Application of hydrogen molecules in preparation of product for promoting stem cell activation

Abstract

The present disclosure relates to an application of hydrogen molecules in the preparation of a product for promoting stem cell activation. Various hydrogen intervention means and generation means may be adopted when hydrogen molecules are applied, promoting the activation of autologous stem cells such as epidermal stem cells, basal stem cells, hair follicle stem cells, bone marrow-derived stem cells, mesenchymal stem cells, spermatocytes and neural stem cells. The related content may be applied to various fields such as wound healing, tissue / organ repair, tumor treatment, nervous system disease treatment, infertility treatment and aesthetic medicine.

Description

Application of hydrogen molecules in the preparation of products for promoting stem cell activation

[0001] Cross-references to related applications

[0002] This disclosure claims the Chinese application with application number 202311061585.3, application date August 22, 2023, and patent name “Application of Hydrogen Molecules in the Preparation of Products for Promoting Stem Cell Activation” 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 stem cell activation. Background Art

[0004] Stem cells are primitive cells with the potential for self-renewal and multidirectional differentiation. They originate from tissues such as the placenta, uterine membranes, fat, and dental pulp. Under certain conditions, they can differentiate into a variety of functional cells, with potential functions such as regenerating organs and tissues and treating or ameliorating serious illnesses. Stem cells have gradually been applied in the medical field, with applications in conditions such as organ failure, cardiovascular disease, infertility, spinal cord injury, and Parkinson's disease. A growing number of successful clinical applications demonstrates the enormous potential of stem cells as a new approach to treating disease and restoring health, while also driving a major revolution in traditional medical methods and concepts.

[0005] According to their differentiation potential, stem cells can be divided into totipotent stem cells, pluripotent stem cells, and unipotent stem cells.

[0006] (1) Totipotent stem cells

[0007] Totipotent stem cells possess the ability to self-renew and differentiate into any cell type, potentially forming a complete individual. Embryonic stem cells, for example, possess morphological characteristics similar to early embryonic cells and possess a strong differentiation capacity. They can proliferate indefinitely and differentiate into over 200 cell types throughout the body, further forming all tissues and organs. Embryonic stem cells are cells selected through in vitro suppressed culture of embryonic inner cell mass or primordial germ cells. Embryonic stem cells possess developmental totipotency and can theoretically be induced to differentiate into all cell types in the body. Embryonic stem cells can be expanded, screened, cryopreserved, and revived in large quantities in vitro without losing their original properties. Furthermore, embryonic stem cells can be obtained using somatic cell nuclear transfer.

[0008] (2) Pluripotent stem cells

[0009] Pluripotent stem cells have the ability to produce multiple types of cells, but they have lost the ability to develop into a complete individual, and their developmental potential is subject to certain restrictions.

[0010] ① Multipotent hematopoietic stem cells (HSCs) in the bone marrow possess the unique potential for enhanced self-renewal and production of offspring, which can differentiate into mature cells of both the myeloid and lymphoid lineages. HSCs possess two key characteristics: a high capacity for self-renewal or self-replication; and the ability to differentiate into all blood cell types. HSCs divide asymmetrically, splitting into two cells. One of these cells retains all the biological properties of a stem cell, thereby maintaining a relatively stable stem cell population within the body. This is stem cell self-renewal. The other cell then further proliferates and differentiates into various blood cells, precursors, and mature blood cells, which are released into the peripheral blood to perform their respective functions until aging and death. In embryonic and rapidly regenerating bone marrow, HSCs are mostly in a proliferative cycle. In normal bone marrow, however, most are in a quiescent phase (G0 phase). When needed, one portion matures while the other continues to differentiate and proliferate, maintaining a relatively stable HSC population. HSCs further differentiate and develop into committed stem cells of different blood cell lineages. Most committed stem cells are in the proliferation cycle and further differentiate into various blood cell lineages, such as erythrocytes, granulocytes, mononuclear phagocytes, megakaryocytes, and lymphocytes. Lymphocytes derived from hematopoietic stem cells have two developmental pathways: one mediated by the thymus, where they mature into thymus-dependent lymphocytes (T cells) under the catalysis of thymosin. The other, independent of the thymus, is influenced by the bursa (birds) or cystoids (mammals), where they mature into cyst-dependent or bone marrow-dependent lymphocytes (B cells). T cells and B cells, respectively, contribute to cellular and humoral immunity. A deficiency in hematopoietic stem cells can lead to severe immunodeficiency. Hematopoietic stem cell transplantation is a major breakthrough in modern life sciences. Hematopoietic stem cell transplantation can treat 75 fatal diseases, including hematologic malignancies, some malignant tumors, and some genetic disorders. Including acute leukemia, chronic leukemia, myelodysplastic syndrome, hematopoietic stem cell disease, myeloproliferative disease, lymphoproliferative disease, macrophage disease, hereditary metabolic disease, tissue cell disease, hereditary red blood cell disease, hereditary immune system disease, hereditary platelet disease, plasma cell disease, thalassemia, non-hematological malignancies, acute radiation sickness, etc.

[0011] Mesenchymal stem cells (HSCs) can differentiate into cells of various mesodermal tissues (such as bone, cartilage, muscle, and fat), as well as cells of other germ layers (such as neurons). Hematopoietic stem cells (HSCs) progressively decline in function over time, leading to aging of the hematopoietic system. Mesenchymal stem cells were first discovered in the bone marrow in 1987. In 1991, Caplan formally named these multipotential cells "mesenchymal stem cells," a term that has been used ever since. Under specific induction conditions in vivo or in vitro, MSCs can differentiate into a variety of tissue cells, including fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, and endothelium. They retain this multipotential potential even after continuous subculture and cryopreservation, making them ideal seed cells for repairing tissue and organ damage caused by aging and disease. MSCs are also the most widely used in clinical practice. Combining them with hematopoietic stem cells can improve transplant success rates and accelerate hematopoietic reconstitution. After high-dose chemotherapy, the co-infusion of MSCs with hematopoietic stem cells significantly accelerates blood cell recovery and is safe and without adverse reactions. In recent years, placenta-derived mesenchymal stem cells have been increasingly used in disease treatment research. Currently, mesenchymal stem cells are the most widely used cell type in clinical research projects worldwide, with nearly ten mesenchymal stem cell-derived drugs currently on the market.

[0012] Adipose-derived stem cells (ADSCs) are stem cells isolated from adipose tissue with multipotential differentiation. Adipose tissue is abundant in the human body. Large numbers of ADSCs, obtained through liposuction, possess the potential for self-renewal and multipotential differentiation into adipocytes, chondrocytes, myocytes, osteoblasts, neurons, glial cells, and pancreatic islet cells. ADSCs also secrete a variety of pro-angiogenic and anti-apoptotic factors, providing anti-inflammatory and antioxidant properties, protecting against damage from oxygen free radicals. ADSCs hold promise as a stem cell source for repairing damaged tissues and organs. The clinical application of ADSCs is primarily in tissue repair and reconstruction. ADSCs can differentiate into various cell lineages, including mesodermal-derived osteocytes, adipocytes, chondrocytes, smooth muscle cells, endothelial cells, neurons, and pancreatic islet cells. Currently, the primary clinical application strategy involves targeted transgenic induction, followed by in vitro expansion of the stem cells and subsequent transplantation into the human body.

[0013] ④ Liver stem cells can differentiate into at least two major epithelial cell types in the liver: hepatocytes and bile duct epithelial cells. The existence of liver stem cells was confirmed by the medical community in 1958. In the event of liver damage, liver stem cells are activated, maintaining normal liver function. However, with persistent liver damage, the regenerative capacity of liver stem cells is suppressed and depleted.

