CN110624096B - Application of ginseng and jujube brain strengthening oral liquid in preparation of medicine for preventing and/or treating Alzheimer disease - Google Patents
Application of ginseng and jujube brain strengthening oral liquid in preparation of medicine for preventing and/or treating Alzheimer disease Download PDFInfo
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Abstract
The invention discloses a new application of ginseng-jujube brain strengthening oral liquid, in particular to an application of the ginseng-jujube brain strengthening oral liquid in preparing a medicament for preventing and/or treating Alzheimer disease. The invention discovers in the experimental process that the medicine can act on a plurality of pathogenic links of AD, can improve the cognitive function of AD, and has the functions of reducing the deposition of Abeta in brain tissues, inhibiting apoptosis, inhibiting oxidative stress, promoting the secretion of neurotrophic factors, improving the environment of the brain tissues, promoting the survival of neurons and improving the cognitive function. The ginseng and jujube brain-strengthening oral liquid can be used for treating AD by being designed into a multi-target medicine, and has great application value.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a new application of ginseng and jujube brain-strengthening oral liquid in preparing a medicine for preventing and/or treating Alzheimer disease (or called senile dementia).
Background
Alzheimer's Disease (AD), also known as senile dementia, is a common central neurodegenerative disease of the middle-aged and elderly people, and mainly manifests progressive memory impairment and cognitive dysfunction. With the aging of the population in China, the incidence of AD is rising year by year, and the AD becomes a social problem and a medical problem to be solved urgently. AD is typically characterized in that senile plaques and neurofibrillary tangles appear in cerebral cortex and hippocampus, and a great amount of neuron degeneration loss and brain tissue atrophy are caused by persistent inflammatory reaction and oxidative stress injury around lesions, wherein the loss of cholinergic neurons of basal forebrain is the most serious to cause learning and memory dysfunction. At present, the FDA approved drugs cholinesterase inhibitors (donepezil, rivastigmine and galantamine) and N-methyl-D-aspartate receptor inhibitors (memantine) act on a single target spot, can only relieve symptoms to a certain extent, are difficult to control the deterioration of the state of an illness, and even can not achieve the effect of radical cure. The collapse and loss of neurons causing atrophy of cerebral cortex and reduction of brain parenchyma are common endpoints of pathological factors such as A beta and tau protein. Therefore, improving cerebral environment, reducing A beta, inhibiting oxidative stress, increasing trophic factor secretion, promoting survival and self-repair of cerebral neurons, can improve cognitive dysfunction of AD animal models or patients.
AD belongs to the categories of dementia and amnesia in traditional Chinese medicine, and the disease location is in the brain and is closely related to the kidney. The traditional Chinese medicine considers that kidney stores essence, essence generates marrow, marrow is communicated with brain, and essence deficiency in kidney is a key factor for AD; in addition, brain depends on the nourishment of acquired food essence, and if the spleen fails to transport and transform, food can not transform essence and qi, and produce phlegm turbidity to mask the clear orifices, so the basic treatment methods are tonifying kidney and replenishing essence, tonifying qi and strengthening spleen, eliminating phlegm and inducing resuscitation, and activating blood and resolving phlegm.
The traditional Chinese medicine has the action characteristics of multiple components and multiple targets, conforms to the characteristic of complex pathogenesis of AD, and is probably an important resource for radically treating AD. The ginseng-jujube brain-strengthening oral liquid (SZJN) is a Chinese patent medicine on the market, is prepared by refining ginseng, spina date seed, epimedium herb, rehmannia root, silktree albizzia, tall gastrodia tuber, chrysanthemum, dried ginger and liquorice, has the effects of tonifying qi and spleen, nourishing heart and soothing nerves, and is currently used for treating symptoms such as myocardial shortness of breath, insomnia and dreaminess, mental fatigue and hypodynamia, soreness and weakness of waist and legs and the like caused by neurasthenia. In SZJN, ginseng can tonify primordial qi, mainly tonify five internal organs, and tonify kidney and middle essence; the medicine is combined with epimedium, rehmannia root and dried ginger, can replenish essence and marrow, warm and tonify kidney yang, lead marrow sea to be active, nourish brain marrow, activate endogenous neural stem cells, promote endogenous nerve regeneration and repair damaged brain tissues; spina date seed and albizia julibrissin durazzini have the effects of nourishing the heart and soothing the nerves, gastrodia elata and chrysanthemum have the effects of calming the liver and suppressing yang, calming endogenous wind and relieving convulsion and increasing the effect of soothing the nerves, and sleep disorder can accelerate the deposition of Abeta in the brain and the degeneration and loss of neurons, and improve sleep and can improve pathological injury of the brain. Therefore, SZJN has the traditional Chinese medicine basis and the modern pharmacology basis for treating AD. Although ginseng, rehmannia root, epimedium, wild jujube seed and the like have reports of improving learning and memory, the medicine has special characteristics and has the wonderful combination of the prescription, and the action of a single Chinese medicine or component cannot represent the action of the whole prescription. The animal experiment or clinical research report about the treatment of AD by SZJN is not found at present by searching domestic and foreign periodicals and patents.
CN109925402A discloses a traditional Chinese medicine composition, a preparation method and an application thereof, wherein the traditional Chinese medicine composition comprises the following raw material medicines in parts by weight: 2-15 parts of ginseng, 2-15 parts of fried spina date seed, 2-15 parts of roasted epimedium herb, 3-18 parts of prepared rehmannia root, 1-12 parts of prepared polygala root, 1-12 parts of bran-fried bighead atractylodes rhizome, 3-18 parts of angelica, 2-15 parts of rhizoma acori graminei and 1-10 parts of roasted liquorice; the formula can cultivate primordial qi, nourish five internal organs, reduce phlegm and activate blood circulation, and refresh brain and benefit intelligence, and the prepared traditional Chinese medicine composition for treating the senile dementia has the advantages of multiple target spots, high safety, good curative effect, wide applicability and the like through animal pharmacodynamics experiments and later clinical feedback. However, the traditional Chinese medicine composition has the following defects: (1) the drug combination shows good therapeutic effect on a single disease model (a goose-actin cysteine-induced rat AD model, a beta-amyloid-induced AD rat model and an APP/PS1 transgenic mouse model), but has poor therapeutic effect on a complex disease model (such as an A beta 42+ scopolamine-induced AD mouse model); (2) the medicine composition can improve the learning and memory ability of AD model animals to a certain extent and improve brain tissue damage, but does not mention whether damaged neurons can be repaired or not, and if the lost neurons cannot be effectively supplemented and replaced, the long-term effect of treating AD is limited.
