Application of hydrogen molecules in preparation of product for promoting extracellular matrix accumulation

Abstract

An application of hydrogen molecules in the preparation of a product for promoting extracellular matrix accumulation. One or more hydrogen molecule intervention means may be used in combination, to increase extracellular matrix accumulation, promote wound healing, or improve skin condition. Hydrogen molecules are used as safe and effective gas molecules, and may be applied to multiple fields such as tissue / organ repair, wound healing and skin care.

Description

Application of hydrogen molecules in the preparation of products for promoting extracellular matrix accumulation

[0001] Cross-references to related applications

[0002] This disclosure claims the Chinese application with application number 202311061582.X, application date August 22, 2023, and patent name “Application of hydrogen molecules in the preparation of products for promoting extracellular matrix accumulation” as the basis, and claims its priority. The disclosure of the Chinese application is hereby introduced into this disclosure as a whole. Technical Field

[0003] The present disclosure belongs to the technical field of hydrogen molecule application, and particularly relates to the application of hydrogen molecules in the preparation of products for promoting extracellular matrix accumulation. Background Art

[0004] The extracellular matrix (ECM) is a three-dimensional structure secreted by cells that surrounds the outside of animal tissues. It is a non-cellular, three-dimensional network of macromolecules composed of various glycoproteins, including collagen, proteoglycans / glycosaminoglycans, elastin, fibronectin, and laminin. The ECM provides cells with the support they need for various life activities, primarily through: 1) serving as a matrix for cell adhesion; 2) providing a supporting scaffold structure and defining tissue boundaries; 3) presenting growth factors to cell receptors; 4) enabling the temporal and spatial regulation of cytokines; and 5) sensing and transmitting mechanical signals. Cells and ECM components interact to form a complex network. Cells reside within the ECM and together form tissues and organs. The ECM determines characteristics such as tissue morphology, texture, and mechanical properties. In addition, the extracellular matrix (ECM) can also serve as a "reservoir" for growth factors and other signaling molecules. Signaling molecules stored in the ECM bind to cell surface receptors to transmit biological signals from outside the cell to inside the cell, thereby regulating various cellular functions such as survival, growth, migration, and differentiation, and is crucial for maintaining normal homeostasis. The extracellular matrix (ECM) can also serve as a site for cell anchoring, providing cell adhesion and crawling migration, as well as maintaining cell polarity.

[0005] The extracellular matrix (ECM) is primarily composed of five types of substances: collagen, non-collagenous proteins, elastin, proteoglycans, and aminoglycosaminoglycans. The ECM at the base of epithelial or endothelial cells forms the basement membrane, while the ECM in intercellular adhesion structures forms the interstitial connective tissue. The ECM does not simply serve as an inert support or connect cells together to form tissues and organs; rather, it contains numerous signaling molecules that actively control cell growth, polarity, shape, migration, and metabolic activity. The most abundant ECM components in the skin are collagen types I and III. Type I collagen provides strong support, maintaining skin firmness and mechanical strength. Type III collagen decreases with aging, and therefore, to some extent, can indicate skin youthfulness. Similarly, type IV collagen is also more commonly found in younger skin. Other important ECM components include collagen XVII, which maintains the epithelial / dermal tight junction, laminin, integrin family proteins, skeletal ECM components such as fibronectin, and other aminoglycosaminoglycan and proteoglycan components.

[0006] ECM plays an important role in various physiological and pathological conditions, such as wound healing, skin care (wrinkles, aging, sagging, etc.), and tissue remodeling.

[0007] (1) Wound healing.

[0008] ECM plays a certain role in all stages of wound healing. Especially in the epithelialization and tissue remodeling stages, ECM plays an important role as a "stepping stone". Through the secretion of cells such as fibroblasts and epithelial cells, a large amount of ECM accumulates in the wound bed, facilitating the crawling of skin cells and the closure and remodeling of wounds. At the same time, there is a cross-talk (interaction) between ECM and stem cells. For example, the hardness of ECM can determine the degree of activation of epidermal stem cells, thereby further regulating the repair of skin wounds. The further stabilization and remodeling of new granulation tissue and subcutaneous tissue also depends on the role of ECM components.

[0009] (2) Skin care.

[0010] Collagen is the most important and well-known ECM component in the skin. As the primary protein component of skin tissue, collagen is considered a crucial component of the dermal support structure. Collagen forms a dense elastic network within the skin, maintaining its support, protection, and elasticity. It also serves as a crucial site for biochemical reactions, providing moisture and nutrients to the epidermis. Loss or damage to collagen can cause skin deformation and moisture loss, leading to dryness, wrinkles, sagging, fine lines, enlarged pores, and other signs of skin aging.

[0011] (3) Tissue repair.

[0012] The ECM is rich in components and possesses numerous functions, including guiding cells, transmitting signals, diagnosing and treating diseases, and promoting regeneration and repair. As a sophisticated and orderly network structure, the extracellular matrix (ECM) possesses unique advantages in tissue regeneration and repair. It provides a microenvironment that closely resembles the in vivo cell growth environment. Furthermore, its rich abundance of active molecules provides a foundation for cellular activity, giving it promising application prospects. The ECM exists in various forms during tissue regeneration. Its advantages include good biodegradability and low immunogenicity, as well as its ability to ameliorate inflammatory responses to a certain extent, effectively supporting tissue regeneration and guiding tissue reconstruction. With the continued in-depth study of the ECM, its application in tissue engineering will further develop, bringing new technologies and treatments to the future of regenerative medicine and tissue engineering. However, the challenges faced by the ECM in tissue repair require further research and exploration.

[0013] (4) Bone collagen supplementation.

