CN114668758A

CN114668758A - Application of artemisinin and derivatives thereof in preparation of ChAT activity enhancer

Info

Publication number: CN114668758A
Application number: CN202210526527.2A
Authority: CN
Inventors: 郑文华; 赵霞
Original assignee: Um Zhuhai Research Institute; University of Macau
Current assignee: Um Zhuhai Research Institute; University of Macau
Priority date: 2021-05-17
Filing date: 2022-05-16
Publication date: 2022-06-28

Abstract

The invention discloses a new application of artemisinin and derivatives thereof. The inventor researches and discovers that the artemisinin and the derivatives thereof remarkably improve the activity of choline acetyltransferase in HT22 hippocampal neuron cells and primary culture neurons, improve the plasticity of cholinergic nerve synapses, reduce the apoptosis of cholinergic neuron cells and further improve the function of cholinergic nervous system. The artemisinin and the derivatives thereof are also found in animal models to improve the activity of choline acetyltransferase and improve learning and memory. In addition, the artemisinin drugs also regulate/improve the activity of choline acetyltransferase of various cell tissues of animals of various ages, which indicates that the artemisinin drugs can also be used for treating other diseases related to cholinergic insufficiency (abnormality), including but not limited to aging, postoperative cognitive dysfunction, dementia, schizophrenia, Huntington's chorea, autism, myasthenia gravis, hyperactivity, respiratory distress syndrome and the prevention and treatment of immune dysfunction of the body.

Description

Application of artemisinin and derivatives thereof in preparation of ChAT activity enhancer

Technical Field

The invention relates to the field of medicines, in particular to a new application of an existing compound, and particularly relates to an application of artemisinin in preparation of a choline acetyltransferase activity enhancer.

Background

Choline is a component of all biological membranes and a precursor of acetylcholine in cholinergic neurons, and mainly has the functions of promoting brain development, improving memory capacity, ensuring information transfer, regulating and controlling apoptosis, forming biological membranes, promoting fat metabolism, reducing serum cholesterol, promoting in vivo transmethylation metabolism and the like. Conventional studies have considered acetylcholine in the brain as a broad spectrum of neuromodulators that homogenize multiple brain regions. However, with the push of the study of the neural circuits in recent years, the correlation between the neural circuits involved in acetylcholine neurons and behaviors such as learning, memory, attention, etc. is gradually resolved, and the function thereof exhibits regiospecific or even neuron-specific function regulation. In addition, cholinergic information transmission abnormalities are accompanied in various complex diseases such as schizophrenia, alzheimer's disease, myasthenia gravis, hyperactivity, respiratory distress syndrome, and immune dysfunction of the body.

In the central cholinergic system, cholinergic neurons play a major role in learning, memory and attention. The central cholinergic nervous system is an important component of the learning and memory neural circuits, and dysfunction and death of cholinergic neurons, particularly in the hippocampal region, are considered to be the major pathophysiological cause of learning and memory deficits. Acetylcholine (ACh) is a neurotransmitter in the central cholinergic system, and its primary function is to maintain consciousness, playing an important role in learning and memory. In nerve cells, acetylcholine is synthesized from choline and acetyl-coa catalyzed by choline acetyltransferase (ChAT), and is taken up and stored by vesicles. Acetylcholine is destroyed by hydrolysis into choline and acetate by cholinesterase (AchE) after acting on postsynaptic membranes to exert physiological effects (which cause receptor membranes to generate action potentials) (AchE is the degradative enzyme of ACh). On the contrary, the AchE inhibitor can inhibit the activity of AchE to reduce ACh degradation and increase the content of ACh to increase the function of cholinergic nerve. Human brain tissue has a large amount of acetylcholine, but the content of acetylcholine decreases with age. The normal old people are reduced by 30 percent compared with the young people, and the senile dementia patients are more seriously reduced by 70 to 80 percent. The decrease of the content of acetylcholine affects the learning and memory ability of the individual. Increasing ChAT activity using ChAT enhancers or inhibiting AchE activity using AchE inhibitors increases the ACh content of neural cells and thus increases cholinergic nerve function. In HD, surviving striatal cholinergic neurons show reduced ACh release, reduced VAChT binding and a marked decrease in ChAT activity. In the early and middle stages of HD, these cholinergic neurons exhibit neuronal dysfunction, possibly associated with early symptoms of HD. The dysfunction observed in striatal cholinergic neurons may affect ACh-dependent processes such as the induction of synaptic plasticity and may significantly affect the function of other neuronal populations, including MSNs. However, in the later stages of HD, loss of spinal cholinergic neurons has been observed. In peripheral diseases, the result of cholinergic activation in the meninges is the release of proinflammatory factor transmitters and cytokines by mast cells, and the extravasation, excitation and sensitization of nociceptive afferent nerves leads to trigeminal neuralgia.

Schizophrenia is a common psychosis characterized by basic personality changes, abruption of thinking, emotion and behavior, and incoordination between mental activities and environments. Studies have shown that schizophrenia is associated with the cholinergic neurotransmitter system, and in particular is closely related to the α 7 neuronal nicotinic acetylcholine receptor (α 7 nAChR). The nAChR subtypes can transmit acetylcholine signals in the limbic and cortical regions of the brain where cholinergic receptors are highly expressed. In addition, researchers have found, by analyzing brain tissue from schizophrenic patients, that α 7 nachrs in hippocampus and dentate gyrus of patients were significantly reduced.

Pathological invasion of cholinergic nuclei and altered expression of acetylcholine receptors, especially nicotinic acetylcholine receptors, occurs in the brain of autistic patients. There was no difference between autistic patients and controls in the activity of choline acetyltransferase or acetylcholinesterase in the cerebral cortex and basal forebrain or in the binding of muscarinic M2 receptor or alpha-mycotoxin in the cortex. In autistic patients, the binding of the cortical M1 receptor was 30% lower than normal, and this difference reached significant levels in the parietal cortex.

