Path research and application of recuperating soup for improving cognitive impairment

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a conditioning decoction for relieving cognitive impairment by regulating a signal channel of hippocampal astrocytes. The invention provides an application of a conditioning decoction, the conditioning decoction is used for preparing a composition or a preparation, and the composition or the preparation is applied to an AQP1 low expression type object so as to improve cognitive impairment of the object. Meanwhile, the invention provides a method for screening the composition or the reagent for improving the cognitive impairment and a kit for evaluating the relieving effect of the composition or the preparation on the cognitive impairment.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and specifically relates to Tiaogeng decoction that alleviates cognitive impairment by regulating the signal pathway of hippocampal astrocytes. Background Art

[0002] With the aging of the population, the incidence of neurodegenerative diseases is increasing year by year. Mild cognitive impairment is the early stage of many neurodegenerative diseases. It takes a long time for patients with neurodegenerative diseases to go from mild cognitive impairment to being unable to live independently. Therefore, if the level of cognitive impairment can be determined in time and early intervention can be carried out, it will be of great significance for the prevention and treatment of related neurodegenerative diseases.

[0003] Men and women experience cognitive decline during andropause and menopause, respectively. Testosterone therapy is used to prevent cognitive decline in men during andropause. Testosterone is not preferred by women due to masculinizing side effects, and its use has not been approved for the preservation of cognition in women. Hormone replacement therapy for menopausal women typically involves treatment with estradiol, conjugated equine estrogens (CEE), or Premarin. However, these therapies are associated with an increased risk of breast and uterine cancer, as well as cardiovascular risk.

[0004] In addition, due to the complex causes of cognitive impairment, there is currently a lack of satisfactory means to effectively classify and diagnose and treat cognitive impairment in this field.

[0005] Therefore, there is an urgent need to develop new methods to effectively classify cognitive impairment and provide targeted diagnosis and treatment plans accordingly. Summary of the invention

[0006] The purpose of the present invention is to provide a method for effectively classifying cognitive impairment and to provide a corresponding targeted diagnosis and treatment plan.

[0007] In a first aspect of the present invention, there is provided a use of a soup for preparing a composition or preparation, wherein the composition or preparation is used for one or more uses selected from the following group:

[0008] (Z0) is administered to a subject with low AQP1 expression to improve cognitive impairment of the subject;

[0009] (Z1) Increase the expression level of water channel protein AQP1;

[0010] (Z2) reduce the expression level of amyloid protein Aβ;

[0011] (Z3) reduce the expression level of nuclear factor NF-kB protein;

[0012] (Z4) Reduce the expression level of estrogen receptor ERα.

[0013] In another preferred embodiment, the AQP1 low expression type refers to: the AQP1 expression level in the astrocytes of the subject is significantly lower than that of the normal control.

[0014] In another preferred embodiment, the “significantly lower than” refers to that the ratio of the AQP1 expression level E1 in the subject's astrocytes to the AQP1 expression level E0 in the normal control's astrocytes (E1 / E0) is ≤2 / 3, preferably ≤1 / 2, and more preferably ≤1 / 3.

[0015] In another preferred embodiment, the subject also has the following typing characteristics:

[0016] (Y1) The expression level of amyloid Aβ protein was significantly higher than that of normal control;

[0017] (Y2) The expression level of nuclear factor NF-kB protein is significantly higher than that of normal control; and / or

[0018] (Y3) The expression level of estrogen receptor ERα was significantly higher than that of normal control.

[0019] In another preferred embodiment, the “significantly higher” refers to a ratio of the amyloid Aβ protein expression level H1 of the subject to the Aβ expression level H0 of the normal control, H1 / H0 ≥ 1.5, preferably ≥ 1.3, more preferably ≥ 1.1;

[0020] The ratio of the NF-kB protein expression level M1 of the subject to the NF-kB protein expression level M0 of the normal control is M1 / M0≥1.5, preferably ≥1.3, more preferably ≥1.1; and / or

[0021] The ratio of the expression level N1 of the estrogen receptor ERα to the expression level N0 of the estrogen receptor ERα of the subject is N1 / N0≥1.5, preferably ≥1.3, and more preferably ≥1.1.

[0022] In another preferred embodiment, the normal control is a healthy young person.

[0023] In another preferred embodiment, the subject is a young person, a middle-aged person, or an elderly person.

[0024] In another preferred embodiment, the subject is female.

[0025] In another preferred embodiment, the subject is a menopausal or postmenopausal woman.

[0026] In another preferred embodiment, the composition or preparation is used for one or more purposes selected from the following group:

[0027] (Z1) Increase the expression level of water channel protein AQP1;

[0028] (Z2) reduce the expression level of amyloid protein Aβ;

[0029] (Z3) reducing the expression level of nuclear factor NF-kB protein; and / or

[0030] (Z4) Reduce the expression level of estrogen receptor ERα.

