A pharmaceutical combination for neuromuscular diseases

Abstract

This invention discloses a drug combination for neuromuscular diseases, relating to the field of biomedical technology. The drug combination includes baicalin and trichodin. Through 2D and 3D culture verification, this invention has ultimately determined that the combined use of baicalin and trichodin can exert a synergistic effect, significantly protecting the neuromuscular axis. This provides a theoretical basis and in vitro screening platform for the treatment of neuromuscular diseases, and opens up a synergistic pathway for the repurposing of existing drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a drug combination for neuromuscular diseases. Background Technology

[0002] Neuromuscular diseases are a heterogeneous group of diseases affecting motor neurons, skeletal muscle, and neuromuscular junctions, with the core pathology being dysfunction of neuromuscular signal transmission / muscle contraction. Based on the affected site, they are classified into four categories: upper / lower motor neuron diseases, peripheral nerve diseases, muscle injury diseases, and neuromuscular junction diseases.

[0003] It is worth noting that most existing treatments are single-target interventions, which can only delay the course of the disease (such as riluzole delaying the course of amyotrophic lateral sclerosis by 3-6 months) and cannot repair the damaged neuromuscular junction structure. Drug delivery is hindered by the blood-brain / blood-nerve barrier, chronic muscle fibrosis is irreversible, and some treatments (such as gene therapy) only cover a small number of mutant individuals, with significant side effects.

[0004] Therefore, screening neuromuscular synergistic protective drugs based on neuromuscular injury models constructed from human induced pluripotent stem cells (hiPSCs) is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] This invention provides a drug combination for neuromuscular diseases.

[0006] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0007] A drug combination for neuromuscular diseases, the drug combination comprising baicalein and trichomoniasisin.

[0008] As a preferred technical solution, the drug combination has a protective effect on both neurons and motor nerves.

[0009] Another object of the present invention is to provide the application of baicalin and trichodin in combination in the preparation of drugs related to neuromuscular diseases.

[0010] Compared with the prior art, the present invention has the following beneficial effects: This invention addresses the lack of effective treatments for neuromuscular diseases in existing technologies. Focusing on neuromuscular junction repair and reconstruction, it utilizes a hiPSC neuromuscular injury model constructed using human induced pluripotent stem cell (hiPSC) directed differentiation technology. Drug combinations for neuromuscular diseases are screened, and after 2D and 3D culture verification, it is ultimately determined that the combined use of baicalein and salvia miltiorrhiza can exert a synergistic effect, significantly protecting the neuromuscular axis. This provides a theoretical basis and in vitro screening platform for the treatment of neuromuscular diseases, and opens up a synergistic pathway for the repurposing of existing drugs. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0012] Figure 1 The results of the establishment of the NILG-hiPSC-derived motor neuron passage stress model and drug screening are shown. Among them, (A) flowchart of the establishment of the NILG-hiPSC-derived motor neuron passage stress model; (B) cell images after treatment with 7 compounds in the passage stress model; (C) cell images of each experimental group after long-term culture (10 days).

[0013] Figure 2 The number of surviving neurons on day 3 after passage stress in NILG-hiPSC-derived motor neurons was significantly different from that in the control group (####P<0.0001) and significantly different from that in the model group ( P<0.05, P<0.01, P<0.001, P<0.0001).

[0014] Figure 3 Map of neural cluster numbers on day 10 after NILG-hiPSC-derived motor neurons undergo passage stress (significantly different from the control group (####P<0.0001); significantly different from the model group (). P<0.0001).

[0015] Figure 4 The results of establishing the M1-hiPSC-derived skeletal muscle cell cardiotoxin model and drug screening are as follows: (A) Flowchart of the establishment of the M1-hiPSC-derived muscle cell cardiotoxin model; (B) Hoechst / PI apoptosis staining images of cells in each group; (C) Quantitative statistical results of PI (significant difference compared with the control group (####P<0.0001); significant difference compared with the model group (…). P<0.01).

[0016] Figure 5 Co-culture conditions for NILG-hiPSC and M1-hiPSC-derived cells. (A) 2D images of motor neurons and muscle cells derived from NILG-hiPSC and M1-hiPSC co-cultured in different culture media; (B) 3D images of motor neurons and muscle cells derived from NILG-hiPSC and M1-hiPSC co-cultured in different culture media.

