CN115154459B - Application of artesunate in preparation of medicine for treating fragile X syndrome - Google Patents

Abstract

The invention discloses an application of artesunate in preparing a medicine for treating hereditary mental and cognitive dysfunction diseases. Experiments prove that the artesunate has remarkable hereditary intelligence and cognitive dysfunction resistance and efficacy and small toxic and side effects, is a safe, efficient and stable-curative medicament for treating fragile X syndrome, is suitable for industrial production and is easy to popularize. The invention provides a new medicine source for treating fragile X syndrome and related diseases caused by changes and mutations of X chromosome in vivo in the genetic process, and provides a new medicinal application of artesunate.

Description

Application of artesunate in preparation of medicine for treating fragile X syndrome

Technical Field

The invention relates to application of a medicine for treating hereditary mental and cognitive dysfunction diseases, in particular to application of a medicine for treating fragile X syndrome, and belongs to the technical field of medicine application.

Background

Fragile X syndrome (also known as Martin-Bell syndrome) is caused by mutations in the fragile X mental retardation gene (Fmr 1) during X chromosome formation in humans. In a stretch of DNA on the X chromosome, one is completely altered and the other is DNA hypermethylated due to genetic relationships that sometimes change. If the extent of these two changes is small, the patient may have no special symptoms or only mild symptoms in terms of clinical manifestations. Conversely, if the degree of these two changes is large, various symptoms of fragile X syndrome, such as cognitive impairment, language impairment, etc., as described below, may occur.

Fragile X syndrome (Fragile X syndrome, FXS) is not only a common genetic intellectual and cognitive dysfunction disease, but also a most common monogenic defective disease causing autism spectrum disorder, mainly manifested as intellectual disorder, social interaction disorder, cognitive disorder and the like, greatly affecting the behavioral ability and quality of life of patients, and an effective clinical treatment scheme is urgently needed.

FXS disease occurs mainly due to mutations in fragile X mental retardation gene (Fmr 1) on the X chromosome, resulting in loss of function of fragile X mental retardation protein (FMRP). Although there is a great deal of research interest in the etiology of fragile X syndrome, particularly as it is likely to be one of the effective pathways for resolving the cause of autism, the exact etiology mechanism behind the disease has not yet been elucidated.

There is still a lack of effective treatment of FXS in the clinic and most of the treatments for FXS are formulated based on their specific symptoms, which also results in a lack of adequate control experiments to demonstrate the effectiveness of these methods, which can only be maximally exerted by psychopharmacological intervention in combination with other supportive strategies, including speech therapy, sensorial integration occupational therapy, personalized educational planning and tailored behavioral intervention.

Agonists are nowadays the most common drugs in treating FXS, but the efficacy of these drugs and their side effects also vary from person to person, the most common therapeutic drugs being agonists. While agonists are effective against the symptoms of hyperactivity, impulsivity, and inattention in young men with FXS, adult men with FXS respond less to agonists.

Although a series of drug studies have been conducted on metabotropic glutamate receptors (metabotropic glutamate receptor, mGluR 5), glycogen synthase kinase-3 (glycogen synthase kinase-3. Beta., GSK-3. Beta.) and gamma-aminobutyric acid (gamma-Aminobutyric Acid, GABA) receptors, the therapeutic effect of this single drug was only evident in animal experiments, but not in clinical experiments. The research result of single drug treatment shows that a gap exists between animal experiments and clinical experiments, and the existing single drug treatment strategy is not up to the requirement of clinical treatment. In addition, selective serotonin reuptake inhibitors (Selective serotonin reuptake inhibitors, SSRIs) are used as drugs for treating mood disorders, anxiety and compulsive behaviors associated with FXS, which have therapeutic effects in alleviating social anxiety, spleen qi development, aggression, and the like; in addition, FXS is also treated with antipsychotics, but side effects associated with antipsychotics such as weight gain, diabetes, nausea, constipation, and tardive dyskinesia lead to poor results. Atypical antipsychotics have also been used to treat self-injury, aggression and autism, and while atypical antipsychotics aripiprazole (aripiprazole) have improved mood stabilization, attention and learning performance, it should be used at low doses to avoid agitation caused by high doses and its side effects should not be small.

The intellectual disability caused by FXS brings great physiological and psychological pain to patients and families thereof, and the long course of disease brings heavy burden to families and society. The therapeutic effect is only maximally exerted by psychopharmacological intervention in combination with other supportive strategies including speech therapy, sensorial integrated occupational therapy, personalized educational planning and tailored behavioral intervention.

However, to date, there is still a lack of effective therapeutic agents that cure or alleviate the major adverse symptoms in patients with this disease. Thus, it is urgent to explore the etiology mechanism of fragile X syndrome and develop new therapeutic drugs. And the genes and proteins which cause abnormal expression after the FMRP is deleted are researched, and the specific influence of the abnormal expression of related targets on the structure, the morphology and the functions of neurons is inspected, so that positive clinical application value and profound social significance can be provided for comprehensively treating FXS and searching new drug intervention targets.

Artesunate (Artesunate, AS, C ₁₉ H ₂₈ O ₈ ) Is one of the derivatives of artemisinin. The molecular weight of the artemisinin derivative is 384.42, and the artemisinin derivative is water-soluble, and is the only water-soluble artemisinin derivative used clinically. Since month 4 of 2019, the world health organization recommended artesunate as a first line drug for treating cerebral malaria and other severe malaria, and also became a first line drug for treating severe malaria in the united states.

Clinical researches show that artesunate has the characteristics of convenient administration mode, good stability, high efficiency, quick acting, low toxicity, good tolerance and the like. Clinically, artesunate intravenous injection can treat severe malaria and cerebral malaria. Modern pharmacological researches have shown that artesunate also has the effects of anti-inflammatory, anti-tumor, immunoregulation, anti-lupus erythematosus and the like. Meanwhile, artesunate has potential treatment and protection effects on central nervous system diseases including neurodegenerative diseases. Although artesunate has been shown to be a multifunctional therapeutic drug with different pharmacological actions, it is still unknown whether artesunate can treat or ameliorate the core symptoms of fragile X syndrome and its mechanism of action.

In order to explore the therapeutic effect and the effect of artesunate on fragile X syndrome diseases, the inventor performs in vitro neural stem cell proliferation and differentiation experiments; in vivo neural stem cell activation, differentiation experiments and cognitive function regulation experiments.

Disclosure of Invention

The primary object of the present invention is to provide the performance and efficacy of artesunate in the treatment of hereditary intellectual and cognitive dysfunction diseases, especially the novel use of artesunate in the preparation of medicines for fragile X syndrome, which aims at the problems of the prior art.

In order to achieve the above purpose, the invention provides an application of artesunate in preparing medicines for treating hereditary mental and cognitive dysfunction diseases.

Wherein the genetic intellectual and cognitive dysfunction disease is autism, fragile X syndrome or cognitive dysfunction related to the neural developmental disorder disease, preferably fragile X syndrome.

In particular, the medicament consists of artesunate and a pharmaceutically acceptable carrier.

The artesunate purity is more than or equal to 60%, preferably more than 90%, and more preferably more than 95%.

The pharmaceutically acceptable carrier is generally approved for this purpose and as an inactive ingredient of the medicament. A compilation of relevant pharmaceutically acceptable carriers can be found in the handbook of pharmaceutical excipients (Handbook of Pharmaceutical excipients, 2 nd edition, edited by A. Wade and P.J. Weller; american Pharmaceutical Association publication Washington and The Pharmaceutical Press, london, 1994) et al.

The artesunate can be used singly or in the form of a pharmaceutical composition containing the artesunate when being used for treating fragile X syndrome.

The medicaments of the present invention are administered by the gastrointestinal route or/and by the parenteral route.

The parenteral route of administration is selected from the group consisting of injection, respiratory tract, skin, mucous membrane or luminal.

