Application of SIRT3 and SIRT5 double-gene co-expression in preparation of medicine for improving Alzheimer's disease

Abstract

The invention discloses an application of SIRT3 and SIRT5 double-gene co-expression in preparation of a medicine for improving Alzheimer's disease. The SIRT3 and SIRT5 double-gene co-expression plasmid is used for treating the Alzheimer's disease, and the result shows that the SIRT3 and SIRT5 double-gene co-expression plasmid can effectively improve the ATP and mtDNA content of damaged microglia and neuronal mitochondria, enhance the antioxidant enzyme activity of cells, reduce ROS, improve the mitochondrial network structure, promote metabolism reprogramming of M1 type microglia to be converted into M2 type microglia, and improve the metabolism reprogramming efficiency of the M1 type microglia. The M2 microglial cells can clear Abeta of neurons and transfer mitochondria to the neurons, so that the Alzheimer's disease is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to the application of SIRT3 and SIRT5 dual gene co-expression to promote microglial metabolic reprogramming in improving Alzheimer's disease. Background Technology

[0002] Disruption of mitochondrial network homeostasis is an early marker of Alzheimer's disease (AD) pathology. Mitochondrial dysfunction impairs neuronal function and vitality, leading to early neuronal death and the onset of AD symptoms. Several interrelated factors, including metabolic dysregulation, oxidative stress, calcium homeostasis disruption, and impaired mitochondrial quality control, are associated with observed mitochondrial dysfunction in AD. Disruption of communication between neurons and glial cells is closely related to the development of Alzheimer's disease. Microglia and neurons cooperate to maintain normal brain function. Microglia can transport healthy mitochondria to energy-depleted neurons via tunneling nanotubes and can engulf damaged mitochondria released by neurons, preventing ROS accumulation. Neuronal glutamate release activates microglia's mGluR5, thereby enhancing Aβ phagocytosis, and can release IL-33, activating microglia's ST2 receptor to release IGF-1, promoting synaptic repair.

[0003] In Alzheimer's disease, the balance between glial cells and neurons is disrupted—glial cells become hyperactive, neuronal function weakens, and inflammation levels rise. Microglia polarize towards the M1 type, releasing pro-inflammatory factors. M1 microglia polarization induces inflammation, while M2 microglia promote anti-inflammatory responses. They exert cytotoxic or anti-inflammatory effects through M1 / M2 polarization under different stimuli, and are key factors influencing brain development and the neuronal microenvironment. M1 cells release the inflammatory factor IL-1β, which phosphorylates Tau, leading to neurofibrillary tangles. The released inflammatory factor TNF-α inhibits synaptic AMPAR membrane localization, leading to LTP impairment. M2 microglia can recognize Aβ aggregates through the TREM2 receptor, activate the SYK signaling pathway to promote endocytosis, break down Aβ monomers with lysosomal enzymes, and secrete ApoE to encapsulate loose Aβ fibers, forming dense core plaques and reducing neurotoxicity. The reversal of microglia from M1 to M2 is considered one of the most promising strategies for treating neurodegenerative diseases, and improving mitochondrial network impairment in AD may promote this transformation.

[0004] Sirtuins are NAD+ cofactor-dependent histone deacetylases that have been shown to play crucial roles in many processes, including mitochondrial function, energy metabolism, and aging. Among them, sirtuin3 (SIRT3), sirtuin4 (SIRT4), and sirtuin5 (SIRT5) are all localized to mitochondria after expression and collectively regulate mitochondrial function. SIRT3 activates several mitochondrial proteins, enhances tricarboxylic acid cycle flux and fatty acid β-oxidation, increases ATP production efficiency, accelerates superoxide clearance, and reduces mitochondrial ROS accumulation by more than 50%. Its potent deacetylation activity plays a vital role in maintaining mitochondrial redox homeostasis, regulating mitochondrial metabolism, controlling stress responses, and maintaining mitochondrial genome stability. SIRT4 promotes autophagy, upregulates the expression of mitochondrial autophagy proteins LC3, LC1, and PINK-1, blocks the conversion of glutamate to α-ketoglutarate (α-KG), inhibits the TCA cycle, and reduces the NADH / NAD+ ratio, thus limiting electron transport chain activity. SIRT5 promotes Arg1 expression and inhibits inflammasome assembly through desuccinylation. Desuccinylation activates SDHA to enhance electron transport chain efficiency, inhibits IDH2 activity to reduce α-ketoglutarate (α-KG) production, regulates epigenetic modifications, and affects the clearance efficiency of damaged mitochondria by modulating the PINK1 / Parkin pathway. Currently, there are no reports of using co-expression of SIRT3 and SIRT5 genes to target microglial mitochondria for the treatment of Alzheimer's disease (AD). Summary of the Invention

[0005] The purpose of this invention is to overcome the defects and shortcomings of the prior art and to provide the application of SIRT3 and SIRT5 dual gene co-expression in the preparation of drugs to improve Alzheimer's disease.

[0006] This invention investigates the effects of SIRT3, SIRT4, and SIRT5 genes, as well as the co-expression of SIRT3 / SIRT4, SIRT3 / SIRT5, and SIRT4 / SIRT5 genes, on mitochondrial function in microglia. The results showed that the SIRT3 and SIRT5 dual-gene plasmid had the most significant effect. It remodeled the mitochondrial network dysfunction in M1 microglia, metabolically reprogramming them into M2-type microglia. M2 microglia can clear neuronal Aβ and transfer mitochondria to neurons, thereby improving Alzheimer's disease.

[0007] Therefore, this application seeks protection for the use of SIRT3 and SIRT5 dual gene co-expression biomaterials in the preparation of drugs to improve Alzheimer's disease; the SIRT3 and SIRT5 dual gene co-expression biomaterials include: a) SIRT3 and SIRT5 dual gene co-expression plasmids; b) cell lines containing SIRT3 and SIRT5 dual gene co-expression plasmids; c) liposomes encapsulating SIRT3 and SIRT5 dual gene co-expression plasmids; d) brain-targeting delivery liposomes encapsulating SIRT3 and SIRT5 dual gene co-expression plasmids.

[0008] Preferably, the SIRT3 and SIRT5 co-expression plasmid is pIRES2-SIRT3-SIRT5, where the template is the parent plasmid pIRES2 carrying the internal ribosome binding site IRES. The construction method involves cloning the SIRT3 gene fragment into the pIRES2 plasmid to construct the pIRES-SIRT3 plasmid; then, the SIRT5 gene fragment is cloned into the pIRES-SIRT3 plasmid to construct the pIRES-SIRT3-SIRT5 plasmid.

[0009] Preferably, the cell line containing the SIRT3 and SIRT5 dual gene co-expression plasmid is a microglia containing the SIRT3 and SIRT5 dual gene co-expression plasmid; the microglia are M1 type microglia.

