New application of pharmaceutical composition in nervous system diseases

Abstract

The invention provides a new application of a pharmaceutical composition in nervous system diseases, the pharmaceutical composition comprises active pharmaceutical ingredients of beta-nicotinamide adenine dinucleotide and PJ34 according to a dosage ratio of (200-400): 1, and particularly relates to a new clinical application of an anti-aging drug for Parkinson's disease, Alzheimer's disease and neurodegenerative disease. And the clinical application indication range of the medicine is expanded, and a new medication choice is provided for clinical patients.

Description

Technical Field

[0001] The present invention relates to a new use of a pharmaceutical composition in the treatment of nervous system diseases, belonging to the field of new pharmaceutical uses. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease (NDs) after Alzheimer's disease (AD), accounting for about 1% in the elderly population over 60 years old. The loss of dopaminergic (DA) neurons in the substantia nigra pars compacta of the midbrain and the dystrophic striatum are the main causes of the motor symptoms such as resting tremor, bradykinesia, rigidity, and postural instability in PD patients. At the same time, other neuronal systems are affected by pathological alpha-synuclein (α-syn), resulting in non-motor symptoms such as anxiety, depression, sleep disorders, autonomic dysfunction, constipation, and cognitive impairment in PD patients. The pathological accumulation and aggregation of α-syn are the basis for the onset of PD. During the onset of PD, monomeric α-syn assembles and misfolds to form pathological α-syn, driving the progressive loss of DA neurons in the substantia nigra pars compacta. Pathological α-syn can also spread from one cell to another, ultimately leading to the progressive onset of PD. However, the mechanisms driving the abnormal assembly of pathological α-syn and the neuronal injury and death mechanisms activated by pathological α-syn remain unclear.

[0003] Oxidative stress injury, mitochondrial dysfunction, autophagy / mtophagy disorders, and neuroinflammation play important roles in the pathophysiological processes of the loss of DA neurons in PD caused by abnormal assembly of α-syn and pathological α-syn. Recent studies have shown that DNA damage induced by oxidative stress response and the overactivation of poly (ADP-ribose) polymerase 1 (PARP1), and subsequent NAD+ (nicotinamide adenine dinucleotide)-dependent or poly (ADP-ribose) (PAR)-dependent cell death are the causes of selective DA neuron loss in PD. In the cerebrospinal fluid and brains of PD patients, the level of PAR produced by the overactivation of PARP1 was significantly increased.

[0004] PARP1 is a key DNA damage sensor protein that responds to various DNA damages to initiate DNA repair. DNA damages usually occur in two forms: DNA single-strand breaks and double-strand breaks. PARP1 is an important DNA repair enzyme for DNA single-strand base excision and repair. In the case of DNA single-strand damage (i.e., mild damage), PARP1 is activated and uses β-NAD+ (a conformation of NAD+) as a donor to hydrolyze β-NAD+ and release nicotinamide and a proton. At the same time, it catalyzes the transfer of the ADP-ribose moiety to the amino acid residues of the receptor protein, forming a covalent poly-ADP-ribosylation modification and generating a highly charged PAR branch. Then, DNA ligase III (LigIII), DNA polymerase β (polβ), and scaffold proteins (such as X-ray repair cross complementing 1 (XRCC1)) are recruited to the damaged site to repair the damaged DNA. After DNA repair, the PAR chain is degraded by PAR glycohydrolase (PARG). Under the condition of severe DNA damage, over-activation of PARP1 leads to the accumulation of PAR polymers. PAR leaves the nucleus and transfers to mitochondria, depolarizing the mitochondria, triggering the release of apoptosis-inducing factor (AIF) from the mitochondrial intermembrane space, and carrying macrophage migration inhibitory factor (MIF) to translocate to the nucleus. Finally, chromosomal DNA is cleaved, and large-scale fragmented DNA appears, thus triggering cell death. During the progression of PD, PAR released into the cytoplasm can convert pathological α-syn into a more toxic strain, accelerating its neurotoxicity in vitro and in vivo. Pathological α-syn can also activate nitric oxide synthase (NOS), causing DNA damage and PARP1 activation. Over-activated PARP1 and the resulting PAR are considered key mediators of the toxicity and spread of pathological α-syn. Therefore, inhibiting PARP1 can significantly reduce the pathological changes of PD caused by the spread of pathological α-syn. In addition, the accumulation of pathological α-syn caused by over-activation of PARP1 can also mediate neuroinflammation and DNA damage by regulating the activation of NLRP3 inflammasome, ultimately forming a self-perpetuating cycle to exacerbate PD. At the same time, over-activated PARP1 can also impair autophagy by regulating Sirt1 and transcription factor EB (TFEB), and exacerbate mitochondrial damage by regulating oxidative stress in mitochondria, ultimately accelerating neuron death.

[0005] β-NAD+ analogues, which target PARP1 and mimic the structure of the nicotinamide moiety of β-NAD+, are PARP1 inhibitors. Its molecular mechanism includes the following two aspects: ① Competing with β-NAD+ at the C-terminal catalytic domain (CAT) of the active site of PARP1 to inhibit the catalytic activity of PARP1. The auto-PAR modification of PARP1 is inhibited and the formation of PAR is reduced. Therefore, single-strand breaks in DNA cannot be repaired in a timely manner, further resulting in double-strand breaks in DNA and ultimately leading to cell death; ② Binding to the β-NAD+ binding pocket of PARP1, causing allosteric changes in PARP1, stabilizing the reversible dissociation of DNA-PARP1, and enabling PARP1 to maintain its binding to DNA, that is, PARP1 is "trapped" on damaged DNA, resulting in the long-term existence of the DNA-PARP1 complex. The remaining PARP1 in the cell nucleus is difficult to bind to damaged DNA, further blocking possible repair pathways for DNA double-strand breaks and promoting apoptosis.

[0006] Nicotinamide adenine dinucleotide (NAD+) is a coenzyme that plays a key role in cellular energy metabolism and signal transduction. ① Nicotinamide adenine dinucleotide (NAD+) is a coenzyme present in all living cells and participates in various biochemical reactions, such as glycolysis, fatty acid oxidation, etc. In these processes, NAD+ acts as a hydrogen carrier, accepting electrons and hydrogen from substrates to maintain the intracellular redox balance. The effects of nicotinamide adenine dinucleotide include the following: 1. Improving cognitive function: NAD+ improves brain cell function, enhances memory and learning ability by activating the SIRT3 protein. 2. Regulating metabolism: NAD+ participates in various physiological activities such as cellular material metabolism, energy synthesis, and cell DNA repair. In particular, the drugs for treating neurological diseases and Parkinson's disease have unsatisfactory curative effects. Therefore, it is very necessary to find a drug with good curative effect and few side effects. Summary of the Invention

[0007] The object of the present invention is to provide a new use of a pharmaceutical composition in nervous system diseases, specifically involving providing new clinical uses for Parkinson's disease, Alzheimer's disease, neurodegenerative diseases, and anti-aging drugs, and expanding the scope of clinical application indications of this drug to provide new drug options for clinical patients.

