Coordination of photobiomodulation promotes neuroregeneration in spinal cord injury by facilitating mitochondrial energy supply

Abstract

This invention discloses the application of coordinated photobiological regulation in promoting nerve regeneration through enhanced mitochondrial energy supply in spinal cord injury, belonging to the field of nerve regeneration and rehabilitation medicine. A coordinated photobiological regulation therapy device includes: a first light-emitting unit configured to emit light with a peak wavelength of 680 nm ± 20 nm; a second light-emitting unit configured to emit light with a peak wavelength of 850 nm ± 20 nm; and a control module electrically connected to both the first and second light-emitting units. The control module is configured to execute a treatment program, which includes: controlling the first light-emitting unit to turn on and continuously irradiate for a first predetermined time, while keeping the second light-emitting unit off; at the end of the first predetermined time, controlling the first light-emitting unit to turn off, and controlling the second light-emitting unit to turn on and continuously irradiate for a second predetermined time.

Description

Technical Field

[0001] This invention relates to the field of nerve regeneration and rehabilitation medicine, specifically to the application of coordinated photobiological regulation to promote nerve regeneration by enhancing mitochondrial energy supply in spinal cord injury. Background Technology

[0002] Spinal cord injury (SCI) is a severe neurological trauma that often leads to permanent motor, sensory, and autonomic dysfunction. Current clinical treatments are limited, and surgery and rehabilitation are insufficient to achieve nerve regeneration and functional reconstruction. Therefore, developing novel treatment strategies that can effectively induce nerve regeneration, repair damaged neural connections, and restore lost nerve function has become a core scientific challenge urgently needing breakthroughs in the field of spinal cord injury research.

[0003] Mitochondrial energy metabolism disorders are a key factor in the failure of neuroregeneration after spinal cord injury (SCI). Mitochondrial energy metabolism homeostasis is fundamental for cellular adaptation to external stress and maintenance of normal function. Disruption of this balance can lead to neuronal dysfunction and drive disease progression. Therefore, targeted regulation of mitochondrial metabolism has become a highly promising research direction in the treatment of spinal cord injury.

[0004] Photobiological modulation (PBM) is a non-invasive therapeutic approach that modulates cellular and tissue function through the emission of light of specific wavelengths (usually low-intensity lasers or light-emitting diodes (LEDs)). During illumination, photons are absorbed by photosensitive receptors within cells, most notably cytochrome c oxidase (CCO) in mitochondria, triggering a series of biochemical reactions. While PBM has shown potential in nerve repair as a non-invasive physical therapy, the effects of single-wavelength therapy are limited, and the mechanisms and effects of synergistic effects of dual-wavelength therapy remain unclear. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes an application of coordinated photobiological regulation to promote nerve regeneration by enhancing mitochondrial energy supply in spinal cord injury.

[0006] The objective of this invention can be achieved through the following technical solutions: A first aspect of the present invention relates to a coordinated photobiological modulation therapy device, comprising: The first light-emitting unit is configured to emit light with a peak wavelength of 680 nm ± 20 nm; The second light-emitting unit is configured to emit light with a peak wavelength of 850 nm ± 20 nm; And a control module, which is electrically connected to the first light-emitting unit and the second light-emitting unit respectively; The control module is configured to execute a treatment procedure, which includes: controlling the first light-emitting unit to turn on and continuously irradiate for a first predetermined time, while keeping the second light-emitting unit in a closed state; at the end of the first predetermined time, controlling the first light-emitting unit to turn off, and controlling the second light-emitting unit to turn on and continuously irradiate for a second predetermined time.

[0007] Optionally, the light emitted by both the first and second light-emitting units is a continuous wave, and the modulation frequency is 50 kHz; or, the device is configured to output light illumination such that the irradiance reaching the surface of the treatment target is 10 mW / cm². 2 Up to 100 mW / cm 2 .

[0008] Optionally, both the first predetermined time and the second predetermined time are 30 minutes; the first light-emitting unit and the second light-emitting unit are LED arrays.

[0009] A second aspect of the invention relates to the use of the aforementioned coordinated photobiological modulation therapy device in the preparation of an apparatus for promoting nerve regeneration in spinal cord injury.