[0014] ⑤Neural stem cells (NSC) can differentiate into a variety of cells of the nervous system, including neurons, astrocytes, oligodendrocytes, and so on. Neural stem cells can be isolated from different areas of the mammalian brain and the nervous system such as the spinal cord. They have the ability to rebuild neural circuits and repair neural tissue. Therefore, neural stem cells have extensive research value in animal models of neurodegenerative diseases, hereditary central nervous system diseases, stroke and spinal cord injury. Neural stem cells play an important role in neural development and repair of damaged neural tissue. Neural stem cell transplantation is an effective method to repair and replace damaged brain tissue, and can rebuild some circuits and functions. In addition, neural stem cells can be used as gene carriers for gene therapy of intracranial tumors and other neurological diseases. The use of neural stem cells as gene therapy carriers makes up for some of the shortcomings of viral vectors.

[0015] ⑥ Pancreatic stem cells are pluripotent stem cells with the ability to divide and self-renew indefinitely. Under the influence or induction of specific factors, they can differentiate into specific pancreatic tissue types such as ducts, islets, and exocrine acini, and can even differentiate into hepatocytes. Based on their source, pancreatic stem cells can be divided into embryonic-derived pancreatic stem cells and adult-derived pancreatic stem cells. Due to the self-renewal and directed differentiation properties of pancreatic stem cells, they can provide a rich donor source for cell replacement therapy, which is of epoch-making significance for the treatment of various pancreatic injuries, pancreatitis, and especially diabetes.

[0016] (3) Unipotent stem cells

[0017] Unipotent stem cells (also called specialized or partial stem cells): often used to describe a type of cell in adult tissues and organs, meaning that such cells can only differentiate in a single direction and produce one type of cell. Adult stem cells in many differentiated tissues are typical unipotent stem cells, which can only produce one type of cell under normal circumstances. For example, the stem cells in the basal layer of epithelial tissue and the myoblasts in muscles are also called satellite cells. This type of tissue is in a stable state of self-renewal. However, if this tissue is damaged and requires multiple types of cells to repair, it is necessary to activate pluripotent stem cells to repair the injured tissue.

[0018] ① Muscle stem cells can develop and differentiate into myoblasts, which fuse to form multinucleated muscle fibers, the fundamental structure of skeletal muscle. Embryonic and fetal muscle stem cells proliferate to develop muscle tissue. Adult muscle stem cells, also known as satellite cells, lie dormant and are distributed along muscle fibers. After intense exercise or injury, adult muscle stem cells become activated and begin to proliferate, thereby increasing or restoring muscle tissue. In older adults, muscle stem cells no longer have the ability to replicate, resulting in muscle atrophy.

[0019] Cardiac stem cells (CSCs) are multipotent stem cells that are present in embryonic and adult tissues and have committed to the myocardial lineage. They are capable of differentiating into cardiomyocytes, endothelial cells, and smooth muscle cells, among other cells. They play a crucial role in cardiac embryogenesis and development, as well as normal postnatal myocardial renewal. Cardiac progenitor cells (CPCs) are generally multipotent stem cells with restricted differentiation and proliferation capabilities. They can be derived from CSCs and play a crucial role in cardiomyocyte renewal and repair. Recent studies have revealed a pool of CSCs within the adult heart, distributed in the atrial groove and apex. Under physiological conditions, CSCs and CPCs within the heart participate in normal cardiomyocyte renewal, albeit at a slow rate. Under pathological conditions, such as myocardial ischemia and necrosis, CSCs within the heart can be mobilized from the CSC pool and accumulate around the necrotic area, where they differentiate into cardiomyocytes, endothelial cells, or smooth muscle cells, thereby repairing the necrotic myocardium and improving blood circulation.

[0020] ③ Cartilage stem cells are stem cells found in the human ear that can develop into cartilage. These stem cells can be used for plastic surgery caused by facial fractures. Cartilage stem cells are derived from early adult articular cartilage tissue and possess the ability to self-proliferate and differentiate, maintaining their stemness after multiple passages. They are primarily used to treat congenital rickets and cartilage degenerative diseases, and to improve the lifespan of normal chondrocytes.

[0021] Endothelial progenitor cells (EPCs), also known as angioblasts, are precursors to endothelial cells. In response to physiological or pathological factors, they can be mobilized from the bone marrow into the peripheral blood to participate in the repair of damaged blood vessels. In 1997, Asahara et al. first demonstrated the presence of circulating peripheral blood precursor cells capable of differentiating into endothelial cells, naming them endothelial progenitor cells. Studies have shown that EPCs play a crucial role in cardiovascular and cerebrovascular diseases, peripheral vascular diseases, tumor angiogenesis, and wound healing.

[0022] ⑤ Germline stem cells (GSC), commonly known as spermatogonial stem cells, are a type of unipotent stem cell that can only produce gametes.

[0023] Epidermal stem cells (ESCs) are a type of cell population with indefinite proliferation capacity, capable of differentiating into various functional cells of the epidermis. Primarily located in the basal layer of the epidermis, they are adult stem cells with a strong ability to divide and proliferate, replenishing aging and shed keratinocytes. ESCs exhibit stem cell characteristics: immaturity, small size, small organelles, high colony-forming capacity, a slow cell cycle, and unlimited division. ESCs are relatively fixed within the tissue structure. The bulge of the hair follicle is currently believed to be the primary habitat for ESCs, where they play a crucial role not only in hair follicle growth but also in skin repair after injury. Stem cells and transient proliferating cells are distributed in a sheet-like pattern within the basal layer of the epidermis. In hairless areas, such as the palms and soles, ESCs reside in the basal layer of the skin, where 1% to 10% of basal cells are stem cells. In hairy areas, ESCs are located in the bulge of the hair follicle. Due to their unique biological advantages, ESCs play a vital role in skin renewal, tissue repair, tumor formation, gene therapy, and cell therapy. The outer root sheath of hair follicles, between the opening of the sebaceous glands and the attachment of the arrector pili muscles, is rich in stem cells. These can be used for autologous and allogeneic transplantation to treat severe burns, chronic ulcers, and other conditions. Stem cells isolated from mouse skin can be transformed into neurons, glial cells, smooth muscle cells, and adipocytes.

[0024] Although the research on stem cells has been relatively in-depth, there are limited ways to activate stem cells, and most of them have certain limitations and defects. Therefore, the development of a new type of efficient and safe stem cell activation strategy and method is of great significance for the widespread clinical application of stem cells.

[0025] The hydrogen molecule is the smallest molecule in nature. According to the law of gas diffusion, the diffusion rate of a gas in a uniform liquid is inversely proportional to the square root of the gas's molecular weight. Therefore, hydrogen has a very large diffusion capacity and can penetrate the skin barrier to reach all parts of the body. Furthermore, hydrogen has no polarity, making it easier for it to cross the cell membrane structure and enter the organelles within the cell, fundamentally adjusting the cell state. Hydrogen is extremely safe for the human body. Diving medicine has proven that high concentrations of hydrogen do not pose a hazard to the human body, indicating that hydrogen is a very safe gas for the human body. Its effectiveness and non-toxicity are also its advantages. Hydrogen has a wide range of synthesis pathways, low application costs, and can be used on a large scale, suggesting that hydrogen has great advantages in clinical disease treatment applications.

[0026] Since the only metabolic product of hydrogen molecules is water, it is safe and has no toxic side effects; it has strong permeability and can easily reach the stem cell areas of various tissues and organs in the body, so it is very likely to develop a method to activate stem cells using hydrogen.

[0027] Public content

[0028] In response to the limitations and deficiencies of current stem cell activation methods, the present disclosure provides an application of hydrogen molecules in the preparation of products for promoting stem cell activation. This method utilizes different forms of hydrogen intervention to promote the activation of various types of stem cells for applications in skin health, anti-aging, tissue and organ repair, regenerative medicine, and other fields.

[0029] Application of hydrogen molecules in the preparation of products for promoting stem cell activation.

[0030] Preferably, the hydrogen molecules are used in the preparation of products for promoting organ and nerve regeneration and repair by promoting stem cell activation, improving the body's hematopoietic capacity and lymphoid and myeloid differentiation capacity, improving reproductive capacity, or for skin care, damage repair and hair regeneration.

