Application of ginseng-radix aconiti lateralis preparata injection in preparation of medicine for improving Alzheimer's disease

Abstract

The invention belongs to the technical field of ginseng-radix aconiti lateralis preparata injection, and particularly relates to application of ginseng-radix aconiti lateralis preparata injection in preparation of medicine for improving Alzheimer's disease. Wherein the radix ginseng rubra-radix aconiti lateralis preparata injection is a water extract of radix ginseng rubra and radix aconiti lateralis preparata slices, and the main medicinal components are ginsenoside and aconite alkaloid. The Shenfu injection provided by the invention can maintain nitric oxide homeostasis in the serum of a mouse with Alzheimer's disease through an NOS / NO way to reduce nerve cell lesion in a CA1 area of brain tissue, so that pathological symptoms of Alzheimer's disease are improved.

Description

Technical Field

[0001] This invention belongs to the technical field of Shenfu injection, specifically relating to the application of Shenfu injection in the preparation of drugs to improve Alzheimer's disease. Background Technology

[0002] Alzheimer's disease, abbreviated as AD, is a neurodegenerative disease of the central nervous system, clinically characterized primarily by decreased memory and learning abilities and behavioral abnormalities. AD is a neurodegenerative disease of the central nervous system caused by oxidative stress in the brain, leading to the loss or apoptosis of neurons. AD can cause changes in brain tissue structure, mainly manifesting as memory impairment. In the early stages of AD, before cognitive function declines, non-cognitive symptoms such as behavioral abnormalities, sleep disturbances, and sensory dysfunction may appear. In the late stages of AD, the disease spreads to the entire cerebral cortex, with severe accumulation of neuritis plaques and neurofibrillary tangles, leading to progressive functional and cognitive impairment, and in severe cases, brain death. AD is currently the leading cause of dementia in the elderly.

[0003] The pathogenesis of Alzheimer's disease (AD) is generally believed to include abnormal deposition of β-amyloid protein, excessive phosphorylation of tau protein, abnormal mitochondrial function, neuroinflammatory responses, and synaptic conduction dysfunction. β-amyloid protein is abbreviated as Aβ. Abnormal deposition of Aβ forms insoluble amyloid plaques, abbreviated as APs. APs have strong neurotoxicity and can cause severe damage to the nervous system. Furthermore, Chen et al. found that Aβ can also exacerbate AD pathogenesis through mechanisms such as influencing oxidative stress, abnormal phosphorylation of tau protein, and neurofibrillary tangles.

[0004] Abnormalities in nitric oxide synthase (NOS) may be associated with the pathogenesis of Alzheimer's disease. NOS is a major source of nitric oxide in the nervous system, and NO is a vasoactive substance that regulates blood circulation in brain tissue, inhibiting or promoting the synthesis and release of neurotransmitters. Nitric oxide (NO) also exhibits neuronal excitotoxicity and is correlated with physiological functions such as memory and learning. However, to date, there are no reported studies demonstrating that Shenfu injection improves Alzheimer's disease in mice via the NOS / NO pathway, thus achieving a therapeutic effect on Alzheimer's disease (AD).

