Application of stress-induced stem cell extract in treatment of cerebral apoplexy

Abstract

The invention belongs to the field of medicines, and discloses application of a stress-induced stem cell extract in treatment of cerebral apoplexy. The inventor finds that stem cells are cultured under proper stress conditions through screening, and the separated stem cell extract (also known as Alritoin) can increase the axon length and dendritic length of normal primary neurons and is beneficial to growth and conduction of nerve fibers; in a pdMCAO model, the volume of cerebral infarction in the acute stage of ischemia of a mouse can be remarkably reduced, the neurological function score can be improved, and a relatively good improvement trend is achieved in the aspect of cognition; in the acute stage of cerebral ischemia reperfusion, the pharmaceutical composition has remarkable effects on reducing the cerebral infarction volume of a mouse, reducing relative edema and improving the neurological function, and is relatively long in medication time window; in a cerebral ischemia reperfusion model, the trend of improving local cerebral blood flow is shown. The allitoin is expected to be developed into a promising drug for cerebral apoplexy, especially a drug for treating ischemic cerebral apoplexy.

Description

Technical Field

[0001] The present application belongs to the field of medicine, and specifically relates to the use of a stress-induced stem cell extract in treating stroke. Background Art

[0002] Stroke, commonly known as stroke, is divided into two types: ischemic stroke and hemorrhagic stroke. It is a disease caused by multiple reasons that damage cerebral blood vessels and produce focal or overall brain tissue damage. The incidence of stroke in China is increasing year by year, from 0.40% in 1993 to 1.23% in 2013, and the average age of onset is also decreasing. Stroke has the characteristics of high incidence, disability rate, recurrence rate and mortality rate, and is the leading cause of death among Chinese residents. The recurrence rate of acute stroke patients in China is as high as 17.7% within one year, and the cumulative recurrence rate in five years exceeds 30%. Among them, ischemic stroke accounts for 75% to 90% of all strokes, while hemorrhagic stroke accounts for 10% to 25%. The incidence of stroke is higher in cold seasons, and the peak of incidence usually occurs near noon. Men, obese people and diabetic patients are high-risk groups. Common symptoms of stroke include sudden numbness or weakness, especially abnormal body shape on one side, language or understanding problems, vision problems, headaches, dizziness, etc.

[0003] Treatment for ischemic stroke usually involves vascular dilation, endarterectomy, or arterial bypass surgery. Treatment for hemorrhagic stroke usually focuses on clearing hemangiomas and blood clots. Immediate treatment after a stroke can significantly improve prognosis. Prevention of stroke includes controlling high blood pressure, exercising regularly, limiting alcohol intake, and avoiding smoking.

[0004] The treatment of stroke has very large limitations. The main treatments in the acute phase include thrombolysis, intravascular interventional therapy, and neuroprotection. Thrombolysis benefits only a small number of patients due to its short treatment window (no more than 9 hours) and many contraindications. Intravascular interventional therapy has many contraindications, is difficult to perform, and is expensive. Neuroprotective agents can theoretically rescue the ischemic penumbra and expand the treatment window, but the neuroprotective agents currently approved for marketing have poor efficacy in treating stroke. A drug for the treatment of stroke with good efficacy needs to be developed. Summary of the invention

[0005] The inventors of the present application discovered that after culturing stem cells under appropriate stress conditions, multiple proteins were isolated from the supernatant of cell culture medium separation or the lysate of stem cells cultured under lysis stress, and the isolated proteins have the efficacy of repairing nervous system functions and treating nervous system diseases, and can be applied to neuroprotection in the acute onset of stroke.

[0006] The technical solution adopted in this application is:

[0007] A use of a stress-induced stem cell extract for treating stroke, wherein the preparation of the stem cell extract comprises the following steps:

[0008] S1) culturing stem cells and creating stress conditions to stimulate the stem cells;

[0009] S2) collecting the culture supernatant of the stem cells in S1) or lysing the stem cells in S1) to collect lysates, and separating and purifying the culture supernatant or lysates to obtain the stem cell extract;

[0010] The separation and purification comprises the following steps:

[0011] S21) filtering the lysate through a 0.22 μm filter membrane to obtain a mother solution, and storing the mother solution at 4° C.;

[0012] S22) filtering and concentrating the mother solution using an ultrafiltration concentration tube with a molecular weight cutoff of 3KD to obtain a crude sample of the stem cell extract;

[0013] S23) purifying the crude sample by molecular sieve exclusion chromatography or purifying the mother solution by reverse phase chromatography to prepare a purified sample of the stem cell extract; wherein the purified sample also satisfies the following characteristics:

[0014] S231) in the molecular sieve exclusion chromatography, taking the volume of the eluent as a reference, collecting the 10th to 40th ml of the eluent as the purified sample;

[0015] S232) In the reverse phase chromatography, taking the elution time as a reference, collecting the eluate with the elution time between 10 min and 40 min as the purified sample.

[0016] In some application examples, in molecular sieve exclusion chromatography, the instrument used is AKTA explorer, and the chromatography column is a superdex150 chromatography column with a size of 8×500 and a column volume of about 30 mL; the operation steps are:

[0017] S2311) rinse the chromatography column with 2CV of pure water, then balance the chromatography column with 2CV of PBS, and make the ultraviolet absorption value of the chromatography column at 280nm return to zero; and S2312) take a predetermined injection amount of the crude sample for loading, the loading flow rate is 0.4mL / min, the PBS elution flow rate is 0.1~0.3mL / min, elute to the peak, and start receiving the separated elution liquid when the volume of the eluent reaches 10ml to prepare the purified sample.

[0018] In some application examples, the purified sample of the stem cell extract also satisfies the following characteristics: a predetermined volume segment of the purified sample is collected for SDS-PAGE electrophoresis analysis, and it is measured that the purified sample contains at least protein molecules with a molecular weight mainly distributed between 11KD and 100KD, wherein, from large to small molecular weight, the first band is located between 75KD and 100KD, and the second band is located between 63KD and 75KD.

[0019] In some application examples, the purified sample of the stem cell extract also meets the following characteristics:

[0020] According to the order of elution, the eluate is divided into five elution volume segments, and the first component sample with an elution volume segment of 12.0 to 13.2 mL, the second component sample with an elution volume segment of 15.2 to 17.0 mL, the third component sample with an elution volume segment of 17.0 to 20.0 mL, the fourth component sample with an elution volume segment of 29.0 to 31.0 mL, and the fifth component sample with an elution volume segment of 31.0 to 34.0 mL are collected respectively according to the order of elution. The sample used for the SDS-PAGE electrophoresis analysis is the third component sample.

[0021] In some application examples, in reverse phase chromatography, the instrument used is a high performance liquid chromatograph, the chromatographic column is XBridge Protein BEH C4, and the pore size of the filler is The filler particle size is 3.5 μm, the inner diameter*length is 4.6 mm*150 mm, and the mobile phase is PBS; the operation steps are: S2321) adding PBS to dilute the mother solution;

[0022] S2322) Take 80-120 μL of the diluted mother solution for sample loading, control the column temperature of the chromatographic column to 20-30°C, the flow rate to 0.2-0.8 mL / min, the detection wavelength to 260 nm and / or 280 nm, set the separation time to 48 min, collect the eluate with the peak time at 10-40 min, and prepare the purified sample.

[0023] In some application examples, there are ten characteristic peaks within the elution time of 10 to 40 minutes in the reverse phase chromatography, among which the elution time of characteristic peak 1# is 13.3 to 14.5 minutes, the elution time of characteristic peak 2# is 21.5 to 22 minutes, and the elution time of characteristic peak 3# is 22 to 22.8 minutes; the elution time of characteristic peak 4# is 23 to 23.7 minutes, the elution time of characteristic peak 5# is 26.8 to 27.8 minutes, the elution time of characteristic peak 6# is 27.8 to 28.7 minutes, the elution time of characteristic peak 7# is 28.7 to 30 minutes, the elution time of characteristic peak 8# is 30 to 30.6 minutes, the elution time of characteristic peak 9# is 30.6 to 32 minutes, and the elution time of characteristic peak 10# is 33.8 to 34.8 minutes; the elution liquids of the characteristic peaks 1# to 10# are collected respectively as the purified samples.

[0024] In some application examples, the stress condition is ultraviolet irradiation, infrared irradiation, electromagnetic field, high temperature, low temperature, low oxygen, high oxygen, oxidation, high pH, ​​low pH, ultrasound, terahertz electromagnetic wave, X-ray, microwave, radiation, radiation, radiation, ion beam, high CO 2 and low CO 2 One or more stimulus conditions.

[0025] In some application examples, the ultraviolet irradiation satisfies at least one of the following conditions:

[0026] a. The irradiation time is 1 to 30 hours;

[0027] b. Irradiation intensity is 10~100μW / cm 2 ;

[0028] c. The wavelength of ultraviolet light is 290~340nm.

[0029] In some application examples, the culture medium used to culture the stem cells is a serum-free stem cell culture medium; and / or the stem cell source can be one or more of mesenchymal stem cells, embryonic stem cells, in vitro induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells and MUSE cells, and the mesenchymal stem cells can be one or more of human umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells and skin-derived mesenchymal stem cells.

[0030] In some application examples, the method for preparing the lysate includes at least one of chemical lysis, enzymatic lysis, mechanical lysis, repeated thawing and freezing, boiling, osmotic pressure, liquid nitrogen and ultrasonic fragmentation; the method for separating and extracting the stem cell extract from the lysate also includes at least one of chromatography, spectroscopy, volumetric method, dialysis, organic solvent precipitation, acid extraction, alkaline extraction, ultrafiltration, chromatography, electrophoresis and centrifugation.

[0031] In some application examples, the drug prepared from the stem cell extract is an injection, a nasal spray preparation or an oral preparation; or the drug is administered by a method selected from intrathecal administration, intravenous administration, a combination of intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, and oral administration.

[0032] In some application examples, the drug is in the form of lyophilized powder, injection, gel, pill, tablet, powder or paste.

[0033] In some application examples, the stroke is ischemic stroke or hemorrhagic stroke.

[0034] The beneficial effects of this application are:

[0035] The inventors found that the extract isolated from stem cells cultured under stress conditions (denoted as alituo) has good biological activity. In rat primary neuron culture, alituo 25ng / mL can increase the axon length and dendrite length of normal primary neurons; after oxygen-glucose deprivation, the number of axon and dendrite branches is increased, suggesting that alituo helps nerve fiber growth and conduction.

[0036] In the pdMCAO model, 10 μg / kg, 20 μg / kg, 40 μg / kg, and 60 μg / kg of aritol could significantly reduce the volume of cerebral infarction in the acute phase of ischemia in mice ( Fig.14 ); in female mice, although the effect of alitoxan on cerebral infarction was not statistically significant, it showed a trend of reducing cerebral infarction ( Fig.15 During the recovery period, Alito was able to improve neurological function scores and had a good trend of improvement in cognition ( Fig.21 ).

[0037] In the acute phase of cerebral ischemia-reperfusion, the 20 μg / kg dose group of Alitol had a significant effect on reducing the volume of cerebral infarction, reducing relative edema, and improving neurological function in mice ( Fig. 22 ), and this dose was used in subsequent experiments. In the study of the medication time window, the administration of Alito within 2h and 3h of transient cerebral ischemia reperfusion can still reduce the volume of cerebral infarction, and there is still a statistical trend when the treatment is 4h and 5.5h of ischemia reperfusion ( Fig.23 ).

