A pharmaceutical composition for inducing reprogramming of astrocytes into neurons after cerebral hemorrhage and application thereof

Abstract

The application relates to the technical field of biological medicine, and particularly discloses a drug composition for inducing reprogramming of astrocytes after cerebral hemorrhage into neurons and application thereof. The drug composition provided by the application has simple components and safe components, can reprogram mouse striatal astrocytes into neurons under in-vitro culture conditions, and the neurons induced to generate are close to the distribution type of the striatal neuron subtypes in vivo. The drug composition can comprehensively cover the signal pathway regulation required for inducing cell fate conversion, cell metabolism pathway regulation and epigenetic regulation mechanism, and plays a synergistic role in reprogramming astrocytes after cerebral hemorrhage into neurons, thereby improving the effect of improving nerve function damage.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage and its application. Background Technology

[0002] Cerebral hemorrhage is a severe type of cerebrovascular disease characterized by high morbidity, high mortality, and high disability rates. The striatum is a common site of cerebral hemorrhage. It is usually caused by the rupture of blood vessels in the brain, leading to the accumulation of blood in the brain tissue, forming a hematoma, which in turn triggers a series of complex pathophysiological reactions, such as cerebral edema, neuroinflammation, and cell apoptosis. These pathological changes not only cause direct mechanical damage to brain tissue but also further exacerbate neuronal damage and death by triggering a series of cascade reactions.

[0003] Neural regeneration refers to the ability of damaged neural tissue to self-repair and restore function through a series of biological processes. Following striatal hemorrhage, neural regeneration plays a crucial role in restoring neurological function and improving prognosis. However, due to the complexity of the central nervous system and the multifactorial regulation of the neural regeneration process, neural regeneration in adults faces numerous challenges. In recent years, with the continuous advancement of regenerative medicine research, new technological changes have occurred in neural regeneration methods, providing new directions for developing therapeutic strategies that promote neural regeneration.

[0004] Somatic cell reprogramming technology offers a new approach to neural regeneration. By employing specific gene regulation methods or small molecule compound interventions, it alters the gene expression patterns of cells, transforming differentiated somatic cells (such as fibroblasts and glial cells) into neurons. This provides new possibilities for neural regeneration and the treatment of neurodegenerative diseases. Astrocytes are the most abundant type of glial cells in the brain, accounting for approximately 40% of the total brain cells. They play crucial roles in maintaining normal neuronal function, the integrity of the blood-brain barrier, regulating synaptic function, and supporting immune cells. However, in central nervous system injury or disease, astrocytes are reactively activated. These reactively activated astrocytes possess stem cell potential and, under appropriate gene regulation and chemical intervention, can be transformed into neurons. Therefore, astrocytes are excellent initiating cells for neuronal reprogramming. Currently, numerous reports have documented the use of viral vector-mediated gene regulation to reprogram astrocytes into neurons in neurological diseases such as Alzheimer's, spinal cord injury, Parkinson's, and ischemic stroke. However, a study published in Cell by Professor Chunli Zhang of the University of Texas, using rigorous astrocyte lineage tracing techniques, points out that these studies may have significant false-positive issues. Specifically, during the targeted infection and labeling of astrocytes using viral vectors, the virus's targeting may be lost, leading to the simultaneous infection and labeling of endogenous neurons. This results in a large number of endogenous neurons being mistakenly identified as neurons induced by reprogramming techniques, ultimately leading to erroneous conclusions. Furthermore, viral vector-mediated gene regulation carries immunogenicity and potential teratogenic risks, hindering clinical translation.

[0005] Small molecule compounds have become an important means of somatic cell reprogramming due to their advantages of ease of operation, low cost, and high safety. The mechanisms of action of these small molecule compounds include regulation of signaling pathways related to cell fate transformation, regulation of cellular metabolic pathways, and epigenetic regulation. Currently, studies have shown that the combined use of multiple small molecule compounds can reprogram astrocytes into neurons under in vitro culture conditions. Reprogramming astrocytes into neurons based on somatic cell reprogramming technology is a current hot topic in the field of neuroregenerative medicine. Among them, small molecule compound-mediated chemical reprogramming technology has broad application value due to its high safety, but its induction efficiency in the brain is still relatively low. Patent CN117815239A discloses a small molecule drug composition and its application, which includes VPA, CHIR99021, RepSox, RG108, DAPT, KC7f2, and forskolin. However, this small molecule drug composition contains too many types, making drug compounding complex, and the cell state is prone to deterioration.

[0006] A study (In vivo chemical reprogramming of astrocytes into neurons) induced mouse cortical and striatal astrocytes to be reprogrammed into neurons by in vivo continuous infusion of six small molecule compounds (DBcAMP, Forskolin, ISX9, CHIR99021, I BET151, and Y-27632) for 14 days. However, the induction efficiency was still low, with only 23 and 39 induced neurons detected near the small molecule infusion sites, respectively.

[0007] Following striatal hemorrhage, the small molecule drug composition disclosed in patent CN117815239A successfully induced the chemical reprogramming of astrocytes into neurons in vivo, with 24 neurons converted per unit field of view. Although this efficiency is significantly improved compared to the aforementioned six small molecule compounds, there is still room for improvement. Summary of the Invention

[0008] The purpose of this application is to overcome the shortcomings of the prior art and provide a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage and its application. The pharmaceutical composition of this application can efficiently convert astrocytes after striatal hemorrhage into neurons, thereby improving neurological function impairment after cerebral hemorrhage. Furthermore, this pharmaceutical composition can be extended to the treatment of cerebral ischemia, reprogramming astrocytes after cerebral ischemia into neurons to improve neurological function.

[0009] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage, the pharmaceutical composition comprising calcitriol, DAPT, LDN193189 and RG108; The molar concentration of calcitriol in the pharmaceutical composition is 0.375 μM to 0.75 μM; The molar concentration of DAPT in the pharmaceutical composition is 3.75 μM to 7.5 μM; The molar concentration of LDN193189 in the pharmaceutical composition is 0.1875 μM ~ 0.375 μM; The molar concentration of RG108 in the pharmaceutical composition is 7.5 μM to 15 μM.

