Application of trichosanthes peel injection in preparation of medicine for treating spinal cord injury

Abstract

The invention discloses application of a snakegourd peel injection in preparation of a medicine for treating spinal cord injury, and relates to the technical field of biological medicines. SCI mouse model research finds that TPI can promote motor function recovery after SCI, relieve tissue damage, protect neuron structures, inhibit neuroinflammation, promote polarization of microglial cells to anti-inflammatory phenotypes, relieve oxidative stress after spinal cord injury, inhibit neuronal apoptosis and promote axonal regeneration, and can be used for preparing the medicine for treating the spinal cord injury. The potential action mechanism of the TPI is disclosed through network pharmacology and molecular docking, and the high consistency of an in-vivo experiment result and an in-vitro prediction result enhances the rationality of taking the TPI or an active component thereof as a candidate drug for SCI treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of a Trichosanthes peel injection in the preparation of drugs for treating spinal cord injury. Background Technology

[0002] Spinal cord injury (SCI) is one of the most severe central nervous system injuries, often leading to permanent neurological deficits and paralysis, imposing a heavy burden on individuals and society. SCI is generally classified into primary and secondary injuries. In the secondary pathological process, neuroinflammation and oxidative stress are particularly crucial. Activated microglia and infiltrating immune cells release pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, inducing neuronal apoptosis, axonal degeneration, and glial scarring. Simultaneously, mitochondrial dysfunction and ischemia-driven oxidative stress produce excessive reactive oxygen species and reactive nitrogen species (ROS / RNS), exceeding the endogenous antioxidant defense capacity of superoxide dismutase (SOD) and glutathione. These processes reinforce each other, forming a vicious cycle that exacerbates SCI pathology. Existing drugs, such as methylprednisolone for anti-inflammation and edaravone and N-acetylcysteine ​​for antioxidation, can provide some protection, but their clinical efficacy is limited. Therefore, simultaneously targeting inflammation and oxidative stress, especially by regulating microglia polarization, is considered a promising therapeutic strategy.

[0003] Trichosanthes peel (TP), also known as gualoupi, is the dried, mature pericarp of Trichosanthes skirilowii Maxim. It is a traditional Chinese medicine often used for clearing heat and resolving phlegm. Phytochemical studies have shown that TP contains active ingredients such as flavonoids (e.g., luteolin and rutin), polysaccharides, and triterpenoids, exhibiting anti-inflammatory, antioxidant, and cardiovascular protective effects. Trichosanthes peel injection (TPI) is a standardized preparation made from TP and has shown significant pharmacological activity. Studies have shown that TPI can inhibit pro-inflammatory factors and apoptosis signaling, enhance antioxidant defense, and alleviate cardiovascular and metabolic diseases. However, whether TPI can regulate neuroinflammation and oxidative stress in SCI, particularly by improving functional recovery through microglial polarization, remains unclear. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an application of Trichosanthes kirilowii peel injection in the preparation of drugs for treating spinal cord injury. Through in vivo experiments and in vitro prediction, it has been confirmed that TPI or its active ingredient can be used as a candidate drug for the treatment of SCI.

[0005] The technical problem to be solved by this invention is achieved by the following technical solution:

[0006] The first objective of this invention is to provide the use of Trichosanthes kirilowii peel injection or its active ingredient in the preparation of a drug for treating spinal cord injury.

[0007] A second object of the present invention is to provide a pharmaceutical composition comprising Trichosanthes kirilowii peel injection or its active ingredient. Trichosanthes kirilowii peel injection or its active ingredient can be used alone to prepare a drug for treating spinal cord injury, or it can be combined with existing spinal cord injury treatments for the same purpose.

[0008] Furthermore, the pharmaceutical composition further includes pharmaceutically acceptable excipients. Even further, the excipients are one or more of the following: diluents, dispersants, binders, wetting agents, solubilizers, disintegrants, lubricants, coating agents, controlled-release agents, pH adjusters, osmotic pressure adjusters, antioxidants, preservatives, flavoring agents, coloring agents, etc.