[0008] The technical solution of this invention patent application is as follows: The drug active ingredients and dosage ratio of the pharmaceutical composition are: β-nicotinamide adenine dinucleotide:PJ34 = 200 - 400:1.

[0009] Preferably, the drug active ingredients and dosage ratio of the pharmaceutical composition are: β-nicotinamide adenine dinucleotide:PJ34 = 400:1.

[0010] Preferably, the chemical name of the active pharmaceutical ingredient of PJ34 is: 2-(dimethylamino)-N-(6-oxo-5H-phenanthridin-2-yl)acetamide.

[0011] Preferably, it is characterized in that the pharmaceutical composition is used in the preparation of a medicament for treating Parkinson's disease.

[0012] Preferably, the pharmaceutical composition is used in the preparation of a medicament for treating Alzheimer's disease.

[0013] Preferably, the pharmaceutical composition is used in the preparation of a medicament for treating neurodegenerative diseases.

[0014] Preferably, the pharmaceutical composition is used in the preparation of an anti-aging medicament.

[0015] The dosage form of the pharmaceutical composition is: oral liquid, granule, tablet, capsule, dropping pill.

[0016] In the process of the research of the present invention, it is found that the combination of PJ34 and β-NAD produces exciting experimental results. PD and other NDs are closely related to the overactivation of PARP1. As a novel and effective PARP1 inhibitor, PJ34 inhibits the overactivated PARP1, which is beneficial to the protection of short-term PD-like neuronal damage. However, PD and other NDs all require long-term treatment. During the long-term application of PJ34, it will inevitably lead to over-inhibition of PARP1, which has a negative impact on the repair of DNA damage and thus aggravates PD. Supplementing NAD + can activate PARP1 and contribute to the repair of damaged DNA. Therefore, we speculate that the combination of PJ34 and NAD + may have better cytoprotective effects. NAD + has β-NAD + and α-NAD + two conformations, and PJ34 inhibits PARP1 by competing with β-NAD + Therefore, we carried out the inhibitory effect of the combination of PJ34 and β-NAD + on H2O2-induced SH-SY5Y cell damage.

[0017] Beneficial effects of the pharmaceutical composition of the present invention ⑴ By detecting cell viability through the MTT method, the combination of PJ34 and β-NAD + has a better effect on improving cell viability than the separate application of the two (* ** P <0.001). Further, through the Hoechst 33342 / PI staining method, the cell mortality was detected. The experimental results are as Figure 1-B shown. Compared with the separate application of PJ34 and β-NAD +, PJ34 and β-NAD + Combined use showed better inhibitory effect on cell death. In summary, the combined use of PJ34 and β-NAD+ can better inhibit cell damage caused by oxidative stress response induced by H2O2. The combined use of PJ34 and β-NAD+ inhibits H2O2-induced DNA damage in SH-SY5Y cells, which also indicates that the pharmaceutical composition of the present invention can provide experimental basis for the treatment of Alzheimer's disease.

[0018] ⑵. The combined use of the active components PJ34 and β-NAD+ of the pharmaceutical composition of the present invention can better avoid the accumulation of PAR polymers caused by overactivation of PARP1 and prevent cell damage caused by PAR accumulation. The combined use of PJ34 and β-NAD+ can better inhibit the formation of intracellular ROS. Although PJ34 has no antioxidant activity, it inhibits PARP1, reduces mitochondrial damage caused by PAR accumulation, and thus reduces secondary ROS release caused by mitochondrial damage. The above pharmacological and pharmacodynamic results suggest that the pharmaceutical composition of the present invention has a therapeutic effect on neurodegenerative diseases.

[0019] ⑶. The combined use of the active components PJ34 and β-NAD of the pharmaceutical composition of the present invention + increases the mitochondrial membrane potential in 6-OHDA-induced PD (Parkinson's disease)-like cell damage. Their combined use shows a better effect of improving mitochondrial function. After PJ34 or β-NAD + , the mitochondria showed fragmented dots and the length was somewhat restored (** P <0.01, *** P <0.001). After their combined use, there were fewer fragmented dots in the mitochondria, and most were linear, and the length was significantly restored (*** P <0.001). It is suggested that the combined use of PJ34 and β-NAD + can better improve mitochondrial morphology than their separate use. The above pharmacological and pharmacodynamic results suggest that the pharmaceutical composition of the present invention has an obvious therapeutic effect on Parkinson's disease.

[0020] ⑷. In senescent and inflammatory cell models, the combined use of the active components PJ34 and β-NAD of the pharmaceutical composition of the present invention + can better delay aging, which can also indirectly indicate that it can better improve mitochondrial function. The combined use of PJ34 and β-NAD+ can also reduce the activation of NLRP3 inflammasome. It also shows that the pharmaceutical composition of the present invention has an obvious therapeutic effect in delaying aging.

[0021] To more fully understand the implementation of the present invention, the present invention will be further described below through typical examples. The following and through pharmacodynamics prove the clinically beneficial therapeutic effect of the pharmaceutical composition of the present invention. Brief Description of the Drawings