[0017] Figure 6 To illustrate the co-differentiation and co-culture of NILG-hiPSC and M1-hiPSC in skeletal muscle induction medium. (A) Co-differentiation and co-culture of NILG-hiPSC and M1-hiPSC in skeletal muscle induction medium under 2D conditions; (B) Co-differentiation and co-culture of NILG-hiPSC and M1-hiPSC in skeletal muscle induction medium under 3D conditions.

[0018] Figure 7 Establishment of a neurobotulinum toxin model: experimental procedure and cell images after treatment with different concentrations of neurobotulinum toxin.

[0019] Figure 8 To demonstrate the synergistic effect of baicalin and trichodin in a neurobotulin model. (A) Cell images of each experimental group in the neurobotulin model; (B) Immunofluorescence identification of each experimental group in the neurobotulin model; (C) Fluorescent staining of the neuromuscular junction in each experimental group in the neurobotulin model.

[0020] Figure 9 To investigate the effect of baicalin-trichosanthesin on skeletal muscle cell contraction. (A) Graphs of skeletal muscle cell contraction and contraction in each experimental group under the neurobotulin model; (B) Quantitative analysis of skeletal muscle cell contraction and contraction in each experimental group under the neurobotulin model (significant difference compared with the control group (####P<0.0001); significant difference compared with the model group (). P<0.05, P<0.0001).

[0021] Figure 10 To investigate the synergistic protective effect of baicalin-trihylcholine in a 3D model of neuromuscular injury mediated by neurobotox. (A) Images of each experimental group in the 3D model of neurobotox; (B) Immunofluorescence staining identification of each experimental group in the 3D model of neurobotox. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0024] The construction methods of the NILG-hiPSC cell line and M1-hiPSC cell line involved in the embodiments of the present invention are described in CN122542477A.

[0025] Example 1 1. Establish a single-cell injury model and screen compounds. (1) Construction of a motor neuron injury model: The NILG-hiPSC cell line successfully constructed in the laboratory was seeded into Matrigel (Corning) coated plates. Y-27632 (5 μM, 16 h, MCE) was added to the hiPSC maintenance medium mTeSR. Then, the medium was replaced with mTeSR containing doxycycline (Dox, Sigma, 2 μg / mL) to induce differentiation for 4 days to form motor neurons (TUJ1). On the 5th day, the medium was replaced with motor neuron maturation medium [based on neuralbase medium (Gibco) and Advanced DMEM / F12 (Gibco) in a volume ratio of 1:1, with the addition of B27 additive (Gibco), N2 additive (Gibco), retinoic acid (Sigma), Purmorphamine (Sigma), BDNF (PeproTech), GDNF (PeproTech), and NT3 (PeproTech) to make the final concentration of B27 additive in the medium 2%, N2 additive 1%, retinoic acid 1 μM, ... Purmorphamine (0.5 μM), BDNF (10 ng / mL), GDNF (10 ng / mL), and NT3 (10 ng / mL) were administered. On day 6, motor neurons were digested and passaged (Accutase, Stemcell Technologies, 4 min) to construct a passage stress model (a 60% neuronal mortality rate after passage was considered a successful model construction criterion).

[0026] One day before passage (i.e., day 5), cells were pretreated with seven compounds (baicalin, cannabidiol, berberine, luteolin, quercetin, and tuftrin, all purchased from MCE) for 24 hours (different concentration groups were set: the first six compounds were at concentrations of 1, 5, and 10 μM; tuftrin was at concentrations of 5, 10, and 20 μM), and photos were taken continuously to record the cell viability and status under different magnifications.