[0014] Collagen, also known as structural protein, accounts for 30% to 40% of the body's total protein. It is found in the tendons connecting muscles, the cartilage and connective tissue connecting joints, and the dermis of the skin. After bone is boiled and cooled, it transforms into a jelly-like, elastic substance, which in the living body is collagen. Collagen promotes the deposition of inorganic substances such as calcium and phosphorus in bone, thereby repairing bone tissue, improving osteoporosis symptoms, and promoting overall health. Collagen significantly contributes to maintaining bone toughness, coordination of movement, and skin elasticity.

[0015] Calcium in bones is deposited as calcium hydroxyphosphate, fixed by collagen. The relationship between collagen and calcium in the body includes two aspects: (1) Hydroxyproline in plasma derived from collagen is the vehicle that transports calcium from plasma to bone cells; (2) Collagen in bone tissue is the adhesive for calcium hydroxyphosphate. Calcium hydroxyphosphate and collagen together constitute the main body of the skeleton.

[0016] Collagen is a type of collagen found in humans and animals. It is the primary component of articular cartilage, epiphyseal cartilage, and trabecular bone. 70% to 86% of bone matter is collagen. Collagen is also a key component of human muscle and skin, contributing significantly to bone toughness, coordination of movement, and skin elasticity.

[0017] In adult skin, ECM content decreases at a rate of approximately 2% per year, gradually undermining the structural integrity of the dermis. A healthy extracellular matrix maintains a certain level of mechanical tension, thereby stabilizing the extracellular microenvironment. Compositionally, the three main extracellular matrix components of the dermis are collagen, elastin, and hyaluronic acid. As these ECM components are gradually degraded by matrix metalloproteinases, they are also gradually depleted. This microenvironment is crucial for cells to maintain a balance between ECM and ECM-degrading enzymes. When this balance is disrupted, the production of various ECM components decreases and their loss accelerates, accelerating skin aging and impairing wound healing.

[0018] Therefore, the extracellular matrix needs to be continuously replenished or maintained. The existing technical means to replenish / maintain ECM mainly rely on the following aspects:

[0019] (1) Reduce the negative impact of exogenous factors on ECM.

[0020] A variety of external factors trigger the degradation of ECM, accelerating skin aging and causing poor skin healing. For example, exposure to light can lead to the production of free radicals in the skin, downregulating the expression of ECM proteins such as collagen, resulting in a decrease in ECM content. Harmful components of air pollution induce increased expression of matrix metalloproteinases, leading to collagen degradation, increased facial wrinkles, and impaired wound healing. Therefore, reducing exogenous factors mainly includes physical / chemical sun protection, adjusting diet, reducing tobacco intake, isolating from air pollutants, and cultivating a good work and rest schedule. Although these methods have a certain effect on improving ECM loss, they are often difficult to implement and maintain, and there is no new ECM production to replenish it.

[0021] (2) Exogenous pathways promote the secretion of endogenous ECM.

[0022] For the skin: Extracellular matrix (ECM) replenishment is primarily achieved through topical skincare products. Skincare products may contain certain anti-aging substances, such as retinol, peptides, and phosphatidylcholine, which can promote ECM secretion and accumulation to a certain extent. However, these molecules all have drawbacks such as poor penetration and low bioavailability, making exogenous approaches to promoting endogenous ECM secretion ineffective.

[0023] Other aspects: Throughout the human lifespan, the body's cell populations are subjected to constant impact and damage from the internal and external environments, causing tissue function to gradually degenerate and age. The most fundamental approach to cellular anti-aging is to repair cells, improve cell metabolism, and activate the function of aging cells. Repairing cell function is, to a certain extent, the optimal path to prolonging the body's ability to produce its own collagen. Current approaches to cellular anti-aging include stem cell therapy, exosome therapy, and extracellular matrix (ECM) therapy. However, these approaches are expensive and can only moderate collagen production to a certain extent, failing to fundamentally increase ECM secretion.

[0024] (3) Direct exogenous supplementation of ECM.

[0025] Direct exogenous ECM supplementation is another common approach, primarily involving the topical application of natural or recombinant ECM proteins or peptides, as well as subcutaneous injections of ECM and its analogs. However, the skin generally cannot absorb macromolecules like collagen and hyaluronic acid, only their hydrolysis products. Furthermore, direct topical application of ECM can cause adverse symptoms such as allergies and hair follicle blockage, negatively impacting skin health. On the other hand, ECM injections offer rapid results. For example, exogenous collagen can promote skin repair and support the dermis, preventing sagging; exogenous hyaluronic acid can fill small cavities, giving the skin a plump and radiant appearance. However, side effects include potential post-injection skin infections, allergic reactions, swelling and redness, hyperpigmentation, vascular and nerve damage, and even blindness. Furthermore, exogenous ECM injections require continuous replenishment and are consumed by the skin over time. Long-term use can accelerate the loss of the skin's original ECM, resulting in facial hollowing. In trauma treatment, the commonly used external ECM supplement method is recombinant collagen dressing, but this type of dressing is expensive and has poor functionality. It only provides ECM and other substances to the wound and cannot play multiple roles such as anti-inflammatory and cell activation.

[0026] (4) Other ways to promote ECM accumulation.

[0027] In the field of medical aesthetics, invasive procedures such as fractional laser, laser ablation, ion beam, microneedling, and Thermage leverage the skin's inherent "break-to-rebuild" nature, inducing ECM regeneration during the repair process through controlled, minimally invasive procedures. While these methods can achieve some success, they are expensive and invasive. Improper use or poor care can easily lead to redness, burns, and localized inflammation, posing a high risk.

[0028] In addition, the above methods are mostly used in the field of medical aesthetics to promote ECM secretion or supplement exogenous ECM, but are less used in trauma medicine and cannot achieve multi-scenario and multi-functionality. Therefore, there is an urgent need to find a new technology to promote ECM accumulation.