[0010] Through extensive research and experimentation, the inventors of this application have discovered that a combination of four components—calcitriol, DAPT, LDN193189, and RG108—used at specific molar concentrations can efficiently convert astrocytes following striatal hemorrhage into neurons. This combination of small molecule drugs, using Aldh1l1CreER, [further details needed].T2 Rigorous lineage tracing of astrocytes was performed in Ai9 transgenic mice to verify the reliability of the drug composition in in vivo chemical reprogramming of astrocytes into neurons after striatal hemorrhage in rodents, and its ability to improve neurological function damage after cerebral hemorrhage.

[0011] Furthermore, the pharmaceutical composition of this application can comprehensively cover the signaling pathway regulation, cell metabolic pathway regulation and epigenetic regulation mechanisms required to induce cell fate transformation, thereby synergistically reprogramming astrocytes after cerebral hemorrhage into neurons and thus improving neurological function damage.

[0012] In this application, the addition of calcitriol can promote neuronal survival and axonal growth.

[0013] Adding DAPT can regulate the level of Notch-related signaling pathways in astrocytes, downregulate glial cell fate-related genes, and upregulate the expression level of pro-neural transcription factors.

[0014] Adding LDN193189 can regulate the level of BMP1-related signaling pathways in astrocytes, and synergistically downregulate glial cell fate-related genes and upregulate the expression level of pro-neural transcription factors in conjunction with Notch pathway inhibitors.

[0015] Adding RG108 alters the methylation state of astrocytes, promoting the initiation of reprogramming.

[0016] By inhibiting classic glial cell fate-related signaling pathways (Notch pathway, BMP pathway), regulating epigenetic characteristics (RG108 promotes cell cycle restart), and combining calcitriol to promote neuronal survival and axon growth, astrocytes are promoted to transform into neurons and survive from multiple dimensions.

[0017] Experiments have shown that the pharmaceutical composition of this application can efficiently reprogram striatal astrocytes into neurons under in vitro culture conditions, with a reprogramming efficiency of 75-85%. The reprogramming efficiency decreases when any component of the pharmaceutical composition is missing.

[0018] When the drug composition uses calcitriol, DAPT, LDN193189 and RG108 in a synergistic combination, it can reprogram astrocytes after cerebral hemorrhage into neurons. The induced neurons are similar in distribution to the subtypes of neurons in the striatum in vivo, and the reprogramming efficiency is higher. Furthermore, when the above components are used at the above molar concentrations, striatal astrocytes can be reprogrammed into neurons more efficiently under in vitro culture conditions.

[0019] In a preferred embodiment of the pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage as described in this application, the molar concentration of calcitriol in the pharmaceutical composition is 0.5 μM to 0.75 μM. The molar concentration of DAPT in the pharmaceutical composition is 5 μM to 7.5 μM; The molar concentration of LDN193189 in the pharmaceutical composition is 0.25 μM ~ 0.375 μM; The molar concentration of RG108 in the pharmaceutical composition is 10 μM to 15 μM.

[0020] When calcitriol, DAPT, LDN193189, and RG108 are preferably used in the above molar ratio, striatal astrocytes can be reprogrammed into neurons more efficiently under in vitro culture conditions, resulting in higher reprogramming efficiency.

[0021] In a preferred embodiment of the pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage as described in this application, the molar concentration of calcitriol in the pharmaceutical composition is 0.5 μM. The molar concentration of DAPT in the pharmaceutical composition is 5 μM; The molar concentration of LDN193189 in the pharmaceutical composition is 0.25 μM; The molar concentration of RG108 in the pharmaceutical composition is 10 μM.

[0022] When calcitriol, DAPT, LDN193189, and RG108 are more preferably in the above molar concentration ratio, the reprogramming efficiency of striatal astrocytes into neurons under in vitro culture conditions is higher, and the synergistic effect of the four components is better.

[0023] This application also provides the use of the above-mentioned pharmaceutical composition in the preparation of products that induce in vitro astrocyte reprogramming into neurons.

[0024] In a preferred embodiment of the application described in this application, the astrocytes are derived from the striatum.

[0025] This application also provides the use of the above-mentioned pharmaceutical composition in the preparation of drugs for the prevention and / or treatment of cerebral hemorrhage or cerebral ischemia.

[0026] This application also provides a drug for the prevention and / or treatment of cerebral hemorrhage or cerebral ischemia, the drug comprising the above-described pharmaceutical composition.

[0027] This application also provides a method for reprogramming astrocytes into neurons, the method comprising: culturing isolated astrocytes in an induction medium containing the aforementioned pharmaceutical composition, and then culturing them in a differentiating medium, thereby transforming the astrocytes into neuronal cells.

[0028] This application, through extensive screening, discovered a pharmaceutical composition of four small molecule compounds that can efficiently induce the reprogramming of striatal astrocytes into neurons under induction culture conditions. Combined with astrocyte lineage tracing technology, the reliability of this pharmaceutical composition in in vitro inducing the chemical reprogramming of astrocytes into neurons after striatal hemorrhage was verified. Furthermore, this pharmaceutical composition can effectively treat cerebral hemorrhage and improve neurological deficits in mice with cerebral hemorrhage.

[0029] In some specific embodiments, the conditions for culturing isolated astrocytes in an induction medium containing the pharmaceutical composition include a temperature of 35-38°C, preferably 37°C.

[0030] In some specific embodiments, the conditions for culturing by changing the differentiation medium include: a temperature of 35~38℃, preferably 37℃.

[0031] In some specific embodiments, isolated astrocytes are cultured in Am-a medium to a density of 80-90%.

[0032] In some specific embodiments, isolated astrocytes are cultured in an induction medium containing the drug composition for 4 to 7 days, and then cultured in a differentiation medium for another 4 to 7 days.

[0033] Preferably, Map2 is first observed by culturing isolated astrocytes in an induction medium containing the drug composition for 4 days, followed by culturing in a differentiation medium for another 4 days. + Neurons.

