Stem cell-negative hydrogen ion compound preparation for treating Alzheimer disease

Abstract

The invention discloses a stem cell-negative hydrogen ion compound preparation for treating Alzheimer's disease, and belongs to the technical field of biomedicine, the compound preparation comprises stem cells, a negative hydrogen ion donor, piceatannol glucoside and a pharmaceutically acceptable carrier. Wherein the concentration of the stem cells is 1 * 10 < 6 >-1 * 10 < 8 > / mL, the concentration of the negative hydrogen ion donor is 25-45mg / mL, and the concentration of piceatannol glucoside is 4-8mu g / mL. The composite preparation integrates neural restoration, anti-inflammatory and immunoregulation functions of stem cells, an efficient antioxidant protection effect of a negative hydrogen ion donor and synergistic interaction and safety guarantee effects of piceatannol glucoside. Animal experiments prove that the composite preparation can significantly improve cognitive impairment of Alzheimer's disease model rats and relieve neuroinflammation, oxidative stress and beta-amyloid protein deposition through synergistic anti-inflammatory, anti-oxidation and neuroprotection mechanisms.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a stem cell-negative hydrogen ion composite preparation for the treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive decline. Its pathological process is complex, with core features including β-amyloid (Aβ) plaque deposition, neurofibrillary tangles formed by tau protein hyperphosphorylation, and persistent chronic neuroinflammation. Oxidative stress is considered a key driver of this pathological cascade: Aβ oligomers and abnormal tau protein can directly induce mitochondrial dysfunction, leading to excessive production of reactive oxygen species (ROS); excessive ROS further exacerbates the pathological accumulation of Aβ and tau, impairs synaptic function, and triggers neuronal apoptosis, creating a vicious cycle.

[0003] Currently, clinical drug treatment for Alzheimer's disease (AD) mainly includes two classes: acetylcholinesterase inhibitors, represented by donepezil, and N-methyl-D-aspartate (NMDA) receptor antagonists, represented by mimantine. These traditional therapies can only relieve symptoms and cannot slow or stop disease progression. In recent years, the emergence of monoclonal antibody drugs (adunatumab, lencanemab, and donepemab) that work by clearing Aβ plaques marks a breakthrough in disease-modifying therapy. However, their clinical efficacy is limited, and they may cause side effects such as amyloid-related imaging abnormalities. This highlights the limitations of single-target intervention.

[0004] Therefore, research has shifted to multiple intervention strategies targeting the complex pathological network of Alzheimer's disease (AD), mainly including: (1) anti-neuroinflammatory therapy, such as targeting microglial receptors such as TREM2 and CD33; (2) metabolic and immune regulation, including intranasal insulin, metformin, and gut microbiota intervention; (3) neural repair and regeneration, such as mesenchymal stem cell transplantation, which demonstrates repair potential by secreting neurotrophic factors, regulating inflammation, and promoting homeostasis; and (4) direct antioxidant intervention, such as negative hydrogen ions, which can effectively reduce oxidative damage and support mitochondrial function due to their excellent biopenetration and selective antioxidant capacity. However, these strategies have limitations when applied alone: ​​the survival rate and functional impairment of stem cells in the high oxidative stress environment of the lesion area are easily affected; and simple antioxidants lack active repair capabilities and have limited targeted delivery efficiency.

[0005] In recent years, multi-target small molecule compounds derived from natural products have attracted attention due to their broad effects and relatively high safety. For example, studies on stilbene compounds such as astringin have shown that it can simultaneously intervene in cholinergic deficiency, Aβ deposition, and Tau pathology in Alzheimer's disease by inhibiting multiple targets, including acetylcholinesterase (AChE), β-amyloid precursor protein cleavage enzyme 1 (BACE1), and glycogen synthase kinase-3β (GSK-3β), while also possessing antioxidant and anti-inflammatory activities. However, there are currently no research reports on the systematic integration of astringin with cell therapy and highly effective antioxidants to construct a synergistic therapeutic system of "cell repair-antioxidation-multi-target regulation."

[0006] Based on this, a ternary compound formulation composed of stem cells, negative hydrogen ions, and paclitaxel glucoside was developed. The aim is to achieve comprehensive treatment of nerve repair, anti-oxidative stress, and immune regulation through multiple synergistic mechanisms. This not only helps to deeply understand the mechanism of action of synergistic intervention in multiple pathological links of AD, but also provides a new strategy with translational potential to promote the transformation of AD from symptomatic treatment to disease modification treatment. Summary of the Invention

[0007] Therefore, the present invention provides a stem cell-negative hydrogen ion composite preparation for the treatment of Alzheimer's disease, in order to overcome the shortcomings of the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, a stem cell-negative hydrogen ion composite preparation for the treatment of Alzheimer's disease is provided, comprising stem cells, a negative hydrogen ion donor, and paclitaxel glucoside, wherein the concentration of the stem cells is 1×10⁻⁶. 6 -1×10 8 The concentration of the negative hydrogen ion donor is 25-45 mg / mL, and the concentration of the leucopicrin glucoside is 4-8 μg / mL.