[0010] A third aspect of the invention relates to the use of an Hspa8 protein expression activator or functional enhancer in the preparation of a medicament for treating spinal cord injury, said medicament being configured for use in conjunction with dual-wavelength coordinated photobiological modulation therapy.

[0011] Optionally, the Hspa8 protein comprises the amino acid sequence shown in SEQ ID NO: 1, or a sequence having at least 90% homology with it; the expression activator is selected from: an expression vector containing an Hspa8 gene, a recombinant Hspa8 protein, or a small molecule compound that specifically upregulates Hspa8 expression.

[0012] Optionally, the dual-wavelength coordinated photobiological modulation therapy is performed using the device described above.

[0013] A third aspect of the invention relates to the use of reagents for detecting the expression level of the Hspa8 gene or protein in the preparation of reagents or kits for evaluating the efficacy of photobiological modulation therapy for spinal cord injury.

[0014] Optionally, the reagent includes an antibody that specifically binds to the Hspa8 protein, an antigen-binding fragment, or a primer pair or probe that specifically amplifies Hspa8 mRNA.

[0015] A fourth aspect of the present invention relates to a method for in vitro screening of photobiological regulatory parameters that have a protective effect on spinal cord neurons, comprising the following steps: (1) Provide in vitro cultured spinal cord neurons or neuron-like cells and treat them with candidate light parameters; (2) Detect the expression level or activity level of Hspa8 in the treated cells; (3) Compare the level detected in step (2) with the control group. If the expression level or activity level of Hspa8 is significantly increased, the candidate light parameter is determined to have a neuroprotective effect.

[0016] Optionally, in step (1), the cells are pretreated with an acidic environment to simulate the spinal cord injury microenvironment, wherein the pH value of the acidic environment is 6.0-6.8.

[0017] The beneficial effects of this invention are: This invention reveals the application of CPBM in the treatment of spinal cord injury, which can most effectively improve the morphology of mitochondria in neurons in the injured area, increase CCO activity and ATP content. Simultaneously, CPBM is superior to single-wavelength therapy in promoting neuronal survival, axonal regeneration, and myelin repair. Furthermore, CPBM exhibits strong immunomodulatory capabilities, most significantly reducing pro-inflammatory cytokine levels, increasing anti-inflammatory cytokine levels, and promoting microglial polarization towards the beneficial M2 phenotype. Attached Figure Description

[0018] The invention will now be further described with reference to the accompanying drawings.

[0019] Figure 1 To coordinate the schematic diagram and spectral characteristics of the photobiological regulatory system; Figure 2 The experimental results show that CPBM promotes the recovery of motor function after spinal cord injury; Figure 3 Experimental results demonstrating how CPBM improves mitochondrial morphology and function; Figure 4 Experimental results showing that CPBM promotes axonal regeneration and neuronal survival; Figure 5 Experimental results on CPBM regulating the immune microenvironment; Figure 6 The experimental results of CPBM protecting neuronal mitochondria in an in vitro model; Figure 7 Experimental results showing that CPBM upregulates Hspa8 expression; Figure 8 This highlights the crucial role of Hspa8 in CPBM-mediated mitochondrial protection. Detailed Implementation

[0020] 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.

[0021] Example 1: Implementation of Coordinated Photobiological Modulation (CPBM) Therapy One core embodiment of the present invention relates to a photobiological modulation method for treating spinal cord injury (SCI). Specifically, it requires the use of a device capable of emitting specific dual-wavelength light. This device integrates a first light-emitting unit (such as an LED array) emitting 680 nm red light and a second light-emitting unit emitting 850 nm near-infrared light.