[0027] Test results: as attached Figure 1 As shown in Figure 1A, after 6 days of Dox-induced differentiation to generate motor neurons, the NILG-hiPSC cell line was pretreated with different concentrations of monomeric compounds for 1 day, followed by passage to evaluate its protective effect. Cell viability, synaptic integrity, and neurofilament morphology were observed under a microscope. As shown in Figure 1B, after 3 days of digestion and passage, passage stress caused approximately 90% neuronal death, with a large number of neurofilaments detaching and breaking. Except for the oleuropein group, where almost all neurons died, the other six groups still retained a certain number of neurons, and adherent neurofilaments were observed in all of them. Notably, in most compound groups, the neuronal condition was poor, with severe neurofilament breakage and detachment. Therefore, we conducted long-term (10 days) culture observation after passage. During continuous observation, the neuronal state in the cannabidiol, berberine, luteolin, and quercetin treatment groups gradually deteriorated, with cell bodies becoming rounded, clumping together, and detaching, making it impossible to observe adherent neurofilaments, indicating massive cell death. In contrast, the baicalein and trichodin treatment groups maintained good growth on day 10 after passage, with intact cell morphology and continuous axonal extension (see Figure 1C). Further quantitative analysis of neuronal survival revealed that baicalein, cannabidiol, berberine, luteolin, quercetin, and trichodin all significantly improved neuronal survival after passage stress (see Figure 1C). Figure 2 Similarly, statistical analysis of neural clusters on day 10 of passage revealed that the control group formed a very complex neural network with intact and strong neurites; in the model group, almost no neural clusters were formed, while the baicalin and trichosine groups formed 10-20 complete neural clusters (see appendix). Figure 3 This demonstrates that both baicalin and trichosanthesin exhibit significant neuroprotective effects.

[0028] (2) Construction of muscle injury model: The M1-hiPSC cell line successfully constructed in the laboratory was induced to differentiate for 4 days in skeletal muscle induction medium containing Dox (1 μg / mL) (the basic medium was DMEM (Gibco), with the addition of L-glutamine (Gibco), penicillin-streptomycin antibiotic (Gibco), β-mercaptoethanol (Gibco), insulin (Sigma) and retinoic acid (Sigma), so that the final concentration in the medium was 1% for L-glutamine, 1% for penicillin-streptomycin antibiotic, 50 μM for β-mercaptoethanol, 7 μg / mL for insulin and 1 μM for retinoic acid) to form skeletal muscle cells (MYH).

[0029] One day prior to the procedure, seven compounds (baicalin, cannabidiol, berberine, luteolin, quercetin, and trichoderma) were added for pretreatment (1 μM for baicalin, and 5 μM for cannabidiol, berberine, luteolin, quercetin, and trichoderma). Then, 20 μM cardiotoxin (CTX, MCE) was added to construct an injury model (a 60% apoptosis rate was used as the criterion for successful model construction). Apoptosis was detected by Hoechst / PI double staining, and the apoptosis rate was statistically analyzed.

[0030] Experimental Results: We further constructed a muscle injury model using muscle cells differentiated from M1-hiPSCs after Dox induction, aiming to screen for small molecule compounds with protective effects on muscle cells. (See attached image) Figure 4 As shown in Figure A, after differentiating M1-hiPSCs in skeletal muscle-inducing medium for 4 days, we pretreated them with various compounds for 1 day, followed by treatment with cardiotoxin for 1 day to induce cell damage and assess their protective effect. We detected cell apoptosis using the Hoechst / PI double staining method (see Appendix). Figure 4 (See Appendix B) The results showed that, compared with the control group, the cardiotoxin model group had a large number of apoptotic cells, confirming the successful establishment of the model; after administration of different compounds, the apoptosis rate was reduced compared with the model group; among them, the experimental groups pretreated with baicalin and trichosanthes showed a significant reduction in the number of apoptotic cells compared with other groups, indicating that these two compounds can effectively alleviate toxin-induced muscle cell apoptosis (see Appendix B). Figure 4 (C). Based on previous screening results in a passaged stress-induced motor neuron injury model, this study further verifies that baicalin and trichomoniasis not only have protective effects on neurons, but also exhibit significant cytoprotective activity in a muscle injury model.