[0010] Preferably, the liposomes encapsulating the SIRT3 and SIRT5 dual gene co-expression plasmid are cationic liposomes encapsulating the SIRT3 and SIRT5 dual gene co-expression plasmids.

[0011] Preferably, the brain-targeting delivery liposome encapsulating the SIRT3 and SIRT5 dual-gene co-expression plasmid is a cationic liposome conjugated with transferrin Tf and encapsulating the SIRT3 and SIRT5 dual-gene co-expression plasmid. Transferrin Tf can target transferrin receptors on brain capillary endothelial cells (blood-brain barrier BBB) and neurons / glial cells, enabling the drug to cross the blood-brain barrier and better achieve the treatment of AD.

[0012] Preferably, the cationic liposomes encapsulating the SIRT3 and SIRT5 dual-gene co-expression plasmid are prepared by preparing a uniform film of cationic lipids, neutral helper lipids, and phospholipids, encapsulating the SIRT3 and SIRT5 dual-gene co-expression plasmid, and eluting. The cationic liposomes coupled with transferrin Tf are obtained by further coupling thiolized transferrin to the above-mentioned cationic liposomes.

[0013] Preferably, the nucleotide sequence of the SIRT3 gene is shown in SEQ ID No. 1, and the nucleotide sequence of the SIRT5 gene is shown in SEQ ID No. 3.

[0014] Specifically, the drug internalizes the SIRT3 and SIRT5 dual-gene co-expression plasmid into the subject's M1 microglia, regulates the activity of mitochondrial antioxidant enzymes in the subject's microglia to clear ROS, increases ATP and mtDNA levels, enhances the generation of microglia mitochondrial networks and restores mitochondrial function, and metabolically reprograms them into M2 microglia. M2 microglia improve the neuronal mitochondrial network by clearing neuronal Aβ and delivering mitochondria to damaged neurons, thereby achieving the treatment of Alzheimer's disease.

[0015] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of SIRT3 and SIRT5 dual gene co-expression in improving Alzheimer's disease. The invention uses a SIRT3 and SIRT5 dual gene co-expression plasmid in the treatment of Alzheimer's disease. Results show that the SIRT3 and SIRT5 dual gene co-expression plasmid can effectively increase the ATP and mtDNA content of damaged microglia and neuronal mitochondria, enhance the activity of antioxidant enzymes in microglia and reduce ROS, improve the mitochondrial network structure of microglia, promote the metabolic reprogramming of M1-type microglia to M2-type, and M2 microglia can clear Aβ from neurons and transfer mitochondria to neurons, thereby improving Alzheimer's disease. Attached Figure Description

[0017] Figure 1 The results of PCR amplification after transfection of SIRT3, SIRT4, and SIRT5 plasmids in Example 1 are shown (M: DNA Marker, lane 1: SIRT3, lane 2: SIRT4, lane 3: SIRT5).

[0018] Figure 2 The image shows the sequencing results of pSIRT3, pSIRT4, and pSIRT5 plasmids in Example 1.

[0019] Figure 3 The expression levels of mitochondrial regeneration protein DRP1, fusion protein OPA1, and autophagy protein PINK1 in Aβ-treated HMC3 cells transfected with SIRT3, SIRT4, and SIRT5 plasmids in Example 1 were detected by ELISA.

[0020] Figure 4 The results show the ATP content in HMC3 cells treated with Aβ and transfected with single-gene and double-gene plasmids, respectively, as described in Example 1.

[0021] Figure 5The results show the ROS content in HMC3 cells treated with Aβ and transfected with single-gene and double-gene plasmids, respectively, in Example 1.

[0022] Figure 6 The relative mRNA expression levels of IDH and α-KGDH in HMC3 cells treated with Aβ and transfected with single-gene and double-gene plasmids in Example 1 are shown (n=3, ***p<0.001).

[0023] Figure 7 The results show the survival rates of HMC3 and SH-SY5Y at Aβ concentration gradients in Example 2.

[0024] Figure 8 Western blot analysis of HMC3 and SH-SY5Y target proteins after Aβ induction by pSIRT3 / 5 transfection in Example 2 (n=3, *p<0.05, **p<0.01, ***P<0.001 vs. WT; ns: no significant vs. Aβ #p<0.05, ##p<0.01, ###P<0.001 vs. Aβ).

[0025] Figure 9 The Aβ level is the result of transfection of pSIRT3 / 5 plasmid with HMC3 and SH-SY5Y in Example 2.

[0026] Figure 10 The ATP level of HMC3 transfected with pSIRT3 / 5 was detected in Example 2.

[0027] Figure 11 This study describes the detection of ROS levels in HMC3 transfected with pSIRT3 / 5 in Example 2.

[0028] Figure 12 The red-green ratio fluorescence intensity of the JC-1 fluorescent probe for HMC3 transfected with pSIRT3 / 5 in Example 2 was used to quantify the fluorescence intensity.

[0029] Figure 13 The relative content of mitochondrial DNA in each group of HMC3 cells transfected with pSIRT3 / 5 in Example 2.

[0030] Figure 14 In Example 2, Western blot was used to detect the expression of marker proteins related to M1 and M2 types in HMC3 microglia transfected with pSIRT3 / 5.

[0031] Figure 15 Western blot analysis was performed on HMC3 and SH-SY5Y cells transfected with pSIRT3 / 5 in Example 2 to detect mitochondrial biogenesis and autophagy protein expression.

[0032] Figure 16 To detect the expression levels of neuronal functional proteins in the co-culture of M2 microglia and Aβ neurons in Example 2 (n=3, *p<0.05, **p<0.01, ***P<0.001 vs. WT; ns: no significant vs. Aβ #p<0.05, ##p<0.01, ###P<0.001 vs. Aβ).

[0033] Figure 17 This is a quantitative fluorescence image of mitochondrial transfer in the co-culture of M2 microglia and Aβ neurons in Example 2 (Mito-Tracker Red fluorescent probe, Cell Mask Deep Red Actin Tracker fluorescent probe).

[0034] Figure 18 The image shows the nanoparticle size curve of Tf-pSIRT3 / 5 liposomes in Example 3.

[0035] Figure 19 The latency time for mice to find the platform in the Morris water maze experiment in Example 3 (n=3, *p<0.05, **p<0.01, ***P<0.001 vs. WT; ns: no significant vs. Aβ #p<0.05, ##p<0.01, ###P<0.001 vs. Aβ).

[0036] Figure 20 The number of times the Morris water maze mouse traversed the original platform within 60 seconds in Example 3.

[0037] Figure 21 The average Recognition Index of the NOR experimental mice in Example 3.

[0038] Figure 22 This shows the expression of M1 and M2 type-related marker proteins in microglia of the mouse cerebral cortex in Example 3.

[0039] Figure 23 The expression of SIRT3 and SIRT5 in the cerebral cortex and hippocampus of mice in Example 3.

[0040] Figure 24 Fluorescence microscopy imaging and fluorescence intensity quantification of amyloid plaque β in the mouse cerebral cortex in Example 3.

