Method for constructing and evaluating asthenic stasis toxin type Alzheimer's disease animal model

Abstract

The invention relates to a construction and evaluation method of an animal model of asthenic stasis toxin type Alzheimer's disease. The model is based on APP / PS1 mice, sleep deprivation, hydrocortisone and carrageenan are used for induction of qi deficiency, kidney deficiency and blood stasis, the total time of modeling is 28 days, and the animal model of the asthenic stasis toxin type Alzheimer disease is obtained. The model is constructed in a multi-factor combination mode, the syndrome characterization is consistent with a clinical observation result, the model is simple, effective and easy to operate, and a reliable experimental carrier can be provided for research and development and action mechanism research of asthenic stasis toxin type Alzheimer disease innovative drugs.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a construction and evaluation method of an animal model of Alzheimer's disease of deficiency, stasis and toxin type. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease with progressive memory loss and cognitive impairment as its main clinical manifestations, and amyloid β protein deposition (Aβ) and Tau protein hyperphosphorylation as its main pathological features. Epidemiological data reveal that the number of AD patients in China is increasing year by year. By 2030, the number of dementia patients in my country may increase sharply to about 78 million, and by 2050, this number will rise to 139 million, highlighting the severe challenges and continuous growth 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 the body, storing essence and producing marrow, and the brain is the sea of ​​marrow. If the kidney essence is deficient, the sea of ​​marrow will not be nourished. In addition, qi deficiency is another important pathological link in the pathogenesis of AD. If the qi is insufficient, the body will be unable to move, blood stasis will stagnate, and the stagnation will cause the brain meridians to be blocked, and blood stasis and toxins will be generated internally, further blocking the qi mechanism, obscuring the clear orifices, and damaging the brain meridians, eventually forming various clinical manifestations of AD. AD is caused by the interweaving of multiple pathogenic factors, and clinical AD patients are mostly elderly people, who generally show the characteristics of kidney essence deficiency and weak qi and blood, accompanied by the decline of organ function, and the sluggish circulation of blood and body fluids, showing a complex situation in which multiple pathological factors such as deficiency, stasis, and toxins coexist and influence each other.

[0004] As a bridge between traditional medicine and modern medicine, TCM animal models provide experimental basis for TCM research by simulating certain characteristics or pathological processes of human diseases, and play an important role in the development of new TCM drugs. However, current AD animal models are mainly concentrated in natural aging models, rapid aging mouse models, cholinergic system damage models, transgenic animal models, and aluminum poisoning models. AD animal models related to TCM syndrome classification are extremely scarce. Although some researchers have constructed a kidney deficiency AD rat model by combining hydrocortisone with intracerebral Aβ injection, the model only simulates kidney deficiency syndrome and fails to include other important syndromes such as blood stasis. In addition, the uncertainty of multifactorial modeling and high mortality rates have brought considerable challenges to the construction of multifactorial TCM AD animal models.

[0005] There are considerable challenges in the establishment of multi-factorial TCM animal models. On the one hand, the current animal models based on TCM etiology and pathogenesis are mostly combined with Western medicine surgery, physics, chemistry and other methods, which are not in line with the natural onset of the disease. On the other hand, some chemical reagents, surgical operations and other modeling factors cause excessive trauma and infection, which makes the survival rate of modeling animals very low, and it is also easy to interfere with the purity of syndrome characteristics. Therefore, surgical modeling is not used as much as possible [Zhao Hui. On the idea of ​​multi-factorial composite animal modeling of disease and syndrome combination [J]. Journal of Anhui University of Traditional Chinese Medicine, 2001, (05): 57-59.]. In addition, the experimental grouping is not scientific enough, the single modeling method is not recognized enough, the repeatability is poor, and the correlation of the detection indicators is poor. These are all problems existing in the current multi-factorial modeling. This also highlights the urgency and importance of constructing a TCM AD animal model that can fully simulate the TCM multi-factor pathological characteristics such as deficiency, stasis, and toxicity. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to construct a deficiency and stasis type AD animal model in view of the scarcity of TCM-related AD animal models, the uncertainty under multi-factor modeling and the high mortality rate, etc., by combining multiple factors. The present invention selects APP / PS1 mice as experimental subjects. As a classic animal model for AD research, this mouse breed can naturally form Aβ plaques in the brain at 4 to 6 months of age, thereby avoiding the additional damage to mice caused by invasive operations such as intracerebral injection of Aβ, and ensuring the high survival rate of AD mice. Further, in order to explore the feasibility of multi-factor combined modeling of APP / PS1 mice, the present invention first explored the conditions for carrageenan to induce blood stasis on APP / PS1 background mice. Finally, sleep deprivation, hydrocortisone gavage and carrageenan intraperitoneal injection were selected to induce qi deficiency, kidney deficiency and blood stasis respectively. This strategy not only simplifies the experimental process, reduces complicated and repeated experimental operations, but also ensures the high survival rate and modeling success rate of mice under multi-factor combined modeling. The present invention provides a method for constructing and evaluating an animal model of AD of the asthenia and stasis type. The animal model of AD of the asthenia and stasis type is constructed by a method combining multiple factors such as sleep deprivation and chemical stimulation. The construction method is simple and easy to operate, can reduce the mortality rate of multi-factor combined modeling, can effectively simulate the symptom manifestations of AD patients of the asthenia and stasis type, and provides a reliable experimental carrier for the research and development of innovative drugs for AD of the asthenia and stasis type and the study of their mechanism of action.