CN104784605A discloses a traditional Chinese medicine composition for treating senile dementia and a preparation method thereof, wherein the traditional Chinese medicine comprises the following components: 15-30 parts of gastrodia elata, 5-10 parts of uncaria, 10-15 parts of poria cocos, 6-8 parts of albizia flower, 1-3 parts of polygala tenuifolia, 9-17 parts of rhodiola rosea, 6-9 parts of fructus alpiniae oxyphyllae, 2-6 parts of dogwood, 2-5 parts of ophiopogon root, 3-7 parts of mirabilitum dehydratum, 2-4 parts of dendrobium, 3-9 parts of prepared rehmannia root, 4-6 parts of semen cuscutae, 2-8 parts of bighead atractylodes rhizome, 3-5 parts of monkshood, 10-20 parts of astragalus membranaceus, 2-3 parts of cinnamon, 6-9 parts of lithospermum, 2-9 parts of radix angelicae pubescentis and 1-3 parts of noto. The formula has the effects of strengthening yin and replenishing vital essence, promoting qi circulation, benefiting qi for tranquillization, promoting resuscitation and removing dampness, can effectively improve the functions of cranial nerve, cerebral vessels and cerebral blood circulation, and is helpful for treating senile dementia. However, the traditional Chinese medicine composition has the following defects: the pharmaceutical composition can effectively relieve vascular dementia model rat dementia symptoms, but has poor effect on more common senile dementia (namely Alzheimer disease) with more complex pathogenesis.
Therefore, an AD mouse model prepared from Abeta 42+ scopolamine and a APP/PS1 transgenic mouse model are utilized respectively, after the SZJN is applied to the gavage, the improvement effect of the SZJN on the cognitive function of the AD mouse is investigated through a behavioural test (Morris water maze); then, on an AD mouse model prepared from Abeta 42+ scopolamine, observing pathological injury conditions of brain tissues of the AD mouse by using a HE and Nissl staining method; the immune tissue fluorescence method is used for detecting the A beta 42 clearance condition, the expression condition of apoptosis protein Caspase-3, neurotrophic factor BDNF, Synaptophysin (SYP) and the neuron survival condition. The result shows that SZJN can obviously improve the learning and memory abilities of an AD mouse model prepared from Abeta 42+ scopolamine and an APP/PS1 transgenic mouse; meanwhile, the medicine can promote A beta 42 to be eliminated, inhibit nerve cell apoptosis, increase neurotrophic factors, improve synaptic function and increase the number of neurons, thereby proving the effectiveness and possible action links of the medicine and increasing new indications for the clinical application of the medicine.
Disclosure of Invention
The invention aims to provide a new application of the ginseng-jujube brain strengthening oral liquid, in particular to an application of the ginseng-jujube brain strengthening oral liquid in preparing a medicament for preventing and/or treating Alzheimer disease, and adds a new medicament for clinically preventing and treating Alzheimer disease and a new indication for clinical application of the ginseng-jujube brain strengthening oral liquid.
The technical scheme of the invention is as follows:
application of brain-strengthening oral liquid containing ginseng and jujube in preparation of medicines for preventing and/or treating Alzheimer disease.
Further, the action mechanism of the ginseng-jujube brain-strengthening oral liquid in preventing and/or treating the alzheimer disease comprises the following steps: reducing A beta deposition in brain tissue, inhibiting nerve cell apoptosis in brain, inhibiting oxidative stress, promoting brain-derived neurotrophic factor BDNF expression, promoting synapsin SYP expression, improving brain tissue environment, and increasing mature neuron (NeuN)+) And the pathological injury of brain tissues is further improved, the cognitive function of AD animals/patients is improved, and the effect of treating AD is achieved.
Furthermore, the active ingredients of the prepared medicine for preventing and/or treating Alzheimer disease are ginseng and jujube brain-strengthening oral liquid or medicines prepared according to a traditional Chinese medicine formula of the ginseng and jujube brain-strengthening oral liquid.
Furthermore, the medicine for preventing and/or treating Alzheimer disease contains one or more pharmaceutically acceptable carriers or auxiliary materials. Further, the auxiliary materials include, but are not limited to, sustained release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants or lubricants.
Further, the medicament for preventing and/or treating alzheimer disease can be prepared into various dosage forms according to the conventional method in the field, including but not limited to tablets, pills, capsules, granules and the like; the dosage forms are different, and the functions of the medicament for preventing and/or treating the Alzheimer disease in the prevention and treatment of the AD are not influenced.
Furthermore, the dosage form of the medicament for preventing and/or treating the Alzheimer disease is preferably oral liquid.
The brain-strengthening ginseng-jujube oral liquid is a Chinese patent medicine prepared by refining 9 traditional Chinese medicines of ginseng, spina date seed, epimedium herb, rehmannia root, albizia julibrissin, gastrodia elata, chrysanthemum, dried ginger and liquorice through a modern pharmaceutical technology, and is purchased from Tonghua Rimin pharmaceutical industry Co. At present, the traditional Chinese medicine composition is used for treating symptoms such as myocardial shortness of breath, insomnia and dreaminess, mental fatigue and hypodynamia, soreness and weakness of waist and legs and the like caused by neurasthenia.
The invention adopts 2 AD models, namely Abeta 42+ scopolamine simulates non-hereditary scattered AD, and APP/PS1 mice simulate familial AD, thereby being beneficial to fully proving the anti-AD effect of the ginseng and jujube brain-strengthening oral liquid. The application of the brain-strengthening ginseng-jujube oral liquid as a medicine for improving AD comprises the following steps: the ginseng-jujube brain-strengthening oral liquid can reduce the A beta deposition in brain tissues, inhibit apoptosis, inhibit oxidative stress, promote the secretion of neurotrophic factors, improve the environment of the brain tissues and promote the survival of neurons, thereby improving the pathological damage of the brain tissues, improving the learning and memory ability of AD model mice and promoting the recovery of AD.