[0031] In another preferred embodiment, the expression level includes protein level and / or nucleic acid level.

[0032] In another preferred embodiment, the soup is a liquid preparation, an ointment, a solid preparation (such as granules, tablets, pills, powders, capsules and microcapsules).

[0033] In another preferred example, the Tiaogeng Decoction includes components in the following weight ratio: 1-2 weight parts of Anemarrhena asphodeloides, 1-2 weight parts of Phellodendron chinense, 1-2 weight parts of Bupleurum chinense, 1.5-2.5 weight parts of White Peony Root, 2.5-3.5 weight parts of Rehmannia glutinosa, 2.5-3.5 weight parts of Epimedium brevicornum, 1-2 weight parts of Morinda officinalis, 5-7 weight parts of Raw Dragon Bone, 5-7 weight parts of Raw Osmanthus fragrans and 5-7 weight parts of Polygonum multiflorum.

[0034] In another preferred example, the Tiaogeng Decoction comprises components in the following weight ratio: 1.8 parts by weight of Anemarrhena asphodeloides, 1.8 parts by weight of Phellodendron chinense, 1.8 parts by weight of Bupleurum chinense, 2 parts by weight of White Peony Root, 3 parts by weight of Rehmannia glutinosa, 3 parts by weight of Epimedium brevicornum, 1.8 parts by weight of Morinda officinalis, 6 parts by weight of Raw Dragon Bone, 6 parts by weight of Raw Osmanthus fragrans and 6 parts by weight of Polygonum multiflorum.

[0035] In another preferred embodiment, the composition or preparation is a pharmaceutical composition, a food composition or a dietary supplement.

[0036] In another preferred embodiment, the form of the composition or preparation includes but is not limited to: liquid reagent, granule, tablet, pill, powder, capsule and microcapsule.

[0037] In another preferred embodiment, the composition or preparation is preferably in the form of freeze-dried powder of soup.

[0038] In another preferred embodiment, the preparation method of the freeze-dried powder is as follows:

[0039] (1) All the raw materials of Tiaogeng decoction are decocted according to conventional Chinese medicine decoction methods to obtain effective components of Chinese medicine, and Chinese medicine liquid is obtained. The Chinese medicine is continuously decocted at high temperature until the Chinese medicine evaporates and concentrates, and the volume of the liquid is reduced to 0.3-0.6 of the original volume. The remaining liquid is concentrated again to about 1 / 10 of the original volume by a rotary evaporator to obtain a concentrated liquid.

[0040] (2) After the concentrated drug solution is packaged, freeze-dried to obtain dry blocks.

[0041] (3) The dried mass is crushed to obtain freeze-dried powder.

[0042] In a second aspect of the present invention, a method for up-regulating the expression of AQP1 in astrocytes in vitro is provided, comprising the steps of: culturing astrocytes in the presence of Tiaogeng Tang, thereby up-regulating the expression level of AQP1 protein in astrocytes.

[0043] In another preferred embodiment, the method described in the second aspect of the present invention is a non-diagnostic and non-therapeutic method.

[0044] In another preferred embodiment, in the culture system, the concentration of the broth is 200-2000 μg / mL, preferably 300-1500 μg / mL, and most preferably 312.5-1250 μg / mL.

[0045] In another preferred embodiment, the administration time of the modified soup is 12-48 hours, preferably 24 hours.

[0046] In a third aspect of the present invention, a companion diagnostic reagent combination is provided, comprising: (a) a first detection reagent for detecting AQP1; and (b) shaogeng decoction.

[0047] A fourth aspect of the present invention provides a method for screening a potential therapeutic agent for alleviating cognitive impairment, comprising the following steps:

[0048] (a) in an experimental group, astrocytes were cultured in the presence of a test substance; and in a control group, astrocytes were cultured under the same conditions but in the absence of the test substance;

[0049] (b) respectively determining the expression levels of aquaporin AQP1 in astrocytes of the experimental group and the control group and comparing them; if the expression level Y1 of aquaporin AQP1 in the experimental group is significantly increased compared with the expression level Y0 of aquaporin AQP1 in the control group, the test substance is a potential therapeutic agent for alleviating cognitive impairment.

[0050] In another preferred embodiment, the test substance is a single compound, a mixture, an extract, or a compound preparation.

[0051] In another preferred embodiment, the method further comprises: (c) further performing activity testing or functional testing on the potential therapeutic agent selected in the previous step.

[0052] In another preferred embodiment, the “significant increase” refers to Y1 / Y0≥1.2, preferably ≥1.5, and more preferably ≥2.0.

[0053] In another preferred embodiment, the expression level of the water channel protein AQP1 includes protein expression level and / or mRNA level.