ACh is widely expressed in non-neural cells of lower organisms, animals and humans, and participates in activities such as proliferation and differentiation of cells, formation of cytoskeleton and the like. The cholinergic anti-inflammatory pathway is a neurophysiological mechanism for regulating the immune system, and controls inflammatory reaction by releasing acetylcholine to inhibit the synthesis of cytokines in the reticuloendothelial systems such as spleen, liver and gastrointestinal tract. Acetylcholine interacts with macrophages and other alpha 7 nicotinic acetylcholine receptors on the cell surface of cell secreting cytokines to inhibit the synthesis and release of proinflammatory cytokines and prevent tissue damage.

Acute Respiratory Distress Syndrome (ARDS) is a serious disease complication associated with high mortality. There is currently no effective drug treatment for ARDS. Recent advances point to an important role for vagally mediated inflammatory reflex and neurocholinergic signaling. With a clinically approved (for myasthenia gravis) cholinergic drug, the acetylcholinesterase inhibitor pyridostigmine improved endotoxin-induced pulmonary and systemic inflammatory responses in ARDS mice, mediating acute respiratory distress syndrome. Changes in nicotinic acetylcholine receptor (nAChR) function may lead to symptoms of hyperactivity, and proposals to stimulate these receptors to relieve symptoms have been studied for more than two decades, but there are currently no approved nAChR drugs for hyperactivity. Clinical studies, including initial treatment trials and acute laboratory studies, have shown that stimulation of nAChRs can alleviate symptoms of hyperactivity and improve executive function.

Myasthenia Gravis (MG) is a recognized autoimmune disease affecting the neuromuscular junction. Recently, studies on the immunomodulatory properties of acetylcholine (ACh) derived from non-neuronal cells have led to an increasing interest in cholinergic anti-inflammatory pathways. It was found that the level of peripheral blood mononuclear cell-derived ACh was significantly higher in patients with myasthenia gravis compared to the healthy control group, and that ACh levels were positively correlated with QMG scores and anti-MuSK Ab levels.

In summary, abnormalities in the cholinergic system are related to the pathological features of a number of complex diseases. It is therefore of particular importance to find suitable drugs to improve the function of the cholinergic system. AchE inhibitors are currently used clinically primarily to increase neuronal ACh levels but no effective ChAT enhancer is currently available clinically. Therefore, the effective ChAT is searched for to enhance and regulate the functional activity of the cholinergic neurons in the brain, and is expected to provide new clues and ideas for various related diseases with cholinergic system abnormality.

The artemisinin is a sesquiterpene lactone medicine with peroxy groups extracted from stems and leaves of a composite inflorescence plant artemisia annua, and is characterized by high efficiency, low toxicity, fat solubility and easy crossing of a blood brain barrier. Artemisinin is well known for its potent antimalarial effect and has been found to have a variety of other functions including anti-apoptotic, anti-cancer, anti-inflammatory etc.

The inventor's previous research shows (CN109364064A, CN103948585A, CN112107576A), artemisinin has the ability of preventing and treating cerebral apoplexy, nervous diseases, nerve cell protection and learning and memory promotion, but no research shows that artemisinin has the function of enhancing choline acetyltransferase activity at present.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of artemisinin in preparing a choline acetyltransferase activity enhancer.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

application of artemisinin in preparation of choline acetyltransferase activity enhancer is provided.

In some examples, the choline acetyltransferase is a choline acetyltransferase of the central or peripheral nervous system.

In some examples, the enhancer is for a mammal.

In some examples, the enhancer is used for experimental purposes, not for therapeutic purposes.

In some examples, the enhancer is used for preventing or treating a disease associated with cholinergic hypofunction.

In some examples, the disorder is selected from the group consisting of aging, post-operative cognitive dysfunction, dementia, schizophrenia, huntington's disease, autism, myasthenia gravis, hyperactivity, respiratory distress syndrome, and immune dysfunction in the body.

In a second aspect of the present invention, there is provided:

use of artemisinin or its derivatives in preparing cholinergic nerve function improving agent is provided.

In some examples, the cholinergic nerve is a cholinergic neuron of the central or peripheral nervous system.

In some examples, the ameliorating agent is used for preventing or treating a disease associated with cholinergic hypofunction.

In some examples, the ameliorating agent is used to:

improving the learning and memory disorder related to the choline nerve hypofunction;

improving the plasticity of the nerve synapse;

reducing the death of cholinergic neuronal cells; or

Improving the diseases related to the abnormal immune function of brain, lung, liver, muscle, heart and kidney tissues and organisms caused by the dysfunction of cholinergic system.

In a third aspect of the present invention, there is provided:

use of low-dose artemisinin or its derivatives in preparation of cognitive function improving agent or medicine for preventing cognitive disorder is provided. Specifically, the dosage of the artemisinin and the derivatives thereof is not more than 9 mg/kg/d, preferably 0.1-10 mg/kg/d, and more preferably 0.1-5mg/kg/d, based on the dosage of the mouse. When used in humans, a corresponding conversion is required. Generally, the amount of the compound is not more than 2 mg/kg/d, preferably 0.02-2 mg/kg/d, and 0.02-1 mg/kg/d.

In some examples, the low dose of artemisinin or derivatives thereof improves cognitive, learning and memory abilities of transgenic mice model for senile dementia.

In some examples, the low dose artemisinin or a derivative thereof increases choline acetyltransferase activity.

In some examples, the low dose artemisinin or derivative thereof pre-treatment improves cognitive, learning and memory abilities of C57 brain stereotactic injection amyloid model mice.

The above effects of artemisinin show that artemisinin and derivatives thereof have a prevention effect on senile dementia, and the artemisinin and derivatives thereof can be used as clinical preventive drugs for senile dementia.