[0031] Preferably, the organs include bone tissue, cartilage tissue, muscle tissue and intestine; 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.

[0032] Preferably, the stem cells include epidermal stem cells, hematopoietic stem cells, skeletal stem cells, neural stem cells, muscle stem cells, intestinal stem cells and germ cells.

[0033] 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.

[0034] Preferably, the application of hydrogen molecules includes one or more of breathing hydrogen, oral hydrogen, injection hydrogen, implant hydrogen or surface hydrogen.

[0035] Preferably, when the hydrogen molecules are used in the preparation of products for promoting nerve regeneration and repair, improving reproductive ability, skin care, damage repair and hair regeneration by promoting stem cell activation, the hydrogen molecules are applied in the form of surface hydrogen administration and / or hydrogen administration through breathing;

[0036] Preferably, when the hydrogen molecules are used in the preparation of a product for promoting the activation of skeletal stem cells and muscle stem cells by promoting the activation of stem cells, the hydrogen molecules are applied in a manner selected from one or more combinations of surface hydrogen administration, inhalation hydrogen administration, and implantation hydrogen administration;

[0037] When the hydrogen molecules are used in the preparation of products for improving the body's hematopoietic ability, improving the lymphoid and myeloid differentiation ability, and promoting intestinal regeneration by promoting stem cell activation, the hydrogen molecules are applied in the form of inhalation and / or oral administration.

[0038] Preferably, the surface hydrogen delivery method includes bathing, soaking or fumigation with a hydrogen-rich solution, local application of a hydrogen-generating device or a hydrogen-containing patch;

[0039] The implantation hydrogen supply method includes implanting bone screws or stents containing hydrogen storage materials into bones, or implanting hydrogen storage sheets into muscles;

[0040] The hydrogen storage material is a mesoporous or alloy hydrogen slow-releasing material.

[0041] Preferably, the product is a mixed gas comprising hydrogen and oxygen, and the volume concentration of hydrogen in the mixed gas is not greater than 79%.

[0042] Preferably, the article is a hydrogen molecule source material that directly contains hydrogen molecules or is capable of generating hydrogen molecules when used.

[0043] 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.

[0044] Based on the biological functions of hydrogen molecules, this disclosure proposes the use of hydrogen molecules in the preparation of products for promoting stem cell activation. Hydrogen molecules can be used through various hydrogen introduction and generation methods to promote the activation of autologous stem cells, including epidermal stem cells, basal stem cells, hair follicle stem cells, bone marrow-derived stem cells, mesenchymal stem cells, spermatocytes, and neural stem cells. This information can be applied to a variety of industries, including wound healing, tissue and organ repair, tumor therapy, neurological disease treatment, infertility treatment, and medical aesthetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG1 is a hydrogen release curve of the hydrogen-containing skin care product of Example 1 of the present disclosure;

[0046] FIG2 is a comparison of the back skin moisture content of different groups of aged mice after treatment with Example 1 of the present disclosure for 30 days (unpaired-T test was performed between all groups, and the bar graph data are expressed as Mean±SD. *P-value<0.05; **P-value<0.01; ***P-value<0.001; ns P-value>0.05; the same below);

[0047] FIG3 is a comparison of the wrinkle levels on the back skin of different groups of elderly mice after treatment for 30 days in Example 1 of the present disclosure;

[0048] FIG4 is a comparison of back skin blood flow in different groups of aged mice after treatment for 30 days in Example 1 of the present disclosure;

[0049] FIG5 is a comparison of the elasticity levels of the back skin of elderly mice in different groups after treatment for 30 days in Example 1 of the present disclosure;

[0050] FIG6 is a hydrogen release curve of a suspension containing a solid hydrogen storage material according to Example 2 of the present disclosure;

[0051] FIG7 is a graph showing changes in the thickness of the epidermal layer of the back skin of different groups of aged mice after treatment for 30 days in Example 2 of the present disclosure;

[0052] FIG8 is a graph showing that hydrogen increases the collagen content in the epidermis and dermis of the skin of different groups of aging mice according to Example 2 of the present disclosure;

[0053] FIG9 is a diagram showing hydrogen promoting the proliferation and activation of epidermal stem cells in aging skin according to Example 3 of the present disclosure;

[0054] FIG10 is a graph showing the secretion of the basal stem cell activator Col-XVII promoted by hydrogen in aging mice according to Example 3 of the present disclosure;

[0055] FIG11 is a diagram showing that hydrogen promotes the accumulation of Col-III and Col-IV in the epidermis of aging mice and promotes the activation of epidermal stem cells according to Example 3 of the present disclosure;

[0056] FIG12 is a diagram showing hydrogen promoting proliferation and activation of dermal fibroblasts in aged skin according to Example 4 of the present disclosure;

[0057] FIG13 is a diagram showing hydrogen promoting the proliferation and activation of myofibroblasts in the dermis of aged skin according to Example 4 of the present disclosure;

[0058] FIG14 is a diagram showing how hydrogen promotes the secretion and accumulation of collagen and elastin in the dermis of aged skin according to Example 4 of the present disclosure;

[0059] FIG15 is a hydrogen release curve of the hydrogen storage patch of Example 5 of the present disclosure;

[0060] FIG16 is a diagram showing that hydrogen promotes the proliferation and activation of different subpopulations of epidermal stem cells (basal stem cells, follicular mesenchymal stem cells, and hair follicle stem cells) in the early stage of wound healing (2 days) and promotes wound repair in Example 5 of the present disclosure;

[0061] FIG17 is a diagram showing that hydrogen promotes the proliferation and activation of proliferative fibroblasts and myofibroblasts and promotes wound repair 2 days after wounding according to Example 5 of the present disclosure;

[0062] FIG18 is a hydrogen release curve of a hair mask containing a hydrogen storage material according to Example 6 of the present disclosure;

[0063] FIG19 is a hydrogen release curve of the hydrogen patch of Example 7 of the present disclosure;

[0064] FIG20 is a hydrogen release curve of the hydrogen patch of Example 8 of the present disclosure;

[0065] FIG21 is a hydrogen release curve of the hydrogen-containing dressing of Example 12 of the present disclosure;

[0066] FIG22 is a hydrogen release curve of the hydrogen-containing patch of Example 13 of the present disclosure;

[0067] FIG23 is a hydrogen release curve of the hydrogen-containing dressing of Example 14 of the present disclosure;

[0068] FIG24 is a diagram showing hydrogen molecules promoting satellite cell activation after muscle injury in Example 14 of the present disclosure;

[0069] FIG25 is a diagram showing hydrogen promoting early myotube repair in mice with gastrocnemius muscle injury according to Example 14 of the present disclosure;

[0070] FIG26 is a diagram showing that hydrogen reduces fat droplet accumulation in gastrocnemius muscle-injured mice and promotes early muscle repair according to Example 14 of the present disclosure;

[0071] FIG27 is a diagram showing hydrogen promoting the proliferation and activation of FAP stem cells in vitro in Example 14 of the present disclosure. DETAILED DESCRIPTION

[0072] Human aging is a process of cellular aging, and tissue and organ damage also requires stem cell activation. Generally speaking, with aging or pathological changes, the number of stem cells in the body decreases, the repair rate of damaged tissue gradually slows, stem cell activity decreases, and even enters a dormant state. This can be manifested as decreased cytokine synthesis and cellular metabolism, adipocyte shrinkage, collagen loss, elastic fiber rupture, skin wrinkling, decreased vision, bone loss, intestinal degeneration, hair loss, decreased hematopoietic function, and decreased sperm motility. Furthermore, after tissue and organ damage and loss, the body's own stem cells need to be rapidly activated for tissue repair and regeneration. Therefore, there is a need to find a method to activate the body's own stem cells to promote tissue regeneration, accelerate cell metabolism, and combat cellular aging.