Parenteral formulations are selected from the group consisting of injections, sprays, aerosols, patches, and the like; the gastrointestinal tract administration preparation is selected from tablet, capsule, powder, granule, pill, solution, emulsion or syrup, etc.

The invention provides a medicine and a corresponding pharmaceutical dosage form for treating fragile X syndrome, which take artesunate as an active ingredient.

Wherein the medicament is in the form of an oral preparation, an injection or a topical preparation.

In particular, the oral formulation includes tablets, capsules, pills, powders, granules, syrups or solutions; the injection comprises an injection formulation or a freeze-dried powder injection formulation for injection; topical formulations include creams, ointments, sprays, aerosols or patches.

Wherein, the pharmaceutical preparation takes artesunate as an effective active ingredient and comprises other carrier components acceptable in pharmacy.

The carrier in the medicine comprises excipient such as starch, water, etc.; lubricants such as magnesium stearate and the like; disintegrants such as microcrystalline cellulose and the like; fillers such as lactose and the like; binders such as pregelatinized starch, dextrin, etc.; a sweetener; an antioxidant; a preservative; a flavoring agent; perfume, etc.

The carrier which can be selected in the preparation of the oral preparation can be starch, dextrin or cyclodextrin, various chemically modified cyclodextrin, sucrose, stearate and other conventional pharmaceutical auxiliary materials. The freeze-dried powder injection can be prepared by methods such as aseptic spray drying, low-temperature vacuum drying, freeze drying and the like. The post-preparation process and equipment of each preparation belong to the conventional technology in the pharmaceutical field, and the invention is not limited to the technology.

The medicine in the invention is in the form of tablets, capsules, pills, powder, granules, syrup, solution, injection, spray, aerosol, patch, gel and cataplasm, namely the medicine preparation comprises the forms of tablets, capsules, pills, powder, granules, syrup, solution, injection, spray, aerosol, patch, gel, cataplasm and the like, but is not limited to the forms.

Compared with the prior art, the invention has the following advantages and benefits:

1. the invention develops a new medicinal value for the artesunate which is a known compound, can be used for treating fragile X syndrome, can be prepared into a medicament for treating fragile X syndrome, and opens up a new application field for the application of medicinal materials of the artesunate.

2. In the invention, artesunate is used for in vitro neural stem cell test, and the result shows that: artesunate significantly inhibits proliferation of Fmr1 KO mouse somatic neural stem cells in vitro, while having no effect on proliferation of WT mouse somatic neural stem cells; artesunate significantly promoted neuronal differentiation of Fmr1 KO mouse adult neural stem cells in vitro, while it had no effect on the differentiation of WT mouse adult neural stem cells.

3. According to the invention, a large number of animal experiments are carried out by adopting different animal models through injection ways, and the experimental results show that: artesunate significantly inhibits abnormal activation of adult neural stem cells in Fmr1 KO mice; promote the maturation of the adult neural stem cell neogenesis neurons in Fmr KO mice.

Artesunate inhibits abnormal proliferation of neural stem cells at the Dentate Gyrus (DG) of the hippocampus caused by FMRP deficiency, promotes differentiation of the neural stem cells to neurons, relieves the damage of hippocampal-dependent learning, and corrects cognitive dysfunction caused by FMRP deficiency.

4. The artesunate has strong pharmacological action, obvious efficacy for treating fragile X syndrome, quick response, small toxic and side effects and good safety, can be taken for a long time, and has good medicinal prospect.

5. The product of the invention has the advantages of abundant raw material sources, low price, safe clinical use, simple preparation process, small dosage and convenient use, can be prepared into various dosage forms, and is easy to popularize.

6. It is well known that one of the most complex and critical systems in the human body is the nervous system, so the pathophysiological mechanisms of nervous system diseases are often complex and the pathogenesis is diverse. The invention can not only prepare the medicine for treating fragile X syndrome by adopting the active ingredient of artesunate with single ingredient, but also prepare the compound medicine for treating fragile X syndrome by using artesunate and other active ingredients together.

The Artesunate (AS) is used for treating FXS, and has low toxicity and small side effect, and high safety for treating FXS; AS also has reliable pharmacokinetic characteristics, good water solubility, easy absorption, low ionization degree of AS in acidic body fluid, and can be dissolved in weak alkaline solution, and in clinical application, AS is prepared into various dosage forms such AS tablets, suppositories, injection and the like to fully exert the efficacy. AS can be maintained at a high concentration in the brain, and even though the drug level of AS in each tissue is significantly reduced one hour after administration, the proportion thereof in the brain, fat, intestine and serum is still high, suggesting that AS may be excellent in treating neurological diseases.

Drawings

FIG. 1A is a diagram of an experimental scheme for regulating abnormal activation of adult mouse brain adult neural stem cells by artesunate;

FIG. 1B shows adult WT/Fmr1 KO Nestin-GFP mouse DG region-activated adult neural stem cells (GFP ⁺ GFAP ⁺ MCM2 ⁺ ) Representative micrograph, blue: DAPI; green: GFP; red: MCM2; grey: GFAP; scale bar: 20 μm;

FIG. 1C is a graph of the results of analysis of activated neural stem cell data from a neural stem cell activation experiment;

FIG. 1D is a diagram of an experimental scheme of artesunate for regulating the differentiation of neonatal neurons of brain neural stem cells of adult mice;

FIG. 1E shows the WT/Fmr1 KO mouse DG region neonatal mature neuron (NeuN) ⁺ BrdU ⁺ ) Is a representative micrograph of (2); the effect of artesunate on neuronal differentiation in Fmr KO mice was assessed by immunostaining with the neuronal markers NeuN (green) and BrdU (red); scale bar: 20 μm;

FIG. 1F is a graph of the results of analysis of neonatal mature neuronal data from neural stem cell differentiation experiments;

FIG. 2A is an in vitro proliferation microscope view of neural stem cells;

FIG. 2B is a statistical graph of neural stem cell proliferation in vitro;

FIG. 2C is an in vitro differentiation microscope view of neural stem cells;

FIG. 2D is a statistical graph of neural stem cell differentiation in vitro;

FIG. 3A is a schematic diagram of a new mouse position identification experiment;

FIG. 3B is a statistical graph of the discrimination indexes of the new position identification experiment of the mice;

FIG. 4A is a schematic diagram of a mouse model separation experiment;

FIG. 4B is a graph showing the discrimination index statistics of a mouse model separation experiment;

FIG. 5A is a schematic diagram of a new object recognition experiment of a mouse;

FIG. 5B is a statistical graph of discrimination indexes of a new object recognition experiment of a mouse;

FIG. 6A is a schematic diagram of a social interaction experiment of mice;

fig. 6B is a graph of the discrimination index statistics of the social interaction experiment of mice.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

Experimental example 1 in vivo neural stem cell activation and differentiation experiments

1. Experimental materials

1. Experimental animal

Male Wild type (Wild-type, WT, or Fmr1 ^+/y ) Mice, purchased from the institute of medical laboratory animals, national academy of medical science.

Female Fmr Gene knockout (K) nock out, KO) mice (Fmr KO, or Fmr1 ^-/- ) Male mice labeled with nestin Green Fluorescent Protein (GFP) were purchased from Jackson laboratories.

Fmr1 Gene Knockout (KO) can be represented by Fmr KO or Fmr1 ^-/- (female mouse)/Fmr 1 ^-/y (Male mouse). Male Wild Type (WT), which may be represented by WT or Fmr1 ^+/+ (female mouse)/Fmr 1 ^+/y (Male mouse)

All mice were bred in the Tianjin university of traditional Chinese medicine laboratory animal center, with no specific pathogen grade (SPF grade) animal breeding room, at 22-25deg.C, with 12 hours illumination/12 hours dark circadian cycle, and were free to take water and food, approved and accepted by Tianjin university of traditional Chinese medicine animal ethics committee. As previously described, fragile X syndrome occurs mainly due to mutation of Fmr1 gene on the X chromosome, resulting in loss of function of FMRP. In order to avoid deviation of experimental data and conclusion caused by different expression of Fmr1 genes on the physiological cycle and double X chromosomes of female mice, only adult wild type male mice and Fmr1 gene defective male mice are selected as experimental samples in the study.