The beneficial effects of the invention are:

the inventor researches and discovers that the artemisinin and the derivatives thereof remarkably improve the activity of choline acetyltransferase in HT22 hippocampal neuron cells and primary culture neurons, improve the plasticity of cholinergic nerve synapses, reduce the apoptosis of cholinergic neuron cells and further improve the function of cholinergic nervous system.

In addition, the artemisinin and the derivatives thereof are also found to improve the activity of choline acetyltransferase and improve the learning and memory in animal models. The inventor also finds that the low-dose artemisinin and the derivatives thereof improve the cognition, learning and memory abilities of the transgenic mice with the Alzheimer's disease and improve the activity of choline acetyltransferase. In addition, low-dose artemisinin pretreatment improves learning and memory of C57 brain stereotactic model mice. The above effects indicate that the artemisinin has the effects of preventing and treating the Alzheimer disease. In addition, artemisinin and its derivatives also increase choline acetyltransferase activity in various (young, adult and elderly) brain, lung, liver, muscle, heart, kidney, oligodendrocyte and immune system tissues (fig. 11-13), suggesting that it may be useful in the prevention and treatment of other cholinergic deficiency-related diseases, including but not limited to aging, post-operative cognitive dysfunction, dementia, schizophrenia, huntington's chorea, autism, myasthenia gravis, hyperactivity, respiratory distress syndrome, and immune dysfunction in the body.

Drawings

FIG. 1 is a graph of the effect of artemisinin on choline acetyltransferase activity.

FIG. 2 is a graph of the effect of artemether on choline acetyltransferase activity.

FIG. 3 is a graph of the effect of artemisinin on cholinergic neuronal apoptosis and neurosynaptic plasticity.

FIG. 4 is a graph of the effect of artemether on cholinergic neuronal apoptosis and neuronal synaptic plasticity.

FIG. 5 is a graph showing the effect of artemisinin on learning and memory in scopolamine model mice.

FIG. 6 shows the effect of artemether on learning and memory of scopolamine model mouse

FIG. 7 is the effect of artemisinin on ChAT expression in scopolamine model mice.

FIG. 8 the effect of artemether on the CHAT expression of the scopolamine model.

FIG. 9 is a graph of the effect of artemisinin on synaptic plasticity in scopolamine model mice.

FIG. 10 is a graph of the effect of artemisinin and its derivatives on ChAT expression in young and old (6 weeks/18 months) and nude C57 mice.

FIG. 11 is the effect of artemisinin on CHAT expression in 4-month old C57 mice and 16-month old C57 mice.

FIG. 12 is a graph of the effect of different concentrations of artemisinin on CHAT expression in lung, liver, muscle, heart and kidney tissues of 7-month-old C57 mice.

Figure 13 effect of artemisinin on CHAT expression in oligodendrocyte (a172), Macrophage (Macrophage), Mesenchymal Stem Cell (MSC), U251 and B16 cells.

FIG. 14 Low doses (0.1-1mg) of artemisinin and its derivatives improved learning and memory in 3xTg model mice.

FIG. 15 is a graph of the effect of low doses (0.1-10mg) of artemisinin on the activity of acetylcholine transferase from brain tissue of 3xTg mice.

FIG. 16 is a graph of the effect of low dose artemisinin (1 mg) on pre-treatment on learning and memory in C57 brain stereotactic injection amyloid model mice.

Detailed Description

The technical scheme of the invention is further explained by combining experiments.

The cells possess characteristics of cholinergic neurons, express cholinergic-related genes, and are useful as in vitro models of hippocampal cholinergic neurons. In this experiment, the inventors used an HT22 neuron in vitro model to determine the effect of artemisinin on choline nerve cells.

Scopolamine is an acetylcholine muscarinic receptor blocker, and is a commonly used molding agent for inducing cognitive impairment of animals. A certain dose of scopolamine (2 mg/kg) is injected into ventricles of brain or abdominal cavity, which can cause cholinergic system abnormality and dysmnesia of organism. The research of the inventor provides experimental basis for preparing the clinical medicine for treating the cognitive impairment. In addition, the inventor researches show that low dose of artemisinin and derivatives thereof significantly improve cognitive impairment and reverse various pathological changes of senile dementia transgenic mice (3 xTg AD mice). Meanwhile, the low-dose artemisinin and the derivative thereof are pretreated to obviously improve the learning and memory and pathological characteristics of the C57 brain stereotaxic model mouse. Low dose artemisinin and its derivatives are suggested to have prevention and treatment effects on AD.

In addition, the inventors 'studies showed that artemisinin also increases choline acetyltransferase activity in various (young, adult and elderly) brain, lung, liver, muscle, heart, kidney, retinal melanin endothelial cells, oligodendrocytes and immune system tissues (fig. 11-13), suggesting that it may be useful in the prevention and treatment of other cholinergic insufficiency-related disorders, including but not limited to aging, post-operative cognitive dysfunction, dementia, schizophrenia, huntington's chorea, autism, myasthenia gravis, hyperactivity disorder, respiratory distress syndrome, and immune dysfunction in the body.

Materials and methods

1.1 Primary reagents

Analytical grade artemisinin was purchased from melphalan biology (Dalian, China). Dimethyl sulfoxide (DMSO) DMEM media and Fetal Bovine Serum (FBS) were ordered from Sigma (st louis, missouri). poly-D-lysine, 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT), 5,5', 6,6' -tetrachloro-1, 1', 3, 3' -tetraethyl-benzimidazolyl-carbocyanoiodide (JC-1) and Hoechst 33258 were purchased from Molecular Probes (ewing, oregon, usa). Primary and rabbit anti- β -actin antibodies, such as ChAT, GAPDH, caspase3, are available from CST. LDH cytotoxicity assay kits were purchased from petunia. CellROX deep red reagent was ordered from Thermo Fisher Scientific (Rockford, Ill., USA). The Annexin V-FITC/PI apoptosis assay kit was obtained from BD Biosciences (san Diego, Calif., USA). 0.25% trypsin was purchased from Life Technologies (Greenland, N.Y., USA).