The ginseng and jujube brain tonic oral liquid can reduce A beta deposition in brain tissues, inhibit apoptosis, inhibit oxidative stress, promote secretion of neurotrophic factors, improve brain tissue environment and promote neuron survival. Therefore, the ginseng and jujube brain tonic oral liquid can be designed into a multi-target medicine for preventing or treating AD.
The ginseng and jujube brain strengthening oral liquid improves cognitive functions, and a detection method can be selected from a water maze experiment (Morris water maze).
The molecular biological detection method can select a protein imprinting method (Western blot) and an immunofluorescence method.
Compared with the prior art, the invention has the beneficial effects that:
(1) the ginseng-jujube brain-strengthening oral liquid is a Chinese patent medicine on the market and has the characteristics of multi-component and multi-target function.
(2) The brain-strengthening ginseng-jujube oral liquid can act on a plurality of pathogenic links of AD, including clearing important pathogenic factor Abeta, reducing oxidative stress, reducing apoptosis, improving neurotrophic factors, and obviously improving the number of neurons in cortex and hippocampus, and has obvious treatment effect on AD. Makes up the deficiency of the current medicine acting on a single disease link.
(3) The invention utilizes modern bioinformatics (mainly utilizes network pharmacology and molecular docking technology) to carry out preliminary analysis on the function of the ginseng-jujube brain-strengthening oral liquid for treating AD, and finds that the medicine can act on a plurality of pathogenic links of AD. Therefore, 2 AD mouse models are repeatedly used for verification, so that the effect of the medicine on improving the AD cognitive function is determined; the medicine is further found to have the effects of reducing A beta deposition in brain tissues, inhibiting apoptosis, inhibiting oxidative stress, promoting secretion of neurotrophic factors, improving brain tissue environment, promoting neuron survival and improving cognitive function, adds new indications for the clinical use of the medicine, can be designed into a multi-target medicine for treating AD, and has great application value.
Drawings
FIG. 1 is a typical diagram of a swimming track of a positioning sailing experiment.
FIG. 2 is a graph showing HE staining results and a picture of a ginseng and jujube brain tonic oral liquid for alleviating pathological damage of brain tissues; wherein: a is a graph of HE staining results of the cortical areas of the mice in each group, and B is a graph of HE staining results of the CA3 areas of the hippocampus of the mice in each group.
FIG. 3 is a graph showing the Nissl staining results and the increase of the Nishi body number of the brain-strengthening oral liquid; wherein: a is the result of Nissl staining of the skin layer area of each group of mice, B is the result graph of Nissl staining of the CA1 area of the hippocampus of each group of mice, C is the statistical graph of the result of Nissl staining of the skin layer area of each group of mice, and D is the statistical graph of the result of Nissl staining of the CA1 area of the hippocampus of each group of mice.
FIG. 4 is a graph showing the result of staining hippocampal Abeta 42 of each group of mice; wherein: a is a staining fluorescence photo picture of hippocampal Abeta 42 of each group of mice, and B is a staining fluorescence intensity statistical picture of hippocampal Abeta 42 of each group of mice.
FIG. 5 is a graph showing the result of staining hippocampal Caspase-3 positive cells of each group of mice; wherein: a is a fluorescent photograph of the hippocampal Caspase-3 positive cells of each group of mice, and B is a statistical chart of the hippocampal Caspase-3 positive cells of each group of mice.
FIG. 6 is a graph showing the result of BDNF staining of hippocampus of each group of mice; wherein: a is a fluorescence photograph of BDNF staining of the hippocampus of each group of mice, and B is a fluorescence intensity statistical chart of the BDNF staining of the hippocampus of each group of mice.
FIG. 7 is a graph showing SYP staining results of hippocampal synaptophysin in each group of mice, wherein: a is a fluorescent photograph of synaptophysin SYP staining of the hippocampus of each group of mice, and B is a fluorescent intensity statistical chart of the synaptophysin SYP staining of the hippocampus of each group of mice.
FIG. 8 is a graph showing the result of staining the cortex and hippocampus mature neurons of each group of mice; wherein: a is a fluorescent photograph of cortex mature neurons of each group of mice, B is a fluorescent photograph of hippocampal mature neurons of each group of mice, C is a statistical chart of the number of cortex mature neurons of each group of mice, and D is a statistical chart of the number of hippocampal mature neurons of each group of mice.
FIG. 9 is a graph showing the results of Bax and Bcl-2 protein expression in each group of mice; wherein: a is a photograph of the expression gray scale of Bax and Bcl-2 proteins, B is a statistical chart of the expression gray scale of Bax proteins, and C is a statistical chart of the expression gray scale of Bcl-2 proteins.
FIG. 10 is a graph showing the result of staining the cortex and hippocampus mature neurons of each group of mice in a comparative experiment; wherein: a is a fluorescent photograph of cortex mature neurons of each group of mice, B is a fluorescent photograph of hippocampal mature neurons of each group of mice, C is a statistical chart of the number of cortex mature neurons of each group of mice, and D is a statistical chart of the number of hippocampal mature neurons of each group of mice.
Detailed Description
In order to realize the application of the brain-strengthening oral liquid of ginseng and Chinese date in the preparation of the drugs for preventing or treating AD, the following specific embodiments are provided for illustration, but not limited to the embodiments.
The technical scheme of the invention is as follows:
application of brain-strengthening oral liquid containing ginseng and jujube in preparation of medicines for preventing and/or treating Alzheimer disease.
Further, the action mechanism of the ginseng-jujube brain-strengthening oral liquid in preventing and/or treating the alzheimer disease comprises the following steps: reducing A beta deposition in brain tissue, inhibiting nerve cell apoptosis in brain, inhibiting oxidative stress, promoting brain-derived neurotrophic factor BDNF expression, promoting synapsin SYP expression, improving brain tissue environment, and increasing mature neuron (NeuN)+) And the pathological injury of brain tissues is further improved, the cognitive function of AD animals/patients is improved, and the effect of treating AD is achieved.