[0022] The accompanying drawings described herein are used to provide a further understanding of the present invention, and constitute a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0023] Figure 1, (A): PJ34 combined with β-NAD+ inhibits the decrease in SH-SY5Y cell viability induced by H2O2, ***P<0.001; (B): PJ34 combined with β-NAD + combined inhibits the death of SH-SY5Y cells induced by H2O2, scale bar: 200 μm (×10). *** P <0.001; Figure 2, (A): PJ34 combined with β-NAD + combined inhibits the formation of γH2A.X foci in SH-SY5Y cells induced by H2O2. Scale bar: 100 μm (×20), * P <0.05, *** P <0.001; (B): PJ34 combined with β-NAD + combined inhibits the expression of γH2A.X and 53BP1 in SH-SY5Y cells induced by H2O2, scale bar: 32 μm (×63); Figure 3 , PJ34 combined with β-NAD + combined inhibits the formation of PAR in SH-SY5Y cells induced by H2O2, scale bar: 50 μm (×40), *** P <0.001; Figure 4 , PJ34 combined with β-NAD + combined inhibits the formation of ROS in SH-SY5Y cells induced by H2O2, scale bar: 200 μm (×10). * P <0.05, *** P <0.001; Figure 5 , PJ34 combined with β-NAD + combined increases the mitochondrial membrane potential of SH-SY5Y cells induced by 6-OHDA. Scale bar: 100μm (×20). * P <0.05, ** P <0.01, *** P <0.001; Figure 6 , PJ34 combined with β-NAD + combined improves the mitochondrial morphology of SH-SY5Y cells induced by 6-OHDA, scale bar: 32 μm (×63). ** P< 0.01, *** P < 0.001; Figure 7, (A): Detection of PJ34 and β-NAD co-treatment inhibiting bleomycin-induced senescence of A549 cells by β-galactosidase staining. Scale bar: 100 μm (×20). ** + Scale bar: 100 μm (×20). ** P < 0.01, *** P < 0.001; (B): Detection of PJ34 and β-NAD co-treatment inhibiting bleomycin-induced senescence of A549 cells by SPiDER-βGal staining. Scale bar: 200 μm (×10). * + Scale bar: 200 μm (×10). * P < 0.05, ** P < 0.01, *** P < 0.001; (C): Detection of PJ34 and β-NAD co-treatment inhibiting 6-OHDA-induced senescence of SH-SY5Y cells by β-galactosidase staining. Scale bar: 100 μm (×20), ** + Scale bar: 100 μm (×20), ** P < 0.01, *** P < 0.001; Figure 8, (A): Inhibition of NLRP3 expression in BV2 cells induced by 6-OHDA by co-treatment of PJ34 and β-NAD. Scale bar: 200 μm (×10), * + Scale bar: 200 μm (×10), * P < 0.05, ** P < 0.01, *** P < 0.001; (B): Inhibition of caspase-1 expression in BV2 cells induced by 6-OHDA by co-treatment of PJ34 and β-NAD. Scale bar: 200 μm (×10), * + Scale bar: 200 μm (×10), * P < 0.05, *** P < 0.001; (C): Inhibition of ASC expression in BV2 cells induced by 6-OHDA by co-treatment of PJ34 and β-NAD. Scale bar: 200 μm (×10). * + Scale bar: 200 μm (×10). * P < 0.05, ** P < 0.01, *** P < 0.001; (D): Inhibition of GSDMD activation on the membrane of BV2 cells induced by 6-OHDA by co-treatment of PJ34 and β-NAD. Scale bar: 32 μm (×63); (E): WB detection of the effect of co-treatment of PJ34 and β-NAD on the activation of NLRP3 inflammasome in BV2 cells induced by 6-OHDA, * + Scale bar: 32 μm (×63); (E): WB detection of the effect of co-treatment of PJ34 and β-NAD on the activation of NLRP3 inflammasome in BV2 cells induced by 6-OHDA, * + Scale bar: 32 μm (×63); (E): WB detection of the effect of co-treatment of PJ34 and β-NAD on the activation of NLRP3 inflammasome in BV2 cells induced by 6-OHDA, * P < 0.05, ** P < 0.01.

[0024] The relevant terms in this study are explained herein so that those skilled in the art can better understand the present invention. 3-AB - 3-aminobenzamide, 6-OHDA - 6-hydroxydopamine; α-syn - α-synuclein; β-NAD+ - β-nicotinamide adenine dinucleotide, AD - Alzheimer's disease, AIF - apoptosis-inducing factor, BBB - blood-brain barrier, CAT - catalytic domain, DA - dopamine, DAT - dopamine transporter, L-Dopa - levodopa, MAO - monoamine oxidase, MAOB - monoamine oxidase B, MIF - macrophage migration inhibitory factor, MPTP - 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, NDs - neurodegenerative diseases, NO - nitric oxide, NOS - nitric oxide synthase, ONO - peroxynitrite, OS - oxidative stress, PAR - poly(ADP-ribose), PARP1 - poly(ADP-ribose) polymerase 1, PBS - phosphate buffer solution, PD - Parkinson's disease, ROS - reactive oxygen species, SNpc - substantia nigra pars compacta, TFEB - transcription factor EB, TH - tyrosine hydroxylase, WB - Western blot, XRCC1 - X-ray repair complementing defective repair in Chinese hamster cells 1. Detailed implementation manners

[0025] To better understand the present invention, several experimental examples are listed below to illustrate its new uses in the pharmaceutical field. The following experiments are intended to illustrate the present invention rather than limit it.

[0026] Example 1 Pharmacological and efficacy experiments of the pharmaceutical composition of the present invention 1 Experimental materials and methods 1.1 Experimental materials 1.1.1 Experimental cell lines SH-SY5Y cells (human bone marrow neuroblastoma cell line) were purchased from the ATCC cell bank. SH-SY5Y cells (human bone marrow neuroblastoma cell line) stably transfected with the pLVX-H2A.X-AcGFP1 plasmid were constructed by ourselves. A549 cells (human non-small cell lung cancer cells) were purchased from the ATCC cell bank. RFP-GFP-LC3 U87 cells stably transfected were gifted by Dr. Zhu Xiaoming (Macau University of Science and Technology, Macau, China). BV2 microglial cells were purchased from the ATCC cell bank.

[0027] 1.1.2 Experimental reagents and equipment The experimental reagents and equipment used in this part of the experiment are shown in Tables 1 and 2.

[0028] Table 1 Experimental reagents and manufacturers

[0029] Table 2 Experimental Equipment and Manufacturers

[0030] 1.1.3 Preparation of Solutions PBS Solution: Add 2 L of pure water to 1 bag of PBS powder, dissolve it, and sterilize it in a high-pressure steam sterilizer.

[0031] Complete DMEM Medium: Prepare according to the ratio of serum: penicillin-streptomycin-gentamicin solution: DMEM basal medium = 10:1:89. Complete 1640 Medium: Prepare according to the ratio of serum: penicillin-streptomycin-gentamicin solution: 1640 basal medium = 10:1:89. Complete MEM Medium: Prepare according to the ratio of serum: penicillin-streptomycin-gentamicin solution: MEM basal medium = 10:1:89. PBST Solution: Add 2 L of pure water to 1 bag of PBS powder, dissolve it, then add 2 mL of Tween 20 and stir evenly.

