Construction and Evaluation Method of an Animal Model of Alzheimer's Disease with Deficiency and Blood Stasis

Abstract

This invention relates to a method for constructing and evaluating an animal model of Alzheimer's disease characterized by deficiency and blood stasis. Based on APP / PS1 mice, the model was induced by sleep deprivation, hydrocortisone, and carrageenan to induce qi deficiency, kidney deficiency, and blood stasis, respectively, for a total modeling period of 28 days, resulting in an animal model of Alzheimer's disease with deficiency and blood stasis. This model was constructed using a multi-factor combined approach, and its symptom manifestations are consistent with clinical observations. It is simple, effective, and easy to operate, providing a reliable experimental vehicle for the development of innovative drugs and the study of mechanisms of action in Alzheimer's disease with deficiency and blood stasis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for constructing and evaluating an animal model of Alzheimer's disease of deficiency and blood stasis and toxin type. BACKGROUND

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive impairment. The main pathological features are β-amyloid deposition (Aβ) and Tau protein hyperphosphorylation. Epidemiological data reveals that the number of AD patients in China is increasing year by year. By 2030, the size of the dementia patient population in China will expand dramatically to about 78 million, and by 2050, this number will rise to 139 million, highlighting the severe challenges and growing trend of AD in China.

[0003] Traditional Chinese medicine believes that the root cause of AD is the kidney. The kidney is the foundation of congenital, storing essence and marrow, and the brain is the marrow sea. If the kidney essence is deficient, the marrow sea will not be nourished. In addition, deficiency of Qi is another important pathological link of AD. If the Qi is insufficient, the blood stasis will be stagnant, the blood stasis will not be transformed, the brain collaterals will not be unblocked, the blood stasis will be generated, and the Qi will be further blocked, which will block the clear orifices and damage the brain collaterals, eventually forming various clinical manifestations of AD. AD is caused by a variety of pathogenic factors, and most of the clinical AD patients are elderly people, who generally show the characteristics of kidney essence deficiency, Qi and blood weakness, accompanied by the decline of zang-fu function, and the slow circulation of blood and body fluid, showing a complex situation of coexistence and mutual influence of various pathological factors such as deficiency, blood stasis and toxin.

[0004] Traditional Chinese medicine animal models serve as a bridge between traditional medicine and modern medicine, simulating certain characteristics or pathological processes of human diseases, providing experimental basis for traditional Chinese medicine research, and playing an important role in the development of new drugs of traditional Chinese medicine. However, current AD animal models mainly focus on natural aging model, rapid aging mouse model, cholinergic system damage model, transgenic animal model and aluminum poisoning model, and AD animal models related to TCM syndrome typing are extremely scarce. Although some researchers have used a combination of hydrocortisone and intracerebral Aβ injection to construct a kidney deficiency type AD rat model, this model only simulates the kidney deficiency syndrome and does not include other important syndromes such as blood stasis. In addition, the uncertainty of multi-factor modeling and high mortality rate pose great challenges to the construction of multi-factor TCM AD animal models.

[0005] There are many challenges in multi-factor TCM animal models. On the one hand, current animal models are made according to TCM etiology and pathogenesis, combined with western surgical, physical, chemical and other methods, which is not consistent with the natural onset of the disease. On the other hand, some chemical reagents, surgical operation and other modeling factors cause too much trauma and infection, which makes the survival rate of modeling animals very low and easily interferes with the purity of the syndrome characteristics, so surgery modeling is not used as much as possible [Zhao Hui. On the multi-factor combined animal model of disease and syndrome [J]. Journal of Anhui University of Chinese Medicine, 2001, (05): 57-59.]. In addition, the experimental grouping is not scientific enough, the single modeling method lacks recognition, the repeatability is poor, and the correlation of the detection index is poor, which are the problems of multi-factor modeling at present. This also highlights the urgency and importance of constructing a TCM AD animal model that can fully simulate the pathological characteristics of TCM multiple factors such as deficiency, blood stasis and toxicity. SUMMARY

[0006] The technical problem to be solved by the present application is to construct a virtual loss blood stasis and toxicity type AD animal model by using multi-factor combination method in view of the current situation of the scarcity of AD animal models related to TCM, the uncertainty and high mortality rate of multi-factor modeling. The present application selects APP / PS1 mice as experimental objects. This mouse variety is a classic animal model for AD research. At the age of 4 to 6 months, it can naturally form intracerebral Aβ plaques, thereby avoiding additional damage to the mouse caused by invasive operations such as intracerebral injection of Aβ, and ensuring a high survival rate of AD mice. Further, to explore the feasibility of multi-factor combined modeling of APP / PS1 mice, the present application first explores the conditions for inducing blood stasis by carrageenan on APP / PS1 background mice. Finally, sleep deprivation, hydrocortisone gavage and intraperitoneal injection of carrageenan are selected to induce qi deficiency, kidney deficiency and blood stasis, respectively. This strategy not only simplifies the experimental process and reduces complex and repetitive experimental operations, but also ensures a high survival rate and modeling success rate of mice under multi-factor combined modeling. The present application provides a construction and evaluation method for a virtual loss blood stasis and toxicity type AD animal model. The virtual loss blood stasis and toxicity type AD animal model is constructed by using multi-factor combination method such as sleep deprivation and chemical stimulation. The construction method is simple and has strong operability, can reduce the mortality rate of multi-factor combined modeling, and can effectively simulate the syndrome manifestations of virtual loss blood stasis and toxicity type AD patients, thereby providing a reliable experimental carrier for the research and development of innovative drugs for virtual loss blood stasis and toxicity type AD and the study of their action mechanisms.