2. Experimental medicine

Artesunate (AS) 88495-63-0 Pichia pharmaceutical Co

5-Bromodeoxyuridine (BrdU) 27847 MCE (MedChemexpress) Inc

2. Experimental method

1. Experimental mouse breeding method

1-1, mouse propagation for neural Stem cell activation experiments

Male mice with nestin Green Fluorescent Protein (GFP) markers (Fmr 1) ^+/y ) With Fmr HET female mouse (Fmr 1) ^+/- ) Mating is carried out, and the following genotype mice can be bred and respectively marked as follows: (1) fmr1 ^+/y Nestin-GFP (Male); (2) fmr1 ^-/y Nestin-GFP (Male); (3) fmr1 ^-/+ Nestin-GFP (female); (4) fmr1 ^+/+ Nestin-GFP (female).

Male mice labeled (1) and (2) in offspring were selected for administration 7-8 weeks later for subsequent cell activation experimental study.

1-2, mouse propagation for neural Stem cell differentiation experiments

Male mice (Fmr 1) ^+/y ) With Fmr HET female mouse (Fmr 1) ^+/- ) Mating is carried out, and the following genotype mice can be bred and respectively marked as follows: (5) fmr1 ^+/y (male); (6) fmr1 ^-/y (male); (7) fmr1 ^-/+ (female); (8) fmr1 ^+/+ (female).

Male mice labeled (5) and (6) in offspring were selected for administration 7-8 weeks for subsequent cell differentiation experimental study.

2. Experimental grouping, administration and sampling

2-1, neural Stem cell activation experiments

Male mice labeled (1) and (2) after 7-8 weeks of feeding were divided into 4 groups of 3 mice each, specifically:

Group A: fmr1 ^+/y Nestin-GFP+ physiological saline group; (WT-gfp+veh); group B: fmr1 ^-/y Nestin-GFP+ physiological saline group; (KO-gfp+veh); group C: fmr1 ^+/y A Nestin-GFP+ artesunate group; (WT-gfp+as); group D: fmr1 ^-/y A Nestin-GFP+ artesunate group; (KO-GFP+AS).

Each group of mice was administered by intraperitoneal injection, respectively, in which: the dosage of artesunate is 20mg/kg; the volume of the physiological saline is calculated according to the administration concentration of artesunate, and the physiological saline with the same volume is administered; intraperitoneal administration was performed at the same time every day, 1 time per day for 14 days.

After 14 days of continuous administration of artesunate and physiological saline, mice were anesthetized on day 15, perfused to take brains, and then sectioned using a frozen microtome to obtain frozen sections for use in subsequent neural stem cell activation experiments, the experimental procedure being as shown in fig. 1A.

Performing immunohistochemical (frozen section) (IHC-Fr) staining treatment on brain frozen sections of four groups of mice, performing patch treatment according to the sequence from small sea horse to large sea horse, and sealing with anti-fluorescence quenching agent to obtain stained brain sections of the mice in each group; then using the neurosphereThe quantitative analysis method is that the stained brain slice is placed under a positive microscope, stereo Investigator neuro-stereoscopic software is opened, and a fluorescent channel is arranged for DG region in the stained brain slice: green-GFP ⁺ Gray-GFAP ⁺ Red-MCM 2 ⁺ blue-DAPI, scanning according to brain slice sequence on glass slide, after scanning, determining GFP by fluorescent staining of different channels ⁺ GFAP ⁺ MCM2 ⁺ The role of blue-DAPI, cell count, was to determine the location and status of cells, and positive targets were quantified on stained mouse brain sections.

Brain sections were scanned under a microscope, and representative pictures of neural stem cell activation are shown in fig. 1B, which shows blue: DAPI; green: GFP; red: MCM2; grey: GFAP; scale bar: 20 μm.

2-2, differentiation experiment of neural Stem cells

Male mice labeled (5) and (6) after 7-8 weeks of feeding were divided into 4 groups of 3 mice each, specifically:

group E: fmr1 ^+/y +saline group; (wt+vehiche); group F: fmr1 ^-/y +saline group; (KO+Vehiche); group G: fmr1 ^+/y +artesunate group; (wt+as); group H: fmr1 ^-/y +artesunate group; (KO+AS).

Each group of mice was administered by intraperitoneal injection, respectively, in which: the dosage of artesunate is 20mg/kg; the volume of the physiological saline is calculated according to the administration concentration of artesunate, and the physiological saline with the same volume is administered; intraperitoneal administration was performed at the same time every day, 1 time per day for 14 days.

The artesunate and the normal saline are injected and administrated intraperitoneally at the same time of each day for 14 days for 1 time/day; each mouse was given an intraperitoneal injection of BrdU on days 7 and 8, respectively, of artesunate and physiological saline for 2 times/day for two days at a BrdU dose of 200mg/kg.

Artesunate was continuously administered for 14 days, and BrdU administration at intermediate 7 and 8 days was completed, mice were anesthetized after waiting four weeks, brains were perfused, the entire rat brain hippocampus area was continuously sectioned at a thickness of 40 μm, and the hippocampus was systematically randomly sampled with 1 slice selected for every 6 slices. For the subsequent neural stem cell differentiation experiment, the experimental flow is shown in fig. 1D.

The metering range of the nerve cells in the dentate gyrus region of the hippocampus is from-0.94 mm to-2.30 mm wide on the dorsal side and from-2.30 mm to-3.80 mm wide on the ventral side relative to the pre-halogen. Performing immunohistochemical (frozen) staining (IHC-Fr) on the frozen brain sections of the mice in each experimental group, and sealing the sections by using an anti-fluorescence quenching agent to obtain stained brain sections of the mice in each experimental group; then using the quantitative analysis method of neurosurgery, namely placing the stained brain slice under a normal microscope, opening Stereo Investigator neurosurgery software, setting fluorescent channels, scanning DG areas in the stained brain slice according to the sequence of brain slices, and determining NeuN through fluorescent staining of different channels after the scanning is finished ⁺ And BrdU+ positive targets, blue-DAPI is used for determining the position of cells, and NeuN is used after scanning ⁺ And BrdU ⁺ Cell count, metering of positive targets on brain sections of mice that were stained, where green-NeuN ⁺ The method comprises the steps of carrying out a first treatment on the surface of the Red-BrdU ⁺ The method comprises the steps of carrying out a first treatment on the surface of the blue-DAPI.

Brain sections were scanned microscopically and mice were stained with DG region neonatal mature neurons (NeuN ⁺ BrdU ⁺ ) As shown in fig. 1E, DAPI in fig. 1E represents all cells, brdU labeled proliferating cells, neuN labeled mature neurons; green-NeuN ⁺ The method comprises the steps of carrying out a first treatment on the surface of the Red-BrdU ⁺ The method comprises the steps of carrying out a first treatment on the surface of the blue-DAPI.