[0007] In order to solve the above technical problems, the present invention discloses a method for constructing and evaluating an animal model of AD with deficiency, stasis and toxicity. The specific technical scheme is as follows:

[0008] A method for constructing an animal model of AD of the deficiency and stasis type, wherein AD double transgenic mice are subjected to continuous sleep deprivation and carrageenan injection every day for 28 consecutive days; hydrocortisone is administered orally starting on the 20th day, once every other day, for a total of 4 times. Preferably, the continuous sleep deprivation is a method for inducing qi deficiency, and if the difference in qi deficiency indicators between the sleep deprivation-induced mice and the blank control group mice is statistically significant, it means that qi deficiency can be successfully induced; the hydrocortisone gavage is a method for inducing kidney deficiency, and if the difference in kidney deficiency indicators between the hydrocortisone-induced mice and the blank control group mice is statistically significant, it means that kidney deficiency can be successfully induced; the carrageenan injection is used to induce blood stasis, and if the difference in blood stasis indicators between the carrageenan-induced mice and the blank control group mice is statistically significant, it means that blood stasis can be successfully induced; if the difference in toxic damage indicators between the transgenic mice and the blank control mice is statistically significant, it means that toxic damage can be successfully induced; if the multi-factor composite model group shows statistical differences in various evaluation indicators such as qi deficiency, kidney deficiency, blood stasis, toxic damage, etc. compared with the blank control group mice, it means that the deficiency and stasis type AD animal model is successfully constructed.

[0009] Wherein, the AD double transgenic mice are SPF-grade APP / PS1 mice of 5-12 months old and 20-40 g. Preferably, the brain tissue pathological feature of the AD double transgenic mice is the appearance of progressive amyloid deposition in the cerebral cortex or hippocampus, and more preferably, the mice are 5 months old.

[0010] The continuous sleep deprivation condition is that the machine rotates forward 1.5 times and then reverses 1.5 times, which is counted as one rotation. The rotation speed is 6-8 rpm, the interval between each rotation is 1-3 minutes, and the duration of sleep deprivation per day is 8-12 hours. Preferably, the rotation speed is 8 rpm, the interval between each rotation is 2 minutes, and the duration of sleep deprivation per day is 10 hours.

[0011] Wherein, the injection is intraperitoneal injection, and the injection dose of the carrageenan is 4-8 mg / kg / day, once a day. Preferably, the carrageenan is injected in the form of a carrageenan solution, and its solvent is normal saline; the injection concentration of the carrageenan is 0.6 mg / mL, and the injection dose is 0.1 mL / 10 g.

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

[0013] Among them, the construction method also includes evaluation indicators of deficiency and stasis type AD, and the evaluation includes qi deficiency indicators, kidney deficiency indicators, blood stasis indicators, toxic damage indicators and AD model evaluation indicators.

[0014] Among them, the Qi deficiency indexes include body weight, grip strength index and spleen index;

[0015] The kidney deficiency indexes include body weight, kidney index, testicular index and cortisol level;

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

[0017] The toxicity indicators include the Aβ deposition in the hippocampus tissue and the expression levels of Aβ generation-related proteins APP, PS1, and BACE-1 in the hippocampus tissue.

[0018] The AD model evaluation index includes learning and memory ability evaluation and brain tissue Nissl pathology. The learning and memory ability evaluation includes water maze evaluation.

[0019] Compared with the blank control mice, the weight change, grip strength index and spleen index of AD mice induced by multi-factor treatment were significantly decreased.

[0020] Compared with the blank control mice, the AD mice induced by kidney deficiency and blood stasis by multifactor treatment had lower body weight, significantly lower kidney index, significantly lower testicular index, and significantly lower serum cortisol content.

[0021] Compared with blank control mice, the content of vascular cell adhesion factor VCAM-1, inflammatory cytokines TNF-α, IL-6, and IL-1β in AD mice induced by kidney deficiency and stasis induced by multifactor treatment was increased.

[0022] Compared with the blank control mice, the immunofluorescence intensity of Aβ in brain tissue of AD mice induced by kidney deficiency and blood stasis was significantly enhanced, and the expression of APP, PS1 and BACE-1 proteins in hippocampus tissue was significantly increased.