[0038] At 24h of tMCAO, cerebral infarction and edema in female castrated mice and male aged mice were evaluated by TTC staining and nuclear magnetic resonance imaging, respectively. The results showed that alituo had a good trend of reducing cerebral infarction volume in castrated female mice and aged mice with cerebral ischemia-reperfusion ( Fig.24 , Fig.25 ), and it also plays a certain role in improving the survival rate of elderly mice.

[0039] In the cerebral ischemia-reperfusion model, laser speckle was used to observe the local blood flow in the mouse brain. Ten minutes after reperfusion, Alito showed a trend of increasing local cerebral blood flow ( Fig.26 ), suggesting that alituo may have a certain vasodilatory effect. Three days after tMCAO, MRI scans of the brains of cerebral ischemic mice showed that alituo could significantly reduce the volume of cerebral infarction and relative brain edema ( Fig. 27 In long-term observations during the recovery period, alituo helped mice regain body weight after transient cerebral ischemia ( Fig.28 ), and the neurological function score was improved to a certain extent ( Fig.29 ), which has a tendency to promote long-term motor function recovery ( Fig.30 ), which may have a certain improvement effect on cognitive function ( Fig.31 ).

[0040] In summary, Alitus is expected to be developed into a promising drug for stroke, especially for the treatment of ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Figure : Time flow chart of the effect of alitox on the survival of primary neurons after oxygen-glucose deprivation.

[0042] Figure 2 Figure : Time flow chart of the effect of alitox on the survival of primary neurons pretreated with microglia after oxygen-glucose deprivation.

[0043] Figure 3 This is a schematic diagram and time flow chart of the study on the effects of alitox on the processes of primary neurons after oxygen-glucose deprivation.

[0044] Figure 4 For: Dosage evaluation of alituo in pdMCAO - technology roadmap.

[0045] Figure 5 For: Pharmacodynamic evaluation of alituo in pdMCAO female mice - technology roadmap.

[0046] Figure 6 For: Effects of alituo on neurological function and cognition in ppMCAO-35d mice - technical roadmap.

[0047] Figure 7For: Dosage evaluation of alitox in tMCAO - technology roadmap.

[0048] Figure 8 To: Evaluation of the time window for the use of alitox in tMCAO - technology roadmap.

[0049] Fig. 9 Pharmacodynamic evaluation of alituo in tMCAO female mice - technology roadmap.

[0050] Fig.10 For: Pharmacological evaluation of alituo in tMCAO male aged mice - technology roadmap.

[0051] Fig.11 For: Effects of alituo on neurological function and cognition in tMCAO mice - technical roadmap.

[0052] Fig.12 Figure 3 Effects of alitox on the survival of primary neurons after oxygen glucose deprivation (OGD): A and B are the survival rates of neurons at 24h and 72h after OGD, respectively; C and D are the survival rates of neurons at 24h and 72h after OGD after inflammation induced by supernatant of microglia culture fluid, respectively; n=5-6 / group; *: p<0.05.

[0053] Fig.13 Figure : Effects of alitate on the processes of primary neurons after oxygen-glucose deprivation: A is a representative image of Tuj1 / MAP2 immunofluorescence staining of neurons in the control group treated with different concentrations of alitate, green represents Tuj1, red represents MAP2, and blue represents DAPI; B is a representative image of Tuj1 / MAP2 immunofluorescence staining of neurons in the OGD group treated with different concentrations of alitate; C is the axon length (μm); D is the number of axon branch points; E is the dendrite length (μm); F is the number of dendrite branch points; **: p < 0.01; ***: p < 0.001.

[0054] Fig.14 Figure 3: Cerebral infarction volume and neurological function score of mice in each group 24 hours after pdMCAO: A is a representative brain slice stained with TTC in each group of mice, B is a statistical chart of cerebral infarction volume (mm3), and C is a statistical chart of neurological function score; *: p<0.05; **: p<0.01; sham group, n=5; pdMCAO model group, n=17-18 mice / group.

[0055] Fig.15 Figure 2: Cerebral infarction volume and neurological function scores of female ovariectomized mice 24 hours after pdMCAO: A is a representative brain slice stained with TTC of mice in each group, and B is a statistical chart of cerebral infarction volume (mm 3 ), C is the statistical diagram of neurological function score; NS: p>0.05; n=15 / group.

[0056] Fig.16 Figure 2: Body weight of mice after ppMCAO: A is the body weight of mice in each group that were dosed 0-14 days after the model was established; B is the body weight of mice in each group that were dosed 8-21 days after the model was established; the same data were used for both Sham and ED groups, and the data of dead mice were taken from the last data before death; NS: p>0.05; 0.9% NS and alitol 0-14 days administration groups, n=37 / group; the rest of the groups, n=9-15 / group.

[0057] Fig.17 Figure 2: Neurological function scores of mice after ppMCAO: A is the neurological function scores of mice in each group that were administered 0-14 days after the model was established; B is the neurological function scores of mice in each group that were administered 8-21 days after the model was established; the same data were used for both Sham and ED groups, and the data of dead mice were used as the last data before death; *: p<0.05; ***: p<0.001; 0.9% NS and alitol 0-14 days administration groups, n=35-36 mice / group; the rest of the groups, n=10-15 mice / group.

[0058] Fig.18 Figure 2: Corner turning test of mice after ppMCAO: A is the right turning rate of mice in each group dosed 0-14 days after the model was established; B is the right turning rate of mice in each group dosed 8-21 days after the model was established; the same data were used for the Sham and ED groups, and the data of dead mice were continued from the last data before death; 0.9% NS and alitol 0-14 days administration group, n=33-34 mice / group; the rest of the groups, n=10-15 mice / group.

[0059] Fig.19 Figure 2: Water maze results of mice after ppMCAO: A, D are the escape latency (s) of mice in the training phase, B, E are the percentage (%) of the time mice stayed in the target quadrant in the test phase, C, F are the average swimming speed (cm / s) of mice in the test phase, the same data were used for the Sham and ED groups; 0.9% NS and alitol 0-14d administration groups, n=23-27 / group; the rest of the groups, n=7-10 / group.

[0060] Fig. 20 The results of open field in ppMCAO mice: A is the time (s) that mice in each group dosed with drugs 0-14 days after the model was established stays in the center of the open field; B is the time (s) that mice in each group dosed with drugs 8-21 days after the model was established stays in the center of the open field; the same data were used for the Sham and ED groups; for the 0.9% NS and alitol 0-14 days administration groups, n=23-27 mice / group; for the other groups, n=7-10 mice / group.

[0061] Fig.21Figure 2: Y-maze results of ppMCAO mice: A is the time (s) that the mice in each group who were dosed 0-14 days after the model was established, B is the time (s) that the mice in each group who were dosed 8-21 days after the model was established, the same data were used for the Sham and ED groups; NS: p>0.05; 0.9% NS and alitol 0-14 days administration groups, n=22-27 mice / group; other groups, n=7-10 mice / group.

[0062] Fig. 22 Figure 2: Cerebral infarction, edema and neurological function scores of mice in each group 24 hours after tMCAO: A is a representative brain slice stained with TTC of mice in each group, B is a statistical chart of cerebral infarction volume, C is a statistical chart of brain edema degree (ischemic side brain tissue volume / healthy side brain tissue volume), D is a statistical chart of neurological function score; *: p<0.05; **: p<0.01; ***: p<0.001; n=8-15 mice / group.

[0063] Fig.23 Figure 2: Medication time window after 24h tMCAO: A, E, I, M are different medication time axes, B, F, J, N are representative brain slices stained with TTC of mice in each group, C, G, K, O are statistical graphs of cerebral infarction volume, D, H, L, P are statistical graphs of neurological function scores; NS: p>0.05; *: p<0.05; **: p<0.01; n=16-18 mice / group.

[0064] Fig.24 Figure 3: Cerebral infarction volume and neurological function score of female ovariectomized mice 24 hours after tMCAO: A is a representative brain slice stained with TTC of mice in each group, B is a statistical chart of cerebral infarction volume, C is a statistical chart of brain edema degree, and D is a statistical chart of neurological function score; NS: p>0.05; *: p<0.05; n=15-16 mice / group.

[0065] Fig.25 Figure 4: Acute effects of tMCAO in male aged mice: A is a representative brain slice of 24h MRI T2 of mice in each group, B is the experimental timeline, C is the survival status of mice in each group, D is a statistical chart of cerebral infarction rate, E is a statistical chart of brain edema degree, and F is a statistical chart of neurological function score; NS: p>0.05; n=5-6 mice / group.

[0066] Fig.26 Figure 2: Cerebral blood flow in mice after tMCAO: A is a representative graph of cerebral blood flow at each time point in each group of mice, and B is a statistical graph of relative cerebral blood flow at different time points (with the cerebral blood flow before ischemia as the baseline); n = 11-12 mice / group.

[0067] Fig. 27Figure 3: Cerebral infarction in mice 3 days after tMCAO: A is the representative T2 MRI image of mice in each group, B is the cerebral infarction situation, C is the cerebral infarction rate, and D is the degree of brain edema; *: p<0.05; **: p<0.01; n=5 mice / group.

[0068] Fig.28 Figure 2: Body weight of mice in the recovery period after tMCAO: **: p<0.01; ***: p<0.001; Sham group, n=11; tMCAO group, n=28-29 mice / group.

[0069] Fig.29 Figure 2: Neurological function scores of mice after tMCAO: A is the neurological function scores of mice in each group that were given the drug 0-14 days after the model was established; B is the neurological function scores of mice in each group that were given the drug 8-21 days after the model was established. The same data were used for both the Sham and ED groups; *: p<0.05; Sham group, n=11; tMCAO group, n=28-29 / group.

[0070] Fig.30 Figure 2: Treadmill test results of mice after tMCAO: NS: p>0.05; Sham group, n=6 mice; tMCAO group, n=12-14 mice / group.

[0071] Fig.31 Figure 3: Results of new location recognition in mice after tMCAO: percentage of time (%) spent by mice exploring new locations; n = 5-11 mice / group.

[0072] Fig.32 Figure 2: mRNA expression of brain tissues of mice in different experimental groups. 6 A. Changes in modification spectrum. A is a cluster heat map of differentially expressed genes between 7d-ALT and 7d-NS, red represents up-regulation and green represents down-regulation; B is a volcano map of differentially expressed genes between 7d-ALT and 7d-NS, red represents up-regulation and green represents down-regulation; C is the m 6 A is a schematic diagram of the distribution of modification sites; D is the differential m 6 A is a volcano diagram of modified sites, red represents upregulation and green represents downregulation; E is a Venn diagram, indicating that 7d-ALT vs 7d-NS contain differential m 6 A. Modified differential mRNAs.

[0073] Fig.33 For: mRNA and m 6 A is the functional enrichment analysis of 7d-ALT vs 7d-NS mRNA, including the biological processes, molecular functions, and cell composition involved; B is the functional enrichment analysis of 7d-ALT vs 7d-NS mRNA 6A mRNA functional enrichment analysis, including biological process, molecular function, and cellular composition.