3. Experimental results

1. Results of neural Stem cell activation experiments

GFP ⁺ And GFAP ⁺ Double positive cells represent neural stem cells, MCM2 ⁺ 、GFP ⁺ And GFAP ⁺ Triple positive cells represent activated neural stem cells and count MCM2 using Stereo Investigator neural stereology software ⁺ 、GFP ⁺ 、GFAP ⁺ Is a cell number of the cell line. MCM2 ⁺ 、GFP ⁺ And GFAP ⁺ Cell number and GFP of (F) ⁺ And GFAP ⁺ The ratio of the number of cells in the activated state (average of 3 times) is shown as follows:

group A: fmr1 ^+/y Stem cells (MCM 2) in activated form in the Nestin-GFP+ saline group ⁺ GPAP ⁺ GFP ⁺ /GFAP ⁺ GFP ⁺ ) The ratio is 12.56%; group B: fmr1 ^-/ Stem cells (MCM 2) in the activated state in the Nestin-GFP+ saline group ⁺ GPAP ⁺ GFP ⁺ /GFAP ⁺ GFP ⁺ ) The ratio is 21.84%; group C: fmr1 ^+/y Stem cells (MCM 2) in the active state in the Nestin-GFP+ artesunate group ⁺ GPAP ⁺ GFP ⁺ /GFAP ⁺ GFP ⁺ ) The ratio is 10.94%; group D: fmr1 ^-/y Stem cells (MCM 2) in the active state in the Nestin-GFP+ artesunate group ⁺ GPAP ⁺ GFP ⁺ /GFAP ⁺ GFP ⁺ ) The ratio was 11.57%.

The results of the neural stem cell activation assay data analysis are shown in figure 1C. Analysis of the above data shows that the proportion of activated stem cells in group B is high compared to that in group A, but that the proportion of activated stem cells in group D is almost restored to a level similar to that in group A immediately after artesunate administration. It is shown that artesunate significantly inhibits abnormal activation of adult neural stem cells in Fmr KO mice.

The fragile X syndrome model mice exhibit abnormal activation of adult neural stem cells, and the disease phenotype can be corrected after artesunate administration, so that: artesunate has certain therapeutic significance on fragile X syndrome and can be used for treating fragile X syndrome.

2. Neural stem cell differentiation test results

NeuN is a marker of mature stem cells, brdU marks neural stem cells in the dividing phase, i.e. marks stem cells in the proliferating state. NeuN ⁺ And BrdU ⁺ Double positive cells represent the new differentiation of neural stem cells into mature neurons. Counting NeuN using Stereo Investigator neuro-stereoscopic software ⁺ And BrdU ⁺ Is a cell number of the cell line. NeuN ⁺ And BrdU ⁺ Cell number and BrdU of (E) ⁺ The ratio of the cell numbers of the activated cells is shown as follows:

group E: fmr1 ^+/y Mature neonatal neurons in the +saline group (NeuN ⁺ BrdU ⁺ /BrdU ⁺ ) The ratio is 64%; group F: fmr1 ^-/y Mature neonatal neurons in the +saline group (NeuN ⁺ BrdU ⁺ /BrdU ⁺ ) The ratio is 44%; group G: fmr1 ^+/y Mature neonatal neurons in the +artesunate group (NeuN ⁺ BrdU ⁺ /BrdU ⁺ ) The ratio is 60%; group H: fmr1- ^/y Mature neonatal neurons in the +artesunate group (NeuN ⁺ BrdU ⁺ /BrdU ⁺ ) The ratio was 61%.

The results of the neural stem cell differentiation assay data analysis showed that, as shown in fig. 1F, the number of mature neoneurons in group F was smaller than that in group E, but it was seen that the ratio of the number of neoneurons in group H was almost restored to a similar level as in group E immediately after artesunate administration. It was demonstrated that artesunate promoted maturation of adult neural stem cell neogenesis neurons in Fmr KO mice.

The fragile X syndrome model mice show the phenomenon that the number of adult neural stem cells differentiated into mature neurons is reduced, so that the cognitive function of the fragile X syndrome model mice is affected, the disease phenotype can be corrected after artesunate is administrated, the number of the adult neural stem cells differentiated into mature neurons of the fragile X syndrome is improved, and therefore the artesunate has a certain therapeutic significance on the fragile X syndrome, and the artesunate can be used for treating the fragile X syndrome.

From the quantitative analysis results, it can be seen that: MCM2 ⁺ GPAP ⁺ GFP ⁺ /GPAP ⁺ GFP ⁺ The percentage shows that artesunate can reduce abnormal activation and proliferation of neural stem cells in Fmr KO mice according to NeuN ⁺ BrdU ⁺ /BrdU ⁺ The percentages show that artesunate promotes the differentiation and maturation of its neurons. From this, it can be seen that: artesunate can reduce the abnormality of Fmr KO mouse brain nerve stem cellsProliferation and neural differentiation are promoted, and the neurogenesis disorder of the Fmr KO mouse can be corrected.

As described above, FMRP depletion results in abnormal proliferation of DG region neural stem cells, but reduces their differentiation into neurons, and impairs hippocampal-dependent learning. After artesunate is administrated, the neural stem cells can be recovered to normal proliferation and differentiation, normal neurogenesis is promoted, and damage caused by FMRP deficiency is reduced.

Experimental example 2 neural stem cell proliferation and differentiation experiments

In vitro primary cultured Wild Type (WT) mice and Fmr gene-deficient (Fmr 1 KO) mice were quantitatively analyzed by immunofluorescent staining combined neurosphere visual quantitative analysis after cell administration. The results show that artesunate significantly reduces the abnormal proliferation of Fmr KO neural stem cells and promotes the differentiation into neurons.

1. Experimental materials

1. Cells

3 male Fmr KO mice and WT mice each of 8-10 weeks old were isolated under a split microscope for the mouse DG region, and then primary adult neural stem cells were cultured in a cell culture room at 37℃in incubator at 5% CO ₂ 。

2 types of stem cells, namely WT stem cells, fmr1 KO stem cells, were obtained, and 3 (i.e., n=3) of each type of stem cells were labeled WT1, WT2, WT3, respectively; KO1, KO2, KO3.

2. Reagent (as shown in Table 1)

TABLE 1 list of reagents for in vitro neural stem cell proliferation and differentiation assays

Neural cell basal medium: a Neural basal medium;

culture medium during stem cell culture: the culture medium of the biological basal medium +B27+L-Glu+diabody is added with growth factors before each cell replacement after the cell culture medium is prepared: EGF and FGF; wherein:

B-27 ^TM additive (50X), serum-free; the use concentration is as follows: 10mL of B27 was added to 500mL of Neurobasal;

penicillin/streptomycin diabody solution (diabody, gibco, 100X) using concentration: 8mL of the diabody was added to 500mL of Neurobasal containing 10000 units of penicillin (base) and 10000. Mu.g of streptomycin per mL;

glutamine L-Glutamine (Gibco, 200 mM), used at a concentration of: 8mL of L-glutamic acid was added to 500mL of Neurobasal.

2. Experimental method

First, preparation in the early stage

1. Experimental grouping and administration

Group A: wt+dmso, WT adult neural stem cells+0.005% DMSO; group B: ko+dmso, fmr1 KO adult neural stem cells+0.005% DMSO; group C: wt+as, WT adult neural stem cells+as at final concentration of 0.5 μm; group D: KO+AS, fmr1 KO-type adult neural stem cells+AS with a final concentration of 0.5. Mu.M.

Blank and dosing treatments were performed on two WTs and Fmr1 KO type adult neural stem cells, respectively, wherein: blank treatment (blank group) 0.005% DMSO, i.e., wt+dmso group, WT adult neural stem cells+0.005% DMSO was added to the medium; KO+DMSO group, fmr1 KO adult neural stem cells+0.005% DMSO; administration treatment (administration group) AS with a final concentration of 0.5. Mu.M, namely WT+AS group, WT type adult neural stem cells+AS with a final concentration of 0.5. Mu.M was added to the medium; KO+AS group, fmr1 KO-type adult neural stem cells+AS with a final concentration of 0.5. Mu.M.

2. Slide coating treatment

A cell slide with a diameter of 14mm was placed in a 24-well plate, and then a proper volume (200-300. Mu.l) of polyornithine (poly-l-orthonine) was added to coat overnight; after recovering polyornithine, adding a proper amount (200-300 microliter) of laminin (0.01068 mg/mL) into a 24-well plate containing a cell climbing plate, and coating again until laminin is recovered before cell plating; wherein, the laminin with the concentration of 0.01068mg/mL is prepared according to the following method: diluting 2.67mg/mL of laminin stock solution with sterilized ultrapure water for 250 times to obtain; the recovered poly-l-ornithine, laminin can be recycled.