[0054] In a fifth aspect of the present invention, there is provided a use of a detection reagent for detecting AQP1, namely, for preparing a kit for evaluating the therapeutic effect of Tiaogeng Decoction on cognitive impairment.

[0055] In another preferred embodiment, the detection reagent for detecting AQP1 detects AQP1 protein level and / or mRNA level.

[0056] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The effects of shaogeng decoction on cognitive abilities characterized by a water maze test are shown.

[0058] Figure 2 Shown is the effect of Tiaogeng decoction on the expression of AQP1 mRNA level characterized by next-generation gene sequencing.

[0059] Figure 3 Shown is the effect of Tiaogeng Decoction on Aβ expression level detected by immunofluorescence.

[0060] Figure 4 Shown is the effect of Tiaogeng Decoction on AQP1 expression level detected by immunofluorescence.

[0061] Figure 5 Shown is the effect of Tiaogengtang on the expression of Aβ mRNA level characterized by qRT-PCR.

[0062] Figure 6 Shown is the effect of Tiaogeng decoction on the expression of AQP1 mRNA level characterized by qRT-PCR.

[0063] Figure 7 Shown is the effect of Tiaogengtang on the expression of NF-kB mRNA level characterized by qRT-PCR.

[0064] Figure 8 Shown is the effect of Tiaogeng decoction on the expression of ER-β mRNA level characterized by qRT-PCR.

[0065] Fig. 9 Shown is the effect of Tiaogeng decoction on the expression of ER-α mRNA level characterized by qRT-PCR.

[0066] Fig.10Western Blot analysis shows the effect of Tiaogeng Decoction on the expression level of AQP1 in hippocampal tissue.

[0067] Fig.11 The effect of Tiaogeng decoction on the expression of AQP1 mRNA level in astrocytes is shown.

[0068] Fig.12 The effect of Tiaogeng decoction on the expression level of AQP1 protein in astrocytes is shown. DETAILED DESCRIPTION

[0069] After extensive and in-depth research, the inventors unexpectedly discovered for the first time through a large number of screening and bioinformatics studies that an existing Chinese medicine preparation, Tiaogeng Decoction, can effectively improve cognitive impairment in specific subjects. Experiments also show that Tiaogeng Decoction can upregulate the expression of the water channel protein AQP1 in hippocampal neuronal cells. Therefore, Tiaogeng Decoction is particularly suitable for subjects with low expression of AQP1 to improve cognitive impairment of the subjects, especially female subjects in the perimenopausal or menopausal period. The present invention was completed on this basis.

[0070] the term

[0071] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0072] As used herein, the terms "comprising", "including", and "comprising" may be open, semi-closed, and closed. In other words, the terms also include "consisting essentially of" or "consisting of".

[0073] As used herein, "TGD" and "TGD" are used interchangeably and both refer to the TGD described in the present invention.

[0074] Soup

[0075] The present invention provides a use of a shogen soup, wherein the shogen soup is used to prepare a composition or a preparation, and the composition or the preparation is administered to a subject with low AQP1 expression type to improve the cognitive impairment of the subject.

[0076] Tiaogeng decoction is a traditional Chinese medicine preparation made from Anemarrhena asphodeloides, Phellodendron chinense, Bupleurum chinense, White Peony Root, Raw Rehmannia glutinosa, Epimedium brevicornum, Morinda officinalis, Raw Dragon Bone, Raw Oyster and Polygonum multiflorum. It can improve patients' symptoms such as hot flashes, sweating, restlessness and insomnia. In addition, Tiaogeng decoction has been shown to have a protective effect in hypothalamic neurons and can improve the cognitive ability of menopausal women. However, the regulation of cognitive ability by Tiaogeng decoction is a broad regulation, and its mechanism is still unclear. Therefore, the effect of Tiaogeng decoction in regulating a specific subtype of cognitive impairment needs to be further explored.

[0077] In another preferred example, the Tiaogeng Decoction comprises the following components in the following weight ratio: 9g of Anemarrhena asphodeloides, 9g of Phellodendron chinense, 9g of Bupleurum chinense, 10g of White Peony Root, 15g of Rehmannia glutinosa, 15g of Epimedium brevicornum, 9g of Morinda officinalis, 30g of Raw Dragon Bone, 30g of Raw Oyster and 30g of Polygonum multiflorum.

[0078] In another preferred embodiment, a typical method for preparing freeze-dried powder of tofu soup comprises:

[0079] (1) All the Chinese medicines in the Tiaogeng Decoction were decocted according to the conventional Chinese medicine decoction method to obtain the effective ingredients of the Chinese medicines, and about 5000 ml of Chinese medicine liquid was obtained. The Chinese medicines were continuously decocted at high temperature until they evaporated and concentrated, and the volume of the liquid was reduced to 2500 ml. The remaining liquid was concentrated again by a rotary evaporator, and finally about 450 ml of liquid was obtained.