[0074] Fig.34 This is a photograph of the cell state after ultraviolet irradiation in Example 1.

[0075] Fig.35 This is the SDS-PAGE electrophoresis diagram of the mother solution of the stem cell extract harvested in Example 1.

[0076] Fig.36 This is the elution curve of Example 2.

[0077] Fig.37 It is the SDS-PAGE electrophoresis diagram of sample No. 12 (pure-12#) after purification in Example 3.

[0078] Fig.38 This is the elution curve of Example 3. DETAILED DESCRIPTION

[0079] The technical solution of the present application is further explained below in conjunction with embodiments.

[0080] For the convenience of explanation, mesenchymal stem cells are used as examples in the embodiments, and the sources of the mesenchymal stem cells include but are not limited to human umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells. In addition, in practical applications, it is not limited to mesenchymal stem cells, and other stem cells can also produce similar stress proteins after stress induction. Other stem cells that can be used include but are not limited to at least one of embryonic stem cells, in vitro induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, and MUSE cells.

[0081] After undergoing stress conditions, mesenchymal stem cells can theoretically induce the production of corresponding stress proteins. For the convenience of explanation, ultraviolet irradiation is used as a stress condition in the embodiment to prepare a mesenchymal stem cell extract containing stress proteins that can be used to treat stroke. In practical applications, other stress conditions can achieve similar effects. Other stress conditions include, but are not limited to, at least one of the following stress methods: ultraviolet irradiation, infrared irradiation, electromagnetic field, high temperature, low temperature, hypoxia, hyperoxia, oxidation (under conditions containing oxidants such as hydrogen peroxide or hypochlorous acid), high pH, ​​low pH, ultrasound, terahertz electromagnetic waves, X-rays, microwaves, rays, rays, rays, ion beams, high CO 2 , low CO 2 . Various stress management methods can be carried out one after another or simultaneously when they do not conflict.

[0082] The preparation method of the lysate includes at least one of chemical lysis, enzyme lysis, mechanical lysis, repeated thawing and freezing, boiling, osmotic pressure, liquid nitrogen and ultrasonic fragmentation.

[0083] Theoretically, any method that can be used to separate and extract cell extracts from stress-induced stem cells can be used in this application. For ease of explanation, chromatography and volumetric methods (molecular sieve exclusion chromatography) are used in some embodiments of this application. In practical applications, any method that can separate and extract the target extract from the lysate can also be used, including but not limited to at least one of spectroscopy, dialysis, organic solvent precipitation, acid extraction, alkali extraction, ultrafiltration, chromatography, electrophoresis, centrifugation, etc.

[0084] Spectroscopic method, using the absorbance of the target protein under a specific wavelength of light to screen out the target extract; dialysis method is to use a dialysis bag to exchange solutions with a buffer solution, and finally obtain a purified and concentrated target extract; organic solvent precipitation method, by adding an organic solvent to the lysate, to achieve a precipitation effect similar to that of high-concentration salt, and the precipitate is filtered out, which is the target extract; acid extraction method, by adding concentrated hydrochloric acid to lyse stem cells to obtain the target extract; alkaline extraction method, by adding sodium hydroxide or potassium hydroxide to lyse cells to obtain the target extract; ultrafiltration method, adding cell lysate to an ultrafiltration column, continuously centrifuging, adding buffer, and ultrafiltration filtration multiple times, and finally concentrating to obtain the target extract; chromatography method, including ion exchange chromatography, gel filtration chromatography, affinity chromatography, different chromatography methods are selected according to the characteristics of the target extract; electrophoresis method, by applying electricity to separate protein molecules in a dispersed matrix, thereby obtaining the target extract; centrifugation method, by centrifuging the lysate, filtering the target extract.

[0085] Example 1: Cultivation of human umbilical cord mesenchymal stem cells

[0086] Resuscitate a small crystal of P8 HUC-MSC (human umbilical cord-derived mesenchymal stem cells) into a T25 culture flask and add 2.5 mL of Huakai mesenchymal stem cell serum-free culture medium.

[0087] The cells were irradiated with 300nm LED UV for 6h. The cells were carefully cleaned of supernatant, washed twice with 1mL physiological saline, added with 660μl pure water and repeatedly blown for 10min, filtered through a 0.22μm filter membrane, and stored at 4℃.

[0088] UV irradiation conditions are as follows:

[0089] Intermediate medium T25 bottle <![CDATA[UVA(μW / cm 2 )]]> 11.71 <![CDATA[UVB(μW / cm 2 )]]> 26.81 Peak wavelength (nm) 320.4

[0090] The cell status after irradiation is Fig.34As shown. After cell lysis, the lysate of mesenchymal stem cells (i.e., the mother solution of stem cell extract, also called ALEETO stock solution, abbreviated as ALEETO stock solution or ALT stock solution) was filtered and harvested. The protein concentration was measured to be 0.718 mg / mL, and the volume was 500 μl. The SDS-PAGE test results of the harvested ALEETO stock solution are shown as follows: Fig.35 shown.

[0091] In this embodiment, the stem cell extract is a protein extracted from cells. In practical applications, it can also be extracted from the cell culture supernatant after cell culture.

[0092] Example 2: Molecular sieve purification and activity detection of mesenchymal stem cell lysate

[0093] 2-1 Molecular sieve purification of mesenchymal stem cell lysate:

[0094] Instrument: AKTA explorer

[0095] Chromatographic column: Nano-micron superdex150 molecular sieve 8×500, column volume is about 30mL

[0096] Reagents: 0.1M NaOH, 20% ethanol, 1× PBS, purified water

[0097] UV absorption wavelength: 280nm, 260nm as reference

[0098] Equilibration column sequence:

[0099] The column was first rinsed with 2CV of pure water, and then equilibrated with 1×PBS for 2CV, and the UV absorption value at 280nm was returned to zero.

[0100] Sample preparation: Repeat the method in Example 1 to harvest a total of 20 mL of protein. Use an ultrafiltration concentration tube with a molecular weight cutoff of 3 KD to reduce the 20 mL sample to about 600 μl to obtain a crude sample.

[0101] Experimental process: After balancing the column, select a 500μl sample loop for sample loading, the sample loading flow rate is 0.4mL / min, the 1×PBS elution flow rate is 0.2mL / min, and the elution is eluted to the peak. Collect the separated elution liquid from the UV absorbance value of 4mAU. For details of the elution curve, see Fig.36 .Pick Fig.36 The eluted liquids at positions 2, 9, 12, 16, and 18 are prepared into freeze-dried mesenchymal stem cell extracts (AliTu) and stored.

[0102] 2-2 Biological Activity Detection of Mesenchymal Stem Cell Extracts

[0103] Day 1: Cell plating: Take complete medium (5% FBS + DMEM) to dilute PC12 poorly differentiated cells, 6000 cells / well plating (96-well plate); 37°C, 5% CO 2 Concentration overnight;

[0104] The next day: dilute the above lyophilized samples and ALEETO stock solution samples (about 800 μg / mL) with DMEM+5% FBS medium;

[0105] Take 30% hydrogen peroxide and dilute it 15,000 times with DMEM + 5% FBS;

[0106] Hydrogen peroxide treatment: discard 80 μL / well culture supernatant, add 50 μL / well diluted hydrogen peroxide to the corresponding cultured cells, and place at room temperature for 25 min;

[0107] Untreated control: 50 μL / well of DMEM + 5% FBS medium was added as the injury treatment control;

[0108] 50 μL / well of the diluted lyophilized sample and mesenchymal stem cell lysate were added to the wells treated with hydrogen peroxide;

[0109] The supernatant of untreated wells was discarded, and 100 μL / well of complete medium was added as cell growth control (PC);

[0110] Incubate at 37°C for 2 days;

[0111] Day 5: Discard the culture supernatant, add complete culture medium at 100 μL / well, set up a culture medium blank control well, add CCK8 at 10 μL / well, and incubate at 37°C for 3.5 h. OD 450 Read the sample and calculate the OD value after subtracting the culture medium blank.

[0112] The experimental results are shown in Table 1.

[0113] Table 1 Biological activity experimental data of different samples

[0114] Sample No. <![CDATA[OD 450 ]]> <![CDATA[OD 450 ]]> <![CDATA[OD 450 ]]> Average OD Relative survival rate% Difference % Pure-2# 0.486 0.47 0.483 0.48 68.90％ 24.40％ Pure-9# 0.474 0.476 0.464 0.471 67.20％ 22.70％ Pure-12# 0.485 0.472 0.492 0.483 69.50％ 25.00％ Pure-16# 0.482 0.496 0.488 0.489 70.70％ 26.10％ Pure-18# 0.391 0.454 0.479 0.441 61.30％ 16.80％ ALEETO original solution 0.457 0.471 0.441 0.456 64.30％ 19.80％ NC 0.339 0.388 0.341 0.356 44.50％ / PC 0.637 0.632 0.644 0.638 100％ / Medium blank 0.13 0.13 0.13 0.13 / /

[0115] Note: Pure-2#, Pure-9#, Pure-12#, Pure-16# and Pure-18# correspond to Fig.36 Lyophilized mesenchymal stem cell extract prepared from the eluted liquid at positions 2, 9, 12, 16 and 18; NC: no mesenchymal stem cell lysate and extract added.

[0116] As shown in Table 1, in the PC12 cell oxidative damage model, the proteins separated from the above bands all have certain therapeutic effects. The three purified samples 2#, 12#, and 16# all have strong oxidative damage repair capabilities, and their repair capabilities are better than the unpurified ALEETO stock solution. The repair ability of the 18# purified sample is lower than that of the unpurified ALEETO stock solution.

[0117] Take 12# purified sample for SDS-PAGE electrophoresis analysis. The electrophoresis results are as follows: Fig.37 As shown in the figure, the sample bands are mainly distributed in the range of 11KD to 100KD, and the molecular weights are from large to small, with the first band located between 75KD and 100KD, and the second band located between 63KD and 75KD.

[0118] Example 3: HPLC-SEC purification and protein polymer activity detection

[0119] Based on the difference in hydrophobicity, reverse phase chromatography can be used to separate the mesenchymal stem cell extract obtained by ultraviolet irradiation stimulation in this application. Under initial conditions, the concentration of organic components in the mobile phase is low, and the protein in the mesenchymal stem cell extract has a strong hydrophobic interaction with the stationary phase and is almost completely adsorbed by the stationary phase; when the organic components in the mobile phase reach a specific concentration, the protein is completely eluted from the stationary phase and no longer interacts with the stationary phase. Therefore, a very small change in the organic components of the mobile phase can significantly affect the reverse phase retention behavior of the protein in the mesenchymal stem cell extract.

[0120] 3-1 HPLC-SEC purification

[0121] Test product information: The mesenchymal stem cell lysate (ALEETO stock solution) prepared by the method in Example 1 was diluted to 1 mg / mL with PBS.

[0122] Mobile phase: PBS.

[0123] Detection conditions: injection volume 100 μL, column temperature 25°C, flow rate 0.4 mL / min, wavelength 280 nm, 260 nm; time: 48 min.

[0124] The collection time range is approximately: 1#13.3~14.5min; 2#21.5~22min; 3#22~22.8min; 4#23~23.7min; 5#26.8~27.8min; 6#27.8~28.7min; 7#28.7~30min; 8#30~30.6min; 9#30.6~32 (no obvious peak of A280)min; 10#33.8~34.8min.