[0022] The treatment process is as follows: First, the spinal cord injury site (or the corresponding surface projection area, such as the corresponding segment on the back) of the animal (e.g., an SD rat weighing approximately 220g) or patient is exposed to the light source. After the device is turned on, the control module (e.g., a microprocessor) first controls the 680 nm light source to work alone, irradiating for 30 minutes. During this stage, the light penetrates the tissue, and its main target is cytochrome c oxidase (CCO) in the mitochondria of neurons, aiming to directly activate the respiratory chain and initiate energy metabolism. Subsequently, the control module automatically turns off the 680 nm light source and turns on the 850 nm light source, continuing irradiation for another 30 minutes. The 850 nm light, with its stronger tissue penetration ability, can deliver the effective light dose deeper to the core area of ​​the injury, exerting a synergistic effect. The total duration of a single treatment is 60 minutes, and the irradiance irradiated to the body surface is controlled at approximately 100 mW / cm². 2 After attenuation through skin and muscle tissue, the light intensity reaching the spinal cord level is approximately 10 mW / cm². 2 The irradiation mode is continuous wave, with a modulation frequency of 50 kHz. This treatment is recommended to begin on day 1 of the injury, once daily for 14 consecutive days.

[0023] Example 2: Composition of a Coordinated Photobiological Regulation Therapy System The treatment system that implements the above method specifically includes: Light source module: Composed of independent 680 nm LED array board and 850 nm LED array board to ensure spectral purity.

[0024] Control module: This is the core controller (such as a microcontroller or PLC) that stores a preset program. This program precisely controls the on / off sequence of the two light sources (680 nm first, then 850 nm), the duration of each irradiation (30 minutes each), and the operating frequency (50 kHz).

[0025] Power supply and heat dissipation module: Provides a stable current to the LED and is equipped with heat sinks or fans to ensure stable light source performance during long-term operation.

[0026] Mechanical structure: The above components are integrated into a portable housing and designed with an adjustable support suitable for aligning with the treatment site.

[0027] The connection relationship between the components is as follows: the power supply module supplies power to the control module and the light source module; the control module is electrically connected to the two LED arrays through the drive circuit to output control signals; the mechanical structure supports and fixes all functional modules.

[0028] Example 3: Validation of its application effect in an animal model of spinal cord injury This embodiment demonstrates the specific application and effect of the above-mentioned CPBM method in the SCI rat model.

[0029] Following the experimental procedure, after establishing the SCI model, rats were randomly divided into groups and treated accordingly. Behavioral assessments revealed that, compared to the untreated group or the single-wavelength treatment group, rats treated with CPBM showed significantly greater recovery in hindlimb motor function (BBB score), limb coordination (LSS score in swimming test), and muscle strength (angle in ramp test). Electrophysiological testing showed that the CPBM group exhibited the best recovery in motor evoked potential (MEP) amplitude, indicating more effective repair of neural conduction pathways.

[0030] At the histopathological level, CPBM treatment most effectively improves the morphology of mitochondria in damaged neurons, increases CCO activity and ATP content. Simultaneously, CPBM is superior to single-wavelength therapy in promoting neuronal survival (increased NeuN-positive cells), axonal regeneration (increased TUJ1 and NF-positive areas), and myelin repair (increased MBP-positive areas).

[0031] In addition, CPBM exhibits strong immunomodulatory capabilities, most significantly reducing pro-inflammatory factor levels, increasing anti-inflammatory factor levels, and promoting microglia polarization toward the beneficial M2 phenotype.

[0032] Example 4: Validation and Prospects of HSPA8 as a Key Medium in Joint Applications The mechanism of this invention has been thoroughly validated at the cellular level. In a neuronal model simulating the acidic environment of SCI, CPBM treatment also showed optimal protective effects, reducing apoptosis, lowering ROS, restoring mitochondrial membrane potential, and improving mitochondrial network morphology.

[0033] Key mechanism exploration revealed that CPBM can specifically and significantly upregulate the expression of the heat shock protein Hspa8. The Hspa8 protein described in this invention is a member of the rat heat shock protein 70 family, with GenBank accession numbers NP_077327.1 (amino acid sequence) and NM_024351.1 (mRNA sequence), and its amino acid sequence is shown in SEQ ID NO:1. To verify the key role of Hspa8 in CPBM-mediated neuroprotection, a siRNA targeting the rat Hspa8 gene was designed and synthesized. Its sense strand sequence is 5'-GGAUGUUAUCACAAUGCUATT-3' (SEQ ID NO:2), and its antisense strand sequence is 5'-UAGCAUUGUGAUAACAUCCUU-3' (SEQ ID NO:3). A negative control siRNA was used as a control.After transfecting primary cultured spinal cord neurons with siRNA, the expression level of Hspa8 was detected by real-time quantitative PCR. The real-time quantitative PCR used forward primer 5'-GACAAGAAGGTGCTGGACAAG-3' (SEQ ID NO: 4) and reverse primer 5'-CAGTTGCTTCACCTTGTCCA-3' (SEQ ID NO: 5). The results showed that the mRNA expression level of Hspa8 in cells transfected with siRNA was reduced by more than 70% compared to the control group. In this cell model with knocked-down Hspa8 expression, the beneficial effects of CPBM in anti-apoptosis, protection of mitochondrial function and structure, and promotion of ATP production were significantly weakened or even eliminated. This confirms that Hspa8 is an indispensable key downstream molecule for CPBM to exert its neuroprotective effects.