[0009] A third object of the present invention is to provide the use of the pharmaceutical composition in the preparation of a medicament for treating spinal cord injury.

[0010] Furthermore, the dosage form of the medication for treating spinal cord injury is injection, capsule, tablet, granule, oral liquid, aerosol, spray, drops, pills, patch, or ointment, etc.

[0011] Furthermore, the spinal cord injury includes primary injury and secondary injury.

[0012] A fourth object of the present invention is to provide a medicament for treating spinal cord injury, comprising Trichosanthes kirilowii peel injection or its active ingredient.

[0013] The beneficial effects of this invention are as follows: Through research using a mouse model of spinal cord injury (SCI), this invention has discovered that TPI can promote the recovery of motor function after SCI, reduce tissue damage and protect neuronal structure, inhibit neuroinflammation and promote the polarization of microglia to an anti-inflammatory phenotype, alleviate oxidative stress after spinal cord injury, inhibit neuronal apoptosis and promote axonal regeneration. Furthermore, through network pharmacology and molecular docking, the potential mechanism of action of TPI has been revealed. The high consistency between in vivo experimental results and in vitro prediction results enhances the rationality of using TPI or its active ingredients as candidate drugs for the treatment of SCI. Attached Figure Description

[0014] Figure 1 To improve motor function recovery after spinal cord injury using TPI; (A) Schematic diagram of experimental procedure and drug administration timeline; (B) Footprint photographs of mice 28 days after injury; (CD) Quantitative results of stride length and stride width in footprint analysis (6 mice per group); (E) BBB scores at 1, 7, 14, 21 and 28 days after injury (6 mice per group); (FG) Rotation speed and dwell time in the rotarod test 28 days after injury;

[0015] Figure 2 Preservation of spinal cord histological structure for TPI; (A) HE-stained images of spinal cord sections 28 days after injury, scale bar 50 μm; (B) Quantitative results of the area of ​​the injury cavity in HE-stained sections (6 sections from 3 mice per group); (C) Masson trichrome staining images, scale bar 50 μm; (D) Quantitative results of the area of ​​collagen-positive areas in Masson staining (6 sections from 3 mice per group); (E) Nissl-stained sections, scale bar 50 μm; (F) Quantitative results of the density of Nissl-positive neurons (6 sections from 3 mice per group).

[0016] Figure 3 To reduce neuroinflammation and regulate microglial polarization after spinal cord injury using TPIs; (AC) protein levels of TNF-α, IL-6, and IL-1β (ELISA, 3 mice per group, 9 data points in total); (DF) mRNA levels of TNF-α, IL-6, and IL-1β (qPCR, 3 mice per group); (GH) Iba-1 immunostaining and quantification results (3 mice per group, 6 slides in total), scale bar 50 μm; (IJ) CD86 staining and quantification results (3 mice per group, 6 slides in total), scale bar 50 μm; (KL) Arg-1 staining and quantification results (3 mice per group, 6 slides in total), scale bar 50 μm;

[0017] Figure 4 To reduce oxidative stress in the injured spinal cord by TPI; (A) Representative image of DHE fluorescence staining, scale bar 100 μm; (B) Quantitative results of DHE fluorescence intensity (3 mice per group, 4 slices in total); (CD) Quantitative detection results of SOD and MDA content (3 mice per group, 5 data points in total).

[0018] Figure 5 To investigate the effects of TPI on neuronal apoptosis and axonal regeneration: (A) Dual immunofluorescence staining results of NeuN and cleaved caspase-3 14 days after injury, scale bar 50 μm; (BC) Quantitative results of cleaved caspase-3 fluorescence intensity and NeuN-positive area (6 slides from 3 mice per group); (D) Western blot detection of NeuN and cleaved caspase-3 expression; (EF) Western blot grayscale value quantification results (3 data points from 3 mice per group); (GH) Immunostaining and quantification results of MAP2 (6 slides from 3 mice per group), scale bar 50 μm; (IJ) Immunostaining and quantification results of NF200 (6 slides from 3 mice per group), scale bar 50 μm.

