Chemical small molecule compound and method for promoting mesenchymal stem cell mitochondria to be delivered to muscle cells

Abstract

The invention discloses a chemical small molecule compound and method for promoting mesenchymal stem cell mitochondria to be delivered to muscle cells. The chemical small molecule compound is composed of metformin, beta-nicotinamide mononucleotide, nicotinamide, ergothioneine and pyrroloquinoline quinone. The concentration range of each component is respectively as follows: 1 to 5mM of metformin, 0.1 to 1mM of beta-nicotinamide mononucleotide, 0.1 to 1mM of nicotinamide, 0.1 to 1mM of ergothioneine and 0.01 to 0.1 mu M of pyrroloquinoline quinone. The compound regulates and controls the mitochondrial function of mesenchymal stem cells through a staged treatment strategy: in the first stage (0-24 h), an AMPK signal channel is activated through metformin, the NAD + level is increased in combination with beta-nicotinamide mononucleotide and nicotinamide, and mitochondrial biogenesis is started; the pyrroloquinoline quinone is combined to promote DNA replication and protein synthesis of mitochondria, further increase the number of mitochondria and optimize functions.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a chemical small molecule complex and method for promoting the delivery of mitochondria from mesenchymal stem cells to muscle cells. Background Technology

[0002] Muscle injury is a common clinical pathological condition, encompassing structural damage and functional impairment of skeletal and cardiac muscles caused by sports injuries, trauma, and diseases. In the field of sports medicine, muscle injury is one of the most common and frequently recurring diseases, causing significant distress to both athletes and ordinary patients. Statistics show that muscle injury accounts for 10% to 55% of all sports injuries. Currently, there are various clinical treatments for muscle injuries, including nonsteroidal anti-inflammatory drugs (NSAIDs), the PRICE principle (rest, ice, compression bandages, and elevation), and surgical treatment. However, these methods are mostly based on expert opinions or "best practices" and lack sufficient evidence-based medical support. For example, while NSAIDs can suppress inflammatory responses, long-term use may inhibit tissue regeneration and negatively impact bone growth; surgical treatment, although the first choice for severe or chronically painful muscle injuries, requires sacrificing healthy tissue and is not suitable for large-scale tissue defects. Furthermore, after a muscle injury, skeletal muscle tissue undergoes three stages: inflammation, repair, and remodeling. If the first two stages are imbalanced, muscle tissue will be replaced by scar tissue, leading to permanent muscle dysfunction.

[0003] Mesenchymal stem cells (MSCs) have attracted much attention in muscle repair due to their multi-lineage differentiation potential and paracrine function. MSCs can migrate to the site of injury, differentiate into muscle cell components, and secrete various cytokines and growth factors to promote tissue regeneration. In recent years, studies have found that MSCs are effective mitochondrial donors. MSCs from different tissue sources (such as bone marrow, adipose tissue, dental pulp, and umbilical cord) have different mitochondrial donor characteristics, which are related to their inherent respiratory state. For example, adipose-derived MSCs (AD-MSCs) and bone marrow-derived MSCs (BM-MSCs) show higher mitochondrial transfer capacity than dental pulp-derived MSCs (DP-MSCs) and umbilical cord-derived MSCs (UC-MSCs). However, the mitochondrial transfer capacity of MSCs is limited, and the stability and activity maintenance of mitochondria during delivery remain problems that need to be solved in delivering them to damaged muscle cells.

[0004] Mitochondria, as the cell's energy factories, are closely related to the activity of muscle cells. Mitochondrial dysfunction in damaged muscle cells can lead to energy metabolism disorders and affect muscle repair. Mitochondrial replacement therapy has become a research hotspot, involving the delivery of healthy mitochondria into damaged cells to restore their normal physiological functions. However, current mitochondrial delivery technologies face many challenges. For example, physical methods (such as electroporation and microinjection) are complex, cause significant cell damage, and are inefficient, while viral vector-mediated methods pose safety risks, such as triggering immune responses and gene insertion mutations. Furthermore, improving the efficiency and precision of mitochondrial delivery is also an urgent problem to be solved. Studies have shown that mitochondrial transplantation therapy (MTT) can improve muscle regeneration and functional recovery, and reduce fibrosis, but its application is still in the exploratory stage.