[0041] Figure 25 The mitochondrial DNA levels in the mouse cerebral cortex and hippocampus tissue in Example 3. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0043] Unless otherwise specified, all reagents and materials used in the following examples are commercially available. I. Experimental Materials and Methods

[0044] 1. Strains, plasmids and cells: pIRES2 was purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd., Escherichia coli DH5α was purchased from Sangon Biotech, and HMC3 was cryopreserved from the Institute of Genomic Drugs, Jinan University.

[0045] 2. Experimental reagents: as shown in Table 1.

[0046] Table 1 Main Reagents

[0047] 3. DNA extraction Following the instructions of the mammalian genomic DNA extraction kit: After complete trypsin digestion of PBMCs with a growth confluence of 80-90%, centrifuge, add 1 mL of culture medium, and resuspend by pipetting. Centrifuge at 12000 rpm for 1 min and discard the supernatant. Add 250 μL of buffer A to the precipitate and mix by pipetting. Then add 10 μL of proteinase K and mix thoroughly. Add 250 μL of buffer B and incubate the mixture in a 60 ℃ oven for 20 min until the solution becomes clear. Add 250 μL of anhydrous ethanol to the solution again and gently shake for 15 s. A small amount of suspended flocculent precipitate will be observed. Transfer this precipitate to an adsorption column and place the column in a collection tube. Centrifuge rapidly at 12000 rpm for 30 s and discard the waste liquid in the collection tube. Add 500 μL of buffer C, centrifuge, and then add 700 μL of wash buffer W2 (wash buffer W2 should be diluted with anhydrous ethanol beforehand). Centrifuge and repeat this step once more, discarding the waste liquid and centrifuging again. Place the adsorption column on a clean surface and let it stand at room temperature for 2-5 minutes to thoroughly dry any residual wash buffer. Finally, transfer the adsorption to a clean 1.5 mL RNase-free EP tube, add 150 μL of elution buffer TE, and allow the column to stand at room temperature for several minutes to allow the elution buffer to fully wet the precipitate. Centrifuge (12000 rpm, 2 min). The lower layer is the whole-genome DNA extract from PBMCs cells. After concentration detection using a micro-ultraviolet spectrophotometer, store at -20 °C for later use.

[0048] 4. PCR amplification The cDNA coding sequences of the SIRT3, SIRT4, and SIRT5 genes (NM_001370310.1, NM_001385733.1, NM_001193267.2) were retrieved from the NCBI database. Primers were designed based on Primer Blast for amplification. The primers were synthesized by Sangon Biotech, and the primer sequences involved are shown in Table 2. Simultaneously, BstXⅠ and NotⅠ restriction sites were introduced at both ends of the SIRT4 gene, and for SIRT3… and NheⅠ and BamHI restriction sites were introduced at both ends of the SIRT5 gene. PCR was performed according to the system and reaction conditions in Tables 3 and 4.

[0049] Table 2 PCR amplification primers

[0050] Table 3 Reaction System

[0051] Table 4 PCR reaction conditions

[0052] 5. Agarose gel electrophoresis Electrophoresis: Slowly pour the prepared 1% agarose gel solution into the gel casting plate, avoiding air bubbles. Once the solution is level, insert the casting comb and let it stand at room temperature for approximately 30 minutes until the gel solidifies, then remove the comb. Meanwhile, prepare 1×TAE electrophoresis buffer and pour it into the electrophoresis tank. Place the gel plate with the wells facing the negative electrode of the electrophoresis tank. Mix the SIRT3, SIRT4, and SIRT5 gene amplification samples and markers with 10×Loading Buffer (5 μL each) and load the samples. Perform electrophoresis at 160 V for 120 minutes and take photographs.

[0053] Fragment recovery: The target gene was cut and transferred to a centrifuge tube under UV light. After weighing, an equal volume of Binding Buffer was added, and the mixture was incubated in a 56°C water bath for 10 min to accelerate the melting into a DNA-agarose solution. The solution was transferred to an adsorption column, allowed to stand for 1 min, and then placed in a collection tube. The tube was centrifuged at 12000 rpm for 1 min, and the lower layer was discarded. This step was repeated. Wash buffer diluted with anhydrous ethanol was added, and the mixture was centrifuged again and the subsalicylate was discarded. This step was repeated once to remove any residual ethanol solution. The adsorption column was then placed in a centrifuge tube, 50 μL of EB elution buffer was added, and the tube was centrifuged at 12000 rpm for 1 min to collect the DNA. The DNA solution was analyzed using the Nucleic acid function of an ultra-micro UV-Vis spectrophotometer. When the OD value... 260 / OD 280A concentration between 1.8 and 2.0 indicates suitable DNA purity, yielding DNA concentration data in ng / μL. Store at -20 ℃ for later use.

[0054] 6. Construct pSIRT3, pSIRT4 and pSIRT5 plasmids The empty pIRES2 vector was digested with either BstXⅠ / NotⅠ or NheⅠ / BamHI, as shown in Table 5. QuickCut BstXⅠ and QuickCut NotⅠ were used to construct the pSIRT4 plasmid, while QuickCut NheⅠ and QuickCutBamHI were used to construct the pSIRT3 and pSIRT5 plasmids. Agarose gel electrophoresis and recovery were then performed.

[0055] Table 5 Enzyme digestion reaction system

[0056] The reaction system was gently mixed and placed in a 37 ℃ water bath for 1 h, then moved to a 70 ℃ water bath for 10 min.

[0057] According to Trelief TM The SoSoo Cloning Kit instructions describe how to ligate the three single-gene fragments recovered from PCR to plasmid pIRES2 to construct plasmids pSIRT3, pSIRT4, and pSIRT5. The ligation reactions are shown in Table 6, and the mixture was incubated at 50°C for 30 min.

[0058] Table 6 Connection Reaction System

[0059] 7. Plasmid extraction and identification E. coli DH5α Preparation of competent states: The frozen state was removed from liquid nitrogen. E. coli After resuscitation, the bacteria were inoculated onto LB agar plates and allowed to grow stably for 24 hours. Single colonies with good morphology were picked and inoculated into 20 mL of LB liquid medium, and incubated in a constant temperature shaking incubator for 3 hours. When the bacterial OD... 600 When the concentration of β-carboxylic acid (β-C) is 0.4–0.6, transfer the sample to a pre-chilled centrifuge tube and centrifuge at 4000 rpm for 10 min at 4 °C, discarding the supernatant. Add 5 mL of pre-chilled 0.1 mol / L CaCl2 hypotonic solution to the precipitate, resuspend the strain for 30 min, centrifuge, and discard the supernatant. Add 1 mL of ice-cold 0.1 mol / L CaCl2-containing glycerol to the precipitate to resuspend the strain, incubate on ice for 24 h, and obtain *Escherichia coli*. DH5α Competent cells were stored at -80 ℃ for later use.