[0073] There are limited methods for stem cell activation, especially for autologous stem cell activation, as there is currently no widely used and accepted method for clinical activation. Furthermore, existing stem cell activation methods target only one or a few types of stem cells and often have significant drawbacks and side effects. This disclosure, based on the biological effects of hydrogen molecules, provides a variety of hydrogen-based stem cell activation methods for use in multiple tissue and organ damage repair, functional enhancement, and cell regeneration.

[0074] Hydrogen can promote the activation of different subpopulations of stem cells, improve their proliferation and differentiation potential, and promote tissue regeneration and repair. Specifically, hydrogen molecules can be used in the following scenarios:

[0075] (1) Skin damage repair, wound epithelialization, skin care and hair follicle regeneration: Hydrogen molecules can promote the activation of epidermal stem cells, such as basal stem cells, hair follicle stem cells, and fibroblasts, thereby promoting skin damage repair, wound epithelialization, anti-aging, promoting hair regeneration, and promoting collagen accumulation.

[0076] (2) Neural remodeling: Hydrogen molecules can promote the activation of neural stem cells, thereby promoting nerve regeneration and nerve damage repair.

[0077] (3) Tissue and organ repair: Hydrogen molecules can promote the activation of bone stem cells, muscle stem cells, intestinal stem cells, etc., thereby promoting the regeneration and functional repair of bone tissue, muscle tissue, and intestines.

[0078] (4) Vascular remodeling and hematopoietic system activation: Hydrogen molecules can promote the activation of hematopoietic stem cells, thereby improving the body's hematopoietic ability and lymphoid and myeloid differentiation ability.

[0079] (5) Improve reproductive ability and increase sperm motility: Hydrogen molecules can promote the activation of reproductive cells, thereby improving reproductive ability and increasing sperm motility.

[0080] (6)Others.

[0081] The above-mentioned hydrogen molecule intervention methods include but are not limited to one or more of the following types: hydrogen administration by breathing, oral hydrogen administration (including hydrogen-rich solutions, hydrogen storage capsules, hydrogen storage effervescent tablets, etc.), hydrogen administration by injection (including subcutaneous injection, intravenous injection, in situ injection, etc.), hydrogen administration by implantation (including implanting bone screws or stents containing hydrogen storage materials into bones, or implanting hydrogen storage sheets into muscles, etc., and the hydrogen storage materials preferably use mesoporous or alloy hydrogen gas sustained-release materials with high safety performance, such as magnesium-based hydrogen storage materials) or surface hydrogen administration (including bathing, soaking or fumigation with hydrogen-rich solutions, local use of hydrogen-generating devices or hydrogen-containing patches, etc.).

[0082] Preferably, when hydrogen molecules are used to prepare products for promoting nerve regeneration and repair, improving reproductive capacity, skin care, injury repair, and hair regeneration by promoting stem cell activation, the hydrogen molecules are applied by topical hydrogen administration and / or inhalational hydrogen administration. When hydrogen molecules are used to prepare products for promoting the activation of skeletal stem cells and muscle stem cells by promoting stem cell activation, the hydrogen molecules are applied by one or more combinations selected from topical hydrogen administration, inhalational hydrogen administration, or implanted hydrogen administration. When hydrogen molecules are used to prepare products for improving the body's hematopoietic capacity, improving lymphoid and myeloid differentiation capacity, and promoting intestinal regeneration by promoting stem cell activation, the hydrogen molecules are applied by inhalational hydrogen administration and / or oral hydrogen administration.

[0083] Specific hydrogen delivery methods for different application scenarios can facilitate the body's absorption of hydrogen and improve its effectiveness. For example, when promoting the activation of skeletal stem cells, implanting bone screws containing hydrogen storage materials allows the hydrogen released by the hydrogen storage materials to reach the bone directly, acting directly on the stem cells. This results in better results than other interventional methods.

[0084] 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.

[0085] 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.

[0086] The hydrogen molecule source material is preferably a solid hydrogen storage material, which is a combination of one 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, polysiloxanes and derivatives, and other new solid hydrogen storage materials.

[0087] 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 alloy, titanium hydrogen storage material Ti-Fe, Ti-Zr, Ti-Cr, Ti-Mn, Ti-Co, Ti-Mn, Ti-Cr, zirconium alloy C15 cubic Laves phase, C14 hexagonal Laves phase, Zr-V alloy, Zr-Ni alloy, Zr-Cr alloy, Zr-Mn alloy, Zr-Co alloy, metal calcium, CaNi5 alloy or Ca-Ni-Mg series alloy; coordination metal hydrogen storage material is selected from Sc (AlH6), Cr (A lH6)2, Co(AlH6)3, Ti(AlH4)4, Mg(AlH4)2, Fe(AlH4)2, Sn(AlH4)4, MgH2, NaAlH4, LiAlH4, KBH4, NaBH4, LiBH4 or one or more thereof; the inorganic hydrogen storage material is one or more selected from NH3BH3 and bicarbonate; the nano hydrogen storage material is tetrafluoromethane hydrogen storage material, the porous polymer hydrogen storage material is self-microporous polymer PIMs, hypercrosslinked polymer H One or more of CPs or conjugated microporous polymers CMPs; the hydrate hydrogen storage material is a carbon dioxide hydrate hydrogen storage material; the polysiloxane and its derivatives are one or more of methyl hydrogen siloxane, ethyl hydrogen siloxane, propyl hydrogen siloxane, polyphenyl hydrogen siloxane, polymethylphenyl hydrogen siloxane, polymethylsiloxane, polymethylethylsiloxane, polymethylpropylsiloxane, polymethylphenylsiloxane, polymethylphenylethylsiloxane, polymethylpropylsiloxane, polymethylphenylsiloxane, polymethylphenylethylsiloxane, etc.

[0088] This disclosure provides several typical hydrogen molecular products and intervention methods, as follows:

[0089] ① Hydrogen inhalation: Hydrogen can be inhaled 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%, thus ensuring that the oxygen content is above 21% to ensure sufficient oxygen intake.

[0090] Hydrogen can be produced by electrolyzing water. The hydrogen and oxygen produced from the cathode and anode can be collected separately and inhaled, or they can be directly mixed and inhaled. Breathing can be done using a mask, nasal cannula, or in a hydrogen atmosphere.

[0091] ② Drinking hydrogen-rich water: hydrogen-rich water that is pre-configured or prepared before drinking. Hydrogen-rich water can be prepared in the following ways:

[0092] a. Physically saturated hydrogen water: A safer method involves introducing hydrogen directly or through microbubbling (filter device) into pure water or other drinking water, tea, or beverages to dissolve molecular hydrogen. For long-term storage, a hydrogen storage container is required to store the liquid for drinking. Hydrogen-enriched water requires the addition of a certain pressure of hydrogen to produce a hydrogen concentration higher than the solubility of hydrogen in regular water for storage.

[0093] b. Preparation of hydrogen water by reaction of hydrogen storage materials: Metal / non-metal hydrogen storage materials, including but not limited to magnesium metal, magnesium hydride, calcium hydride, boron-based compounds, etc., can react chemically with water to produce hydrogen.

[0094] c. Preparation of hydrogen water by alkaline electrolysis: Hydrogen is dissolved in water through electrolysis.

[0095] ③ Injection of hydrogen-rich saline: Hydrogen is delivered into the body by injecting an isotonic saline solution containing hydrogen into the vein, abdominal cavity, or affected area. The preparation method of hydrogen-rich saline is the same as that of hydrogen-rich water.

[0096] ④ Local use of hydrogen-generating devices or hydrogen-containing patches: Local hydrogen-generating devices or hydrogen-containing patches (patches containing solid hydrogen storage materials that generate hydrogen through contact with the reaction solution when applied) can be used on local or internal damaged skin to bring hydrogen into the body through the skin due to its strong permeability. The preparation method of the hydrogen-containing patch is as follows:

[0097] Use any medical membrane material to load a hydrogen storage material or hydrogen water / hydrogen saline / hydrogen-containing buffer solution to form a hydrogen-containing patch. The hydrogen storage material is a solid hydrogen storage material, and carbon-based hydrogen storage materials, porous polymer hydrogen storage materials, MOF hydrogen storage materials, metal-alloy hydrogen storage materials, hydride hydrogen storage materials, etc. are optional. Preferably, carbon-based hydrogen storage materials, metal / alloy hydrogen storage materials, and hydride hydrogen storage materials are selected. Weigh one or more of the above hydrogen storage materials. For the preparation of hydrogen water, hydrogen saline, and hydrogen-containing buffer solution, refer to the introduction to hydrogen water preparation methods.