[0029] Hydrogen is a gas with relatively stable chemical properties. It is clean, safe, non-toxic and harmless. The small molecular weight of hydrogen allows it to pass through the skin barrier and enter the human body. It has good diffusivity and can exist in the human body and other organisms and quickly diffuse to almost any part, including the skin, mucous membranes, bones, blood-brain barrier, tissues, cells and intracellular structures (nuclei and mitochondria). As an endogenous gas (the human intestinal flora can produce hydrogen), hydrogen has strong biocompatibility. Since 2007, hydrogen has been proven to significantly improve cerebral ischemia-reperfusion injury in rats, which has opened up a wave of research on hydrogen molecular biology. The study of the biological effects of hydrogen has become a hot research area, and hydrogen has been used in the clinical treatment of many diseases. Many existing studies have confirmed that hydrogen has anti-inflammatory, antioxidant and anti-apoptotic biological properties.

[0030] Therefore, hydrogen is likely to act as a new type of gas molecule, promoting the accumulation and synthesis of ECM such as collagen, thereby achieving the effects of repairing damaged tissues and organs, promoting wound healing, improving skin condition, and repairing bone collagen loss and muscle collagen loss.

[0031] Public content

[0032] The purpose of the present disclosure is to solve the above technical problems and provide an application of hydrogen molecules in the preparation of products for promoting extracellular matrix accumulation.

[0033] Application of hydrogen molecules in the preparation of products for promoting extracellular matrix accumulation.

[0034] Preferably, the hydrogen molecules are used in the preparation of products for promoting wound healing, organ repair and regeneration, or skin care by promoting the accumulation of extracellular matrix.

[0035] Preferably, the organs include bone tissue, cartilage tissue, muscle tissue and cornea; the skin care includes promoting the firmness and elasticity of the skin of the face, eyes and other parts of the human body, and reducing wrinkles, dullness and pigment accumulation.

[0036] Preferably, the extracellular matrix includes collagen, non-collagen, elastin, proteoglycan, aminoglycan, and extracellular matrix components in the epithelium, dermis, and epidermis-dermis tight junction layer.

[0037] Preferably, the product is in the form of one or more of gas, aerosol, solution, emulsion, foam, gel, colloidal dispersion, liposome, droplet, microbubble, paste, suppository, chewing gum, granule, pill, powder, lozenge, capsule, microcapsule.

[0038] Preferably, the application of hydrogen molecules includes one or more of hydrogen administration by breathing, oral administration, injection or surface administration.

[0039] Preferably, the surface hydrogen delivery method includes bathing or soaking in a hydrogen-rich solution, or locally using a hydrogen-generating device or a hydrogen-containing patch.

[0040] Preferably, the product is pure hydrogen or a mixed gas comprising hydrogen and oxygen, and the volume concentration of hydrogen in the mixed gas is not greater than 79%.

[0041] Preferably, the article is a hydrogen molecule source material that directly contains hydrogen molecules or is capable of generating hydrogen molecules when used.

[0042] Preferably, the hydrogen molecule source material is a solid hydrogen storage material, which is one or a combination of two or more of the original or modified metal hydrogen storage materials, coordinated metal hydrogen storage materials, inorganic hydrogen storage materials, nano hydrogen storage materials, porous polymer hydrogen storage materials, hydrate hydrogen storage materials and other new solid hydrogen storage materials.

[0043] Based on the biological functions of hydrogen, this disclosure proposes the use of hydrogen molecules in the preparation of products for promoting extracellular matrix accumulation. Hydrogen can be introduced through various methods, including inhalation, permeation of the body surface, hydrogen water, hydrogen spray, hydrogen baths, oral hydrogen storage capsules, and injection of hydrogen-containing solutions. As a safe and effective gas molecule, hydrogen molecules can be applied in various fields, including tissue and organ repair, wound healing, and skin care. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG1 is a schematic diagram of the hydrogen molecules disclosed herein promoting the accumulation of various ECMs in different tissues (taking skin tissue as an example);

[0045] FIG2 is a diagram showing the collagen accumulation results of traumatic mouse skin promoted by breathing at different hydrogen concentrations in Example 1 of the present disclosure (black arrows indicate immature collagen, degraded collagen, and tissue cavities in the control group);

[0046] FIG3 is a diagram showing the results of promoting collagen accumulation and tight arrangement in normal mouse skin by breathing at different hydrogen concentrations according to Example 1 of the present disclosure (black arrows indicate loosely arranged collagen fibers in the control group);

[0047] FIG4 is a hydrogen release curve of the hydrogen storage material patch of Example 2 of the present disclosure;

[0048] FIG5 is a diagram showing the results of promoting overall collagen accumulation in the skin by topical application of a hydrogen storage material patch / dressing according to Example 2 of the present disclosure;

[0049] FIG6 is a diagram showing the results of promoting skin type I collagen accumulation by topical application of a hydrogen storage material patch / dressing according to Example 3 of the present disclosure;

[0050] FIG7 is a diagram showing the results of promoting skin type III collagen accumulation by topical application of a hydrogen storage material patch / dressing according to Example 4 of the present disclosure;

[0051] FIG8 is a diagram showing the results of simultaneous intervention of breathing and local hydrogen in promoting Col-I accumulation in mouse wounds according to Example 5 of the present disclosure;

[0052] FIG9 is a diagram showing the results of simultaneous intervention of breathing and local hydrogen in Example 6 of the present disclosure to promote Col-III accumulation in the wound surface of mice;

[0053] FIG10 is a diagram showing the results of simultaneous intervention of breathing and local hydrogen in Example 7 of the present disclosure to promote Col-XVII accumulation in the wound surface of mice;

[0054] FIG11 is a diagram showing the results of simultaneous intervention of breathing and local hydrogen in Example 8 of the present disclosure to promote Fibronectin accumulation in the wound surface of mice;

[0055] FIG12 is a diagram showing the results of simultaneous intervention of breathing and local hydrogen in promoting the accumulation of Laminin and Integrin in the wound surface of mice according to Example 9 of the present disclosure;