[0034] As a preferred embodiment of the method described in this application, the induction culture medium further includes a mixed basal culture medium and an additive A dissolved in the mixed basal culture medium; Additive A includes bFGF, fetal bovine serum, B27, and L-alanyl-L-glutamine dipeptide; The mixed basal medium includes DMEM / F12 and Neurobasal medium.

[0035] The volume ratio of DMEM / F12 and Neurobasal medium was 1:1.

[0036] In some specific embodiments, the differentiation medium includes Neurobasal medium and additive B dissolved in Neurobasal medium; Additive B includes B27, L-alanyl-L-glutamine dipeptide, and BDNF.

[0037] In a preferred embodiment of the method described in this application, the content of each component in additive A in the induction culture medium is as follows: bFGF at a mass concentration of 100 ng / ml, fetal bovine serum at a volume fraction of 2%, B27 at a volume fraction of 2%, and L-alanyl-L-glutamine dipeptide at a volume fraction of 1%. The components of additive B in the induction medium contain the following amounts: 2% B27 (volume fraction), 1% L-alanyl-L-glutamine dipeptide (volume fraction), and 20 ng / mL BDNF (mass concentration).

[0038] In the in vivo experimental portion of this application, astrocyte lineage tracer mice were used to explore the possibility and reliability of in situ reprogramming striatal astrocytes into neurons after cerebral hemorrhage. This avoids the off-target problem that may be caused by using viral methods to label astrocytes in their initial state, and avoids the false positive problem of misidentifying endogenous neurons that are mislabeled by the virus as newly generated neurons that have been reprogrammed.

[0039] Compared with the prior art, this application has the following beneficial effects: This application provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage and its application. The pharmaceutical composition provided in this application has simple and safe components and can reprogram mouse striatal astrocytes into neurons under in vitro culture conditions with a reprogramming efficiency of up to 85%. The induced neurons have a distribution pattern similar to the subtypes of neurons in the striatal region in vivo. In an in vitro cerebral hemorrhage model, the pharmaceutical composition induced the reprogramming of mouse striatal astrocytes into neurons with an efficiency of 28.3%. The pharmaceutical composition of this application can comprehensively cover the signaling pathway regulation, cellular metabolic pathway regulation, and epigenetic regulatory mechanisms required for inducing cell fate transformation, thereby synergistically reprogramming astrocytes into neurons after cerebral hemorrhage and thus improving neurological function impairment. Attached Figure Description

[0040] Figure 1 The flowchart for inducing reprogramming using a drug composition and the immunofluorescence display of Map2+ mature neurons are shown below. Figure 1 A is a flowchart illustrating the reprogramming process induced by the drug compositions described in Examples 1-3; AM: astrocyte culture medium; DMSO: dimethyl sulfoxide; hemin: heme chloride. Figure 1(Figure B shows the immunofluorescence visualization of Map2+ mature neurons induced by the drug compositions in the normal in vitro model (controlCDLR group), the drug compositions of Examples 1-3 in the Hemin-simulated in vitro brain hemorrhage model, and the drug compositions of Comparative Examples 1-21 in the Hemin-simulated in vitro brain hemorrhage model). Figure 2 This is a dynamic observation diagram of the reprogramming of mouse striatal astrocytes into neurons under in vitro culture conditions induced by a drug composition in Experiment Example 2. Figure 2 Image A shows dynamic images taken in the same field of view on days 2, 4, 6, 8, 10, 12, 14, and 16 of in vitro astrocyte reprogramming induced by the drug composition. Scale bar = 200 μm. Figure 2 Image B is an immunofluorescence staining representation of Map2, a marker of mature neurons, on days 8, 12, and 16 of astrocyte reprogramming induced by the drug composition. Scale bar = 100 μm. Figure 2 The diagram in C is a schematic of the induced neuron sholl analysis on day 16 of reprogramming, with a scale bar of 10 μm. Figure 2 D is a statistical graph of Sholl analysis on days 8, 12, and 16 of astrocyte reprogramming induced by the drug composition (n=16). Figure 2 E represents the immunofluorescence staining of the synaptic protein Synapsin1 and the mature neuron marker Map2 induced by reprogramming on day 16 (scale bar = 100 μm). Figure 3 This is a diagram illustrating the reprogramming of astrocytes and identification of neuronal subtypes following autologous blood-induced brain hemorrhage using a drug composition in Experiment Example 3. Figure 3 Figure A shows the experimental timeline. One day after autologous blood intracerebral hemorrhage (aICH), CDLR was administered in situ via a micro-osmotic pump for 14 days, and brain tissue samples were collected on day 60 after reprogramming. Figure 3 Images B and C show the immunofluorescence staining results on day 60 after autologous blood brain hemorrhage reprogramming; co-localization of tdTOMATO, NeuN, and DAPI. Scale bar = 100 μm; Figure 3 The middle D represents the subtype identification of induced neurons, and the co-localization of DARPP32 (a marker of moderately spinous neurons in the striatum), tdTomato, NeuN, and DAPI. Scale bar = 50 μm. Figure 3 E represents the identification of induced neuron subtypes, and the co-localization of PV (a positive inhibitory neuron marker for parvalbumin), tdTomato, NeuN, and DAPI. Scale bar = 50 μm. Figure 3 In the middle F, the subtypes of induced neurons are identified, and the co-localization of VGLUT1 (a marker of excitatory neurons), tdTomato, NeuN, and DAPI is shown. Scale bar = 50 μm. Figure 3 G represents the identification of induced neuron subtypes, and the co-localization of CHAT (cholinergic neuron marker), tdTomato, NeuN, and DAPI (scale bar = 50 μm). Figure 4 Figure 4 shows the results of the drug composition in improving neurological function impairment after autologous blood cerebral hemorrhage in Experiment Example 4. Figure 4 In the figure A, the proportion of right turns in the cornering experiment was compared between the group receiving the drug composition via in-situ micro-osmotic pump administration and the control group 60 days after autologous blood cerebral hemorrhage. Figure 4 (B represents the percentage of successful placements in the left forelimb placement experiment in the group receiving in-situ micro-osmotic pump administration of the drug composition and the control group 60 days after autologous blood intracerebral hemorrhage). Figure 5 This is a statistical analysis of the total number of neurons generated by astrocyte reprogramming after autologous blood-brain hemorrhage induced by the drug composition in Experiment Example 5. Figure 5 In the diagram, A represents the fMOST imaging process; Figure 5 B is a schematic diagram of a three-dimensional imaging of the whole brain; Figure 5 The number of induced neurons around the striatal cerebral hemorrhage lesion in unit visual field C and D; Figure 5 (E represents the spatial distribution of the induced neurons). Figure 6 To illustrate the improvement of neurological function impairment after cerebral ischemia by the drug composition in Experiment 6 (behavioral differences in mNSS scores, foot slip test, cylinder test, and adhesive removal test among the in-situ microosmotic pump administration group (CDLR), sham operation group, and normal control group). Figure 6 (Chinese AD). Detailed Implementation