[0080] (2) Dispense the liquid medicine into small iron plates, seal and label the plates, and place them in a -80°C freezer for one week. After the liquid medicine is frozen dry, break it into pieces for later use.

[0081] (3) Place the small iron plate containing the dried blocks of each drug solution into the Sihuan freeze-dried powder making machine and take it out after two days. Observe that the texture of the dried blocks has become lighter, crush them and divide them into 50 ml centrifuge tubes for subsequent experiments.

[0082] Soup improves cognitive impairment in certain subjects

[0083] The subject treated with Tiaogengtang has an increased expression level of aquaporin AQP1 and a decreased expression level of amyloid Aβ in hippocampal astrocytes, and the cognitive impairment of the subject is improved. Therefore, the present invention provides a new use of Tiaogengtang, especially for improving cognitive impairment of a specific subject.

[0084] In the present invention, the preferred specific objects have the following typing characteristics:

[0085] (a) AQP1 low expression type;

[0086] (b) female or feminine;

[0087] (c) in the perimenopausal or menopausal period.

[0088] Preferably, the preferred specific object also has one or more of the following typing characteristics:

[0089] (Y1) The expression level of amyloid Aβ protein was significantly higher than that of normal control;

[0090] (Y2) The expression level of nuclear factor NF-kB protein was significantly higher than that of normal control;

[0091] (Y3) The expression level of estrogen receptor ERα was significantly higher than that of normal control.

[0092] Experiments have shown that administering Tiaogeng Soup to subjects with low AQP1 expression can increase the expression of AQP1, reduce the expression level of amyloid Aβ protein, and improve the cognitive impairment of the subjects.

[0093] Tiaogengtang upregulates the expression of AQP1

[0094] The present invention also provides a method for up-regulating the expression of AQP1 in astrocytes in vitro, comprising the steps of: culturing astrocytes in the presence of Tiaogeng Tang, thereby up-regulating the expression level of AQP1 protein in the astrocytes.

[0095] Since the up-regulation of AQP1 expression is correlated with the decreased expression of amyloid Aβ protein and is correlated with the improvement of cognitive impairment, the present invention also provides a method for screening candidate compositions or agents for alleviating cognitive impairment.

[0096] Typically, a method for screening potential therapeutic agents for alleviating cognitive impairment comprises the following steps:

[0097] (a) in an experimental group, astrocytes were cultured in the presence of a test substance; and in a control group, astrocytes were cultured under the same conditions but in the absence of the test substance;

[0098] (b) respectively determining the expression levels of aquaporin AQP1 in astrocytes of the experimental group and the control group and comparing them; if the expression level Y1 of aquaporin AQP1 in the experimental group is significantly increased compared with the expression level Y0 of aquaporin AQP1 in the control group, it indicates that the test substance is a potential therapeutic agent for alleviating cognitive impairment.

[0099] The test substances include (but are not limited to): small molecule compounds, traditional Chinese medicine preparations (single or compound), antibodies, nucleic acid drugs, or combinations thereof.

[0100] In another preferred embodiment, the method further comprises a positive control group, wherein astrocytes are cultured under the same conditions but in the absence of the test substance and in the presence of the soup. If the expression level Y1 of the water channel protein AQP1 in the experimental group is ≥ the expression level Y2 of the water channel protein AQP1 in the positive control group, it indicates that the test substance is a potential therapeutic agent for alleviating cognitive impairment.

[0101] In another preferred embodiment, in the positive control group, the concentration of the shaogeng decoction is 200-2000 μg / mL, preferably 300-1500 μg / mL, and most preferably 312.5-1250 μg / mL.

[0102] AQP1 Assays and Companion Diagnostic Kits

[0103] In the present invention, the shogen soup is particularly suitable for subjects with low AQP1 expression to improve cognitive impairment of the subject. Therefore, the present invention also provides a companion diagnostic method for identifying subjects suitable for the therapy of the present invention.

[0104] Typically, the reagent combination for companion diagnosis of the present invention comprises: (a) a first detection reagent for detecting AQP1; and (b) shokugen soup. The present invention also provides a kit containing the above reagent combination.

[0105] Typically, the kit of the present invention further comprises an instruction sheet for describing the method of companion diagnosis.

[0106] In the present invention, the first detection reagent for detecting AQP1 can be a conventional reagent for detecting AQP1 at the mRNA level or protein level. There is no particular limitation on the method for detecting the expression level of AQP1 that can be used in the present invention, and representative examples include but are not limited to: mRNA sequencing, immunofluorescence detection, qRT-PCR, and Western Blot.