[0125] The elution curve is Fig.38As shown, the eluates at positions 1-10 shown in the figure are taken respectively, freeze-dried and stored.

[0126] 3-2 Biological Activity Detection of Protein Polymers

[0127] Day 1: Cell plating: Take complete medium (5% FBS + DMEM) to dilute PC12 poorly differentiated cells, and plate 6000 cells / well (96-well plate). Incubate at 37°C, 5% CO 2 Concentrate overnight.

[0128] The next day: dilute the above lyophilized sample and ALEETO stock solution (about 800 ug / mL) with DMEM+5% FBS medium.

[0129] Take 30% hydrogen peroxide and dilute it 15,000 times with DMEM + 5% FBS.

[0130] Hydrogen peroxide treatment: discard 80 μL / well culture supernatant of cells, add 50 μL / well diluted hydrogen peroxide to the corresponding cultured cells, and place at room temperature for 25 minutes.

[0131] Untreated control: 50 μL / well of DMEM+5% FBS medium was added as the injury treatment control.

[0132] 50 μL / well of diluted lyophilized samples and ALEETO stock solution samples were added to the wells treated with hydrogen peroxide.

[0133] The supernatant of the untreated wells was discarded, and 100 μL / well of complete culture medium was added as a cell growth control (PC).

[0134] Incubate at 37°C for 2 days.

[0135] Day 5: Discard the culture supernatant, add complete culture medium at 100 μL / well, set up a culture medium blank control well, add CCK8 at 10 μL / well, and incubate at 37°C for 3.5 h. OD 450 Read the sample and calculate the OD value after subtracting the culture medium blank.

[0136] The experimental results are shown in Table 2.

[0137] Table 2 Biological activity experimental data of different samples

[0138] sample <![CDATA[OD 450 ]]> <![CDATA[OD 450 ]]> <![CDATA[OD 450 ]]> Average OD Relative survival rate% Difference % SEC-1# 0.524 0.512 0.499 0.512 70.60％ 11.60％ SEC-2# 0.532 0.49 0.494 0.505 69.50％ 10.40％ SEC-3# 0.504 0.48 0.485 0.49 66.60％ 7.60％ SEC-4# 0.495 0.481 0.483 0.486 66.00％ 7.00％ SEC-5# 0.476 0.482 0.451 0.47 62.90％ 3.90％ SEC-6# 0.489 0.481 0.509 0.493 67.20％ 8.20％ SEC-7# 0.532 0.503 0.489 0.508 69.90％ 10.90％ SEC-8# 0.503 0.483 0.496 0.494 67.40％ 8.40％ SEC-9# 0.483 0.474 0.481 0.479 64.70％ 5.70％ SEC-10# 0.479 0.497 0.506 0.494 67.40％ 8.40％ ALEETO original solution 0.464 0.502 0.461 0.476 64.00％ 5.00％ NC 0.458 0.439 0.448 0.448 59.00％ / PC 0.672 0.673 0.672 0.672 100％ / Medium blank 0.125 0.128 0.124 0.126 / /

[0139] Note: SEC-1#-SEC-10 correspond to Fig.38 The lyophilized mesenchymal stem cell extract was prepared from the eluted liquid at positions 1-10; NC: no mesenchymal stem cell lysate and extract were added.

[0140] As shown in Table 2, in the PC12 cell oxidative damage model, the 1#, 2#, 7#, 8#, and 10# purified samples all have strong oxidative damage repair capabilities, and their repair capabilities are better than those of the ALEETO stock solution. The repair ability of the 5# sample is lower than that of the ALEETO stock solution.

[0141] The technical solution of the present application is further described below in conjunction with biological experiments on the mesenchymal stem cell lysate and the purified extract in Examples 1-3 above.

[0142] Example 4: Study on the therapeutic effect and dosage of mesenchymal stem cell extract on OGD rat model

[0143] The experiment was conducted by injecting the lyophilized preparation of alituo which was the purified sample No. 12 prepared in Example 2 into rats with a primary neuron oxygen and glucose deprivation (OGD) injury model.

[0144] 4-1 Rat primary cell culture

[0145] (1) The in vitro study used 4 SD pregnant mice at day 18 and 20 SD newborn mice (within 24 hours of birth). All animals were provided by Beijing Huafukang Biotechnology Co., Ltd.

[0146] (2) Rat primary neuron culture: 10% chloral hydrate was used to anesthetize SD pregnant mice, and the fetal brain tissue was separated using surgical instruments. The meninges on the surface and inside of the brain tissue were carefully peeled off with the help of a stereomicroscope. The cortical tissue was cut into pieces with surgical scissors, and then digested with an appropriate amount of trypsin. After neutralization with fetal bovine serum, the cells were evenly blown and centrifuged. The cells were resuspended in Neurobasal medium for inoculation. The morphology and growth status of the neurons were observed every day during the culture period. Drug stimulation can be performed on the 10th day of culture.

[0147] (3) Culture of primary microglia in rats: Use 75% alcohol disinfectant to disinfect newborn SD rats within 24 hours of birth, use surgical instruments to separate the brain tissue of newborn rats, and carefully peel off the meninges on the surface and inside of the brain tissue with the help of a stereo microscope. Use surgical scissors to cut the brain tissue into pieces, then add an appropriate amount of trypsin to digest it, neutralize it with fetal bovine serum, blow it evenly and centrifuge it. Use DMEM / F12 medium to resuspend the cells for inoculation. Observe the morphology and growth status of glial cells every day during the culture period. On the 14th day of culture, separate microglia by shaking on a shaker: when no drug stimulation is added, it is M0 (microglia in the resting state), and when 40μg / mL IFN-γ and 100ng / mL LPS are added, it is M1 (microglia with inflammatory phenotype).

[0148] (3) Oxygen and glucose deprivation (OGD) of primary neurons: OGD was performed on the 10th day of neuron culture. In a clean bench, neurons were washed twice with sterile PBS to remove glucose from the culture medium, and then replaced with DMEM culture medium without glucose. Neurons were placed in a CO 2 In sealed tank, 95% N 2 / 5%CO 2 Pass the oxygen into the tank to exhaust it, and seal the CO after 5 minutes of balancing. 2 Seal the tank and remove the CO 2 The sealed jar was placed in a cell culture incubator. After 0.5 h, the neurons were taken out for reoxygenation and replaced with the original neuron basal medium. After 24 h (or 72 h), CCK8 was added and the OD value was detected with an enzyme marker. It should be noted that the corresponding concentration of alitox should be added to the DMEM medium without glucose during OGD and the original neuron basal medium after OGD. The time flow chart of this study is shown in the figure. Figure 1 shown.

[0149] (4) Primary neurons and microglia were co-cultured and then oxygen-glucose deprivation was performed: 24 h before OGD, the supernatant of M0 and M1 microglia culture fluid was used to stimulate the corresponding groups of neurons. Subsequent operations were the same as (3). The time flow chart of this study is shown in the figure. Figure 2 shown.

[0150] (5) Effect of alitox on the processes of primary neurons after oxygen-glucose deprivation: After extracting primary neurons, low-density neurons were inoculated on the glass slide, and high-density neurons were inoculated on the culture wells. After 24 hours, the glass slide was inverted into the culture wells to allow low-density neurons to be in direct contact with high-density neurons. The two were co-cultured until OGD was performed on the 10th day. After 24 hours of OGD, the cells were fixed and double-stained with MAP2 and Tuj1 immunofluorescence. The morphology of neuronal axons and dendrites was observed under a confocal microscope and photographs were taken. The schematic diagram of neuronal co-culture and the time flow chart are shown in the figure. Figure 3 shown.

[0151] (6) The concentration gradient of Alitretolide used in rat primary cells is: 0.39 ng / mL, 1.56 ng / mL, 6.25 ng / mL, 25 ng / mL, 100 ng / mL, 400 ng / mL, 1600 ng / mL, 6400 ng / mL. In the in vitro study, the control solvent of Alitretolide was PBS.

[0152] 4-2. Experimental results

[0153] Effects of 4-2-1Alitol on primary neurons after oxygen-glucose deprivation

[0154] Effects of 4-2-1-1-alitate on the survival of primary neurons after oxygen-glucose deprivation

[0155] We investigated the survival of neurons 24h and 72h after OGD and found that alitox 25ng / mL could significantly increase the survival rate of neurons 72h after OGD ( Fig.12 B, p = 0.0299). After adding microglial cell culture supernatant to neurons to induce inflammation and then performing OGD, 24 hours after OGD, the alitox in each administration group did not exert therapeutic effects, while at 72 hours, alitox 25 ng / mL could significantly increase the survival rate of neurons ( Fig.12 D, p=0.0257).

[0156] Effects of 4-2-1-2Alitol on the growth of primary neurons after oxygen-glucose deprivation

[0157] Alitol 25ng / mL can increase the axon length of normal primary neurons ( Fig.13 C, p = 0.0063) and dendrite length ( Fig.13 E, p<0.0001); after oxygen-glucose deprivation, axonal branching increased ( Fig.13 D, p<0.0001) and dendritic branches ( Fig.13 F, p<0.0001), suggesting that alituo contributes to nerve fiber growth and conduction. Although alituo 6.25ng / mL can increase the length of dendrites in normal primary neurons ( Fig.13 E, p = 0.0004) and the number of axonal branch points after oxygen-glucose deprivation ( Fig.13 D, p = 0.0056), but its effect was not as significant as 25 ng / mL. Therefore, in subsequent in vitro studies, 25 ng / mL of alituo was determined as the concentration for primary cell culture.

[0158] Example 5: Dosage and efficacy evaluation of mesenchymal stem cell extracts for permanent cerebral ischemia

[0159] 5-1 Test drug and positive control drug

[0160] The test drug in this experiment is the lyophilized preparation of the purified sample No. 12 prepared in Example 2 above, provided by Beijing Darwin Cell Biotechnology Co., Ltd. The drug specification is 130 μg / vial, the production batch number is 2022Y-0120, and the shelf life is 24 months. The test drug is prepared and used immediately, and the solvent is 0.9% normal saline (NS).

[0161] The positive control drug in this study is Edaravone and Dexborneol Concentrated Solution for Injection (ED), which has a specification of 5mL / vial: containing 10mg of Edaravone and 2.5mg of Dexborneol, provided by Beijing Darwin Cell Biotechnology Co., Ltd. The manufacturer is Xiansheng Pharmaceutical Co., Ltd., the production batch number is 181-221010, and the validity period is until 2024 / 10 / 13. The dose of ED for C57 mice is 1.95mL / kg, and its diluent is NS. Another positive control drug is the postsynaptic density-95 protein inhibitor (NA-1, HY-P0117, Lot#294330, MCE, USA), and the dose for C57 mice is 10nmol / g.