[0023] Compared with blank control mice, AD mice induced by kidney deficiency and blood stasis by multifactor treatment had significant damage to Nissl pathological neurons in brain tissue, significantly prolonged escape latency in water maze, and significantly reduced learning and memory abilities.

[0024] In a second aspect, the present invention provides an application of the animal model constructed by the construction method described in the first aspect in screening drugs for treating or improving AD of the deficiency and stasis type. Preferably, the application comprises the following steps: administering a candidate drug to mice of the animal model, and performing drug efficacy detection and comparison on the mice administered the drug; wherein the animal model comprises: an animal model of AD of the deficiency and stasis type.

[0025] In a third aspect, the present invention provides an application of the animal model constructed by the construction method described in the first aspect in the study of the pathogenesis of AD of the deficiency and stasis type and in the evaluation of drug efficacy.

[0026] Beneficial effects:

[0027] The present invention provides a method for constructing and evaluating an animal model of AD of the deficiency and stasis type. The animal model of AD of the deficiency and stasis type constructed by the method has symptom manifestations that are consistent with clinical observation results, and can provide a reliable experimental carrier for the development of innovative drugs for AD of the deficiency and stasis type, and for the evaluation and mechanism research of marketed products, thus filling the gap in the prior art of an animal model of TCM AD that comprehensively simulates TCM pathological characteristics such as deficiency, stasis, and toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0029] Figure 1 The figure is a graph of the pathological results of the mouse aorta vascular endothelium after intervention with different concentrations of carrageenan in one embodiment of the present invention, wherein A, B, C, and D represent the control group, 0.02% carrageenan group, 0.04% carrageenan group, and 0.06% carrageenan group, respectively.

[0030] Figure 2 This is a data graph of 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, 0.02% carrageenan group, 0.04% carrageenan group, and 0.06% carrageenan group, respectively.

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

[0032] Figure 4 It is a weight data graph of mice of the present invention. Wherein TW, DS, QX, SX, XS, and FH represent blank control group, brain collateral damage group, qi deficiency group, kidney deficiency group, deficiency group, and deficiency group respectively.

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

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

[0035] Figure 7It is a data graph of mouse kidney index and testis index in one embodiment of the present invention. Wherein TW, DS, QX, SX, XS, and FH represent blank control group, brain collateral damage group, qi deficiency group, kidney deficiency group, deficiency group, and deficiency group, respectively.

[0036] Figure 8 It is a data graph of the Cortisol content in mouse serum in one embodiment of the present invention. Wherein TW, DS, QX, SX, XS, and FH represent the blank control group, the brain network damage group, the qi deficiency group, the kidney deficiency group, the deficiency group, and the stasis group, respectively.

[0037] Fig. 9 It is a data graph of the content of vascular cell adhesion factor VCAM-1 and inflammatory factors TNF-α, IL-6, and IL-1β in mouse serum in one embodiment of the present invention. Wherein TW, DS, QX, SX, XS, and FH represent the blank control group, the toxic brain network group, the qi deficiency group, the kidney deficiency group, the deficiency group, and the stasis group, respectively.

[0038] Fig.10 It is a data graph of escape latency time, platform quadrant residence time, and platform crossing times in a mouse water maze test in one embodiment of the present invention. Wherein TW, DS, QX, SX, XS, and FH represent a blank control group, a toxic brain network damage group, a qi deficiency toxic group, a kidney deficiency toxic group, a deficiency toxic group, and a deficiency toxic group, respectively.

[0039] Fig.11 It is a graph of the pathological results of mouse brain tissue in one embodiment of the present invention. Wherein TW, DS, QX, SX, XS, and FH represent the blank control group, the brain network damage group, the qi deficiency group, the kidney deficiency group, the deficiency group, and the stasis group.

[0040] Fig.12 It is a graph showing the results of Aβ deposition in the hippocampus of mice in one embodiment of the present invention. TW, DS, QX, SX, XS, and FH respectively represent the blank control group, the brain network damage group, the qi deficiency group, the kidney deficiency group, the deficiency group, and the stasis group.

[0041] Fig.13 It is a graph showing the expression results of APP, BACE1 and PS1 proteins in mouse hippocampus tissue in one embodiment of the present invention. TW, DS, QX, SX, XS and FH respectively represent the blank control group, the brain network damage group, the qi deficiency group, the kidney deficiency group, the deficiency group and the stasis group. DETAILED DESCRIPTION

[0042] The specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain 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, if not otherwise specified, can all be purchased.