[0041] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0042] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the raw materials used in each parallel experiment are the same.

[0043] In the following embodiments: Am-a culture medium is from Sciencell; artificial cerebrospinal fluid can be any artificial cerebrospinal fluid commonly used in this field.

[0044] The indwelling needle and Alzert pump are from Alzert Corporation.

[0045] The following LDN193189 is a free base.

[0046] In the following embodiments: The induction medium consists of the following components: A mixed basal medium of DMEM / F12 and Neurobasal at a volume ratio of 1:1, and additive A dissolved in the mixed basal medium, wherein additive A consisted of bFGF nutrient factor at a mass concentration of 100 ng / ml, fetal bovine serum at a volume fraction of 2%, B27 at a volume fraction of 2%, and GlutaMAX at a volume fraction of 1%.

[0047] The differentiation culture medium consists of the following components: Neurobasal medium, and additive B dissolved in Neurobasal medium, which consisted of 2% B27, 1% GlutaMAX, and 20 ng / mL BDNF.

[0048] Example 1 This embodiment provides a pharmaceutical composition (0.75x concentration group) for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, LDN193189 and RG108.

[0049] The molar concentration of calcitriol in the pharmaceutical composition is 0.375 μM, the molar concentration of DAPT in the pharmaceutical composition is 3.75 μM, the molar concentration of LDN193189 in the pharmaceutical composition is 0.1875 μM, and the molar concentration of RG108 in the pharmaceutical composition is 7.5 μM.

[0050] Example 2 This embodiment provides a pharmaceutical composition (1x concentration group) for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, LDN193189 and RG108.

[0051] The molar concentration of calcitriol in the pharmaceutical composition is 0.5 μM, the molar concentration of DAPT in the pharmaceutical composition is 5 μM, the molar concentration of LDN193189 in the pharmaceutical composition is 0.25 μM, and the molar concentration of RG108 in the pharmaceutical composition is 10 μM.

[0052] Example 3 This embodiment provides a pharmaceutical composition (1.5x concentration group) for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, LDN193189 and RG108.

[0053] The molar concentration of calcitriol in the pharmaceutical composition is 0.75 μM, the molar concentration of DAPT in the pharmaceutical composition is 7.5 μM, the molar concentration of LDN193189 in the pharmaceutical composition is 0.375 μM, and the molar concentration of RG108 in the pharmaceutical composition is 15 μM.

[0054] Comparative Example 1 This embodiment provides a pharmaceutical composition (0.5x concentration group) for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, LDN193189 and RG108.

[0055] The molar concentration of calcitriol in the pharmaceutical composition is 0.25 μM, the molar concentration of DAPT in the pharmaceutical composition is 2.5 μM, the molar concentration of LDN193189 in the pharmaceutical composition is 0.125 μM, and the molar concentration of RG108 in the pharmaceutical composition is 5 μM.

[0056] Comparative Example 2 This embodiment provides a pharmaceutical composition (2x concentration group) for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, LDN193189 and RG108.

[0057] The molar concentration of calcitriol in the pharmaceutical composition is 1 μM, the molar concentration of DAPT in the pharmaceutical composition is 10 μM, the molar concentration of LDN193189 in the pharmaceutical composition is 0.5 μM, and the molar concentration of RG108 in the pharmaceutical composition is 20 μM.

[0058] Comparative Example 3 This embodiment provides a pharmaceutical composition (10x concentration group) for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, LDN193189 and RG108.

[0059] The molar concentration of calcitriol in the pharmaceutical composition is 5 μM, the molar concentration of DAPT in the pharmaceutical composition is 50 μM, the molar concentration of LDN193189 in the pharmaceutical composition is 2.5 μM, and the molar concentration of RG108 in the pharmaceutical composition is 100 μM.

[0060] Comparative Example 4 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes DAPT, LDN193189, and RG108.

[0061] The molar concentration of DAPT in the pharmaceutical composition is 5 μM, the molar concentration of LDN193189 in the pharmaceutical composition is 0.25 μM, and the molar concentration of RG108 in the pharmaceutical composition is 10 μM.

[0062] Compared to Example 2, the pharmaceutical composition of Comparative Example 4 does not contain calcitriol.

[0063] Comparative Example 5 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, LDN193189, and RG108.

[0064] The molar concentration of calcitriol in the pharmaceutical composition is 0.5 μM, the molar concentration of LDN193189 in the pharmaceutical composition is 0.25 μM, and the molar concentration of RG108 in the pharmaceutical composition is 10 μM.

[0065] Compared to Example 2, the pharmaceutical composition of Comparative Example 5 does not contain DAPT.

[0066] Comparative Example 6 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage, the pharmaceutical composition comprising calcitriol, DAPT, and RG108.

[0067] The molar concentration of calcitriol in the pharmaceutical composition is 0.5 μM, the molar concentration of DAPT in the pharmaceutical composition is 5 μM, and the molar concentration of RG108 in the pharmaceutical composition is 10 μM.