[0034] Based on this, the present invention also encompasses a combined treatment approach: simultaneously with or before and after CPBM irradiation, a drug capable of activating or enhancing Hspa8 expression or function (i.e., an Hspa8 expression activator or function enhancer) is administered to produce an additive or synergistic therapeutic effect. Furthermore, detecting the expression level of Hspa8 in tissues or cells can serve as a biomarker for screening or evaluating the effectiveness of other photobiological regulation strategies for SCI.

[0035] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0036] 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 claimed invention.

Claims

1. A coordinated photobiological regulation therapy device, characterized in that, include: The first light-emitting unit is configured to emit light with a peak wavelength of 680 nm ± 20 nm; The second light-emitting unit is configured to emit light with a peak wavelength of 850 nm ± 20 nm; And a control module, which is electrically connected to the first light-emitting unit and the second light-emitting unit respectively; The control module is configured to execute a treatment procedure, which includes: controlling the first light-emitting unit to turn on and continuously irradiate for a first predetermined time, while keeping the second light-emitting unit in a closed state; at the end of the first predetermined time, controlling the first light-emitting unit to turn off, and controlling the second light-emitting unit to turn on and continuously irradiate for a second predetermined time.

2. The apparatus according to claim 1, characterized in that, The light emitted by both the first and second light-emitting units is a continuous wave with a modulation frequency of 50 kHz; or, the device is configured to output light illumination such that the irradiance reaching the surface of the treatment target is 10 mW / cm². 2 Up to 100 mW / cm 2 .

3. The apparatus according to claim 1, characterized in that, The first predetermined time and the second predetermined time are both 30 minutes; the first light-emitting unit and the second light-emitting unit are LED arrays.

4. The use of the coordinated photobiological modulation therapy device according to any one of claims 1 to 3 in the preparation of a device for promoting nerve regeneration in spinal cord injury.

5. The use of Hspa8 protein expression activators or functional enhancers in the preparation of drugs for treating spinal cord injuries, characterized in that, The drug is formulated for use in conjunction with dual-wavelength coordinated photobiological modulation therapy.

6. The application according to claim 5, characterized in that, The Hspa8 protein comprises the amino acid sequence shown in SEQ ID NO: 1, or a sequence having at least 90% homology with it; the expression activator is selected from: an expression vector containing an Hspa8 gene, a recombinant Hspa8 protein, or a small molecule compound that specifically upregulates Hspa8 expression.

7. Application of reagents for detecting Hspa8 gene or protein expression levels in the preparation of reagents or kits for evaluating the efficacy of photobiological modulation therapy for spinal cord injury.

8. The application according to claim 6, characterized in that, The reagents include antibodies that specifically bind to the Hspa8 protein, antigen-binding fragments, or primer pairs or probes that specifically amplify Hspa8 mRNA.

9. A method for in vitro screening of photobiological regulatory parameters that have a protective effect on spinal cord neurons, characterized in that, Includes the following steps: (1) Provide in vitro cultured spinal cord neurons or neuron-like cells and treat them with candidate light parameters; (2) Detect the expression level or activity level of Hspa8 in the treated cells; (3) Compare the level detected in step (2) with the control group. If the expression level or activity level of Hspa8 is significantly increased, the candidate light parameter is determined to have a neuroprotective effect.

10. The method according to claim 9, characterized in that, In step (1), the cells are pretreated in an acidic environment to simulate the microenvironment of spinal cord injury, wherein the pH of the acidic environment is 6.0-6.8.

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