Furthermore, the active ingredients of the prepared medicine for preventing and/or treating Alzheimer disease are ginseng and jujube brain-strengthening oral liquid or medicines prepared according to a traditional Chinese medicine formula of the ginseng and jujube brain-strengthening oral liquid.
Furthermore, the medicine for preventing and/or treating Alzheimer disease contains one or more pharmaceutically acceptable carriers or auxiliary materials. Furthermore, the auxiliary materials are sustained-release agents, excipients, fillers, adhesives, wetting agents, disintegrating agents, absorption promoters, surfactants or lubricants.
Furthermore, the medicament for preventing and/or treating alzheimer disease can be prepared into various dosage forms according to the conventional method in the field, including tablets, pills, capsules, granules and the like; the dosage forms are different, and the functions of the medicament for preventing and/or treating the Alzheimer disease in the prevention and treatment of the AD are not influenced.
Furthermore, the dosage form of the medicament for preventing and/or treating the Alzheimer disease is preferably oral liquid.
The brain-strengthening ginseng-jujube oral liquid is a Chinese patent medicine prepared by refining 9 traditional Chinese medicines of ginseng, spina date seed, epimedium herb, rehmannia root, albizia julibrissin, gastrodia elata, chrysanthemum, dried ginger and liquorice through a modern pharmaceutical technology, and is purchased from Tonghua Rimin pharmaceutical industry Co. At present, the traditional Chinese medicine composition is used for treating symptoms such as myocardial shortness of breath, insomnia and dreaminess, mental fatigue and hypodynamia, soreness and weakness of waist and legs and the like caused by neurasthenia.
The invention adopts 2 AD models, namely, Abeta 42 is combined with scopolamine to simulate non-hereditary dispersible AD, and APP/PS1 mice simulate familial AD, so that the anti-AD effect of the ginseng and jujube brain-strengthening oral liquid can be fully proved. The application of the brain-strengthening ginseng-jujube oral liquid as a medicine for improving AD comprises the following steps: the ginseng-jujube brain-strengthening oral liquid can reduce the A beta deposition in brain tissues, inhibit apoptosis, inhibit oxidative stress, promote the secretion of neurotrophic factors, improve the environment of the brain tissues and promote the survival of neurons, thereby improving the pathological damage of the brain tissues, improving the learning and memory ability of AD model mice and promoting the recovery of AD.
The ginseng and jujube brain tonic oral liquid can reduce A beta deposition in brain tissues, inhibit apoptosis, inhibit oxidative stress, promote secretion of neurotrophic factors, improve brain tissue environment and promote neuron survival. Therefore, the ginseng and jujube brain tonic oral liquid can be designed into a multi-target medicine for preventing or treating AD.
The ginseng and jujube brain strengthening oral liquid improves cognitive functions, and a detection method can be selected from a water maze experiment (Morris water maze).
The molecular biological detection method can select a protein imprinting method (Western blot) and an immunofluorescence method.
The invention systematically investigates the improvement effect and possible action mechanism of the ginseng-jujube brain-strengthening oral liquid on the cognitive function of AD mice from the whole animal level to the protein molecule level. The invention firstly utilizes 2 AD mouse models (firstly, lateral ventricle injection of Abeta 42 is combined with intraperitoneal injection of scopolamine, secondly, APP/PS1 transgenic mice), and the water maze experiment confirms that the brain strengthening oral liquid of the ginseng and the jujube can improve the cognitive function of the AD mouse model. Then, an HE and Nissl dyeing method is used for verifying the protection effect of the ginseng and jujube brain tonic oral liquid on the brain tissue morphology of AD mice; by utilizing a protein imprinting method (Western blot) and an immunofluorescence method, a plurality of pathogenic links of the brain-strengthening ginseng-jujube oral liquid to AD are examined, including A beta deposition, oxidative stress, nerve cell apoptosis, neurotrophic factors, synaptic plasticity and the improvement effect of neuron survival conditions. The ginseng and jujube brain-strengthening oral liquid can be fully proved to act on the disease-causing links, so that the survival number of neurons is increased, and the cognitive function of AD mice is improved. The value of the ginseng and jujube brain-strengthening oral liquid in the aspect of treating AD is proved, and the jujube brain-strengthening oral liquid can be prepared into a medicine for treating AD.
The following are descriptions of specific embodiments of the present invention:
example 1
AD mouse model prepared by combining Abeta 42 and scopolamine
1. Animals: SPF-level Kunming mice, each half male and female, with a weight of 28-30 g, are purchased from Liaoning Biotechnology Limited, and have the following production certificate numbers: SCXK (Liao) 2015-0001.
2. Solution preparation: abeta 42 (purchased from Booxsen Bio Inc.) was dissolved in sterile physiological saline to prepare 1. mu.g/. mu.L, and the solution was left at 37 ℃ for 24 hours; scopolamine hydrobromide (purchased from Shanghai Allan Biotechnology, Inc.) was prepared to 0.1mg/mL with physiological saline and was prepared just before use.
3. Anesthesia: after 20% urethane anesthesia, the head is fixed on a stereotaxic instrument, and a bregma is exposed by a middle incision at the top of the head.
4. Injection of a β 42: referring to Franklin and Paxinos mouse stereotaxis, based on Bregma, backward 0.2mm, rightward 1.0mm, and depth 2.5-3 mm. The skull is drilled by a miniature electric drill to reach the dura mater, 3 mu L (1 mu g/mu L) of Abeta 42 protein is slowly injected into the right ventricle by a micro-injector, the same volume of normal saline is injected into a sham operation group, and a needle is left for 2min to ensure that Abeta 42 is fully diffused and reduce backflow.
5. A water maze test was performed 7 days after the operation, and scopolamine (1mg/kg) was intraperitoneally injected 20min before each test.
Second, study and memory ability of AD mice by water maze experiment
1. Influence on learning and memory of AD mouse model prepared by combining Abeta 42 and scopolamine
(1) Animals: kunming mouse
(2) Grouping: mice were randomly divided into the following 6 groups of 10 mice per group by body weight.