[0005] There are three main subtypes of nitric oxide synthase (NOS): neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS). nNOS is primarily expressed in central and peripheral neurons, affecting synapse-related learning and cognitive functions. In a healthy state, iNOS is almost not expressed, but under pathological conditions, it is continuously expressed and produces large amounts of neurotoxic NO, leading to neuroinflammatory damage to nerve cells. eNOS mainly derives NO to regulate endothelial cells and cerebral blood flow, thereby protecting brain neurons. Excessive NO produced by nNOS, under the combined action of amyloid-oligosaccharide B-peptide and neuronal hyperexcitability, leads to abnormal S-nitrosylation of various proteins. This nitrosylation reaction causes AD synaptic damage, reducing learning and cognitive abilities. Studies have found that iNOS can promote neuroinflammatory effects through oxidative and nitrogenous stress, thus playing an important role in neuronal functional impairment and decreased synaptic strength. Studies have shown that inhibiting iNOS gene expression has a significant protective effect against premature death and microglial proliferation in AD transgenic mice. Simultaneously, reducing iNOS expression levels in glial cells can protect against Aβ-induced neurological damage. Under sustained oxidative stress, the TLR4 / NF-κB pathway can induce excessive activation of iNOS, producing NO far exceeding physiological requirements. This excessive NO can induce demyelination, exposing nerve cells to harmful substances and ultimately inducing neuroinflammation. Furthermore, since NO produced by the binding of eNOS and CaM is a key factor affecting cerebral hemodynamics, decreased eNOS levels or activity lead to reduced NO production, resulting in abnormal cerebral blood flow and inducing AD. Partial eNOS deficiency can exacerbate behavioral disorders and Aβ deposition in APP / PS1 mice, further suggesting that reduced eNOS expression contributes to the pathogenesis of AD. Furthermore, the steady-state products of NOS in AD patients promote uncoupling, producing peroxynitrite superoxide. This superoxide not only causes oxidative damage to biological macromolecules, leading to the deposition of cytotoxic substances, but also reacts with Aβ, causing nitration, which is highly toxic to neurons, inducing synaptic transmission disorders and accelerating Aβ accumulation, thus contributing to the pathogenesis of AD. Studies have shown that, under pathological conditions, NO can also lead to characteristic pathological changes in AD, such as Aβ deposition, by regulating various intracellular signaling pathways. In summary, it can be inferred that the NOS / NO signaling pathway is associated with abnormal Aβ deposition and neuroinflammation; therefore, inhibiting NOS activation and reducing NO levels are of great significance for the treatment of AD.

[0006] Shenfu Injection, abbreviated as SFI, is widely used clinically to improve cardiac function, enhance myocardial blood supply, and regulate immune function. Its application has become increasingly widespread in recent years, frequently appearing in the treatment of heart failure, myocardial infarction, shock, ischemia-reperfusion injury, and cerebral infarction, with significant effects. SFI is a traditional Chinese medicine compound formula, with red ginseng and aconite as its main components. Its active ingredients are ginsenosides and aconitine alkaloids.

[0007] Currently, among existing technologies, only acetylcholinesterase inhibitors are approved for the treatment of Alzheimer's disease (AD), making the search for highly effective drugs for AD an urgent matter. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides an application of Shenfu injection in the preparation of drugs to improve Alzheimer's disease.

[0009] The purpose of this invention is to provide an application of Shenfu injection in the preparation of drugs to improve Alzheimer's disease.

[0010] The ginseng and aconite injection is prepared from water extracts of red ginseng and black aconite slices, and its main active ingredients are ginsenosides and aconite alkaloids.

[0011] Preferably, the drug is composed solely of Ginseng and Aconite Injection.

[0012] Preferably, the dosage of the ginseng and aconite injection is 10 mL / kg / d to 20 mL / kg / d.

[0013] Preferably, the drug is prepared by adding pharmaceutically acceptable excipients or auxiliary ingredients, with Shenfu injection as the main component.

[0014] Preferably, the adjuvant ingredient is a known drug for improving Alzheimer's disease.

[0015] Preferably, the excipients are selected from one or more of isotonic agents, buffers, and water for injection.

[0016] Preferably, the drug can be any one of an aqueous solution, an injection, or an infusion.

[0017] Preferably, the Shenfu injection maintains the stability of nitric oxide levels in the serum of Alzheimer's patients.