[0032] Primary Antibody for Immunofluorescence: Prepare using 5% BSA at a ratio of 1:100. Secondary Antibody for Immunofluorescence: Prepare using PBS at a ratio of 1:500. 0.5% TritonX-100: Dilute to 0.5% using PBS according to the ratio. Hoechst 33342 Solution: Prepare a stock solution at 5 mg / mL using sterile water. MTT Solution: Prepare at 5 mg / mL using sterile PBS, then ultrasonically dissolve, filter. PI Solution: Prepare a stock solution at 1 mg / mL using sterile water. 1× Cell Lysis Buffer: Prepare according to the ratio of 10× RIPA: protease inhibitor (100×): sterile water = 10:1:89. 10× Transfer Buffer: Weigh 30.3 g of Tris and 151.1 g of glycine, dilute with pure water to 1 L, and ultrasonically dissolve. 1× Transfer Buffer: Prepare according to the ratio of 10× transfer buffer: methanol: pure water = 1:2:7. 1× Electrophoresis Buffer: Prepare according to the ratio of 10× electrophoresis buffer: pure water = 1:9. Primary and Secondary Antibodies for Western Blotting (WB): Prepare according to the ratio in the corresponding antibody instruction manual using antibody diluent. 6-OHDA Stock Solution: Weigh an appropriate amount of 6-OHDA powder, prepare a 400 mM stock solution using dimethyl sulfoxide according to the relative molecular mass, ultrasonically dissolve it, and store it in a -20 °C refrigerator.

[0033] PJ34 Stock Solution: Weigh an appropriate amount of PJ34 powder, prepare a 20 mM stock solution using dimethyl sulfoxide according to the relative molecular mass, ultrasonically dissolve it, and store it in a -20 °C refrigerator. β-NAD + Stock Solution: Weigh an appropriate amount of β-NAD+ Weigh the powder, dissolve it in sterile water according to the relative molecular mass to prepare a 75 mM stock solution, sonicate it for dissolution, and store it in a -20 °C refrigerator.

[0034] 1.2 Experimental methods 1.2.1 Cell culture, subculture and seeding Aspirate the culture medium in the culture dish, add 2 - 3 mL of sterile PBS solution to wash the cells, then aspirate the PBS, and add fresh complete medium. When the cell growth density reaches 80% or more, perform subculture. The specific operation is to first add sterile PBS solution to wash the cells, then add an appropriate amount of trypsin cell digestive solution to evenly cover the bottom of the dish. When the cells shrink, add 1.5 - 2 mL of complete medium to terminate the digestion. Collect the cell suspension in a centrifuge tube, centrifuge at 700 rpm for 3 min, pour out the mixed solution of the upper layer of medium and trypsin cell digestive solution, add an appropriate amount of complete medium to the centrifuge tube, pipette to resuspend the cell pellet, and transfer it to a new culture dish. For cell seeding, when the cell pellet is resuspended, aspirate 10 μL of the cell suspension, count it on a cell counting plate, and calculate the required volume of the cell suspension and the dilution volume of the complete medium according to the cell density required for seeding.

[0035] 1.2.2 Cell resuscitation and cryopreservation Take out the cells to be resuscitated, immediately put them into a 37 °C water bath, gently shake to accelerate melting. After melting, quickly add the cryopreservation solution to a centrifuge tube containing pre-prepared complete medium, centrifuge at 700 rpm for 3 min. After centrifugation, pour out the upper solution, add 3 - 4 mL of complete medium, pipette to resuspend the cell pellet, and transfer it to a new culture dish.

[0036] For cell cryopreservation, first digest the cells in the culture dish, collect the cell pellet, pour out the upper solution, add an appropriate amount of cell cryopreservation solution to resuspend the cell pellet, transfer it to a cell cryopreservation tube, and put it into a liquid nitrogen tank.

[0037] 1.2.3 Immunofluorescence assay Immunofluorescence assay is a technique that uses fluorescently labeled antibodies (antigens) as probes to localize and quantitatively analyze specific antigens (antibodies) in tissues or cells. The experimental steps are as follows: Seed SH - SY5Y cells in a well plate according to the method in 1.2.1. Mix PJ34 with β - NAD +Pre-protect with separate and combined administration for 8 h, then intervene with the modeling agent for 30 min. After the treatment is completed, add 4% paraformaldehyde to fix at room temperature for 15 min, then wash the slides with PBS. Add 0.5% TritonX-100 to permeabilize at room temperature for 20 min, then add 5% BSA to block at room temperature for 30 min. Then invert the slides onto the antibody and incubate overnight at 4 °C in the refrigerator. The next day, wash the slides with PBST, then add the pre-prepared fluorescent secondary antibody dropwise until the slides are evenly covered, and incubate in the dark at room temperature for 1 h. After washing the slides with PBST, add the pre-prepared Hoechst 33342 solution dropwise and incubate in the dark at room temperature for 15 min. After washing the slides with PBST, add a drop of anti-fluorescence quenching agent, and observe and take pictures under a fluorescence microscope after drying in the dark.

[0038] 1.2.4 MTT method Seed SH-SY5Y cells into 96-well plates according to the method in 1.2.1, and the drug administration method is the same as above. After the drug treatment is completed, add 10 μL of the pre-prepared MTT solution to each well, incubate the plate in a cell culture incubator for 2 - 4 h, then aspirate the culture medium in the plate. Add 100 μL of dimethyl sulfoxide solution to each well, shake the plate until the purple crystals are completely dissolved, and then detect the absorbance at a wavelength of 570 nm using a multifunctional microplate reader. The calculation method of cell viability is: (absorbance of each experimental group - absorbance of the blank control group) / (absorbance of the normal group - absorbance of the blank control group) * 100%.

[0039] 1.2.5 Hoechst 33342 / PI staining Seed SH-SY5Y cells into 96-well plates according to the method in 1.2.1, and the drug administration method is the same as above. After the drug treatment is completed, aspirate the culture medium in the plate, add 100 μL of the pre-prepared Hoechst 33342 / PI staining working solution prepared with complete culture medium, and incubate in the dark in a cell culture incubator for 15 min, then observe and take pictures under a fluorescence microscope. The calculation method of cell mortality is: number of PI-positive cells (red fluorescence) / number of Hoechst 33342-positive cells (blue fluorescence) * 100%.

[0040] 1.2.6 Construction of SH-SY5Y cells stably transfected with pLVX-H2A.X-AcGFP1 plasmid Perform according to the instructions. The specific steps are as follows: Lentivirus packaging: Seed 293T cells in a 10 cm cell culture dish at a density of 3 - 5 million cells and culture for 24 h before cell transfection. The specific steps are as follows. Prepare two EP tubes, labeled A and B respectively. Add 300 μL of Opti-MEM to each tube. Add 40 μL of Transfection Reagent to tube A, mix well, and let it stand at room temperature for 5 min. Add 2.5 μg of the target plasmid to tube B, then add 7.5 μL of the lentivirus packaging plasmid mixture. Transfer the liquid in tube A to tube B and mix well. Let it stand at room temperature for 15 - 30 min, and then slowly add the mixture in tube A dropwise to the cell culture dish and gently shake to mix. After 6 h of transfection, aspirate the medium in the culture dish and replace it with fresh complete medium pre-warmed at 37 °C. After 48 h of transfection, collect the virus supernatant, and then add 10 - 15 mL of fresh complete medium to the culture dish. After 72 h of transfection, collect the virus supernatant again and mix it with the supernatant collected at 48 h to obtain the virus supernatant.