[0007] To solve the above technical problems, the present application discloses a construction and evaluation method for a virtual loss blood stasis and toxicity type AD animal model, and the specific technical solutions are as follows:

[0008] A method for constructing an animal model of AD of deficiency, damage, blood stasis and toxin type, the AD double transgenic mice are subjected to continuous sleep deprivation and injection of carrageenan simultaneously every day for 28 days; starting from the 20th day, hydrocortisone is administered intragastrically once every other day for a total of 4 times. Preferably, the continuous sleep deprivation is performed in a way of inducing qi deficiency, if the difference in qi deficiency indicators between the sleep-deprived mice and the blank control group mice is statistically significant, it indicates that qi deficiency can be induced successfully; the hydrocortisone intragastric administration is performed in a way of inducing kidney deficiency, if the difference in kidney deficiency indicators between the hydrocortisone-induced mice and the blank control group mice is statistically significant, it indicates that kidney deficiency can be induced successfully; the carrageenan injection is performed in a way of inducing blood stasis, if the difference in blood stasis indicators between the carrageenan-induced mice and the blank control group mice is statistically significant, it indicates that blood stasis can be induced successfully; if the difference in toxin damage indicators between the transgenic mice and the blank control mice is statistically significant, it indicates that toxin damage can be induced successfully; if the multi-factor composite model group shows statistical differences in qi deficiency, kidney deficiency, blood stasis, toxin damage and other evaluation indicators compared with the blank control group mice, it indicates that the AD animal model of deficiency, damage, blood stasis and toxin type is constructed successfully.

[0009] Preferably, the brain tissue pathological characteristics of the AD double transgenic mice are progressive amyloid deposition in the cerebral cortex or hippocampus, and more preferably, the AD double transgenic mice are 5 months old.

[0010] Preferably, the rotation speed is 8 rpm, the interval time of each rotation is 2 min, and the sleep deprivation time per day is 10 hours.

[0011] Preferably, the carrageenan is injected in the form of a carrageenan solution, and the solvent is physiological saline; the injection concentration of the carrageenan is 0.6 mg / mL, and the injection dose is 0.1 mL / 10 g.

[0012] Preferably, the intragastric dose of the hydrocortisone is 50 mg / kg / time.

[0013] Preferably, the method further comprises evaluation indicators of AD of deficiency, damage, blood stasis and toxin type, and the evaluation includes qi deficiency indicators, kidney deficiency indicators, blood stasis indicators, toxin damage indicators and AD model evaluation indicators.

[0014] Preferably, the qi deficiency indicators include body weight, grip strength index and spleen index.

[0015] The kidney deficiency indexes include body weight, kidney index, testis index and Cortisol level;

[0016] The blood stasis indexes include VACM-1, IL-6, IL-1β and TNF-α levels;

[0017] The toxin damage indexes include Aβ deposition in hippocampus, expression levels of Aβ generation related proteins APP, PS1 and BACE-1 in hippocampus.

[0018] The AD model evaluation indexes include learning and memory ability evaluation and brain tissue Nissl pathology.

[0019] Compared with the blank control mice, the kidney deficiency and blood stasis toxin AD mice treated by the multi-factor have the decreased body weight, decreased grip strength index and decreased spleen index.

[0020] Compared with the blank control mice, the kidney deficiency and blood stasis toxin AD mice treated by the multi-factor have the decreased body weight, significantly decreased kidney index, significantly decreased testis index and significantly decreased serum Cortisol content.

[0021] Compared with the blank control mice, the kidney deficiency and blood stasis toxin AD mice treated by the multi-factor have the increased VCAM-1 content, increased TNF-α, IL-6 and IL-1β contents.

[0022] Compared with the blank control mice, the kidney deficiency and blood stasis toxin AD mice treated by the multi-factor have the significantly enhanced Aβ immunofluorescence intensity in brain tissue, and the significantly increased APP, PS1 and BACE-1 protein expression in hippocampus.

[0023] Compared with the blank control mice, the kidney deficiency and blood stasis toxin AD mice treated by the multi-factor have the significantly damaged neurons in brain tissue Nissl pathology, significantly prolonged escape latency in water maze and significantly decreased learning and memory ability.

[0024] In the second aspect, the animal model constructed by the construction method in the first aspect is applied to screening drugs for treating or improving the deficiency, damage and blood stasis toxin type AD.

[0025] In the third aspect, the animal model constructed by the construction method in the first aspect is applied to the pathogenesis research and drug efficacy evaluation of the deficiency, damage and blood stasis toxin type AD.

[0026] Beneficial effects:

[0027] This invention provides a method for constructing and evaluating an animal model of AD with deficiency, blood stasis, and toxicity. The animal model of AD with deficiency, blood stasis, and toxicity constructed by this method exhibits syndrome characteristics consistent with clinical observation results. It can provide a reliable experimental vehicle for the innovative drug development, market evaluation, and mechanism of action research of AD with deficiency, blood stasis, and toxicity, filling the gap in the existing technology for TCM AD animal models that comprehensively simulate the TCM pathological characteristics of deficiency, blood stasis, and toxicity. Attached Figure Description

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0029] Figure 1 This is a pathological result diagram of the aortic endothelium of mice after intervention with different concentrations of carrageenan in one embodiment of the present invention. A, B, C, and D represent the control group, the 0.02% carrageenan group, the 0.04% carrageenan group, and the 0.06% carrageenan group, respectively.