[0018] Preferably, the Shenfu injection improves the CA1 region neuronal lesions in the brain tissue of Alzheimer's patients.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The application of the ginseng and aconite injection provided by this invention in the preparation of drugs to improve Alzheimer's disease. The ginseng and aconite injection is an aqueous extract of red ginseng and black aconite slices, with ginsenosides and aconitine alkaloids as its main active ingredients. The active ingredient in the SFI of this invention is mainly ginsenosides. Ginsenosides can weaken oxidative stress and reduce damage to neurons and brain tissue in AD mice. Furthermore, ginsenosides also have protective effects on vascular endothelial cells, anti-inflammatory effects, and anti-apoptotic effects. In Examples 1-3 of this invention, with the increase of SFI dosage, the average dwell time of AD mice was 20.5 s, and the average number of platform crossings within 60 s was 1.14, which is close to the average dwell time of normal mice (21 s) and the average number of platform crossings within 60 s (1.17). However, the average dwell time of AD mice in Example 3 without SFI was 13 s, and the average number of platform crossings within 60 s was 0.17, showing a significant difference from the examples. This indicates that SFI can significantly improve AD symptoms in AD mice. The results of the water maze test showed that mice treated with SFI exhibited significantly improved memory function, indicating that SFI has a significant effect on treating the pathological changes of AD-related memory decline. HE and Nissl staining results showed that SFI can effectively restore the morphological damage of neurons in the CA1 region of the hippocampus in AD mice, as well as the number of neurons and Nissl bodies. Therefore, SFI can effectively alleviate the pathological state of AD mice.

[0020] Therefore, SFI can improve the pathological state of AD patients and can be used to prepare drugs that improve AD.

[0021] 2. The application of the Shenfu injection provided by this invention in the preparation of drugs to improve Alzheimer's disease mainly involves improving inflammatory responses and neurological function damage, and reducing neuronal apoptosis through SFI (Symptom-Free Injection), thereby treating AD caused by cerebral ischemia-reperfusion injury. This invention provides a new approach to the application of SFI and offers a new drug for the treatment of AD. Attached Figure Description

[0022] Figure 1 This is a graph showing the time mice spent in the target quadrant of a water maze according to the present invention.

[0023] Figure 2 This is a graph showing the number of times the mice of this invention passed the target platform in a water maze within 60 seconds.

[0024] Figure 3 This is a graph showing the NO content in the serum of mice according to the present invention.

[0025] Figure 4 This is an HE staining image of mouse brain tissue from the present invention. In the image, A is the control group, B is the AD model group, C is the low-dose SFI group, D is the medium-dose SFI group, and E is the high-dose SFI group.

[0026] Figure 5 This diagram illustrates the effect of Nissl staining on the CA1 region of the mouse hippocampus, as presented in this invention. A represents the control group. B represents the AD model group. C represents the low-dose SFI group. D represents the medium-dose SFI group. E represents the high-dose SFI group.

[0027] Figure 6 This is a diagram of NOS gene expression in mouse brain tissue according to the present invention.

[0028] Figure 7 This is a diagram showing the expression of NOS and Aβ proteins in mouse brain tissue according to the present invention.

[0029] Figure 8 This is a localization map of NOS and Aβ expression in mouse brain tissue according to the present invention. Detailed Implementation

[0030] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the following detailed description, in conjunction with preferred embodiments and accompanying drawings, provides a clear and complete account of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0031] It should be noted that all technical terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0032] Example 1 A method for preparing a ginseng and aconite injection includes the following steps: Take 1 kg of black aconite root slices, crush them, add 4 times their weight of water and soak for 24 hours. Adjust the pH to 4 with hydrochloric acid, add cellulase and amylase, stir, and incubate in a 37℃ water bath for 2 hours. Filter to obtain the black aconite root filtrate. Load the black aconite root filtrate onto a D101 macroporous resin, wash with water to remove sugars, and then elute with a gradient of 5 volumes of 10%, 20%, 30%, 50%, 70%, and 95% ethanol. Collect the 70% and 95% ethanol eluates, recover the ethanol under reduced pressure until no alcohol odor remains, centrifuge, and extract the supernatant 5 times with ethyl acetate. Evaporate the solvent to obtain the black aconite root extract.