Cellular Immunofluorescence (ICC)

After appropriate treatment, HT22 cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then washed 3 times with 1 xPBS. The samples were then incubated with 1xPBS (containing 0.25% Triton X-100) for 10 min and blocked with 3% BSA for 1h at room temperature. Thereafter, cells were incubated with ChAT in PBS containing 1% BSA at 4 ℃ overnight. The following day, cells were washed 3 times with PBS for 5 minutes and incubated with secondary antibody in the dark for 1 hour at room temperature. Nuclei were counterstained and mounted with a drop of DAPI-containing anti-quenching mounting tablets (P0131, Beyotime) and images were taken using a Nikon a1 confocal microscope.

Tissues of brain, lung, liver, muscle, heart, kidney, etc., HT22 cells, retinal melanin endothelial cells, lacolloid cells, and immune system cells were harvested and lysed with ice-cold RIPA lysis buffer. Protein concentration was determined by BCA method. Samples with equal amounts of protein (50. mu.g) were separated by 10% SDS-PAGE and then transferred to PVDF membrane. After blocking with 3% BSA for 1 hour, the membrane was incubated with the selective primary antibody overnight at 4 ℃. The following day, the primary antibody (anti-CHAT, CHE, GAPDH, MAP2 SYP, PSD95) was washed 3 times with 1 × TBST and incubated with the secondary antibody for 2 hours. The intensity of the bands was quantified using Image J software.

Flow cytometry

Following artemisinin treatment HT22 cells were trypsinized, washed twice with 1x PBS, then centrifuged at 1000rpm for 5 minutes, and then resuspended in Annexin VFITC 1x binding buffer (195 μ L). Annexin V-FITC (5. mu.L) was added and cells were incubated in the dark at 37 ℃ for 20 min. The cells were then centrifuged at 1000rpm for 5 minutes and resuspended in 1 Xbinding buffer (190. mu.L). Propidium Iodide (PI) (10 μ L) was further added, followed by incubation in the dark for 5 minutes. Apoptosis was quantified using flow cytometry. Cell Quest Pro software was used to analyze apoptosis.

Dyeing process

Cells were seeded into 96-well plates (1X 10)⁴Individual cells/well). After appropriate artemisinin treatment, 4% PFA blocking for 30 min, PBS washing 3 times, cells were incubated with TUNEL reaction mix (50 μ L) in the dark for 60 min at 37 ℃. PBS was washed 3 times. DAPI counterstain for 3 min, wash well with PBS and observe with microscope.

Measurement of Reactive Oxygen Species (ROS)

Intracellular Reactive Oxygen Species (ROS) production was assessed by Cell ROXs Deep Red reagent or DCFH-DA reagent. Briefly, cells grown in 96-well plates were either pretreated with 1 μ MA β 1-42 or without artemisinin. Cells were then incubated with either Cell ROXs Deep Red reagent or DCFH-DA reagent (5 μ M in fresh DMEM incubated for 1 hour in the dark). Cells were washed with IX PBS. Fluorescence microscopy used a high-content screening system (ArrayScan VTI, Sammer Feishell science, USA) for Cell ROXs Deep Red reagents at 640 nm excitation and 665 nm wavelength, and for DCFH-DA reagents at 488 nm excitation and 525 nm wavelength.

Dyeing process

JC-1 dye was used to monitor mitochondrial integrity. Briefly, HT22 cells were seeded into 96-well plates (1 × 104 cells/well) in the dark. Pretreatment with 12.5 μ M artemisinin for 2h followed by exposure to 1 μ M β 1-42 for another 24h, then cells were treated with JC-1 dye (10 μ g/mL in medium) for 15 min at 37 ℃ and rinsed twice with PBS. To quantify the signal, the intensity of red (excitation 560 nm, emission 595 nm) and green fluorescence (excitation 485 nm, emission 535 nm) was evaluated using an Infinite M200 PRO multimode microplate reader. Δ ψ m was calculated as a ratio of red/green fluorescence intensity, and normalized with respect to the control group. Fluorescence signals in the cells were also recorded with a fluorescence microscope.

Activity detection

The measurement of acetylcholine transferase uses acetyl-CoA and choline as substrates. The reaction product was combined with a color developer under the action of ChAT and the absorbance was measured at 324 nm. ChAT activity was tested using the ChAT assay kit (XFA 079-1, Xin Fan Biological, Shanghai, China) according to the manufacturer's instructions.

Preparation of tissue samples (10% homogenate): the brain, lung, liver, muscle, heart, kidney, etc. tissues of each group were accurately weighed. The weight ratio (g): reagent 1 (mL) = 1: 9 to tissue samples, saline was added and the samples were then homogenized on ice. The homogenate was centrifuged at 2500rpm for 10 minutes at 4 ℃. The supernatant was taken for testing. Reagents were added sequentially according to the instructions. Immediately mixed well and incubated at room temperature for 15 minutes, and then the change in absorbance at 324nm was measured.

Immunohistochemistry (IHC)

Immunohistochemistry (IHC) is used to localize/visualize protein expression in fixed tissue sections using protein-specific antibodies (Zhang et al, 2016). Brain tissue was cut into 5 μm sections using a manual rotary microtome basic instrument (Leica RM2235, Leica, germany). After conventional deparaffinization hydration, antigens were subjected to antigen retrieval by immersion in 0.01M citrate buffer solution. Followed by incubation with 3% H2O2 for 15 minutes to remove endogenous peroxidase activity. After blocking with 10% Bovine Serum Albumin (BSA) for 1 hour, primary antibody was added dropwise to the sections, which were stored overnight at 4 ℃. The next day, brain tissue sections were incubated with secondary antibodies for 60 minutes, followed by DAB visualization.