[0005] Prior Art 1: A method for preparing and applying mitochondrial-enhanced dental pulp mesenchymal stem cells (IPCMS) (Publication No.: CN 119776272 A) provides a method for obtaining mitochondrial-enhanced PPCMS by co-culturing platelet-rich fibrin with PPCMS. Specific steps include: co-culturing PPCMS with platelet-rich fibrin, optimizing co-culturing time, cell-to-platelet-rich fibrin ratio, and other conditions to improve mitochondrial function of the PPCMS. This method mainly enhances the regenerative capacity of PPCMS by transferring mitochondria to PPCMS through platelet-rich fibrin, and is suitable for dental pulp regeneration and related tissue repair.

[0006] Disadvantages of existing technology 1: This method has limitations in its preparation process, requiring strict control over co-culture time, the ratio of cells to platelet-rich fibrin, and other conditions, making it difficult to flexibly apply to mesenchymal stem cells from different sources and to different types of muscle injury treatment scenarios. Simply transferring mitochondria to dental pulp mesenchymal stem cells via platelet-rich fibrin may not effectively regulate the mitochondrial transfer process, thus affecting mitochondrial transfer efficiency. The lack of subsequent regulatory mechanisms for mitochondria transferred to dental pulp mesenchymal stem cells makes it difficult to ensure the stable distribution and function of mitochondria within the cell, thereby affecting the long-term regenerative capacity of the cells.

[0007] Prior art two provides an engineered mitochondria and its preparation method (application publication number: CN 118048300A). The method involves preparing mesenchymal stem cells modified with targeting molecules into a cell homogenate using an extrusion method, followed by differential centrifugation to obtain engineered mitochondria. This method provides a membrane-bound environment for the mitochondria and modifies them with targeting short peptides to increase uptake. Specific steps include: extracting mitochondria from cells or tissues; processing the mitochondria using a liposome extruder to bind exogenous cell membranes to the mitochondrial outer membrane; and separating the mitochondria by differential centrifugation. After extraction, the mitochondria require processing under specific conditions, such as centrifugation at 4°C, to ensure their activity and function. This method primarily enhances the stability and targeting of mitochondria, thereby improving their therapeutic efficacy, and is applicable to the extraction and modification of mitochondria from various tissue sources.

[0008] Disadvantages of existing technology 2: This method is complex to operate, requires sophisticated equipment, and is difficult to apply on a large scale. Mitochondrial extraction requires strict control of experimental conditions, such as temperature, centrifugation time, and speed, demanding high levels of expertise from both laboratory equipment and operators. It focuses on modifying the mitochondria themselves and does not address efficient intercellular delivery mechanisms, making it difficult to meet the precise mitochondrial delivery requirements in muscle injury treatment. Furthermore, the mitochondrial extraction process suffers from low yield and easily compromised activity, further impacting its application prospects. For example, during extraction, mitochondria may experience functional impairment due to mechanical damage or the influence of chemical reagents, reducing their therapeutic effectiveness. Summary of the Invention

[0009] The present invention aims to provide a chemical small molecule complex and method for promoting the delivery of mesenchymal stem cell mitochondria to muscle cells, in order to overcome the shortcomings of the prior art, improve the efficiency of mitochondrial delivery, and provide a new strategy for the treatment of muscle injury.

[0010] Treated mesenchymal stem cells (MSCs) significantly improved mitochondrial delivery efficiency when co-cultured with muscle cells (including skeletal muscle cells, cardiomyocytes, and smooth muscle cells) through direct contact or indirect Transwell culture (37°C, 5% CO2). Experimental data showed that compared to the untreated group, the treated group exhibited a significant increase in the number of mitochondria in muscle cells, with fluorescence microscopy revealing a marked increase in mitochondrial translocation. Simultaneously, intracellular ROS levels in muscle cells were significantly reduced, and oxygen consumption rate (OCR) results indicated a significant enhancement in mitochondrial energy metabolism.

[0011] This invention solves the problems of low mitochondrial delivery efficiency and poor stability in existing technologies by using multi-target synergistic effects and precise staged regulation, providing a new, efficient, and safe strategy for muscle damage repair.