[0098] ⑤ Oral products such as oral hydrogen-containing capsules / effervescent tablets; the preparation method of oral hydrogen-containing products is as follows:

[0099] Oral hydrogen-containing products are prepared by using one or more of hydrogenated coral calcium, magnesium hydride, calcium hydride, nano-ammonia borane, nano-magnesium hydride, nano-calcium hydride, metallic magnesium, etc., combined with capsules or compressed candies.

[0100] ⑥ Hydrogen-rich water bathing / soaking, etc.

[0101] ⑦ Hydrogen fumigation.

[0102] ⑧ Using hydrogen-containing skincare products: Using nano- / micron-sized emulsified hydrogen storage particles, or hydrogen storage material gel microbeads encapsulated, or surface-modified, these hydrogen storage materials (preferably one or more of magnesium metal, magnesium hydride, calcium hydride, coral calcium hydride, or ammonia borane) or hydrogen-containing solutions / water, mixed with conventional skincare products to create hydrogen-containing skincare products. The recommended weight of the hydrogen storage material is 0.1% to 10%.

[0103] 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.

[0104] Example 1. Applying hydrogen-containing skin care products to the skin of aged mice improves skin aging, increases skin moisture, reduces wrinkles, promotes blood flow, and increases skin elasticity

[0105] Aged (18 months) C57BL / 6 mice were used as animal models. The animal epidermis showed varying degrees of aging, such as sagging, wrinkles, water loss, and pigmentation. After preparing the skin on the back of the mice, the samples of each group were evenly applied to the back of the mice by surface application. The groups were: hydrogen-containing skin care product group, skin care product group, and blank control group. Application was performed once a day for 30 days. The hydrogen-containing skin care product group was a 5% medical glycerol aqueous solution containing 0.5% solid hydrogen storage material (mass percentage) (the solid hydrogen storage material and the glycerol aqueous solution were stored separately and mixed before use). The solid hydrogen storage material was a magnesium-based hydrogen storage material. The hydrogen release curve of the hydrogen-containing skin care product is shown in Figure 1 (measured using the liquid phase hydrogen electrode method), and the hydrogen release time exceeded 24 hours; the skin care product group was a 5% medical glycerol aqueous solution; and the blank control group was an aqueous solution.

[0106] The changes in the skin condition of the mice's backs were observed after 30 days. The main evaluations were on the moisture content, wrinkle score, blood flow, and skin elasticity of the mice's back skin. The results showed that after 30 days, the ordinary skin care product group had a slight increase in moisture compared to the control group, but it was far lower than the hydrogen-containing skin care product group (as shown in Figure 2); the wrinkle score showed that the hydrogen-containing skin care product group was 2-3 points lower than the other two groups (as shown in Figure 3); the blood flow statistics showed that local blood flow increased significantly after 30 days of surface application of hydrogen-containing skin care products, indicating that the local skin metabolism level was accelerated and the oxygen and nutrient supply capacity increased (as shown in Figure 4); the skin elasticity results showed that the skin elasticity of hydrogen-containing skin care products was significantly improved compared to the other two groups (as shown in Figure 5). The above results show that hydrogen can improve the skin quality of elderly mice, improve the aging state of the skin, and promote skin rejuvenation, including increased moisture, improved wrinkles, increased elasticity, and increased blood flow and skin metabolism levels.

[0107] Example 2. Hydrogen activates epidermal stem cells, increases epithelial thickness, and resists skin aging

[0108] Using the aged mice described in Example 1 as a model, the mice were skinned and then each sample was evenly applied to the backs of the mice using a surface application method. The groups were divided into: a hydrogen molecule group, a solvent group, and a blank control group. The hydrogen molecule group consisted of a suspension containing a solid hydrogen storage material, a mixture of Mg and MgH2, with a mass percentage concentration of 1%. The suspension solvent was a citric acid buffer solution (the solid hydrogen storage material and the solvent were stored separately and mixed before use). The hydrogen release curve of this sample is shown in Figure 6 (measured using the liquid phase hydrogen electrode method); the solvent group consisted of a citric acid buffer solution, and the blank control group consisted of an aqueous solution.

[0109] Apply once a day for 30 days. Observe the changes in the back skin of the mice after 30 days. After killing the mice, the back skin was fixed, embedded, and stained with HE and Mason. The thickening of the epithelial layer is due to the activation of epidermal stem cells, especially basal stem cells, which form asymmetric divisions, thereby thickening the epithelial layer. HE staining results showed that the thickness of the epithelial layer in the hydrogen group increased significantly (as shown in Figure 7), and the fullness of the skin increased; Mason staining showed that the epidermal collagen and dermal collagen in the hydrogen group increased compared with the other two groups (as shown in Figure 8). The above results suggest that hydrogen can activate epidermal stem cells in aged mice, promote epithelialization and skin fullness, increase subcutaneous collagen accumulation, and resist skin aging.

[0110] Example 3. Hydrogen directly activates the epidermis of aging mouse skin, activates basal stem cells, improves skin aging, and increases related ECM accumulation

[0111] Using the aged mice described in Example 1 as a model, the mice were skinned and then the samples from each group were evenly applied to the backs of the mice. The groups were divided into: hydrogen emulsion group, emulsion group, and blank control group. The emulsion group consisted of a combination of olive oil, purified water, emulsifier, and rose essential oil (mass ratio 10:50:1:0.2). The hydrogen emulsion group also contained 5% (mass fraction) of a hydrogen storage material, a mixture of Mg and MgH2 (the solid hydrogen storage material and emulsion were stored separately and mixed before use). The blank control group was an aqueous solution.

[0112] Apply the solution once daily for 30 days. Observe changes in the dorsal skin of the mice after 30 days. After the mice were sacrificed, the dorsal skin was fixed, embedded, and subjected to immunohistochemistry and immunofluorescence staining. Changes in the expression levels of p63 (a stem cell marker), Ki67 (a cell proliferation marker), Col-XVII (a skin aging marker), Col-IV, and Col-III (skin youthfulness markers) were measured after 30 days.

[0113] The results showed that after 30 days of hydrogen emulsion treatment, the total number of epidermal stem cells in the skin of aged mice increased significantly (approximately 50% more than in the control and emulsion groups). Simultaneously, the number of proliferating epidermal stem cells also increased significantly (as shown in Figure 9). This suggests that the asymmetric division ability of aged skin is enhanced, and the anti-aging ability of the epidermis is strengthened.

[0114] Col-XVII is an important type of collagen specifically expressed by basal stem cells, which can reflect the degree of skin aging. Immunohistochemical staining statistics showed that the expression level of Col-XVII in the hydrogen emulsion group was significantly increased compared with the other two groups, indicating that the basal stem cells are more active and youthful (as shown in Figure 10).

[0115] Col-IV and Col-III are expressed in the epidermis, reflecting the youthfulness of the epidermis and the activation of epidermal stem cells. The results showed that after 30 days, the expression levels of Col-IV and Col-III in the hydrogen emulsion group were significantly increased (as shown in Figure 11).

[0116] The above results collectively indicate that hydrogen can promote the activation of epidermal stem cells and basal stem cells in the skin of aging mice, resist skin aging, and promote skin regeneration.

[0117] Example 4. Hydrogen activates dermal fibroblasts and myofibroblasts, promotes skin collagen accumulation and improves skin elasticity.

[0118] Using the aged mice described in Example 1 as a model, the mice were skinned and then the samples were evenly applied to the mice's backs using a topical application. The groups were divided into: a hydrogen molecule group, a solvent group, and a blank control group. The samples in each group were the same as in Example 2.