[0041] Concentration of the virus solution: Centrifuge the collected virus supernatant in a low-temperature centrifuge at 4 °C and 4000 g for 10 min, then filter it using a 0.45 μm filter to remove cell debris. Add an appropriate volume of the concentration reagent according to the volume ratio of virus supernatant:concentration reagent = 5:1, and let it stand overnight at 4 °C. The next day, centrifuge the mixture at 4 °C and 4000 g for 30 min to obtain an off-white precipitate. Add an appropriate amount of complete medium to dissolve the white precipitate and store it at -80 °C.

[0042] Infection of target cells with the virus solution: Seed SH-SY5Y cells in a 96-well plate according to the method in 1.2.1. Dilute the virus concentrate with complete medium at an appropriate ratio, and at the same time add 10 μg / mL of polybrene to improve the infection efficiency of the virus on the cells. Add the complete medium containing the virus concentrate and polybrene to the wells. After 24 h, replace it with fresh complete medium and continue culturing. After 48 - 72 h of infection, observe the GFP fluorescence of SH-SY5Y cells under a fluorescence microscope.

[0043] Selection of stably transfected cells: Seed SH-SY5Y cells in a 96-well plate according to the method in 1.2.1, and intervene with puromycin at a concentration gradient. Observe the cell death situation in the wells after 24 h. The lowest concentration that can kill all SH-SY5Y cells should be used as the optimal concentration of puromycin for screening stably transfected cells. When the virus solution infects SH-SY5Y cells until fluorescence appears, then add the pre-determined concentration of puromycin to the wells, and re-add puromycin every 24 h for screening until all the surviving cells in the final wells are cells with fluorescence.

[0044] 1.2.7 Plasmid transfection Taking a 6-well plate as an example, inoculate cells into the 6-well plate according to the method in 1.2.1. After the cells adhere and grow for 24 h, prepare the transfection complex. The specific operation is as follows: Prepare two EP tubes, labeled A and B respectively. Add 2.1 mL of Opti-MEM to each tube. Add 9 μg of the target plasmid to tube A and 18 μL of the transfection reagent Lipo2000 to tube B. Flick gently to mix evenly, and let it stand at room temperature for 10 min. Then transfer the liquid in tube B into tube A, flick gently to mix evenly, and let it stand at room temperature for 15 min. Then add 1.8 mL of complete medium to tube A, flick gently to mix evenly. Aspirate the medium in the well plate, and add 1 mL of the transfection complex in tube A to each well. Place the well plate in the cell culture incubator for culturing.

[0045] 1.2.8 Cell morphological observation Inoculate SH-SY5Y cells into a 96-well plate according to the method in 1.2.1. The drug administration method is the same as above. After the treatment is completed, place the well plate under an inverted microscope for observation and photography.

[0046] 1.2.9 DHE staining Carry out according to the instructions. The specific steps are as follows: Inoculate SH-SY5Y cells into a 96-well plate according to the method in 1.2.1. The drug administration method is the same as above. After the treatment is completed, prepare the DHE working solution in the dark according to the ratio of adding 1 μL of DHE dye and 2.5 μL of Hoechst 33342 to every 1 mL of complete medium. Add 100 μL of the DHE working solution to each well, and incubate in the cell culture incubator in the dark for 15 - 20 min. Then observe and photograph under an inverted fluorescence microscope to detect the formation of ROS. The calculation method of the DHE positive cell rate is: the number of DHE positive cells (red fluorescence) / the number of Hoechst 33342 positive cells * 100%.

[0047] 1.2.10 Western blotting (WB) Preparation of samples: SH-SY5Y cells and BV2 cells were seeded in 6-well plates according to the method in 1.2.1. The drug administration method was the same as above. After the drug treatment was completed, 1× cell lysate prepared in advance was added to each well, and the mixture was placed in a -20 °C refrigerator and lysed overnight. The next day, the cells were collected and centrifuged at 12000 rpm for 15 min at 4 °C. The supernatant was aspirated into an EP tube. 300 μL of protein assay solution was added to each well of a 96-well plate, and 2 μL of different sample protein supernatants was added to each well. After mixing, the absorbance at a wavelength of 595 nm was measured using a multi-functional microplate reader. The protein concentration and loading volume of each sample were calculated according to the calculation formula. Loading buffer (6×) was added to the protein supernatant in the EP tube, vortexed and mixed well, and then heated in a 95 °C metal bath for 10 min. Then the samples were quickly placed in a -20 °C or -80 °C refrigerator for rapid cooling and storage.

[0048] Electrophoresis: A gel with an appropriate concentration was selected according to the molecular weight of the target protein. The upper gel solution and the lower gel solution were prepared respectively according to the preparation method in the gel kit. The comb was gently inserted into the upper gel. After waiting for it to solidify, the glass plate was placed in the electrophoresis tank, and 1× electrophoresis buffer was poured into the electrophoresis tank. The comb was pulled out, 2 μL of marker was added to the lane, and the thawed samples were vortexed and added to the corresponding lanes according to the pre-calculated loading volume. Electrophoresis was started at a voltage of 100 V.

[0049] Transfer: The glass plate was soaked in 1× transfer buffer, and the PVDF membrane was pre-soaked in methanol for activation. A "sandwich structure" was formed with the sponge pad, gel, and PVDF membrane. Transfer was started at a current of 300 mA, and the transfer time was determined according to the molecular weight of the target protein. Blocking: The PVDF membrane was taken out and placed in 5% skim milk, and blocked at room temperature on a shaker for 2 h. Antibody incubation: The bands were washed 3 times with PBST on a shaker, 10 min each time. The bands were placed in the corresponding primary antibody against the target protein and incubated overnight at 4 °C. The next day, the primary antibody was recovered, and the bands were washed 3 times with PBST on a shaker, 10 min each time. Then the bands were incubated with the secondary antibody at room temperature on a shaker for 1 h, and the bands were washed again 3 times, 10 min each time. Development: The ECL luminescent solution A and B were mixed in a 1:1 ratio, and the target bands were immersed in the luminescent solution and exposed and developed in a developer.