[0012] A small chemical molecule complex that promotes the delivery of mitochondria from mesenchymal stem cells to muscle cells, comprising the following components: metformin, β-nicotinamide mononucleotide, nicotinamide, ergothionein, and pyrrolidinequinoline quinone.

[0013] The metformin, at a concentration of 1-5 mM, is used to activate the AMPK signaling pathway and promote mitochondrial biogenesis. AMPK is a key regulatory protein for cellular energy sensing; its activation can upregulate the expression of genes related to mitochondrial biosynthesis.

[0014] The β-nicotinamide mononucleotide is at a concentration of 0.1-1 mM and is used to increase NAD+ levels.

[0015] The nicotinamide concentration is 0.1-1 mM, used to assist the NAD+ cycle. It stabilizes NAD+ metabolism, prevents its degradation, and works synergistically with β-nicotinamide mononucleotide to further improve the availability of NAD+ in mitochondria.

[0016] The ergothioneine concentration is 0.1-1 mM, and it is used as a mitochondrial-targeted antioxidant to scavenge ROS.

[0017] The pyrroloquinoline quinone is at a concentration of 0.01 - 0.1 μM and is used to promote mitochondrial biogenesis.

[0018] A method for staged treatment of mesenchymal stem cells with a small chemical molecule complex to promote mitochondrial translocation to muscle cells, comprising the following steps: Step 1, first stage of mesenchymal stem cell treatment, 0-24 h: Metformin, β-nicotinamide mononucleotide, and nicotinamide were added to the culture medium to achieve a metformin concentration of 1–5 mM, a β-nicotinamide mononucleotide concentration of 0.1–1 mM, and a nicotinamide concentration of 0.1–1 mM. The main objective of this stage was to activate AMPK and increase NAD+ levels, thereby initiating mitochondrial biogenesis.

[0019] Upon activation, AMPK upregulates the expression of genes related to mitochondrial biosynthesis by phosphorylating downstream signaling molecules. Simultaneously, β-nicotinamide mononucleotide and nicotinamide synergistically increase intracellular NAD+ concentration, providing essential coenzymes for the normal functioning of the mitochondrial respiratory chain and promoting the initial recovery of mitochondrial function.

[0020] Step 2, second stage of mesenchymal stem cell treatment, 24-48 hours: Ergothioneine and pyrroloquinoline quinone were added to the culture medium to make the ergothioneine concentration 0.1-1 mM and the pyrroloquinoline quinone concentration 0.01-0.1 μM; the focus of this stage was to clear ROS and directly promote mitochondrial synthesis.

[0021] After entering the cell, ergothione selectively accumulates in the mitochondrial matrix and reacts specifically with ROS molecules, reducing them to harmless water and alcohols. This clears excess ROS in the mitochondria and reduces oxidative stress damage to the mitochondria. At the same time, pyrroloquinoline quinone, as a highly efficient redox agent, can stimulate mitochondrial DNA replication and mitochondrial protein synthesis, accelerate mitochondrial biogenesis, and further increase the number and function of mitochondria in the cell.

[0022] Step 3: Co-culture of mesenchymal stem cells and muscle cells: Two methods were employed: direct contact co-culture and indirect co-culture, such as using Transwell. Direct contact co-culture simulates direct cell-to-cell interactions in vivo, promoting direct mitochondrial delivery via the intercellular junction pathway. In indirect co-culture, mesenchymal stem cells were seeded in the upper chamber of the Transwell insert, while muscle cells were seeded in the lower chamber. The two cell layers shared nutrients and signaling molecules through the culture medium, avoiding direct cell-to-cell contact, which is beneficial for investigating the mitochondrial delivery mechanism via the paracrine pathway.

[0023] The treated mesenchymal stem cells and muscle cells (skeletal muscle cells, cardiomyocytes, and smooth muscle cells) were seeded in a co-culture system at a certain ratio. For direct contact co-culture, mesenchymal stem cells and muscle cells were seeded separately in a culture plate so that they could directly contact each other in the same culture medium. For indirect co-culture, mesenchymal stem cells were seeded in the upper chamber of the Transwell insert, and muscle cells were seeded in the lower chamber, ensuring that the culture medium between the two cell layers could flow between them. Co-culture was carried out in an incubator at 37°C and 5% CO2 for 24-48 hours. During co-culture, the culture medium was changed regularly to maintain the cell growth environment and the effective concentration of small chemical molecule complexes. At the same time, the cell growth status and mitochondrial distribution were observed using a fluorescence microscope to preliminarily determine the mitochondrial delivery efficiency.