Immunofluorescence (IF)

Immunofluorescence (IF) is a method of detecting a target antigen in a test tissue or cell using a known antibody labeled with fluorescein as a probe and providing fluorescein to the formed antigen-antibody complex. Direct observation can be performed by fluorescence microscopy. Brain tissue was embedded in OCT (optimal cutting temperature) compounds. The brains were cut into 20 μm sections using a cryostat (Leica CM3050, Leica, germany). Each section was washed 3 times with 1xPBS and then blocked with 10% BSA for 1h at room temperature. Thereafter, the tissue sections were incubated overnight with primary antibody in PBS containing 1% BSA at 4 ℃. The following day, sections were incubated with appropriate secondary antibodies for 1 hour at room temperature in the dark. Nuclei were counterstained with DAPI and images were taken with a Nikon a1 confocal microscope.

Water maze experiment

The Morris water maze consists of a stainless steel round swimming pool, a movable platform is arranged in the Morris water maze, four marking points with equal distance are arranged on the wall of the pool, the pool is divided into four quadrants, and the quadrants are also used as the water inlet points of the mice. In the experimental process, the water temperature is kept constant, the temperature is kept at (22-26) DEG C, and the movable platform is submerged to be about 1.5cm away from the water surface. Before the experiment began, mice were placed in the water maze for swimming in 4 quadrants until they found a platform, and if they could not find a platform within 60 seconds, the mice were guided to the platform and allowed a 10 second rest time. Training is continued for 3 days to adapt to the environment. And then carrying out formal experiments, carrying out the experiments in the morning every day, and recording the time when the mouse finds the platform, namely the incubation period of the mouse. At the beginning of each experiment, mice were placed in water from any one of the four quadrants, facing the pool wall, and four experiments were performed per mouse, with a swimming time of 60s each. If the mouse can find the platform within 60s, the mouse can rest on the platform for 10 s; if the platform can not be found within 60s, an experimenter guides the mouse to the platform, the experimenter also takes a rest for 10s, the latency period of the mouse is 60s, and the time and the movement route of the mouse for finding the platform are recorded through the camera system and the software acquisition system. The above procedure was repeated on

days

2, 3, 4 and 5. And (4) counting the average latency in the 5-day positioning navigation experiment of each group of mice as an index for judging the learning ability of the mice. And on the 6 th day, the platform hidden under the water is removed, and a space exploration experiment is carried out. Randomly selecting a same water inlet point, putting the mouse into water, and recording the movement track of the mouse in 60s, the times of passing through the platform and the residence time of the mouse in the quadrant where the platform is located. And (4) counting the time percentage of each group of mice in the platform quadrant to measure the space positioning capability and the memory capability of the mice. All data acquisition and processing are completed by an automatic Morris water maze image monitoring and processing system.

Brain stereotaxic injection

Four weeks after artemisinin pre-treatment, mice were weighed and anesthetized by intraperitoneal injection of 6% chloral hydrate at a dose of 0.6 mL/100 g. The anesthetized mice were fixed in a stereotaxic apparatus, parietal skin and sterilized with medical alcohol. A1.5 cm long tip was cut in the sagittal middle of the head and the soft tissue wiped with cotton cloth dipped in 1XPBS to clearly expose bregma. According to the three-dimensional positioning diagram of the mouse, the mouse is placed in a mode of ensuring that the front and the back of the mouse are placed on the same horizontal line. The injection point is determined according to the corresponding coordinates and marked with a marker. 2 ul of A β (1-42) in polymerized form (1 μ g/. mu.l) was injected over 5 minutes using a micro-syringe. The needle was held in place for about 5 minutes after injection and then slowly withdrawn over 2 minutes. The skin was sutured and the wound was disinfected with alcohol to prevent infection. The coordinates of the hippocampus were as follows: the previous halo is 0, 1.9 mm behind bregma, 1.2 mm on the side, and the insertion depth of the needle is 2.0 mm.

Primary neuronal cell culture

Taking the newly born one within 24hC57BL/6 mice. The whole body was disinfected with 75% alcohol and the brain surgically removed and then stored in cold HBSS (calcium and magnesium free) balanced solution. The entire hippocampal and cortical regions were dissected using glass rods bent on both sides. The tissue was cleared of blood and mixed blood vessels by 3 washes with HBSS. Then, the tissue was cut to 1 mm with scissors³After washing 3 times with HBSS, the tissue was digested with 0.125% trypsin at 37 ℃ for 15 minutes. Enzyme digestion was stopped with 10% FBS and 5 mL of neural basal medium a was added to the digested tissue in a 15 mL centrifuge tube. The turbid tissue supernatant was collected in another 15 mL centrifuge tube and centrifuged at 1000rpm for 10 minutes. The resulting cell pellet was resuspended in Neurobasal A/B27 at approximately 1-2X 10⁵cells/mL were seeded at a density in poly-D-lysine-treated plates and incubated at 37 ℃ with 5% CO₂Culturing in an incubator, and changing the culture solution half a day.

Results

2.1 Effect of Artemisinin and derivatives on Choline acetyltransferase Activity

Ach is the central neurotransmitter known to be most closely associated with learning and memory. Choline is synthesized from choline acetyl-coa and choline under catalysis by ChAT. Thus, the inventors examined the effect of artemisinin and derivatives on ChAT expression. The inventors' results indicate that artemisinin can increase ChAT expression. Meanwhile, artemisinin inhibited the decrease in ChAT expression caused by a β (1-42) in HT22 (fig. 1, (a) analysis of ICC staining for cholinergic marker protein ChAT the effect of artemisinin on ChAT in ART (B) QPCR detection of the effect of artemisinin on ChAT (C) treatment with different concentrations of ART for 120min, and Western blot analysis of ChAT and GAPDH expression after treatment with 12.5 μ M ART for different times). Similarly, we assessed ARTE cytotoxicity in the artemisinin derivative artemether (fig. 2, (a) chemical structure of ARTE · (B) treatment of cells with different concentrations of ARTE (1, 3, 10, 30, 90 μ M) for 24 hours and using MTT assay for cell viability · (C) Western blot analysis of ChAT and GAPDH expression after 120min treatment with different concentrations of ARTE · quantification of d.c. (E) · Western blot analysis of ChAT and GAPDH expression within 180 min after 30 μ M ARTE treatment · (F) quantification of E · (G) assessment of pressure levels of ChAT, AChE by Western blot · (H) quantification of G. (I) staining analysis of cholinergic marker protein ChAT · (J) ChAT quantification of ChAT).