[0050] 1.2.11 JC-1 staining Performed according to the instructions. The specific steps are as follows: The SH-SY5Y cells were seeded into 96-well plates according to the method in 1.2.1. The drug administration method was the same as above. After the drug treatment was completed, prepare the JC-1 staining working solution. The specific operation is as follows: Take an appropriate amount of JC-1 (200×), dilute and mix it according to the ratio of adding 8 mL of ultrapure water to every 50 μL of JC-1 (200×), and then add 2 mL of JC-1 staining buffer (5×) and mix well to obtain the JC-1 staining working solution. Add 100 μL of complete medium to each well, and then add 100 μL of JC-1 staining working solution. Incubate in the dark for 20 min, and observe and take pictures under an inverted fluorescence microscope to detect the mitochondrial membrane potential. The calculation method of mitochondrial membrane potential is: JC-1 aggregate fluorescence intensity (red fluorescence) / JC-1 monomer fluorescence intensity (green fluorescence).

[0051] 1.2.12 Mito-tracker staining Perform according to the instructions. The specific steps are as follows: The SH-SY5Y cells were seeded into confocal dishes according to the method in 1.2.1. The drug administration method was the same as above. After the drug treatment was completed, prepare the Mito-tracker staining working solution. The specific operation is as follows: Take an appropriate amount of Mito-tracker, dilute and mix it according to the ratio of adding 1 mL of DMEM complete medium to every 1 μL of Mito-tracker, add it to the confocal dish, incubate in the dark in the incubator for 20 min, and then add DAPI staining solution and incubate in the dark for 20 min. Observe and take pictures under a laser confocal microscope to detect the mitochondrial morphology.

[0052] 1.2.13 β-galactosidase staining Perform according to the instructions. The specific steps are as follows: The A549 cells and SH-SY5Y cells were seeded into 96-well plates according to the method in 1.2.1. The drug administration method was the same as above. After the drug treatment was completed, aspirate the medium in the wells. Add 100 μL of β-galactosidase staining fixative to each well and fix at room temperature for 15 min, then wash with PBS. The steps for preparing the staining working solution are as follows: Take 10 μL of β-galactosidase staining solution A, 10 μL of β-galactosidase staining solution B, 930 μL of β-galactosidase staining solution C, and 50 μL of X-Gal solution. After adding the staining working solution, place the well plate in a 37 °C constant temperature incubator without carbon dioxide and incubate overnight. Observe and take pictures under an inverted microscope to detect the cell senescence situation. The calculation method of the positive rate of senescent cells is: the number of β-galactosidase positive cells (blue) / the total number of cells * 100%.

[0053] 1.2.14 PiDER-βGal staining Perform according to the instructions. The specific steps are as follows: A549 cells were seeded in 96-well plates according to the method described in 1.2.1. The drug administration method was the same as above. After the drug treatment was completed, the Bafilomycin A1 dimethyl sulfoxide stock solution was diluted 1000-fold with complete medium to obtain the Bafilomycin A1 working solution. Then, the SPiDER-βGal dimethyl sulfoxide stock solution and the Bafilomycin A1 dimethyl sulfoxide stock solution were diluted 1000-fold with medium to obtain the SPiDER-βGal working solution. Then, 2.5 μL / mL of Hoechst33342 solution was added to the working solution. 100 μL of the Bafilomycin A1 working solution was added to each well and incubated in a cell culture incubator for 1 h. Then, 100 μL of the SPiDER-βGal working solution was added to each well and incubated in a cell culture incubator for 30 min. The cells were observed and photographed under an inverted fluorescence microscope to detect cell senescence. The calculation method for the positive rate of senescent cells was: the number of SA-β-galactosidase positive cells (green fluorescence) / the number of Hoechst 33342 positive cells (blue fluorescence) * 100%.

[0054] 1.2.15 Lysosomal Acidic pH Detection Kit - Green / Deep Red Staining Performed according to the instructions. The specific steps are as follows: SH-SY5Y cells were seeded in 96-well plates according to the method described in 1.2.1. The drug administration method was the same as above. After the drug treatment was completed, the cells in the wells were washed twice with serum-free medium, and then the pHLys Green / LysoPrime Deep Red working solution was added and incubated in a cell culture incubator for 30 min. The cells were observed and photographed under an inverted fluorescence microscope to detect the lysosomal function of the cells.

[0055] 1.2.16 Statistical Methods and Image Result Analysis GraphPad Prism 8.0 software was used for data statistics, and one-way analysis of variance (One-Way ANOVA) was used for statistical analysis to compare all groups. The test level α = 0.05. Image J software was used for quantitative analysis of the pictures.

[0056] 2.1 PJ34 and β-NAD + Combined Use Inhibits H2O2-Induced SH-SY5Y Cell Injury 2.1.1 PJ34 and β-NAD + Combined Use Inhibits H2O2-Induced SH-SY5Y Cell Injury First, the cell viability was detected by the MTT method. The experimental results are as Figure 1-AAs shown, compared with the model group, PJ34 and β-NAD + showed better effects in improving cell viability when used in combination than when used separately (* P <0.001). Further, the Hoechst 33342 / PI staining method was used to detect the cell mortality. The experimental results are as Figure 1-B shown. Relative to the separate use of PJ34 and β-NAD + , the combination of PJ34 and β-NAD + showed better inhibitory effects on cell death. In summary, these results suggest that the combination of PJ34 and β-NAD + can better inhibit the cell damage caused by oxidative stress induced by H2O2.

[0057] 2.1.2 Inhibition of H2O2-induced DNA damage in SH-SY5Y cells by the combination of PJ34 and β-NAD + Alzheimer's disease is a neurodegenerative disease characterized by the formation of amyloid plaques and neurofibrillary tangles in the brain. It has been found that SH-SY5Y cells may exhibit DNA damage when affected by oxidative stress or inflammatory factors, which is related to the pathogenesis of Alzheimer's disease. To demonstrate the inhibitory effect of the combination of PJ34 and β-NAD