[0019] Figure 6 This is a compound-disease-target (CDT) network constructed based on the intersection targets of TPI active ingredients and SCI-related genes; where green squares represent TPI preparations, red nodes represent diseases (SCI), purple nodes represent active ingredients, and blue nodes represent common target genes.

[0020] Figure 7 The following are the network pharmacology analysis results of TPIs in SCI: (A) Venn diagram; (B) Protein-protein interaction (PPI) network; (CE) GO enrichment analysis results of core targets; (F) KEGG pathway enrichment analysis results.

[0021] Figure 8 The results show the molecular docking of key active components of TPI with TNF; (A) TNF-kaempferol complex (binding energy: -6.195 kcal / mol); (B) TNF-luteolin complex (binding energy: -6.727 kcal / mol); (C) TNF-quercetin complex (binding energy: -6.983 kcal / mol); The left figure shows the overall binding conformation; the middle figure shows the specific interaction sites; the right figure is a two-dimensional interaction pattern diagram, showing hydrogen bonds, hydrophobic interactions and other interaction forces. Detailed Implementation

[0022] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments and illustrations.

[0023] Reagents and antibodies:

[0024] Trichosanthes Peel Injection (Cat#Z20027540) was purchased from Shanghai No.1 Biochemical Pharmaceutical Co., Ltd.

[0025] The qPCR reagents included HiScript IIQ RT SuperMix (Cat#R122-01) and AceQ qPCR SYBRGreen Master Mix (Cat#Q111-02), both purchased from Vazyme.

[0026] DAPI (Cat#D9542) was purchased from Sigma-Aldrich.

[0027] Masson staining kit (Cat#C0189S), BCA protein quantification kit (Cat#P001), Nissl staining solution (Cat#C0117), SOD detection kit (Cat#S0101S), DHE fluorescent probe ROS detection kit (Cat#S0064S), and IL-6 ELISA kit (Cat#PI326) were all purchased from Beyotime.

[0028] Anti-CD86 antibody (1:1000, Cat#DF6332, RRID: AB_2838296) and ECL chemiluminescence reagent were purchased from Affinity.

[0029] The MDA test kit (Cat#BC0025) was purchased from Solarbio.

[0030] Anti-β-actin antibody (1:10000, Immunoway, Plano, TX, USA, Cat#YM3028, RRID:AB_2629465), TNF-α ELISA kit (Cat#KE1419), and IL-1β ELISA kit (Cat#KE1416) were purchased from Immunoway.

[0031] Anti-cleaved caspase-3 antibody (1:1000, Abcam, UK, Cat#ab32042, RRID: AB_725947) was purchased from Abcam.

[0032] Other antibodies include anti-Arg-1 (arginase-1, 1:10000, Cat#66129-1-Ig, RRID: AB_2881528), anti-Iba-1 (ionized calcium-binding aptamer protein 1, 1:1000, Cat#10904-1-AP, RRID: AB_2224377), anti-MAP2 (Cat#17490-1-AP), and anti-NF200 (Cat#60331-1-Ig). The secondary antibody CoraLite488-labeled goat anti-mouse IgG (1:400, Proteintech, Cat#SA00013-1, RRID: AB_2810983) and CoraLite594-labeled goat anti-rabbit IgG (1:400, Proteintech, Cat#SA00013-4, RRID: AB_2810984) were all purchased from Proteintech.