[0033] Take 0.5 kg of ginseng, crush it, add 12 times its weight of water and soak for 24 hours. Adjust the pH to 3 with hydrochloric acid, add cellulase and amylase, stir, and incubate in a 37℃ water bath for 5 hours. Filter to obtain red ginseng filtrate. Load the red ginseng filtrate onto D101 macroporous resin, wash with water to remove sugars, and then elute with a gradient of 3 times its volume of 10%, 20%, 30%, 50%, 70%, and 95% ethanol. Collect the 20% and 30% ethanol eluates. Then, extract the treated ginseng four times with 9 times its volume of 70% ethanol by reflux. Combine the ethanol eluates with the reflux extracts, recover the ethanol under reduced pressure until no alcohol odor remains, centrifuge, and extract the supernatant seven times with n-butanol. Evaporate the solvent to obtain the red ginseng extract.

[0034] Take water for injection, heat to boiling, add red ginseng extract and black aconite extract, stir and mix well, adjust pH to about 5, heat to boiling, refrigerate the solution for 12 hours, filter, add activated carbon to the filtrate, boil, filter through a 0.22μm microporous membrane, add Tween-80 to the filtrate, stir to dissolve, add water for injection to make up to volume, fill into 2ml ampoules, and sterilize by steaming at 100℃ for 45 minutes to obtain SFI.

[0035] The application of Shenfu injection in improving Alzheimer's disease includes the following steps: SFI was injected into AD mice at a dose of 10 mL / kg / day for 22 consecutive days. After the administration period, the Morris water maze test was performed to observe the effect of drug treatment on the recovery of learning and memory abilities in mice. Water maze test method: A circular pool was used as the swimming area for mice. A platform was placed in the third quadrant of the pool. The water was filled to cover the platform, and the pool was colored with ink to hide the platform. Mice were placed headfirst into the pool from the first, second, and fourth quadrants, and the time required for the mice to find the platform was recorded. After 5 days of training, a 1-day rest period was given. On the 7th day, the platform was removed, and mice were placed in the pool from the first quadrant, allowing them to find the location of the platform. The time spent in the third quadrant and the number of times the mice crossed the platform within 60 seconds were recorded. The average time spent in the third quadrant was 17.5 seconds, and the average number of times the mice crossed the platform within 60 seconds was 0.82.

[0036] Example 2 The application of Shenfu injection in improving Alzheimer's disease includes the following steps: the dose of SFI in Example 1 was adjusted to 15 mL / kg / d, and other conditions were the same as in Example 1. The average dwell time of mice was 18.5 s, and the average number of times they crossed the platform within 60 s was 1.1.

[0037] Example 3 The application of Shenfu injection in improving Alzheimer's disease includes the following steps: the dose of SFI in Example 1 was adjusted to 20 mL / kg / d, and other conditions were the same as in Example 1. The average dwell time of mice was 20.5 s, and the average number of times they crossed the platform within 60 s was 1.14.

[0038] Comparative Example 1 The application of Shenfu injection in improving Alzheimer's disease includes the following steps: The SFI in Example 1 was adjusted to physiological saline, while other conditions remained the same as in Example 1. The average dwell time for mice was 13 seconds, and the average number of times they crossed the platform within 60 seconds was 0.17.

[0039] Comparative Example 2 The application of Shenfu injection in improving Alzheimer's disease includes the following steps: The AD mice in Example 1 were adjusted to normal mice, and SFI was adjusted to physiological saline; other conditions remained the same as in Example 1. The average dwell time of the mice was 21 seconds, and the average number of times they crossed the platform within 60 seconds was 1.17.

[0040] The results of Examples 1-3 show that as the dosage of SFI increased, the mice gradually recovered to normal. As shown in Comparative Example 2, the average dwell time of the AD mice in Example 3 was 20.5 s, and the average number of platform crossings within 60 s was 1.14, which is close to the average dwell time of the normal mice (21 s) and the average number of platform crossings within 60 s (1.17). In contrast, the average dwell time of the AD mice that did not use SFI was 13 s, and the average number of platform crossings within 60 s was 0.17. Therefore, SFI can significantly improve the AD symptoms in AD mice.

[0041] To illustrate the beneficial effects of the present invention, the following experiments were also conducted.

