Construction method and application of kidney-yang deficiency type senile cognitive impairment rat model

Abstract

The invention discloses a construction method and application of a kidney-yang deficiency type senile cognitive impairment rat model. The construction method provided by the invention comprises the following steps: continuously injecting a D-galactose solution into the neck and back of a rat for 8 weeks in a subcutaneous manner every day, and combining intragastric administration of an adenine suspension from the fifth week to the eighth week. According to the method, the model is evaluated through multiple indexes including appearance, behavioristics, biochemical indexes and histopathology, if all the indexes meet requirements, modeling succeeds, and the accuracy and reliability of the model are improved through multi-aspect and multi-level evaluation of the model. The model can simulate traditional Chinese medicine syndromes and pathological characteristics of cognitive impairment, and can be used for screening traditional Chinese medicines for preventing or treating kidney-yang deficiency type senile cognitive impairment.

Description

Technical Field

[0001] The present invention belongs to the field of research on animal models combining TCM diseases and syndromes, and specifically relates to a method for preparing a rat model of senile cognitive dysfunction of kidney yang deficiency type, which is used to simulate the pathological process of senile cognitive dysfunction of kidney yang deficiency type and provide an experimental basis for mechanism research and drug screening of related diseases. Background Art

[0002] Traditional Chinese Medicine (TCM) believes that the kidneys store essence, which is the foundation of life. Kidney essence is closely related to the production and nourishment of the brain marrow. The brain, the seat of the primordial spirit, governs consciousness and is nourished by kidney essence. The brain is a body of pure yang, and the head is the meeting point of all yang. "Yang Qi depletion leads to forgetfulness." Therefore, kidney yang deficiency is a key pathogenesis of cognitive impairment in the elderly, leading to malnutrition of the brain marrow and, in turn, cognitive decline. Therefore, an animal model of elderly cognitive impairment due to kidney yang deficiency, consistent with TCM syndromes, was established to provide a foundation for further investigation into the molecular pathological mechanisms and the mechanisms of action of TCM prescriptions and herbal formulas in the treatment of cognitive impairment in the elderly.

[0003] The existing animal models are all single kidney yang deficiency syndrome models, or single elderly cognitive dysfunction models. There is no combined disease and syndrome model of kidney yang deficiency type elderly cognitive dysfunction.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention aims to provide a method for constructing and applying a rat model of cognitive dysfunction in the elderly with kidney yang deficiency. This animal model, constructed in accordance with the present invention, adheres to the characteristics of "combining disease and syndrome," is simple and easy to operate, and offers a stable model with a low mortality rate and a high success rate. It can be used to study the pharmacodynamics and mechanism of action of traditional Chinese medicine in treating cognitive dysfunction in the elderly with kidney yang deficiency.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for constructing a rat model of elderly cognitive dysfunction of kidney yang deficiency type, comprising the following steps: injecting D-galactose solution subcutaneously into the back of the neck of the rats every day for 8 consecutive weeks, and administering adenine suspension by gavage from the 5th to the 8th week.

[0008] The dosage of the D-galactose is 500 mg / kg; the concentration of the D-galactose solution is 250 mg / ml.

[0009] The dosage of adenine is 30 mg / kg; the adenine suspension is prepared by adding adenine to 100 ml of 0.5% sodium carboxymethylcellulose solution, with a concentration of 15 mg / ml.

[0010] In the present invention, the dosage of D-galactose and adenine is strictly controlled to ensure successful modeling without causing serious disease damage to the animals.

[0011] In a specific embodiment of the present invention, the construction method comprises the following steps:

[0012] S1. Select 3-4 month old male SD rats;

[0013] S2. Male mice were randomly divided into a model group and a blank control group;

[0014] S3. Rats in the model group were subcutaneously injected with D-galactose 500 mg / kg on the back of the neck every day for 8 consecutive weeks, and adenine 30 mg / kg was administered orally from the 5th to the 8th week. Rats in the blank control group were fed normally and given an equal amount of normal saline.