When stem cells are subjected to proliferation and differentiation experiments, compound holes are needed, stability and authenticity of data are guaranteed, 2 compound holes are formed in each stem cell, and specific groups are shown in table 2, wherein the concentration of DMSO is 0.005%; AS concentration was 0.5. Mu.M.

TABLE 2 Stem cell experiment grouping

2 24-well plates were prepared according to the above method, one for proliferation assay and the other for differentiation assay.

3. Stem cell digestion and enumeration process

3A) Numbering WT1, WT2, WT3; the stem cells of KO1, KO2 and KO3 respectively absorb 8mL of adult neural stem cell suspension from the corresponding culture dishes, respectively transfer the suspension into the corresponding centrifuge tubes (15 mL), centrifuge (3 min,1200 r) and discard the supernatant, and leave cell precipitates;

since the adult neural stem cells are suspension cells, it is necessary to collect the cell-containing medium in a 15mL centrifuge tube at 1200rpm/3min, discard the supernatant, leave the cell pellet, and perform the operation of 3B).

3B) 1 XTrypsin (Trypsin solution, 1 mL) was added to each centrifuge tube at a concentration of 0.05% and incubated for 1-2min;

3C) After incubation, defined Trypsin Inhibitor (well-defined trypsin inhibitor, 1 mL) was added to each centrifuge tube, and the tubes were blown 20-30 times and centrifuged (2 min,1200 rpm); sucking and discarding supernatant fluid, and keeping sediment;

3D) And respectively adding a stem cell culture medium (a biological basal culture medium+B27+L-Glu+diabody+EGF+FGF, 1mL, wherein the final concentration of EGF and FGF is 10 ng/mL) into each centrifuge tube, blowing uniformly, filtering by using a 40-micrometer screen after stem cell resuspension, beating all 1mL of stem cell suspension on the screen, and filtering into a 50-mL centrifuge tube, so that the adult stem cells are in a single cell state, and obtaining the adult stem cell suspension in a single cell state.

3E) Taking 6 EP tubes (1.5 mL), respectively adding 90 microliters of the neural basal culture medium into each EP tube, respectively accurately sucking 10 microliters of the adult stem cell suspension in the single cell state after filtering in the step 3D), adding into the corresponding EP tube, uniformly mixing, respectively sucking 10 microliters of the adult stem cell suspension from each EP tube, respectively dripping on a blood cell counting plate, and observing and counting under a microscope.

The purpose of cell counting is to keep the number of cells per compound well consistent for each group, WT1/WT2/WT3, while the cell plates are being seeded; each cell of KO1/KO2/KO3 was counted separately and averaged to separate plates; wherein the counting results are (respectively):

WT1：16*10 ⁵ ；WT2：16.75*10 ⁵ ；WT3；17.25*10 ⁵ ；

KO1：24.5*10 ⁵ ；KO2：20.7*10 ⁵ ；KO3：21.5*10 ⁵ 。

(II) proliferation treatment

1. Adult stem cells in single cell state after digestion and counting in the preliminary preparation step are prepared according to 1 x 10 ⁵ Is added to a 24-well plate containing a coating slide, and the number of stem cells in one well of the 24-well plate is 100000 (1×10) ⁵ ) The volume of the culture medium in each hole is 1mL, and the culture is carried out in a cell incubator for 18h;

2. after 18h of incubation, 500. Mu.l of medium in each well of a 24-well plate was aspirated, and 500. Mu.l of fresh culture broth containing BrdU (concentration: 5. Mu.M) was added; growth factor required 2 times (final concentration 20 ng/mL); based on this medium, blank: maintaining 0.005% dmso; drug administration group: artesunate (AS) was 0.5. Mu.M final concentration, then incubated for 6h in incubator.

3. Cellular immunofluorescence

3A, taking out the 24-pore plate from the cell culture room after 6h of incubation, sucking out 0.5mL of culture medium from each pore, adding 4% paraformaldehyde (0.5 mL) into each pore, standing at normal temperature, sucking out all liquid in the pore after 20min, adding 1mL of 4% paraformaldehyde into each pore, and standing at normal temperature for 15min; then the liquid in the hole is sucked away and washed by PBS for 5 min/time for three times;

3B, adding HCl (1M) preheated to 37 ℃ into a 24-well plate, 1 mL/well; then placing the mixture into an incubator, standing the mixture (at 37 ℃ for 30 min), and performing acid permeation treatment; then adding borate buffer (pH8.5, 2 mL), standing at room temperature for 15min, sucking out the liquid in each well, and washing with PBS for 3 times and 5 min/time;

Purpose of HCL acid permeation: brdU antibodies recognize single-stranded DNA, so that the double-stranded DNA needs to be first disentangled prior to antibody incubation. The acid denaturation or thermal denaturation effect of the hydrochloric acid is best;

preparation and incubation of 3C and antibodies

3C-1) preparing an antibody diluent: 1 XTBS was added to 0.5g BSA,2mL Trionx-100 and 2mL goat serum until 40mL was dissolved, and stored at-20 ℃;

3C-2) primary antibody, preparing BrdU antibody: preparing BrdU antibodies using the antibody dilutions prepared in step 3C-1), and reacting the primary anti-BrdU according to 1:4000 (typically 1:3000-1:5000) was added to the well plate after acid permeation treatment in step 3B) and washed with PBS, 300 μl was added to each well, and incubated overnight at room temperature; then washing with PBS for 5 min/time for three times;

3C-3) secondary antibody, alexa Fluor 568 coat anti-rate lgG (H+L), preparing a fluorescent secondary antibody (1:2000) by using the antibody diluent of the step 3C-1), adding the fluorescent secondary antibody into a pore plate which is treated by the primary antibody of the step 3C-2) and washed by PBS, adding 300 microliters into each pore, and incubating for 2 hours at normal temperature to obtain the fluorescent secondary antibody;

3C-4) DAPI solution was incubated with the second fluorescent antibody for the last 15min of incubation time, or after waiting for the second fluorescent antibody to end of incubation time, 500. Mu.l DAPI solution was added to each well after the second fluorescent antibody was blotted dry.

( After incubation of the fluorescent secondary antibody, DAPI needs to be incubated: 4', 6-diamidino-2-phenylindole, a fluorescent dye capable of binding DNA strongly, is commonly used for fluorescent microscopy observation and shows blue fluorescence )

After the incubation of the 3C-5) DAPI solution is finished, PBS is used for washing for 5 min/time, and the DAPI solution is removed three times;

4. sealing, fixing, microscopic examination and counting

Taking a common glass slide (long), respectively dripping 15 microliters (usually 10-20 microliters) of anti-fluorescence quenching agent at two different positions on the common glass slide, taking out the cell climbing sheet incubated in the step 3) from a 24-hole plate, fixing the cell climbing sheet with the cell facing downwards on the glass slide, observing the glass slide under a normal microscope, and counting; wherein: fluorescence of the microscope is adjusted to a red channel, and cells with red fluorescence, which are BrdU marked and represent the number of proliferated cells, are observed and counted; fluorescence of the microscope is adjusted to a DAPI channel, and blue fluorescence cells are observed and counted, and all cells are marked by blue fluorescence DAPI; the BrdU and DAPI quantities were calculated separately, and the ratio BrdU/DAPI was calculated.

Microscopic observations of neural stem cell proliferation in vitro are shown in fig. 2A, wherein BrdU-labeled proliferating cells appear red; DAPI marks all nuclei blue in the figure: "-" means that only 0.005% DMSO was added to the solution, and no drug AS was added; "+" indicates that AS was added at a final AS concentration of 0.5. Mu.M.