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

[0044] Mice were uniformly housed in the SPF animal room of the Animal Experiment Center of China Pharmaceutical University with free access to food and water. The ambient temperature was 22-25°C and the relative humidity was 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 County Hongbao Veterinary Drug Co., Ltd., Veterinary Drug No. (2016) 040101269); carrageenan (Shanghai McLean Biochemical Technology Co., Ltd., item number: C804872); physiological saline injection (Anhui Fengyuan Pharmaceutical Co., Ltd., national medicine standard number H20093237); TNF-α, IL-6, IL-1β, VACM-1, Cortisol enzyme-linked immunosorbent assay kit (Shanghai ELISA Biotechnology Co., Ltd., catalog numbers: MM-0132M1, MM-0163M1, MM-0040M1, MM-0129M1, MM-0565M2); APP, PS-1, β-actin primary antibodies (Wuhan Sanying Biotechnology Co., Ltd., catalog numbers: 25524-1-AP, 16163-1-AP, 66009-1-Ig), BACE1 antibody (Hunan Aifang Biotechnology Co., Ltd., catalog number AFW5266).

[0046] Instruments used in the embodiments of the present invention: XR-XS108 sleep deprivation instrument (Shanghai Xinruan Information Technology Co., Ltd.); XR501 rat gripping force measuring instrument (Shanghai Xinruan Information Technology Co., Ltd.); Morris water maze video analysis system (Beijing Zhongshi Di Chuang Technology Development Co., Ltd.); Donatello dehydrator (Italy DIAPATH Company); JB-P5 embedding machine (Wuhan Junjie Electronics Co., Ltd.); RM2016 pathological slicer (Shanghai Leica Instrument Co., Ltd.); G6100 upright fluorescence microscope (Nikon Company, Japan); Pannoramic MID scanner (3DHISTECH Company, Hungary); Tanon-4600 chemiluminescence system (Shanghai Tianneng Life Science Co., Ltd.); EL10A automatic microplate reader (Shandong Bo Ke Holding Group Co., Ltd.)

[0047] The present invention uses Graph Pad Prism 8.4.3 software for statistical analysis, and the data are expressed as mean ± standard deviation. The data that conform to the normal distribution are compared between groups using one-way analysis of variance, and the data that do not conform to the normal distribution are compared using non-parametric tests. P < 0.05 indicates that the difference is statistically significant.

[0048] The animal model provided by the present invention can objectively evaluate the syndrome manifestations from multiple aspects such as kidney deficiency, qi deficiency, blood stasis syndrome symptoms, brain tissue pathology, brain tissue beta-amyloid protein deposition, etc. The animal model can provide a reliable experimental carrier for the development of innovative prescriptions and medicines for kidney deficiency and blood stasis type AD, re-evaluation of marketed varieties and research on the mechanism of action. In the present invention, "the difference is statistically significant" means P<0.05.

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

[0050] The indices of kidney deficiency syndrome of the present invention include body weight, kidney index, testis index and cortisol level.

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

[0052] The toxicity indexes of the present invention include the Aβ deposition in hippocampal tissue and the expression levels of Aβ generation-related proteins APP, PS1 and BACE-1 in hippocampal tissue.

[0053] The evaluation indexes of the AD model of the present invention include Nissl pathology of brain tissue and water maze in behavioral experiments to evaluate the learning and memory ability of mice.

[0054] Example 1

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

[0056] 1.1 Animal grouping and intervention:

[0057] Twenty-four SPF C57BL / 6J mice were randomly divided into four groups, with 6 mice in each group, and the following settings were used:

[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 1 week of adaptive feeding, mice in each group were subjected to modeling for 28 days.

[0063] 1.2 Animal sampling and sample processing:

[0064] After the modeling was completed, the eyeballs were removed to collect blood, and then the mice were killed by cervical dislocation. The aorta and epididymal fat were separated and fixed in 4% paraformaldehyde for 24 hours, then embedded in paraffin, sliced ​​and other operations were performed for histopathological staining observation. After the whole blood was allowed to stand for 20 minutes, it was centrifuged at 3500rpm for 10 minutes, and the serum was collected and stored in a -20℃ refrigerator.

[0065] 1.3 Test indicators and results:

[0066] (1) Histopathological analysis: The prepared paraffin sections of the aorta were taken out and dewaxed and hydrated in sequence, and the sections were stained using the hematoxylin-eosin staining method. After staining, the sections were dehydrated and transparentized, sealed and fixed, and the pathological changes of the aorta were observed under a microscope.

[0067] The results are as follows Figure 1 As shown: In the control group, the vascular endothelial cells were arranged neatly, the nuclei were round or oval, and the chromatin was evenly colored. There was no significant difference in vascular pathology between the 0.02% and 0.04% carrageenan groups and the control group. In the 0.06% carrageenan group, slight vascular endothelial cell arrangement disorder and nucleus deformation were observed, and some vascular walls were thickened and slightly damaged.