[0009] Furthermore, the stem cells are selected from at least one of neural stem cells, mesenchymal stem cells, and neural progenitor cells derived from induced pluripotent stem cells.

[0010] Furthermore, the stem cells are neural stem cells.

[0011] Furthermore, the negative hydrogen ion donor is selected from at least one of negative hydrogen ion powder, negative hydrogen ion tablets, and negative hydrogen ion aqueous solution.

[0012] Furthermore, the negative hydrogen ion donor is a negative hydrogen ion powder.

[0013] Furthermore, it also includes pharmaceutically acceptable carriers.

[0014] Furthermore, the carrier is selected from at least one of physiological saline, phosphate-buffered saline (PBS), Hank's balanced salt solution (HBSS), and Earle's balanced salt solution (EBSS).

[0015] Furthermore, the stem cell-negative hydrogen ion composite preparation is formulated as an injectable preparation or an implantable sustained-release preparation suitable for local administration; the dosage form of the injectable preparation is a suspension injection or a lyophilized powder injection, and the dosage form of the implantable sustained-release preparation suitable for local administration is a gel or a microsphere preparation.

[0016] Furthermore, the stem cell-negative hydrogen ion composite preparation is formulated into an injectable preparation, which is administered via intravenous infusion, local targeted injection, or acupoint injection.

[0017] Furthermore, the administration method of the injectable preparation is acupoint injection.

[0018] Furthermore, the acupoints for the acupoint injection are selected from at least one of Zusanli, Fengchi, Xuehai, Fenglong, Shenshu, Baihui, Neiguan, Taichong, and Yanglingquan.

[0019] Furthermore, the acupoints for injection are Baihui (GV20), Fengchi (GB20), Shenshu (BL23), Zusanli (ST36), and Fenglong (ST40). Among them, Baihui belongs to the Du meridian and has the effects of raising Yang and awakening the brain, calming the mind and opening the orifices, which can improve mental dysfunction and regulate the spirit; Fengchi belongs to the Gallbladder meridian and has the effects of dispelling wind and unblocking the meridians, clearing the head and eyes, which helps to relieve dizziness and memory loss; Shenshu belongs to the Bladder meridian and has the effects of tonifying the kidney and replenishing essence, benefiting the marrow and strengthening the brain, which can target the root cause of AD, "kidney deficiency and marrow depletion"; Zusanli belongs to the Stomach meridian and has the effects of strengthening the spleen and replenishing Qi, regulating Qi and blood. When the spleen and stomach are functioning well, Qi and blood will be supplied to the brain, and it can also resolve phlegm and dampness and remove blood stasis; Fenglong belongs to the Stomach meridian and has the effects of resolving phlegm and dampness, unblocking the meridians and opening the orifices, which is good at clearing phlegm and turbidity to open the brain orifices.

[0020] According to a second aspect of the invention, the use of the said stem cell-negative hydrogen ion complex in the preparation of a medicament for treating Alzheimer's disease is provided.

[0021] Furthermore, the pathological features of Alzheimer's disease include at least one of the following: abnormal deposition of β-amyloid protein, abnormal phosphorylation and aggregation of Tau protein, decreased function of the cholinergic neurotransmitter system, mitochondrial dysfunction, loss of synaptic plasticity, synapse loss, and neuronal dysfunction.

[0022] Compared with the prior art, the present invention has the following advantages: This invention targets the core pathological link of mitochondrial dysfunction and oxidative stress in Alzheimer's disease (AD). It combines stem cells (such as neural stem cells) with tissue repair and immune regulation functions with negative hydrogen ion donors possessing antioxidant potential, and further introduces astringin, a natural stilbene derivative with multi-target regulatory potential, to develop a stem cell-negative hydrogen ion composite formulation. This composite formulation aims to achieve a dual effect of cell repair and antioxidant protection, with specific advantages in the following three aspects: (1) Complementary functions and synergistic effect This compound formulation constructs a synergistic system of "protection-repair-regulation": On the one hand, the active hydrogen components released by the negative hydrogen ion donor can actively clear reactive oxygen species (ROS) in the lesion area, providing a microenvironment with low oxidative stress levels for stem cells and damaged neurons, thereby effectively improving the survival and function of stem cells under pathological conditions; on the other hand, protected stem cells can more effectively exert their nerve repair, anti-inflammatory and immune regulation functions to achieve active repair; in addition, piracetam glucoside, as a multi-target regulatory molecule, can further enhance the regulation of the cholinergic system, amyloid protein and tau pathology by inhibiting key targets such as AChE, BACE, and GSK-3β, consolidating and expanding the long-term benefits of antioxidant therapy. The three form a virtuous cycle, fundamentally overcoming the limitations of the mechanism of action of single therapy.