[0042] I. Materials and Equipment 1. Experimental Materials The main materials used in this invention include ginseng and aconite injection, D-galactose, scopolamine hydrobromide, hematoxylin-eosin staining kit, Nissl staining solution, rabbit anti-nNOS, rabbit anti-eNOS, rabbit anti-iNOS, rabbit anti-Aβ, goat anti-rabbit, RNA extraction kit, immunohistochemistry kit, sodium nitrite, N-1-naphthylethylenediamine acid, and anhydrous p-aminobenzenesulfonic acid.

[0043] Among them, the following were included: Shenfu injection (50mL / vial), purchased from China Resources Sanjiu Pharmaceutical Co., Ltd., National Drug Approval Number Z20043117; D-galactose, Macklin, batch number C17022416; Scopolamine hydrobromide, Macklin, batch number C17268965; Hematoxylin-eosin staining kit, purchased from Beijing Regen Biotechnology Co., Ltd., batch number 0417A25; Nissl staining solution, Solarbio, batch number 240001001; Rabbit anti-nNOS, Origene, batch number TA367542; Rabbit anti-eNOS, RuiYing, batch number E6872; Rabbit anti-iNOS, Origene, batch number TA422856; Rabbit anti-Aβ, Bioss, batch number bs-0107R. Goat anti-rabbit kit, Bioss, batch number BE02187352. RNA extraction kit, Mei5bio, batch number 24MA1114. Immunohistochemistry kit, Boster, batch number 20B20B02. Sodium nitrite, purchased from Shanghai Maitener Chemical Technology Co., Ltd., batch number M10097462. N-1-naphthylethylenediamine acid, purchased from Tianjin Xiens Biochemical Technology Co., Ltd., batch number N13000MCN1. Anhydrous p-aminobenzenesulfonic acid, Shanghai Maitener, batch number M10097842.

[0044] Mice were purchased from Hunan Slack Jingda Laboratory Animal Co., Ltd. They were SPF-grade Kunming mice, weighing 17g-20g. Humane care was provided to laboratory animals according to the 3R principle during the experiment.

[0045] II. Experimental Methods 1. Preparation, grouping, and administration of AD model mice Sixty mice (30 males and 30 females, totaling 60 mice) were acclimatized to their new living environment for three days and then randomly divided into a control group and a model group. The control group consisted of 12 mice (BG group), and the model group consisted of 48 mice. Mice in the model group received subcutaneous injections of D-galactose (300 mg / kg / day) in the neck for 21 consecutive days, followed by intraperitoneal injections of scopolamine hydrobromide (2 mg / kg / day) for 15 consecutive days to induce the model. The control group received the same injection of saline. The modeling effect was assessed by observing the daily condition of the mice, comparing their water and food intake, coat luster and density, and behavior with the control group. After modeling, the mice in the model group were randomly divided into an AD model group and four groups (12 mice each): a low-dose group (AD group), a medium-dose group (SFI-L group), and a high-dose group (SFI-L group). The medium-dose group of Shenfu injection, with a dose of 15 mL / kg / d, was designated SFI-M. The high-dose group of Shenfu injection, with a dose of 20 mL / kg / d, was designated SFI-H. Mice in the BG and AD groups were intraperitoneally injected with 15 mL / kg / d of physiological saline. All Shenfu injection groups were administered the same medications for a total of 22 days.

[0046] 2. Behavioral experiments on mice After drug administration, the Morris water maze test was conducted to observe the effect of drug treatment on the recovery of learning and memory abilities in mice. Water maze test method: A circular pool was used as the swimming area for mice. A platform was placed in the third quadrant of the pool. The pool was filled with water to cover the platform, and ink was used to change the pool color to hide the platform. Mice were placed headfirst into the pool, sequentially from the first, second, and fourth quadrants, and the time required for the mice to find the platform was recorded. After 5 days of training, a 1-day rest period was given. On the 7th day, the platform was removed, and mice were placed back into the pool from the first quadrant, allowing them to search for the previously located platform. The time spent in the third quadrant and the number of times the mice crossed the platform within 60 seconds were recorded.