[0015] S4. The model group rats and the blank control group rats were scored for appearance, subjected to adrenal tissue pathology analysis, HPG (hypothalamus-pituitary-gonad) / HPT (hypothalamus-pituitary-thyroid) axis related biochemical index detection, behavioral testing, serum cholinergic index, oxidative stress index, and hippocampal tissue pathology analysis to confirm the construction of the kidney yang deficiency type elderly cognitive dysfunction rat model.

[0016] In step S4, the kidney yang deficiency type is evaluated using the following indicators: appearance scoring, adrenal tissue pathological analysis, and detection of biochemical indicators related to the HPG / HPT axis.

[0017] The adrenal tissue pathological analysis was performed as follows: renal hematoxylin-eosin staining and Masson's staining were used to observe and analyze the glomerular structure, tubular cell morphology and arrangement, and whether there was inflammatory infiltration or fibrous tissue hyperplasia in the tubular lumen and interstitial structure to evaluate kidney yang deficiency syndrome.

[0018] In the evaluation of kidney yang deficiency, the HPG / HPT axis index detection is performed according to the following operation: the serum TSH, T3, LH, FSH and E2 levels are detected using an Elisa kit. The comparison of the two groups of data is considered to be significantly different when P < 0.05, and the evaluation is passed.

[0019] In step S4, the elderly cognitive dysfunction is evaluated using the following indicators: behavioral testing, hippocampal tissue pathological analysis, serum cholinergic and oxidative stress indicators.

[0020] The behavioral test was performed using the Morris water maze method, according to the following procedures:

[0021] (1) Training experiment: First, the experimental rats were trained in a continuous water maze experiment for 4 days, twice a day, each time for 1 min. The water temperature was maintained at about 24°C. The rats were placed in the water with their heads facing the wall. Four directions, east, west, south, and north, were randomly selected and marked with different colored cardboard. The time taken for the rats to find the underwater platform was recorded. If the rats did not find the platform within the time limit, the experimenter would guide the rats to the platform and let them stay there for 20 s.

[0022] (2) Formal experiment: On the fifth day of the water maze experiment, the underwater platform was removed and the rat was placed into the pool with its head facing the pool wall from the same position. The number of times the rat passed the original platform position and the time it stayed in the quadrant where the platform was located were recorded within 3 minutes.

[0023] (3) Evaluation: The time it takes for rats to find the underwater platform in the two experiments, i.e., the escape latency, as well as the number of times the rats cross the platform and the quadrant retention time in the formal experiment, were recorded. The three data were compared with the blank control group. A P value of < 0.05 between the two groups was considered to be a significant difference, indicating that the cognitive function of the rats in the model group was impaired.

[0024] The hippocampal tissue pathological analysis was performed as follows: the arrangement, number, and structure of neurons were observed and analyzed by hematoxylin-eosin staining and Nissl staining of the hippocampal tissue to determine whether they were abnormal, and whether the model had cognitive impairment by comparing with the blank control group.

[0025] In the evaluation of elderly cognitive dysfunction, the serum cholinergic test was performed according to the following procedure: the serum ACh and AChE levels were detected using an Elisa kit, and a P<0.05 comparison between the two groups of data was considered to be significantly different, and the evaluation passed.

[0026] In the evaluation of elderly cognitive dysfunction, the oxidative stress index detection is performed according to the following operation: serum SOD and MDA levels are detected using an Elisa kit, and a P < 0.05 comparison between the two groups of data is considered to be significantly different, and the evaluation is passed.

[0027] In a second aspect, the present invention provides a method for screening candidate drugs for preventing or treating kidney yang deficiency type elderly cognitive dysfunction, comprising the step of administering the test candidate drug to the kidney yang deficiency type elderly cognitive dysfunction rat model obtained by the established method.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention uses D-galactose to construct an aging animal model and simultaneously administers adenine to cause kidney yang deficiency in rats. The operation is simple, the modeling cycle is short, and the model is stable.