[0030] Figure 2 This is a graph showing the content of vascular cell adhesion factor VCAM-1 and inflammatory factors TNF-α, IL-6, and IL-1β in mice after intervention with different concentrations of carrageenan in one embodiment of the present invention. A, B, C, and D represent the control group, the 0.02% carrageenan group, the 0.04% carrageenan group, and the 0.06% carrageenan group, respectively.

[0031] Figure 3 This is a diagram of a sleep deprivation experiment.

[0032] Figure 4 This is a graph showing the body weight data of mice in this invention. TW, DS, QX, SX, XS, and FH represent the blank control group, the brain toxicity group, the qi deficiency toxicity group, the kidney deficiency toxicity group, the deficiency toxicity group, and the deficiency stasis toxicity group, respectively.

[0033] Figure 5 This is a data graph of mouse gripping index in one embodiment of the present invention. TW, DS, QX, SX, XS, and FH represent the blank control group, the brain toxicity group, the qi deficiency toxicity group, the kidney deficiency toxicity group, the deficiency toxicity group, and the deficiency stasis toxicity group, respectively.

[0034] Figure 6 This is a graph of mouse spleen index data in one embodiment of the present invention. TW, DS, QX, SX, XS, and FH represent the blank control group, the brain toxicity group, the qi deficiency toxicity group, the kidney deficiency toxicity group, the deficiency toxicity group, and the deficiency stasis toxicity group, respectively.

[0035] Figure 7This is a graph showing mouse kidney and testicular index data in one embodiment of the present invention. TW, DS, QX, SX, XS, and FH represent the blank control group, the brain toxicity group, the qi deficiency toxicity group, the kidney deficiency toxicity group, the deficiency toxicity group, and the deficiency stasis toxicity group, respectively.

[0036] Figure 8 This is a graph showing the cortisol content in mouse serum in one embodiment of the present invention. TW, DS, QX, SX, XS, and FH represent the blank control group, the brain toxicity group, the qi deficiency toxicity group, the kidney deficiency toxicity group, the deficiency toxicity group, and the deficiency stasis toxicity group, respectively.

[0037] Figure 9 This is a graph showing the levels of vascular cell adhesion factor VCAM-1 and inflammatory factors TNF-α, IL-6, and IL-1β in mouse serum according to one embodiment of the present invention. TW, DS, QX, SX, XS, and FH represent the blank control group, the brain vascular toxicity group, the qi deficiency toxicity group, the kidney deficiency toxicity group, the deficiency toxicity group, and the deficiency stasis toxicity group, respectively.

[0038] Figure 10 This is a graph showing the escape latency time, platform quadrant dwell time, and platform crossing number in a mouse water maze experiment according to one embodiment of the present invention. TW, DS, QX, SX, XS, and FH represent the blank control group, the brain toxicity group, the qi deficiency toxicity group, the kidney deficiency toxicity group, the deficiency toxicity group, and the deficiency stasis toxicity group, respectively.

[0039] Figure 11 This is a pathological image of mouse brain tissue in one embodiment of the present invention. TW, DS, QX, SX, XS, and FH represent the blank control group, the brain toxicity group, the qi deficiency and toxicity group, the kidney deficiency and toxicity group, the deficiency and toxicity group, and the deficiency and stasis toxicity group, respectively.

[0040] Figure 12 This is an image showing the Aβ deposition results in mouse hippocampal tissue according to one embodiment of the present invention. TW, DS, QX, SX, XS, and FH represent the blank control group, the brain toxicity group, the qi deficiency toxicity group, the kidney deficiency toxicity group, the deficiency toxicity group, and the deficiency stasis toxicity group, respectively.

[0041] Figure 13 This is a graph showing the expression results of APP, BACE1, and PS1 proteins in mouse hippocampal tissue in one embodiment of the present invention. TW, DS, QX, SX, XS, and FH represent the blank control group, the brain toxicity group, the qi deficiency toxicity group, the kidney deficiency toxicity group, the deficiency toxicity group, and the deficiency stasis toxicity group, respectively. Detailed Implementation

[0042] The following is a detailed description of the specific embodiments of the present invention in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only for the purpose of illustrating and explaining the present invention, and are not intended to limit the present invention. The reagents, instruments and equipment involved in the embodiments of the present invention can be obtained by purchase unless otherwise specified.

[0043] The sources of the mice used in the following examples are as follows: SPF-grade APP / PS1 mice, male, 30 mice, 5 months old, with a body weight range of 25-35 g; SPF-grade littermate control mice, male, 6 mice, 5 months old, with a body weight range of 25-35 g; SPF-grade C57BL / 6J mice, male, 24 mice, 2 months old, with a body weight range of 22-28 g. The mice were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd., and the experimental animal production license number is SCXK(Zhe)2019-0004. The animal experiment was approved by the Ethics Committee of the Affiliated Hospital of Nanjing University of Chinese Medicine, and the approval number is 2023DW-037-01.

[0044] The mice were uniformly housed in the SPF-grade animal house of the Animal Experiment Center of China Pharmaceutical University, with free access to food and water, an environmental temperature of 22-25 °C, and a relative humidity of 40%-60%.