[0060] Plasmid transformation: Add 5 μL of DNA plasmid to 200 μL of... DH5α The competent bacterial suspension was thoroughly agitated and then incubated on ice for 30 min. It was then transferred to a 42 ℃ water bath for 90 s and cooled again on ice for 3 min. 1 mL of sterile, antibiotic-free LB medium was mixed with the plasmid-containing bacterial suspension and incubated at 250 rpm and 37 ℃ with shaking for 1 h. 100 μL of colonies was then centrifuged at 3000 rpm for 2 min. The precipitated bacterial cells were inoculated onto an LB plate containing Kan (50 μg / mL, 1:100), incubated upright for 1 h until the plate had completely absorbed the bacterial suspension, and then incubated upside down at 37 ℃ for 24 h. Finally, a single colony was picked with an inoculation loop and inoculated into LB liquid medium containing Kan, and incubated at 37 ℃ with shaking for 12 h.

[0061] Colony PCR detection: The strain to be cultured to OD 600 When the temperature reaches approximately 2°C, collect 1 mL of bacterial culture and incubate on ice for 10 min. Centrifuge at 12000 rpm for 2 min and collect the precipitate. Add 100 μL of 1×TAE buffer to the precipitate and resuspend for 5 min. Immediately incubate at -20 °C for 15 min. Remove the precipitate, allow it to thaw, centrifuge at 3000 rpm for 10 min, and perform PCR detection.

[0062] Plasmid extraction and sequencing: Follow the instructions of the plasmid extraction kit: After thawing the strain, centrifuge at 5000 rpm for 1 min, collect the precipitate, and repeat this step once. Collect 100 mL of the bacterial precipitate, add 5 mL of suspension to the precipitate to resuspend the bacteria, and gently pipette to disperse the precipitate. Add 5 mL of lysis buffer (containing RNase A) to a centrifuge tube, invert the tube 5 times, and incubate at room temperature for 2 min to allow complete lysis of the strain. Then add 7 mL of binding buffer, mix well, and let stand to obtain a white flocculent substance. Centrifuge at 12000 rpm at room temperature for 10 min, aspirate the supernatant to the adsorption column, centrifuge for 2 min, and discard the waste liquid. Add 12 mL of diluted washing buffer, centrifuge for 2 min, and discard the waste liquid. Repeat this step once. The adsorption column was allowed to stand at room temperature for 2-5 minutes to allow the ethanol to evaporate. Then, the column was transferred to a 50 mL centrifuge tube, 2 mL of elution buffer was added, and the column was allowed to stand for 2 minutes. The tube was then centrifuged at 12000 rpm for 2 minutes, and the collected supernatant was the DNA plasmid. The three collected DNA plasmids were sent to Sangon Biotech for sequencing. The cDNA coding sequences and pIRES2 sequences of the three genes were retrieved from NCBI. SnapGene version 6.0.2 software was used to design plasmid maps, resulting in the constructed pSIRT3, pSIRT4, and pSIRT5 plasmid maps.

[0063] 8. Construction of pIRES-SIRT3 / 4, pIRES-SIRT4 / 5 and pIRES-SIRT3 / 5 plasmids The pIRES-SIRT3 plasmid was double-digested with BstXⅠ and NotⅠ, and then the SIRT5 fragment was cloned into the restriction sites of the pIRES-SIRT3 plasmid to construct the pIRES-SIRT3 / 5 plasmid. The dual-gene plasmid was then transformed into E. coli. DH5α Competent cells were extracted, and the collected pIRES-SIRT3 / 5 plasmids were sent to Shanghai Sangon Biotech Co., Ltd. for double enzyme digestion identification and sequencing. The pIRES-SIRT3 / 4 and pIRES-SIRT4 / 5 plasmids were constructed using the same steps as above.

[0064] 9. Western Blot Detection Extraction of total cellular protein: After washing each group of cells with PBS, the cell dishes were placed directly on an ice box, and RIPA lysis buffer (RIPA:PMSF=1 mL:10 μL, 100 mM) was added for lysis for 30 min. After the cells were completely lysed, the cells at the bottom of the cell dish were carefully scraped off with a cell scraper and transferred to an EP tube. The cells were centrifuged at 4 ℃ and 12000 rpm for 20 min. The supernatant was carefully aspirated and transferred to a clean EP tube and stored at -20 ℃ for later use.

[0065] BCA protein quantification: Follow the instructions for the BCA protein concentration assay kit. The specific steps are as follows: Take an appropriate amount of BCA protein standard, dilute it to 0.2 mg / mL with PBS, and mix well. Take a 96-well plate, add 0.5 mg / mL of protein standard 0, 1, 2, 4, 8, 12, 16, and 20, and then make up to 20 μL with PBS, with three replicates per group. Then, add 200 μL of BCA protein quantitative working solution (Solution A:Solution B=50:1) to each well. Cap the plate, seal it with sealing film, and shake it in a constant temperature air bath shaker at 37 ℃ for 30 min to promote the chemical reaction of the solution. After the plate cools to room temperature, immediately place it on a microplate reader to measure the absorbance at 562 nm and plot the standard curve (R). 2 >0.99). Proteins extracted from each group of cells were diluted 10-fold with 2 μL of PBS and then measured using a microplate reader to determine OD. 560nm The absorbance value is used to calculate the protein concentration.

[0066] 10. SDS-PAGE gel electrophoresis Protein denaturation: Add the collected total cellular protein to 1 / 5 volume of Loading Buffer, incubate at 100 ℃ for 5 min to denature the protein, and then cool to room temperature; Preparation of SDS-PAGE gel (top layer: 5% stacking gel, bottom layer: 15% separating gel): Pour the prepared separating gel into the glass clamp, aligning it with one side of the glass plate, avoiding the formation of air bubbles. Then, pour anhydrous ethanol to the top to seal it. Let it stand for about 30 minutes until a relatively clear separation layer appears in the middle, indicating that the separating gel has solidified. Pour off the top layer of anhydrous ethanol, then add more stacking gel to the top, insert the gel casting comb, and let it stand to solidify. Protein electrophoresis: Remove the gel casting comb from the SDS-PAGE gel glass clip and fix it in the electrophoresis tank. Fill the electrophoresis tank with the prepared protein electrophoresis buffer, add 20 μg of denatured protein for loading, and add pre-stained rainbow markers to both sides of the gel. Turn on the power to start electrophoresis. First, stabilize the electrophoresis at 80 V. When the markers enter the stacking gel, increase the voltage to 120 V and continue electrophoresis for about 2 hours until the markers reach the bottom of the gel, indicating that the proteins have been completely separated. The electrophoresis is then complete. Remove the glass plate and take out the gel. Transfer: Transfer the gel to a PVDF membrane of similar size and immerse it in the transfer solution for 2 min. Assemble the transfer clamp in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge". Remove air bubbles in time during the operation. Transfer at 100 V for 2 h. Antibody incubation: After transfer, wash the membrane three times with 1×TBST buffer for 5 min each time, add 5% skim milk and block in a shaker for 2 h, wash the membrane again as above, add the corresponding protein primary antibody dilution (1:1000 or 1:1500), and incubate overnight at 4 ℃; after primary antibody incubation, wash the membrane three times for 10 min each time, add secondary antibody dilution (1:5000), and incubate at room temperature for 2 h; finally, remove the membrane after incubation and wash it three times for 10 min each time. ECL color development: The prepared ECL luminescent liquid is evenly coated on the PVDF film, placed in a multi-functional imager for imaging and exposure, and grayscale is measured using Image J v1.54 software.