[0047] 3. Preparation of mouse tissue samples After the Morris water maze experiment, mice were fasted for one day but allowed free access to water, and were collected the following day. Before serum preparation, mice were anesthetized with 3% sodium barbital, and blood was collected from the orbital venous plexus. Using random sampling, three anesthetized mice were selected from each group, and their thoracic cavities were dissected to expose the heart. The right atrial appendage was then opened. After blood flowed out, a catheter was inserted into the left ventricle. 50 mL of physiological saline was instilled until clear fluid flowed from the right atrial appendage, followed by continuous perfusion with 4% paraformaldehyde until the liver turned white and the neck showed a rigid response. The skull was opened, and the entire brain of the mice was placed in 10% formaldehyde for tissue staining. The remaining mice were anesthetized and euthanized, and their entire brain tissue was removed and stored at -80°C.

[0048] 4. Histopathological examination of hippocampal tissue in mice HE staining: Dewaxed brain tissue sections were sequentially treated with hematoxylin for 5 min, differentiation solution for 30 s, and eosin for 2 min, rinsing with distilled water after each treatment to obtain brain tissue sections. The brain tissue sections were dehydrated in ethanol solution, cleared with xylene, mounted with neutral resin, and the morphology of hippocampal CA1 neurons was observed.

[0049] Nissl staining: Dewaxed brain tissue sections were stained with tar purple in a 56℃ incubator for 1 hour, then differentiated with Nissl differentiation solution for 2 minutes. After each step, the sections were rinsed with distilled water. The brain tissue sections were then dehydrated in ethanol solution, cleared with xylene, mounted with neutral resin, and the morphology of the hippocampus and Nissl bodies was observed.

[0050] 5. Detection of NO content in mouse serum 50 μL of NaNO₂ standard solutions with concentrations of 1.56 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25.0 μM, 50.0 μM, and 100 μM, and 50 μL of serum samples to be tested, were added to different wells of a 96-well plate, respectively. At 37 °C, 50 μL of Griess reagent A and 50 μL of Griess reagent B were added sequentially, reacting for 10 min after each addition. The plate was shaken several times before detection, and the absorbance was measured at 540 nm. Absorbance is abbreviated as OD. A standard curve for NO content was plotted based on the OD values ​​of the standards, and the NO concentration in each serum sample was calculated using the equation corresponding to the curve.

[0051] 6. Detection of nitric oxide synthase gene expression in mice 30 mg of mouse brain tissue was collected, and total RNA was extracted using an RNA extraction kit. The RNA was then reverse transcribed into cDNA and amplified by qPCR. The primers for the RNA extraction were shown in Table 1. For detailed procedures, please refer to the RNA extraction kit instructions. The relative gene expression levels of nNOS, iNOS, and eNOS in nitric oxide synthase were calculated using 2-ΔΔCt.

[0052] Table 1 qPCR gene primers 7. Expression and localization of β-amyloid protein and nitric oxide synthase in mouse brain tissue Mouse brain tissue was collected and subjected to routine fixation, dehydration, embedding, sectioning, and sealing slides. β-amyloid protein is abbreviated as Aβ. The expression of Aβ, nNOS, iNOS, and eNOS in brain tissue was measured according to the kit instructions. Brownish-yellow granules appearing in the field of view were observed and photographed under an optical microscope, indicating enhanced expression of these proteins. The expression levels of each group were compared, and the number of positive staining particles in the field of view was calculated.

[0053] 8. Statistical Analysis All data were analyzed using SPSS 20.0 statistical software, with mean ± standard deviation and α = 0.05 as the criteria for difference testing. One-way ANOVA was used for comparisons among multiple groups, LDS was used for data that conformed to homogeneous variance and normal distribution, Tamhan's ST2 test was used for data that conformed to normal distribution but had unequal variances, and nonparametric tests were used for data that did not conform to normal distribution.