[0023] (2) Multi-target intervention for comprehensive improvement This compound formulation works synergistically through multiple mechanisms to address the complex pathological network of Alzheimer's disease (AD). At the direct level, the highly effective antioxidant properties of the active hydrogen components released by the negative hydrogen ion donor directly alleviate mitochondrial oxidative damage, breaking the vicious cycle between oxidative stress and Aβ deposition and Tau phosphorylation. At the indirect level, stem cells indirectly reduce neuronal damage and promote endogenous repair by secreting neurotrophic factors and inhibiting neuroinflammation. Animal experiments have verified that this compound formulation, while improving cognitive function in AD model rats, effectively reduces the levels of inflammatory factors (IL-6, IL-1β, and TNF-α) in their hippocampus, increases the activity of antioxidant enzymes (SOD and GSH-Px), and reduces pathological Aβ deposition. 1-40 and Aβ 1-42 The content of these substances demonstrates a comprehensive regulatory capacity for multiple key targets, including neuroinflammation, oxidative stress, and protein pathology.

[0024] (3) Natural multi-target, synergistic and safe Paclitaxel glucoside, a naturally derived stilbene compound, possesses favorable pharmacokinetic characteristics and a low risk of drug interactions. Its multi-target mechanism of action can compensate for the limitations of stem cells and negative hydrogen ions in regulating molecular signaling pathways. The addition of this component not only broadens the coverage of the core pathological network of Alzheimer's disease (AD) by stem cell-negative hydrogen ion composite formulations, but also provides a safety guarantee for long-term use due to its natural origin and low toxicity, aligning with the modern concept of multi-target, low-toxicity drug design.

[0025] (4) Integration of Chinese and Western cultures, with clear transformation This invention employs acupoint injection as the preferred drug delivery method, integrating modern targeted therapy with traditional Chinese medicine meridian theory. Modern research shows that acupuncture at specific acupoints (Baihui, Fengchi, Shenshu, Zusanli, and Fenglong) can improve Alzheimer's disease (AD) cognitive function through multiple pathways, including regulating Aβ metabolism and inhibiting neuroinflammation. This invention delivers the compound preparation directly to the effector acupoints, utilizing not only the pharmacological effects of active hydrogen components released by stem cells and negative hydrogen ion donors, but also potentially activating endogenous neuroprotective and repair pathways through the physical stimulation and meridian regulation of acupuncture. This enhances the drug's targeting and bioavailability, providing a promising new paradigm for the integrated treatment of AD using both traditional Chinese and Western medicine. Detailed Implementation

[0026] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] According to a first aspect of the present invention, a stem cell-negative hydrogen ion composite preparation for the treatment of Alzheimer's disease is provided, comprising stem cells, a negative hydrogen ion donor, and paclitaxel glucoside, wherein the concentration of stem cells is 1×10⁻⁶. 6 -1×10 8 The concentration of negative hydrogen ion donors is 25-45 mg / mL, and the concentration of leucopicrin glucoside is 4-8 μg / mL.

[0028] Furthermore, the stem cells are selected from at least one of neural stem cells, mesenchymal stem cells, and neural progenitor cells derived from induced pluripotent stem cells.

[0029] Furthermore, the stem cells are neural stem cells.

[0030] Furthermore, the negative hydrogen ion donor is selected from at least one of negative hydrogen ion powder, negative hydrogen ion tablets, and negative hydrogen ion aqueous solution.

[0031] Furthermore, the negative hydrogen ion donor is negative hydrogen ion powder.

[0032] Furthermore, it also includes pharmaceutically acceptable carriers.

[0033] Furthermore, the carrier is selected from at least one of physiological saline, phosphate-buffered saline (PBS), Hank's balanced salt solution (HBSS), and Earle's balanced salt solution (EBSS).

[0034] Furthermore, the stem cell-negative hydrogen ion composite preparation is formulated as an injectable preparation or an implantable sustained-release preparation suitable for local administration; the dosage form of the injectable preparation is a suspension injection or a lyophilized powder injection, and the dosage form of the implantable sustained-release preparation suitable for local administration is a gel or a microsphere preparation.

[0035] Furthermore, the stem cell-negative hydrogen ion composite preparation is formulated into an injectable preparation, which is administered via intravenous infusion, local targeted injection, or acupoint injection.

[0036] Furthermore, the injection preparation is administered via acupoint injection.

[0037] Furthermore, the acupoints for acupoint injection are selected from at least one of the following: Zusanli, Fengchi, Xuehai, Fenglong, Shenshu, Baihui, Neiguan, Taichong, and Yanglingquan.

[0038] Furthermore, the acupoints for acupoint injection are Baihui, Fengchi, Shenshu, Zusanli, and Fenglong. Among them, Baihui belongs to the Du meridian and has the effects of raising Yang and awakening the brain, calming the mind and opening the orifices. It can improve mental dysfunction and regulate the spirit. Fengchi belongs to the Gallbladder meridian and has the effects of dispelling wind and unblocking the meridians and clearing the head and eyes. It can help relieve dizziness and memory loss. Shenshu belongs to the Bladder meridian and has the effects of tonifying the kidney and replenishing essence, benefiting the marrow and strengthening the brain. It can target the root cause of AD, "kidney deficiency and marrow depletion". Zusanli belongs to the Stomach meridian and has the effects of strengthening the spleen and replenishing Qi, regulating Qi and blood. When the spleen and stomach are functioning well, Qi and blood will be supplied to the brain. It can also resolve phlegm and dampness and remove blood stasis. Fenglong belongs to the Luo-connecting point of the Stomach meridian and has the effects of resolving phlegm and dampness, unblocking the meridians and opening the orifices. It is good at clearing phlegm and turbidity to open the brain orifices.