[0119] Apply the solution once daily for 30 days. Observe changes in the dorsal skin of the mice after 30 days. After the mice were sacrificed, the dorsal skin was fixed, embedded, and subjected to immunohistochemistry and immunofluorescence staining. Changes in the expression levels of vimintin (a fibroblast marker), α-SMA (a myofibroblast marker), Ki67 (a cell proliferation marker), Col-I, and elastin in the dermis were measured after 30 days.

[0120] The results showed that the total number of fibroblasts (as shown on the left side of Figure 12), the number of proliferating fibroblasts (as shown on the right side of Figure 12), the total number of myofibroblasts (as shown on the left side of Figure 13), the number of proliferating myofibroblasts (as shown on the right side of Figure 13), the Col-1 expression level (as shown on the left side of Figure 14), and the elastin expression level (as shown on the right side of Figure 14) were all significantly increased in the hydrogen molecule group and were significantly higher than those in the solvent group and the control group (the fibroblast measurement methods on the left and right sides of Figures 12 and 13 are different. The left side uses the expression level of characteristic markers to evaluate the total number of fibroblasts, and the right side shows the absolute number of fibroblasts per square millimeter, which is evaluated by quantity). This suggests that hydrogen can stimulate the proliferation and activation of dermal stem cell subsets (fibroblasts, myofibroblasts, etc.), promote collagen accumulation and skin elasticity, and resist skin aging.

[0121] Example 5. Hydrogen promotes the activation of early epidermal stem cells and fibroblasts during wound repair, accelerating wound repair

[0122] A full-thickness skin defect on the back of mice was selected as an animal model. A hydrogen storage patch and hydrogen breathing were used to intervene in the wound healing process. The hydrogen storage material in the patch was 5% by mass and was made of medical non-woven fabric. The hydrogen storage material was a mixture of ammonia borane, magnesium metal, magnesium hydride, and hydrated silicon. The hydrogen release curve of the patch is shown in Figure 15 (measured using the liquid phase hydrogen electrode method. Before testing, the patch was soaked in saline, and the ratio of the patch to saline was 2 cm / s.). 2 / 10mL), hydrogen release time>2d, replace the patch every 2d; hydrogen breathing concentration is 66% (volume concentration), breathing 2h a day.

[0123] The activation levels of epidermal stem cells (as shown in FIG16 ) and fibroblasts (as shown in FIG17 ) in the skin tissue at the wound edge of mice were detected 2 days later; and the healing of wounds in each group was monitored 10 days later (as shown in Table 1 ).

[0124] The results showed that in the early stage of wound healing, in the hydrogen patch group and the hydrogen breathing group, p63 + Basal stem cells, proliferation stage k5 + Follicular mesenchymal epithelial stem cells, proliferative stage Lgr6 + The hair follicle stem cell subpopulations showed a higher level of increase compared with the control group and the substrate group (as shown in Figure 16), suggesting that hydrogen molecules in the early stage of wound healing promoted the proliferation and activation of epidermal stem cells and follicular mesenchymal stem cells.

[0125] Dermal staining and positive cell statistics showed that both hydrogen patch and breathing significantly increased the proliferation rate of two stem cell subsets in the dermis, fibroblasts and myofibroblasts, and promoted dermal regeneration and tissue remodeling (as shown in Figure 17).

[0126] Based on the activation levels of these stem cell subsets in the epidermis and dermis, we further analyzed the healing rates of mice in each group at different stages of healing. The results (as shown in Table 1) show that hydrogen can promote tissue repair and wound healing by activating stem cells.

[0127] Table 1 Hydrogen promotes stem cell activation and skin healing in mouse skin lesions

[0128] Example 6. Hydrogen promotes hair growth after chemotherapy by activating hair follicle stem cells

[0129] Chemotherapy-induced alopecia (CIA) is one of the most commonly reported side effects among cancer patients and survivors, with an overall incidence of 65%. Some topical medications (such as minoxidil or bimatoprost) have been shown to effectively stimulate hair regrowth after chemotherapy, but they have no preventive effect on CIA. However, hydrogen therapy for hair loss, through its antioxidant properties, regulation of homeostasis, metabolic stimulation, reduction of scalp inflammation, and hair growth promotion, not only effectively stimulates hair regrowth after chemotherapy but also offers a preventive benefit. A study of 225 CIA patients treated with hydrogen therapy (topically applying a hair mask containing hydrogen storage materials) revealed a significant effect. Hydrogen not only reduced the amount of hair loss after chemotherapy but also played a role in preventing it when used before chemotherapy (as shown in Table 2).

[0130] The chemotherapy group used a hair mask without a hydrogen storage material. The pre-chemotherapy hydrogen group used a hair mask containing a hydrogen storage material once daily for 15 days before chemotherapy. The post-chemotherapy hydrogen group used a hair mask containing a hydrogen storage material once daily for 15 days after chemotherapy. The hair mask consisted of a suspension containing 20% ​​aloe vera extract and 0.8% magnesium-based hydrogen storage microspheres as the hydrogen storage material (the hair mask and hydrogen storage material were stored separately and mixed before use). The hydrogen release curve of the hair mask containing the hydrogen storage material is shown in Figure 18 (measured using the liquid-phase hydrogen electrode method), with a sustained hydrogen release time of >40 minutes. The hair mask was used as follows: After cleansing the head daily, apply the hair mask evenly to the hair, allowing it to contact the scalp. After 20 to 30 minutes, rinse with warm water.

[0131] Table 2 Hair loss status of 225 CIA patients before and after hydrogen treatment

[0132] Example 7. The healing effect of hydrogen on hair transplant surgery

[0133] Common side effects after hair transplant surgery are swelling and pain. The symptoms will gradually ease as the transplant time increases. If there is improper care, it may lead to infection, which can easily cause the transplant to fail. At the same time, some people will form local scars on their scalp after surgery. The use of hydrogen to treat patients after hair transplantation can not only relieve the postoperative swelling and pain of the transplant recipients, but also help repair scars. In addition, the activation effect of hydrogen on hair follicle stem cells can significantly increase the survival rate of hair follicles. Postoperative hydrogen treatment of 27 hair transplant recipients found that the swelling of the patients was significantly reduced after using hydrogen, the effect was good, and the hair follicle survival rate was significantly higher than that after conventional hair transplantation (as shown in Table 3).

[0134] This example uses a combination of hydrogen breathing and a hydrogen patch for 60 consecutive days. Hydrogen breathing involves breathing a 66% hydrogen gas concentration (volume concentration) twice daily for 2 hours each. The hydrogen patch contains a 2% hydrogen storage material by mass, made of a medical nonwoven fabric. The hydrogen storage material is a mixture of a magnesium-based hydrogen storage material and a borohydride hydrogen storage material. Before use, the patch is carefully soaked and applied to the affected scalp once daily for 1 hour. The hydrogen release curve of this patch is shown in Figure 19, with a sustained hydrogen release time of >1 hour.

[0135] Table 3 Swelling sensation and hair follicle survival rate of 27 hair transplant recipients before and after hydrogen treatment

[0136] Example 8. Hydrogen promotes the activation of osteoblasts and accelerates bone regeneration

[0137] To prevent and alleviate osteoporosis, it's crucial to adjust the balance between bone formation and bone resorption when osteoporosis symptoms occur. There are two approaches to adjusting this balance: 1. Inhibiting overactive bone resorption 2. Activating osteoblasts to promote bone formation. Inhibiting bone resorption can prevent osteoporosis symptoms from worsening, but since it doesn't restore bone mass to its original state, promoting bone formation is a more important strategy for treating osteoporosis. Interventional treatment uses a topical hydrogen patch combined with hydrogen breathing.