[0024] Step 4, Mitochondrial-related detection: Mitochondria from treated mesenchymal stem cells or co-cultured cells were stained with the fluorescent probe MitoTracker Red and photographed using a fluorescence microscope to indicate changes in mitochondrial number. Mitochondria were extracted from mesenchymal stem cells and muscle cells, stained with MitoTracker Red, and their fluorescence intensity was analyzed by flow cytometry to quantitatively assess mitochondrial number. Intracellular reactive oxygen species (ROS) levels were detected using the fluorescent probe DCFH-DA to assess oxidative stress and verify the effect of improved mitochondrial function. Mitochondria function and energy metabolism levels were examined using an oxygen consumption rate (OCR) assay.

[0025] The beneficial effects of the technical solution of this invention are as follows: Significantly Improved Mitochondrial Delivery Efficiency: This invention utilizes a small chemical molecule complex to treat mesenchymal stem cells in stages, enabling their mitochondria to migrate naturally to muscle cells in large quantities. In the co-culture system, the mitochondrial membrane potential and metabolic state of the treated mesenchymal stem cells are altered, increasing the likelihood of mitochondrial release. Simultaneously, the optimized co-culture system provides favorable conditions for mitochondrial transfer, such as direct contact with cell gap junctions in co-culture and the simulation of in vivo tissue fluid circulation in indirect co-culture. This significantly improves mitochondrial delivery efficiency compared to existing technologies, overcoming the drawbacks of complex and inefficient physical methods such as electroporation and microinjection, as well as the high safety risks of viral vector-mediated methods.

[0026] Effectively enhances the stability and activity of mitochondria during delivery: Antioxidant components such as ergothionein in the small molecule chemical complex can eliminate ROS generated by mitochondria during delivery, reducing oxidative stress damage to mitochondria. Simultaneously, components such as pyrroloquinoline quinone promote mitochondrial biogenesis, resulting in higher activity of the delivered mitochondria. OCR experiments were used to detect mitochondrial function and energy metabolism levels. The results showed that after treatment with this invention, the energy metabolism function of mitochondria was significantly enhanced, ensuring that mitochondria maintain high activity after reaching muscle cells, providing effective support for muscle cell repair and regeneration, and solving the problems of poor stability and low activity of mitochondria after delivery in existing technologies.

[0027] Achieving a sustained increase in mitochondrial numbers to meet long-term repair needs: The chemical small molecule complex of this invention promotes mitochondrial biogenesis within mesenchymal stem cells through multiple pathways, including activating the AMPK signaling pathway and increasing NAD+ levels, thereby increasing the number of intracellular mitochondria. During co-culture, the continuous release and transfer of mitochondria provides ample mitochondrial support to muscle cells. Compared to existing technologies where the limited number of intracellular mitochondria in mesenchymal stem cells hinders sustained mitochondrial transfer, this invention achieves a sustained increase in mitochondrial numbers, enabling long-term and effective repair of damaged muscle cells and improving the therapeutic effect of muscle injury.

[0028] This invention can also improve mitochondrial delivery efficiency: In existing technologies, mitochondrial delivery efficiency is generally low. For example, physical methods such as electroporation and microinjection are complex and cause significant cell damage, resulting in low efficiency, while viral vector-mediated methods pose safety risks. This invention aims to provide a method for culturing mesenchymal stem cells using a chemical small molecule complex. The mitochondria from the treated mesenchymal stem cells can then be naturally transferred in large quantities to muscle cells, thereby improving mitochondrial delivery efficiency.

[0029] This invention can also improve the stability of mitochondria during delivery: In existing mitochondrial delivery technologies, the stability of mitochondria after leaving the donor cell is difficult to maintain, and they are easily affected by the extracellular environment and lose their activity. This invention provides a relatively stable microenvironment for mitochondria through the action of small chemical molecule complexes, reducing functional damage during delivery and ensuring that mitochondria can be taken up by muscle cells with high activity.