A. Blank group: the ventricles of brain are not injected, the abdominal cavity is not injected, and the stomach is perfused with normal saline;
B. the sham operation group: injecting normal saline into ventricles of brain, injecting normal saline into abdominal cavity, and infusing normal saline into stomach;
C. model group: injecting Abeta 42 into ventricle, injecting scopolamine into abdominal cavity, and infusing normal saline;
low dose of szjn: intracerebroventricular injection of Abeta 42, intraperitoneal injection of scopolamine, gavage of SZJN low dose (0.25 g/kg);
dose in szjn: injecting Abeta 42 into ventricle, injecting scopolamine into abdominal cavity, and injecting SZJN medium dosage (0.5 g/kg);
high dose of szjn: injecting Abeta 42 into ventricle, injecting scopolamine into abdominal cavity, and injecting stomach-irrigation SZJN at high dose (1 g/kg);
2. influence on learning and memory of APP/PS1 transgenic mice
(1) Animals: c57BL/6 mice and APP/PS1 transgenic mice, 9 months of age, male and female.
(2) Grouping: the weight was randomly divided into 5 groups of 10 individuals.
A. Blank group: c57BL/6 mice, gavage normal saline;
B. model group: APP/PS1 transgenic mice, gastric saline;
low dose of szjn: APP/PS1 transgenic mice, gavage SZJN low dose (0.25 g/kg);
dose in szjn: APP/PS1 transgenic mice, gavage SZJN dosage (0.5 g/kg);
high dose of szjn: APP/PS1 transgenic mice, gavage SZJN high dose (1 g/kg);
3 Water maze experiment
After the model is made, the intragastric administration is started, and after 4 weeks of continuous administration, the water maze experiment is carried out.
(1) Positioning navigation experiment: the Morris water maze device consists of a barrel with a black inner wall (the diameter is 120cm multiplied by 60cm in height), a camera and an automatic image acquisition and analysis system. Dividing the interior of the barrel into 4 quadrants, placing an escape platform (with the diameter of 10cm) in one quadrant, controlling the temperature to be 20-21 ℃ and enabling the water surface to submerge the platform by 0.5-1 cm. The training is carried out 1 time in each of 4 quadrants every day, the mouse is placed in water with the curvature of any quadrant 1/2 facing the pool wall, and if the mouse cannot find the platform within 60s, the mouse is led to the platform to stay for 20 s. And continuously training for 5 days, collecting and analyzing swimming images by a shooting system, and evaluating the space learning capacity of the mouse according to the average incubation period of the mouse on the bench every day.
(2) Space exploration experiment: and (3) removing the platform on the 6 th day, putting the mouse into water from the side quadrant opposite to the original platform to the pool wall, detecting the times of the mouse passing through the platform within 120s, the residence time percentage of the quadrant where the platform is located and the swimming distance before the mouse passes through the platform for the 1 st time, and analyzing and evaluating the space memory capacity of the mouse.
4 the results show that:
(1) as shown in table 1 and fig. 1, the results of the water maze localized navigation experiments show that the escape latency of each group of mice decreases day by day after training. During the whole detection period, the model group mice take longer time to find a platform compared with a blank group, which shows that the mice can generate obvious cognitive dysfunction by ventricular injection of Abeta 42 and intraperitoneal injection of scopolamine. Compared with the model group, the escape latency of each dose group of SZJN is shortened to different degrees; wherein, the action effect of the low, medium and high dosage of SZJN is more obvious, and no obvious difference exists among three groups, which indicates that the best effect can be achieved by the low dosage of SZJN; the escape latency of the SZJN medium dose group mice on the 5 th day is 7.38 +/-2.99 s, and the mice have significant difference compared with 25.8 +/-16.71 s of a model group (P < 0.01).
TABLE 1 results of escape latency in water maze orientation navigation experiment (A β 42 AD mouse model prepared by combining scopolamine)
Note:*P<0.05，**P<0.01，***P<0.001vs. model set;#P<0.05#，##P<0.01，###P<0.001vs. blank.
(2) As shown in Table 2, the results of the water maze space exploration experiment show that the swimming speed of each group of mice has no obvious difference (P is more than 0.05); however, compared with the blank group, the model group mice need to swim longer distance to pass through the original platform (P < 0.001); both the number of passes through the plateau and the percentage of time spent in the target quadrant were significantly reduced (P < 0.001). Compared with the model group, the swimming distance of the mice in each dose group of SZJN before finding the platform is obviously shortened, the platform crossing times and the target quadrant time percentage are obviously increased, wherein the effect of the medium dose group (0.5g/kg) is most obvious: the swimming distance before the 1 st platform crossing is 320.91 +/-142.00 cm, and the difference is obvious compared with 1255.75 +/-1008.48 cm of a model group (P is less than 0.01); the number of times of platform crossing is 8.67 +/-5.59 times, and the ratio of the number of times of platform crossing to the number of 4.07 +/-2.16 times of the model group is poor and remarkable (P is less than 0.05); the target quadrant residence time accounts for 34.63 ± 8.42% of the total swimming time, and the difference is significant compared with 27.20 ± 4.57% of the model group (P < 0.01).
TABLE 2 Water maze space exploration experimental results (AD mouse model prepared by Ass 42 in combination with scopolamine)
Note:*P<0.05，**P<0.01，***P<0.001vs. model set;#P<0.05#，##P<0.01，###P<0.001vs. blank.
(3) As shown in table 3, similar to the results in table 1, the escape latency decreased day by day after each group of mice was trained. The escape latency of each dose group of SZJN is shortened to a different extent compared with the APP/PS1 model group; the escape latency of the SZJN high-dose group mice on day 5 is 10.08 +/-4.43 s, and the mice have significant difference (P <0.01) compared with 23.80 +/-12.01 s of a model group.
TABLE 3 Water maze positioning navigation experiment escape latency results (APP/PS1 transgenic mice)
Note:*P<0.05，**P<0.01，***P<0.001vs. model set;#P<0.05#，##P<0.01，###P<0.001vs. blank.