[0045] The sources of the drugs and reagents used in the embodiments of the present invention are as follows: Hydrocortisone injection (Shandong Ruicheng Hongbao Veterinary Drug Co., Ltd., veterinary drug approval number (2016)040101269); Carrageenan (Shanghai Macklin Biochemical Co., Ltd., product number: C804872); Normal saline injection (Anhui Fengyuan Pharmaceutical Co., Ltd., national drug approval number H20093237); TNF-α, IL-6, IL-1β, VACM-1, Cortisol enzyme-linked immunosorbent assay kits (Shanghai Enzyme-Free Biotechnology Co., Ltd., product numbers are: MM-0132M1, MM-0163M1, MM-0040M1, MM-0129M1, MM-0565M2); APP, PS-1, β-actin primary antibodies (Wuhan Sanying Biotechnology Co., Ltd., product numbers are: 25524-1-AP, 16163-1-AP, 66009-1-Ig), BACE1 antibody (Hunan Aifang Biotechnology Co., Ltd., product number AFW5266).

[0046] The instruments used in this invention embodiment are: XR-XS108 sleep deprivation device (Shanghai Xinruan Information Technology Co., Ltd.); XR501 rat and mouse grip strength tester (Shanghai Xinruan Information Technology Co., Ltd.); Morris water maze video analysis system (Beijing Zhongshidichuang Technology Development Co., Ltd.); Donatello dehydrator (DIAPATH, Italy); JB-P5 embedding machine (Wuhan Junjie Electronics Co., Ltd.); RM2016 pathological slide machine (Leica Instruments Co., Ltd., Shanghai); G6100 upright fluorescence microscope (Nikon, Japan); Pannoramic MID scanner (3DHISTECH, Hungary); Tanon-4600 chemiluminescence system (Shanghai Tianneng Life Science Co., Ltd.); EL10A automatic microplate reader (Shandong Boke Holding Group Co., Ltd.).

[0047] This invention uses Graph Pad Prism 8.4.3 software for statistical analysis. Data are expressed as mean ± standard deviation. Data conforming to a normal distribution are compared between groups using one-way ANOVA, while data not conforming to a normal distribution are compared using nonparametric tests. P < 0.05 indicates that the difference is statistically significant.

[0048] The animal model provided by this invention can objectively evaluate the syndrome manifestations from multiple aspects, including symptoms of kidney deficiency, qi deficiency, and blood stasis, brain tissue pathology, and β-amyloid protein deposition in brain tissue. This animal model can provide a reliable experimental vehicle for the innovative drug development, re-evaluation of marketed products, and mechanism of action research of AD with kidney deficiency and blood stasis. In this invention, "statistically significant difference" is defined as P<0.05.

[0049] The indicators for Qi deficiency syndrome described in this invention include body weight, grip strength index, and spleen index.

[0050] The indicators for kidney deficiency syndrome described in this invention include body weight, kidney index, testicular index, and cortisol level.

[0051] The indicators of blood stasis syndrome described in this invention include the levels of VCAM-1, IL-6, IL-1β, and TNF-α.

[0052] The toxicity indicators described in this invention include the Aβ deposition status in the hippocampus and the expression levels of Aβ-related proteins APP, PS1, and BACE-1 in the hippocampus.

[0053] The evaluation indicators of the AD model described in this invention include Nissl pathology of brain tissue and the water maze test in behavioral experiments to evaluate the learning and memory abilities of mice.

[0054] Example 1

[0055] This example illustrates the selection of intervention conditions for carrageenan-induced blood stasis in mice. The different concentrations of carrageenan described in this example were prepared using physiological saline at a mass-to-volume ratio (g / mL).

[0056] 1.1 Animal grouping and intervention:

[0057] Twenty-four SPF-grade C57BL / 6J mice, six in each group, were randomly divided into four groups, as follows:

[0058] Control group (Group A): normal saline, intraperitoneal injection, 0.1 mL / 10 g;

[0059] 0.02% carrageenan group (Group B): 0.02% carrageenan, intraperitoneal injection, 0.1 mL / 10 g;

[0060] 0.04% carrageenan group (Group C): 0.04% carrageenan, intraperitoneal injection, 0.1 mL / 10 g;

[0061] 0.06% carrageenan group (Group D): 0.06% carrageenan, intraperitoneal injection, 0.1 mL / 10 g;

[0062] After one week of acclimatization feeding, mice in each group underwent a 28-day modeling process.

[0063] 1.2 Animal sampling and sample processing:

[0064] After modeling, blood was collected by enucleating the eyeballs, and the mice were then euthanized by cervical dislocation. The aorta and epididymal fat were separated, fixed in 4% paraformaldehyde for 24 hours, and then embedded in paraffin and sectioned for histopathological staining. Whole blood was allowed to stand for 20 minutes, then centrifuged at 3500 rpm for 10 minutes, and the serum was collected and stored at -20°C.

[0065] 1.3 Detection indicators and results:

[0066] (1) Histopathological analysis: Prepared paraffin sections of the aorta were taken out and dewaxed and hydrated in sequence. The sections were then stained using the hematoxylin-eosin staining method. After staining, the sections were dehydrated, cleared, mounted and fixed, and then placed under a microscope to observe the pathological changes of the aorta.