[0162] 5-2 Reagents, consumables and instruments and equipment

[0163] Main reagents: 10% chloral hydrate, 75% ethanol disinfectant, poly-D-lysine hydrobromide, sterile water for cell culture, HBSS buffer, PBS buffer, Neurobasal medium, DMEM / F12 medium, B27 supplement, trypsin, fetal bovine serum, MEM NEAA (100X, non-essential amino acids), Sodium Pyruvate (100mM, sodium pyruvate), GlutaMAX TM -1Supplement (100X, glutamine supplement), Penicillin-Streptomycin (penicillin-streptomycin double antibody), LPS (lipopolysaccharide), IFN-γ (interferon γ), MAP2 (sc-20172, Santa Cruz, USA), Tuj1 (T5201, Sigma, USA); iodine tincture, isoflurane for animals (R510-22, Shenzhen Ruiwode), normal saline (NS), 5% glucose (Glu), phosphate buffered saline (PBS), TTC dye (T8877, Sigma, USA), tissue fixative, etc.

[0164] Main consumables: pipettes, cell culture dishes, cell culture plates, cell counting plates, cell strainers, OGD tanks, etc.; cotton swabs, disposable syringes, thread plugs (602256PK5Re, Doccol, USA), suture needles, needle holders, sutures, cranial drills, surgical scissors, surgical curved forceps, gun tips, 50mL centrifuge tubes, 15mL centrifuge tubes, 5mL centrifuge tubes, 1.5mL centrifuge tubes, etc.

[0165] Main instruments: cell culture clean bench (ESCO, Singapore), stereo microscope, high-speed refrigerated centrifuge (Eppendorf, Germany), carbon dioxide incubator (SANYO, Japan), inverted microscope (Zeiss, Germany), desktop pressure steam sterilizer (SANYO, Japan), microplate reader (Tecan, Austria); anesthesia machine (Shenzhen Ruiwode), stereotaxic instrument (Shenzhen Ruiwode), analytical balance (Mettler Toledo, Switzerland), laser speckle blood flow imaging system (Shenzhen Ruiwode), constant temperature water bath (Shanghai Boxun Medical), EthoVision XT tracking system (Beijing Nordus), mouse and rat gripping instrument, small animal rotarod instrument (Panlab under Harvard Apparatus, USA), etc.

[0166] 5-3 Experimental Animals

[0167] (1) The experimental animals used in this experiment are as follows: 733 clean and healthy C57 BL / 6 male mice (8-10 weeks old), 61 female mice (8-10 weeks old), and 11 elderly male mice (15 months old). The above animals were provided by Beijing Huafukang Biotechnology Co., Ltd. C57 BL / 6 mice were kept at 22℃-26℃, humidity 50%-60%, separated into cages, fed with standard feed and purified water, and randomly divided into groups after 1 week of adaptive feeding.

[0168] (2) Animal culling standards

[0169] Animal exclusion criteria: (1) death during anesthesia; (2) death before drug administration; (3) mice remained inactive at the starting position during cognitive memory tests; (4) death during multiple time point studies.

[0170] (3) Animal administration

[0171] The drug administration method in this example is tail vein injection. In order to ensure the smooth implementation of the blind method in the project research, we unified the volume of each injection of solvent or drug to 150 μL, and used physiological saline to make up to 150 μL when it was less than 150 μL. Among them, the edaravone and dexamethasone concentrated solution group was administered twice a day, with an interval of ≥6h between the two administrations, and the other groups were administered once a day.

[0172] (4) Treatment of model mice

[0173] The pdMCAO model and ppMCAO model were processed as follows: the mice were weighed, anesthetized with isoflurane, and the common carotid artery was isolated. The skull of the mouse was exposed in the right prone position, and the skull was drilled through with a cranial drill to fully expose the distal end of the middle cerebral artery. The distal end of the middle cerebral artery was coagulated and cut with a high-frequency electric knife, and the common carotid artery was clamped with an artery clamp. After 20 minutes, the artery clamp was removed, the wound was sutured, and the drug was injected into the tail vein. The body temperature maintenance device maintained the rectal temperature of the mouse at 36℃~38℃ during the operation.

[0174] The tMCAO model was prepared as follows: the mice were weighed, anesthetized with isoflurane, the right common carotid and internal and external carotid arteries were separated, and the tMCAO model was established by thread embolism. The thread embolism was inserted from the external carotid artery. After 1 hour of ischemia, the embolism was removed to achieve reperfusion. The drug was injected into the tail vein immediately after the model was established or 2 hours (3 hours, 4 hours, 5.5 hours) after ischemia. The rectal temperature of the mice was maintained at 36℃~38℃ during the operation by a temperature maintenance instrument.

[0175] 5-4 Neurological function scoring test method

[0176] 5-4-1 Rules and standards for neurological function scoring 24 hours after ischemia

[0177] It includes seven scoring items, including autonomous movement ability, open field test, proprioception, whisker touch, limb symmetry, lateral turning, and forelimb walking, with a total score of 21 points.

[0178] (1) Spontaneous activity: Place the mouse in an empty cage and observe its activity trajectory within 3 minutes. Scoring criteria: 0 points - basically no activity, 1 point - very little activity and no touching of any wall of the cage, 2 points - touching any one or two walls, 3 points - touching three or more walls.

[0179] (2) Open field test (Motor tests): Place the mouse on the ground or platform and observe whether it can walk normally and turn direction. Scoring criteria: 0 points - falling to the affected side, 1 point - turning in circles to the affected side, 2 points - unable to walk in a straight line, 3 points - normal walking.

[0180] (3) Body proprioception: Use a fine brush to brush the left and right sides of the mouse's trunk and observe its reaction ability. Scoring criteria: 0 points - no reaction, 1 point - unilateral reaction, 2 points - weak bilateral reaction or rapid reaction on the same side and weak reaction on the contralateral side, 3 points - rapid reaction on both sides.

[0181] (4) Whisker touch: Use a fine brush to brush the left and right sides of the mouse's whiskers and observe its reaction ability. Scoring criteria: 0 points - no reaction, 1 point - unilateral reaction, 2 points - weak reaction on both sides or rapid reaction on the same side and weak reaction on the contralateral side, 3 points - rapid reaction on both sides.

[0182] (5) Limb symmetry: The mouse was lifted by its tail and the symmetry of its forelimbs and hindlimbs was assessed with the head of the mouse 5 cm away from the table. Scoring criteria: 0 points - complete flexion of the forelimbs and hindlimbs, 1 point - forelimbs flexed but hindlimbs extended or forelimbs extended and hindlimbs flexed, 2 points - forelimbs mid-flexed or hindlimbs mid-flexed, 3 points - forelimbs and hindlimbs extended.

[0183] (6) Lateral turning: The mouse's tail was lifted and its lateral turning ability was assessed with the head of the mouse 5 cm away from the table. Scoring criteria: 0 points - no turning, 1 point - unequal turning, 2 points - equal turning on both sides and less than 45 degrees, 3 points - equal turning on both sides and greater than 45 degrees.

[0184] (7) Forelimb walking: The mouse's tail is lifted, and its hind limbs are 10 cm above the table. The mouse is allowed to walk with its hind limbs suspended in the air. Scoring criteria: 0 points - unable to move with forelimbs, 1 point - turning in circles, 2 points - moving to one side, 3 points - walking forward symmetrically and quickly.

[0185] (8) Total neurological assessment: The total score of each mouse's autonomous activity, open field test, proprioception, whisker touch, limb symmetry, lateral turning, and forelimb walking.

[0186] 5-4-2 Rotational acceleration test

[0187] Turn on the mouse rotarod in advance and run it at a constant speed (4 rpm / min), place the mice on their respective tracks and keep them in their respective positions. Press the start button of the instrument to accelerate the track from 4 rpm / min to 40 rpm / min within 300 s, and record the time when the mouse falls from the track. Each mouse is tested 3 times, with an interval of 15 minutes between each test.

[0188] 5-4-3 Corner Test

[0189] Two wooden boards were placed at an angle of 30°, and the mice were placed between the two boards, with their heads facing the angle. The mice were allowed to walk forward autonomously and turn at the angle. The number of times the mice turned left and right was observed and recorded. Each mouse was placed 10 times, with an interval of at least 30 seconds each time, and the ratio of the mice turning right was recorded. The corner turning experiment was used to evaluate the postoperative neurological function recovery of each group of mice.

[0190] 5-4-4 Grip Test

[0191] Place the mouse on the grip strength meter and gently pull the mouse's tail so that the mouse's forelimbs grasp the instrument grid probe with force and avoid the hind limbs on the grip strength meter. When the experimenter exerts the maximum force on the mouse, record the reading on the grip strength meter, repeat the measurement 6 times, and calculate the average grip strength value.

[0192] 5-4-5 Water Maze

[0193] The Morris water maze (MWM) was used to test the spatial learning and memory ability of mice. The device consisted of a circular pool with a diameter of 109 cm, a circular platform 2 cm above the water surface and a diameter of 11 cm, and spatial markers around the pool. A camera was installed above the device. The mice were trained for spatial learning every day at an appropriate time before sampling: the mice were placed in the pool from four directions (northwest, north, east, and southwest) to observe whether the mice could find the platform within 60 seconds, and the time taken for the mice to reach the platform was recorded. After each test, the mice were placed on the platform for 15 seconds; the training lasted for 4 days. The mice were tested for spatial memory the next day after the training: the circular platform was removed, and the mice were placed in the pool from the farthest distance from the original platform. The time the mice stayed in the quadrant where the original platform was located within 60 seconds and the average swimming speed were recorded.

[0194] 5-4-6Y Maze

[0195] The three arms of the Y maze are defined as the starting arm, the new arm, and the other arms. The Y maze is divided into two stages: training and testing. During the training stage, the new arm is blocked with a baffle, and the mice are allowed to move freely in the starting arm and the other arms for 5 minutes, after which the mice are returned to the cage. The test is performed 1 hour later, and the baffle of the new arm is removed, allowing the mice to move freely in the three arms for 5 minutes. The total distance of the mouse's movement, the movement time in each arm, and the movement distance, etc. are recorded.

[0196] 5-4-7 Open Field

[0197] The open field test is used to evaluate the anxiety and short-term memory ability of mice after cerebral ischemia. The device consists of a 40cm×40cm×35cm plexiglass box. The mouse is placed in the center of the box and allowed to explore freely for 10 minutes, after which the mouse is returned to the cage. Its exploration trajectory, total movement distance, and movement time in the center area are recorded.

[0198] 5-4-8 New Object Recognition

[0199] The novel object recognition (NOR) device is made of a 50cm x 50cm plexiglass plate with black walls and a matte white bottom. The device is placed in a dark room with a camera and a light on top. On the day of the experiment, two identical objects (A and B) were placed in the device, and the mice were allowed to explore freely for 10 minutes, and then the mice were returned to the cage. After 6 hours, object B was replaced with object C (object C was different from objects A and B), and the mice were placed in the device again, and the time they explored the new object and the total exploration time were recorded.

[0200] 5-4-9 New location identification

[0201] New location recognition (NLR) uses the same set of equipment as NOR. On the day of the experiment, two identical objects (A and B) were placed in the device, and the mice were allowed to explore freely for 10 minutes, and then returned to the cage. After 6 hours, the position of object A was changed, and the mice were allowed to continue exploring for another 10 minutes. The time they explored the new location and the total exploration time were recorded.

[0202] 5-4-10 Evaluation of cerebral infarction

[0203] After 24 h of cerebral ischemia and neurological function scoring, the mice were killed and the brains were removed. Fresh brain coronal sections were stained with TTC, and the infarct volume of the mice was measured using Image J software. The infarct volume of the mice (mm) was calculated according to the following formula 3 ):

[0204] Cerebral infarction volume = healthy side brain volume - affected side healthy brain volume.