[0030] 2. The present invention evaluates the model through multiple indicators, including appearance, behavior, biochemical indicators, and tissue pathology. The model is successful if all indicators meet the requirements. The multi-faceted and multi-level evaluation model improves the accuracy and reliability of the model.

[0031] 3. The model obtained by the construction method provided by the present invention can simulate the pathological characteristics of TCM syndromes and cognitive impairment, and can be used to screen traditional Chinese medicines (such as Guilingji) for preventing or treating cognitive dysfunction in the elderly with kidney yang deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a diagram showing the analysis of hematoxylin-eosin staining and Masson's staining of rat kidney tissue in an embodiment of the present invention.

[0033] Figure 2 The following are the kidney-related serum biochemical indicators of kidney yang deficiency in rats in the present invention; in the figure: (a) serum follicle-stimulating hormone (FSH) level; (b) serum luteinizing hormone (LH) level; (c) serum estradiol (E2) level; (d) serum thyroid-stimulating hormone (TSH) level; (e) serum triiodothyronine (T3) level; the model group was significantly lower than the blank control group, *P<0.05, **P<0.01, ***P<0.005.

[0034] Figure 3 Figure 2 is a diagram of the Morris water maze test for rats in an embodiment of the present invention; in the figure: (a) escape latency; (b) target quadrant retention time; (c) platform crossing times; *P<0.05, **P<0.01 for the model group compared with the blank control group.

[0035] Figure 4 This is a diagram showing hematoxylin-eosin staining and Nissl staining of rat brain tissue in an embodiment of the present invention; in the figure: blue represents the nerve cell count of the blank group; red represents the nerve cell count of the model group; *P<0.05 for the model group compared with the blank control group.

[0036] Figure 5 These are the serum biochemical indicators related to aging and cognitive dysfunction in rats in the examples of the present invention; in the figure: (a) serum acetylcholine (ACh) level; (b) serum acetylcholinesterase (AChE) level; (c) serum superoxide dismutase (SOD) level; (d) serum malondialdehyde (MDA) level; *P<0.05, **P<0.01 in the model group compared with the blank control group.

[0037] Figure 6 This is a diagram showing the analysis of hematoxylin-eosin staining and Masson's staining of renal tissues of rats in each group after drug administration in the examples of the present invention.

[0038] Figure 7The following are the kidney-related serum biochemical indicators of kidney yang deficiency in rats in each group after drug administration in the embodiment of the present invention; in the figure: (a) serum follicle-stimulating hormone (FSH) level; (b) serum luteinizing hormone (LH) level; (c) serum estradiol (E2) level; (d) serum thyroid-stimulating hormone (TSH) level; (e) serum triiodothyronine (total T3) level; the model group was compared with the blank control group #P<0.05, ##P<0.01, ###P<0.005, and the drug-treated group was compared with the model group *P<0.05, **P<0.01, ***P<0.005.

[0039] Figure 8 The Morris water maze test diagram of rats in each group after drug administration in the embodiment of the present invention; in the figure: (a) escape latency; (b) target quadrant retention time; (c) platform crossing times; #P<0.05, ##P<0.01 for the model group compared with the blank control group, *P<0.05, **P<0.01 for the drug administration group compared with the model group.

[0040] Figure 9 This is a display of hematoxylin-eosin staining and Nissl staining of the brain tissue of rats in each group after drug administration in the examples of the present invention; in the figure, blue represents the nerve cell count of the blank group; red represents the nerve cell count of the model group; green represents the nerve cell count of the Guilingji low-dose group; purple represents the nerve cell count of the Guilingji high-dose group; #P<0.05 for the model group compared with the blank control group, *P<0.05 for the drug-treated group compared with the model group.