[0107] In addition, during the treatment process, the change in AQP1 levels can be used as an indicator to evaluate the degree of improvement of cognitive impairment by Tiaogeng Soup. Generally, an increase in AQP1 levels indicates a better improvement effect or degree of cognitive impairment.

[0108] The main advantages of the present invention include:

[0109] (a) The present invention provides a use of shogeng tang for improving cognitive impairment of AQP1 low expression type. The cognitive level of the subject treated with shogeng tang is improved.

[0110] (b) The present invention provides a non-diagnostic, non-therapeutic method for upregulating the expression of AQP1 in astrocytes in vitro.

[0111] (c) The present invention provides a method for screening a composition or agent for improving cognitive impairment.

[0112] (d) The present invention provides a kit for evaluating the effect of a composition or preparation on alleviating cognitive impairment, wherein the kit comprises a diagnostic reagent for AQP1.

[0113] The present invention will be described in further detail below in conjunction with specific embodiments and with reference to data. It should be understood that these embodiments are intended to illustrate the present invention and are not intended to limit the scope of the invention in any way. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as the conditions described in Sambrook et al. Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0114] Experimental methods:

[0115] 1. Construction of sham-operated mice (control group)

[0116] The same volume of fat tissue near the bilateral ovaries of C57BL young mice was removed as a sham operation group.

[0117] 2. Construction of model mice

[0118] The APP / PS1 mouse model was purchased directly from Beijing Huafukang Biotechnology Co., Ltd. The ovariectomized mouse model was obtained by removing both ovaries.

[0119] 3. Construction of estradiol group mice (Western medicine group)

[0120] After bilateral ovariectomy, the daily estradiol dose required for each mouse (weight about 20g) was 0.003mg / mouse (about 0.15mg / kg), and the daily oral dose was 0.3mL, once a day, for 3 consecutive months. In the astrocyte experiment, the estradiol concentration was 1μM.

[0121] 4. Construction of Tiaogeng Decoction Group Mice

[0122] For the APP / PS1 mouse model with bilateral ovariectomy, the daily amount of the soup required for each mouse (weighing about 20g) is 64mg / mouse (calculated on dry weight) (about 3200mg / kg), with a daily oral dose of 0.4mL, once a day for 3 consecutive months.

[0123] In the astrocyte experiment, the concentrations of Tiaogeng decoction in the low-dose, medium-dose, and high-dose groups were 312.5 μg / mL, 625 μg / mL, and 1250 μg / mL, respectively.

[0124] 5. Immunofluorescence detection of Aβ and AQP1 expression levels in mouse brain tissue

[0125] The brain tissue of each group of mice was isolated, and 3 intact brain tissues were randomly immersed in 4% paraformaldehyde for 24 hours. Subsequently, the brain tissue of each group of mice was embedded in paraffin and stored at -20 °C. It was cut into 5 μm thick sections, baked in a drying oven at 60 °C for 60 minutes, dewaxed to water, and then the antigen retrieval solution was heated to boiling in a microwave oven at high temperature, and the sections were immersed in the retrieval solution, heated for 20 minutes, taken out and cooled for 20 minutes, incubated with hydrogen peroxide for 10 minutes, washed three times with PBS, added goat serum blocking solution, incubated at room temperature for 10 minutes, added an appropriate amount of diluted primary antibody, overnight at 4 °C, washed three times with PBS, added an appropriate amount of secondary antibody, incubated at room temperature for 10 minutes, washed three times with PBS, and then added TSA fluorescent dye and hydrogen peroxide for 10 minutes each, and washed three times with PBS. Add an appropriate amount of DAPI and stain again for 5 minutes, seal the film with anti-quencher, and then scan with Zeiss Axio Scan Z1.

[0126] Example 1 Preparation of freeze-dried powder of Tiaogeng Soup

[0127] This embodiment provides the preparation of freeze-dried powder of Tiaogengtang, wherein the following formula is used:

[0128] Recipe: 9g of Anemarrhena asphodeloides, 9g of Phellodendron chinense, 9g of Bupleurum chinense, 10g of White Peony Root, 15g of Rehmannia glutinosa, 15g of Epimedium brevicornum, 9g of Morinda officinalis, 30g of Raw Dragon Bone, 30g of Raw Oyster and 30g of Polygonum multiflorum.

[0129] The production method is as follows:

[0130] (1) All Chinese medicines in the Tiaogeng Decoction were decocted according to the conventional Chinese medicine decoction method to obtain the effective ingredients of the Chinese medicines, and about 5000 ml of Chinese medicine liquid was obtained. The Chinese medicines were continuously decocted at high temperature until they evaporated and concentrated, and the volume of the liquid was reduced to 2500 ml. The remaining liquid was concentrated again by a rotary evaporator, and the final liquid was about 450 ml.