[0068] (2) Enzyme-linked immunosorbent assay: According to the kit instructions, the levels of vascular cell adhesion factor VCAM-1 and inflammatory 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 expressions of vascular cell adhesion factor VCAM-1 and inflammatory related factors TNF-α, IL-6, and IL-1β in the serum of different doses of carrageenan groups were increased, among which the expression of adhesion factors and inflammatory factors in the 0.06% carrageenan injection group was the highest, and the difference was significant (P<0.05, P<0.01).

[0070] 1.4 Experimental Summary

[0071] According to TCM theory, the causes of blood stasis syndrome are poor 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 or even systemic inflammatory responses, directly interfering with the normal circulation of blood. The vascular cell adhesion molecule VCAM-1 is easily stimulated by inflammatory factors and upregulated. This process enhances the adhesion and infiltration of immune cells in the blood vessel wall, deepens inflammation and vascular damage in the blood vessel wall, further blocks blood circulation, and forms blood stasis.

[0072] During the process of constructing blood stasis syndrome by intraperitoneal injection of carrageenan, no mice died. The levels of proinflammatory cytokines such as TNF-α, IL-6, IL-1β and the content of vascular cell adhesion factor VCAM-1 increased with the increase of the intraperitoneal injection concentration of carrageenan. Among them, after 0.06% carrageenan injection, the expression levels of various serum indicators were significantly different from those of the control group (P<0.05). Based on the experimental results of this part, the present invention selected 0.06% carrageenan as the optimal dose, and the single injection dose was 0.1mL / 10g to establish a blood stasis syndrome mouse model.

[0073] Example 2

[0074] This example is used to illustrate the operation method and control group setting for constructing an animal model of AD of kidney deficiency and blood stasis type.

[0075] (1) Animal grouping and intervention:

[0076] Among them, 6 mice from the same litter were set as the blank control group; the other 30 APP / PS1 mice, 6 mice in each group, were randomly divided into 5 groups, namely, the brain network damage group, the qi deficiency group, the kidney deficiency group, the deficiency group, the deficiency group, and the deficiency group.

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

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

[0079] Qi deficiency and toxicity group (QX group): equal volume of saline gavage + saline intraperitoneal injection + sleep deprivation:

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

[0081] XS group: hydrocortisone gavage + saline intraperitoneal injection + sleep deprivation;

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

[0083] After 1 week of adaptive feeding, the mice in each group were subjected to modeling for 28 days. After modeling, the grip strength of the mice in each group was measured using a grip strength meter, and the learning and memory ability of the mice was tested using a water maze.

[0084] (2) Animal model construction:

[0085] The modeling method of the FH group is as follows: the transgenic AD model mice are subjected to continuous sleep deprivation and intraperitoneal injection of carrageenan (concentration of 0.06% g / mL, dosage of 0.1 mL / 10 g) for 28 consecutive days; the continuous sleep deprivation is used to induce qi deficiency syndrome, and the injection of carrageenan is used to induce blood stasis syndrome; hydrocortisone is given intragastrically starting from the 20th day (dosage of 50 mg / kg / time, once every other day, a total of 4 times) to further induce kidney deficiency syndrome;

[0086] The modeling method of the TW group was as follows: an equal amount of normal saline (0.1 mL / 10 g) was injected intraperitoneally for 28 consecutive days, and an equal amount of normal saline was gavaged starting on the 20th day, once every other day, for a total of 4 times;

[0087] The modeling method of the DS group was as follows: an equal amount of normal saline (0.1 mL / 10 g) was injected intraperitoneally for 28 consecutive days, and an equal amount of normal saline was gavaged starting on the 20th day, once every other day, for a total of 4 times;

[0088] The modeling method of the QX group is as follows: continuous sleep deprivation and intraperitoneal injection of an equal amount of saline (0.1 mL / 10 g) for 28 days, and oral gavage of an equal amount of saline starting on the 20th day, once every other day, for a total of 4 times;

[0089] The SX group modeling method was as follows: an equal amount of normal saline (0.1 mL / 10 g) was intraperitoneally injected for 28 consecutive days, and hydrocortisone was gavaged starting on the 20th day at a dose of 50 mg / kg / time, once every other day, for a total of 4 times;

[0090] The modeling method of the XS group is as follows: continuous sleep deprivation and intraperitoneal injection of an equal amount of saline (0.1 mL / 10 g) for 28 days, and hydrocortisone gavage starting on the 20th day, at a dose of 50 mg / kg / time, once every other day, for a total of 4 times;

[0091] Wherein, the continuous sleep deprivation is as follows: Figure 3 As shown, the sleep deprivation condition is to rotate forward 1.5 times and then reverse 1.5 times (recorded as 1 time), the speed is 8 rpm, the interval between each rotation is 2 minutes, and the sleep deprivation time is 10 hours per day. The equal amount of normal saline gavage is the same as the hydrocortisone gavage (dose is 50 mg / kg / time).