[0031] 2. Co-culture method of NILG-hiPSC and M1-hiPSC (2D / 3D) To optimize the co-differentiation and co-culture medium for motor neurons and muscle cells, we screened the medium under 2D conditions. M1-hiPSCs and NILG-hiPSCs were mixed at a 1:1 ratio and seeded in 24-well cell culture plates (10,000 cells per well). mTeSR containing Y-27632 was added and cultured for 16 h to form flat-layered cells. Then, mTeSR containing Dox (1 μg / mL) was used for induction for 4 days. Finally, the cells were transferred to three different culture media with different formulations and cultured for 5 days to observe the cell growth status. The three formulations are as follows: Formulation 1: mTeSR; Formulation 2: a 1:1 volume ratio mixture of motor neuron maturation medium and skeletal muscle induction medium. The motor neuron maturation medium formulation uses neuralbasel medium and Advanced DMEM / F12 as basal media in a 1:1 volume ratio, supplemented with B27, N2, retinoic acid, purmorphamine, BDNF, GDNF, and NT3, resulting in a final concentration of 2% B27, 1% N2, 1 μM retinoic acid, 0.5 μM purmorphamine, 10 ng / mL BDNF, 10 ng / mL GDNF, and 10 ng / mL NT3. The skeletal muscle induction medium formulation uses DMEM as a base, supplemented with L-glutamine, penicillin-streptomycin antibiotics, β-mercaptoethanol, insulin, and retinoic acid, resulting in a final concentration of 1% L-glutamine, 1% penicillin-streptomycin antibiotics, 50 μM β-mercaptoethanol, and 7 μM insulin. Formula 3: Skeletal muscle organoid culture medium and skeletal muscle induction medium were mixed at a volume ratio of 1:1. The skeletal muscle organoid culture medium formula (based on neuralbasel medium and DMEM / F12 at a volume ratio of 1:1, with the addition of B27 additive, N2 additive, L-glutamine, non-essential amino acids, penicillin-streptomycin antibiotics, β-mercaptoethanol, BDNF, GDNF, and NT3, resulting in a final concentration of 2% B27 additive, 1% N2 additive, 1% L-glutamine, 1% non-essential amino acids, 1% penicillin-streptomycin antibiotics, 10 ng / mL BDNF, 10 ng / mL GDNF, and 10 ng / mL NT3) was used for neuromuscular co-culture in the above three formulas. Microscopic observation of cell morphology revealed that with prolonged culture time, regardless of the formula, large-scale cell death occurred in the co-cultured cells, with muscle cells being the predominantly dead cells (see Appendix). Figure 5 (A)

[0032] We performed similar co-culture condition screening under 3D culture conditions. Half an hour before seeding, a low-adhesion buffer (Anti-Adherence Rinsing Solution, Stemcell Technologies 07010) was added to 96-well round-bottom plates, and the plates were incubated at 37°C for half an hour. After removing the buffer, M1-hiPSCs and NILG-hiPSCs were seeded into 96-well low-adhesion U-shaped plates, respectively, and cultured with mTeSR containing Y-27632 for 16 h to form embryoid spheres. These were then induced with mTeSR containing Dox (1 μg / mL) for 4 days, and finally transferred to three different culture media with the same formulations as the 2D culture for 5 days to observe changes in the spheres. The results showed that neurospheres expressing green fluorescence were structurally intact and dense; while muscle spheres not expressing green fluorescence were loose and showed cell detachment, indicating significant muscle cell death (see Appendix). Figure 5 (B)

[0033] 3. Neuromuscular co-differentiation and co-culture conditions based primarily on skeletal muscle induction medium. Since a large number of muscle cells died under the above conditions, we adjusted the co-culture conditions to primarily use skeletal muscle induction medium for the co-differentiation and co-culture of M1-hiPSCs and NILG-hiPSCs. Thirty minutes before cell seeding, a basement membrane matrix pretreatment was initiated: Matrigel working solution was evenly spread on the bottom of a 24-well plate, then transferred to a 37°C, 5% CO2 culture environment and incubated for 30 min to form a gel-like coating layer. The Matrigel was removed, and mTeSR containing Y-27632 was added. NILG-hiPSCs and M1-hiPSCs were mixed at a 1:1 ratio, totaling 1×10⁻⁶ cells. 4 Cells were seeded into 24-well plates, gently shaken, and incubated at 37°C. After 16 h, the medium was replaced with mTeSR, and cultured for another 24 h. Then, skeletal muscle induction medium containing Dox (1 μg / mL) (based on DMEM, supplemented with L-glutamine, penicillin-streptomycin, β-mercaptoethanol, insulin, and retinoic acid, with final concentrations of 1% L-glutamine, 1% penicillin-streptomycin, 50 μM β-mercaptoethanol, 7 μg / mL insulin, and 1 μM retinoic acid) was added to initiate differentiation. Under the influence of the differentiation medium, the cells dispersed from a clonal pattern and differentiated into neurons and muscle cells. Cells exhibiting green fluorescence were motor neurons, while those not expressing green fluorescence were muscle cells (see Appendix). Figure 6 (A)