[0205] 5-4-11 MRI evaluation of cerebral infarction volume and edema degree

[0206] After ischemia, the mouse brain was scanned with magnetic resonance imaging, and the infarction volume and relative brain edema degree of the mice were counted.

[0207] Infarct volume (mm 3 ) = healthy volume of the healthy side - healthy volume of the affected side;

[0208] Relative brain edema degree (%) = ischemic side brain tissue volume / healthy side brain tissue volume × 100%.

[0209] 5-4-12 Cerebral blood flow monitoring

[0210] The cerebral blood flow (CBF) of mice was monitored by laser speckle imaging system at four time points: before ischemia, 10 minutes after ischemia, 10 minutes after reperfusion, and 24 hours after reperfusion. The relative CBF (%) of each group of mice relative to the baseline level was calculated.

[0211] 5-4-13 Treadmill Test

[0212] The treadmill test is used to perform forced training on mice and evaluate the motor coordination ability of mice in the acute phase of ischemia. After giving appropriate learning time, the mice are placed on the treadmill belt, and the transmission speed is slowly increased to the predetermined test speed, and the length of time the mice stay on the treadmill is recorded.

[0213] 5-4-14 RNA-Sequence

[0214] The RNA-Sequence sequencing part was commissioned to Shanghai Shupu Biological Company. The total RNA of the sample was tested for integrity using agarose gel electrophoresis, and was quantified and further quality checked using NanoDrop ND-1000. 1-2 μg of total RNA was selected from each sample for sequencing library construction. The constructed library was quality checked by Agilent 2100Bioanalyzer (library concentration, fragment size 400-600bp, whether it contains adapters, etc.), and the final quantification of the library was performed by qPCR. According to the quantitative results and the final sequencing data volume requirements, the sequencing libraries of different samples were mixed and entered into the sequencing process, n=3 / group.

[0215] 5-4-15MeRIP-Sequence

[0216] The MeRIP-Sequence sequencing was commissioned by Shanghai Shupu Biotechnology Co., Ltd. The total RNA was tested by agarose gel electrophoresis and then quantified by Nanodrop. After extracting mRNA, the separated mRNA was broken into fragments of about 300 nt using chemical methods, and then quantified by m 6 A antibody was used for enrichment, and the main process included immunoprecipitation, washing and elution. 6 A mRNA and input mRNA were enriched separately, and sequencing libraries were constructed and quality checked at the same time, and finally sequenced on a sequencer, n=3 / group.

[0217] 5-4-16 Statistical Methods

[0218] GraphPad Prism 8.0.2 software was used for statistical analysis. The measured data were in accordance with normal distribution and expressed as mean ± standard deviation (Mean ± SD). Unpaired t-test was used to compare two independent groups. One-way or two-way analysis of variance was used for comparison of more than two groups, and Tukey's multiple comparison test was used. P < 0.05 was considered statistically significant.

[0219] Evaluation of drug dosage and efficacy in permanent cerebral ischemia in 5-5pd MCAO model mice

[0220] 5-5-1 PdMCAO cerebral infarction and neurological function evaluation in male mice

[0221] (1) The pdMCAO model animals were divided into the following groups: ① sham operation group (Sham, n = 5), ② model control group (pdMCAO, n = 18), ③ solvent group (0.9% NS, n = 18), ④ Aleeto 5 μg / kg group (Aleeto 5 μg / kg, n = 17), ⑤ Aleeto 10 μg / kg group (Aleeto 10 μg / kg, n = 18), ⑥ Aleeto 20 μg / kg group (Aleeto 20 μg / kg, n = 18), ⑦ Aleeto 40 μg / kg group (Aleeto 40 μg / kg, n = 18), ⑧ Aleeto 60 μg / kg group (Aleeto 60 μg / kg, n = 18), ⑨ Aleeto 80 μg / kg group (Aleeto 80 μg / kg, n=18), ⑩ edaravone and dextran concentrated solution for injection group (ED, n=18), postsynaptic density-95 protein inhibitor group (NA-1, n=18).

[0222] (2) Experimental route: see Figure 4 .

[0223] (3) Experimental results

[0224] Fig.14 AB are the cerebral infarction conditions of mice in each group 24 hours after pdMCAO. 3 ) compared with the alituo 10 μg / kg group (15.92±8.123 mm 3 , p=0.0036), Alitol 20 μg / kg group (17.06±5.634 mm 3 , p = 0.0154), aritol 40 μg / kg (17.77 ± 5.237 mm 3 , p = 0.0346), aritol 60 μg / kg (17.76 ± 8.4 mm 3 , p = 0.0341), positive control drug ED (17.12 ± 7.356 mm 3 , p = 0.0164), positive control drug NA-1 (16.67 ± 7.439 mm 3 , p=0.0095) mice had significantly reduced cerebral infarction volume. Fig.14C is the neurological function score of mice in each group 24 hours after pdMCAO. Compared with the 0.9% NS group, the score of the 10μg / kg group of alituo (p=0.009) was significantly higher. The above results suggest that the test drug alituo has a clear therapeutic effect on acute brain injury in pdMCAO ischemic stroke in mice, and alituo may have a certain improvement effect on the recovery of neurological function in pdMCAO ischemic stroke, which has positive guiding significance for the treatment of clinical ischemic stroke.

[0225] 5-5-2 PdMCAO cerebral infarction and neurological function evaluation in female castrated mice

[0226] (1) The pdMCAO model animals were divided into two groups: a solvent group (pdMCAO+0.9% NS, n=15) and an Aleeto injection 20 μg / kg group (pdMCAO+Aleeto 20 μg / kg, n=15).

[0227] (2) Experimental route: 14 days before the model was established, the ovaries of female mice were surgically removed and a randomized controlled pdMCAO model was established. Drugs were administered immediately after the model was established to evaluate the neurological function scores of ovariectomized female mice after cerebral ischemia for 24 hours. The volume of cerebral infarction was determined by TTC staining to determine whether alituo injection had a protective effect on female mice with permanent cerebral ischemia. Figure 5 .

[0228] (3) Experimental results

[0229] Fig.15 AB are the cerebral infarction conditions of mice in each group 24 hours after pdMCAO. 3 ) compared with the alituo 20 μg / kg group (20.51±8.873mm 3 , p = 0.0621) mice had a reduced cerebral infarction volume, with no statistically significant difference. Fig.15 C, There was no statistical difference in neurological function score 24h after pdMCAO in the 20μg / kg group compared with the control group. The above results suggest that Alituo may have a certain effect on reducing the volume of cerebral infarction in the acute phase of pdMCAO ischemic stroke in ovariectomized female mice, but has no obvious effect on the recovery of neurological function.

[0230] Effects of 5-6 ppMCAO model on neurological and cognitive functions in male mice at 35 days

[0231] (1) The animals of the ppMCAO model were divided into the following groups: ① sham operation group (Sham, n = 10), ② solvent 0-14d group (ppMCAO + 0.9% NS 0-14d, n = 37), ③ Aleeto injection 20 μg / kg 0-14d group (ppMCAO + Aleeto 20 μg / kg 0-14d, n = 37), ④ solvent 8-21d group (ppMCAO + 0.9% NS 8-21d, n = 15), ⑤ Aleeto injection 20 μg / kg 8-21d group (ppMCAO + Aleeto 20 μg / kg 8-21d, n = 15), ⑥ Edaravone and dextromethorphan concentrated solution for injection group (ppMCAO + ED, n = 15). Neurological function scores were performed on days 1, 7, 14, 21, 28, and 35 after modeling; corner turning tests were performed on days 1, 7, 14, 21, 28, and 35; water maze training was performed on days 20, 21, 22, 23, and 24, and a water maze test was performed on day 25; an open field test was performed on day 26; and a Y maze test was performed on day 27.

[0232] (2) Experimental route: The ppMCAO model was studied by sampling at 35 days, and the drug was administered immediately after the model was established until 14 days, and then from 8 days after the model was established until 21 days, to evaluate the effects of alituo injection on the neurological function and cognition of male mice during the recovery period of permanent cerebral ischemia. Figure 6 .

[0233] (3) Experimental results

[0234] (3.1)Weight

[0235] Fig.16 The weight of mice in each group after ppMCAO. There was no significant difference in weight between the 0-14d group and the 0.9% NS 0-14d group. Compared with the 0.9% NS 8-21d group, the weight of mice in the 8-21d group increased to a certain extent, but there was no statistical difference.

[0236] (3.2) Neurological function score

[0237] The neurological function of mice was scored at 1, 7, 14, 21, 28, and 35 days after ppMCAO. Fig.17 Compared with the mice in the 0.9% NS 0-14d group, the neurological function scores of mice in the alituo 0-14d group (p=0.0004) were significantly improved, and the neurological function of mice in the alituo 8-21d group (p=0.0241) was also significantly higher than that in the 0.9% NS 8-21d group. This suggests that alituo may improve the neurological function of mice in the recovery period of ppMCAO ischemic stroke.

[0238] (3.3) Corner test

[0239] The mice were tested for corner turning at 1, 7, 14, 21, 28, and 35 days after ppMCAO. The results of the right turning rate of the mice are shown in Fig.18 There was no statistically significant difference in the right turning rate between the alituo 0-14d group and the 0.9% NS 0-14d group, and between the alituo 8-21d group and the 0.9% NS 8-21d group.

[0240] (3.4) Evaluation of cognitive memory function

[0241] (3.4.1) Water maze

[0242] The mice were trained in the water maze test 20-24 days after ppMCAO and tested in the water maze test 25 days later. Fig.19 As shown. During the training phase, Fig.19 A. Fig.19 D is the escape latency of mice, that is, the time it takes for mice to find the platform. There was no statistical difference between the alituo group and the 0.9% NS group at each single time point and the overall level. During the test phase, the percentage of time that mice in each group stayed in the target quadrant ( Fig.19 B, E), average swimming speed ( Fig.19 C, F) had no statistical difference.

[0243] (3.4.2) Open field

[0244] The mice were subjected to open field test 26 days after ppMCAO. Fig. 20 As shown, Fig. 20 A and B are the time that mice in each group stayed in the central area of ​​the open field. There was no statistical difference between the alituo group and the 0.9% NS group.

[0245] (3.4.3) Maze

[0246] The mice were subjected to Y-maze test 27 days after ppMCAO. Fig.21 As shown, A and B are the time that mice in each group stayed in the new arm in the Y maze. Compared with 0.9% NS 0-14d, mice in the Alito 0-14d group (p=0.1279) stayed longer in the new arm, with no statistical difference. Compared with 0.9% NS 8-21d, mice in the Alito 8-21d group (p=0.0777) stayed longer in the new arm, with no statistical difference.

[0247] Evaluation of drug dosage and efficacy in permanent cerebral ischemia in 5-7t MCAO model mice

[0248] 5-7-1 tMCAO cerebral infarction and neurological function evaluation in male mice

[0249] (1) The tMCAO model animals were divided into the following groups: ① sham operation group (Sham, n = 8), ② model control group (tMCAO, n = 8), ③ solvent group (tMCAO + 0.9% NS, n = 12), ④ Aleeto injection 10 μg / kg group (tMCAO + Aleeto 10 μg / kg, n = 15), ⑤ Aleeto injection 20 μg / kg group (tMCAO + Aleeto 20 μg / kg, n = 14), ⑥ Aleeto injection 40 μg / kg group (tMCAO + Aleeto 40 μg / kg, n = 13), ⑦ Edaravone and dexamethasone concentrated solution for injection group (tMCAO + ED, n = 14), ⑧ postsynaptic density-95 protein inhibitor group (tMCAO + NA-1, n = 12).