(4) As shown in Table 4, similar to the results in Table 2, there was no significant difference in swimming speed between the groups of mice (P > 0.05); however, compared with the blank group, the swimming distance of the mice in the model group is obviously prolonged (P < 0.001); both the number of passes through the plateau and the percentage of time spent in the target quadrant were significantly reduced (P < 0.001). Compared with the model group, the swimming distance of the mice in each dose group of SZJN before finding the platform is obviously shortened, the platform crossing times and the target quadrant time percentage are obviously increased, wherein the effect of the high dose group (1g/kg) is most obvious: the swimming distance before the 1 st crossing of the platform is 389.82 +/-134.87 cm, and the difference is obvious compared with 1305.35 +/-868.44 cm of a model group (P is less than 0.01); the number of times of passing through the platform is 9.08 +/-3.07 times, and the difference of the number of times of passing through the platform and the number of times of passing through the platform is worse and more remarkable (P is less than 0.05) compared with the number of times of passing through the platform of 3.87 +/-2.11 times of the model group; the target quadrant residence time accounts for 34.28 + -6.11% of the total swimming time, which is significantly different (P <0.01) compared to the model group's 26.25 + -3.65%.
TABLE 4 Water maze space exploration experimental results (APP/PS1 transgenic mice)
Note:*P<0.05，**P<0.01，***P<0.001vs. model set;#P<0.05#，##P<0.01，###P<0.001vs. blank.
The results fully show that SZJN can obviously improve the learning and memory ability of AD mice and improve the symptoms of AD cognitive dysfunction.
III, HE staining and Nissl staining
1. The experimental steps are as follows:
(1) HE staining: after the water maze experiment is finished, 3 mice are taken from each group to prepare brain slices (10 mu m), HE is carried out, the brain tissue slices are respectively fixed for 5s by 70 percent, 80 percent and 90 percent of alcohol, washed, then stained by 50 ℃ hematoxylin staining solution for 30s, washed, differentiated for 5s by 1 percent of hydrochloric acid alcohol, washed, put into 0.5 percent of ammonia water anti-blue solution for 10s, washed, stained for 5s by eosin staining solution, washed by running water, dehydrated for 5s by 70 percent, 80 percent and 90 percent of alcohol respectively, dried in the air, and finally sealed by neutral gum. And observing the pathological damage condition of the tissue under a microscope.
(2) Nissl staining: placing brain tissue slices in 75% ethanol, fully degreasing, performing gradient ethanol hydration, placing in tar violet staining solution, stopping reaction after Nissel is clear, cleaning, differentiating with 1% hydrochloric acid ethanol, dehydrating, air drying, adding xylene for transparency, and sealing with neutral gum. Each group of mice selects 2 section specimens, each section specimen selects three visual fields, and the change of the nissl body is observed under an optical lens.
2. The results show that:
(1) HE staining results: blank groups of nerve cells visible under the microscope are large in quantity, clear in layers, rich in cytoplasm, large and round in cell nucleus, uniform in cytoplasm and cell nucleus staining and normal in tissue morphological structure; the nerve cells of the model group are obviously reduced, and the model group is loose in arrangement, irregular in shape, deeply stained and condensed in cell nucleus, loose in arrangement, irregular in shape and obvious in vacuolation of the nerve cells, so that the brain tissue of the model mouse is obviously damaged; the number of nerve cells of the SZJN group is obviously increased compared with that of the model group, the layers are clear, cytoplasm and nucleus are uniformly dyed, and the cell nucleus shrinkage is obviously reduced, which shows that the SZJN obviously reduces the pathological damage of the brain tissue of the mouse and increases the number of nerve cells (figure 1).
(2) Nissl staining results: the cortex of the blank mouse and the nerve cells in the hippocampal CA1 area are densely and regularly arranged, and the nissl in cytoplasm is rich and is dark blue; the number of nerve cells of a mouse in a model group is reduced, the arrangement is sparse, the cell gap is increased, the intracytoplasmic Niger is reduced, the boundary is not clear, and the mouse is dyed into light blue; the number of the SZJN (7.5g/kg) group Nile bodies is obviously increased, the arrangement is neat, and the dyeing is clear; the quantitative analysis result shows that the number of cortex and hippocampus nissl of the SZJN group is obviously increased compared with that of the model group (P <0.05), which indicates that the number of neurons in AD brain is obviously increased after the SZJN treatment (figure 2).
Fourth, the immunofluorescence method detects the expression of AD related protein in brain
1. The experimental steps are as follows: fixing the slices with 4% paraformaldehyde for 30min, permeabilizing with 0.1% Triton X-100 for 15min, adding 5% BSA solution, sealing for 30min, adding primary anti-diluent (Abeta, Caspase-3, BDNF, SYP and NeuN), placing at 4 ℃ overnight, adding a secondary anti-diluent chamber marked with Cy3, incubating in the dark for 1h, washing, adding DAPI, incubating in the dark for 15min, washing, adding an anti-fluorescence quencher, sealing, and observing under a fluorescence microscope. Fluorescence intensity scans were performed for each field using Image J software.
2. The results show that:
(1) a beta 42 is not detected in the blank group and the sham operation group, a large amount of A beta 42 protein exists in the model group, and A beta 42 in the SZJN administration group is obviously reduced (vs. model, P <0.01, figure 3), which indicates that SZJN can promote the clearance of A beta 42;
(2) only a small amount of Caspase-3 was present in the blank group+Positive cells, model group Caspase-3+The number of positive cells is obviously increased, and the difference is very obvious compared with a blank group (P)<0.01), whereas after SZJN administration, Caspase-3 positive cells were significantly reduced in number and very significantly different from the model group (vs. model, P)<0.01, FIG. 4), suggesting that SZJN reduces Caspase-3+Positive cell number, inhibition of apoptosis;
(3) a large amount of BDNF proteins exist in visible cells and around a blank group, the BDNF protein amount of a sham operation group is reduced, the BDNF protein expression amount of a model group is obviously reduced, and has extremely obvious difference (P <0.01) compared with the blank group, and after SZJN is administrated, the BDNF protein expression amount is obviously increased (vs. the model, P <0.01, and figure 5), which indicates that the SZJN can increase the BDNF expression of a brain-derived nutrition factor;
(4) synaptophysin is an important protein in brain and an important index for reflecting synaptic function. SYP in a hippocampal part of a blank group is expressed in a large amount, while SYP expression in a model group is obviously reduced (P is less than 0.01), SYP expression is obviously increased after SZJN is administrated, (vs. model, P is less than 0.01, and figure 6) shows that the SZJN can increase synapsin SYP expression;
(5) a large number of mature neurons were found in both the cortex and hippocampus of the blank group (NeuN)+Positive cells), whereas both the model cortex and hippocampus were significantly reduced (vs. blank, P)<0.01), NeuN after administration of SZJN+Positive cells are significantly elevated (vs. model, P)<0.01 fig. 7), suggesting that SZJN can activate endogenous nerve regeneration, resulting in a significant increase in neuronal numbers.