[0068] Compared to Example 2, the pharmaceutical composition of Comparative Example 6 does not contain LDN193189.

[0069] Comparative Example 7 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, and LDN193189.

[0070] The molar concentration of calcitriol in the pharmaceutical composition is 0.5 μM, the molar concentration of DAPT in the pharmaceutical composition is 5 μM, and the molar concentration of LDN193189 in the pharmaceutical composition is 0.25 μM.

[0071] Compared to Example 2, the pharmaceutical composition of Comparative Example 7 does not contain RG108.

[0072] Comparative Example 8 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage, the pharmaceutical composition comprising LDN193189 and RG108.

[0073] The molar concentration of LDN193189 in the pharmaceutical composition is 0.25 μM, and the molar concentration of RG108 in the pharmaceutical composition is 10 μM.

[0074] Compared to Example 2, the pharmaceutical composition of Comparative Example 8 does not contain calcitriol and DAPT.

[0075] Comparative Example 9 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage, the pharmaceutical composition comprising DAPT and RG108.

[0076] The molar concentration of DAPT in the pharmaceutical composition is 5 μM, and the molar concentration of RG108 in the pharmaceutical composition is 10 μM.

[0077] Compared to Example 2, the pharmaceutical composition of Comparative Example 9 does not contain calcitriol and LDN193189.

[0078] Comparative Example 10 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage, the pharmaceutical composition comprising calcitriol and RG108.

[0079] The molar concentration of calcitriol in the pharmaceutical composition is 0.5 μM, and the molar concentration of RG108 in the pharmaceutical composition is 10 μM.

[0080] Compared to Example 2, the pharmaceutical composition of Comparative Example 10 does not contain DAPT and LDN193189.

[0081] Comparative Example 11 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage, the pharmaceutical composition comprising calcitriol and DAPT.

[0082] The molar concentration of calcitriol in the pharmaceutical composition is 0.5 μM, and the molar concentration of DAPT in the pharmaceutical composition is 5 μM.

[0083] Compared to Example 2, the pharmaceutical composition of Comparative Example 11 does not contain RG108 and LDN193189.

[0084] Comparative Example 12 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage, the pharmaceutical composition comprising calcitriol and LDN193189.

[0085] The molar concentration of calcitriol in the pharmaceutical composition is 0.5 μM, and the molar concentration of LDN193189 in the pharmaceutical composition is 0.25 μM.

[0086] Compared to Example 2, the pharmaceutical composition of Comparative Example 12 does not contain DAPT and RG108.

[0087] Comparative Example 13 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage, the pharmaceutical composition comprising DAPT and LDN193189.

[0088] The molar concentration of DAPT in the pharmaceutical composition is 5 μM, and the molar concentration of LDN193189 in the pharmaceutical composition is 0.25 μM.

[0089] Compared to Example 2, the pharmaceutical composition of Comparative Example 13 does not contain calcitriol and RG108.

[0090] Comparative Example 14 This comparative example provides a pharmaceutical composition (0.1x concentration group) that induces the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, LDN193189 and RG108.

[0091] The molar concentration of calcitriol in the pharmaceutical composition is 0.050 μM, the molar concentration of DAPT in the pharmaceutical composition is 0.5 μM, the molar concentration of LDN193189 in the pharmaceutical composition is 0.025 μM, and the molar concentration of RG108 in the pharmaceutical composition is 1 μM.

[0092] Comparative Example 15 This comparative example provides a pharmaceutical composition (20x concentration group) that induces the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, LDN193189, and RG108.

[0093] The molar concentration of calcitriol in the pharmaceutical composition is 10 μM, the molar concentration of DAPT in the pharmaceutical composition is 100 μM, the molar concentration of LDN193189 in the pharmaceutical composition is 5 μM, and the molar concentration of RG108 in the pharmaceutical composition is 200 μM.

[0094] Comparative Example 16 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, LDN193189, and RG108.

[0095] The molar concentration of calcitriol in the pharmaceutical composition is 10 μM, the molar concentration of DAPT in the pharmaceutical composition is 5 μM, the molar concentration of LDN193189 in the pharmaceutical composition is 0.25 μM, and the molar concentration of RG108 in the pharmaceutical composition is 10 μM.

[0096] Comparative Example 17 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, LDN193189, and RG108.

[0097] The molar concentrations of calcitriol, DAPT, LDN193189, and RG108 in the pharmaceutical composition are 0.5 μM, 5 μM, 5 μM, and 10 μM respectively.

[0098] Comparative Example 18 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, LDN193189, and RG108.

[0099] The molar concentration of calcitriol in the pharmaceutical composition is 0.5 μM, the molar concentration of DAPT in the pharmaceutical composition is 5 μM, the molar concentration of LDN193189 in the pharmaceutical composition is 0.25 μM, and the molar concentration of RG108 in the pharmaceutical composition is 200 μM.

[0100] Comparative Example 19 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, LDN193189, RG108, and VPA.

[0101] The molar concentrations of calcitriol, DAPT, LDN193189, RG108, and VPA in the pharmaceutical composition are 0.5 μM, 5 μM, 0.25 μM, 10 μM, and 0.5 mM respectively.

[0102] Comparative Example 20 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes VPA, DAPT, LDN193189, and RG108.

[0103] The molar concentrations of VPA, DAPT, LDN193189, and RG108 in the pharmaceutical composition are 0.5 μM, 5 μM, 0.25 μM, and 10 μM respectively.

[0104] Comparative Example 21 This comparative example provides a pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage. The pharmaceutical composition includes calcitriol, DAPT, forskolin, and RG108.

[0105] The molar concentration of calcitriol in the pharmaceutical composition is 0.5 μM, the molar concentration of DAPT in the pharmaceutical composition is 5 μM, the molar concentration of forskolin in the pharmaceutical composition is 0.25 μM, and the molar concentration of RG108 in the pharmaceutical composition is 10 μM.