[0131] (2) Divide the liquid medicine into small iron plates and place them in a -80°C freezer for one week. After the liquid medicine is frozen dry, break it into pieces for later use.

[0132] (3) Crush the dried blocks containing the various medicinal solutions to obtain freeze-dried powder of the Tiaogeng decoction, and divide them into portions for later use.

[0133] Example 2: Tiaogeng decoction improves the learning ability of model animals

[0134] A water maze experiment was performed using mice in the sham operation group, model group, western medicine group and Tiaogeng Decoction group, and the escape latency time of the mice in each group was recorded on the first, second, third and fourth days respectively.

[0135] The results are shown in Table 1.

[0136] Table 1. Escape latency of each group (s)

[0137]

[0138] Note: ** P<0.01, there is a statistical difference compared with the sham operation group; *P<0.05, there is a statistical difference compared with the sham operation group; # P<0.05, ## P<0.01 was statistically different compared with the model group.

[0139] The escape latency of mice in each group in Table 1 shows that on the 4th day, the escape latency of the model group was significantly longer than that of the sham operation group, while the latency of the western medicine group and the Tiaogeng Decoction group was significantly shorter than that of the model group, with statistical differences. This shows that the treatment of Tiaogeng Decoction and the western medicine group can significantly improve the learning speed of mice.

[0140] Example 3: Tiaogeng decoction improves cognitive impairment in model animals

[0141] A water maze experiment was performed using mice in the sham operation group, model group, western medicine group and Tiaogeng Decoction group. The trajectories of mice in each group were recorded, and the number of times they crossed the platform was statistically compared.

[0142] The results are as follows Figure 1 The trajectory diagrams of mice in each group showed that compared with the sham operation group, the number of times the model group crossed the platform was significantly reduced (P < 0.05), and the difference between the two groups was statistically significant.

[0143] Compared with the model group, the number of platform crossings in the western medicine group and the Tiaogeng Decoction group increased, but there was no statistically significant difference between the two groups (P>0.05).

[0144] The above results show that the cognitive ability of mice in the model group was significantly lower than that in the sham operation group, and the intervention or treatment of Tiaogeng Decoction can significantly reverse the cognitive impairment, and its reversal effect is comparable to that of positive drugs (estradiol).

[0145] Example 4: Tiaogeng decoction intervention significantly increased AQP1 mRNA levels

[0146] In this example, based on a bilaterally ovariectomized mouse model, the gene expression profile of the bilaterally ovariectomized mouse model and the effect of Tiaogeng Decoction intervention on the gene expression profile were studied by mRNA sequencing analysis.

[0147] The hippocampal tissues of C57BL young mice (control group), APP / PS1 transgenic mice (bilaterally ovariectomized model group) and APP / PS1 transgenic mice treated with Tiaogengtang (Tiaogengtang group) (3 mice in each group) were obtained and subjected to second-generation gene sequencing and analysis.

[0148] The results showed that the expression of multiple different genes changed (up-regulated or down-regulated) after bilateral ovarian removal compared with the control group. In addition, the intervention with Tiaogeng Decoction also had an effect on the expression of some genes (not shown).

[0149] Surprisingly, if Figure 2 As shown in the data, compared with the control group, the AQP1 expression in the model group was significantly decreased, while Tiaogeng Decoction intervention could completely reverse the decrease in AQP1 expression.

[0150] Specifically, the AQP1 mRNA level in APP / PS1 mice was significantly reduced compared to that in young C57BL mice. The AQP1 mRNA level in APP / PS1 mice (2.61) was 3.07 times lower than that in young C57BL mice (8.08).

[0151] After the intervention of Tiaogeng Decoction, the AQP1 mRNA level of APP / PS1 mice (9.72) increased by 3.74 times compared with that before Tiaogeng Decoction intervention (2.61), and was even higher than the AQP1 mRNA level of C57BL young mice (8.08). This shows that Tiaogeng Decoction intervention can significantly increase the AQP1 mRNA level.

[0152] Example 5 Relationship between hippocampal astrocyte reduction and low expression of AQP1 and high expression of Aβ

[0153] In this example, immunofluorescence and other methods were used to detect the relationship between the expression levels of Aβ and AQP1 and the number of hippocampal astrocytes in the mice of the sham operation group, model group, western medicine group and Tiaogeng decoction group.

[0154] The results are as follows Figure 3 and Figure 4 shown.

[0155] The results showed that GFAP-labeled astrocytes accumulated in the hippocampal region compared with intact brain tissue areas assessed by immunofluorescence staining.

[0156] Compared with the sham operation group, the number of hippocampal astrocytes in the model group was reduced, accompanied by a decrease in AQP1 and an increase in Aβ expression.