[0056] FIG13 is a diagram showing the results of promoting collagen accumulation in aged mice by applying hydrogen-rich water and hydrogen-rich saline according to Example 10 of the present disclosure;

[0057] FIG14 is a diagram showing the results of promoting Fibronectin accumulation in aged mice by applying hydrogen-rich water and hydrogen-rich saline according to Example 10 of the present disclosure;

[0058] FIG15 is a diagram showing the results of promoting Laminin accumulation in aged mice by applying hydrogen-rich water and hydrogen-rich saline according to Example 10 of the present disclosure;

[0059] FIG16 is a hydrogen release curve of the hydrogen storage membrane material of Example 11 of the present disclosure;

[0060] FIG17 is a diagram showing the results of promoting total collagen accumulation in the mouse dermis by the hydrogen storage material according to Example 11 of the present disclosure;

[0061] FIG18 is a diagram showing the results of the hydrogen storage material in Example 11 of the present disclosure promoting the accumulation of Col-I in the dermis and epithelium of mice;

[0062] FIG19 is a diagram showing the results of the hydrogen storage material in Example 11 of the present disclosure promoting Col-III accumulation in the mouse epithelium;

[0063] FIG20 is a diagram showing the results of promoting the accumulation of Col-XVII in the epithelial-dermal tight junction layer of mice using the hydrogen storage material of Example 11 of the present disclosure;

[0064] FIG21 is a hydrogen release curve of the hydrogen storage material of Example 12 of the present disclosure;

[0065] FIG22 is a hydrogen release curve of the hydrogen storage material of Example 13 of the present disclosure;

[0066] FIG23 is a hydrogen release curve of the hydrogen storage material of Example 14 of the present disclosure. DETAILED DESCRIPTION

[0067] The present disclosure relates to a hydrogen molecule intervention method, which is used to increase the accumulation of extracellular matrix, promote wound healing, or improve skin condition through the combined intervention of one or more hydrogen molecules. Specifically, the intervention of hydrogen molecules can activate the self-skin ECM secretion ability, promote the accumulation of self-ECM, thereby promoting the wound healing rate and healing effect, and can improve the skin condition, promote skin firmness, filling, etc. Hydrogen molecules promote ECM accumulation and are suitable for all kinds of skin problems and all kinds of trauma people, truly realizing multi-functional and multi-scenario applications. At the same time, hydrogen promotes the secretion and accumulation of ECM, not only limited to one or several types such as collagen or laminin, fibronectin, etc., but promotes the accumulation of the entire ECM, including the ECM components in the epithelium, dermis, and epidermal-dermal tight junction layer (as shown in Figure 1). In the fields of bone loss, muscle damage, corneal repair, etc., hydrogen molecules can also achieve the purpose of ECM supplementation and damage repair of bone tissue and muscle tissue by promoting the accumulation of ECM such as self-bone collagen, muscle collagen, corneal collagen, etc.

[0068] Hydrogen molecules have significant effects in promoting wound healing and skin care applications. Specifically, hydrogen molecules can be used in the following scenarios:

[0069] 1. Hydrogen molecules promote the accumulation of ECM such as collagen and tissue remodeling under traumatic conditions.

[0070] In the case of a skin defect, epithelialization must first be completed, whereby epithelial cells crawl forward to cover the entire wound bed. Subcutaneous tissue repair and remodeling then proceed. During these epithelialization and tissue remodeling phases, hydrogen molecules promote the accumulation of ECM and reduce its degradation, thereby improving wound healing. These wounds include acute sterile wounds, chronic, difficult-to-heal wounds, and internal trauma. The pathologies involved include surgical sutures, open skin lesions, difficult-to-heal diabetic wounds, burns, and blunt soft tissue contusions.

[0071] 2. Hydrogen molecules promote the accumulation of normal skin collagen and other ECM.

[0072] ECM is primarily expressed in the dermis, with a small amount also distributed in the epithelium. Under the action of hydrogen molecules, normal skin can increase the accumulation of ECM in the epithelium and dermis, achieving skin firming, lifting, anti-wrinkle, and plumping effects.

[0073] 3. Types of ECM accumulation promoted by hydrogen molecules: mainly including collagens (Col-I, Col-III, Col-IV, Col-XVII, etc.), fibronectins (Fibronectin, etc.), laminins (Laminin family proteins), integrin proteins (Integrin family), etc.

[0074] 4. Hydrogen molecules promote ECM accumulation and block ECM degradation, mainly by inhibiting the expression of matrix metalloproteinases and preventing ECM degradation.

[0075] 5. Hydrogen molecules can promote collagen secretion in fibroblasts, mesenchymal stem cells and epithelial cells at the cellular level.

[0076] The above-mentioned hydrogen molecule intervention methods include but are not limited to one or more of the following types: hydrogen administration through breathing, oral hydrogen administration, hydrogen injection (including subcutaneous injection, intravenous injection, in situ injection, etc.) or hydrogen administration through the body surface (including bathing or soaking in hydrogen-rich solutions, local use of hydrogen-generating devices or hydrogen-containing patches, etc.).

[0077] The hydrogen molecule product may be a hydrogen molecule source material that directly contains hydrogen molecules or is capable of generating hydrogen molecules when used.

[0078] The product can be in the form of one or more of gas, aerosol, solution, emulsion, foam, gel, colloidal dispersion, liposome, droplet, microbubble, paste, suppository, chewing gum, granule, pill, powder, lozenge, capsule, microcapsule.

[0079] The hydrogen molecule source material is preferably a solid hydrogen storage material, which is one or a combination of two or more of the original or modified metal hydrogen storage materials, coordinated metal hydrogen storage materials, inorganic hydrogen storage materials, nano hydrogen storage materials, porous polymer hydrogen storage materials, hydrate hydrogen storage materials and other new solid hydrogen storage materials.