[0106] Experimental Example 1: Verification of astrocyte reprogramming into neurons after cerebral hemorrhage induced by a drug composition This experimental example provides a method for reprogramming astrocytes into neurons, including the following steps: 1) Seeding cells: Third-generation or higher mouse striatal astrocytes are seeded in 24-well plates incubated with matrix gel and cultured in Am-a medium to a density of 80-90%.

[0107] 2) Construction of in vitro brain hemorrhage model: Am-a medium supplemented with 10 μM hemin was used to simulate the in vitro brain hemorrhage model. After 24 h of treatment, the medium was replaced with normal Am-a medium.

[0108] 3) Induction of reprogramming: After primary astrocytes were stimulated with DMSO (control group) or hemin, they were induced in induction medium for four days with the drug compositions of Examples 1-3 and Comparative Examples 1-21, and then the medium was changed to differentiation medium. Six days after differentiation, the mature neuron marker Map2 was immunofluorescence stained, as follows: Figure 1 As shown in Figure A.

[0109] 4) MAP2 immunofluorescence: Cell smears were collected on day 18 of induction and reprogramming for fluorescent staining.

[0110] The reprogramming efficiency (number of Map2 positive cells / number of all nucleated cells in a 300μm×300μm field of view) of the drug combinations of Examples 1-4 and Comparative Examples 1-21 in the normal in vitro environment (control CDLR, i.e., the drug composition of Example 2) and the drug compositions of Examples 1-4 and Comparative Examples 1-21 in the hemin simulated in vitro brain hemorrhage environment was tested and is shown in Table 1.

[0111] Table 1 The results are as follows Figure 1 As shown, the pharmaceutical composition of Example 2 can efficiently ( Figure 1 In the control group (CDR group B), the conversion efficiency was as high as 85%, reprogramming astrocytes into Map2+ neuron-like cells. In the in vitro brain hemorrhage model simulated by hemin, the drug composition of the examples also reprogrammed astrocytes into neuron-like cells, but the conversion efficiency was lower than that of the control group. Figure 1 (The conversion efficiency of Examples 1-3 was 23.4%-28.3%). On day 16 of reprogramming, the pharmaceutical compositions of Examples 1-3 could induce the reprogramming of mouse striatal astrocytes into neurons under in vitro culture conditions.

[0112] like Figure 1 As shown in Figure B, during the astrocyte reprogramming induction phase following hemin stimulation, the reprogramming efficiency of astrocytes reprogramming into neurons was significantly reduced in the drug compositions of Comparative Examples 5-7 when one of DAPT, LDN193189, and RG108 was missing, and in the drug compositions of Comparative Examples 8-13 when two of calcitriol, DAPT, LDN193189, and RG108 were missing, compared to the drug compositions of Examples 1-3. Although the reprogramming efficiency of astrocytes reprogramming into neurons was not significantly reduced compared to the drug compositions of Examples 1-3 when calcitriol was missing in the drug composition of Comparative Example 4, the number of surviving cells was significantly increased compared to Comparative Examples 5-7, suggesting that calcitriol played a role in promoting cell survival and creating a more favorable reprogramming environment in the composition.

[0113] In Comparative Examples 1-3 and 14-15, changing the overall concentrations of calcitriol, DAPT, LDN193189, and RG108 to 0.1, 0.5, 2, 10, and 20 times respectively significantly reduced the reprogramming efficiency of astrocytes into neurons.

[0114] In Comparative Examples 16-18, increasing the concentration of any one of the compounds among calcitriol, LDN193189, and RG108 resulted in a decrease in the reprogramming efficiency of astrocytes into neurons. In Comparative Example 19, adding another small molecule, VPA, to calcitriol, DAPT, LDN193189, and RG108 resulted in a decrease in the reprogramming efficiency of astrocytes into neurons. In Comparative Example 20, replacing calcitriol with VPA resulted in a decrease in the reprogramming efficiency of astrocytes into neurons. In Comparative Example 21, replacing LDN193189 with Forskolin resulted in a decrease in the reprogramming efficiency of astrocytes into neurons.

[0115] Experiment Example 2: Dynamic observation of the reprogramming of mouse striatal astrocytes into neurons under in vitro culture conditions. In the following experimental examples, the drug composition of Example 2 was used to induce the reprogramming of mouse striatal astrocytes into neurons.

[0116] Specific experiments include: 1) Dynamic observation under an optical microscope: The live-cell dynamic imaging microscope was set to automatically capture images at 1-hour intervals, and the focusing plane was determined to record the dynamic changes in the plane containing the cells. The captured image data was automatically recorded and transferred to a computer for storage. Results are shown below. Figure 2 A.

[0117] The results showed that on day 4 of reprogramming, astrocytes first exhibited a slender spindle shape, and on day 8, scattered highly refractive neuron-like cells began to appear. Subsequently, these neuron-like cells gradually developed longer processes and matured into more mature neurons. Throughout the reprogramming process, no nested or clustered neurogenesis occurred, which to some extent suggests that no significant atavism occurred during the reprogramming process.

[0118] 2) Dynamic observation of neuronal marker expression: Mouse striatal astrocytes were cultured in Am-a medium to a density of 80-90%; after 4 days of induction medium, the cells were replaced with differentiation medium and cultured for another 4, 8, and 12 days. Cell smears were collected for fluorescence staining to identify Map2+ expression, and Sholl analysis was performed using ImageJ software to statistically analyze the trend of neurite richness changes in induced neurons. The results showed that from 8 to 16 days after reprogramming, neuronal neurite richness gradually increased (…). Figure 2 China B to Figure 2 In the middle D, the longest neurite of the induced neurons on day 16 of reprogramming reached 200 μm, and they highly expressed the neuronal synaptic protein Synapsin1 ( Figure 2 (E).

[0119] Experimental Example 3: Astrocyte reprogramming and neuronal subtype identification after autologous blood-induced brain hemorrhage induced by a drug composition. The following experimental examples used the drug composition of Example 2 to induce astrocyte reprogramming and neuronal subtype identification after autologous blood brain hemorrhage.