[0067] The results are as follows Figure 1 As shown: In the control group, vascular endothelial cells were neatly arranged with round or oval nuclei and uniform chromatin staining. No significant differences were observed in vascular pathology between the 0.02% and 0.04% carrageenan groups and the control group. In the 0.06% carrageenan group, slight disordered arrangement of vascular endothelial cells, nucleus deformation, and some areas showed thickening of the vessel wall and minor damage.

[0068] (2) Enzyme-linked immunosorbent assay: According to the kit instructions, the levels of vascular cell adhesion factor VCAM-1 and inflammation-related factors TNF-α, IL-6 and IL-1β in serum were detected.

[0069] The results are as follows Figure 2 As shown, compared with the control group, the expression of vascular cell adhesion factor VCAM-1 and inflammation-related factors TNF-α, IL-6 and IL-1β in serum were increased in different carrageenan groups. Among them, the expression of adhesion factor and inflammation factor was the highest in the 0.06% carrageenan injection group, and the difference was statistically significant (P<0.05, P<0.01).

[0070] 1.4 Experiment Summary

[0071] Traditional Chinese medicine theory holds that blood stasis syndrome arises from two main aspects: impaired circulation of qi and blood, and internal obstruction of blood stasis. Excessive release of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β can trigger local and even systemic inflammatory responses, directly interfering with normal blood flow. Meanwhile, the vascular cell adhesion molecule VCAM-1 is easily upregulated by inflammatory factors. This process enhances the adhesion and infiltration of immune cells on the blood vessel wall, deepening inflammation and vascular damage, further obstructing blood flow and leading to blood stasis.

[0072] No mice died during the intraperitoneal injection of carrageenan to establish a blood stasis syndrome model. The levels of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, and the content of the vascular cell adhesion factor VCAM-1, increased with increasing carrageenan concentration. Specifically, after injection of 0.06% carrageenan, the expression levels of all serum indicators were significantly different from the control group (P<0.05). Based on these experimental results, this invention selected 0.06% carrageenan as the optimal dose, with a single injection dose of 0.1 mL / 10 g, to establish a blood stasis syndrome mouse model.

[0073] Example 2

[0074] This embodiment illustrates the operation method for constructing an AD animal model of kidney deficiency and blood stasis type and the setting of control groups.

[0075] (1) Animal grouping and intervention:

[0076] Six littermates served as the blank control group; the remaining 30 APP / PS1 mice (n=6 per group) were randomly divided into five groups: brain toxicity group, qi deficiency leading to toxicity group, kidney deficiency leading to toxicity group, deficiency leading to toxicity group, and deficiency leading to blood stasis group. The intervention methods are as follows:

[0077] Blank control group (TW group): equal volume of normal saline by gavage + normal saline by intraperitoneal injection + no sleep deprivation;

[0078] Brain damage group (DS group): equal volume of normal saline by gavage + normal saline by intraperitoneal injection + no sleep deprivation;

[0079] Qi deficiency leading to toxicity (QX group): equal volume of normal saline administered by gavage + normal saline injected intraperitoneally + sleep deprivation:

[0080] Kidney deficiency leading to toxicity group (SX group): hydrocortisone gavage + intraperitoneal injection of normal saline + no sleep deprivation;

[0081] Depletion and poisoning group (XS group): hydrocortisone gavage + intraperitoneal injection of normal saline + sleep deprivation;

[0082] Group with deficiency and stasis (FH group): hydrocortisone gavage + carrageenan intraperitoneal injection + sleep deprivation;

[0083] After a week of acclimatization feeding, mice in each group underwent a 28-day modeling process. Following modeling, grip strength was measured using a grip strength meter, and learning and memory abilities were assessed using a water maze test.

[0084] (2) Animal model construction:

[0085] The modeling method for the FH group is as follows: Transgenic AD model mice were subjected to continuous sleep deprivation and intraperitoneal injection of carrageenan (concentration of 0.06% g / mL, dose of 0.1 mL / 10 g) for 28 consecutive days. The continuous sleep deprivation was used to induce qi deficiency syndrome, and the carrageenan injection was used to induce blood stasis syndrome. Starting from day 20, hydrocortisone was administered by gavage (dose of 50 mg / kg / time, once every other day, for a total of 4 times) to further induce kidney deficiency syndrome.

[0086] The modeling method for the TW group is as follows: equal volume of physiological saline (0.1 mL / 10 g) was continuously injected intraperitoneally for 28 days, and equal volume of physiological saline was administered by gavage starting on the 20th day, once every other day, for a total of 4 times;

[0087] The modeling method for the DS group is as follows: equal volume of physiological saline (0.1 mL / 10 g) was continuously injected intraperitoneally for 28 days, and equal volume of physiological saline was administered by gavage starting on the 20th day, once every other day, for a total of 4 times;

[0088] The modeling method for the QX group is as follows: continuous sleep deprivation and intraperitoneal injection of an equal volume of physiological saline (0.1 mL / 10 g) for 28 consecutive days. Starting from day 20, an equal volume of physiological saline was administered by gavage once every other day for a total of 4 times.

[0089] The SX group modeling method is as follows: equal volume of physiological saline (0.1 mL / 10 g) was continuously injected intraperitoneally for 28 days. Starting from day 20, hydrocortisone was administered by gavage at a dose of 50 mg / kg / time, once every other day, for a total of 4 times.