[0080] Among them, the metal hydrogen storage material is selected from metal Mg, Mg-Ni series alloys, Mg2Ni alloys, Mg-Pd alloys, Mg-Co alloys, Mg-Fe alloys, Mg-V-Al-Cr-Ni multi-element high entropy alloys, metal vanadium, vanadium-based alloys V-Ti-Cr, V-Ti-Fe, V-Ti-Mn, V-Ti-Ni, rare earth hydrogen storage materials AB5 type alloys, LaNi5 alloys, superlattice La-Mg-Ni alloys, La-Y-Ni alloys, MmNi5 alloys, titanium hydrogen storage materials Ti-Fe, Ti-Zr, Ti-Cr, Ti-Mn, Ti-Co, Ti-Mn, Ti-Cr, zirconium alloys C15 cubic Laves phase, C14 hexagonal Laves phase, Zr-V alloys, Zr-Ni alloys, Zr-Cr alloys, Zr-Mn alloys , Zr-Co alloy, one or more of metallic calcium, CaNi5 alloy or Ca-Ni-Mg series alloy; the coordination metal hydrogen storage material is one or more selected from Sc(AlH6), Cr(AlH6)2, Co(AlH6)3, Ti(AlH4)4, Mg(AlH4)2, Fe(AlH4)2, Sn(AlH4)4, MgH2, NaAlH4, LiAlH4, KBH4, NaBH4, LiBH4; the inorganic hydrogen storage material is one or more selected from NH3BH3 and bicarbonate; the nano hydrogen storage material is carbon tetrafluoride hydrogen storage material, the porous polymer hydrogen storage material is one or more of self-microporous polymer PIMs, hyper-crosslinked polymer HCPs, or conjugated microporous polymer CMPs; the hydrate hydrogen storage material is carbon dioxide hydrate hydrogen storage material.

[0081] This disclosure provides several typical hydrogen molecular products and intervention methods, as follows:

[0082] ① Hydrogen inhalation: Inhale hydrogen directly through breathing. Hydrogen can be mixed with oxygen or air, or it can be inhaled as pure hydrogen. Considering the oxygen demand, the volume concentration of hydrogen should not exceed 79% to ensure sufficient oxygen intake.

[0083] ② Drinking hydrogen-rich water: hydrogen-rich water that is pre-configured or prepared before drinking.

[0084] ③ Injection of hydrogen-rich saline: Hydrogen is delivered into the body by injecting isotonic saline solution dissolved in hydrogen into the vein, abdominal cavity or patient area.

[0085] ④ Local use of hydrogen-producing devices or hydrogen-containing patches: Local hydrogen-producing devices or hydrogen-containing patches (patches containing solid hydrogen storage materials, which produce hydrogen through contact with the reaction liquid when used) can be used on local or internal damaged skin to bring hydrogen into the body through the skin due to its strong permeability.

[0086] ⑤ Oral administration of hydrogen capsules / effervescent tablets.

[0087] ⑥ Hydrogen-rich water bathing / soaking, etc.

[0088] In order to better understand the above technical solution, the above technical solution will be described in detail below with reference to the accompanying drawings and specific embodiments, but the present disclosure is not limited to these specific embodiments.

[0089] Example 1 Hydrogen breathing promotes collagen accumulation in normal skin and post-traumatic skin

[0090] Hydrogen breathing was used to treat normal mice and mice with a trauma model. Hydrogen concentrations were adjusted to varying ratios of hydrogen to air (volume concentration) at 80% (20% oxygen), 66% (21% oxygen, the remainder air), 30% (21% oxygen, the remainder air), and 5% (21% oxygen, the remainder air). The control group breathed air. Hydrogen breathing was performed once daily for one hour, and subcutaneous collagen accumulation was observed after 10 days. Mouse skin samples were collected, embedded, fixed, and sectioned for Mason staining. Results showed that in the trauma model (Figure 2), hydrogen exposure at various concentrations increased overall subcutaneous collagen accumulation, resulting in a relatively neat arrangement, with no significant degradation or cavitation. In contrast, the control group showed significant immature collagen, degraded collagen, and cavitation (indicated by the black arrows in Figure 2). Similar results were observed in normal mouse skin treated with hydrogen (Figure 3): breathing at various hydrogen concentrations significantly promoted collagen accumulation and tight arrangement in the mouse skin. These results suggest that breathing hydrogen at varying concentrations can promote the accumulation of ECM, such as collagen, in normal skin, improving skin quality. It can also promote ECM accumulation during wound repair, enhancing healing outcomes. Furthermore, there is a dose-response effect with hydrogen, with high-concentration hydrogen achieving a greater healing effect than low-concentration hydrogen. Low-concentration hydrogen can also achieve comparable effects to high-concentration hydrogen by increasing breathing time.

[0091] Example 2 Topical application of hydrogen storage material patches / dressings promotes the accumulation of ECM such as skin collagen

[0092] The hydrogen storage material was prepared into a patch / dressing. The patch substrate was medical PU, and the hydrogen storage material was PLGA-wrapped metal Mg (0.1 g / patch). The sustained release time was 12 to 18 hours. The hydrogen release curve of the patch is shown in Figure 4 (measured by liquid hydrogen electrode method. Before the test, it was soaked with physiological saline. The ratio of patch to physiological saline was: 2 cm 2 / 10mL). Male C57BL / 6 mice were selected as the animal model, and the mouse skin was prepared and then applied with a patch / dressing. The control group was treated with a patch / dressing base material. The patch / dressing was applied once daily for 12 hours, and the changes in ECM accumulation in the mouse skin were observed after 14 days. Masson staining results showed (Figure 5) that local application of the hydrogen storage material hydrogen molecule patch / dressing significantly promoted the overall collagen accumulation level in the mouse skin, reduced cavitation in the dermis, and promoted ECM arrangement and remodeling.