[0041] Figure 10 These are the serum biochemical indicators related to aging and cognitive dysfunction in rats in each group after drug administration in the examples of the present invention; (a) serum acetylcholine (ACh) level; (b) serum acetylcholinesterase (AChE) level; (c) serum superoxide dismutase (SOD) level; (d) serum malondialdehyde (MDA) level; #P<0.05, ##P<0.01 in the model group compared with the blank control group; *P<0.05, **P<0.01 in the drug administration group compared with the model group. DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to specific examples, but the present invention is not limited to the following examples.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0044] Unless otherwise specified, the reagents, materials, instruments, etc. used in the following examples can be obtained from commercial sources.

[0045] Example

[0046] This embodiment provides a method for preparing a rat model of elderly cognitive dysfunction of kidney yang deficiency type, comprising the following steps:

[0047] (1) Animal grouping:

[0048] Male SD rats were randomly divided into a blank control group and a model group (kidney yang deficiency + elderly cognitive dysfunction), with 10 rats in each group.

[0049] (2) Model preparation:

[0050] Model group: D-galactose 500 mg / kg was injected subcutaneously in the neck and back every day for 8 consecutive weeks, and adenine 30 mg / kg was administered by gavage from the 4th to the 8th week;

[0051] Blank control group: The mice were given an equal amount of normal saline by gavage.

[0052] (3) Evaluation of Kidney Yang Deficiency:

[0053] a. Scoring criteria were fur dullness, decreased diet, locomotor activity, weight gain, loose stools, and frequent chills. Higher scores indicated more pronounced kidney yang deficiency. The evaluation criteria are shown in Table 1, and the scores for each group are shown in Table 2.

[0054] Table 1 Quantitative scoring table of kidney yang deficiency in rats

[0055]

[0056]

[0057] Table 2 Kidney Yang deficiency index scores of rats in the blank control group and model group

[0058]

[0059] b. Histopathological analysis:

[0060] The rat kidney tissue was fixed with paraformaldehyde, embedded in paraffin, sliced, and stained with hematoxylin-eosin and Masson's staining. The glomerular structure, tubular cell morphology and arrangement, tubular lumen, and interstitial structure were observed under a light microscope to see if there was inflammatory infiltration or fibrous tissue proliferation. Figure 1 As shown in the data, the glomeruli of rats in the CON group were regular in shape, the cells in the glomeruli were neatly arranged, the number and cell matrix were uniform, the renal tubular epithelial cells were plump, and the brush borders were neatly arranged. The glomeruli of rats in the MOD group were irregular in shape, the cells in the glomeruli were disordered, and the renal tubular epithelial cells showed vacuolar degeneration, loose cytoplasm, and detached brush borders.

[0061] c. Serum biochemical index detection:

[0062] Blood was collected from the abdominal aorta of rats and centrifuged after 30 minutes of rest to obtain the supernatant. The serum TSH, T3, LH, FSH and E2 levels were detected by Elisa. If the model group was compared with the blank control group with P < 0.05, the model was considered to have kidney yang deficiency syndrome. Figure 2 As shown in the results, the levels of TSH and T3, the HPG axis related indicators, in the model group were significantly lower than those in the blank group, indicating that the rats in the model group had hypothyroidism, which was consistent with the symptoms of kidney yang deficiency. The levels of LH, FSH and E2, the HPT axis related indicators, in the model group were significantly lower than those in the blank group, indicating that the rats in the model group had hypopituitarism, which was consistent with the symptoms of kidney yang deficiency.

[0063] (4) Evaluation of cognitive dysfunction in the elderly:

[0064] a. Behavioral testing:

[0065] The Morris water maze was used to test the model group rats' escape latency, number of platform crossings, and platform quadrant retention time to evaluate the rats' learning and memory abilities. Figure 3 As shown in the data, starting from the second day of training, the escape latency of the model group rats was significantly prolonged, the retention time in the target quadrant was significantly shortened, and the number of times crossing the underwater platform was significantly reduced compared with the blank group rats, indicating that the learning and memory ability of the model group rats was impaired.