[0067] 11. Determination of ATP content The specific procedures according to the ATP assay kit are as follows: Lyse cells and centrifuge to collect the supernatant. Dilute the ATP standard solution with ATP assay lysis buffer to concentration gradients of 0.01, 0.04, 0.1, 0.4, 1, 4, and 12 μM, respectively; dilute 200 μL of ATP assay reagent with 1.8 mL of diluent. Add 100 μL of ATP assay working solution to an opaque 96-well plate and incubate at room temperature for 3 min to completely consume background ATP. Add 20 μL of the test solution or standard solution to each well, mix quickly, and then use the luminometer function of a full-wavelength multi-mode microplate reader to detect the chemiluminescence value. Plot a standard curve and calculate the ATP content of each group of cells based on the chemiluminescence value.

[0068] 12. Determination of ROS content ROS content was measured using the DCFH-DA probe method, and the fluorescence intensity of 10,000 cells was detected by flow cytometry (excitation 488 nm, emission 525 nm).

[0069] 13. RT-qPCR detection Total RNA extraction from cells: Discard the upper culture medium layer of cells and wash twice with PBS. Add 1 ml of TRIzol evenly to a 6 mm cell culture dish, and repeatedly aspirate and pipette to mix thoroughly. Transfer the mixture to an RNase-free EP tube, incubate at room temperature for 5 min, add 200 μL of chloroform, vortex vigorously for 15 s, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 15 min at 4 ℃, collect the supernatant, add 500 μL of isopropanol, invert 5 times to mix, incubate at room temperature for 10 min, and centrifuge again for 10 min under the same conditions to precipitate RNA. Discard the supernatant, add 1 mL of anhydrous ethanol, vortex, centrifuge for 5 min, discard the supernatant again, and dry the precipitate in an oven. Finally, dissolve the precipitate with an appropriate amount of DEPC water and check the RNA purity.

[0070] cDNA synthesis: cDNA synthesis was performed according to the kit instructions, as detailed in Table 7 below. Table 7 Preparation of reaction solution

[0071] The above solution was treated at 65 °C for 5 min and then immediately transferred to ice for cooling. Reverse transcription was then performed, and the reaction solution was prepared as shown in Table 8. Table 8 Reverse Transcription Reaction Solution

[0072] After slow mixing, the reverse transcription reaction needs to be carried out immediately. That is, place the mixed sample in a PCR instrument, set the temperature to 42 ℃ and incubate for 1 h, and finally heat at 70 ℃ for 15 min to terminate the reaction.

[0073] Establishment and execution of the PCR reaction system: The reverse-transcribed cDNA was diluted 10-fold with sterile water, and a 20 μL PCR reaction system was constructed in eight-tube sets. The components are shown in Table 9 below, and the PCR reaction conditions are shown in Table 10 below. Table 9 PCR Reaction System

[0074] Table 10 PCR Reaction Conditions

[0075] The primers used in the PCR reaction system were designed based on NCBI's Primer Blast and synthesized by Sangon Biotech. The primer sequences are shown in Table 11 below. Table 11 PCR Amplification Primers

[0076] 14. Statistical Analysis All data are presented as arithmetic mean ± standard deviation (SD). Data analysis was performed using GraphPad Prism version 9.3. One-way ANOVA was used to compare multiple groups of samples, and P < 0.05 indicates that the difference is statistically significant.

[0077] II. Experimental Results Amplification of human-derived SIRT3, SIRT4, and SIRT5 genes ( Figure 1 ) and perform sequencing ( Figure 2 The results were consistent with expectations. The pIRES gene vector, containing an internal ribosome entry site, was loaded into plasmids pSIRT3, pSIRT4, and pSIRT5 carrying the target gene. Detection revealed that SIRT3 and SIRT5 both act on different protein factors involved in mitochondrial fusion and regeneration, and that mitochondrial network remodeling depends on the combined effects of mitochondrial fusion and regeneration. pSIRT4 significantly increased the level of mitophagy proteins, exacerbating mitochondrial network disruption. Figure 3 In addition, pIRES-SIRT3 / 4, pIRES-SIRT4 / 5 and pIRES-SIRT3 / 5 dual-gene plasmids (abbreviated as pSIRT3 / 4, pSIRT4 / 5 and pSIRT3 / 5) were constructed.

[0078] Three single-gene plasmids (pSIRT3, pSIRT4, and pSIRT5) and three dual-gene plasmids (pSIRT3 / 4, pSIRT4 / 5, and pSIRT3 / 5) were transfected into Aβ-damaged HMC3 cells. The transfected pSIRT3 / 5 plasmid significantly increased ATP levels compared to the other groups. However, the SIRT4 protein did not significantly increase ATP levels. Figure 4 All groups transfected with plasmids showed a significant reduction in ROS levels, while pSIRT3 / 5 showed a significantly higher level compared to other groups. Figure 5 RT-PCR was used to detect key enzymes in mitochondrial oxidative phosphorylation. It was found that SIRT3 and SIRT5 significantly improved the expression levels of isocitrate dehydrogenase (IDH) and α-ketoglutarate dehydrogenase (α-KGDH) compared to SIRT4, and the pSIRT3 / 5 ratio was significantly higher than that of pSIRT3 / 4 and pSIRT4 / 5. Figure 6 ).

[0079] Example 2: pSIRT3 / 5 plasmid improves Aβ-induced mitochondrial dysfunction in HMC3, causing it to transform into the M2 type and improves damaged neurons. I. Experimental Materials and Methods

[0080] 1. Cells: HMC3 and SH-SY5Y were cryopreserved from the Institute of Genomic Drugs, Jinan University.

[0081] 2. Experimental reagents: DMEM cell culture medium was purchased from Gibco; trypsin was purchased from Sigma; ELISA kits for JC-1, TUBBY3, SYP and MAP2 were purchased from RayBiotech.

[0082] 3. Construction of Alzheimer's disease (AD) microglia and neuron models Aβ-induced Alzheimer's disease models of SH-SY5Y and HMC3 cells were established. Synthesized Aβ peptides were dissolved in DMSO to prepare high-concentration stock solutions. Neurons and microglia were cultured to the logarithmic growth phase, then replaced with culture media containing 0, 5, 10, 20, and 30 μM Aβ peptides, incubated for 24 hours, and Aβ expression in neurons and microglia at different concentrations was detected. To promote Aβ aggregation, the Aβ peptides were incubated at 37°C for a period of time (1-7 days) to form oligomers or fibrous structures before cell treatment, thereby enhancing the toxic effect.