[0157] Compared with the model group, the number of astrocytes in the hippocampus of mice in the western medicine group and the TGD intervention group increased, accompanied by increased AQP1 expression and decreased Aβ expression.

[0158] The results showed that TGD intervention could reverse the decline in astrocyte number associated with memory impairment, while increasing AQP1 expression and reducing Aβ expression.

[0159] The above results suggest that for subjects with low AQP1 expression (and / or high Aβ expression), intervention with Tiaogeng Decoction can more specifically and effectively reverse the decrease in the number of astrocytes associated with memory impairment, thereby improving learning ability and / or cognitive ability.

[0160] Example 6 Tiaogeng decoction upregulates AQP1 expression via ERs / NF-kB / AQP1 signaling pathway

[0161] 6.1 Studies at the mRNA level

[0162] In this example, qRT-PCR technology was used to detect the mRNA expression of genes such as Aβ, AQP1, NF-kB, ER-β and ER-α in mice in the sham operation group, model group, western medicine group and Tiaogeng Decoction group, so as to further study the mechanism of action of Tiaogeng Decoction.

[0163] The results are as follows Figure 5 , Figure 6 , Figure 7 , Figure 8 and Fig. 9 shown.

[0164] Compared with the sham operation group, the expression level of AQP1 mRNA in the model group decreased by 24% (P<0.01), the expression level of NF-kB mRNA increased by 25% (P<0.05), the expression level of Aβ mRNA increased significantly by 21% (P<0.01), the expression level of ERβ mRNA decreased by 48%, and the expression level of ERα mRNA decreased by 45%.

[0165] Compared with the model group, the expression level of AβmRNA in the Tiaogengtang group decreased by 36%, and the expression level of AQP1 mRNA increased by 13% (P<0.01). Compared with the model group, the expression of NF-kB mRNA in the Tiaogengtang group decreased by 44% (P<0.01); compared with the model group, the expression level of ERβmRNA in the Tiaogengtang group increased by 18% (P<0.05); compared with the model group, the expression level of ERαmRNA in the Tiaogengtang group increased by 13% (P<0.01). This suggests that Tiaogengtang upregulates AQP1 expression through the ERs / NF-kB / AQP1 signaling pathway, thereby downregulating the expression of Aβ.

[0166] 6.2 Protein level studies

[0167] Western Blot was further used to detect and quantitatively analyze the expression of AQP1 protein in the hippocampal tissue of mice in the blank group, model group, estradiol group and Tiaogengtang group.

[0168] The results are as follows Fig.10 The results showed that the expression of AQP1 protein in the hippocampus of the model group was significantly lower than that of the blank group. The expression of AQP1 protein in the estradiol group and the Tiaogengtang group was significantly higher than that in the model group. This confirmed the ability of Tiaogengtang to upregulate the expression of AQP1 protein.

[0169] The results of this example show that Tiaogeng decoction activates AQP1 and reduces Aβ deposition through the ERs / NF-kB / AQP1 signaling pathway, thereby improving the hippocampal function (including learning ability and / or cognitive ability, etc.) of the model mice.

[0170] Example 7 Effects of different doses of Tiaogeng decoction on AOP1 expression

[0171] In this example, the effects of different doses of Tiaogeng Decoction on the expression of AOP1 in astrocytes were evaluated using an in vitro model. The method is as follows:

[0172] Primary astrocytes were cultured for 24 hours under different culture conditions. The experimental groups included: control group (blank control group, astrocytes were derived from the hippocampal tissue of newborn 24h C57BL mice, no western medicine or Tiaogeng Decoction was added), Aβ model group (Aβ-induced neuroinflammation model, astrocytes were derived from the hippocampal tissue of newborn 24h C57BL mice, no western medicine or Tiaogeng Decoction was added), western medicine group (Aβ-induced neuroinflammation model, 17β-estradiol was added at a concentration of 1uM), Tiaogeng Decoction low-dose group (Aβ-induced neuroinflammation model, Tiaogeng Decoction was added at a dose of 312.5μg / mL), Tiaogeng Decoction medium-dose group (Aβ-induced neuroinflammation model, Tiaogeng Decoction was added at a dose of 625μg / mL) and Tiaogeng Decoction high-dose group (Aβ-induced neuroinflammation model, Tiaogeng Decoction was added at a dose of 1250μg / mL).

[0173] After the culture, the AQP1 mRNA and protein levels in astrocytes of mice in the control group, model group, western medicine group and different doses of Tiaogeng Decoction groups were measured.

[0174] The results are as follows Fig.11 and Fig.12 shown.

[0175] like Fig.11 As shown in the figure, compared with the control group, the expression level of AQP1 mRNA and protein in astrocytes in the model group were significantly decreased (P<0.01).