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

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

[0094] After the behavioral test, the eyeballs were removed to collect blood, and then the mice were killed by cervical dislocation. The spleen, kidneys and testicles of the mice were separated, and the weights of the corresponding organs were weighed. In addition, 3 mice were randomly selected from each group to separate the brain tissue, which was fixed in 4% paraformaldehyde for 24 hours, and then paraffin-embedded, sectioned, and other operations were performed for histopathological staining observation. The hippocampal tissue of the remaining 3 mice was isolated, quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator. After the whole blood was allowed to stand for 20 minutes, it was centrifuged at 3500rpm for 10 minutes, and the serum was taken and stored in a -20°C refrigerator.

[0095] Example 3

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

[0097] (1) Body weight: During the mouse modeling period, the mouse body weight was measured every week. After the mice were numbered, they were gently picked up and placed on a calibrated electronic scale to read and record their body weight. Figure 4 Shown: Compared with before modeling, the body weight of the QX group increased.

[0098] (2) Grip strength index: The grip strength of mice in each group was measured using the XR501 rat grip strength tester. Figure 5 As shown: Compared with the TW group, the grasping force index of mice in the QX group, XS group and FH group was reduced (P<0.01).

[0099] (3) Spleen index: Weigh the wet weight of the spleen and the body weight of the mouse. The spleen index is calculated as follows: Spleen index % = spleen weight / mouse body weight × 100%. Figure 6As shown: Compared with the TW group, the spleen index of mice in the QX group, XS group and FH group was decreased (P<0.01).

[0100] Experimental summary: According to the theory of traditional Chinese medicine, Qi deficiency is the decline of physiological functions of the body and the lack of Qi in the organs and meridians. On the one hand, Qi deficiency will lead to the decline of the spleen's function of transportation and transformation, and the insufficient biochemical transformation of Qi and blood will make it difficult to expel evil Qi such as blood stasis and phlegm in the body, which will accumulate in the body, leading to obesity. On the other hand, Qi deficiency syndrome is often manifested as symptoms such as general fatigue, mental depression, loss of appetite, and changes such as weight loss. In addition, the mouse grip index is one of the important indicators for evaluating muscle strength and physical fitness. Due to the decline in physical strength and weakened muscle strength, the grip index of Qi-deficient mice is usually lower than that of normal mice. At the same time, due to the low physiological function of the body, Qi-deficiency mice are often accompanied by weakened immune function, and their spleens may atrophy. Therefore, the spleen index of mice is often used as an important indicator for evaluating their immune function and spleen development.

[0101] In this embodiment, body weight, grip index and spleen index were used as evaluation indicators of Qi deficiency syndrome, and the statistical differences in Qi deficiency indicators between single Qi deficiency syndrome mice and composite model mice were verified. Studies have shown that compared with mice in the TW group, the grip index and spleen index of mice in the QX group were reduced and accompanied by increased body weight, which was considered to be caused by spleen deficiency and inability to transport and transform, and the accumulation of evil qi in the body, indicating that Qi deficiency syndrome was successfully induced. At the same time, it was found that compared with mice in the TW group, the XS group and the FH group were significantly reduced in indicators such as weight, grip index and spleen index, and the differences were statistically significant, indicating that Qi deficiency syndrome was successfully induced under the composite model of deficiency and blood stasis.

[0102] Example 4

[0103] This example is used to illustrate the detection of hydrocortisone-induced kidney deficiency indexes and to prove that kidney deficiency induction is successful.

[0104] (1) Body weight: During the mouse modeling period, the mouse body weight was measured every week. After the mice were numbered, they were gently picked up and placed on a calibrated electronic scale to read and record their body weight. 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, among which the difference between the SX group and the FH group was statistically significant.

[0105] (2) Kidney index and testicular index: Weigh the wet weight of both kidneys (testes) and the body weight of the mouse. The formula for calculating the kidney (testicular) index is as follows: Kidney (testicular) index % = bilateral kidney (testicular) weight / mouse body weight × 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: According to the kit instructions, the content of cortisol in serum was detected. Figure 8 As shown: Compared with the TW group, the serum Cortisol level of mice in the SX group, XS group and FH group was increased (P<0.01).

[0107] Experimental summary: In the kidney deficiency model, mice usually lose weight due to the influence of factors such as decreased body function, decreased metabolic rate and loss of appetite caused by kidney deficiency. In addition, the lack of kidney yin fluid may affect the normal physiological function of the kidney and cause changes in kidney tissue; it may also cause decreased testicular function, thereby affecting testicular weight and testicular index. On the other hand, the lack of body yin fluid causes the yang heat to be relatively hyperactive, which may affect the function of the hypothalamus-pituitary-adrenal axis (HPA axis) and cause changes in the secretion of hormones such as adrenocorticotropic hormone and cortisol.