[0120] One day after the cerebral hemorrhage, the drug composition of Example 2 was administered in situ via a micro-osmotic pump for 14 days. Brain tissue was collected on day 60 of reprogramming for immunofluorescence staining. Figure 3 (A). The results showed that a large number of tdTOMATO+NeuN+ induced neurons existed around the autologous blood brain hemorrhage lesion. Figure 3 (B)

[0121] Further identification of neuronal subtypes revealed that these induced neurons primarily expressed Darpp32, a marker of moderately spinous neurons in the striatum. Figure 3 (C), but does not express parvalbumin (PV), a positive inhibitory neuronal marker for parvalbumin (C). Figure 3 D), excitatory neuronal marker Vglut1 ( Figure 3 (E) and cholinergic neuron marker Chat ( Figure 3 (F). This indicates that the neurons derived from astrocytes induced by the drug composition after autologous blood-brain hemorrhage are mostly Darpp32-positive GABAergic neurons.

[0122] Experimental Example 4: The effect of the drug composition on the improvement of neurological function impairment after autologous blood cerebral hemorrhage. Sixty days after autologous blood intracerebral hemorrhage, the angle-turning test and left forelimb placement test were performed on the group receiving the drug composition via in-situ micro-osmotic pump and the control group to assess behavioral improvement. Figure 4 China A Figure 4 (B)

[0123] Corner Experiment: I. Experimental Preparation: 1. Apparatus: Two 30 cm × 20 cm × 1 cm wooden boards / plexiglass boards are fixed at a 30° angle to form a V-shaped channel. A small opening is left at the connection point, and the opening end is flush with the edge of the experimental table to prevent escape.

[0124] 2. Environment: Soundproof and light-proof, dim lighting. It is recommended to conduct the experiment at the beginning of the dark period to reduce stress.

[0125] 3. Adaptation: Place the mouse in the center of the device for 5-10 minutes before the test to allow it to become familiar with the environment.

[0126] II. Test Steps: 1. Place the mouse: Place the mouse facing the angle into the center of the far end of the V-shaped channel, and gently hold its tail to help it move towards the corner.

[0127] 2. Triggered behavior: When the mouse enters the corner, the touch of its whiskers on both sides to the board wall will induce the hind legs to stand upright (rearing), and then complete a 180° turn to exit the corner.

[0128] 3. Recording criteria: Only valid turns that meet the following conditions will be counted: After the hind leg is fully upright, turn to the left or right. When turning, keep your back against the wall, avoiding curling up your abdomen or turning horizontally.

[0129] 4. Repeated testing: Each mouse was tested 10 times consecutively, with a 30-second interval between each test, to avoid fatigue or aversion.

[0130] III. Data Analysis: Calculate the steering index (CTS): CTS = [Number of right turns (R) / (R + Number of left turns L)] × 100%.

[0131] Left forelimb placement experiment: I. Experimental Preparation: 1. Apparatus: The experimental table should be flat and free from interference. A transparent plexiglass plate (approximately 20cm × 30cm) should be vertically fixed to one edge. The edge of the plate should be smooth and without sharp corners.

[0132] 2. Environment: Quiet, dimly lit, avoid visual disturbances, room temperature 22-25℃.

[0133] 3. Adaptation: Place the mice on the experimental table and let them move freely for 5-10 minutes before the test to reduce stress.

[0134] II. Operating Procedures: 1. Tentacle-induced placement: Fixed posture: The experimenter gently pinches the middle section of the mouse's trunk (behind the sternum and in front of the pelvis) with the thumb and forefinger, allowing the mouse's forelimbs to suspend and move freely in the air, with the trunk parallel to the ground and the head facing forward.

[0135] Trigger response: Slowly move the mouse horizontally so that its left whiskers lightly touch the edge of the plexiglass plate, and observe whether the left forelimb immediately lifts up and rests on the plate.

[0136] Test parameters: 10 consecutive tests, with 10-15 second intervals to avoid fatigue. Successful placement: The left forelimb is accurately placed on the board within 1 second of the tentacle contact. Failure: No response, delay (>1 second), or placement of only the right forelimb. Scoring: Number of successful tests / total number of tests × 100%, with a success rate of ≥80% for normal mice.

[0137] like Figure 5As shown, the pharmaceutical composition of this application embodiment can improve neurological function impairment after autologous blood cerebral hemorrhage. Figure 5 In the figure A, the proportion of right turns in the cornering experiment was compared between the group receiving the drug composition via in-situ micro-osmotic pump administration and the control group 60 days after autologous blood cerebral hemorrhage. Figure 5 In the left forelimb placement experiment, B represents the percentage of successful placements in the CDLR in situ micro-osmotic pump administration group and the control group 60 days after autologous blood cerebral hemorrhage.

[0138] Experiment Example 5: Statistical Experiment on the Total Number of Neurons Generated by Astrocyte Reprogramming After Autologous Blood-Induced Brain Hemorrhage Following Drug Composition The following experimental examples used the drug composition of Example 2 to induce autologous blood brain hemorrhage and reprogrammed astrocytes, and then counted the total number of neurons induced.

[0139] The experimental group (CDLR group) received the drug composition of Example 2 in situ via a micro-osmotic pump for 14 days 1 day after cerebral hemorrhage, while the solvent control group received the solvent in situ via a micro-osmotic pump for 14 days 1 day after cerebral hemorrhage. Both groups received AAV.CAP-B10 hSyn-GFP virus-labeled whole-brain neurons via the tail vein on day 48 of reprogramming, and whole-brain tissue was harvested on day 60 of reprogramming. The whole-brain tissue underwent a series of treatments including fixation, dehydration, and resin embedding, followed by fluorescence optical section tomography (fMOST) imaging to obtain a high-resolution three-dimensional dataset of the whole brain with a voxel resolution of 0.35 × 0.35 × 2 μm³. After seamless stitching, inter-channel image registration, illumination correction, and noise reduction preprocessing of the dual-channel imaging data, 3D reconstruction was performed in Imaris software. Figure 5 (A and B). The results showed that the experimental group (CDLR group) had a large number of tdTOMATO+GFP+ induced neurons around the autologous blood brain hemorrhage lesion. Figure 5 (C and D). The total number of tdTOMATO+GFP+ induced neurons in the experimental group (CDLR group) was 1647. Of these, 5 were located within 50-100 μm of the hematoma center, 783 within 100-200 μm, and 859 within more than 200 μm. Figure 5 (E).