[0176] Compared with the AQP1 mRNA expression level in the model group, the AQP1 mRNA expression levels in the high, medium and low doses of Tiaogeng Decoction groups were significantly increased (P<0.01).

[0177] In addition, the AQP1 mRNA expression level in the low-dose Tiaogeng Decoction group was comparable to that in the Western medicine group. In addition, the high-dose Tiaogeng Decoction group had a better upregulation effect on the AQP1 mRNA expression level than the Western medicine estradiol.

[0178] like Fig.12 As shown, at the protein level, compared with the AQP1 protein expression level in the model group, the AQP1 protein expression levels in the high, medium and low doses of Tiaogeng Decoction groups and the Western medicine group were significantly increased (P<0.01), and with the increase of the dose of Tiaogeng Decoction, the AQP1 protein expression level gradually increased.

[0179] The experimental results of the above-mentioned Examples 2-7 of the present invention suggest that Tiaogeng Decoction can improve the hippocampal function of model mice by regulating the ERs / NF-kB / AQP1 signaling pathway, activating AQP1 and reducing Aβ deposition, thereby improving the learning ability and / or cognitive ability of ovariectomized mice, improving astrocyte neuritis, etc. During the menopausal window period, it prevents and blocks the progression of memory loss to Alzheimer's disease.

[0180] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of soup, characterized in that: For preparing a composition or preparation for one or more uses selected from the following group: (Z0) is administered to a subject with low AQP1 expression to improve cognitive impairment of the subject; (Z1) Increase the expression level of water channel protein AQP1; (Z2) reduce the expression level of amyloid protein Aβ; (Z3) reduce the expression level of nuclear factor NF-kB protein; (Z4) Reduce the expression level of estrogen receptor ERα.

2. The use according to claim 1, characterized in that The AQP1 low expression type means that the AQP1 expression level in the astrocytes of the subject is significantly lower than that of the normal control.

3. The use according to claim 2, characterized in that The “significantly lower” means that the ratio of the AQP1 expression level E1 in the astrocytes of the subject to the AQP1 expression level E0 in the astrocytes of the normal control is E1 / E0≤2 / 3, preferably ≤1 / 2, and more preferably ≤1 / 3.

4. The use according to claim 1, characterized in that The object also has the following typing characteristics: (Y1) The expression level of amyloid Aβ protein was significantly higher than that of normal control; (Y2) The expression level of nuclear factor NF-kB protein is significantly higher than that of normal control; and / or (Y3) The expression level of estrogen receptor ERα was significantly higher than that of normal control.

5. The use according to claim 4, characterized in that The ratio of the amyloid Aβ protein expression level H1 of the subject to the Aβ expression level H0 of the normal control is H1 / H0 ≥ 1.5, preferably ≥ 1.3, more preferably ≥ 1.1; The ratio of the NF-kB protein expression level M1 of the subject to the NF-kB protein expression level M0 of the normal control is M1 / M0≥1.5, preferably ≥1.3, more preferably ≥1.1; and / or The ratio N1 / N0 of the estrogen receptor ERα expression level N1 of the subject to the estrogen receptor ERα expression level N0 of the normal control is ≥1.5, preferably ≥1.3, and more preferably ≥1.

1.

6. The use according to claim 1, characterized in that The composition or preparation is used for one or more purposes selected from the following group: (Z1) Increase the expression level of water channel protein AQP1; (Z2) reduce the expression level of amyloid protein Aβ; (Z3) reducing the expression level of nuclear factor NF-kB protein; and / or (Z4) Reduce the expression level of estrogen receptor ERα.

7. A method for upregulating the expression of AQP1 in astrocytes in vitro, characterized in that: Includes steps: Astrocytes were cultured in the presence of Tiaogeng Tang, thereby upregulating the expression level of AQP1 protein in the astrocytes.

8. A reagent combination for companion diagnosis, characterized in that: The reagent combination comprises: (a) a first detection reagent for detecting AQP1; and (b) a soup.

9. A method for screening potential therapeutic agents for alleviating cognitive impairment, characterized in that: The following steps are involved: (a) In the experimental group, astrocytes were cultured in the presence of the test substance; and a control group, in which astrocytes are cultured under the same conditions but in the absence of the test substance; (b) respectively determining the expression levels of aquaporin AQP1 in astrocytes of the experimental group and the control group and comparing them; if the expression level Y1 of aquaporin AQP1 in the experimental group is significantly increased compared with the expression level Y0 of aquaporin AQP1 in the control group, it indicates that the test substance is a potential therapeutic agent for alleviating cognitive impairment.

10. A use of a detection reagent for detecting AQP1, characterized in that: Used to prepare a kit for evaluating the therapeutic effect of Tiaogeng Decoction on cognitive impairment.

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