[0039] According to a second aspect of the invention, the use of a stem cell-negative hydrogen ion complex in the preparation of a medicament for treating Alzheimer's disease is provided.

[0040] Furthermore, the pathological features of Alzheimer's disease include at least one of the following: abnormal deposition of β-amyloid protein, abnormal phosphorylation and aggregation of Tau protein, decreased function of the cholinergic neurotransmitter system, mitochondrial dysfunction, loss of synaptic plasticity, synapse loss, and neuronal dysfunction.

[0041] Preparation Example Preparation of neural stem cell (NSC) suspension (1) Resuscitation and vaccination of NSCs A 15 mL centrifuge tube containing NE-4C mouse neural stem cells (purchased from Tianjin Yousai Life Science Development Co., Ltd.) was disinfected by spraying its outer surface with 75% alcohol. After observing the cell state under a microscope, the centrifuge tube was placed in a 37℃ incubator for equilibration for 2 hours. After equilibration, the tube was centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded. The cell pellet was resuspended in 5 mL of complete culture medium (DMEM / F12, containing 20 ng / mL recombinant human epidermal growth factor, 20 ng / mL recombinant human basic fibroblast growth factor, 1% non-essential amino acids, 1% L-glutamine, 2% B27, and 1% penicillin-streptomycin solution) and seeded into T25 cell culture flasks. The T25 cell culture flasks were incubated in a 37℃, 5% CO2 incubator for 7 days, with the complete culture medium replaced every 2.5 days.

[0042] (2) Cell passage and culture When the cell density reaches 80% or higher, passage the cells. Discard the old complete culture medium in the T25 cell culture flask, add 2 mL of sterile PBS (calcium and magnesium ions-free) to wash the cell pellet once, and discard the sterile PBS. Then add 1 mL of 0.25% trypsin-EDTA digestion solution, gently agitate to cover the bottom of the flask, and digest at 37°C for 3 min. Under a microscope, when most cells become rounded, the gaps between them increase, and they detach from the bottom of the flask, immediately add 3 mL of fresh complete culture medium to stop the digestion. Gently pipette the bottom of the flask to detach all cells, forming a single-cell suspension. Transfer this suspension to a new 15 mL centrifuge tube, centrifuge at 1000 rpm for 3 min, and discard the supernatant. Resuspend the cells in 3 mL of fresh complete culture medium, seed them at a 1:3 ratio into a new T25 cell culture flask, and replenish with fresh complete culture medium to continue culturing. Repeat the above steps, passage continuously to the 3rd generation (P3), to obtain a P3 generation NSCs suspension, store at 4°C for use within 2 hours.

[0043] Example 1 Stem cell-negative hydrogen ion compound preparation 25 mg of medical negative hydrogen ion powder (purchased from Rizhao Life Valley Biotechnology Development Co., Ltd.) and 4 μg of leucopicrin glucoside (CAS No.: 29884-49-9, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were added to an appropriate amount of HBSS (free of calcium and magnesium ions and phenol red) pre-chilled at 4°C and gently vortexed to form a homogeneous suspension. This suspension was then mixed thoroughly with the P3 generation NSCs suspension prepared in the preparation example and incubated on ice for 30 min to obtain the stem cell-negative hydrogen ion premix. Finally, the volume was adjusted to 1 mL with HBSS pre-chilled at 4°C, and the NSCs concentration was adjusted to 1 × 10⁻⁶. 6Cells / mL, keep on ice. Before use, check the pH to ensure it is within the range of 7.2-7.4 and ensure cell viability >95%.

[0044] Example 2 Stem cell-negative hydrogen ion compound preparation 30 mg of medical negative hydrogen ion powder (purchased from Rizhao Life Valley Biotechnology Development Co., Ltd.) and 6 μg of leucopicrin glucoside (CAS No.: 29884-49-9, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were added to an appropriate amount of HBSS (free of calcium and magnesium ions and phenol red) pre-chilled at 4°C and gently vortexed to form a homogeneous suspension. This suspension was then mixed thoroughly with the P3 generation NSCs suspension prepared in the preparation example and incubated on ice for 30 min to obtain the stem cell-negative hydrogen ion premix. Finally, the volume was adjusted to 1 mL with HBSS pre-chilled at 4°C, and the NSCs concentration was adjusted to 1 × 10⁻⁶. 7 Cells / mL, keep on ice. Before use, check the pH to ensure it is within the range of 7.2-7.4 and ensure cell viability >95%.