[0108] In this example, body weight, kidney index, testicular index and cortisol content were used as evaluation indicators of kidney deficiency syndrome, and the statistical differences in kidney deficiency indicators between single kidney deficiency syndrome mice and compound model mice were verified. The study showed that compared with the TW group mice, the SX group, XS group and FH group mice had significant differences in body weight, kidney index, testicular index and cortisol content, and the differences were statistically significant, indicating that the induction of kidney deficiency syndrome was successful.

[0109] Example 5

[0110] This example is used to illustrate the detection of blood stasis index induced by carrageenan and prove the successful induction of blood stasis.

[0111] ELISA: According to the kit instructions, the levels of VCAM-1, TNF-α, IL-6, and IL-1β in serum were detected. Fig. 9 As shown: Compared with the TW group, the expressions of VCAM-1, TNF-α, IL-6 and IL-1β in the serum of mice in the FH group were increased (P<0.01).

[0112] Experimental summary:

[0113] In the state of blood stasis, due to poor blood circulation, local tissue hypoxia and ischemia, inflammatory and immune responses may be triggered. TNF-α, IL-6 and IL-1β, as important inflammatory cytokines, often have increased expression levels in patients with blood stasis. At the same time, under the influence of inflammatory immunity, the expression of VCAM-1 is often upregulated to promote the adhesion and infiltration of leukocytes, thereby participating in inflammatory responses and tissue repair.

[0114] In this example, the expression levels of VCAM-1, TNF-α, IL-6 and IL-1β are evaluation indicators of blood stasis syndrome, which verifies the statistical differences in blood stasis indicators in the mice of the deficiency and stasis type model. The study shows that compared with the mice in the TW group, the expression of serum 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 example illustrates the detection of toxic damage and AD animal model indicators, and proves that the toxic damage and AD animal model were successfully induced.

[0117] (1) Water maze test: The water maze experiment is divided into three parts. Before the experiment begins, the mice are placed in the experimental room in advance to adapt to the surrounding environment. After determining the position of the platform and quadrant, first, place the platform in the water and expose it to the water surface. Allow the mice to swim freely and find the platform, so that the mice can adapt to the water environment to reduce stress. Next, hide the platform 1-2 cm underwater and conduct a positioning navigation experiment for 5 days. Place the mice in the water from 4 different positions and record the time it takes to find the platform. Finally, remove the platform and conduct a spatial exploration experiment. Record the time it takes for the mouse to cross the platform and the time it stays in the platform quadrant within 1 minute. In addition, record the distance and time the mouse swims in the water to calculate the mouse's swimming rate. The results are as follows: Fig.10 As shown: In the positioning navigation stage, compared with the first day of training, the escape latency of mice in the TW group, DS group, and QX group on the fifth day was significantly shortened (P<0.05), while the escape latency of the XS group and FH group was shortened, but the difference was not statistically significant; in the spatial exploration stage, compared with the blank group, the number of times the mice in the other groups crossed the platform decreased (P<0.01), and the time they stayed in the platform quadrant was shortened (P<0.01). Among them, the number of times the mice in the FH group crossed the platform was the smallest, and the time they stayed in the platform quadrant was the shortest.

[0118] (2) Brain tissue pathology: Take out the prepared brain tissue paraffin sections, dewax and hydrate them in sequence, and stain them with Nissl staining solution. After staining, dehydrate and make them transparent, seal and fix them, and observe the pathological changes of brain tissue under a microscope. Fig.11 As shown in the figure, the neurons in the hippocampus of mice in the TW and DS groups were closely arranged, with complete morphology and clear Nissl body staining. Compared with the DS group, with the superposition of modeling factors, the neurons in the hippocampus of the QX, SX, XS and FH groups showed pathological manifestations such as damage, loose arrangement, irregular morphology, nuclear condensation, dissolution or even disappearance of Nissl bodies. Among them, the brain tissue pathology of the FH group deteriorated the most seriously.

[0119] (3) Immunofluorescence: The prepared brain tissue paraffin sections were taken out and antigen repair, cell membrane permeabilization, blocking of nonspecific binding sites, primary antibody incubation (Aβ 1-42 ), secondary antibody incubation, cell nucleus staining, and sealing. Finally, the fluorescence images were obtained under a fluorescence microscope. Fig.12 As shown: Compared with the DS group, with the superposition of modeling factors, Aβ deposition appeared in the brain tissue of mice in the QX group, SX group, XS group and FH group, among which Aβ deposition was the most obvious in the SX group and XS group.

[0120] (4) Protein immunoblotting: Take out some mouse hippocampal tissue from -80℃, add tissue lysis buffer, grind and centrifuge, collect the supernatant, add an appropriate amount of protein loading buffer, and boil the protein at 95℃ for 10 minutes to denature the protein. Add the boiled protein to gel electrophoresis, and perform electrophoresis, transfer, blocking, primary antibody incubation (APP, BACE1, PS1), secondary antibody incubation and exposure in sequence. Finally, use Image J to analyze the gray value of the exposed image. The results are shown in Figure 2. Fig.13 As shown: Compared with the DS group, with the superposition of modeling factors, the expression levels of APP, BACE1, and PS1 increased, among which the expression levels of APP, BACE1, and PS1 in the XS group and FH group increased and the differences were significant (P<0.01).