[0034] Similarly, under 3D co-culture conditions, NILG-hiPSCs and M1-hiPSCs were co-differentiated. Half an hour before seeding, anti-adhesion buffer (Anti-Adherence Rinsing Solution, Stemcell Technologies 07010) was added to 96-well round-bottom plates, and the plates were incubated at 37°C for half an hour. The anti-adhesion buffer was removed, and mTeSR containing Y-27632 was added. NILG-hiPSCs and M1-hiPSCs were then cultured in a 1:1 ratio, with a total density of 5 × 10⁶ cells / well. 3 Cells were seeded into 96-well plates and incubated at 37°C. After 16 h, the medium was replaced with mTeSR without Y-27632, and cultured for another 24 h. Then, skeletal muscle induction medium containing Dox (1 μg / mL) was added to initiate differentiation. Differentiation continued for 4 days, with the status of the 3D spheres and any cell shedding observed and recorded daily. Results are as follows: Figure 6 As shown in Figure B, the NILG-hiPSC+M1-hiPSC neuromuscular spheres did not exhibit spheroid dispersion or cell detachment and death, demonstrating excellent structural stability. After co-differentiation, the medium was replaced with skeletal muscle induction medium, and N2, B27, BDNF, GDNF, and NT-3 were added for co-culture.

[0035] 4. Establishment of a model of neuromuscular junction injury mediated by botulinum neurotoxin (BNT, MCE) and its synergistic effects with other drugs. (1) A neuromuscular injury model was established based on the strategy of 2D / 3D NILG-hiPSC and M1-hiPSC co-differentiation and co-culture in “Neuromuscular Co-differentiation and Co-culture Conditions Based on Skeletal Muscle Induction Medium”.

[0036] Neurotoxin is one of the most potent neurotoxins known and also the most widely used neuromuscular blocking agent in clinical practice. To simulate the damage and pathological state caused by neuromuscular diseases, we used neurotoxin to establish a neuromuscular injury model. Different concentrations (0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM) of neurotoxin were added on day 6 of co-differentiation and co-culture. After 2 days, cell viability and status were observed to select an appropriate concentration.

[0037] The results are as follows Figure 7 The results showed that, compared with the control group, after 2 days of treatment with 2 μM BNT, significant death of both neurons and muscle cells was observed in the co-culture system. This indicates that 2 μM of neurobotulinum toxin is sufficient to establish a neuromuscular junction injury model, providing a reliable basis for subsequent evaluation of the drug's protective effect.

[0038] (2) Synergistic effect of drugs: On the 4th day of differentiation, compounds (1 μM baicalein, 5 μM trichoderma, 0.5 μM baicalein + 2.5 μM trichoderma) were added for pretreatment. Two days later, 2 μM neurotoxin was added. After two days of treatment, the cell survival rate and the state of neurofilaments and muscle cells were observed under different magnifications of a microscope. The images were recorded, and immunofluorescence staining was performed to examine the formation of nerves, muscles and neuromuscular junctions.

[0039] As attached Figure 8 As shown in Figure A, compared with the model group, the addition of baicalin significantly improved neuronal survival and muscle cell survival. The addition of trichodin allowed for stable muscle cell survival, and the number of surviving neurons was also higher than in the model group. Notably, the simultaneous addition of both baicalin and trichodin resulted in better neuronal survival and a higher number of surviving muscle cells compared to the single-drug treatment group, approaching the control group, suggesting that these two compounds may work synergistically to combat cell damage.

[0040] Next, we performed immunofluorescence staining on each experimental group, as follows: In 24-well plates containing cell smears, the culture medium was first washed three times with DPBS to remove the cells. 4% PFA was added to each well, and the plates were fixed at room temperature for 20 min. The 4% PFA was then removed, and the plates were washed three times with DPBS for 15 min each time. 500 μL of tissue blocking buffer (3% BSA + 0.1% Triton) was added to each well, and the plates were blocked at room temperature for 2 h. After removing the blocking buffer, 400 μL of primary antibody working solution diluted with the blocking buffer (TUJ1 1:1000, MYH 1:1000) was directly applied to the sample surface, ensuring a uniform coating of the cell monolayer. The plates were then transferred to a 4°C environment for slow incubation for 12–16 h to promote the specific formation of antigen-antibody complexes. After 16 h of incubation, the primary antibody was discarded, and the plates were washed three times with DPBS for 15 min each time. Secondary antibody incubation: After thoroughly washing to remove non-specifically bound primary antibody, immediately prepare the fluorescent secondary antibody working solution: Based on the primary antibody source, dilute goat anti-mouse (555nm) and goat anti-rabbit (488nm) fluorescently labeled secondary antibodies with 3% BSA buffer at a ratio of 1:2000, and add 400 μL / well evenly to the cell surface. Incubate at room temperature in the dark for 2 hours. Aspirate the secondary antibody, wash three times with DPBS for 15 min each time. Add DAPI working solution, incubate at room temperature in the dark for 5 min, then wash with DPBS for 5 min. Remove the cell slides, place them cell-side up on a sealing film, add 50% glycerol, and invert a glass slide onto the cell slides. After mounting, store in the dark and photograph under a fluorescence microscope. Results are attached. Figure 8As shown in Figure B, more neurons survived under baicalein treatment, and muscle cells survived better under trichodin treatment. The dual-drug treatment group showed the best protective effect.