[0250] (2) Experimental route: see Figure 7 .

[0251] (3) Experimental results: Fig. 22 As shown, Fig. 22 A is a representative brain slice of each group of mice stained with TTC. B is the cerebral infarction of each group of mice after tMCAO for 24 hours: compared with the 0.9% NS group (91.52±22.64mm 3 ) compared with the alituo 20 μg / kg group (57.36±17.76 mm 3 , p = 0.0004), aritol 40 μg / kg (68.37 ± 20.66 mm 3 , p = 0.0286), positive control NA-1 group (69.74 ± 21.52 mm 3 , p = 0.0495) mice had significantly reduced cerebral infarction volume. C is the edema of mice in each group 24h after tMCAO: compared with the 0.9% NS group, the relative edema degree of the alituo 20μg / kg group (p = 0.0419) was significantly reduced. D is the neurological function score of mice in each group 24h after tMCAO: compared with the 0.9% NS group, the scores of the alituo 10μg / kg group (p = 0.0054), alituo 20μg / kg group (p = 0.0086), and alituo 40μg / kg group (p = 0.0389) were significantly improved. The above results suggest that the test drug alituo has a clear therapeutic effect on the acute phase of tMCAO ischemic reperfusion brain injury in mice, and alituo may have a certain improvement effect on the recovery of neurological function of tMCAO ischemic stroke, and when its dose is 20μg / kg, it can significantly reduce the cerebral infarction volume and edema degree of mice.

[0252] 5-7-2 Evaluation of the medication time window for tMCAO in male mice

[0253] (1) The animals of the tMCAO model were divided into the following groups: drug administration 2 h after ischemia, including the solvent group (tMCAO+0.9% NS, n=17) and the Aleeto injection 20 μg / kg group (tMCAO+Aleeto 20 μg / kg, n=18); drug administration 3 h after ischemia, including the solvent group (tMCAO+0.9% NS, n=17) and the Aleeto injection 20 μg / kg group (tMCAO+Aleeto 20 μg / kg, n=16); drug administration 4 h after ischemia, including the solvent group (tMCAO+0.9% NS, n=17) and the Aleeto injection 20 μg / kg group (tMCAO+Aleeto 20 μg / kg, n=17); drug administration 5.5 h after ischemia, including the solvent group (tMCAO+0.9% NS, n=17) and the Aleeto injection 20 μg / kg group (tMCAO+Aleeto 20 μg / kg, n=17).

[0254] (2) Experimental route: see Figure 8 .

[0255] (3) Experimental results: Fig.23 The drug administration at different time points after tMCAO. In the figure, C, injection into the tail vein 2 hours after ischemia, compared with the 0.9% NS group (116.0±17.69mm 3 ) compared with the alituo 20 μg / kg group (94.44±27.45 mm 3 , p = 0.0098) mice had a significantly reduced cerebral infarction volume. G, 3 h after ischemia, the cerebral infarction volume of the mice was significantly reduced compared with that of the 0.9% NS group (102.9 ± 19.65 mm 3 ) compared with the alituo 20 μg / kg group (85.54±21.31 mm 3 , p = 0.0210) mice had significantly reduced cerebral infarction volume, and the neurological function score of aritol (p = 0.0332) was higher than that of NS group (H), with statistical difference. K, O, 4h and 5.5h after ischemia, the cerebral infarction volume of mice in aritol 20μg / kg group had a decreasing trend compared with 0.9% NS group, with no statistical difference.

[0256] 5-7-3 Cerebral infarction and neurological function in female castrated mice induced by tMCAO

[0257] (1) The animals in the tMCAO model were divided into the following groups: 14 days before the model was established, the ovaries of female mice were surgically removed, and a randomized controlled tMCAO model study was performed. Immediately after the model was established, the mice were given drugs and divided into two groups: a solvent group (tMCAO+0.9% NS, n=16) and an Aleeto injection 20 μg / kg group (tMCAO+Aleeto 20 μg / kg, n=15).

[0258] (2) Experimental route: see Fig. 9 .

[0259] (3) Experimental results: Fig.24 As shown, Fig.24 B shows the cerebral infarction of mice in each group 24 hours after tMCAO. 3 ) compared with the alituo 20 μg / kg group (82.02±20.77 mm 3 , p = 0.0247) mice had a significant reduction in cerebral infarction volume, but the degree of brain edema in the alituo group was not significantly different from that in the solvent control group. Fig.24 As shown in D, the neurological function score of ovariectomized female mice 24 hours after tMCAO showed an upward trend in the 20 μg / kg group compared with the control group, with no statistical difference. The above results suggest that aritol may have a certain effect on reducing the volume of cerebral infarction in the acute phase of tMCAO ischemia-reperfusion in ovariectomized female mice and improving neurological function.

[0260] 5-7-4 Effects of tMCAO in male aged mice 3 days after induction of tMCAO

[0261] (1) The animals of the tMCAO model were divided into the following groups: solvent group (tMCAO+0.9% NS, n=6), Aleeto injection 20 μg / kg group (tMCAO+Aleeto 20 μg / kg, n=5).

[0262] (2) Experimental route: see Fig.10 Neurological function scores were performed 1 day and 3 days after the model was established, and magnetic resonance imaging was used to evaluate the infarction volume and edema degree on 1 day.

[0263] (3) Experimental results: Fig.25 As shown in Figure 2, alitox has a tendency to reduce the survival rate of elderly mice in the acute phase of cerebral ischemia-reperfusion injury ( Fig.25 C), there was no statistical difference. Compared with the 0.9% NS group, the cerebral infarction rate and brain edema degree of the elderly mice in the aritol 20 μg / kg group showed a downward trend ( Fig.25 D, E), there is no significant difference. Fig.25 As shown in D, the neurological function score of the castrated female rats in the 20 μg / kg group of alituo showed an upward trend compared with the control group 24 hours after tMCAO, but there was no statistical difference. The above results suggest that alituo may have a certain effect on reducing the volume of cerebral infarction in the acute phase of tMCAO ischemia-reperfusion in male elderly rats and improve neurological function.

[0264] 5-7-5 Effects of tMCAO in male aged mice 7 days after induction of tMCAO

[0265] (1) The animals of the tMCAO model were divided into the following groups: solvent group (tMCAO+0.9% NS, n=18) and Aleeto injection 20 μg / kg group (tMCAO+Aleeto 20 μg / kg, n=16).

[0266] (2) Experimental route: see Fig.11 .

[0267] (3) Experimental results: The laser speckle imaging system was used to monitor the cerebral blood flow of mice at four time points: before ischemia, 10 minutes after ischemia, 10 minutes after reperfusion, and 24 hours after reperfusion. Magnetic resonance imaging was used to evaluate the infarct volume and edema degree 3 days after the model was established. Fig.26 The representative graphs and statistical results of cerebral blood flow in mice at various time points show that compared with the 0.9% NS group, the cerebral blood flow in the alituo group 10 minutes after reperfusion showed an increasing trend, but there was no statistical difference.

[0268] Three days after tMCAO, the mice in each group were subjected to MRI T2 scanning, and the cerebral infarction and edema were shown in Fig. 27 The cerebral infarction volume of mice in the 0.9% NS group was 64.88±17.46mm 3 , Alitol 20μg / kg group was 34.03±20.29mm 3 The cerebral infarction rate of mice in the 0.9% NS group was 21.04±5.623 mm 3 , the aritol 20μg / kg group was 10.43±6.178mm 3 Compared with the 0.9% NS group, the cerebral infarction volume, cerebral infarction rate and relative edema of mice in the alituo 20 μg / kg group were significantly reduced ( Fig. 27 BD, p=0.0328, p=0.0218, p=0.0038), which was statistically significant.

[0269] 5-7-6 Effects of tMCAO in male aged mice 35 days after tMCAO

[0270] (1) The animals of the tMCAO model were divided into six groups as follows: ① sham operation group (Sham, n = 11), ② solvent 0-14d group (tMCAO + 0.9% NS 0-14d, n = 28), ③ Aleeto injection 20 μg / kg 0-14d group (tMCAO + Aleeto 20 μg / kg 0-14d, n = 29), ④ solvent 8-21d group (tMCAO + 0.9% NS 8-21d, n = 28), ⑤ Aleeto injection 20 μg / kg 8-21d group (tMCAO + Aleeto 20 μg / kg 8-21d, n = 28), ⑥ Edaravone and dexamethasone concentrated solution for injection group (tMCAO + ED, n = 29). Neurological function scores were performed on days 1, 3, 5, 7, 9, 11, 14, and 21 after modeling; treadmill tests were performed on days 3, 7, 9, 11, 14, and 21; and a new location recognition experiment was performed on day 25.

[0271] (2) Experimental route: see Fig.11 .

[0272] (3) Experimental results:

[0273] (3.1)Weight

[0274] Fig.28 The weight of mice in each group during the recovery period after tMCAO. Compared with 0.9% NS 0-14d, the weight of mice in the Alitol 0-14d and positive control ED groups increased significantly; and compared with 0.9% NS 8-21d, the weight of mice in the Alitol 8-21d and positive control ED groups increased significantly, with statistical differences.

[0275] (3.2) Neurological function score

[0276] The neurological function of mice was scored after tMCAO. The scoring results are shown in Fig.29 As shown. Compared with the 0.9% NS 0-14d group, the neurological function scores of mice in the alituo 0-14d group showed an upward trend, with no statistical difference; the neurological function of mice in the alituo 8-21d group (p=0.0127) was significantly higher than that in the 0.9% NS 8-21d group. This suggests that alituo may improve the neurological function of mice in the recovery period of tMCAO ischemic stroke.

[0277] (3.3) Treadmill test

[0278] Treadmill tests were performed on days 3, 7, 9, 11, 14, and 21 after tMCAO in mice to evaluate motor function. Fig.30 Compared with 0.9% NS 0-14d, mice in the alituo 0-14d group stayed on the treadmill longer overall, but there was no statistical difference.

[0279] (3.4) Evaluation of cognitive memory function

[0280] After tMCAO, mice were subjected to a novel location recognition experiment. Fig.31 The figure shows the percentage of time that mice spent exploring the same object in a new location. The percentage of time in the alituo group and the 0.9% NS group showed an upward trend, but there was no statistical difference.