[0045] Example 3 Stem cell-negative hydrogen ion compound preparation 45 mg of medical negative hydrogen ion powder (purchased from Rizhao Life Valley Biotechnology Development Co., Ltd.) and 8 μg of leucopicrin glucoside (CAS No.: 29884-49-9, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were added to an appropriate amount of HBSS (free of calcium and magnesium ions and phenol red) pre-chilled at 4°C and gently vortexed to form a homogeneous suspension. This suspension was then mixed thoroughly with the P3 generation NSCs suspension prepared in the preparation example and incubated on ice for 30 min to obtain the stem cell-negative hydrogen ion premix. Finally, the volume was adjusted to 1 mL with HBSS pre-chilled at 4°C, and the NSCs concentration was adjusted to 1 × 10⁻⁶. 8 Cells / mL, keep on ice. Before use, check the pH to ensure it is within the range of 7.2-7.4 and ensure cell viability >95%.

[0046] Comparative Example 1 Stem cell and leucopicrin glucoside compound preparation 4 μg of leucopicrin glucoside (CAS No.: 29884-49-9, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to an appropriate amount of pre-chilled HBSS (free of calcium and magnesium ions and phenol red) at 4°C and gently vortexed to form a homogeneous suspension. This suspension was then mixed thoroughly with the P3 generation NSCs suspension prepared in the preparation example and incubated on ice for 30 min to obtain a premix of stem cells and leucopicrin glucoside. Finally, the volume was adjusted to 1 mL with pre-chilled HBSS at 4°C, and the NSCs concentration was adjusted to 1 × 10⁻⁶. 6 Cells / mL, keep on ice. Ensure cell viability >95% before use.

[0047] Comparative Example 2 Stem cell and negative hydrogen ion compound preparation 25 mg of medical-grade negative hydrogen ion powder (purchased from Rizhao Life Valley Biotechnology Development Co., Ltd.) was added to an appropriate amount of HBSS (free of calcium and magnesium ions and phenol red) pre-cooled at 4°C and gently vortexed to form a homogeneous suspension. This suspension was then mixed thoroughly with the P3 generation NSCs suspension prepared in the preparation example and incubated on ice for 30 min to obtain a stem cell and negative hydrogen ion premix. Finally, the volume was adjusted to 1 mL with HBSS pre-cooled at 4°C, and the NSCs concentration was adjusted to 1 × 10⁻⁶. 6 Cells / mL, keep on ice. Ensure cell viability >95% before use.

[0048] Comparative Example 3 stem cell preparations The P3 generation NSCs suspension prepared in the preparation example was mixed thoroughly with HBSS (containing no calcium or magnesium ions and phenol red) pre-cooled at 4°C, and the volume was adjusted to 1 mL with HBSS pre-cooled at 4°C. The NSCs concentration was then adjusted to 1 × 10⁻⁶. 6 Cells / mL, keep on ice. Ensure cell viability >95% before use.

[0049] Test case 1. Establishment of a rat model of Alzheimer's disease (AD) 1.1 Laboratory Animals and Grouping 6-9 week old SPF-grade SD rats, weighing 230-260g, were acclimatized for 1 week and then uniformly numbered and weighed.

[0050] 1.2 AD Model Construction Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 50 mg / kg, and their heads were fixed in a stereotaxic instrument. After routine skin preparation and disinfection, the scalp was incised along the sagittal line of the skull to expose the anterior and posterior fontanelles. Referring to the "Rats' Stereotaxic Brain Atlas," holes were drilled bilaterally at the CA1 coordinates of the hippocampus (3.6 mm posterior to the anterior fontanelle and 2 mm lateral to the sagittal suture) using a motorized mini-skull drill. Using a microinfusion pump, 4 μL of Aβ was injected bilaterally at the target sites in the CA1 region of the hippocampus (depth 3.86 mm) at a rate of 1 μL / min. 23-35 The solution (1 μg / μL, prepared with deionized water and incubated at 37°C for 96 h) was used. After injection, the needle was left in place for 5 minutes, then slowly withdrawn. The rat's head wound was sealed with bone wax, the scalp was sutured, and the area was disinfected. Post-operatively, the rats were placed in a 26°C incubator for recovery and observed for 3 consecutive days. The blank control group received an equal volume of physiological saline instead of Aβ. 23-35 The solution is prepared using the same steps as the rest.

[0051] 1.3 Experimental grouping and drug administration Intervention began on day 7 after modeling. Rats with successful modeling were randomly divided into 7 groups: model group (saline), control group 1 (stem cell and piracetam glucoside compound preparation), control group 2 (stem cell and negative hydrogen ion compound preparation), control group 3 (stem cell preparation), and Examples 1-3 (low, medium, and high doses of stem cell-negative hydrogen ion compound preparation, respectively). A sham-operated rat was set up as a blank control group (except for the modeling surgery, it underwent the same handling and fixation as the other groups, but was not injected with any preparation). A total of 8 groups were formed, with 8 rats in each group.

[0052] Table 1 below shows the grouping and intervention protocols for all experimental rats (n=8): Table 1

[0053] Note: All groups underwent continuous intervention for 4 weeks. The acupoints for injection were Baihui, Fengchi, Shenshu, Zusanli, and Fenglong.

