after washing the membrane, it was blocked with 5% skim milk powder on a horizontal shaker at room temperature for 1 hour. After washing the membrane, the primary antibody was incubated in a 4°C refrigerator overnight. After washing the membrane, the secondary antibody was incubated on a horizontal shaker at room temperature for 1 hour. The membrane was washed, stained with ECL chemiluminescence, exposed in a gel imaging system, and the protein gray value was analyzed using ImageJ software.

[0147] The above primary antibodies are:

[0148] (1) BACE1 (Rabbit), brand: proteintech, catalog number: 12807-1-AP;

[0149] (2) ADAM10 (Rabbit), brand: proteintech, catalog number: 25900-1-AP;

[0150] (3) β-Actin (Mouse), Brand: Youkang, Product No.: UM4001.

[0151] The above secondary antibodies are:

[0152] Secondary antibody goat anti-rabbit IgG-HRP, brand: Affinity, catalog number: S0001;

[0153] Secondary antibody: goat anti-mouse IgG-HRP, brand: Affinity, catalog number: S0002.

[0154] The results are as follows Fig.12As shown: CON group is the healthy control group, MOD group is the Alzheimer's disease model group, PQQ group is the pyrroloquinoline quinone administration group, B group is the bovine bone marrow peptide administration group, and PB group is the pyrroloquinoline quinone and bovine bone marrow peptide combined administration group.

[0155] Depend on Fig.12 It can be seen that Figures B, D, and E show the expression of BACE1 protein and ADAM10 protein in the cerebral cortex of mice. The expression level of BACE1 protein in the model group was significantly higher than that in the control group, and there was no significant difference between the PQQ group and the B group and the model group, and PB was significantly lower than the model group, the PQQ group, and the B group. The expression level of ADAM10 protein in the model group was significantly lower than that in the control group, and there was no significant difference between the PQQ group and the B group and the model group, and the PB group was significantly higher than the model group, the PQQ group, and the B group.

[0156] Figures A, C, and F show the expression of BACE1 protein and ADAM10 protein in the hippocampus of mice. The expression level of BACE1 protein in the model group was significantly higher than that in the control group, and there was no significant difference between the PQQ group, group B and the model group, and the PB group was significantly lower than the model group, PQQ group and group B. The expression level of ADAM10 protein in the model group was significantly lower than that in the control group, and there was no significant difference between the PQQ group, group B and the model group, and the PB group was significantly higher than the model group, PQQ group and group B.

[0157] Fig.12 The results showed that pyrroloquinoline quinone and bovine bone marrow peptide had a synergistic effect. Their combined use could significantly reduce the BACE1 protein level in the cerebral cortex and hippocampus of mice and increase the ADAM10 protein level.

[0158] Test Example 7

[0159] Inflammatory factor mRNA expression level (RT-qPCR) experiment

[0160] The cerebral cortex and hippocampus of the mice in the above five groups were respectively obtained, and the total RNA was extracted using a kit and reverse transcribed into cDNA. The two-step PCR fluorescence quantitative detection was performed (the reaction conditions were pre-denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, and annealing & extension at 60°C for 30 s). β-actin was used as the internal reference gene, and the 2-ΔΔCt method was used to calculate the relative expression levels of the target genes TNF, IL-1β, and IL-6 (the primer sequences are shown in Table 3). The experiment was repeated three times.

[0161] Table 3 Primer sequence list

[0162]

[0163] The experimental results are as follows Fig.13As shown: CON group is the healthy control group, MOD group is the Alzheimer's disease model group, PQQ group is the pyrroloquinoline quinone administration group, B group is the bovine bone marrow peptide administration group, and PB group is the pyrroloquinoline quinone and bovine bone marrow peptide combined administration group.

[0164] Depend on Fig.13 It can be seen that: Figures AC are the mRNA expressions of inflammatory factors TNF, IL-1β, and IL-6 in the cerebral cortex of mice, respectively. The results show that the model group is higher than the control group, the PQQ group and the B group have no significant changes compared with the model group, and the PB group is significantly lower than the model group, the PQQ group and the B group. Figures D and E are the mRNA expressions of TNF and IL-1β in the hippocampus of mice. The results show that the model group is higher than the control group, the PQQ group and the B group have no significant changes compared with the model group, and the PB group is significantly lower than the model group, the PQQ group and the B group. Figure F is the mRNA expression of IL-6 in the hippocampus of mice. The results show that the model group is higher than the control group, the PQQ group and the B group have no significant difference from the model group, and the PB group is significantly lower than the model group, the PQQ group and the B group. This shows that the PB group can reduce the relative expression levels of TNF, IL-1β, and IL-6 genes in the cerebral cortex and hippocampus of mice, and has an anti-inflammatory effect, while the anti-inflammatory effects of the PQQ group and the B group are not significant, and the PB group shows the synergistic effect of PQQ and bovine bone marrow peptide.