5. Data analysis and results

The analysis was performed using the neurospectoral software Stereo Investigator, the 3 analysis data for each group were averaged, and the statistical analysis results were shown in fig. 2B.

Group A: proliferation of adult neural stem cells in the wt+dmso group (BrdU ⁺ /BrdU ⁺ DAPI ⁺ ) The ratio is 31.40%; group B: proliferation of adult neural stem cells in KO+DMSO group (BrdU ⁺ /BrdU ⁺ DAPI ⁺ ) The ratio is 55.91%; group C: in the group of WT+Artesunate (WT+AS) in adult neural stem cell proliferation (BrdU) ⁺ /BrdU ⁺ DAPI ⁺ ) The ratio is 33.07%; group D: adult neural stem cell proliferation (BrdU) in KO+Artesunate (KO+AS) group ⁺ /BrdU ⁺ DAPI ⁺ ) The ratio is 31.61%;

BrdU ⁺ the new stem cells are labeled and the new stem cells are labeled,BrdU ⁺ /DAPI ⁺ representing the duty cycle of the novacells. From the above data, artesunate significantly inhibited proliferation of adult neural stem cells in vitro in Fmr KO mice. And the proliferation level of Fmr KO type adult neural stem cells is reduced to be similar to that of WT, and artesunate has no influence on the proliferation of WT mouse in-vitro adult neural stem cells, so that artesunate can be used for treating diseases caused by mutation of fragile X mental retardation gene (Fmr 1) in the formation process of X chromosome.

(III) differentiation treatment

1. Adult stem cells in single cell state after digestion and counting in the preliminary preparation step were prepared according to 2×10 ⁵ Is added to a 24-well plate containing a coating slide, and when the cells are seeded, the number of stem cells in one well in the 24-well plate is 200000 (2 x 10) ⁵ ) The volume of the culture medium in each hole is 2mL, and the culture is carried out in a cell incubator for 18h;

2. after 18h of incubation, 500. Mu.l of the medium in each well of the 24-well plate was aspirated, and 500. Mu.l of fresh medium containing forskolin (final concentration 5. Mu.M) was added; tretinoin (final concentration 1 μm); blank group: maintaining 0.005% dmso; drug administration group: artesunate was kept at a final concentration of 0.5 μm, then incubated in incubator for 3 days, and liquid exchange and administration were performed three consecutive days.

3. Cellular immunofluorescence

3A, taking out the incubated 24-well plate from the cell culture room, sucking out 0.5mL of culture medium from each well, keeping 0.5mL of culture medium in each well, adding 4% paraformaldehyde (0.5 mL) into each well, standing at normal temperature, sucking out all liquid in the well after 20min, adding 1mL of 4% paraformaldehyde into each well, and standing at normal temperature for 15min; then sucking the liquid in the hole, washing with PBS for 5 min/time for three times;

preparation and incubation of 3B antibodies

3B-1) preparing an antibody dilution: the same as in step 3C-1) of "second, proliferation treatment";

3B-2) primary antibody, formulated as Tuj1 antibody: preparing Tuj1 antibody by using the antibody diluent prepared in the step 3B-1), adding a primary antibody Tuj1 into the pore plate treated in the step 3A) according to a ratio of 1:3000, adding 300 microliters into each pore, and incubating overnight at normal temperature; then washing with PBS for 5 min/time for three times;

3B-3) a secondary antibody, alexa Fluor 568 coat anti-rate lgG (H+L), preparing a fluorescent secondary antibody (1:2000) by using the antibody diluent prepared in the step 3B-1), then adding 300 microliters into a pore plate, and incubating for 2 hours at normal temperature to obtain the fluorescent secondary antibody;

3B-4) DAPI solution may be incubated with the last 15min of incubation time for the fluorescent secondary antibody or after waiting for the end of incubation time for the fluorescent secondary antibody, 500 μl of DAPI solution may be added to each well after the secondary antibody is blotted clean.

( After incubation of the fluorescent secondary antibody, DAPI needs to be incubated: 4', 6-diamidino-2-phenylindole, a fluorescent dye capable of binding DNA strongly, is commonly used for fluorescent microscopy observation and shows blue fluorescence )

After the incubation of the 3B-5 and DAPI solutions is finished, the solution is washed by PBS for 5 min/time for three times;

4. sealing, fixing, microscopic examination and counting

Taking a common glass slide (long), respectively dripping 15 microliters (usually 10-20 microliters) of anti-fluorescence quenching agent at two different positions on the common glass slide, taking out the cell climbing sheet incubated in the step 2) from a 24-hole plate, fixing the cell climbing sheet with the cell facing downwards on the glass slide, observing the glass slide under a normal microscope, and counting; fluorescence of the microscope is adjusted to a red channel, and cells with red fluorescence, which are the number of cells marked by Tuj1 and representing differentiated neural stem cells, are observed and counted; fluorescence of the microscope is adjusted to a DAPI channel, and blue fluorescence cells are observed and counted, and all cells are marked by blue fluorescence DAPI; tuj1 and DAPI quantities are calculated separately, and the ratio of Tuj1/DAPI is calculated.

Microscopic observations after neural stem cell differentiation are shown in fig. 2C, wherein Tuj 1-labeled differentiated cells appear red; DAPI marks all nuclei blue in the figure: "-" means that only 0.005% DMSO was added to the solution, and no drug AS was added; "+" indicates that AS was added at a final AS concentration of 0.5. Mu.M.

5. Data analysis and results

The analysis was performed using the neurospectoral software Stereo Investigator, the 3 analysis data for each group were averaged, and the statistical analysis results were shown in fig. 2D.

Group A: in the wt+dmso group, adult neural stem cell differentiation (Tuj 1 ⁺ /Tuj1 ⁺ DAPI ⁺ ) The ratio is 13.96%; group B: differentiation of adult neural stem cells in KO+DMSO group (Tuj 1 ⁺ /Tuj1 ⁺ DAPI ⁺ ) The ratio is 4.32%; group C: in the wt+as group, adult neural stem cell differentiation (Tuj 1 ⁺ /Tuj1 ⁺ DAPI ⁺ ) The ratio is 12.71%; group D: differentiation of adult neural stem cells in KO+AS group (Tuj 1 ⁺ /Tuj1 ⁺ DAPI ⁺ ) The duty cycle is 13.74.

Tuj1 ⁺ Labeling neurons, tuj1 ⁺ /DAPI ⁺ Representing the duty cycle of the conversion from neural stem cells to neurons. From the above data, it can be seen that artesunate significantly promoted neuronal differentiation of the adult neural stem cells in vitro in Fmr KO mice. And the differentiation level of the neural stem cells of Fmr KO is increased to be similar to that of WT, and artesunate has no influence on the differentiation of the adult neural stem cells of the WT mouse in vitro, so that the artesunate can be used for treating diseases caused by mutation of fragile X mental retardation gene (Fmr 1) in the formation process of X chromosome.

Test example 3 cognitive function control test

Animal cognitive function detection method proves that artesunate can correct cognitive dysfunction and social interaction dysfunction of Fmr gene-deficient mice (Fmr 1 KO). To further investigate whether artesunate's improving effect on cognitive dysfunction functions by promoting adult neurogenesis, the present invention was intervened using the neurogenesis specific blocker Temozolomide (TMZ). The results indicate that temozolomide can block the therapeutic effect of artesunate on Fmr gene-deficient mice (Fmr 1 KO) for cognitive dysfunction. The research result shows that artesunate can treat cognitive dysfunction of fragile X syndrome mice by regulating neurogenesis.

1. New position identification experiment

The new position identification experiment is a classical behavioural for testing the spatial memory capacity of mice. In order to explore whether artesunate can correct the spatial memory capacity of fragile X syndrome model mice, dry prognosis of mice was administered by intraperitoneal injection of artesunate, and a new position identification experimental test was performed. To further investigate whether artesunate functions by promoting adult neurogenesis, neurogenesis was blocked in advance using Temozolomide (TMZ), a neurogenesis specific blocker, and administered in combination with artesunate, followed by a new position recognition experimental test.