[0033] Mouse SCI model and drug treatment

[0034] Male C57BL / 6 mice (8-10 weeks old, weighing 20-22g) were purchased from the Experimental Animal Center of the First Affiliated Hospital of the University of Science and Technology of China and housed under standard conditions (22-24℃, 60-80% humidity). All experiments were approved by the Animal Ethics Committee of Anqing Normal University (Approval No.: AQNU2024147). Under 2% isoflurane anesthesia, a spinal cord incision model (SCI) was established by striking the exposed spinal cord with a 10g metal rod dropped from a height of 8cm after laminectomy at T9-10 vertebrae. The sham-operated group underwent only laminectomy without the striking. Mice were randomly divided into a Sham group, an SCI group, and an SCI+TPI group. Postoperative care included daily artificial bladder compression and local antibiotic treatment. TPI (1.0mL / kg) was administered intraperitoneally once daily for 28 consecutive days postoperatively; the SCI control group received an equal volume of physiological saline (0.5mL).

[0035] Behavioral assessment:

[0036] 1. BBB score

[0037] Hindlimb motor function was assessed using the 21-point Basso-Beattie-Bresnahan (BBB) ​​scoring system at 0, 1, 7, 14, 21, and 28 days post-surgery. Assessments included joint mobility, gait coordination, and trunk stability, and were independently scored by two observers.

[0038] 2. Footprint Analysis

[0039] Red and blue ink were applied to the hind feet of mice, respectively, and the mice were then allowed to run across a track covered with white paper. Stride length and stride width were measured to assess hind limb coordination.

[0040] 3. Rotating bar experiment

[0041] Motor coordination and balance were assessed using a rotarod accelerator (Mouse Rotarod Fatigue Meter, model: ZF54-YLS-4D, Ca#M407469, Beijing, China), gradually accelerating from 10 rpm to 40 rpm for 5 minutes. Each mouse underwent three tests at 20-minute intervals, and the average value was taken.

[0042] Histological analysis:

[0043] Under deep anesthesia, the spinal cord was perfused with normal saline and 4% paraformaldehyde (PFA). A 6 mm segment of spinal cord tissue, centered at the injury center, was harvested, fixed overnight in 4% PFA, graded dehydration, and embedded in paraffin. Sections were 5 μm thick. HE staining was performed using standard methods; Nissl staining was performed using 0.05% desiccant solution at 40°C for 10 min; Masson staining was performed sequentially using Wiegand hematoxylin, acid fuchsin, phosphomolybdic acid, and aniline blue according to the kit instructions. All sections were dehydrated, cleared, mounted, and observed under a microscope.

[0044] Reactive oxygen species (ROS) detection:

[0045] The ROS level in spinal cord tissue was detected using a dihydroethidium (DHE) fluorescent probe method (Beyotime, Cat#S0064S). After DHE staining, the sections were observed under a fluorescence microscope, and the fluorescence intensity (Ex 520nm / Em 605nm) was measured using a microplate reader.

[0046] ELISA test:

[0047] The levels of TNF-α, IL-6, and IL-1β in spinal cord tissue were determined using a commercial ELISA kit (Immunoway) according to the instructions. Absorbance was measured at 450 nm using a microplate reader (Multiskan MK3, Thermo Fisher).

[0048] Detection of malondialdehyde (MDA) and superoxide dismutase (SOD):

[0049] The MDA content and total SOD activity in spinal cord tissue were detected using appropriate kits. All results were normalized to the total protein content determined by the BCA method.

[0050] Real-time quantitative polymerase chain reaction (qPCR):

[0051] Total RNA was extracted using the Trizol method, and cDNA was synthesized by reverse transcription using a HiScript IIQ RT SuperMix (R122-01, Vazyme, China). qPCR was performed using an AceQ SYBR Green Master Mix (Q111-02, Vazyme) on a Roche LightCycler 480 II real-time PCR instrument (Roche LightCycler 480 II, Germany). Relative mRNA levels were calculated using the 2^-ΔΔCT method, with β-actin as the internal reference gene. Primer sequences are shown in Table 1.