[0281] 5-8 Preliminary study on the mechanism of alitox in treating subacute stroke

[0282] 5-8-1 The mechanism of action of Alito in treating subacute stroke may be related to synapse formation and nerve conduction

[0283] In order to explore the therapeutic mechanism of alituo in the subacute phase of stroke, we performed mRNA and mRNA expression analysis in different experimental groups of mice (a group treated with alituo for 7 days after stroke and a stroke control group). 6 A Sequencing analysis. The distribution of the mRNA differential expression profile dataset is shown in the form of a dendrogram ( Fig.32 A), using the volcano map ( Fig.32 B) to filter and identify the statistically significant differentially expressed mRNAs between the two groups. Fig.32 C) to represent the mRNA mRNA of mice in different experimental groups 6 A site distribution, found differential m 6 The A sites are mainly distributed at the junction of the 5'UTR region and the CDS region. Fig.32 D) to filter and identify statistically significant differences between the two groups 6 A site. Therefore, in order to find the possible target of aritol in the treatment of subacute stroke ( Fig.32 E), respectively, after treatment with Alito 6 A 2924 differentially upregulated genes by methylation and 69 genes with differentially upregulated mRNA expression, m 6 A 946 differentially down-regulated genes and 94 genes with differentially up-regulated mRNA expression, m 6 A. 2943 differentially upregulated genes and 75 differentially downregulated genes in mRNA expression 6 The intersection of 942 differentially upregulated genes in A methylation and 77 differentially upregulated genes in mRNA expression was obtained, and 28 m 6 A up-regulated mRNA up-regulated genes, 3 m 6 A down-regulated mRNA up-regulated genes, 9 m 6 A up-regulated mRNA down-regulated genes and 7 m 6A. The genes whose mRNA was downregulated were all possible target genes of aritol in the treatment of subacute stroke.

[0284] In order to study the differentially expressed mRNA and m 6 A potential function, we performed GO enrichment analysis to reveal the functions of the predicted differentially expressed genes, including the biological processes involved in the mRNA, molecular functions, cellular composition ( Fig.33 A). Similarly, we have differentiated m 6 A. mRNA was also subjected to GO analysis to reveal the predicted differential m 6 A. mRNA functions, including biological processes, molecular functions, and cell composition ( Fig.33 B). The above sequencing analysis shows that the mechanism of action of Alito in treating subacute stroke may be related to synapse formation, nerve conduction, and cell signaling.

[0285] 6. Summary and discussion

[0286] (1) In rat primary neuron culture, 25 ng / mL of alitate can increase the axon length and dendrite length of normal primary neurons; after oxygen-glucose deprivation, the number of axon and dendritic branches is increased, suggesting that alitate helps nerve fiber growth and conduction.

[0287] (2) This study used a randomized, controlled, blinded, and multi-model approach to conduct a preliminary exploration of the dosage and neurocognitive function of Alito in ischemic brain injury in mice. This report can provide data support for further in-depth research on the mechanism of action of Alito. The research results only represent the test results of this batch of Alito in C57 mice.

[0288] In the pdMCAO model, 10 μg / kg, 20 μg / kg, 40 μg / kg, and 60 μg / kg of aritol could significantly reduce the volume of cerebral infarction in the acute phase of ischemia in mice ( Fig.14 ); in female mice, although the effect of alitoxan on cerebral infarction was not statistically significant, it showed a trend of reducing cerebral infarction ( Fig.15 During the recovery period, Alito was able to improve neurological function scores and had a good trend of improvement in cognition ( Fig.21 ).

[0289] In the acute phase of cerebral ischemia-reperfusion, the 20 μg / kg dose group of Alitol had a significant effect on reducing the volume of cerebral infarction, reducing relative edema, and improving neurological function in mice ( Fig. 22 ), and this dose was used in subsequent experiments. In the study of the medication time window, the administration of Alito within 2h and 3h of transient cerebral ischemia reperfusion can still reduce the volume of cerebral infarction, and there is still a statistical trend when the treatment is 4h and 5.5h of ischemia reperfusion ( Fig.23), based on the experimental results of this part, the administration time point can be extended to continue exploring the possible application time of Alitol.

[0290] At 24h of tMCAO, cerebral infarction and edema in female castrated mice and male aged mice were evaluated by TTC staining and nuclear magnetic resonance imaging, respectively. The results showed that alituo had a good trend of reducing cerebral infarction volume in castrated female mice and aged mice with cerebral ischemia-reperfusion ( Fig.24 , Fig.25 ), and it also plays a certain role in improving the survival rate of elderly mice.

[0291] In the cerebral ischemia-reperfusion model, laser speckle was used to observe the local blood flow in the mouse brain. Ten minutes after reperfusion, Alito showed a trend of increasing local cerebral blood flow ( Fig.26 ), suggesting that alituo may have a certain vasodilatory effect. Three days after tMCAO, MRI scans of the brains of cerebral ischemic mice showed that alituo could significantly reduce the volume of cerebral infarction and relative brain edema ( Fig. 27 In long-term observations during the recovery period, alituo helped mice regain body weight after transient cerebral ischemia ( Fig.28 ), and the neurological function score was improved to a certain extent ( Fig.29 ), which has a tendency to promote long-term motor function recovery ( Fig.30 ), which may have a certain improvement effect on cognitive function ( Fig.31 ).

[0292] For ordinary technicians in the technical field to which the present application belongs, simple deduction or substitution without departing from the concept of the present application is within the protection scope of the present application.

Claims

1. A use of a stress-induced stem cell extract for treating stroke, characterized in that: The preparation of the stem cell extract comprises the following steps: S1) culturing stem cells and creating stress conditions to stimulate the stem cells; S2) collecting the culture supernatant of the stem cells in S1) or lysing the stem cells in S1) to collect lysates, and separating and purifying the culture supernatant or lysates to obtain the stem cell extract; The separation and purification of the culture supernatant or the lysate comprises the following steps: S21) filtering the culture supernatant or the lysate through a 0.22 μm filter membrane to obtain a mother solution, and storing the mother solution at 4° C.; S22) filtering and concentrating the mother solution using an ultrafiltration concentration tube with a molecular weight cutoff of 3KD to obtain a crude sample of the stem cell extract; S23) purifying the crude sample by molecular sieve exclusion chromatography or purifying the mother solution by reverse phase chromatography to prepare a purified sample of the stem cell extract; wherein the purified sample also satisfies the following characteristics: S231) in the molecular sieve exclusion chromatography, taking the volume of the eluent as a reference, collecting the 10th to 40th ml of the eluent as the purified sample; S232) In the reverse phase chromatography, taking the elution time as a reference, collecting the eluate with the elution time between 10 min and 40 min as the purified sample.

2. The use according to claim 1, characterized in that: In the molecular sieve exclusion chromatography, the instrument used is AKTAexplorer, and the chromatography column is a superdex150 chromatography column with a size of 8×500 and a column volume of about 30 mL; the operation steps are: S2311) washing the chromatography column with 2CV of pure water, then balancing the chromatography column with 2CV of PBS, and returning the ultraviolet absorption value of the chromatography column at 280nm to zero; as well as S2312) Take a predetermined injection amount of the crude sample for loading, the loading flow rate is 0.4mL / min, the PBS elution flow rate is 0.1-0.3mL / min, and elute to the peak. When the volume of the eluent reaches 10ml, the separated elution liquid is received to prepare the purified sample.

3. The use according to claim 2, characterized in that: The purified sample of the stem cell extract also meets the following characteristics: The purified sample of a predetermined volume segment is collected for SDS-PAGE electrophoresis analysis, and it is measured that the purified sample at least contains protein molecules with molecular weights mainly distributed between 11KD and 100KD, wherein, from large to small molecular weights, the first band is located between 75KD and 100KD, and the second band is located between 63KD and 75KD.

4. The use according to claim 3, characterized in that: The purified sample of the stem cell extract also meets the following characteristics: According to the order of elution, the eluate is divided into five elution volume segments, and the first component sample with an elution volume segment of 12.0 to 13.2 mL, the second component sample with an elution volume segment of 15.2 to 17.0 mL, the third component sample with an elution volume segment of 17.0 to 20.0 mL, the fourth component sample with an elution volume segment of 29.0 to 31.0 mL, and the fifth component sample with an elution volume segment of 31.0 to 34.0 mL are collected respectively according to the order of elution. The sample used for the SDS-PAGE electrophoresis analysis is the third component sample.

5. The use according to claim 1, characterized in that: In the reverse phase chromatography, the instrument used was a high performance liquid chromatograph, the chromatographic column was XBridge Protein BEH C4, and the pore size of the filler was The filler particle size is 3.5 μm, the inner diameter*length is 4.6 mm*150 mm, and the mobile phase is PBS; the operation steps are as follows: S2321) adding PBS to dilute the mother solution; S2322) Take 80-120 μL of the diluted mother solution for sample loading, control the column temperature of the chromatographic column to 20-30°C, the flow rate to 0.2-0.8 mL / min, the detection wavelength to 260 nm and / or 280 nm, set the separation time to 48 min, collect the eluate with the peak time at 10-40 min, and prepare the purified sample.

6. The use according to claim 5, characterized in that: There are ten characteristic peaks in the elution time of 10 to 40 minutes of the reverse phase chromatography, among which the elution time of characteristic peak 1# is 13.3 to 14.5 minutes, the elution time of characteristic peak 2# is 21.5 to 22 minutes, and the elution time of characteristic peak 3# is 22 to 22.8 minutes; the elution time of characteristic peak 4# is 23 to 23.7 minutes, the elution time of characteristic peak 5# is 26.8 to 27.8 minutes, the elution time of characteristic peak 6# is 27.8 to 28.7 minutes, the elution time of characteristic peak 7# is 28.7 to 30 minutes, the elution time of characteristic peak 8# is 30 to 30.6 minutes, the elution time of characteristic peak 9# is 30.6 to 32 minutes, and the elution time of characteristic peak 10# is 33.8 to 34.8 minutes; the elution liquids of the characteristic peaks 1# to 10# are collected respectively as the purified samples.

7. The use according to claim 1, characterized in that: The stress conditions are one or more of ultraviolet irradiation, infrared irradiation, electromagnetic field, high temperature, low temperature, hypoxia, high oxygen, oxidation, high pH, ​​low pH, ultrasound, terahertz electromagnetic waves, X-rays, microwaves, rays, rays, rays, ion beams, high CO2 and low CO2 stimulation conditions.

8. The use according to claim 7, characterized in that: The ultraviolet irradiation satisfies at least one of the following conditions: a. The irradiation time is 1 to 30 hours; b. Irradiation intensity is 10~100μW / cm 2 ; c. The wavelength of ultraviolet light is 290~340nm.

9. The use according to claim 1, characterized in that: The culture medium used to culture the stem cells is a serum-free stem cell culture medium; and / or the stem cell source can be one or more of mesenchymal stem cells, embryonic stem cells, in vitro induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells and MUSE cells, and the mesenchymal stem cells can be one or more of umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells and skin-derived mesenchymal stem cells.

10. The use according to claim 1, characterized in that: The method for preparing the lysate includes at least one of chemical lysis, enzymatic lysis, mechanical lysis, repeated thawing and freezing, boiling, osmotic pressure, liquid nitrogen and ultrasonic fragmentation; the method for separating and obtaining the stem cell extract from the lysate may also include at least one of chromatography, spectroscopy, dialysis, organic solvent precipitation, acid extraction, alkali extraction, ultrafiltration, chromatography, electrophoresis and centrifugation.

11. The use according to claim 1, wherein the drug prepared from the stem cell extract is an injection, a nasal spray or an oral preparation; or the drug is administered by a method selected from intrathecal administration, intravenous administration, a combination of intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, and oral administration.

12. The use according to claim 11, characterized in that: The dosage form of the drug is lyophilized powder, injection, gel, pill, tablet, powder or paste.

Images