The results fully show that the SZJN can promote the clearing of the A beta, inhibit apoptosis, promote the secretion of neurotrophic factor BDNF and increase the number of neurons, thereby relieving the structural damage of brain tissues, effectively relieving the clinical symptoms of AD and preventing or delaying the disease process of AD.
Fifth, detecting the expression conditions of Bax and Bcl-2 by Western blot method
1. The experimental steps are as follows:
(1) tissue protein extraction
The mice are sacrificed and the brains are taken out, after the hippocampus and the cortex are stripped, the mice are put into a centrifuge tube to be cut into pieces, weighed, 100mL of lysate is added into each 20mg of tissues to be fully homogenized, the mixture is centrifuged at 12000rmp at 4 ℃ for 15min, and the supernatant is taken, so that the total protein extract is obtained.
(2) Sample preparation
Mixing protein sample with 5 × loading buffer solution at ratio of 4: 1, heating with boiling water at 100 deg.C for 5min to completely denature protein, and quenching. Freezing and storing at minus 80 ℃.
(3) Electrophoresis
Preparing SDS-PAGE gel, adding the sample into each hole, switching on a power supply, carrying out 80V electrophoresis, changing to 120V after the sample indicator is over-concentrated, and stopping electrophoresis when the indicator runs to the edge of the gel.
(4) Rotary film
And (3) sequentially placing filter paper-PVDF membrane-SDS-PAGE gel-filter paper, discharging air bubbles, and carrying out electrotransformation at 25V for 10-20 minutes until the marker is transferred to PVDF.
(5) Immunoblotting
Placing the target protein strip into 5% BSA, shaking at low speed and 7 ℃ for sealing for 1h, incubating overnight at 4 ℃ with a primary antibody diluted by TBST solution, washing the membrane at room temperature, adding a secondary antibody diluted by TBST, placing on a shaking table, incubating for 1h at 37 ℃, and washing the membrane at room temperature;
(6) development
Preparing ECL working solution according to the instruction, dripping the ECL working solution to a PVDF film, tabletting and developing. The photographs were recorded and analyzed using Image J.
2. The results show that: the blank group has lower expression level of the pro-apoptotic protein Bax and higher expression level of the anti-apoptotic protein Bcl-2, while the model group has obviously increased expression level of the pro-apoptotic protein Bax (vs. blank, P <0.01) and obviously decreased expression level of the anti-apoptotic protein Bcl-2 (vs. blank, P < 0.01). After the administration of SZJN, the expression of a pro-apoptotic protein Bax (vs. model, P <0.01) is obviously reduced, the expression of an anti-apoptotic protein Bcl-2 (vs. model, P <0.01) is improved, and the combination of the results of Caspase-3 in immunofluorescence fully shows that the SZJN can inhibit the apoptosis of nerve cells in the brain of an AD model mouse (figure 8).
Sixthly, detecting the levels of SOD, MDA and GSH in oxidative stress indexes in brain
1. The experimental steps are as follows: after the completion of the water maze experiment, mice fasted for 12 hours, freely drunk water, killed by removing neck, and brain prepared into brain homogenate, which was diluted to the corresponding concentration according to the kit instructions. And respectively detecting and measuring the levels of SOD, MDA and GSH in the brain homogenate, and evaluating the lipid peroxidation condition in the brain of the mouse.
2. The results show that: the results are shown in table 5, each dose group of SZJN can effectively improve the brain tissue SOD activity (P <0.05), GSH level (P <0.05), and significantly reduce MDA level (P <0.01), which indicates that SZJN can enhance the scavenging ability of oxygen free radicals in brain of AD model mice, and reduce lipid peroxidation injury.
TABLE 5 Effect of SZJN on brain tissue SOD, MDA and GSH
Note:*P<0.05，**P<0.01，***P<0.001vs. model set;#P<0.05#，##P<0.01，###P<0.001vs. blank.
In conclusion, the results of the present example fully demonstrate the significant efficacy of the ginseng-jujube brain-strengthening oral liquid in preventing or treating alzheimer's disease, and the ginseng-jujube brain-strengthening oral liquid can achieve the effect of improving AD cognitive function by eliminating a β 42, inhibiting apoptosis, increasing neurotrophic factors, inhibiting oxidative stress, promoting neuron survival, and repairing damaged brain tissue. Fully indicates that the ginseng-jujube brain-strengthening oral liquid has great potential and application value in the aspect of preventing or treating the Alzheimer disease. The new application of the ginseng and jujube brain-strengthening oral liquid expands the indications of the ginseng and jujube brain-strengthening oral liquid, can be applied to the preparation of the medicines for preventing and/or treating Alzheimer's disease, and has wide prospects.
Example 2
Comparative experiment
The traditional Chinese medicine composition is prepared according to the traditional Chinese medicine composition disclosed in CN109925402A and the preparation method, and the traditional Chinese medicine composition comprises the following raw material medicines in parts by weight: 2-15 parts of ginseng, 2-15 parts of fried spina date seed, 2-15 parts of roasted epimedium herb, 3-18 parts of prepared rehmannia root, 1-12 parts of prepared polygala root, 1-12 parts of bran-fried bighead atractylodes rhizome, 3-18 parts of angelica, 2-15 parts of rhizoma acori graminei and 1-10 parts of roasted liquorice; in particular, the preparation is carried out according to the water decoction group of the traditional Chinese medicine composition given in the formula: (1) consists of the following components: 6g of ginseng, 6g of fried spina date seed, 6g of fried epimedium herb, 9g of prepared rehmannia root, 5g of bran-fried bighead atractylodes rhizome, 5g of prepared polygala root, 6g of rhizoma acori graminei, 9g of angelica and 3g of honey-fried licorice root; (2) the preparation method comprises the following steps: pulverizing radix Angelicae sinensis and semen Ziziphi Spinosae, decocting twice with water, adding 120g water for the first time, soaking for 0.5 hr, decocting for 30min, adding 90g water for the second time, and decocting for 20 min. Sieving the decoction with 120 mesh sieve, filtering, mixing filtrates, vacuum concentrating at 55 deg.C until each 1ml concentrate contains 1g crude drug, to obtain control water decoction, and performing experiment according to the patent with optimal drug effect of 3.80 g/kg.