[0138] This embodiment adopts the combined use of hydrogen breathing and hydrogen patch, and is used continuously for 90 days. Hydrogen breathing is to breathe the gas containing hydrogen every day (nasal inhalation), and the hydrogen concentration in the gas is 5% (volume concentration), and breathe once a day, each time for 2 hours; The hydrogen patch contains 3% hydrogen storage material by mass fraction, and the membrane cloth adopts medical non-woven fabric. The hydrogen storage material is a mixture of magnesium hydride, calcium hydride, metallic magnesium, and ammonia borane. Before using the patch, the patch is carefully soaked and applied to the affected part body surface. It is applied once a day for 12 to 20 hours each time. The hydrogen release curve of this patch is shown in Figure 20, and the hydrogen continuous release time is> 24 hours.

[0139] The bone mineral density (BMD) of the second middle finger joint of osteoporosis patients in the control group and the hydrogen group was measured by absorbance spectrophotometry. The BMD (bone density) value of the hydrogen group tended to increase compared with the control group (as shown in Table 4). Osteoblasts are the main cells responsible for bone formation. Multiple osteoblasts interact with each other to form a bone unit called an osteon. Hydrogen can significantly stimulate the proliferation of osteoblasts, which is significantly different from the control group. ALP and BGP, which reflect osteoblast activity, also increase with increasing hydrogen concentration, which is significantly different from the control group (as shown in Table 5). In summary, hydrogen has a certain activating effect on osteoblast stem cell subsets.

[0140] Table 4 Bone mineral density (BMD) values ​​determined by spectrophotometry

[0141] Table 5 Determination of osteoblast number and activity after hydrogen treatment

[0142] Example 9. Hydrogen Activates Intestinal Stem Cells

[0143] Intestinal stem cells (ISCs) within the crypts play an important role in maintaining intestinal homeostasis and promoting intestinal regeneration after injury. To determine whether hydrogen can directly regulate the proliferation of intestinal crypt stem cells, the morphology of the intestinal crypts and the number of various stem cell subsets in a mouse IBD model were examined (Tables 6 and 7).

[0144] This example treated mice by drinking hydrogen water combined with breathing hydrogen-containing gas for 15 consecutive days. The hydrogen content in the hydrogen water was 1.4±0.2ppm, and the hydrogen water was consumed daily and replaced twice daily. The hydrogen-containing gas was breathed at a volume concentration of 5% at a flow rate of 600mL / min, with breathing occurring twice daily for 2 hours each. A control group drank pure water and breathed normally.

[0145] Lgr5 is the most important surface marker of intestinal stem cells. Other intestinal stem cell-specific markers include Olfm4, Axin2, Ascl2, Olfm4, and Bmi1. + The cells can form intestinal organoids in vitro without mesenchymal cells. The intestinal epithelial cells produced are polarized and orderly arranged with crypt-like and villus-like domains. Compared with the control group, hydrogen treatment significantly increased Lgr5 + The number of ISCs was significantly improved and the expression of Lgr5 mRNA in intestinal crypts was significantly improved. Oflm4 is specifically expressed in intestinal stem cells, and its positive cells can generate all major cell types in the intestine. Ascl2 can activate genes that maintain the stem cell state by synergizing with target gene promoters and enhancers to regulate Lgr5. +The expression levels of the above marker genes increased significantly after hydrogen treatment, proving that hydrogen can effectively activate intestinal stem cells.

[0146] Table 6 Intestinal crypt morphology and cell status after hydrogen treatment

[0147] Table 7 Relative expression fold changes of intestinal ISC marker genes regulated by hydrogen treatment

[0148] Example 10. Hydrogen promotes the activation and proliferation of bone marrow-derived stem cells

[0149] Using aged (18-month-old) male C57BL / 6 mice as an animal model, hydrogen breathing (66% H₂ by volume, once daily for 2 hours at a flow rate of 1000 mL / min) was used to test the effects of hydrogen on the activation of various hematopoietic stem cell subsets and bone marrow-derived mesenchymal stem cells. These two types of stem cells in the bone marrow are important adult stem cells that play a crucial role in myeloid / lymphoid differentiation, hematopoiesis, tissue repair, and immune response.

[0150] After 30 days of treatment, the mice's tibial bone marrow, thymus, and spleen were embedded and sectioned, and stained and counted using immunofluorescence. The results showed a significant increase in the number and proportion of stem cell subsets within the bone marrow (as shown in Table 8). After hydrogen treatment, the proportion of undifferentiated thymic cells increased by more than 2% (as shown in Table 9), and the number of differentiated and mature T cells also increased (as shown in Table 10), suggesting that hydrogen promotes the activation of thymic stem cells. Statistics of various stem cell subsets in the spleen also showed that hydrogen can promote the increase in the proportion of various splenic stem cell subsets.

[0151] Table 8 Hydrogen respiration promotes the activation of bone marrow stem cells in aged mice

[0152] Table 9 Hydrogen respiration promotes the increase in the proportion of undifferentiated cells in the thymus of aged mice and accelerates T cell maturation

[0153] Table 10 Hydrogen respiration promotes the increase in the proportion of undifferentiated cells in the spleen of aged mice

[0154] Example 11. Hydrogen promotes bone marrow cell activity and improves hematopoietic function in mice with esophageal cancer after chemotherapy

[0155] Due to its non-targeted nature, chemotherapy usually causes a decrease in the activity of multiple stem cell subsets in patients, which can be manifested in clinical symptoms such as bone marrow suppression, gastrointestinal reactions, hair loss, nerve damage, etc. Among them, bone marrow suppression after chemotherapy mainly leads to anemia, and some patients experience bleeding spots. Bone marrow suppression affects the hematopoietic function of the bone marrow, and a decrease in blood cells may occur. A significant decrease in red blood cells and hemoglobin can lead to anemia, manifested as symptoms such as fatigue and pale complexion. Some patients may experience a decrease in platelets, leading to an increased risk of bleeding, skin ecchymoses or bleeding spots. In addition, leukopenia can lead to a decrease in resistance and easily induce infection. In this example, BALB / C male nude mice were used as experimental animals, and the mouse esophageal cancer cell line (tumor sphere) AKR was inoculated under the forelimb armpits to establish an esophageal cancer model. The chemotherapy drug used was 5-FU, which was injected once a week for a total of 4 weeks of chemotherapy. During chemotherapy, mice were treated with either hydrogen breathing or hydrogen water drinking. The chemotherapy group received 5-FU chemotherapy alone after tumor modeling and maintained with air breathing and pure water drinking. The negative control group received no treatment after tumor modeling. Hydrogen breathing was performed at a concentration of 100% at a flow rate of 600 mL / min for two hours daily. The hydrogen water drinking consisted of purified water saturated with hydrogen (1.4 ± 0.1 ppm) and was freely available for daily drinking (100 mL). After chemotherapy (i.e., 28 days after hydrogen treatment), the white blood cell (WBC), red blood cell (RBC), hemoglobin (Hb), and plasminogen activator (PLT) counts in each group were measured using a hematology analyzer to assess hematopoietic function. Bone marrow nucleated cell (BMC) counts were also microscopically counted and compared across groups. Results showed that the BMC, RBC, Hb, PLT, and WBC counts in the hydrogen breathing and hydrogen water drinking groups were significantly higher than those in the chemotherapy and tumor model groups alone (Table 11). The above results suggest that hydrogen molecules can reduce bone marrow suppression caused by chemotherapy drugs, promote the activation of bone marrow stem cells, and improve bone marrow hematopoietic function.

[0156] Table 11 Comparison of bone marrow nucleated cell counts and blood routine results of mice in each group

[0157] Example 12 Hydrogen promotes neural stem cell activation and induces neural regeneration

[0158] The brain impact model mice were used as the experimental model. Immediately after model establishment, interventional treatment was performed using hydrogen breathing (pure hydrogen breathing, flow rate 300 mL / min, each time for 2 hours, twice a day) and hydrogen-containing dressing application. The control group served as the modeling group.