[0090] The XS group modeling method is as follows: continuous sleep deprivation and continuous intraperitoneal injection of equal volume of physiological saline (0.1mL / 10g) for 28 days. Starting from day 20, hydrocortisone was administered by gavage at a dose of 50mg / kg / time, once every other day, for a total of 4 times.

[0091] Among them, the aforementioned persistent sleep deprivation, such as Figure 3 As shown, the sleep deprivation conditions were: 1.5 clockwise rotations followed by 1.5 counterclockwise rotations (recorded as one cycle), at a rotation speed of 8 rpm, with a 2-minute interval between each rotation, and a daily sleep deprivation duration of 10 hours. The aforementioned equal volume of physiological saline administered via gavage was equivalent to the volume of hydrocortisone administered via gavage (dose of 50 mg / kg / time).

[0092] The AD mouse model of deficiency, blood stasis and toxicity was evaluated by comparing the statistical differences in syndrome indicators such as qi deficiency, kidney deficiency, blood stasis and toxicity between the treatment group and the control group.

[0093] (3) Animal sampling and sample processing:

[0094] After behavioral testing, blood was collected by enucleation of the eyeballs. Mice were then euthanized by cervical dislocation, and the spleen, kidneys, and testes were isolated and weighed. Additionally, brain tissue was isolated from three mice in each group, fixed in 4% paraformaldehyde for 24 hours, and then embedded in paraffin, sectioned, and prepared for histopathological staining. Hippocampal tissue was isolated from the remaining three mice, flash-frozen in liquid nitrogen, and stored at -80°C. Whole blood was allowed to stand for 20 minutes, then centrifuged at 3500 rpm for 10 minutes, and serum was collected and stored at -20°C.

[0095] Example 3

[0096] This embodiment is used to illustrate the detection of Qi deficiency indicators induced by sleep deprivation and to prove the success of Qi deficiency induction.

[0097] (1) Body weight: During the mouse modeling period, the body weight of the mice was measured weekly. After the mice were numbered, they were gently picked up and placed on a calibrated electronic scale, and their weight was read and recorded. The results are as follows: Figure 4 As shown: Compared with before modeling, the QX group gained weight.

[0098] (2) Grasping force index: The gripping force of each group of mice was measured using an XR501 rat and mouse gripping force meter. The results are as follows: Figure 5 As shown: Compared with the TW group, the grip strength index of mice in the QX, XS and FH groups was reduced (P<0.01).

[0099] (3) Spleen Index: The wet weight of the spleen and the body weight of the mouse were measured. The spleen index was calculated using the following formula: Spleen Index % = Spleen weight / Mouse body weight × 100%. Results are as follows... Figure 6As shown: Compared with the TW group, the spleen index of mice in the QX, XS and FH groups was decreased (P<0.01).

[0100] Experimental Summary: Traditional Chinese medicine theory explains that Qi deficiency is a decline in the body's physiological functions and a lack of Qi in the internal organs and meridians. On the one hand, Qi deficiency leads to a decline in the spleen's function of transporting and transforming Qi, resulting in insufficient production of Qi and blood. This makes it difficult to expel pathogenic factors such as blood stasis and phlegm, causing them to accumulate in the body and leading to obesity. On the other hand, Qi deficiency often manifests as symptoms such as general weakness, lethargy, and loss of appetite, and may also show changes such as weight loss. In addition, the grip strength index in mice is one of the important indicators for assessing muscle strength and physical condition. Due to decreased physical strength and weakened muscle strength, mice with Qi deficiency usually have a lower grip strength index than normal mice. At the same time, due to the decline in physiological functions, mice with Qi deficiency often have weakened immune function, and their spleen may atrophy. Therefore, the spleen index in mice is often used as an important indicator for assessing their immune function and spleen development.

[0101] In this embodiment, body weight, grip strength index, and spleen index were used as evaluation indicators for Qi deficiency syndrome to verify the statistical differences in Qi deficiency indicators between mice with single Qi deficiency syndrome and mice with a combined model. The study showed that compared with the TW group mice, the QX group mice had lower grip strength index and spleen index, accompanied by higher body weight, which was considered to be due to spleen deficiency and impaired digestion, leading to the accumulation of pathogenic factors in the body, indicating successful induction of Qi deficiency syndrome. Simultaneously, compared with the TW group mice, the XS and FH groups showed significant reductions in body weight, grip strength index, and spleen index, with statistically significant differences, indicating successful induction of Qi deficiency syndrome under the combined model of deficiency, depletion, and toxicity.

[0102] Example 4

[0103] This embodiment is used to illustrate the detection of indicators of kidney deficiency induced by hydrocortisone and to prove the success of kidney deficiency induction.

[0104] (1) Body weight: During the mouse modeling period, the body weight of the mice was measured weekly. After the mice were numbered, they were gently picked up and placed on a calibrated electronic scale, and their weight was read and recorded. The results are as follows: Figure 4 As shown: Compared with before modeling, the average body weight of mice in the SX group, XS group and FH group was reduced, with the difference between the SX group and FH group being statistically significant.

[0105] (2) Kidney Index and Testicular Index: The wet weight of both kidneys (testes) and the body weight of the mouse were measured, respectively. The formula for calculating the kidney (testicular) index is as follows: Kidney (testicular) index % = (Weight of both kidneys (testes) / Body weight of mouse) × 100%. The results are as follows: Figure 7 As shown: Compared with the TW group, the kidney index and testis index of mice in the SX group, XS group and FH group were decreased (P<0.05, P<0.01).