[0093] Example 3 Topical application of hydrogen storage material patches / dressings promotes the accumulation of type I collagen in the skin

[0094] Using the animal model and the hydrogen molecule dressing / patch intervention described in Example 2, changes in type I collagen accumulation in the newly formed skin of mice were observed 14 days later. As shown in Figure 6, type I collagen was significantly darker in the hydrogen molecule patch / dressing group, and Col-I accumulation in both the dermis and epithelium was significantly increased. Col-I is closely related to the support and structural integrity of the skin. Therefore, the accumulation of Col-I in the dermis / epithelium suggests that the hydrogen molecule dressing / patch can improve the integrity and support of skin tissue, promote skin firming, and improve healing.

[0095] Example 4 Topical application of hydrogen storage material patches / dressings promotes the accumulation of type III collagen in the skin.

[0096] Using the animal model and the hydrogen molecule dressing / patch intervention described in Example 2, changes in type III collagen accumulation in the newly healed skin of mice were observed 14 days later. Figure 7 shows a significant increase in type III collagen expression in newly healed mouse tissue, particularly in the epithelial layer (arrowed). Because Col-III has a softer strength and better flexibility, these results suggest that hydrogen molecule dressings / patches can increase the elasticity of newly healed tissue (epithelial layer), promoting and improving healing outcomes.

[0097] Example 5 Hydrogen breathing and local action (hydrogen chamber) promote the accumulation of type I collagen in the early stage of traumatic skin

[0098] A full-thickness skin defect wound model was used as a mouse model, and 66% H2 was used for both hydrogen breathing and local action. That is, after modeling, the mice were placed in a sealed hydrogen chamber, which was filled with 66% H2 (the rest was O2 to ensure a non-hypoxic environment). Under the conditions of hydrogen chamber treatment, traumatized mice can absorb hydrogen through breathing and absorb H2 in the environment through the body surface. Immediately after modeling, they were placed in a hydrogen chamber and treated once a day for 1 hour each time. Three days later, the wounds and proximal parts of the wounds were obtained and stained, showing a significant increase in Col-I accumulation in the dermis and epithelium of the mice (Figure 8, black arrows), suggesting that breathing and local hydrogen promote the accumulation of ECM such as skin collagen, promote wound healing, and improve skin quality.

[0099] Example 6 Hydrogen breathing and local action (hydrogen chamber) promote the accumulation of type III collagen in the early stage of traumatic skin

[0100] Experiments were conducted using the animal model and hydrogen chamber treatment conditions described in Example 5. Three days later, Col-III expression levels in mouse skin were measured. Figure 9 shows significant Col-III accumulation in the skin proximal to the wound in the hydrogen chamber-treated mice, particularly in the epithelium. This suggests that hydrogen molecules promote the early accumulation of collagen and other ECM in the epithelium during wound healing, improving healing quality and increasing skin epidermal elasticity.

[0101] Example 7 Hydrogen breathing and local action (hydrogen chamber) promote the accumulation of Col-XVII collagen in the early stage of traumatic skin

[0102] Experiments were conducted using the animal model and hydrogen chamber treatment conditions described in Example 5 above, and Col-XVII expression levels in mouse skin were measured three days later. As shown in Figure 10, Col-XVII accumulation in the proximal wound skin of mice in the hydrogen chamber treatment group increased significantly (dark areas), with the accumulation primarily concentrated in the epithelial-dermal tight junction layer. The function of Col-XVII is closely related to the epithelial-dermal tight junction and the activity of basal stem cells. Therefore, this example suggests that hydrogen molecules can promote epithelial-dermal junctions, maintain skin tissue integrity, and promote the activation of basal stem cells.

[0103] Example 8 Hydrogen breathing and local action (hydrogen chamber) promote the accumulation of fibronectin in the dermis of skin in the early stage of trauma

[0104] Experiments were conducted using the animal model and hydrogen chamber treatment conditions described in Example 5. Fibronectin expression in mouse skin was measured three days later. Figure 11 shows a significant increase in fibronectin accumulation in the dermis of the proximal wound skin of mice in the hydrogen chamber treatment group, suggesting that hydrogen promotes the accumulation of fibronectin and other ECM in the dermis during the early stages of wound healing, increasing the skin's support capacity and improving healing quality.

[0105] Example 9 Hydrogen breathing and local action (hydrogen chamber) promote the accumulation of laminin and integrin in the early stage of traumatic skin

[0106] Experiments were conducted using the animal model and hydrogen chamber treatment conditions described in Example 5. Three days later, the expression levels of laminin and integrin in mouse skin were measured. Figure 12 shows a significant increase in the accumulation of laminin and integrin in the epidermis of the proximal wound skin of mice in the hydrogen chamber treatment group (black arrows indicate positive cells), suggesting that hydrogen promotes early epithelial repair and ECM accumulation in wounds, improving healing quality.

[0107] Example 10: Hydrogen-rich water and hydrogen-rich saline application promotes the accumulation of collagen and other ECM in the skin of aged mice

[0108] Preparation method of hydrogen-rich water and hydrogen-rich saline: Pure hydrogen gas (nanobubbling) was introduced into sterile purified water / saline respectively for about 30 minutes to obtain hydrogen-rich water / saline with a hydrogen molecule concentration of about 0.5ppm. The experimental animals were 12-month-old C57BL / 6 male aged mice. After the back skin was prepared, hydrogen-rich water and hydrogen-rich saline were applied (medical sponges were soaked in hydrogen-rich water or hydrogen-rich saline) once a day for 15 days. After the application was completed, the back skin of each group of mice was fixed, embedded and sectioned, and Mason staining, Fibronectin and Laminin staining were performed. As shown in Figure 13, after 15 days, the subcutaneous collagen accumulation in the hydrogen-rich water group and the hydrogen-rich saline group was significantly higher than that in the control group; immunohistochemical staining showed that the expression levels of Fibronectin (Figure 14) and Laminin (Figure 15) in the dermis and epithelium of the hydrogen-rich water and hydrogen-rich saline groups were significantly higher than those in the control group. The above results suggest that hydrogen-rich water and hydrogen-rich saline significantly increase the accumulation of ECM such as collagen under the skin and improve the skin quality of aging skin.