[0054] III. Experimental Results 1. Behavioral experiments on mice Behavioral results of mice as follows Figure 1 and Figure 2 As shown in the figure. The results showed that compared with the BG group, the AD group had significantly less time spent in the third quadrant and fewer times to the plateau within 60 seconds (P < 0.05). However, compared with the AD group, the Shenfu injection group had significantly more time spent in the third quadrant and more times to the plateau within 60 seconds (P < 0.05), and the learning and memory abilities of the mice were enhanced to varying degrees with increasing Shenfu injection dosage. This indicates that Shenfu injection can improve the memory ability of mice.

[0055] 2. NO content in mouse serum The effect of Shenfu injection on NO content in mouse serum is as follows: Figure 3As shown in the figure. The results showed that compared with the BG group, the NO content in the serum of mice in the AD group was significantly increased (P < 0.05). However, compared with the AD group, the NO content in the serum of mice in each dose group of Shenfu injection was significantly decreased (P < 0.05), indicating a statistically significant difference. Furthermore, due to different dosages, the NO content in the mouse serum also showed varying degrees of decrease. This indicates that Shenfu injection reduced the NO content in mouse serum.

[0056] 3. Pathological examination of hippocampal tissue in mice Effects of Shenfu injection on hippocampal tissue in AD model mice: Figure 4 and Figure 5 As shown. HE staining Figure 4 As shown, the results indicated that, compared with the BG group, the CA1 region of the hippocampus in mice with the AD model exhibited significant pathological features, specifically a marked decrease in neuronal cell density, discontinuity of cell layers in some areas, and nuclear pyknosis and intensified cytoplasmic staining in neurons. However, after intervention with different concentrations of Shenfu injection, the degree of hippocampal tissue damage in all Shenfu injection groups was reduced, and the orderly arrangement of neuronal cells was significantly improved.

[0057] Nissl staining, such as Figure 5 As shown, the results indicated that compared to the AD group, the AD group mice had a significantly decreased number of neurons and Nissl bodies in the CA1 region of the hippocampus, and a significantly increased proportion of vacuolated cells. The AD group mice exhibited abnormal cell morphology, characterized by shrinkage. With increasing dosage of Shenfu injection, the thickness of the hippocampal neuronal cell layer increased in the Shenfu injection group mice, the cell arrangement became more orderly, the morphology of most neurons recovered to a state closer to normal, and the number of Nissl bodies also showed a significant improvement.

[0058] HE and Nissl staining results indicate that Shenfu injection can improve the severity of lesions in the CA1 region of the mouse hippocampus.

[0059] 4. Detection of nitric oxide synthase gene expression in mouse brain tissue The effects of Shenfu injection on the expression of nNOS, iNOS, and eNOS genes in the brain tissue of AD model mice are as follows: Figure 6 As shown in the figure. The results showed that compared with the BG group, the expression of eNOS gene in the brain tissue of mice in the AD group was significantly reduced, while the expression of nNOS and iNOS genes was significantly increased (P < 0.05). Different doses of Shenfu injection could reduce the expression of nNOS and iNOS genes in mouse brain tissue to varying degrees, while increasing the expression of eNOS gene (P < 0.05). The results of nitric oxide synthase gene expression indicate that Shenfu injection can reduce the expression of nNOS and iNOS genes in mouse brain tissue, while increasing the expression of eNOS gene.

[0060] 5. Expression and localization of β-amyloid protein and nitric oxide synthase in mouse brain tissue The effects of Shenfu injection on the expression of nNOS, iNOS, eNOS and Aβ proteins in the brain tissue of AD model mice are as follows: Figure 7 and Figure 8 As shown. Figure 7 The results showed that Shenfu injection significantly improved the expression of various NOS subtypes and Aβ in the brain tissue of AD mice. Compared with the BG group, the positive staining of nNOS, iNOS, and Aβ in the brain tissue of AD mice was significantly increased, while the positive staining of eNOS was significantly decreased (P < 0.05). Figure 8 The results showed that, compared with the AD group mice, the positive staining of nNOS, iNOS, and Aβ in the brain tissue of the Shenfu injection group mice was significantly decreased, and the positive staining gradually decreased with increasing Shenfu injection dosage, while the positive staining of eNOS showed the opposite trend (P < 0.05). Shenfu injection can reduce the expression of nNOS, iNOS, and Aβ in mouse brain tissue.