Effect of artemisinin and derivatives on apoptosis of cholinergic nerve cells and plasticity of nerve synapses

Neuronal damage and apoptosis affect cholinergic function. HT22 cells were treated appropriately with artemisinin and the effect of artemisinin on apoptosis was examined using Tunel staining, flow cytometry and ICC. As a result of Tunel staining, the A beta (1-42) can cause neuron apoptosis, and the artemisinin can improve the neuron apoptosis caused by the A beta (1-42). The inventors have further confirmed this with flow cytometry and obtained similar results. Artemisinin can protect neurons from damage by A β (1-42). Caspase3 is a key factor in apoptosis. The inventor uses ICC to detect the expression of chat and caspase3, and finds that artemisinin reduces the expression of apoptosis factor caspase 3. The mechanism of formation of hippocampal synaptic transmission and long-term potentiation (LTP), which increases plasticity of synaptic transmission, is one of the factors that increase learning and memory. Thus, the inventors examined the expression of the synaptic related proteins MAP2, PSD95 and SYP using western blots. The inventor's results found that artemisinin can increase the expression of MAP2, PSD95 and SYP. In addition, the proportion of synapses in the branches increased after artemisinin treatment (fig. 3, (a) cells were pretreated with different concentrations of artemisinin for 2 hours followed by incubation for 10 μ M A β 24 hours, and the level of apoptosis was assessed by Tunel staining assay: (C) immunofluorescence co-stained with cholinergic marker proteins ChAT and caspase 3: (D) treatment with different concentrations of ART for 120min, and expression of MAP2, YAP, PSD95 and GAPDH after different times of treatment with 12.5 μ M ART:thelevel of expression of MAP2 and ChAT was assessed using immunofluorescence staining). The artemisinin can improve synaptic plasticity and is expected to increase learning and memory ability. Similarly, the results obtained were consistent when validated with artemether (fig. 4, (a) assessment of Α β 42 cytotoxicity HT22 cells incubated with different concentrations (1.25, 2.5, 5, 10, 20 μ M) of Α β 42 for 24 hours and cell viability determined using MTT method (B) assessment of the neuroprotective effect of ARTE treatment on Α β 42-induced neurotoxicity pre-treated with different concentrations of ARTE for 2 hours followed by incubation for 10 μ M A β 24 hours and MTT assay assessment of cell viability (C) western blot analysis of the expression of cholinergic marker proteins ChAT, ACh hydrolase AChE and GAPDH · (D) quantification of immunofluorescence of ChAT and AChE (E-F) western blot analysis using immunofluorescence staining to assess the expression levels of MAP2 and ChAT).

Influence of artemisinin and derivatives on learning and memory of scopolamine model mouse

The inventors used 48 6-week-old C57 mice, divided into 4 groups of 12 mice each. Before administration, the body weights are respectively weighed, and the dosage is calculated according to the body weights. The artemisinin is administrated by intraperitoneal injection every day, the control group is administrated with physiological saline, the positive control group is administrated with donepezil (3 mg/kg/d), after one month of administration, the positive control group is administrated with scopolamine (3 mg/kg), after 20min of each treatment, the Morris water maze experiment is carried out, and then the materials are obtained for subsequent experiments. The results show that artemisinin treatment significantly improved the learning memory of C57 scopolamine model mice (fig. 5, (a) representative swimming trajectories and time to find the platform before and after withdrawal of the table: (D) percentage of time in target quadrant: (E) number of crossings of the platform). Also, our pretreatment with artemether gave consistent results, with artemether significantly improving the learning memory of the C57 scopolamine model mouse (fig. 6, (a-B) the number of crossings of the platform (D) the time percentage of the target quadrant by four consecutive days of training, swim trajectory and latency (C)).

Artemisinin and its derivatives reduce the loss of choline acetyltransferase in scopolamine-model mice

Its expression was detected by immunofluorescence. Artemisinin was found to increase ChAT expression significantly more than the positive control. In addition, the inventors tested ChAT activity in the blood of each group of mice using the ChAT kit. The inventors found that both artemisinin and donepezil increased ChAT expression, with artemisinin acting better than donepezil (fig. 7). The same results were verified in artemether (figure 8).

Artemisinin and derivatives can improve synaptic plasticity of scopolamine model mice

Impairment of brain synaptic structure and functional plasticity is a central link in AD pathological progression and is also one of the pathological mechanisms of AD learning and memory decline. Therefore, increasing synaptic plasticity has become one of the major measures to prevent AD. As a result of immunofluorescence, both artemisinin and donepezil were found to improve synaptic plasticity. The curative effect of the artemisinin is better than that of the positive drug donepezil. Western blot results show that artemisinin and donepezil both increase the expression of synaptic related proteins MAP2, PSD95 and SYP, whereas artemisinin has superior effects to donepezil (FIG. 9).