[0066] b. Hippocampal tissue pathological analysis:

[0067] The rat brain tissue was fixed with paraformaldehyde, embedded in paraffin, sliced, and stained with hematoxylin-eosin and Nissl stain. Figure 4 As shown, light microscopic observation revealed that the neurons of the model group rats were disordered, their number decreased, and their Nissl bodies dissolved.

[0068] c. Serum cholinergic and oxidative stress indicators:

[0069] Biochemical indicators were measured by Elisa method to detect serum SOD, MDA, ACh, and AChE levels. Figure 5 As shown in the figure, the model group was compared with the blank control group, P < 0.05, which was considered to be a model with cognitive dysfunction.

[0070] (5) Application of the model:

[0071] After the model was successfully established, the 10 rats in the model group were randomly divided into two groups. Drug administration began from the 5th week. One group was given a high dose of Guilingji (150 mg / Kg) by gavage, and the other group was given a low dose of Guilingji (75 mg / Kg) by gavage. The drugs were administered continuously for 4 weeks. Finally, the efficacy of different doses of drugs was evaluated using the evaluation indicators of kidney yang deficiency and elderly cognitive dysfunction.

[0072] a. Efficacy evaluation - improving kidney yang deficiency:

[0073] The appearance status scores are shown in Table 3.

[0074] Table 3 Kidney Yang deficiency index scores of rats in each group after administration

[0075]

[0076] From the results in Table 3, we can see that high-dose Guilingji has a significant improvement effect on kidney yang deficiency, while the improvement effect of low-dose Guilingji is slightly worse than that of high-dose.

[0077] like Figure 6 As shown, renal tissue pathology showed that high-dose Guilingji could significantly improve the glomerular structure, tubular cell morphology and arrangement, and tubular lumen and interstitial inflammatory infiltration in the model group rats.

[0078] like Figure 7 As shown in the results, the serum biochemical indicators TSH, T3, LH, FSH and E2 showed that both high and low doses of Guilingji could significantly restore these indicators to normal levels.

[0079] b. Pharmacological evaluation - improving cognitive dysfunction in the elderly:

[0080] like Figure 8 As shown in the Morris water maze experiment, both high and low doses of Guilingji could significantly regulate the escape latency, the number of times crossing the platform and the time spent in the platform quadrant.

[0081] like Figure 9 As shown, the results of hematoxylin-eosin staining and Nissl staining of rat brain tissue showed that both high and low doses of Guilingji could significantly improve the disordered arrangement and reduced number of neurons and the dissolution of Nissl bodies in the model group rats, and the high dose had a better improvement effect.

[0082] like Figure 10 As shown in the figure, the test results of serum biochemical indicators SOD, MDA, ACh and AChE content showed that both high and low doses of Guilingji could significantly regulate the above indicators.

[0083] The above experiments show that the rat model of kidney yang deficiency type elderly cognitive dysfunction combined with disease syndrome established in the present invention can be used to evaluate the pharmacological effects of the traditional Chinese medicine Guilingji and screen the optimal dosage.

[0084] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for constructing a rat model of elderly cognitive dysfunction of kidney yang deficiency type, comprising the following steps: injecting D-galactose solution subcutaneously into the back of the neck of the rats every day for 8 consecutive weeks, and administering adenine suspension by gavage from the 5th to the 8th week.

2. The construction method according to claim 1, characterized in that The dosage of the D-galactose is 500 mg / kg; the concentration of the D-galactose solution is 250 mg / ml.

3. The construction method according to claim 1 or 2, characterized in that The dosage of adenine is 30 mg / kg; the adenine suspension is prepared by adding adenine to 100 ml of 0.5% sodium carboxymethylcellulose solution, with a concentration of 15 mg / ml.