[0083] 4. mtDNA level detection Total DNA was extracted from cells. Primers were designed based on Primer Blast and synthesized by biotechnology. Among them, NADH dehydrogenase... ND1 Quantitative analysis of subunit genes in mitochondrial DNA (mtDNA). β-actinQuantitative analysis of nuclear DNA (nDNA) was performed. SYBR Green Master Mix was added to each group, and qPCR was conducted according to the primer configuration system. β-actin As an internal reference gene, the corresponding primer sequences and PCR reaction conditions are shown in Tables 12 and 13, and three independent biological replicates were performed. According to 2 -△△Ct Calculate the relative gene expression levels.

[0084] Table 12 PCR Amplification Primers

[0085] Table 13 PCR Reaction Conditions

[0086] 5. Measurement of mitochondrial membrane potential (JC-1 method) The mitochondrial membrane potential of each group was measured according to the instructions of the JC-1 detection kit. The specific steps are as follows: First, take an appropriate amount of JC-1 (200×) and dilute it with ultrapure water at a ratio of 1:160. Shake vigorously to fully dissolve the JC-1 solution in water, then add 2 mL of JC-1 staining buffer (5×) and mix well to obtain the JC-1 staining working solution for later use. Discard the old culture medium in the culture dish, replace it with the JC-1 staining working solution, and incubate in an incubator for 20 min. After incubation, discard the supernatant, wash twice with pre-cooled JC-1 staining buffer (1×) (JC-1 staining buffer (5×): H2O = 1:4), then add fresh culture medium, place under an inverted fluorescence microscope, set excitation light at 490 nm and 525 nm respectively, observe and take pictures, and use ImageJ software to quantitatively analyze the ratio of red and green fluorescence intensity of cells.

[0087] 6. ROS content detection ROS levels in cells cultured for 3 days were detected using the H2DCFDA probe method. A 10 μM working solution was prepared by diluting the cells 1:1000 according to the manufacturer's instructions, transferred to the cells, and incubated in a cell culture incubator in the dark for 30 min. The working solution was discarded, and the cells were rinsed twice with DMEM medium, followed by incubation with Hoechst stain in the dark for 10 min. Finally, the stain was discarded, and the cells were washed twice with basal medium and observed under an inverted microscope. Fluorescence intensity was quantitatively analyzed using flow cytometry and ImageJ software.

[0088] 7. Western Blot Detection The expression of SIRT3 and SIRT5 proteins and mitochondrial biogenesis transcription factors (OPA1, DRP1, PINK1, BNIP3) in cell lysates of each group were extracted and detected.

[0089] 8. Mitochondrial transfer assessment The transfer of mitochondria from microglia to AD neurons was assessed. AD microglia mitochondria were labeled with MitoTracker Red CMXRos, diluted in growth medium at 37°C for 30 min, and then seeded for co-culture. AD neurons were co-cultured with AD microglia (donor cells) at a 1:1 ratio and grown for 24 h prior to fixation, as previously described. Cells were imaged, and the number of MitoTracker Red positive recipient cells was manually counted along different z-planes of the optical section images to ensure that the counted particles were intracellular.

[0090] 9. ELISA protein detection kit Neuronal functional proteins (TUBB3, SYP, and MAP2), M1-related secretion markers (IL-1β, TNF-α, and iNOS), and M2-related secretion markers (Arg-1, TGF-β, and IL-10) were detected using ELISA kits. Supernatants from stably cultured cells in each group were collected, and protein concentrations were determined using ELISA kits.

[0091] 10. Statistical Analysis All data are presented as arithmetic mean ± standard deviation (SD). Data analysis was performed using GraphPad Prism version 9.3. One-way ANOVA was used to compare multiple groups of samples, and P < 0.05 indicates that the difference is statistically significant.

[0092] II. Experimental Results To determine the optimal concentration of Aβ solution for inducing aplastic anemia (AD) in HMC3 cells, this experiment used 0, 5, 10, 20, and 30 mmol / L Aβ solutions to co-incubate SH-SY5Y neurons and HMC3 microglia for 24 h, and cell viability was measured. Aβ concentrations above 20 mmol / L significantly reduced cell viability. Therefore, a 20 mmol / L Aβ solution was selected as the AD model construction protocol for treating cells for 24 h. Figure 7 ), and successfully detected the target proteins SIRT3 and SIRT5 in transfected microglia and neurons ( Figure 8 ).

[0093] After transfection with pSIRT3 / 5, the Aβ level in AD microglia was significantly reduced compared to other groups, while the Aβ level in AD neurons transfected with pSIRT3 / 5 did not decrease significantly. This indicates that microglia have an Aβ-phagocytic effect and are more effective in treating Aβ deposition than neurons. Figure 9Aβ induces mitochondrial dysfunction in neurons and microglia, resulting in a significant decrease in cellular ATP levels. Compared to the empty plasmid group, the SIRT3 / 5 group of microglia successfully transfected with pSIRT3 / 5 showed the most significant increase in intracellular ATP levels. Figure 10 This indicates that co-expression of SIRT3 and SIRT5 proteins significantly enhances intracellular oxidative phosphorylation levels. Our results show that, compared to the Aβ group with mitochondrial dysfunction in microglia, the empty plasmid group showed no significant changes, but the ROS level in the SIRT3 / 5 group was significantly reduced. Figure 11 The JC-1 fluorescent probe was used to assess changes in mitochondrial membrane potential. The results showed that the Aβ group exhibited enhanced green fluorescence after Aβ induction, indicating a significant decrease in mitochondrial membrane potential. The SIRT3 / 5 microglia transfected with pSIRT3 / 5 showed a more pronounced decrease in green fluorescence, suggesting that SIRT3 and SIRT5 proteins can significantly attenuate the Aβ-induced decrease in mitochondrial membrane potential. Figure 12 The results of mitochondrial mtDNA content assays showed that, compared with the WT group, the Aβ group had decreased mitochondrial mtDNA content, suggesting a significant defect in Aβ-induced neuronal mitochondrial biogenesis. In contrast, the SIRT3 / 5 group significantly increased mitochondrial mtDNA content, indicating that SIRT3 and SIRT5 proteins can significantly enhance mitochondrial biogenesis. Figure 13 Detection of M1 and M2 type-related proteins in microglia of each group showed that, compared with the WT group, Aβ-induced microglia showed an increase in M1 type-related markers (IL-1β, TNF-α, and iNOS), while pSIRT3 / 5 transfected microglia showed a decrease in M1 type-related protein levels and an increase in M2 type-related protein levels (Arg-1, TGF-β, and IL-10) compared with the Aβ group. Figure 14 The results indicate that Aβ can induce M1 polarization in microglia, while SIRT3 and SIRT5 proteins can effectively transform AD microglia from M1 to M2 type. These results suggest that transfection with the dual-gene pSIRT3 / 5 gene has a significant effect on treating mitochondrial dysfunction in Aβ microglia and can promote the transformation of Aβ microglia from M1 to M2 type.