[0058] on DNA damage in oxidative stress cells induced by H2O2, we first observed the effect of the combination of PJ34 and β-NAD + on H2A.X phosphorylation in SH-SY5Y cells stably transfected with the pLVX-H2A.X-AcGFP1 plasmid. The experimental results are as + shown. Relative to the control group, the formation of γH2A.X foci was significantly increased in the H2O2 model group (*** Figure 2-A <0.001), indicating a large accumulation of DNA damage. When PJ34 or β-NAD P was used alone, the formation of γH2A.X foci decreased. When PJ34 and β-NAD + were used in combination, the formation of γH2A.X foci further decreased, indicating that the combination of PJ34 and β-NAD + can better inhibit DNA damage. In addition to observing the formation of γH2A.X foci, 53BP1 foci are also an important indicator for evaluating the kinetic changes of DNA damage, and the formation of γH2A.X and 53BP1 foci is highly consistent during DNA damage. Therefore, we further used immunofluorescence to detect the expression of γH2A.X and 53BP1 to evaluate the inhibitory effect of the combined drug on DNA damage. The experimental results are as + shown. Figure 2-BAs shown, the expressions of γH2A.X and 53BP1 in the H2O2 model group were significantly increased compared with the control group. When PJ34 or β-NAD was applied alone, + the expressions of γH2A.X and 53BP1 were both decreased. When PJ34 and β-NAD were + used in combination, the expressions of γH2A.X and 53BP1 were further decreased, further verifying that the combination of PJ34 and β-NAD + can better inhibit DNA damage, which also indicates that the pharmaceutical composition of the present invention can provide an experimental basis for the treatment of Alzheimer's disease.

[0059] 2.1.3 Inhibition of PAR formation in H2O2-induced SH-SY5Y cells by the combination of PJ34 and β-NAD + As shown in the results, compared with the model group, the formation of PAR was reduced when PJ34 or β-NAD was applied alone, but the effect of β-NAD on the formation of PAR was relatively weak. The combination of PJ34 and β-NAD Figure 3 more significantly inhibited the formation and expression of PAR. Although β-NAD had no inhibitory effect on PARP1 activity, + the antioxidant effect of β-NAD could reduce DNA damage, and then inhibit the activation of PARP1 and reduce the formation of PAR. There are literature reports that + supplementation with β-NAD can activate PARP1 to promote the repair of damaged DNA, which may be the reason why the reduction of PAR formation by β-NAD alone in this study was less than that by PJ34 alone. + The combination of PJ34 and β-NAD more significantly inhibited the formation and expression of PAR. Although β-NAD had no inhibitory effect on PARP1 activity, + the antioxidant effect of β-NAD could reduce DNA damage, and then inhibit the activation of PARP1 and reduce the formation of PAR. There are literature reports that + supplementation with β-NAD can activate PARP1 to promote the repair of damaged DNA, which may be the reason why the reduction of PAR formation by β-NAD alone in this study was less than that by PJ34 alone. + The above results suggest that the combination of PJ34 and β-NAD + can better avoid the accumulation of PAR polymers caused by overactivation of PARP1 and prevent cell damage caused by PAR accumulation. This also indicates that the pharmaceutical composition of the present invention can treat neurodegenerative diseases.

[0060] The above results suggest that the combination of PJ34 and β-NAD + can better avoid the accumulation of PAR polymers caused by overactivation of PARP1 and prevent cell damage caused by PAR accumulation. This also indicates that the pharmaceutical composition of the present invention can treat neurodegenerative diseases.

[0061] 2.1.4 Inhibition of ROS production in H2O2-induced SH-SY5Y cells by the combination of PJ34 and β-NAD + As reported in many literatures, H2O2 can induce SH-SY5Y cells to produce ROS; ROS is one of the key factors causing DNA damage. To evaluate the effect of the combination of PJ34 and β-NAD on intracellular ROS production, we evaluated the formation of ROS by DHE staining, observing the number of DHE-positive cells (stained red) and calculating the DHE-positive cell rate. The results are as + shown, Figure 4As shown, compared with the control group, the DHE positive cell rate in the H2O2 model group was significantly increased (*** P <0.001), indicating that H2O2 induced a large amount of ROS production in SH-SY5Y cells. When PJ34 or β-NAD was applied alone + , the DHE positive cell rate decreased, indicating that both could inhibit the production of ROS in SH-SY5Y cells induced by H2O2. After combining PJ34 and β-NAD + , the DHE positive cell rate decreased more significantly than that of single use (*** P <0.001), suggesting that the combination of the two could better inhibit the formation of intracellular ROS. Although PJ34 has no antioxidant activity, it inhibits PARP1 and reduces the mitochondrial damage caused by PAR accumulation, thereby reducing the secondary ROS release caused by mitochondrial damage. The above pharmacological and efficacy results suggest that the pharmaceutical composition of the present invention has a therapeutic effect on neurodegenerative diseases.

[0062] 2.1.5 PJ34 and β-NAD + Combined use increases the mitochondrial membrane potential in 6-OHDA-induced PD-like cell damage As an important index for evaluating mitochondrial function, the mitochondrial membrane potential reflects the functions of mitochondrial energy conversion, antioxidant, etc. JC-1 is a fluorescent probe for detecting the mitochondrial membrane potential, which exists in two states: monomer and polymer. The reversible transformation between monomer and polymer depends on the concentration change of JC-1. At low concentration of JC-1, the green fluorescence in the form of monomer can be detected; at high concentration, the red fluorescence in the form of polymer can be detected. When the mitochondrial membrane potential is normal, the intracellular JC-1 concentration increases to form polymers with red fluorescence; when the cells are damaged, the JC-1 concentration decreases, forming monomers with green fluorescence. Therefore, the change of mitochondrial membrane potential can be judged by the red / green fluorescence of JC-1 or the ratio of the two.

[0063] The experimental results are as Figure 5 shown. Compared with the control group, the ratio of red / green fluorescence in the 6-OHDA model group was significantly decreased, indicating that the mitochondrial membrane potential decreased significantly at this time and the mitochondrial function was significantly damaged. After applying PJ34 or β-NAD alone + and their combined use, the ratio of red / green fluorescence increased significantly (* P <0.05, ** P <0.01, *** P <0.001), and the increase was more obvious with their combined use (*** P <0.001). The experimental results suggest that PJ34 and β-NAD +The combination shows better effects in improving mitochondrial function compared to their separate applications.

[0064] 2.1.6 PJ34 and β-NAD + The combination improves mitochondrial morphology in 6-OHDA-induced PD-like cell injury Normal mitochondria are filamentous, and when damaged, they become fragmented. Mito-tracker is a mitochondrial fluorescent probe that can be used for specific fluorescence staining of mitochondria in living cells to detect changes in mitochondrial morphology. The results of photographing using a laser confocal microscope show ( Figure 6 ), compared with the control group, mitochondria in the 6-OHDA model group are fragmented and punctate, and the length is significantly reduced (*** P <0.001), indicating mitochondrial morphological damage. After separate application of PJ34 or β-NAD + , some mitochondria show a fragmented and punctate appearance, and the length is somewhat restored (** P <0.01, *** P <0.001). After the combination of the two, there are fewer fragmented and punctate mitochondria, and most are linear, and the length is significantly restored (*** P <0.001). It is suggested that the combination of PJ34 and β-NAD + can better improve mitochondrial morphology compared to their separate applications. The above pharmacological and efficacy results suggest that the pharmaceutical composition of the present invention has an obvious therapeutic effect on Parkinson's disease.