[0109] Example 11 Hydrogen storage membrane materials promote the accumulation of ECM such as collagen in mouse subcutaneous tissue

[0110] Mice were selected from Kunming mice aged 6 to 8 weeks, and their skins were prepared for processing. The hydrogen storage material was selected from micron-sized magnesium and nano-sized ammonia borane, with a mixing ratio of 1:1. After adsorption by the membrane material (the membrane material was medical silk material), it was soaked in sterile deionized water. The hydrogen release curve of the hydrogen storage membrane material is shown in Figure 16 (measured using the liquid phase hydrogen electrode method, soaked in deionized water before testing, the ratio of the patch to deionized water was: 2cm 2 The control group used a membrane material without hydrogen storage material soaked in sterile deionized water for application. The back application was performed once for 30 minutes each time for 15 days. After the experiment, mice in each group were sacrificed, and the skin at the application site was harvested, fixed, embedded, and sectioned. Mason staining and Col-I, Col-III, and Col-XVII staining were performed.

[0111] Figure 17 shows that the hydrogen storage material promotes the overall collagen accumulation in the subcutaneous tissue of mice, especially in the dermis. Compared with the control group, the collagen in the dermis of the mice in the hydrogen storage material application group is more neatly arranged and has significantly fewer cavities.

[0112] Immunohistochemical staining results showed that compared with the control group, the hydrogen storage material group showed significantly darker staining of Col-I in the dermis and epithelium (Figure 18), increased accumulation of Col-III in the epithelium (Figure 19), and increased expression of Col-XVII in the epithelial-dermal tight junction layer (Figure 20). This suggests that hydrogen storage material application can promote skin ECM accumulation, increase skin firmness and elasticity, enhance the tight junction between the epithelium and dermis, and improve skin quality.

[0113] Example 12: Different hydrogen intervention methods promote the accumulation of ECM such as collagen in muscle tissue

[0114] The muscle injury model mice and normal mice were treated by hydrogen breathing (the control group was air breathing) (hydrogen volume concentration of 66%, each breathing for 2 hours, once a day), hydrogen water drinking (the control group was pure water drinking) (500 μL each time, once a day, the hydrogen molecule concentration in the hydrogen water was about 0.5 ppm), hydrogen storage material application (the control group was patch base material application) (applied once a day, each application for 12 to 16 hours, the hydrogen storage material was a 1:1 mixture of magnesium hydride and calcium hydride, the hydrogen sustained release time was more than 18 hours, the hydrogen release curve of the patch was shown in Figure 21, and the test method was the same as Example 11), and hydrogen saline intramuscular injection (hydrogen concentration of about 0.5 ppm, the control group was conventional saline intramuscular injection) (100 μL each time, once a day), with 10 mice in each group. After 20 days, the expression levels of Col-I, Col-II, Col-III, Col-IV, Col-VII, Col-XVII, Fibronectin, Integrin-β, Laminin, and Elastin in the gastrocnemius muscles of mice in each group were compared to evaluate changes in ECM accumulation in normal and injured muscle tissue. As shown in Tables 1 and 2, after different hydrogen treatments, expression of these proteins increased to a certain extent in both normal and injured gastrocnemius muscles of mice, suggesting that hydrogen promotes ECM accumulation in muscle tissue, improving muscle function and accelerating injury repair.

[0115] Table 1 Effects of different hydrogen intervention methods on ECM accumulation levels in gastrocnemius muscle tissue of normal mice ↑ indicates that the proportion of positive cells in the treatment group increased relative to that in the control group, and ↓ indicates that the proportion of positive cells in the treatment group decreased relative to that in the control group.

[0116] Table 2 Effects of different hydrogen intervention methods on ECM accumulation levels in injured gastrocnemius muscle tissue of mice ↑ indicates that the proportion of positive cells in the treatment group increased relative to that in the control group, and ↓ indicates that the proportion of positive cells in the treatment group decreased relative to that in the control group.

[0117] Example 13: Different hydrogen intervention methods promote ECM accumulation in bone tissue

[0118] A mouse model of osteoarthritis was used for research, and cartilage and tibial ECM loss was observed after modeling. Interventional therapy was performed using hydrogen breathing (hydrogen concentration of 66% by volume, air breathing in the control group) (2 hours per breath, once daily), drinking hydrogen water (hydrogen concentration of approximately 0.6 ppm in hydrogen water, pure water drinking in the control group) (500 μL per drink, once daily), a hydrogen storage material application (made of medical PU film and medical non-woven fabric, the hydrogen release curve of the patch is shown in Figure 22, the test method is the same as in Example 11, the hydrogen release time exceeds 20 hours, the control group is a patch base material application) (applied once daily, each application for more than 12 hours), and hydrogen saline joint injection (hydrogen concentration of approximately 0.6 ppm, conventional saline joint injection in the control group) (50 μL per injection, once daily). Changes in different types of collagen in cartilage and tibia were observed after 20 days. Total collagen content was observed and quantified using Mason staining, and different collagen types (Col-I, Col-III, and Col-V) were stained and quantified using immunohistochemistry. After 20 days, the collagen positivity rates in the hydrogen-treated and control groups were compared. It was found that the expression levels of total collagen, Col-I, Col-III, and Col-V in cartilage tissue (Table 3) and tibial tissue (Table 4) were increased in all hydrogen-treated groups, suggesting that hydrogen can promote ECM accumulation in bone tissue.