1 materials and methods

1.1 materials:

a square open field: object A, object B, facial tissue, 75% alcohol, two timers.

1.2 experimental animals, drugs:

fmr1 KO and WT mice 7-8 weeks old, fmr KO and WT mice 6 weeks old.

Artesunate (AS), purity not less than 99%, purchased from Pichia medical CAS:88495-63-0;

temozolomide (Temozolomide; TMZ), available from selectk, CAS No.85622-93-1.

2 experimental group and administration

2.1 experimental grouping: wt+vehicle: WT mice were intraperitoneally injected with physiological saline; ko+vehicle: fmr1 KO mice were intraperitoneally injected with physiological saline; wt+as: WT mice were injected intraperitoneally with artesunate; ko+as: fmr1 KO mice were injected intraperitoneally with artesunate; wt+as/TMZ: WT mice were intraperitoneally injected with temozolomide and artesunate; KO+AS/TMZ: fmr1 KO mice were intraperitoneally injected with temozolomide and artesunate in 6 groups of 7-10 (i.e., n=7-10).

2.2 modes of administration:

(1) the method comprises the following steps Wt+vehicle, ko+vehicle group: the 7-8 week old Fmr KO and WT mice were intraperitoneally injected with physiological saline once daily for 14 consecutive days.

(2) The method comprises the following steps Wt+as, ko+as group: the artesunate is injected into the abdominal cavity of the mice of Fmr KO and WT of 7-8 weeks old once daily for 14 days, and the administration dosage is 20mg/kg.

(3) The method comprises the following steps wt+as/TMZ, ko+as/TMZ group: fmr1 KO and WT mice of 6 weeks of age were intraperitoneally injected with Temozolomide (TMZ) once daily for the first three days of weeks 6, 7, 8 and 9, and Artesunate (AS) once daily for the 8 and 9 weeks, for 14 consecutive days, with AS administered at a dose of 20mg/kg.

3 experimental procedure

The test measures the spatial memory of rodents by assessing their ability to identify new locations of familiar objects based on spatial cues.

First, the laboratory animal is taken to a behavioural test laboratory, adapted for at least 1 hour. During the training phase, each animal was placed individually in a square field, two identical objects were placed at the same distance from one side with colored wallpaper, allowing the mice to explore freely for 6min, and the training process was repeated 3 times. During the test phase, the position of one of the already familiar objects is moved to another new position. And recording the exploration time of the experimental animal on the objects at the new and old positions within 6 min. A normal animal should take more time to explore a new location of an object. The criterion is calculated from the difference between the percentage of time spent identifying the newly located object and the time spent identifying the in-situ located object. In the test phase, detection of an object is defined as any exploratory behavior of the animal's nose within 1cm of the object, including head direction, climbing, sniffing, etc. The method for calculating the discrimination index comprises the following steps: discrimination index= (new position object recognition time/total recognition time×100) - (home position object recognition time/total recognition time×100).

The new position identification experiment is shown in fig. 3A, and after the test of each mouse is finished, the field needs to be wiped clean with 75% alcohol to eliminate the influence of the similar odor interference on the test behavior of the next experimental mouse.

4 experimental results

Data were analyzed by Two-way ANOVA using GrapaPad Prism software, data processing results were expressed as mean±sem, and significant level differences were considered statistically significant when P < 0.05.

Wt+vehicle; KO+Vehicle; wt+as; ko+as; wt+as/TMZ; the discrimination indexes of the KO+AS/TMZ group are AS follows: 40.19%; -30.00%;47.50%;50.94%; -11.48%; -19.53%. The statistics of the mouse identification experiment for the new location are shown in fig. 3B.

From the experimental results, it can be seen that: WT mice tended to explore new objects while Fmr KO mice tended to explore old objects, indicating that Fmr KO mice had a defective spatial memory capacity, and artesunate could correct this defect after administration, while TMZ could block the spatial memory improvement effect of artesunate on Fmr KO mice. It was demonstrated that artesunate can improve spatial memory in Fmr KO mice by modulating neurogenesis.

2. Mode separation experiment

The pattern separation experiment is a superior way to explore the ability of mice to differentiate between similar events. The experiment will investigate whether artesunate can correct the pattern separation defect of fragile X syndrome model mice by promoting neurogenesis.

1 materials and methods

1.1 materials: a square open field; objects a (2); objects B (2), object a being different from object B; facial tissues; 75% alcohol; two timers.

1.2 experimental animals, drugs: the method is the same as that of experimental animals and medicines for' one and new position identification experiments

2 experimental group and administration

2.1 experimental grouping: wt+vehicle: WT mice were intraperitoneally injected with physiological saline; ko+vehicle: fmr1 KO mice were intraperitoneally injected with physiological saline; wt+as: WT mice were injected intraperitoneally with artesunate; ko+as: fmr1 KO mice were injected intraperitoneally with artesunate; wt+as/TMZ: WT mice were intraperitoneally injected with temozolomide and artesunate; KO+AS/TMZ: fmr1 KO mice were intraperitoneally injected with temozolomide and artesunate in 6 groups of 8-10 (i.e., n=8-10).

2.2 modes of administration: the same drug administration mode as that of the first and the new position identification experiments

3 experimental procedure

During training, the numbered mice were placed in an empty field for training for a period of time, then placed individually in the first square field, with a specific floor pattern and two identical objects (a objects), and allowed to explore for 10min, as shown in fig. 4A. After 30 minutes, the same animal was placed in a second square field, with a different floor pattern and two completely different sets of objects (B objects) than the first experiment, and allowed to explore for 10 minutes.

In the test, one of the training wallpaper is randomly taken as a background, an A object and a B object are respectively placed at the same position in the test, the interaction condition of each mouse on the A object and the B object in the scene is recorded by adopting a video recorder, the test time is 10min, the interaction condition of the mice is counted and analyzed by a timer, and in the test stage, the detection of the object is defined as any exploring behavior of the nose of the animal within 1cm from the object, including head direction, climbing, sniffing and the like.

Each mouse was cleaned with purified water after the end of the test so as not to affect the experimental results. And calculating a discrimination index according to a formula, wherein the discrimination index is = (new object detection time/total detection time) - (old object detection time/total detection time) multiplied by 100%, and judging the preference of the mouse to the new object and the old object.

4 experimental results

Data were analyzed by Two-way ANOVA using GrapaPad Prism software, data processing results were expressed as mean±sem, and significant level differences were considered statistically significant when P < 0.05.

Wt+vehicle; KO+Vehicle; wt+as; ko+as; wt+as/TMZ; the discrimination indexes of the KO+AS/TMZ group are AS follows: 24.91%; -10.81%;24.73%;32.65%; -7.85%; -11.10%. The statistical graph of the results of the mouse model separation experiment is shown in fig. 4B.

Experimental results showed that WT mice were more prone to exploring new objects, while Fmr KO performed in contrast to WT mice, indicating that the pattern separation of Fmr KO mice was defective and that artesunate could correct this defect after administration. And TMZ can block the mode separation improving effect of artesunate on Fmr KO mice. It was demonstrated that artesunate can improve pattern isolation defects in Fmr KO mice by modulating neurogenesis.

From the above experiments, it can be seen that: artesunate can improve the pattern separation defect of fragile X syndrome model mice by regulating adult neurogenesis.

Three new object identification experiment

This test is based on the natural tendency of rodents to like to explore novel objects rather than familiar ones. The experiment detects the new object recognition capability of the mice, and is used for exploring whether artesunate can correct learning and memory disorder of fragile X syndrome model mice by regulating and controlling adult neurogenesis.