[0052] Table 1 Primer sequences used for quantitative real-time PCR analysis

[0053]

[0054]

[0055] Western blot:

[0056] Total protein was extracted from spinal cord tissue, quantified by BCA, and then loaded onto a plate for separation by SDS-PAGE electrophoresis. After transfer to a PVDF membrane, the sample was blocked at room temperature for 1 hour and incubated overnight at 4°C with primary antibodies (NeuN, cleaved-caspase-3, β-actin). The following day, the sample was incubated with HRP-labeled secondary antibody, and ECL was used for colorimetric analysis. Detection was performed using a chemiluminescence imaging system (FluorChem R, ProteinSimple). Grayscale analysis was performed using ImageJ software. All experiments were repeated three times.

[0057] Immunofluorescence staining:

[0058] After dewaxing and hydration of paraffin sections, antigen retrieval was performed using EDTA buffer. Sections were blocked with 10% BSA and incubated overnight at 4°C with primary antibodies (CD86, Arg-1, Iba-1, cleaved-caspase-3, MAP2, NF200). The following day, sections were incubated with CoraLite fluorescently labeled secondary antibody and counterstained with DAPI. Fluorescence images were acquired using a panoramic tissue scanner (Pannoramic DESK, 3D HISTECH, Hungary), and signal quantification was performed in three regions of interest (ROIs).

[0059] Network pharmacology:

[0060] TPI-related components were obtained from the TCMBank database, and SCI-related genes were obtained from the GeneCards and OMIM databases. Overlapping targets were screened using Venn diagrams, and a PPI network was constructed from the STRING database (Homo sapiens) and visualized in Cytoscape. GO and KEGG pathway enrichment analysis was performed using clusterProfiler and ggplot2 (R package), with a threshold set at p < 0.05.

[0061] Molecular docking:

[0062] Core active ingredients (such as luteolin and quercetin) were obtained from PubChem and processed using RDKit and Open Babel. The TNF three-dimensional structure (PDB:1TNF) was downloaded from the PDB database and preprocessed using PyMol and MGLTools. Molecular docking was performed using AutoDock Vina v1.2.7 (mesh parameters: x = 23, y = 61.0, z = 42.0). Binding energy was evaluated by docking scoring; lower binding energy indicates stronger affinity.

[0063] Statistical analysis:

[0064] All data are expressed as mean ± SEM and are from at least three independent experiments. After tests for normality and homogeneity of variance, comparisons between groups were performed using one-way or two-way ANOVA, with a Tukey post-hoc test. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 26.0 and GraphPad Prism 8.4.2.

[0065] Results analysis:

[0066] 1. TPI promotes motor function recovery after SCI.

[0067] To evaluate the therapeutic effect of TPI on motor function after SCI, this invention conducted a series of behavioral assessments, the flowchart of which is shown in Figure 1A.

[0068] Footprint analysis showed that, compared with the SCI group, the TPI treatment group exhibited a more coordinated and regular hindlimb gait pattern 28 days after injury. Figure 1 B). Quantitative analysis further confirmed that TPI treatment significantly improved both stride length and stride width in mice. Figure 1 C, D). From day 7 post-injury, the BBB motor score in the PI group was significantly higher than that in the SCI group, and continued to improve throughout the 28-day observation period. Figure 1 E). Furthermore, the results of the rotator bar experiment showed that the TPI-treated group was significantly superior to the SCI group in both rotational speed and drop time. Figure 1 F, G).

[0069] The above results indicate that TPI can promote the recovery of motor function after SCI.

[0070] 2. TPIs reduce tissue damage and protect neuronal structure.

[0071] To evaluate the protective effect of TPI on spinal cord morphology after SCI, histological analysis was performed. HE staining results showed that 28 days post-injury, the SCI group exhibited significant lacunar infarction and tissue loss, while the TPI-treated group had smaller lacunar areas. Figure 2 A). Quantitative analysis confirmed that the cavity area in the TPI group was significantly lower than that in the SCI control group ( Figure 2 B). Masson's trichrome staining was used to assess fibrous scar formation, and the results showed extensive collagen deposition at the injury site in the SCI group, while TPI treatment significantly reduced collagen accumulation. Figure 2 C, D). Nissl staining results showed that the neuronal structure in the peri-injury area was better preserved in the TPI group, and the number of Nissl-positive neurons was significantly higher than that in the SCI group, suggesting that it has a neuroprotective effect. Figure 2 E, F).