Effect of artemisinin and derivatives on ChAT expression in different tissues and cells of mice

To further determine the effect of artemisinin compounds on cholinergic nerve function, the inventors studied young C57 mice (6 weeks old), middle aged and aged mice (7-18 months old), and found that artemisinin increased ChAT expression after pretreatment for one month with artemisinin compounds and then material collection (FIG. 10). In addition, the applicant also treated C57 with artemisinin for a short period of time (2 h) to verify whether short-term stimulation with artemisinin compounds could cause an increase in CHAT expression, and found that short-term stimulation with artemisinin compounds could likewise cause an increase in CHAT expression in brain tissue (fig. 11). Further, the inventors treated C57 mice with artemisinin (0.5, 1, 5 mg/kg) at various concentrations for a short period of time (2 h), then collected tissues of brain, lung, liver, muscle, heart, kidney, etc., and examined the CHAT expression, and found that artemisinin increased the ChAT expression in various tissues to various degrees (FIG. 12), indicating that artemisinin drugs can regulate the function of CHAT of neuro/non-neuro systems, and be used for the treatment of various diseases of neuro/non-neuro systems (aging, postoperative cognitive dysfunction, dementia, schizophrenia, Huntington's chorea, autism, myasthenia gravis, hyperactivity, respiratory distress syndrome, and immune dysfunction of the body). In addition to HT22 cells, the inventors also found that artemisinin drugs affect CHAT expression in oligodendrocytes (a172), macrophages (Macrophage), mesenchymal stem cells and T cells, suggesting that artemisinin drugs may be useful in the modulation of immune function and treatment of various immune and inflammatory diseases (fig. 13, effect of artemisinin on CHAT expression in oligodendrocytes (a172), macrophages (Macrophage), mesenchymal stem cells MSC, U251, B16 and T cells).

Low-dose artemisinin and derivatives thereof improve learning and memory abilities of 3xTg model mice

Alzheimer model mice (12 months old) were subjected to analysis of the results of the Morris water maze experiment after one month of high, medium and low dose artemisinin treatment (0.1 mg/kg/d, 0.5mg/kg/d, 1mg/kg/d, 5mg/kg/d, 10 mg/kg/d). The results show that 1 month of artemisinin or its derivatives administration in 3xTg AD mice can significantly improve its ability to localize the platform on MWM and can also enhance the spatial learning task. The mean escape latency of the artemisinin or derivative treated group was significantly lower than that of the 3xTg group. And exhibits higher retention performance in the learning test. The probe tests performed without the platform on the following day 6 further enhanced the above results. There was more platform position crossing time in the artemisinin or derivative treated group compared to the 3xTg group and the percentage of target quadrant search time increased. These results indicate that artemisinin or its derivatives (0.1-10mg, 1-5>10 mg) can improve cognitive impairment in 3 × tg AD mice (figure 14).

Low dose (0.1-5mg/kg/d) of artemisinin and its derivatives can improve the activity of 3xTg mouse acetylcholine transferase

The reduction of ChAT is an important feature and contributing factor to AD. Thus, the inventors tested the expression of ChAT in each group of mice. ChAT expression was found to be reduced in the brains of model mice, whereas ChAT expression was increased after low dose artemisinin treatment (0.1-5mg/kg/d) (FIG. 15).

Pretreatment of low-dose artemisinin and derivatives thereof (0.1-5mg/kg/d) improves learning and memory of C57 brain stereotaxic injection amyloid model mice

The inventors analyzed the effect of low doses of artemisinin (0.1-5mg/kg/d) on cognitive function in C57 model mice using the Morris Water Maze (MWM). The mean escape latency was found to be significantly lower in the artemisinin-pretreated group than in the model group. The longer the platform position crossing time was in the artemisinin-pretreated group after platform withdrawal on day five, the percentage of search time in the target quadrant increased, indicating that artemisinin could improve learning and memory function in C57 model mice (fig. 16). The results show that artemisinin can improve cognitive impairment of model mice.

Discussion:

with aging, learning and memory abilities are gradually reduced, cerebral cortex, hippocampus and the like are widely degenerated to cause cognitive impairment, and serious troubles are brought to independent life and activity of the old. With the increasing aging of the population, the method becomes a big problem affecting the whole world. The objective wish of human dreaming is to find a medicine with the function of improving learning and memory. Currently, there is an increasing body of research data that suggests that the normal function of central cholinergic neurons is a necessary condition for learning and memory in mammals. The cholinergic nerve cells of a patient with cognitive impairment are obviously lost, cholinergic nerve fibers are degenerated to cause central cholinergic system dysfunction, the expression of choline acetyltransferase of cortex and basal forebrain is reduced, the hippocampus is most obvious, and the generation and release amount of acetylcholine (ACh) are reduced, so that the cholinergic nerve cells are direct causes of cognitive impairment such as learning and memory. Meanwhile, the early stage of the cognitive disorder disease is accompanied by synaptic dysfunction, neuron death and the like. Therefore, the search for effective drugs for regulating the functional activities of cholinergic neurons in the brain is expected to provide new clues and ideas for preventing and treating cognitive disorder diseases and enhancing the learning and memory efficiency of human beings.

Currently, acetylcholinesterase inhibitors represent the most common pharmacological compounds for the treatment of AD, however, acetylcholinesterase inhibitors do not restore dying or degenerated neurons [6 ]. However, at present, no related research of a target ChAT reinforcing agent exists, and whether the ChAT reinforcing agent can effectively treat the cholinergic system related diseases or not has innovative characteristics. The artemisinin is a sesquiterpene lactone medicine with peroxy groups extracted from stems and leaves of a composite inflorescence plant artemisia annua, and is characterized by high efficiency, low toxicity, fat solubility and easy crossing of a blood brain barrier. In contrast, artemisinin-related therapeutics are predictive of maintaining cholinergic morphology, survival and function.

The above research results show that: the artemisinin and the derivatives thereof remarkably improve the activity of choline acetyltransferase in HT22 hippocampal neuronal cells, improve the plasticity of cholinergic nerve synapses, reduce the apoptosis of cholinergic neuronal cells and further improve the function of cholinergic nervous system. The inventor further verifies in a scopolamine model that artemisinin can improve the learning and memory functions of a model mouse, and further shows that artemisinin and derivatives thereof can improve the cholinergic system function. The artemisinin and the derivatives thereof not only increase the expression of ChAT and promote the survival of cholinergic neurons, but also have low toxic and side effects, can pass through the blood brain barrier and have low price, can be well used as experimental drugs for the research of animal models and finally provide the experimental basis of clinical medication for the human beings to overcome the cholinergic dysfunction diseases.