[0054] 2. Evaluation of intervention effectiveness 2.1 Behavioral Experiment (Morris Water Maze Experiment) One week after the last administration, a water maze test was performed to assess the spatial learning and memory abilities of the rats. The specific method is as follows: A circular pool (150cm in diameter and 70cm in height) was divided into four quadrants. A hidden platform was placed in the center of the first quadrant (the target quadrant), with its surface 1cm below the water surface. Rats from each experimental group were placed into the water from different quadrants and asked to find the hidden platform, with a maximum time limit of 90 seconds. If they could not find it, they were guided to the platform and allowed to stay there for 20 seconds. After training, they were dried and returned to the pool. Training was conducted for four consecutive days, and the total distance traveled and the time spent in the central region were recorded for each experimental group to assess their spatial learning ability. On the fifth day, the platform was removed, and the percentage of distance traveled in the target quadrant (the percentage of the total distance traveled in the target quadrant) and the central region distance (the total distance traveled in the central region) for each experimental group were recorded to assess their memory retention ability.

[0055] 2.2 Sample Collection After the behavioral tests were completed, rats in each experimental group were anesthetized by intraperitoneal injection of 10% urethane. 10 mL of blood was collected via the abdominal aorta, centrifuged at 1500 r / min for 15 min at 4℃, and the serum was separated and stored at -80℃. Subsequently, the rats in each experimental group were sacrificed, and the bilateral hippocampal tissue was quickly removed and stored at -80℃.

[0056] 2.3 ELISA detection Collect the supernatant from the homogenate of hippocampal tissue, and strictly follow the instructions of the corresponding reagent kit to detect the following indicators: (1) Inflammatory factors: The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were measured; (2) Oxidative stress indicators: The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured.

[0057] (3) Aβ protein content: Determination of β-amyloid protein Aβ 1-40 and Aβ 1-42 The content of.

[0058] 2.4 Statistical Analysis Data were processed using IBM SPSS Statistics 29.0 software. Quantitative data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used for comparisons among multiple groups; the LSD test was used for post-hoc testing when variances were homogeneous, and Dunnett's T3 test was used when variances were unequal. p <0.05 indicates a statistically significant difference.

[0059] 3. Results and Conclusions 3.1 General condition of rats During the experiment, the vital signs of rats in all groups remained stable, and there were no deaths.

[0060] Compared with the blank control group, the weight gain of rats in the model group was significantly reduced. p <0.01), and accompanied by reduced activity, sparse and dull fur, yellow urine, and dry feces, as well as other appearance and behavioral abnormalities; at the same time, these rats were irritable and showed strong stress responses such as piloerection, screaming, and violent struggle when grabbed.

[0061] Compared with the model group, the general condition of rats in all treatment groups improved after drug intervention, manifested as increased activity, improved hair, urine and fecal characteristics, and reduced resistance during capture. Regarding weight gain, comparative group 3 (containing only stem cells) showed no significant improvement; comparative group 1 (stem cells and piracetam glucoside compound preparation) and comparative group 2 (stem cells and negative hydrogen ion donor compound preparation) both showed significant improvement. p <0.05); while the improvement effects of groups 1-3 (low, medium, and high dose stem cell-negative hydrogen ion compound preparations, respectively) were all more significant ( p <0.01), and the effect was better than the formulation group containing two components (Comparative Example 1 and Comparative Example 2), and even better than the formulation group containing only stem cells (Comparative Example 3), and showed a dose-dependent increasing trend.

[0062] Table 2 below shows the changes in body weight of rats in each experimental group (n=8, mean±SD): Table 2

[0063] Note: Weight difference = Post-treatment weight - Pre-modeling weight; ## Compared with the blank control group, p <0.01; ** Compared with the model group, p <0.01; * Compared with the model group, p <0.05.

[0064] 3.2 Results of the behavioral experiment Compared with the blank control group, the model group rats showed significantly lower total distance traveled, time spent in the central region, percentage of distance traveled in the target quadrant, and distance traveled in the central region. p <0.01), indicating that its spatial exploration and memory abilities are impaired.

[0065] Compared with the model group, rats in all treatment groups treated with drugs showed an improvement trend in all four behavioral indicators. Specifically, Comparative Group 3 (containing only stem cells) showed no significant improvement in any of the four behavioral indicators; Comparative Group 2 (containing a combination of stem cells and negative hydrogen ion donors) showed significant improvement in both total distance traveled and central distance traveled. p <0.05); Comparative Group 1 (stem cell and piracetam glucoside combination preparation) showed significant improvement in all four behavioral indicators ( p <0.05); while groups 1-3 of Examples (low, medium, and high dose stem cell-negative hydrogen ion compound preparations, respectively) showed more significant improvements in all four behavioral indicators ( p The concentration of the drug in the high-dose group was <0.01%, and the effect was superior to the two-component formulation (Comparative Example 1 and Comparative Example 2), and even better than the formulation containing only stem cells (Comparative Example 3), showing a dose-dependent increasing trend. Notably, the high-dose group rats recovered to levels similar to the blank control group in four behavioral indicators.

[0066] Table 3 below shows the test results of Morris water maze behavioral indicators in rats of each experimental group (n=8, mean±SD): Table 3

[0067] Note: ## Compared with the blank control group, p <0.01; ** Compared with the model group, p <0.01; * Compared with the model group, p <0.05.