[0041] (3) Staining of neuromuscular junction α-BTX (α-clade krait venom): Fluorescently labeled α-BTX was used as a histochemical probe to locate and visualize the postsynaptic membrane region of the neuromuscular junction by labeling the aggregation and distribution of nAChR, thereby indirectly assessing the formation and integrity of the neuromuscular junction structure. In 24-well plates containing cell spreaders, cells were washed three times with 500 μL DPBS in each well, followed by the addition of 250 μL 4% PFA and fixation at room temperature for 15–20 min. The 4% PFA was removed, and each well was washed three times with 500 μL DPBS for 15 min each time. α-BTX incubation: The blocking solution was discarded, and diluted α-BTX and Hoechst were added directly, followed by incubation for 10 min. Cell spreaders were removed, placed cell-side up on a sealing film, and 50 μL of 50% glycerol was added per spread. The slide was then inverted onto the cell spreader. After mounting, the slides were immediately transferred to a dark box or a light-protected environment wrapped in aluminum foil to prevent fluorescence quenching. The images were then taken using a Zeiss laser scanning confocal microscope.

[0042] As attached Figure 8 As shown in Figure C, in the α-BTX immunofluorescence identification, the control group cells successfully constructed a dense neuromuscular network and exhibited a high level of α-BTX signal, indicating the formation of mature acetylcholine receptor clusters. In contrast, the neurobotulinum toxin group suffered severe synaptic structure damage due to large-scale apoptosis of neuromuscular cells, resulting in a significant attenuation or even absence of α-BTX labeling signal. However, drug intervention effectively reversed this pathological process. Both baicalein and trichosanthesin alone could partially restore α-BTX expression levels, suggesting the reconstruction of neuromuscular connections. The combined treatment with both drugs restored the α-BTX fluorescence intensity to levels close to the control group, indicating that this combined drug strategy not only significantly improved cell survival but also effectively promoted the regeneration and maturation of the neuromuscular junction.

[0043] 5. Baicalein-trichosamine promotes the contraction and twitching of skeletal muscle cells. Basement membrane matrix pretreatment was initiated 30 minutes before cell seeding: Matrigel working solution was evenly spread on the bottom surface of a 24-well plate, then transferred to a 37°C, 5% CO2 culture environment and incubated for 30 min to form a gel coating layer. The Matrigel was removed, and NILG-hiPSCs and M1-hiPSCs were mixed at a 1:1 ratio, totaling 1×10⁻⁶ cells. 4Cells were seeded into 24-well plates, and mTeSR containing Y-27632 was added. The plates were gently shaken and placed in an incubator at 37°C. After 16 h, the cells were replaced with mTeSR and cultured for another 24 h. Then, skeletal muscle induction medium containing Dox (1 μg / mL) (based on DMEM, supplemented with L-glutamine, penicillin-streptomycin antibiotics, β-mercaptoethanol, insulin, and retinoic acid, with final concentrations of 1% L-glutamine, 1% penicillin-streptomycin antibiotics, 50 μM β-mercaptoethanol, 7 μg / mL insulin, and 1 μM retinoic acid) was added to initiate differentiation. On day 4 of differentiation, monomeric compounds (1 μM baicalein, 5 μM trichosine, 0.5 μM baicalein + 2.5 μM trichosine) were added for pretreatment. Two days later, 2 μM BNT was added and treated for another two days. Then, the culture medium was changed to co-culture medium (skeletal muscle induction medium with added N2, B27, BDNF, GDNF, and NT-3). The muscle cell beating was observed, and videos were taken and recorded.