Furthermore, our results also show that artemisinin drugs also enhance choline acetyltransferase activity in various (young, adult and elderly) brain, lung, liver, muscle, heart, kidney, retinal melanocytes, oligodendrocytes and immune system tissues (fig. 11-13), suggesting that artemisinin drugs may also be used for the prevention and treatment of other cholinergic insufficiency related disorders, including but not limited to aging, post-operative cognitive dysfunction, dementia, schizophrenia, huntington's chorea, autism, myasthenia gravis, hyperactivity, respiratory distress syndrome, and immune dysfunction in the body.

Reference:

[1] LI Y，HAI S，ZHOU Y，et al. Cholinesterase inhibitors for rarer dementias associated with neurological conditions[J]. Cochrane Database of Systematic Reviews，2015(3)： CD009444.

[2] Zeng Z, Xu J, Zheng W (2017). Artemisinin protects PC12 cells against β-amyloid-induced apoptosis through activation of the ERK1/2 signaling pathway. Redox biology, 12:625-633.

[3] TUSZYNSKI M H，YANG J H，BARBA D，et al. Nerve growth factor gene therapy activation of neuronal responses in Alzheimer disease[J]. JAMA Neurol，2015，72(10)： 1139-1147.

[4]ZHANG L，FANG Y，LIAN Y J，et al. Brain-derived neurotrophic factor ameliorates learning deficits in a rat model of Alzheimer’s disease induced by a beta 1-42[J]. PLoS One，2015，10(4)： e0122415.

[5] TAN C C，YU J T，WANG H F，et al. Efficacy and safety of donepezil，galantamine，rivastigmine，and memantine for the treatment of Alzheimer’s disease： a systematic review and meta-analysis[J]. Journal of Alzheimers Disease，2014，41(2)： 615-631.

[6] Haake, Andrea, et al. "An update on the utility and safety of cholinesterase inhibitors for the treatment of Alzheimer’s disease." Expert Opinion on Drug Safety 19.2 (2020): 147-157.

[7] Stanciu, Gabriela Dumitrita, et al. "Alzheimer’s disease pharmacotherapy in relation to cholinergic system involvement." Biomolecules 10.1 (2020): 40.

[8] D’Souza, G. X., & Waldvogel, H. J. (2016). Targeting the cholinergic system to develop a novel therapy for Huntington’s disease. Journal of Huntington's disease, 5(4), 333-342.

Shelukhina, I., Mikhailov, N., Abushik, P., Nurullin, L., Nikolsky, E. E., & Giniatullin, R. (2017).

[9] Cholinergic nociceptive mechanisms in rat meninges and trigeminal ganglia: potential implications for migraine pain. Frontiers in neurology, 8, 163. 27 April 2017

[10] Berman, J. A., Talmage, D. A., & Role, L. W. (2007). Cholinergic circuits and signaling in the pathophysiology of schizophrenia. International review of neurobiology, 78, 193-223.

[11] Corradi, J., & Bouzat, C. (2016). Understanding the bases of function and modulation of α7 nicotinic receptors: implications for drug discovery. Molecular pharmacology, 90(3), 288-299.

[12]Potter, A. S., Newhouse, P. A., & Bucci, D. J. (2006). Central nicotinic cholinergic systems: a role in the cognitive dysfunction in attention-deficit/hyperactivity disorder. Behavioural brain research, 175(2), 201-211.

[13]Potter, A. S., Schaubhut, G., & Shipman, M. (2014). Targeting the nicotinic cholinergic system to treat attention-deficit/hyperactivity disorder: rationale and progress to date. CNS drugs, 28(12), 1103-1113.

[14]Bricher Choque, P. N., Vieira, R. P., Ulloa, L., Grabulosa, C., Irigoyen, M. C., De Angelis, K., . . . Consolim-Colombo, F. M. (2021). The Cholinergic Drug Pyridostigmine Alleviates Inflammation During LPS-Induced Acute Respiratory Distress Syndrome. Frontiers in pharmacology, 12, 966Front. Pharmacol., 04 May 2021.

[15] Han, B., Zhang, C., Liu, S., Xia, Y., Sun, H., Gong, Z., ... & Hao, J. (2017). Non-neuronal cholinergic activity is potentiated in myasthenia gravis. BMC neurology, 17(1), 1-9.。

Claims

1. the application of artemisinin or its derivatives in preparing choline acetyltransferase activity enhancer is disclosed, wherein the derivatives are selected from artemisinin, artemether, artesunate, dihydroartemisinin and other compounds with structure similar to artemisinin.

2. Use according to claim 1, characterized in that: the enhancer is for a mammal.

3. Use according to claim 1 or 2, characterized in that: the enhancer is used for preventing or treating diseases related to choline hypofunction.

4. Use according to claim 3, characterized in that: the disease is selected from the group consisting of aging, post-operative cognitive dysfunction, dementia, schizophrenia, Huntington's disease, autism, myasthenia gravis, hyperactivity, respiratory distress syndrome, and immune dysfunction in the body.

5. Application of artemisinin or its derivatives in preparing cholinergic nerve function improving agent is provided.

6. The use according to claim 5, wherein: the improver is used for preventing or treating diseases related to choline nerve hypofunction.

7. Use according to claim 6, characterized in that: the disease is selected from the group consisting of aging, post-operative cognitive dysfunction, dementia, schizophrenia, Huntington's disease, autism, myasthenia gravis, hyperactivity, respiratory distress syndrome, and immune dysfunction in the body.

8. Use according to claim 6, characterized in that: the improving agent is used for:

improving the plasticity of the nerve synapse;

reducing the death of cholinergic neuronal cells; or

9. Use of low-dose artemisinin or its derivatives in preparation of cognitive function improving agent or medicine for preventing cognitive disorder is provided.

10. Use according to claim 9, characterized in that: the amount of artemisinin and its derivatives is not more than 9 mg/kg/d based on the amount of mouse.