[0119] Table 3 Effects of different hydrogen intervention methods on ECM accumulation levels in cartilage tissue damaged by osteoarthritis in mice ↑ indicates that the proportion of positive cells in the treatment group increased relative to that in the control group, and ↓ indicates that the proportion of positive cells in the treatment group decreased relative to that in the control group.

[0120] Table 4 Effects of different hydrogen intervention methods on ECM accumulation levels in tibial tissue of mice with osteoarthritis ↑ indicates that the proportion of positive cells in the treatment group increased relative to that in the control group, and ↓ indicates that the proportion of positive cells in the treatment group decreased relative to that in the control group.

[0121] Example 14 Different hydrogen intervention methods improve the accumulation of collagen and other ECM in the cornea, corneal stroma, lens, sclera, and vitreous body of mice, and improve eye function

[0122] The content of ECM such as collagen in the eye is relatively high. Adequate ECM can maintain normal eye structure and function, such as maintaining corneal thickness, scleral thickness, corneal stroma thickness, lens capsule stability, vitreous integrity and conjunctival tightness. C57BL / 6 mice were used as experimental animals. Different hydrogen intervention methods were used, including hydrogen breathing (hydrogen volume concentration of 5%, control group air breathing) (each breathing for 2 hours, once a day), hydrogen water drinking (hydrogen molecule concentration in hydrogen water was about 0.5 ppm, control group pure water drinking) (each drinking 500 μL, once a day), hydrogen storage material application (the hydrogen storage material selected PVP-wrapped Mg microbeads as the main material, the load membrane was medical non-woven fabric, and the control group was patch base non-woven fabric application) (the hydrogen release curve of the patch is shown in Figure 23, the test method is the same as Example 11, the hydrogen storage material hydrogen release time is not less than 3 hours, once a day, each application for more than 2 hours), hydrogen saline extraocular muscle injection (control group conventional saline joint injection) (each injection 50 μL, once a day) interventional treatment was performed, and the changes in the content of ECM such as collagen in different tissue structures of the eye were detected after 20 days. The results are shown in Table 5. The levels of collagen accumulation in eye tissue increased in all groups treated with hydrogen molecules, indicating that hydrogen molecules can improve eye function.

[0123] Table 5 Improvement of collagen accumulation levels in various eye tissues of mice by different hydrogen intervention methods

[0124] Based on the above experimental results, it can be seen that different hydrogen intervention methods can promote the accumulation of extracellular matrix in normal mouse skin, post-traumatic skin, eye skin, skin of elderly mice, injured muscle tissue, arthritic bone, cartilage tissue, etc.

[0125] When hydrogen molecules are introduced by breathing hydrogen, it is preferred to use a hydrogen concentration of 5% or higher.

[0126] When promoting the accumulation of extracellular matrix in injured muscle tissue or arthritic bone tissue, a hydrogen storage material application is preferred. This method is more effective than hydrogen breathing, oral hydrogen water, and intramuscular injection in promoting extracellular matrix accumulation, and its application method is more convenient. Hydrogen storage materials with high biosafety properties, such as magnesium-based hydrogen storage materials, are preferred.

[0127] When promoting the accumulation of extracellular matrix in the eye skin, it is preferred to use intramuscular injection of hydrogen molecules, which is more effective in promoting the accumulation of extracellular matrix than hydrogen breathing, oral hydrogen water and application of hydrogen storage materials.

[0128] Although preferred embodiments of the present disclosure have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present disclosure. Obviously, those skilled in the art may make various changes and modifications to the present disclosure without departing from the spirit and scope of the present disclosure. Thus, if such changes and modifications of the present disclosure fall within the scope of the claims of the present disclosure and their equivalents, the present disclosure is intended to include such changes and modifications.

Claims

1. Application of hydrogen molecules in the preparation of products for promoting extracellular matrix accumulation.

2. The use according to claim 1, characterized in that The hydrogen molecules are used in preparing a product for promoting wound healing, organ repair and regeneration, or skin care by promoting extracellular matrix accumulation.

3. The use according to claim 2, characterized in that The organs include bone tissue, cartilage tissue, muscle tissue and cornea; the skin care includes promoting the firmness and elasticity of the skin of the face, eyes and other parts of the human body, and reducing wrinkles, dullness and pigmentation.

4. The use according to claim 1, characterized in that The extracellular matrix includes collagen, non-collagen, elastin, proteoglycan, aminoglycan, and extracellular matrix components in the epithelium, dermis, and epidermis-dermis tight junction layer.

5. The use according to claim 1, characterized in that The product is in the form of one or more of gas, aerosol, solution, emulsion, foam, gel, colloidal dispersion, liposome, droplet, microbubble, paste, suppository, chewing gum, granule, pill, powder, lozenge, capsule, and microcapsule.

6. The use according to claim 1, characterized in that The application methods of hydrogen molecules include one or more of breathing hydrogen, oral hydrogen, injection hydrogen or surface hydrogen.

7. The use according to claim 6, characterized in that The surface hydrogen delivery method includes bathing or soaking in a hydrogen-rich solution, and locally using a hydrogen-generating device or a hydrogen-containing patch.

8. The use according to claim 1, characterized in that The product is pure hydrogen or a mixed gas including hydrogen and oxygen, and the volume concentration of hydrogen in the mixed gas is not greater than 79%.

9. The use according to claim 1, characterized in that The article is a hydrogen molecule source material that directly contains hydrogen molecules or is capable of generating hydrogen molecules when used.

10. The use according to claim 9, characterized in that The hydrogen molecule source material is a solid hydrogen storage material, which is one or a combination of two or more of a metal hydrogen storage material, a coordinated metal hydrogen storage material, an inorganic hydrogen storage material, a nano hydrogen storage material, a porous polymer hydrogen storage material, a hydrate hydrogen storage material and other new solid hydrogen storage materials in their original state or after modification.

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