[0068] 3.3 ELISA test results 3.3.1 Levels of inflammatory factors in hippocampal tissue (IL-6, IL-1β, TNF-α) Compared with the blank control group, the levels of IL-6, IL-1β and TNF-α in the hippocampus of rats in the model group were significantly increased. p <0.01), indicating that the neuroinflammation model was successfully constructed.

[0069] Compared with the model group, the levels of IL-6, IL-1β, and TNF-α in the hippocampus of rats in all treatment groups treated with drugs were decreased. Specifically, the decrease in the levels of the three inflammatory factors (IL-6, IL-1β, and TNF-α) was not significant in Comparative Group 3 (containing only stem cells); the decreases were significant in Comparative Group 1 (stem cells and piracetam glucoside combination preparation) and Comparative Group 2 (stem cells and negative hydrogen ion donor combination preparation). p <0.05); while the reduction effect of groups 1-3 (low, medium, and high dose stem cell-negative hydrogen ion compound preparations, respectively) was more significant ( p The levels of IL-6, IL-1β, and TNF-α in the high-dose group were significantly lower than those in the two-component formulation (Comparative Example 1 and Comparative Example 2), and significantly higher than those in the stem cell-only formulation (Comparative Example 3), showing a dose-dependent increasing trend. Notably, the levels of IL-6, IL-1β, and TNF-α in the hippocampus of rats in the high-dose group recovered to levels similar to those in the blank control group.

[0070] Table 4 below shows the test results of inflammatory factors IL-6, IL-1β and TNF-α in rats of each experimental group (n=8, mean±SD): Table 4

[0071] Note: ## Compared with the blank control group, p <0.01; ** Compared with the model group, p <0.01; * Compared with the model group, p <0.05.

[0072] 3.3.2 Oxidative stress indicators in hippocampal tissue (SOD and GSH-Px) Compared with the blank control group, the levels of SOD and GSH-Px in the hippocampus of rats in the model group were significantly reduced. p <0.01), indicating that the oxidative stress damage model was successfully constructed.

[0073] Compared with the model group, the levels of SOD and GSH-Px in the hippocampus of rats in all treatment groups after drug intervention were increased. Among them, the increase was not significant in comparative group 3 (containing only stem cells); the levels of both oxidative stress indicators (SOD and GSH-Px) were significantly increased in comparative group 1 (stem cells and piracetam glucoside compound preparation) and comparative group 2 (stem cells and negative hydrogen ion donor compound preparation). p <0.05); while the enhancing effects of groups 1-3 (low, medium, and high dose stem cell-negative hydrogen ion compound preparations, respectively) were all more significant ( p The concentration of SOD and GSH-Px in the high-dose group was <0.01%, and the enhancing effect was superior to that of the two-component formulation (Comparative Example 1 and Comparative Example 2), and even superior to that of the formulation containing only stem cells (Comparative Example 3), showing a dose-dependent increasing trend. Notably, the SOD and GSH-Px levels in the hippocampus of rats in the high-dose group recovered to levels similar to those in the blank control group.

[0074] Table 5 below shows the test results of SOD and GSH-Px levels (n=8, mean±SD) in rats of each experimental group: Table 5

[0075] Note: ## Compared with the blank control group, p <0.01; ** Compared with the model group, p <0.01; * Compared with the model group, p <0.05.

[0076] 3.3.3 Hippocampal Aβ protein content (Aβ) 1-40 and Aβ 1-42 ) Compared with the blank control group, the Aβ content in the hippocampus of rats in the model group was higher. 1-40 and Aβ 1-42 The content increased significantly ( p The value <0.01 indicates that the core protein pathological feature model of AD has been successfully constructed.

[0077] Compared with the model group, the Aβ levels in the hippocampus of rats in each treatment group after drug intervention were significantly higher. 1-40 and Aβ 1-42 The content of all proteins decreased. Among them, the decrease was not significant in Comparative Example 3 (containing only stem cells); the content of the two Aβ proteins (Aβ) in Comparative Example 1 (stem cells and leucopicrin glucoside compound preparation) and Comparative Example 2 (stem cells and negative hydrogen ion donor compound preparation) decreased. 1-40 and Aβ 1-42 All significantly reduced ( p<0.05); while the reduction effect of groups 1-3 (low, medium, and high dose stem cell-negative hydrogen ion compound preparations, respectively) was more significant ( p <0.01), and the reducing effect was superior to the two-component formulation (Comparative Example 1 and Comparative Example 2), and even superior to the stem cell-only formulation (Comparative Example 3), showing a dose-dependent increasing trend. Notably, Aβ in the hippocampus of rats in the high-dose group was significantly higher than that in the high-dose group. 1-40 and Aβ 1-42 The content has recovered to a level similar to that of the blank control group.

[0078] Table 6 below shows the Aβ β-amyloid protein levels in rats from each experimental group. 1-40 and Aβ 1-42 Content test results (n=8, mean±SD): Table 6

[0079] Note: ## Compared with the blank control group, p <0.01; ** Compared with the model group, p <0.01; * Compared with the model group, p <0.05.