[0072] The above results indicate that TPI can effectively reduce secondary tissue damage and promote neuronal survival after SCI.

[0073] 3. TPIs inhibit neuroinflammation and promote the polarization of microglia toward an anti-inflammatory phenotype.

[0074] To investigate the anti-inflammatory effects of TPIs, this invention detected the levels of pro-inflammatory factors in spinal cord tissue. ELISA results showed that TNF-α, IL-6, and IL-1β were significantly elevated in the SCI group, while TPI treatment significantly reduced the levels of these factors. Figure 3 AC). qPCR results further confirmed that TPI can downregulate the mRNA expression of inflammatory factors TNF-α, IL-6, and IL-1β. Figure 3 DF).

[0075] Given the central role of microglia in neuroinflammation, this invention examined their activation and polarization states. Iba-1 immunostaining showed that microglia in the SCI group were significantly activated, while TPI significantly reduced this activation. Figure 3 G, H). Furthermore, the M1 biomarker CD86 was highly expressed in the SCI group, but significantly reduced in the TPI treatment group (G, H). Figure 3 I, J); Conversely, the M2 marker Arg-1 was upregulated in the TPI group ( Figure 3 K, L).

[0076] The above results indicate that TPI can not only reduce the level of inflammatory factors, but also promote the transformation of microglia from a pro-inflammatory phenotype to an anti-inflammatory phenotype.

[0077] 4. TPI alleviates oxidative stress after spinal cord injury

[0078] To assess the effect of TPI on oxidative stress after SCI, this invention performed DHE staining to detect ROS levels. The results showed that DHE fluorescence was significantly enhanced in the SCI group, indicating a large accumulation of ROS, while ROS levels were significantly reduced in the TPI group. Figure 4 A, B).

[0079] Further analysis was conducted to determine the activity of the antioxidant enzymes SOD and MDA levels. Compared to the SCI group, TPI significantly increased SOD activity and decreased MDA levels. Figure 4 C, D).

[0080] The above results indicate that TPI alleviates oxidative damage after SCI by restoring redox homeostasis.

[0081] 5. TPIs inhibit neuronal apoptosis and promote axonal regeneration.

[0082] To assess whether TPI protects neurons after SCI, dual immunofluorescence staining for NeuN and cleaving caspase-3 was performed on day 14 post-injury. Results showed significantly enhanced fluorescence of cleaving caspase-3 and a reduced area of ​​NeuN-positive neurons in the SCI group, while TPI significantly reversed these changes. Figure 5 AC). Western blot results further confirmed that TPI can reduce cleavage caspase-3 and maintain NeuN protein levels (AC). Figure 5 DF).

[0083] To assess neuronal regeneration, this invention detected dendritic and axon-related markers. Immunofluorescence results showed that MAP2 and NF200 expression were reduced in the SCI group, while TPI treatment significantly enhanced MAP2 signaling. Figure 5 G, H) and increase NF200 level ( Figure 5 I, J), thus demonstrating that TPI can promote dendritic repair and axon regeneration.

[0084] 6. Network pharmacology and molecular docking reveal the potential mechanism of action of TPIs

[0085] To identify the active ingredients that exert neuroprotective effects of TPIs, this invention screened 19 representative active compounds based on OB and DL thresholds, including various flavonoids such as rutin, kaempferol, quercetin, and phenylalanine, as shown in Table 2.