[0044] Three days after the establishment of the neuromuscular co-culture system, mature motor neurons successfully innervated muscle fibers and induced spontaneous contraction; the results recorded under a microscope showed (see Appendix) Figure 9 In the control group (A), regular and powerful rhythmic twitching of muscle fibers was observed, indicating intact neuromuscular transmission. However, in the neurobotulinum toxin group, large-scale cell death led to complete loss of contractile function, and twitching was barely observable in the field of vision. Notably, drug intervention significantly reversed this functional impairment. While baicalein / trichoderma monotherapy could partially restore muscle fiber contractile activity, the combined baicalein-trichoderma group showed superior efficacy, effectively reconstructing the frequency and amplitude of muscle fiber twitching. Quantitative analysis of spontaneous contraction of skeletal muscle fibers revealed no significant contraction in the model group, indicating that the observed contraction depended on the transmission of neural electrical activity. In contrast, the control group experienced more than 10 spontaneous contractions per minute. After drug treatment, skeletal muscle was again innervated by motor neurons, producing significant muscle contraction and twitching (see Appendix). Figure 9 (Chinese B), confirming that this treatment strategy not only improved cell survival rate, but more importantly, it suggests that the synergistic treatment of two drugs restored the functional contractile ability mediated by the neuromuscular junction.

[0045] 6. Investigate the synergistic protective effect of baicalin-trihrenulin in a neurobotulin 3D model. To verify the protective effects of baicalin and trichodin in a 3D microenvironment, this study established a neuromuscular junction injury model in a 3D co-culture system. NILG-hiPSCs and M1-hiPSCs were co-seeded in low-adhesion 96-well plates and co-differentiated for 4 days in skeletal muscle induction medium to form a 3D organoid structure with fused neurospheres and myosoids. Mature organoids were treated with 2 μM BNT for 2 days on day 6 of differentiation to establish a BNT-3D neuromuscular junction rupture model. (See attached...) Figure 10 As shown in Figure A, the model group exhibited structural damage, with loose edges and disintegration of the original dense spherical structure. A large number of cells detached from the core and entered the culture medium, suggesting that the neuromuscular connections in the 3D microenvironment underwent functional disintegration under toxin attack. Organoids pretreated with baicalein (1 μM, 48 h) alone maintained a relatively intact neurosphere core structure, and the TUJ1-positive neurofilament network was preserved and maintained a certain 3D architecture. However, partial detachment of the surrounding MYH-positive myofibril layer occurred. While the myofibril layer showed some improvement compared to the model group, it was not ideal (see Appendix). Figure 10 (See Figure B) indicates that baicalin mainly plays a neuroprotective role in the 3D environment, with limited effect on the structural stability of muscle fibers. Organoids pretreated with baicalin (5 μM, 48 h) alone exhibited different protective characteristics. MYH-positive muscle fiber layers had a denser structure, significantly reduced cell shedding, and more intact myospheric morphology, but obvious cavitation was visible in the center of the neurosphere, and the continuity of TUJ1-positive neurofilaments was interrupted and their density decreased (see Appendix B). Figure 10 (See Figure B). This suggests that while trichodin maintains muscle cell survival in the three-dimensional system, its long-term stabilizing effect on neural structures is insufficient. Crucially, the combined treatment of baicalin (0.5 μM, 48 h) and trichodin (2.5 μM, 48 h) exhibited a significant synergistic structural stabilizing effect. The organoid structure was dense, the fusion interface between the neurosphere and muscle ball was clearly discernible, and there was almost no cell shedding. Immunofluorescence showed that TUJ1-positive neurofilaments penetrated deep into the myofiber layer and formed a complex neural network, while MYH-positive myofibers were arranged in an orderly manner and tightly connected around the neurosphere. Both together maintained the original structural integrity of the 3D organoid (see Appendix). Figure 10 (B)

[0046] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0047] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A pharmaceutical combination for neuromuscular diseases, characterized in that, The drug combination includes baicalin and salvia miltiorrhiza.

2. The pharmaceutical combination for neuromuscular diseases according to claim 1, characterized in that, The drug combination has a protective effect on both neurons and motor nerves.

3. Application of baicalin and trichosanthesin in the preparation of drugs related to neuromuscular diseases.

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