Then the concentrated solution prepared above is tested together with the ginseng and jujube brain-strengthening oral liquid
AD mouse model prepared by combining Abeta 42 and scopolamine
1. Animals: SPF-level Kunming mice, each half male and female, with a weight of 28-30 g, are purchased from Liaoning Biotechnology Limited, and have the following production certificate numbers: SCXK (Liao) 2015-0001.
2. Solution preparation: abeta 42 (purchased from Booxsen Bio Inc.) was dissolved in sterile physiological saline to prepare 1. mu.g/. mu.L, and the solution was left at 37 ℃ for 24 hours; scopolamine hydrobromide (purchased from Shanghai Allan Biotechnology, Inc.) was prepared to 0.1mg/mL with physiological saline and was prepared just before use.
3. Anesthesia: after 20% urethane anesthesia, the head is fixed on a stereotaxic instrument, and a bregma is exposed by a middle incision at the top of the head.
4. Injection of a β 42: referring to Franklin and Paxinos mouse stereotaxis, based on Bregma, backward 0.2mm, rightward 1.0mm, and depth 2.5-3 mm. The skull is drilled by a miniature electric drill to reach the dura mater, 3 mu L (1 mu g/mu L) of Abeta 42 protein is slowly injected into the right ventricle by a micro-injector, the same volume of normal saline is injected into a sham operation group, and a needle is left for 2min to ensure that Abeta 42 is fully diffused and reduce backflow.
5. A water maze test was performed 7 days after the operation, and scopolamine (1mg/kg) was intraperitoneally injected 20min before each test.
Second, study and memory ability of AD mice by water maze experiment
1. Animals: kunming mouse
2. Grouping: mice were randomly divided into the following 7 groups of 10 mice per group by body weight.
A. Blank group: the ventricles of brain are not injected, the abdominal cavity is not injected, and the stomach is perfused with normal saline;
B. the sham operation group: injecting normal saline into ventricles of brain, injecting normal saline into abdominal cavity, and infusing normal saline into stomach;
C. model group: injecting Abeta 42 into ventricle, injecting scopolamine into abdominal cavity, and infusing normal saline;
group szjn: injecting Abeta 42 into ventricle, injecting scopolamine into abdominal cavity, and injecting stomach-irrigation SZJN at high dose (1 g/kg);
E. control water-decocted group: injecting Abeta 42 into ventricle, injecting scopolamine into abdominal cavity, and controlling water decoction (3.80 g/kg).
3. The results show that:
(1) as shown in table 6, the results of the experiments on the mapping navigation of the water maze showed that the escape latency of the mice in the SZJN group was significantly reduced in the 2d, 3d, 4d and 5d of training compared with the model group (P <0.05 or P < 0.01); the escape latency of the mice in the control water decoction group is not obviously different from that of the model group in the first 3 days of training (P is more than 0.05); under the same training days, the latency of the SZJN group is shorter than that of the control water decoction group, which shows that the SZJN group has quick effect and better effect than that of the control water decoction.
TABLE 6 results of water maze positioning navigation contrast experiment escape latency
Note:*P<0.05，**P<0.01, model group;#P<0.05#，##P<0.01, vs. blank set.
(2) As shown in table 7, the results of the water maze space exploration comparison experiment show that, compared with the model group, the swimming distance before the platform is found by the mice in the SZJN group is obviously shortened (P <0.01), and the times of passing through the platform and the percentage of the target quadrant time are obviously increased (P < 0.01); although the swimming distance of the control water decoction group is obviously shortened (P <0.01) and the frequency of passing through the platform is obviously reduced (P <0.05) compared with the model group, the target quadrant time has no significant difference (P >0.05) compared with the model group, which indicates that SZJN is superior to the control water decoction in improving the AD cognitive function and is more suitable for a complex disease model (A beta combined with scopolamine induced AD).
TABLE 7 Water maze space exploration contrast experiment results
Note:*P<0.05，**P<0.01vs. model set;#P<0.05#，##P<0.01vs. blank set.
Third, the mature neuron (NeuN) in the brain of AD mouse is investigated by immunofluorescence experiment+) Number of
1. The experimental steps are as follows: after the completion of the water maze experiment, the mice were anesthetized, perfused with paraformaldehyde, embedded with a freezing embedding medium, and cut into brain tissue sections with a thickness of 10 μm by a freezing microtome, and then frozen at-80 ℃. The immunohistochemical method was as above, and NeuN was used to label mature neurons, and the number of mature neurons in the cortex and hippocampus of each group of mice was determined.
2. The results show that: as shown in FIG. 10, a large number of mature neurons (NeuN) were visible in both the cortex and hippocampus of the blank group+Positive cells), whereas both the model cortex and hippocampus were significantly reduced (vs. blank, P)<0.01), NeuN after administration of SZJN+Positive cells are significantly elevated (vs. model, P)<0.01)(ii) a The number of cortex and hippocampal mature neurons in the control water decoction group was increased, but there was no significant difference compared with the model group (vs. model, P)>0.05). It is shown that SZJN can increase the number of neurons and repair damaged nerve cells, and the effect is better than that of the control water decoction (FIG. 10).
Claims (1)
1. Application of brain-strengthening oral liquid containing ginseng and jujube in preparation of medicines for preventing and/or treating Alzheimer disease.
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Denomination of invention: The use of Shen Zao Jian Nao oral liquid in the preparation of drugs for the prevention and/or treatment of Alzheimer's disease Granted publication date: 20210629 Pledgee: Jilin Bank Co.,Ltd. Tonghua Liuhe Branch Pledgor: DIAOYUTAI MEDICAL GROUP JILIN TIANQIANG PHARMACEUTICAL Co.,Ltd. Registration number: Y2024220000126 |
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