[0094] Compared with the Aβ group, the expression levels of mitochondrial fusion protein OPA1 and mitochondrial splitting protein DRP1 were significantly increased in the SIRT3 / 5 group, indicating that the synergistic effect of the two genes can promote the balance between mitochondrial fusion and splitting. Figure 15 The results above all indicate that transfection with the dual-gene pSIRT3 / 5 gene has a more significant effect on treating Aβ-induced mitochondrial dysfunction in microglia.

[0095] By co-culturing microglia and Aβ-induced neurons and detecting neuronal functional proteins, the results showed that M2-type microglia, compared to M1-type microglia, effectively improved the functional proteins of damaged neurons. Figure 16 To investigate the mechanisms by which microglia improve neuronal function through mitochondrial transfer, we labeled the cytoskeleton (Cell Mask Deep RedActin Tracker) and stained M2 microglia mitochondria with MitoTracker Red CMXRos staining before co-culturing them with damaged neurons for 24 hours. Red-positive signals from microglia mitochondria appeared in neurons, indicating that microglia mitochondria can enter neurons. Quantitative analysis of red fluorescence revealed that M2 microglia transferred more mitochondria to damaged neurons compared to M1 microglia. Figure 17 ).

[0096] Example 3: Tf-pSIRT3 / 5 liposomes improve AD symptoms in mice I. Experimental Materials and Methods

[0097] 1. Cell, plasmid, and animal sources are shown in Table 14, and the main reagent sources are shown in Table 15: Table 14 Cells, Plasmids, and Animals

[0098] Table 15 Main Reagents

[0099] 2. Preparation of Tf-pSIRT3 / 5 targeted liposomes 10 mg DC-chol, 20 mg DOPE, 0.2 mg DiR, and 4 mg DSPE-PEG 2000 were dissolved in dichloromethane, mixed, and rotary evaporated to prepare a homogeneous film. The film was hydrated and swollen with 25 mL of deionized water, and 1 mL of PBS solution containing 0.2 mg / mL pSIRT3 / 5 was added. The film was rotary evaporated again, and the uncoated plasmids were degraded by DNase I (10 U) and Exonuclease III (25 U). The liposome-DNA and the nuclease-degraded plasmids were separated by gel column chromatography, and eluted with PBS solution (pH=7.4). The eluent contained the pSIRT3 / 5-coated liposomes.

[0100] The prepared cationic liposomes coated with plasmids were coupled with thiolized transferrin as follows: Transferrin was dissolved in borate solution at pH 8.0, then mixed with 2-iminothione hydrochloride at a molar ratio of 1:75 and reacted at room temperature for 60 min to obtain thiolized transferrin. The liposomes coated with pSIRT3 / 5 were incubated with thiolized transferrin for 12 h. Separation was performed using agarose gel CL-4B; the eluted liposomes were the thiolized transferrin-coupled pSIRT3 / 5 liposomes, abbreviated as Tf-pSIRT3 / 5 liposomes. The coupling rate of transferrin was determined using a BCA kit. The particle size and zeta potential of the liposomes were measured using a nanoparticle size analyzer and a zeta potential meter.

[0101] 3. Animals and Animal Therapy APP / PS1 double transgenic Alzheimer's disease mouse model and normal mice were purchased from the Animal Experiment Center of Southern Medical University. Each mouse weighed 18-20g, was approximately 6 months old, and had a female-to-male ratio of 1:1. APP / PS1 mice were randomly divided into three groups (N=8): AD group (AD mice were injected with 1 mL of physiological saline); Tf-SIRT3 / 5 group (AD mice were injected with 1 mL of 50 μg / mL Tf-SIRT3 / 5 liposomes); and pSIRT3 / 5 group (AD mice were injected with 1 mL of 50 μg / mL pSIRT3 / 5 liposomes). One month later, the Morris water maze test and NOR test were performed as usual.

[0102] 4. Morris water maze experiment The Morris water maze experiment involves fixing a transparent platform, 10 cm in diameter and 25 cm high, 2 cm underwater in a circular pool with a diameter of 120 cm and a height of 40 cm. The platform is covered with milk powder. The pool is divided into four quadrants, and the water temperature is maintained between 24-28°C. On the first day, mice are allowed to swim freely for 60 seconds to familiarize themselves with the environment. On the second day, after a tail vein injection, mice are placed in the water from any quadrant. A mouse is considered to have found the platform if it locates it within 60 seconds and stays there for 10 seconds. If it stays there for more than 60 seconds, it is considered not to have found the platform, and the mouse is manually guided to the platform. The latency period is recorded. Each mouse is tested four times per day for five consecutive days, and the average time to reach the platform is calculated for each group. After the experiment, the platform is removed, and the mice are placed in the water from the opposite direction. The number of times a mouse crosses the original platform location within 60 seconds is recorded to assess its spatial memory ability.

[0103] 5. NOR Experiment Two days before the NOR experiment, mice were placed in a glass box measuring 50 cm long, 40 cm wide, and 25 cm high, allowing them to freely move around and become familiar with their environment. Two identical objects were then placed in the box, and the mice were allowed to explore freely for 6 minutes at the same distance from the objects. After 24 hours, the mice were returned to their original cages and placed back in the box. One of the objects was then replaced, and the exploration time for the new and old objects was recorded for 6 minutes, denoted as T1 and T2 respectively. The Recognition Index (RI) was calculated based on the recorded time using the formula: RI = T1 / (T1+T2) × 100%.

[0104] 6. Preparation of animal brain tissue One month after liposome injection and completion of animal behavioral experiments, mice were anesthetized by intraperitoneal injection of 10% chloral hydrate and euthanized by decapitation. Half of the mice in each group were euthanized. Cortical and hippocampal tissues were separated, immediately frozen on dry ice, and stored at -80°C. 0.1 g of cortical and hippocampal tissues were taken separately, and 1 mL of lysis buffer containing phosphatase inhibitor, protease inhibitor, and PMSF was added, followed by homogenization. The obtained tissue homogenate was centrifuged (4°C, 2500 rpm, 5 min), and the supernatant was collected.

[0105] 7. Detection of target proteins and mitochondrial function in cortical and hippocampal tissues Take appropriate amounts of supernatant from the cortex and hippocampus of each group of mice, and determine the total protein concentration in each sample using the BCA kit. Detect the relative mtDNA content in the cortex and hippocampus supernatant according to the kit's instructions.