1 Experimental materials

1.1 materials: a square open field; an object A; an object B; facial tissues. 75% alcohol; two timers.

1.2 experimental animals, drugs: the method is the same as that of experimental animals and medicines for' one and new position identification experiments

2 experimental group and administration

2.1 experimental grouping: wt+vehicle: WT mice were intraperitoneally injected with physiological saline; ko+vehicle: fmr1 KO mice were intraperitoneally injected with physiological saline; wt+as: WT mice were injected intraperitoneally with artesunate; ko+as: fmr1 KO mice were injected intraperitoneally with artesunate; wt+as/TMZ: WT mice were intraperitoneally injected with temozolomide and artesunate; KO+AS/TMZ: fmr1 KO mice were intraperitoneally injected with temozolomide and artesunate, 9-11 per group (i.e., n=9-11).

2.2 modes of administration: the same drug administration mode as that of the first and the new position identification experiments

3 experimental procedure

The test measures the new object recognition capabilities of rodents by preferentially exploring for strange objects rather than familiar objects. New object recognition is a form of memory task that does not rely on spatial cues. In this task, animals are trained to recognize specific objects.

As in fig. 5A, first, the laboratory animal is taken to the behavioural test laboratory, for at least 1 hour. During the training phase, each animal was placed individually in an L-shaped field with the same corridor length, and two identical objects were placed at the ends of the two corridor sides and allowed to explore for 10min. In the test stage, one of the familiar objects is replaced by a new object, and the exploration time of the experimental animal on the new object and the old object is recorded within 10 minutes. A normal animal should take more time to explore strange objects. The discriminant criteria is calculated from the difference between the percentage of time spent identifying strange objects and the time spent identifying familiar objects. In the test phase, detection of an object is defined as any exploratory behavior of the animal's nose within 1cm of the object, including head direction, climbing, sniffing, etc. The method for calculating the discrimination index comprises the following steps: discrimination index= (strange object recognition time/total recognition time×100) - (familiar object recognition time/total recognition time×100).

After each mouse test was completed, the scene was cleaned with purified water so as not to affect the results of the subsequent mouse experiments.

4 experimental results

Data were analyzed by Two-way ANOVA using GrapaPad Prism software, data processing results were expressed as mean±sem, and significant level differences were considered statistically significant when P < 0.05.

The statistics of the results of the mouse new object recognition experiment are shown in fig. 5B. Wt+vehicle; KO+Vehicle; wt+as; ko+as; wt+as/TMZ; the discrimination indexes of the KO+AS/TMZ group are AS follows: 23.32%; -21.76%;18.72%;20.52%; -3.21%; -23.94%.

The experimental results show that: WT mice were more prone to exploring new objects, whereas Fmr KO mice performed in contrast to WT mice, indicating that Fmr KO mice had a defective learning and memory capacity, and that artesunate could correct this defect after administration. And TMZ can block the learning and memory improving effect of artesunate on Fmr KO mice.

From the above experiments, it can be seen that: artesunate can improve the memory of fragile X syndrome model mice by modulating adult neurogenesis.

4. Social interaction experiment

Social interaction disorder is one of the most prominent manifestations of autistic patients. The experiment uses three cases of interactive social behavior test experiments to detect whether artesunate can correct social behavior defects of a fragile X syndrome model mouse through regulating neurogenesis.

1 Experimental materials

1.1 materials, a rectangular operation box and a metal cage; two mice; facial tissues; 75% alcohol; two timers.

1.2 experimental animals, drugs: the method is the same as that of experimental animals and medicines for' one and new position identification experiments

2 experimental group and administration

2.1 experimental grouping: wt+vehicle: WT mice were intraperitoneally injected with physiological saline; ko+vehicle: fmr1 KO mice were intraperitoneally injected with physiological saline; wt+as: WT mice were injected intraperitoneally with artesunate; ko+as: fmr1 KO mice were injected intraperitoneally with artesunate; wt+as/TMZ: WT mice were intraperitoneally injected with temozolomide and artesunate; KO+AS/TMZ: fmr1 KO mice were intraperitoneally injected with temozolomide and artesunate, 8-10 per group (i.e., n=8-10).

2.2 modes of administration: the same drug administration mode as that of the first and the new position identification experiments

3 experimental procedure

The test is to detect changes in social behavior of rodents by their natural liking to colonise and exploring the new object. The social behavior testing device is a three-chamber box, the partition wall is made of black organic glass, a small round door with the diameter of 7cm is arranged in the partition wall, and animals can freely move in the three chambers.

As in fig. 6A, first, the laboratory animal is taken to the behavioural test laboratory, for at least 1 hour. The experiment comprises three stages, namely habituation, social interaction test and social novelty identification. In the test phase, the experimental animals were placed in the middle box for the first time and allowed to freely explore the left, middle and right three chambers for 10min; next, a strange animal was placed in the left-hand cage, a toy was placed in the right-hand cage, and the time of exploration of the toy and strange animal by the experimental animal was recorded within 10 minutes during the social interaction test. In the social novelty recognition test, the animals that had interacted before were kept unchanged (left-hand side), the toys in the right-hand side were replaced with a new stranger (right-hand side), and the time of exploration of the familiar animals (left-hand side, from the previous social interaction stage) and the stranger animals (right-hand side, new replacement animals) by the experimental animals was recorded within 10 min. The criterion is calculated from the difference between the percentage of time spent identifying strangers and the time spent identifying toys (or familiar animals). In the test phase, the detection of an object or another animal is defined as any exploratory activity within 1 cm of the animal's nose from the object wire cage or another animal wire cage, including head direction, climbing, sniffing, etc. The method for calculating the discrimination index comprises the following steps: discrimination index= (strange animal recognition time/total recognition time×100) - (toy or familiar animal recognition time/total recognition time×100).

After each mouse test was completed, the scene was cleaned with purified water so as not to affect the results of the subsequent mouse experiments.

4 experimental results

Data were analyzed by Two-way ANOVA using GrapaPad Prism software, data processing results were expressed as mean±sem, and significant level differences were considered statistically significant when P < 0.05.

The statistics of the results of the mouse social interaction experiment are shown in fig. 6B. Wt+vehicle; KO+Vehicle; wt+as; ko+as; wt+as/TMZ; the discrimination indexes of the KO+AS/TMZ group are AS follows: 30.46%; -29.55%;43.81%;47.32%; -22.62%; -42.29%.

The experimental results show that: WT mice tended to interact more with new mice, whereas Fmr KO mice tended to interact more with old mice, indicating that Fmr KO mice had a defective social capacity, which could be corrected after artesunate administration. And TMZ can block the social interaction improving effect of artesunate on Fmr KO mice. It follows that artesunate can correct social interaction disorder of fragile X syndrome model mice by modulating neurogenesis.

From the above experiments, it can be seen that: artesunate can correct social defects in fragile X syndrome model mice by modulating neurogenesis.

The above-described embodiments of the present invention are merely exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

Claims (6)

1. Use of artesunate in the manufacture of a medicament for the treatment of hereditary intellectual and cognitive dysfunction disease, wherein the hereditary intellectual and cognitive dysfunction disease is fragile X syndrome.

2. The use according to claim 1, wherein the medicament consists of artesunate and a pharmaceutically acceptable carrier.

3. The use according to claim 1 or 2, wherein the medicament is administered by the gastrointestinal route or/and by the parenteral route.

4. The use according to claim 1 or 2, wherein the medicament is in the form of an oral formulation, an injectable formulation or a topical formulation.

5. The use according to claim 4, wherein the oral formulation is selected from the group consisting of tablets, capsules, pills, powders, granules, syrups and solutions; the injection is selected from injection dosage forms or freeze-dried powder injection dosage forms for injection; the topical preparation is selected from cream, ointment, spray, aerosol or patch.

6. The use according to claim 1 or 2, wherein the artesunate has a purity of more than 60%.

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