[0080] 4. Conclusion The above experimental results indicate that the stem cell-negative hydrogen ion composite formulation of the present invention can significantly improve Aβ-related diseases. 23-35 The cognitive impairment in an AD rat model induced by bilateral hippocampal injection of oligomers was significantly improved, with behavioral indicators of spatial learning and memory abilities showing significant improvement.

[0081] Mechanistic studies have shown that this compound formulation can synergistically regulate multiple key pathological processes in Alzheimer's disease (AD): effectively reducing the levels of key inflammatory factors (IL-6, IL-1β, and TNF-α) in hippocampal tissue, increasing the activity of antioxidant enzymes (SOD and GSH-Px), and reducing Aβ pathological proteins (Aβ... 1-40 and Aβ 1-42 The content) deposition, and the above effects all show a clear dose-dependent effect.

[0082] It is worth noting that, in all assessment indicators, including behavioral, inflammation, oxidative stress, and Aβ pathological protein, the improvement effect of each dosage group of this compound preparation was better than that of the two-component preparation group (stem cells and negative hydrogen ion donor, and stem cells and paclitaxel glucoside), and even better than that of the preparation group containing only stem cells (stem cells). This fully demonstrates that the combination of stem cells, negative hydrogen ions and paclitaxel glucoside has a synergistic effect.

[0083] In summary, this invention not only verifies the therapeutic potential of stem cell-negative hydrogen ion composite preparation in AD model rats, but also elucidates its mechanism of action from multiple target levels, such as anti-inflammatory, antioxidant, and Aβ pathological protein clearance, providing valuable preclinical experimental evidence for a new multi-target synergistic treatment strategy for AD.

[0084] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A stem cell-hydron complex preparation for Alzheimer's disease treatment, characterized by, comprising stem cells at a concentration of 1 x 10 6 -1 x 10 8 mg / mL, and a concentration of 4-8 pg / mL of podophyllotoxin glucoside.

2. The stem cell-hydron complex preparation for Alzheimer's disease treatment according to claim 1, wherein the stem cell-hydron complex preparation is prepared by mixing stem cells and hydrons in a ratio of 1: 1 to 1:

10. The stem cells are selected from at least one of neural stem cells, mesenchymal stem cells, and induced pluripotent stem cell-derived neural precursor cells.

3. The stem cell-hydron complex preparation for Alzheimer's disease treatment according to claim 1, wherein the stem cell-hydron complex preparation is prepared by mixing stem cells and hydrons in a ratio of 1: 1 to 1:

10. The negative hydrogen ion donor is selected from at least one of a negative hydrogen ion powder, a negative hydrogen ion tablet, and a negative hydrogen ion aqueous solution.

4. The stem cell-hydron complex preparation for the treatment of Alzheimer's disease according to any one of claims 1 to 3, wherein the stem cell-hydron complex preparation is administered in an amount of 1 x 106 cells / kg to 1 x 108 cells / kg. A pharmaceutically acceptable carrier is further included.

5. The stem cell-hydron complex preparation for Alzheimer's disease treatment according to claim 4, wherein the stem cell-hydron complex preparation is prepared by mixing stem cells and hydrons in a ratio of 1: 1 to 1:

10. The carrier is selected from at least one of physiological saline, a phosphate buffer, Hank's balanced salt solution, and Earle's balanced salt solution.

6. The stem cell-hydron complex preparation for Alzheimer's disease treatment according to claim 5, wherein the stem cell-hydron complex preparation is prepared by mixing stem cells and hydrons in a ratio of 1: 1 to 1:

10. The stem cell-negative hydrogen ion complex preparation is formulated as an injection preparation or an implant-type sustained-release preparation suitable for local administration; the injection preparation is in the form of a suspension-type injection or a lyophilized powder injection, and the implant-type sustained-release preparation suitable for local administration is in the form of a gel or a microsphere preparation.

7. The stem cell-hydron complex preparation for Alzheimer's disease treatment according to claim 6, wherein the stem cell-hydron complex preparation is prepared by mixing stem cells and hydrons in a ratio of 1: 1 to 1:

10. The stem cell-negative hydrogen ion complex preparation is formulated as an injection preparation, and the injection preparation is administered by intravenous infusion, local targeted injection, or acupoint injection.

8. The stem cell-hydron complex preparation for Alzheimer's disease treatment according to claim 7, wherein the stem cell-hydron complex preparation is prepared by the method according to claim 7. The injection preparation is administered by acupoint injection.

9. The stem cell-hydron complex preparation for Alzheimer's disease treatment according to claim 8, wherein the stem cell-hydron complex preparation is prepared by mixing stem cells and hydrons in a ratio of 1: 1 to 1:

10. The acupoint of the acupoint injection is selected from at least one of Zusanli, Fengchi, Xuehai, Fenglong, Shenshu, Baihui, Neiguan, Taichong, and Yanglingquan.

10. Use of the stem cell-negative hydrogen ion complex preparation according to claim 9 in the preparation of a medicament for the treatment of Alzheimer's disease.

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