[0106] 8. Immunofluorescence detection of amyloid plaques Frozen sections (approximately 10–15 µm) of mouse brains from each group were prepared. The sections were treated with 70% formic acid for 10 min at room temperature to repair antigens. They were then treated with 2% H₂O₂ for 15 min. The sections were incubated with PBS containing 5% goat serum and 0.5% Triton X-100 for 30 min. Mouse monoclonal antibody 6E10 (1:500; 803014, BioLegend, America) was added, and the sections were incubated at 4°C for 12 h. Goat anti-mouse IgG (1:200; ab96879, Abcam) was added, and the sections were incubated at room temperature for 1 h. Biotin-streptolysin (1:150; ZSGB-BIO, Beijing, China) was added, and the sections were incubated at room temperature for 1 h. DAB chromogenic solution (34002, Thermo Scientific™) was added to the sections, and the sections were reacted at room temperature in the dark for 20 min. The sections were washed three times with PBS (pH=7.4) between each step. All images were acquired using a fluorescence confocal microscope, and the fluorescence intensity of the starch spots was quantified using Image-Pro Plus 5.1 software.

[0107] 9. ELISA protein detection kit M1-related secretion markers (IL-1β, TNF-α, and iNOS) and M2-related secretion markers (Arg-1, TGF-β, and IL-10) were detected using ELISA kits. Supernatants from stably cultured cells were collected, and protein concentrations were determined using ELISA kits.

[0108] 10. Statistical Analysis All data in the figures and text are presented as arithmetic mean ± standard deviation (SD) and analyzed using GraphPad Prism version 5.0. Data from animal experiments represent four independent biological replicates, while data from all other experiments represent three independent biological replicates. One-way ANOVA was used to calculate the significance of differences between groups. In all cases, p < 0.05 was considered statistically significant.

[0109] II. Experimental Results Liposome particle size and zeta potential are important in vitro evaluation indicators for liposomes because they affect their stability. Therefore, we measured the particle size, polydispersity index (PDI), and zeta potential of the prepared targeted cationic liposomes. The results are as follows: Figure 18As shown in Table 16, the average particle size of the Tf-pSIRT3 / 5 cationic liposomes was 182 nm, the PDI was 0.212, and the Zeta potential was 7.9 mV. Based on the standard curves, we calculated the encapsulation efficiency and coupling efficiency of the prepared cationic liposomes. The results showed that the encapsulation efficiency of the plasmid was greater than 85% (Table 17), and the coupling efficiency of the targeted cationic liposomes reached more than 50% (Table 17).

[0110] Table 16 Characterization of liposome particle size, PDI, and Zeta potential

[0111] Table 17 Determination of liposome encapsulation efficiency and coupling rate

[0112] To investigate the efficacy of drug liposomes in AD mice, Tf-pSIRT3 / 5 liposomes were injected intravenously into APP / PS1 double transgenic AD model mice. Behavioral experiments were conducted one month later. Compared with the group without transferrin pSIRT3 / 5 liposomes and the AD group, the Tf-pSIRT3 / 5 group showed a significantly shorter escape latency in the Morris water maze test. Figure 19 ), and the number of times the platform is traversed increases ( Figure 20 This indicates that Tf-pSIRT3 / 5 can enhance spatial learning ability and strengthen the formation and consolidation of spatial memory in AD model mice. NOR experiments showed that the RI value of the Tf-pSIRT3 / 5 group was significantly increased compared to other groups, indicating that injection of Tf-pSIRT3 / 5 can significantly enhance the recognition and memory of new objects in AD mice. Figure 21 ).

[0113] After completing the animal behavioral experiments, we euthanized half of the mice in each group and tested the secretory factors related to M1 and M2 types of microglia in the mice. We found that the secretory factor of M2 type was increased and the secretory factor of M1 type was decreased. Figure 22 Western blotting was used to validate the expression of SIRT3 and SIRT5 proteins in mouse cerebral cortex and hippocampal cells. Only the Tf-pSIRT3 / 5 group of mice successfully expressed high levels of SIRT3 and SIRT5 proteins. Figure 23 Immunofluorescence staining was used to detect amyloid plaques β-formed in the cerebral cortex. Results showed that 6-month-old AD mice exhibited amyloid senile plaques similar to those seen in Alzheimer's disease on the brain surface. The fluorescence intensity of these plaques was significantly reduced in the Tf-pSIRT3 / 5 treatment group compared to the AD group. Figure 24These results suggest that Tf-pSIRT3 / 5 may inhibit the production of amyloid plaque β in the brains of AD mice by improving mitochondrial function and activity.

[0114] Compared to the AD group and the pSIRT3 / 5 liposome group without transferrin, the mtDNA content in the cerebral cortex and hippocampal cells of AD mice was increased in the Tf-pSIRT3 / 5 group injected with Tf-pSIRT3 / 5. Figure 25 These results demonstrate that Tf-pSIRT3 / 5 can improve mitochondrial function and homeostasis, playing an important role in improving neurons.

Claims

1. The application of SIRT3 and SIRT5 dual-gene co-expression biomaterials in the preparation of drugs to improve Alzheimer's disease, characterized in that, The SIRT3 and SIRT5 co-expression biomaterials include: a) a SIRT3 and SIRT5 co-expression plasmid; b) a cell line containing the SIRT3 and SIRT5 co-expression plasmid; c) liposomes encapsulating the SIRT3 and SIRT5 co-expression plasmid; and d) brain-targeted delivery liposomes encapsulating the SIRT3 and SIRT5 co-expression plasmid.

2. The application according to claim 1, characterized in that, The plasmid for co-expression of the SIRT3 and SIRT5 genes is pIRES2-SIRT3-SIRT5.

3. The application according to claim 1, characterized in that, The cell line containing the SIRT3 and SIRT5 dual gene co-expression plasmid is a microglia containing the SIRT3 and SIRT5 dual gene co-expression plasmid.

4. The application according to claim 1, characterized in that, The liposomes encapsulating the SIRT3 and SIRT5 dual-gene co-expression plasmid are cationic liposomes encapsulating the SIRT3 and SIRT5 dual-gene co-expression plasmids.

5. The application according to claim 1, characterized in that, The brain-targeted delivery liposomes encapsulating the SIRT3 and SIRT5 dual-gene co-expression plasmids are cationic liposomes containing transferrin Tf coupled to the SIRT3 and SIRT5 dual-gene co-expression plasmids.

6. The application according to claim 1, characterized in that, The nucleotide sequence of the SIRT3 gene is shown in SEQ ID No. 1, and the nucleotide sequence of the SIRT5 gene is shown in SEQ ID No.

3.

7. The application according to claim 1, characterized in that, The drug internalizes the SIRT3 and SIRT5 dual-gene co-expression plasmid into the subject's M1 microglia, regulates the activity of mitochondrial antioxidant enzymes in the subject's microglia to clear ROS, increases ATP and mtDNA levels, enhances the generation of microglia mitochondrial networks and restores mitochondrial function, and metabolically reprograms them into M2 microglia. M2 microglia improve the neuronal mitochondrial network by clearing neuronal Aβ and delivering mitochondria to damaged neurons, thereby achieving the treatment of Alzheimer's disease.

8. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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