[0106] (3) Enzyme-linked immunosorbent assay (ELISA): The level of cortisol in serum was detected according to the kit instructions. Results are as follows: Figure 8 As shown: Compared with the TW group, the serum Cortisol level of mice in the SX, XS and FH groups was increased (P<0.01).

[0107] Experimental Summary: In the kidney deficiency model, mice typically experience weight loss due to decreased bodily function, reduced metabolic rate, and decreased appetite caused by kidney deficiency. Furthermore, insufficient kidney yin fluid may impair normal kidney function, leading to changes in kidney tissue; it may also cause decreased testicular function, affecting testicular weight and testicular index. On the other hand, insufficient yin fluid can lead to relative yang heat, potentially affecting the function of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in altered secretion of hormones such as adrenocorticotropic hormone (ACTH) and cortisol.

[0108] This embodiment uses body weight, kidney index, testicular index, and cortisol content as evaluation indicators for kidney deficiency syndrome. It verifies the statistical differences in kidney deficiency indicators between mice with single kidney deficiency syndrome and mice with combined kidney deficiency syndrome. The study shows that compared with the TW group mice, the SX group, XS group, and FH group mice have significant differences in indicators such as body weight, kidney index, testicular index, and cortisol content, and the differences are statistically significant, indicating that the induction of kidney deficiency syndrome was successful.

[0109] Example 5

[0110] This embodiment is used to illustrate the detection of carrageenan-induced blood stasis indicators and to prove the success of blood stasis induction.

[0111] Enzyme-linked immunosorbent assay (ELISA): Serum levels of VCAM-1, TNF-α, IL-6, and IL-1β were measured according to the kit instructions. Results are as follows: Figure 9 As shown: Compared with the TW group, the expression of VCAM-1, TNF-α, IL-6 and IL-1β in the serum of mice in the FH group was increased (P<0.01).

[0112] Experiment Summary:

[0113] In the state of blood stasis syndrome, due to poor blood circulation, local tissue hypoxia and ischemia may be triggered, which may lead to inflammatory and immune responses. TNF-α, IL-6, and IL-1β, as important inflammatory cytokines, often show elevated expression levels in patients with blood stasis syndrome. Simultaneously, influenced by inflammatory immunity, VCAM-1 expression is often upregulated to promote leukocyte adhesion and infiltration, thereby participating in the inflammatory response and tissue repair.

[0114] In this embodiment, the expression levels of VCAM-1, TNF-α, IL-6, and IL-1β were used as evaluation indicators for blood stasis syndrome. The study verified the statistical differences in blood stasis indicators in mice with deficiency and blood stasis-toxin type. The study showed that compared with the TW group mice, the serum expression of VCAM-1, TNF-α, IL-6, and IL-1β in the FH group mice was increased, and the difference was statistically significant, indicating that blood stasis syndrome was successfully induced in the composite model.

[0115] Example 6

[0116] This embodiment illustrates the detection of toxicity and AD animal model indicators, and demonstrates the successful induction of toxicity and AD animal models.

[0117] (1) Water Maze Test: The water maze test consisted of three parts. Before the experiment, mice were placed in the experimental room to acclimatize to their surroundings. After determining the platform and quadrant locations, the platform was first placed in the water, exposed on the surface, allowing the mice to swim freely and find the platform, thus acclimatizing them to the aquatic environment and reducing stress. Next, the platform was hidden 1-2 cm underwater for a navigational test lasting 5 days. Mice were placed in the water from four different locations, and the time it took for them to find the platform was recorded. Finally, the platform was removed, and a spatial exploration test was conducted. The time it took for the mice to cross the platform and the time they spent in the platform quadrant within 1 minute were recorded. Additionally, the distance and time the mice swam in the water were recorded to calculate their swimming speed. The results are as follows: Figure 10 As shown: During the orientation and navigation phase, compared with the first day of training, the escape latency of mice in the TW, DS, and QX groups was significantly shortened on the fifth day (P<0.05), while the escape latency of the XS and FH groups was shortened, but the difference was not statistically significant. During the spatial exploration phase, compared with the control group, the number of times mice crossed the platform decreased in all other groups (P<0.01), and the time spent in the platform quadrant was shortened (P<0.01). Among them, the FH group had the fewest platform crossings and the shortest time spent in the platform quadrant.

[0118] (2) Brain tissue pathology: Prepared paraffin sections of brain tissue were removed and subjected to dewaxing and hydration processes. The sections were then stained with Nissl stain. After staining, the sections were dehydrated, cleared, mounted, and fixed, and then observed under a microscope for pathological changes in the brain tissue. Results are as follows: Figure 11 As shown, neurons in the hippocampus of mice in the TW and DS groups were tightly arranged, morphologically intact, and clearly stained with Nissl bodies. Compared with the DS group, with the superposition of modeling factors, neurons in the hippocampus of the QX, SX, XS, and FH groups showed damage, loose arrangement, irregular morphology, nuclear pyknosis, and Nissl body dissolution or even disappearance. Among them, the FH group showed the most severe pathological deterioration in brain tissue.