[0086] Table 2. Network pharmacologically active components of TPIs

[0087]

[0088] Subsequently, this invention constructed a compound-disease-target (CDT) network, revealing that TPIs possess a multi-component, multi-target pharmacological action mode. Network visualization was performed using Cytoscape 3.9.1 software. Figure 6 As shown, the core components (such as rutin, quercetin, and arginine) interact with multiple inflammation and apoptosis-related targets (such as TNF, IL1B, CASP3, and MAPK1), supporting the hypothesis that TPIs exert their effects through multi-target synergy.

[0089] Furthermore, Venn diagrams were used to screen for overlap between TPI predicted targets and SCI-related genes. Figure 7 A), and construct a PPI network based on these targets ( Figure 7B), the color intensity of the nodes reflects the connectivity, with darker colors indicating higher connectivity. Key pivotal proteins such as TNF, IL6, and TP53 are highlighted. These key pivotal genes may play important roles in the pathogenesis of SCI and the regulation of TPI. GO enrichment analysis results show that these targets are mainly involved in biological processes such as vascular circulation, epithelial cell proliferation, cellular responses to chemical stress, blood-brain barrier transport, hypoxia, and lipopolysaccharide responses. Figure 7 C). Cell component analysis results ( Figure 7 (D) indicates that these targets are mainly distributed in the membrane rafts, vesicle cavities, apical and basal regions, and the outer side of the plasma membrane, suggesting that membrane signaling, immune molecule transport, and cellular structural polarity may be involved. Molecular functional analysis results ( Figure 7 E) showed that these targets were significantly enriched in functions such as cytokine activity, receptor binding, transcription factor interaction, and transmembrane transport, pathways closely related to inflammatory signaling, oxidative stress, and gene regulation. KEGG pathway enrichment analysis results ( Figure 7 F) shows that these targets are associated with multiple immune and inflammation-related pathways, including the TNF signaling pathway, the AGE-RAGE signaling pathway (a complication of diabetes), and the hepatitis B / C pathway. These pathways are all closely related to oxidative stress, neuroinflammation, and apoptosis.

[0090] Molecular docking results confirmed that key TPI components (such as rutin, kaempferol, and quercetin) have strong binding activity with TNF-α, revealing their potential direct regulation of inflammatory and apoptotic mediators. Figure 8 Consistent with this, the results of this invention indicate that TPI significantly reduces the mRNA and protein levels of TNF-α in SCI tissues. Figure 3 A and D) provide in vivo evidence for the predicted interactions.

[0091] In summary, TPIs may exert neuroprotective effects by regulating oxidative stress and inflammatory pathways, altering immune responses, and directly acting on key proteins related to nerve injury and repair.

[0092] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. The application of Trichosanthes peel injection or its active ingredients in the preparation of drugs for treating spinal cord injury.

2. The application according to claim 1, characterized in that: The spinal cord injury includes primary injury and secondary injury.

3. The application according to claim 1, characterized in that: The dosage forms of the medication for treating spinal cord injury are injections, capsules, tablets, granules, oral liquids, aerosols, sprays, drops, pills, patches, or ointments.

4. A pharmaceutical composition, characterized in that: This includes Trichosanthes peel injection or its active ingredients.

5. The pharmaceutical composition according to claim 4, characterized in that: The pharmaceutical composition also includes pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 5, characterized in that: The excipients are one or more of the following: diluent, dispersant, binder, wetting agent, solubilizer, disintegrant, lubricant, coating agent, controlled-release agent, pH adjuster, osmotic pressure adjuster, antioxidant, preservative, flavoring agent, and coloring agent.

7. Use of the pharmaceutical composition according to any one of claims 4 to 6 in the preparation of a medicament for treating spinal cord injury.

8. The application according to claim 7, characterized in that: The spinal cord injury includes primary injury and secondary injury.

9. The application according to claim 7, characterized in that: The dosage forms of the medication for treating spinal cord injury are injections, capsules, tablets, granules, oral liquids, aerosols, sprays, drops, pills, patches, or ointments.

10. A medicine for treating spinal cord injury, comprising Trichosanthes kirilowii peel injection or its active ingredient.

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