[0065] 2.1.7 PJ34 and β-NAD + The combination inhibits 6-OHDA-induced SH-SY5Y Mitochondrial dysfunction is an important marker of cellular senescence, and senescence is one of the major risk factors for the development of PD. The characteristics of cellular senescence are an increase in cell volume, an increase in the activity of senescence-associated β-galactosidase, and overexpression of p21 protein, etc. The β-galactosidase staining method (SA-β-gal) forms a dark blue product through in situ staining with X-Gal as the substrate under the catalysis of the above enzyme, and then observes the senescence status with the aid of an ordinary optical microscope. To better evaluate the improvement of mitochondrial function by the combination of PJ34 and β-NAD + , we first induced an A549 cell (type II alveolar epithelial cell) senescence model with bleomycin (a drug causing pulmonary fibrosis) to study the inhibitory effect of the combination of PJ34 and β-NAD + on cellular senescence, and indirectly illustrate the improvement of mitochondrial function by their combination. The experimental results are shown in Figure 7A. Compared with the control group, the blue products in the model group are significantly increased, indicating obvious cellular senescence. After separate application of PJ34 or β-NAD +After their separate or combined use, the blue products in the cells were significantly reduced, with the combined use group showing a more obvious effect. This suggests that PJ34 and β-NAD + combined use can better delay the senescence of A549 cells than their separate use.

[0066] SPiDER-βGal is a new reagent for detecting β-galactosidase, which has the characteristics of high cell permeability and long intracellular fluorescence maintenance time, and can better illustrate the cell senescence situation. We found in the experimental results (Figure 7B) that compared with the control group, the green fluorescence of the cells in the model group was significantly increased compared with the control group, indicating obvious cell senescence. After the separate use of PJ34 or β-NAD + or their combined use, the green fluorescence in the cells was significantly reduced (** P <0.01, *** P <0.001), and the combined use had a better effect than their separate use.

[0067] The above experimental results demonstrated the anti-senescence effect of the combined use of PJ34 and β-NAD in the bleomycin-induced A549 cell model. Next, we further verified it in the 6-OHDA-induced SH-SY5Y cell model. The results showed ( + ), that compared with the model group, after the separate use of PJ34 or β-NAD Figure 7-C or their combined use, the blue products in the cells were significantly reduced, with the combined use group showing a more obvious effect. +

[0068] In summary, the above results suggest that the combined use of PJ34 and β-NAD + can better delay senescence, and can also indirectly indicate that it can better improve mitochondrial function. It also shows that the pharmaceutical composition of the present invention has an obvious effect in delaying senescence.

[0069] 2.1.8 Inhibition of NLRP3 inflammasome activation by the combined use of PJ34 and β-NAD + in 6-OHDA-induced BV2 cells Some studies have shown that during the pathogenesis of PD, the overactivation of PARP1 can activate the NLRP3 inflammasome through multiple pathways, leading to neuroinflammation. To prove whether the combined use of PJ34 and β-NAD + can inhibit NLRP3 inflammasome activation, we first transiently transfected pGFP-N1-NLRP3 plasmid, pEGFP-N1-caspase-1 plasmid, pmCherry-C1-ASC and pLVX-AcGFP1-N1-GSDMD plasmid into BV2 cells respectively to study the effect of PJ34 or β-NAD + ​Effects of single use and combined use of both on NLRP3 inflammasome activation. As shown in Figures 8A, 8B, 8C and 8D, the GFP green fluorescence of NLRP3, the GFP green fluorescence of caspase-1 and the mCherry red fluorescence of ASC were significantly enhanced in the 6-OHDA model group, and the GFP green fluorescence of GSDMD aggregated on the cell membrane, suggesting that 6-OHDA could activate the NLRP3 inflammasome in BV2 cells. After single application of PJ34 or β-NAD + , the GFP green fluorescence of NLRP3, the GFP green fluorescence of caspase-1 and the mCherry red fluorescence of ASC were all weakened to a certain extent, and the aggregation of GFP green fluorescence of GSDMD on the cell membrane decreased, suggesting that the inhibitory effects of single use of the two on NLRP3 inflammasome activation were not ideal; After the combination of PJ34 and β-NAD + , the GFP green fluorescence of NLRP3, the GFP green fluorescence of caspase-1 and the mCherry red fluorescence of ASC were significantly weakened compared with the model group (** P <0.01, *** P <0.001), and the aggregation of GFP green fluorescence of GSDMD on the cell membrane was significantly reduced, indicating that the combination of the two could significantly inhibit NLRP3 inflammasome activation in BV2 cells.

[0070] In the WB detection results (Figure 8E), the expressions of NLRP3 protein, cleaved-caspase-1 protein, IL-1β protein and IL-18 protein were significantly increased in the model group, indicating that the NLRP3 inflammasome was activated. Single use of PJ34 or β-NAD + and their combination could all lead to the down-regulation of the above proteins, and the combination was more obvious.

[0071] In summary, the above results suggest that single use of PJ34 or β-NAD + and their combination can inhibit NLRP3 inflammasome activation.

Claims

1. A pharmaceutical composition, characterized in that, The drug active ingredients and dosage ratio of the pharmaceutical composition are as follows: β-nicotinamide adenine dinucleotide: PJ34 = 200-400:

1.

2. The pharmaceutical composition according to claim 1, wherein The drug active ingredients and dosage ratio of the pharmaceutical composition are as follows: β-nicotinamide adenine dinucleotide: PJ34 = 400:

1.

3. The pharmaceutical composition according to claim 1, characterized in that, The chemical name of the drug active component of PJ34 is: 2-(dimethylamino)-N-(6-oxo-5H-phenanthridin-2-yl)acetamide.

4. Use of the pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The application of the pharmaceutical composition in the preparation of drugs for treating Parkinson's disease.

5. Use of the pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The application of the pharmaceutical composition in the preparation of drugs for treating Alzheimer's disease.

6. Use of the pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The application of the pharmaceutical composition in the preparation of drugs for treating neurodegenerative diseases.

7. Use of the pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The application of the pharmaceutical composition in the preparation of anti-aging drugs.

8. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The dosage form of the pharmaceutical composition is: oral liquid, granule, tablet, capsule, dropping pill.

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