[0159] The hydrogen-containing dressing contains 4% hydrogen storage material by weight. The dressing base is made of medical non-woven fabric, and the hydrogen storage material is a mixture of metallic magnesium, magnesium hydride, and hydrated silicon. Before use, the patch should be carefully moistened and applied to the affected area. The patch should be applied once daily for 12 to 20 hours. The hydrogen release curve of this dressing is shown in Figure 21.

[0160] Six days later, tissue from the traumatic brain area was embedded and sectioned, and the sections were stained using immunofluorescence, photographed, and counted. The detection indicators were Nestin and SOX2, which are indicators of neural stem cells, and BrdU, which is an indicator of proliferation. The results showed that the total number of neural stem cells and the number of proliferating neural stem cells in the brain injury tissue increased significantly in both the hydrogen breathing and hydrogen-containing dressing groups (as shown in Table 12). Further statistics on the percentage of dormant and activated neurons suggested that hydrogen can promote neural stem cell activation and induce neural regeneration.

[0161] Table 12 Effects of hydrogen molecules on the activation of neural stem cells

[0162] Example 13 Hydrogen promotes sperm cell activation in male aged mice, improving sperm motility and fertility

[0163] Twenty-month-old Kunming mice were selected as experimental animals and divided into a hydrogen breathing group (66% hydrogen, once a day for 2 hours each time), a hydrogen water drinking group (saturated hydrogen water, concentration of approximately 1.5±0.2ppm), a hydrogen-containing patch application group (Mg-based hydrogen storage material + medical non-woven fabric, soaked with physiological saline before use, applied to the testicles, once a day for 12-18 hours each time, the hydrogen release curve of the patch is shown in Figure 22) and a control group, and the treatment was continuous for 30 days.

[0164] Sertoli cells provide protection and nutrition to developing sperm, and all stages of sperm development are dependent on them. Tissues from the testes, epididymis, and seminiferous tubules of mice were fixed, embedded, and stained with hematoxylin and eosin (HE) to analyze relevant parameters. As shown in Table 13, the number of Sertoli cells in the cross-section of the seminiferous tubules in the hydrogen-treated group was significantly increased compared to the control group. The ratio of spermatids to Sertoli cells in each subpopulation was also significantly increased in all hydrogen-treated groups.

[0165] Table 13 Effects of hydrogen molecules on the number of supporting cells in the cross section of the seminiferous tubules of mice and the ratio of spermatogenic cells to supporting cells at each level

[0166] Further analysis of parameters related to sperm motility in the caput epididymis of mice revealed that the hydrogen molecule intervention group showed improvements in all parameters compared to the negative control group, including increased sperm density, survival rate, motility rate, and normal morphology rate. No significant differences were observed between the hydrogen breathing group, the hydrogen patch group, and the hydrogen water drinking group. This suggests that hydrogen can promote sperm cell activation in aged mice (as shown in Table 14).

[0167] Table 14 Effects of hydrogen molecules on the mirror parameters of the epididymal caput of aging mice

[0168] Example 14. Hydrogen promotes muscle damage repair and regeneration by activating muscle satellite cells

[0169] Skeletal muscle is composed of bundles of contractile myofibers, each of which is surrounded by satellite cells—muscle stem cells that generate new myofibers. Thanks to the action of these satellite cells, muscle fibers can regenerate even after being bruised or torn during strenuous exercise. Satellite cells also play a crucial role in muscle growth during development and hypertrophy during strength training. However, in certain muscle diseases, the number and function of satellite cells decrease, leading to poor muscle regeneration.

[0170] Fibroadipocyte precursor (FAP) cells normally play a beneficial role in muscle regeneration after injury by clearing debris and promoting the fusion of muscle cells into new myofibers. These FAP cells can differentiate into both fibrocytes and adipocytes, but differentiation into adipocytes can hinder the regeneration and repair of muscle tissue.

[0171] In this embodiment, the mouse gastrocnemius muscle blow model is selected as the animal model: a 25g weight is dropped from a height of 60cm onto the medial surface of the left gastrocnemius muscle. The modeling method has a medium intensity and does not cause bone damage or gait abnormality. The experimental groups are: a negative control group (only blow without treatment), a positive control group (treated with Yunnan Baiyao, a conventional spray drug), a hydrogen breathing group (66% hydrogen breathing is performed immediately after the blow, once a day, each time for 2 hours), and a hydrogen-containing dressing group (a hydrogen-containing patch is applied immediately after the blow, Mg-based hydrogen storage material + medical non-woven fabric, soaked with physiological saline before use, applied once a day, each time for 18 to 24 hours, and the hydrogen release curve of the patch is shown in Figure 23). After 3 days, each group of mice was killed, and the muscle tissue of the leg blow site was fixed and embedded, and immunofluorescence staining was performed. Pax7+ / MyoD+ can represent activated satellite cells, and myotube tissue is stained with phalloidin, and Perilipin-1 is used as a lipid droplet marker.

[0172] The results showed that after three days of treatment, the number of activated satellite cells in the gastrocnemius muscles of the two groups of mice treated with hydrogen molecules increased significantly compared to the negative control group, and the effect was superior to that of the positive drug group (as shown in Figure 24). Myotube area (phalloidin staining) and lipid droplet area (perilipin-1 staining) statistics (as shown in Figures 25 and 26) showed that hydrogen significantly promoted myotube regeneration and repair in damaged muscle tissue in mice and reduced lipid droplet secretion. These results suggest that hydrogen can reduce the differentiation of FAP cells into adipocytes, further improving muscle damage repair and functional reconstruction.

[0173] FAP cells were cultured in vitro for verification, and were treated with conventional culture medium and hydrogen-containing culture medium (saturated hydrogen gas was introduced into the culture medium, with a hydrogen concentration of 100%, pure hydrogen) for 24 hours, respectively. BrdU staining was used, and the results showed that the hydrogen treatment group significantly promoted the proliferation of FAP cells in vitro and increased their stem cell activity (as shown in Figure 27).

[0174] 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 stem cell activation.

2. The use according to claim 1, characterized in that The hydrogen molecules are used in the preparation of products for promoting organ and nerve regeneration and repair by promoting stem cell activation, improving the body's hematopoietic ability and lymphoid and myeloid differentiation ability, improving reproductive ability, or skin care, damage repair and hair regeneration.

3. The use according to claim 2, characterized in that The organs include bone tissue, cartilage tissue, muscle tissue and intestine; 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 stem cells include epidermal stem cells, hematopoietic stem cells, skeletal stem cells, neural stem cells, muscle stem cells, intestinal stem cells and germ cells.

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 of hydrogen molecules includes one or more of breathing hydrogen, oral hydrogen, injection hydrogen, implant hydrogen or surface hydrogen; Preferably, when the hydrogen molecules are used in the preparation of products for promoting nerve regeneration and repair, improving reproductive ability, skin care, damage repair and hair regeneration by promoting stem cell activation, the hydrogen molecules are applied in the form of surface hydrogen supply and / or breath hydrogen supply; Preferably, when the hydrogen molecules are used in the preparation of a product for promoting the activation of bone stem cells and muscle stem cells by promoting the activation of stem cells, the hydrogen molecules are applied in a manner selected from one or more combinations of hydrogen delivery through the body surface, hydrogen delivery through breathing, or hydrogen delivery through implantation; Preferably, when the hydrogen molecules are used in the preparation of products for improving the body's hematopoietic ability, improving lymphoid and myeloid differentiation ability, and promoting intestinal regeneration by promoting stem cell activation, the hydrogen molecules are applied by breathing hydrogen and / or oral hydrogen administration.

7. The use according to claim 6, characterized in that The surface hydrogen delivery method includes bathing, soaking or fumigation with hydrogen-rich solution, local use of hydrogen-generating device or hydrogen-containing patch; The implantation hydrogen supply method includes implanting bone nails or stents containing hydrogen storage materials into bones, or implanting hydrogen storage sheets into muscles; The hydrogen storage material is a mesoporous or alloy hydrogen slow-release material.

8. The use according to claim 1, characterized in that The product is a mixed gas comprising 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 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, polysiloxanes and derivatives, and other new solid hydrogen storage materials.

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