[0061] In summary, Shenfu injection can reduce the expression of nNOS and iNOS genes in mouse brain tissue while increasing the expression of eNOS gene, thus lowering the NO content in mouse serum. Shenfu injection can increase the thickness of the neuronal cell layer in the CA1 region of the mouse hippocampus, resulting in a more orderly arrangement of neurons, a restoration of most neurons to a more normal morphology, and a significant improvement in the number of Nissl bodies, thereby improving the severity of lesions in the CA1 region of the mouse hippocampus. Therefore, Shenfu injection can improve the memory ability of mice by reducing the expression of nitric oxide synthase and the severity of lesions in the CA1 region of the hippocampus.

[0062] IV. Discussion of Results Compared to the control group, mice in the AD model constructed by this invention showed a significant decline in memory function and a marked increase in brain tissue pathological damage, with abnormal Aβ deposition in the brain tissue. Experimental results from this invention showed that the levels of nNOS and iNOS genes and proteins in the brain tissue and serum NO content were significantly increased in the AD model group mice, while the expression of eNOS genes and proteins was decreased. Therefore, AD mice exhibit abnormal NO utilization due to changes in the expression of NOS subtypes, leading to abnormal Aβ deposition and neuroinflammation, inhibiting cerebral blood flow, damaging neurons, and consequently affecting learning, memory, and cognitive abilities. By observing changes in cognitive ability and oxidative stress response in mice, it can be concluded that SFI can treat AD symptoms caused by Aβ deposition. This invention found that SFI injection significantly reduced Aβ expression in the brain tissue of AD mice, therefore SFI can reduce abnormal Aβ deposition.

[0063] In summary, our experimental results indicate that Shenfu injection may exert its therapeutic effect on Alzheimer's disease (AD) through the NOS / NO signaling pathway. The mechanism involves regulating the expression of various NOS subtypes, influencing NO bioavailability, and reducing abnormal Aβ deposition, thereby protecting cerebral blood vessels and neurons, and ultimately improving AD. This invention provides a novel drug, theoretical basis, and therapeutic target for SFI-related traditional Chinese medicine treatment of AD.

[0064] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0065] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. The application of a ginseng and aconite injection in the preparation of a drug for improving Alzheimer's disease, characterized in that, in, The ginseng and aconite injection is prepared from water extracts of red ginseng and black aconite slices, and its main active ingredients are ginsenosides and aconite alkaloids.

2. The application of the Shenfu injection according to claim 1 in the preparation of a drug for improving Alzheimer's disease, characterized in that, The drug is composed solely of Ginseng and Aconite Injection.

3. The application of the Shenfu injection according to claim 1 in the preparation of a drug for improving Alzheimer's disease, characterized in that, The drug is prepared by adding pharmaceutically acceptable excipients or auxiliary ingredients, with Shenfu injection as the main component.

4. The application of the Shenfu injection according to claim 3 in the preparation of a drug for improving Alzheimer's disease, characterized in that, The adjuvant ingredient is a known drug for improving Alzheimer's disease.

5. The application of the Shenfu injection according to claim 3 in the preparation of a drug for improving Alzheimer's disease, characterized in that, The excipients are selected from any one or more of isotonic agents, buffers, and water for injection.

6. The application of the Shenfu injection according to claim 3 in the preparation of a drug for improving Alzheimer's disease, characterized in that, The drug can be any one of aqueous solution, injection, or infusion.

7. The application of the Shenfu injection according to claim 1 in the preparation of a drug for improving Alzheimer's disease, characterized in that, The aforementioned Shenfu injection maintains the stability of nitric oxide levels in the serum of Alzheimer's patients.

8. The application of the Shenfu injection according to claim 1 in the preparation of a drug for improving Alzheimer's disease, characterized in that, The aforementioned Shenfu injection improves neuronal lesions in the CA1 region of the brain tissue of Alzheimer's patients.

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