[0119] (3) Immunofluorescence: Prepared paraffin sections of brain tissue were removed and subjected to antigen retrieval, cell membrane permeability treatment, blocking of non-specific binding sites, and incubation with primary antibody (Aβ). 1-42 The cells were incubated with secondary antibody, stained with nuclear cells, and mounted. Finally, the cells were observed under a fluorescence microscope, and fluorescence images were acquired. The results are as follows: Figure 12 As shown: Compared with the DS group, with the superposition of modeling factors, Aβ deposition in mouse brain tissue was observed in the QX group, SX group, XS group and FH group, with the most obvious Aβ deposition in the SX group, XS group and Aβ deposition.

[0120] (4) Western blotting: A portion of mouse hippocampal tissue was taken from -80℃, ground with tissue lysis buffer, centrifuged, and the supernatant was collected. An appropriate amount of protein loading buffer was added, and the protein was boiled at 95℃ for 10 min to denature it. The boiled protein was then added to a gel for electrophoresis, followed by electrophoresis, transfer, blocking, primary antibody incubation (APP, BACE1, PS1), secondary antibody incubation, and exposure. Finally, the grayscale values ​​of the exposed images were analyzed using ImageJ. The results are as follows: Figure 13 As shown: Compared with the DS group, the expression levels of APP, BACE1, and PS1 increased with the superposition of modeling factors. Among them, the expression levels of APP, BACE1, and PS1 in the XS group and FH group were significantly increased (P<0.01).

[0121] Experimental Summary: The most commonly used AD mouse model is the transgenic mouse, which carries gene mutations associated with human AD, such as mutations in genes like APP (amyloid precursor protein) and PSEN1 (presenilin 1). These mutations lead to excessive deposition of β-amyloid protein (Aβ) in the mice, mimicking the pathological changes in the brains of AD patients. Furthermore, AD mouse models typically exhibit significant cognitive impairment and neuronal damage, manifesting in the Morris water maze test as prolonged time to find hidden platforms and disorganized swimming paths. In Traditional Chinese Medicine, AD toxicity is often described as the accumulation of pathological products such as blood stasis and turbidity; in modern medicine, this can be reflected in Aβ deposition and increased expression of inflammatory factors. Since blood stasis indicators and inflammatory factor expression were evaluated in Example 3, this example primarily uses Aβ deposition as an indicator for evaluating toxicity.

[0122] In this embodiment, the learning and memory abilities of mice were assessed using a water maze, and the pathology of mouse brain tissue was observed using Nissl staining. Simultaneously, the deposition of Aβ and the expression of Aβ-related proteins in the mouse brain were detected. The study showed that mice in the FH group had prolonged escape latency, reduced number and duration of platform crossings, and shorter time spent in the platform quadrant (P<0.01). Damage was observed in hippocampal neurons, with loose arrangement, irregular morphology, nuclear condensation, and Nissl body dissolution or even disappearance, exhibiting pathological manifestations consistent with the characteristics of an AD mouse model. Furthermore, increased Aβ deposition and increased expression of Aβ-related proteins in the mouse brain (P<0.01) indicated that this composite model met the characteristics of toxic damage, and the model was successfully constructed.

[0123] In summary, Examples 2-6 of this invention demonstrate that the present invention successfully constructed an animal model of AD with deficiency and blood stasis, and the syndrome manifestations of the animal model are consistent with the clinical observation results. This provides a reliable experimental vehicle for the innovative drug development, re-evaluation of marketed products, and study of the mechanism of action of AD with deficiency and blood stasis.

[0124] This invention provides a method for constructing and evaluating an animal model of Alzheimer's disease characterized by deficiency and stagnation. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for constructing an animal model of Alzheimer's disease of the deficiency and blood stasis and toxin type, characterized in that, The Alzheimer's disease double transgenic mice are subjected to continuous sleep deprivation and injection of carrageenan simultaneously every day for 28 days; starting from the 20th day, hydrocortisone is administered by gavage once every other day, and the gavage is performed for 4 times in total; The Alzheimer's disease double transgenic mice are 5-12 months old and 20-40 g of SPF level APP / PS1 mice; The injection is intraperitoneal injection, and the injection dose of carrageenan is 4-8 mg / kg / day, once a day; The gavage dose of hydrocortisone is 50 mg / kg / time.

2. The construction method according to claim 1, characterized in that, The continuous sleep deprivation is performed under the condition that the sleep deprivation is first performed by rotating clockwise for 1.5 turns and then by rotating counterclockwise for 1.5 turns, which is recorded as one rotation, and the rotation speed is 6-8 rpm, and the interval time between each rotation is 1-3 min, and the sleep deprivation time per day is 8-12 hours.

3. The construction method of claim 1, wherein, The construction method comprises evaluation indexes of Alzheimer's disease of deficiency and damage with blood stasis, and the evaluation indexes comprise indexes of qi deficiency, indexes of kidney deficiency, indexes of blood stasis, indexes of damage and indexes of AD model.

4. The construction method according to claim 3, characterized in that, The indexes of qi deficiency comprise body weight, grip strength index and spleen index; The indexes of kidney deficiency comprise body weight, kidney index, testis index and cortisol level; The indexes of blood stasis comprise levels of VACM-1, IL-6, IL-1β and TNF-α; The indexes of damage comprise Aβ deposition in hippocampus, expression levels of Aβ generation related proteins APP, PS1 and BACE-1 in hippocampus; The AD model evaluation indexes comprise evaluation of learning and memory ability and Nissl pathology of brain tissue.

5. Application of the animal model constructed by the construction method of any one of claims 1-4 in screening of drugs for treating or improving Alzheimer's disease of deficiency and damage with blood stasis.

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