[0165] TNF is a cytokine secreted by immune cells that has pro-inflammatory effects. High levels of TNF can directly or indirectly lead to neuronal damage and death. It can increase the permeability of the blood-brain barrier, allowing more inflammatory cells and factors to enter the central nervous system, thereby aggravating the inflammatory response.

[0166] IL-1β is secreted by activated immune cells such as microglia and macrophages. It can activate multiple downstream signaling pathways such as NF-κB and MAPK pathways through its receptor IL-1R1, leading to the production of other proinflammatory factors, which can maintain and amplify neuroinflammatory responses and lead to chronic nervous system damage. It can promote the activation of glial cells, causing them to release more proinflammatory factors and neurotoxic substances, thereby further aggravating neuroinflammation.

[0167] IL-6 forms a trimeric complex with the membrane-bound IL-6 receptor (mIL-6R) and gp130 through the classical signaling pathway, activates downstream signaling pathways (such as JAK / STAT, MAPK, and PI3K / Akt), promotes the production of other proinflammatory factors such as IL-1β and TNFα, and thus aggravates the inflammatory response. It activates microglia and astrocytes, causing these cells to release more proinflammatory factors and free radicals, further aggravating neuroinflammation.

[0168] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A combination drug for preventing and / or treating neurodegenerative diseases, characterized in that: The combination drug comprises the following active pharmaceutical ingredients: bone-derived polypeptide; or Any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives, and bone-derived polypeptide.

2. The combination drug according to claim 1, characterized in that: The combination drug is a single drug preparation containing a bone-derived polypeptide; or, A single-drug preparation comprising any one of pyrroloquinoline quinone and a pyrroloquinoline quinone derivative and a single-drug preparation comprising a bone-derived polypeptide; or, The combined drug is a drug composition containing any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives, and a bone-derived polypeptide.

3. The combination drug according to claim 1, characterized in that: When the active pharmaceutical ingredients in the combination drug include component A and bone-derived polypeptide, component A is any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives; the mass ratio of component A to bone-derived polypeptide is (1-5):(75-150).

4. The combination drug according to claim 1, characterized in that: The pyrroloquinoline quinone derivatives include one or more of pyrroloquinoline quinone in pharmaceutically acceptable salts or esters.

5. The combination drug according to claim 4, characterized in that: The pharmaceutically acceptable salt of pyrroloquinoline quinone includes one or more of disodium salt, dipotassium salt, dicalcium salt, dimagnesium salt, hydrochloride, sulfate, tartrate, oxalate, maleate, fumarate and citrate of pyrroloquinoline quinone.

6. The combination drug according to claim 1, characterized in that: The bone-derived polypeptide is one or more of bovine bone marrow polypeptide, bovine collagen polypeptide, sheep bone marrow polypeptide or horse bone marrow polypeptide.

7. The combination drug according to claim 2, characterized in that: The dosage form of the single-drug preparation containing any one of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives is powder, pill or oral solution; the dosage form of the single-drug preparation containing bone-derived polypeptide is powder, pill or oral solution; The combined drug is a pharmaceutical composition containing any one or more of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives and a bone-derived polypeptide, and its dosage form is aqueous solution injection, powder injection, pill, powder, tablet, patch, suppository, emulsion, cream, gel, granule, capsule, aerosol, spray or powder spray.

8. Use of the combination drug according to any one of claims 1 to 7 in the preparation of drugs for preventing and / or treating neurodegenerative diseases.

9. The use according to claim 8, characterized in that: The neurodegenerative disease is Alzheimer's disease, Lewy body dementia or Parkinson's disease; Prevention and / or treatment of Alzheimer's disease may include at least one or more of the following: A) Improve spatial learning and memory abilities in patients with Alzheimer's disease; B) Relieve anxiety in patients with Alzheimer's disease; C) reduce the damage to neuronal cells in the brains of patients with Alzheimer's disease; D) reduce Aβ deposition in the brains of patients with Alzheimer's disease and alleviate the inflammatory response caused by microglia; E) Reduce the expression of inflammatory factors in the brain of patients with Alzheimer's disease and alleviate the inflammatory response.

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