4. The construction method according to claim 1, characterized in that The construction method also includes performing appearance scoring, adrenal tissue pathology analysis, HPG / HPT axis-related biochemical index detection, behavioral testing, serum cholinergic index, oxidative stress index, and hippocampal tissue pathology analysis on the rats in the model group and the blank control group to confirm whether the model is constructed.

5. The construction method according to claim 4, characterized in that In step S4, the kidney yang deficiency type is evaluated using the following indicators: appearance scoring, adrenal tissue pathological analysis, and detection of biochemical indicators related to the HPG / HPT axis.

6. The construction method according to claim 5, characterized in that: In the evaluation of kidney yang deficiency, the adrenal tissue pathological analysis is performed according to the following procedures: kidney hematoxylin-eosin staining and Masson staining are used to observe and analyze the glomerular structure, tubular cell morphology and arrangement, and whether there is inflammatory infiltration or fibrous tissue hyperplasia in the tubular lumen and interstitial structure to evaluate kidney yang deficiency syndrome; In the evaluation of kidney yang deficiency, the HPG / HPT axis index detection is performed according to the following operation: the serum TSH, T3, LH, FSH and E2 levels are detected using an Elisa kit. The comparison of the two groups of data is considered to be significantly different when P < 0.05, and the evaluation is passed.

7. The construction method according to claim 4, characterized in that: In step S4, the elderly cognitive dysfunction is evaluated using the following indicators: behavioral testing, hippocampal tissue pathological analysis, serum cholinergic index and oxidative stress index.

8. The construction method according to claim 7, characterized in that: In the evaluation of elderly cognitive dysfunction, the behavioral test adopts the Morris water maze method and is performed according to the following procedures: (1) Training experiment: First, the experimental rats were trained in a continuous water maze experiment for 4 days, twice a day, each time for 1 min. The water temperature was maintained at about 24°C. The rats were placed in the water with their heads facing the wall. Four directions, east, west, south, and north, were randomly selected and marked with different colored cardboard. The time taken for the rats to find the underwater platform was recorded. If the rats did not find the platform within the time limit, the experimenter would guide the rats to the platform and let them stay there for 20 s. (2) Formal experiment: On the fifth day of the water maze experiment, the underwater platform was removed and the rat was placed into the pool with its head facing the pool wall from the same position. The number of times the rat passed the original platform position and the time it stayed in the quadrant where the platform was located were recorded within 3 minutes. (3) Evaluation: The time it takes for rats to find the underwater platform in the two experiments, i.e., the escape latency, as well as the number of times the rats cross the platform and the quadrant retention time in the formal experiment, were recorded. The three data were compared with the blank control group. A P value of < 0.05 between the two groups was considered to be a significant difference, indicating that the cognitive function of the rats in the model group was impaired.

9. The construction method according to claim 7, characterized in that: In the evaluation of elderly cognitive dysfunction, the hippocampal tissue pathological analysis is performed according to the following procedures: the arrangement, number, and structure of neurons are observed and analyzed by hematoxylin-eosin staining and Nissl staining of hippocampal tissue to see if they are abnormal, and by comparing with the blank control group, whether the model has cognitive impairment; In the evaluation of elderly cognitive dysfunction, the serum cholinergic test was performed as follows: serum ACh and AChE levels were detected using an Elisa kit. A P<0.05 comparison between the two groups of data was considered to indicate a significant difference, and the evaluation was passed. In the evaluation of elderly cognitive dysfunction, the oxidative stress index detection is performed according to the following operation: serum SOD and MDA levels are detected using an Elisa kit, and a P < 0.05 comparison between the two groups of data is considered to be significantly different, and the evaluation is passed.

10. A method for screening candidate drugs for preventing or treating kidney yang deficiency type elderly cognitive dysfunction disease, characterized in that: The method comprises the step of administering a test candidate drug to the kidney yang deficiency type elderly cognitive dysfunction rat model obtained by the establishment method according to any one of claims 1 to 9.

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