[0140] Experiment Example 6: Experiment on the improvement of neurological function damage after cerebral ischemia by a drug composition Three days after photochemical embolization for cerebral ischemia, the drug composition of Example 2 was administered in situ via a micro-osmotic pump for 14 days. On day 60 of reprogramming, the mNSS score, foot slip test, cylinder test, and adhesive removal test were performed on the drug composition administered via micro-osmotic pump in situ (CDLR) group, sham operation group, and normal control group to assess behavioral improvement. Figure 6 (Chinese AD).

[0141] mNSS score: Before the experiment, an observer who was unaware of the grouping situation conducted a blind assessment of the animals, and tested motor function (tail lifting to observe forelimb flexion / hindlimb extension, walking on flat ground), sensory function (vision, touch, proprioception), and reflex / balance function (auricular reflex, corneal reflex, startle reflex, walking on a balance beam, etc.) in sequence. Each abnormality was scored from 0 to 3 according to the established criteria, and the higher the total score, the more severe the neurological deficit.

[0142] Foot error test: The animal is placed on a metal grid platform and allowed to move freely while being videotaped. The number of times the animal's front / hind paws slipped and fell into the grid gaps within a specified time or number of steps, as well as the total number of steps, are counted. The percentage of foot errors to total steps is used as an indicator to reflect the animal's limb coordination and sensorimotor integration ability.

[0143] Cylinder test: Animals are placed in a transparent cylinder to explore freely, and the number of times their forelimbs touch the inner wall of the cylinder is recorded. The use of the left forelimb, right forelimb, and both forelimbs at the same time is counted separately. The asymmetry index (i.e. (uninjured side - injured side) / total number of times the wall is touched) is calculated to assess the lateralization of forelimb use and the degree of motor function recovery after unilateral brain injury.

[0144] Adhesive removal test: Animals were given 3–5 days of acclimatization training to reach a stable baseline level before the experiment. Then, adhesive tags of equal area were attached to the palms of both forepaws. After the animals were returned to their cages, the time it took for them to completely remove the tags was recorded. The ratio of the time taken on the damaged side to that on the undamaged side was calculated to assess the sensory function and fine motor coordination of the forepaws.

[0145] The improvement of neurological function impairment after cerebral ischemia by the pharmaceutical composition in Example 2 (results of behavioral differences in mNSS scores, foot slip test, cylinder test, and adhesive removal test in the in-situ microosmotic pump administration group (CDLR), sham operation group, and normal control group) are as follows: Figure 6 As shown.

[0146] The pharmaceutical composition provided in this application is simple in composition, consisting of only four small molecule compounds. Compared with most other formulations, it is concise and safe. It can reprogram mouse striatal astrocytes into neurons under in vitro culture conditions. The induced neurons closely resemble the subtype distribution of neurons in the striatal region in vivo, with a reprogramming efficiency of up to 85%. The pharmaceutical composition of this application can comprehensively cover the signaling pathway regulation, cellular metabolic pathway regulation, and epigenetic regulatory mechanisms required to induce cell fate transformation, thereby synergistically reprogramming astrocytes after cerebral hemorrhage into neurons and thus improving neurological function impairment.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A pharmaceutical composition for inducing the reprogramming of astrocytes into neurons after cerebral hemorrhage, characterized in that, The pharmaceutical composition includes calcitriol, DAPT, LDN193189, and RG108; The molar concentration of calcitriol in the pharmaceutical composition is 0.375 μM to 0.75 μM; The molar concentration of DAPT in the pharmaceutical composition is 3.75 μM to 7.5 μM; The molar concentration of LDN193189 in the pharmaceutical composition is 0.1875 μM ~ 0.375 μM; The molar concentration of RG108 in the pharmaceutical composition is 7.5 μM to 15 μM.

2. The pharmaceutical composition according to claim 1, characterized in that, The molar concentration of calcitriol in the pharmaceutical composition is 0.5 μM to 0.75 μM; The molar concentration of DAPT in the pharmaceutical composition is 5 μM to 7.5 μM; The molar concentration of LDN193189 in the pharmaceutical composition is 0.25 μM ~ 0.375 μM; The molar concentration of RG108 in the pharmaceutical composition is 10 μM to 15 μM.

3. The use of the pharmaceutical composition of claim 1 or 2 in the preparation of a product for in vitro inducing astrocyte reprogramming into neurons.

4. The application as described in claim 3, characterized in that, The astrocytes are derived from the striatum.

5. The use of the pharmaceutical composition as described in claim 1 or 2 in the preparation of a drug for the prevention and / or treatment of cerebral hemorrhage or cerebral ischemia.

6. A drug for the prevention and / or treatment of cerebral hemorrhage or cerebral ischemia, characterized in that, The drug comprises the pharmaceutical composition as described in claim 1 or 2.

7. A method for reprogramming astrocytes into neurons for non-therapeutic purposes, characterized in that, The method includes: culturing isolated astrocytes in an induction medium containing the pharmaceutical composition as described in claim 1 or 2, and then culturing them in a differentiating medium to transform the astrocytes into neurons.

8. The method as described in claim 7, characterized in that, The induction medium also includes a mixed basal medium and additive A dissolved in the mixed basal medium; Additive A includes bFGF, fetal bovine serum, B27, and L-alanyl-L-glutamine dipeptide; The mixed basal medium includes DMEM / F12 and Neurobasal medium.

9. The method as described in claim 7, characterized in that, The differentiation medium includes Neurobasal medium and additive B dissolved in Neurobasal medium; Additive B includes B27, L-alanyl-L-glutamine dipeptide, and BDNF.

Images