[0121] Experimental summary: The most commonly used AD mouse model is transgenic mice, which carry gene mutations related to human AD, such as mutations in genes such as APP (amyloid precursor protein) and PSEN1 (presenilin 1). These mutations lead to excessive deposition of β-amyloid protein (Aβ) in mice, simulating the pathological changes in the brains of AD patients. In addition, AD mouse models usually show obvious cognitive dysfunction and neuronal damage, which are manifested in the Morris water maze test as symptoms of cognitive impairment such as prolonged time to find the hidden platform and confusion in the swimming path. AD toxicity is mostly manifested in traditional Chinese medicine as the accumulation of pathological products such as blood stasis, and in modern medicine it can be manifested as the deposition of Aβ, increased expression of inflammatory factors, etc. In Example 3, the expression of blood stasis indicators and inflammatory factors has been evaluated, so in this example, the deposition of Aβ is mainly used as a toxicity evaluation indicator for elaboration.

[0122] In this example, the learning and memory ability of mice was evaluated by a water maze, and the pathology of mouse brain tissue was observed by Nissl staining. At the same time, the deposition of Aβ in the mouse brain and the expression of Aβ-related proteins were detected. The study showed that the escape latency of mice in the FH group was prolonged, the number and time of crossing the platform were reduced, and the stay time in the platform quadrant was shortened (P<0.01). The neurons in the hippocampus were damaged, the arrangement was loose, the morphology was irregular, the nucleus was condensed, and the Nissl bodies were dissolved or even disappeared, which was consistent with the characteristics of the AD mouse model; at the same time, the Aβ deposition in the mouse brain increased, and the expression of Aβ-related proteins increased (P<0.01), indicating that the composite model met the characteristics of toxic damage and the model was successfully constructed.

[0123] In summary, Examples 2-6 of the present invention show that the present invention successfully constructs an animal model of AD with deficiency and stasis, and the symptom manifestations of the animal model of the present invention are consistent with the clinical observation results, which can provide a reliable experimental carrier for the development of innovative drugs for AD with deficiency and stasis, re-evaluation of marketed products and research on the mechanism of action.

[0124] The present invention provides a method and idea for the construction and evaluation of an animal model of Alzheimer's disease with deficiency and stasis type. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A method for constructing an animal model of Alzheimer's disease of deficiency and stasis type, characterized in that: The Alzheimer's disease double transgenic mice were subjected to continuous sleep deprivation and carrageenan injection every day for 28 consecutive days. On the 20th day, hydrocortisone was gavaged every other day for a total of 4 times.

2. The construction method according to claim 1, characterized in that: The Alzheimer's disease double transgenic mice are SPF-grade APP / PS1 mice of 5-12 months old and 20-40g.

3. The construction method according to claim 1, characterized in that: The continuous sleep deprivation condition is that the machine rotates forward 1.5 times and then reverses 1.5 times, which is counted as one rotation. The rotation speed is 6-8 rpm, the interval between each rotation is 1-3 minutes, and the sleep deprivation duration is 8-12 hours per day.

4. The construction method according to claim 1, characterized in that: The injection is intraperitoneal injection, and the injection dosage of the carrageenan is 4-8 mg / kg / day, once a day.

5. The construction method according to claim 1, characterized in that: The intragastric dose of hydrocortisone is 50 mg / kg / time.

6. The construction method according to claim 1, characterized in that: The construction method includes evaluation indicators of deficiency and stasis type Alzheimer's disease, and the evaluation indicators include qi deficiency index, kidney deficiency index, blood stasis index, toxic damage index and AD model evaluation index.

7. The construction method according to claim 6, characterized in that: The Qi deficiency indexes include body weight, grip strength index and spleen index; The kidney deficiency indexes include body weight, kidney index, testicular index and cortisol level; The blood stasis indicators include VACM-1, IL-6, IL-1β and TNF-α levels; The toxicity indicators include Aβ deposition in hippocampal tissue, and the expression levels of Aβ production-related proteins APP, PS1, and BACE-1 in hippocampal tissue; The AD model evaluation indexes include learning and memory ability evaluation and brain tissue Nissl pathology.

8. Use of the animal model constructed by the construction method according to any one of claims 1 to 7 in screening drugs for treating or improving Alzheimer's disease of the deficiency and stasis type.

9. Application of the animal model constructed by the construction method according to any one of claims 1 to 7 in studying the pathogenesis of Alzheimer's disease of deficiency and stasis type.

Images