[0030] This invention can also increase the number of mitochondria to meet the requirements of continuous delivery: Due to the limited number of mitochondria within mesenchymal stem cells, existing technologies struggle to continuously supply mitochondria, impacting the long-term repair efficacy for damaged muscle cells. This invention utilizes a small chemical molecule complex to activate the mitochondrial biogenesis pathway in mesenchymal stem cells, increasing the number of intracellular mitochondria and thus providing continuous mitochondrial support to muscle cells, enhancing the long-term repair effect on damaged muscle cells. Attached Figure Description

[0031] Figure 1 The cell morphology of cardiomyocytes co-cultured with mesenchymal stem cells in Comparative Example 1 and Example 1 is shown under a bright-field microscope.

[0032] Figure 2 The image shows the mitochondrial fluorescence staining of cardiomyocytes co-cultured with mesenchymal stem cells in Comparative Example 1 and Example 1.

[0033] Figure 3 This is a diagram showing the number of mitochondria in cardiomyocytes cultured with the same amount as mesenchymal stem cells in Comparative Example 1 and Example 1.

[0034] Figure 4 This is a fluorescence staining image of mesenchymal stem cell mitochondria transferred to cardiomyocytes in Comparative Example 2.

[0035] Figure 5 This is a fluorescence staining image of mesenchymal stem cell mitochondria transferred to cardiomyocytes in Example 2.

[0036] Figure 6 The ROS expression in muscle cells is shown in Comparative Example 1 and Example 1.

[0037] Figure 7 The oxygen consumption rate of muscle cells co-cultured with mesenchymal stem cells in Comparative Example 1 and Example 1 is shown. Detailed Implementation

[0038] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0039] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0040] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments. Example 1

[0041] Preparation of small molecule chemical complexes: Based on serum-free mesenchymal stem cell culture medium, a drug combination was added. The final concentrations were: metformin 1 mM, β-nicotinamide mononucleotide 0.5 mM, nicotinamide 0.1 mM, ergothioneine 0.1 mM, and pyrroloquinoline quinone 0.01 μM. The compounds were stirred to dissolve, ensuring thorough mixing.

[0042] Mesenchymal stem cell culture: Collected umbilical cord-derived mesenchymal stem cells were cultured in GMP-grade serum-free mesenchymal stem cell medium at 1×10⁻⁶ ppm. 4 Cells were seeded at a density of 6 cells / cm² in 6-well culture plates and incubated at 37°C in a 5% CO2 incubator.

[0043] Mesenchymal stem cell treatment: The collected mesenchymal stem cells were processed at a rate of 1×10⁻⁶. 4 Cells were seeded into culture dishes or flasks at a density of 1 / cm². After the cells adhered, the prepared compound culture medium was added in two stages. In the first stage (0-24 h), metformin, β-nicotinamide mononucleotide, and nicotinamide were added. In the second stage (24-48 h), ergothionein and pyrroloquinoline quinone were added.

[0044] Co-culture of mesenchymal stem cells and cardiomyocytes: An indirect co-culture method was used, in which treated mesenchymal stem cells were cultured in serum-free medium at 2×10⁻⁶ mg / L. 4 Cells were precisely seeded at a density of 10 cells / cm² in the upper chamber of a Transwell culture plate. The plate was gently shaken to ensure even distribution of cells, and then incubated for 10 minutes to promote uniform cell dispersion. Simultaneously, cardiomyocytes in the logarithmic growth phase were cultured in a specialized medium at 1×10⁻⁶... 4 cells / cm 2Cells were evenly seeded at a uniform density in the lower chamber of a 6-well culture plate to ensure uniform cell distribution. The plate was then incubated at 37°C in a 5% CO2 incubator for 48 hours. During incubation, cell morphology changes could be observed using an inverted microscope, and cell growth status, adhesion, and signs of contamination could be recorded to ensure the stability of the co-culture system and the reliability of the experimental results. The cultured cells can then be used for subsequent assays.

[0045] Mitochondrial-related assays: Mitochondria of co-cultured cardiomyocytes were stained with the fluorescent probe MitoTracker Red and photographed using a fluorescence microscope to indicate changes in mitochondrial number. Mitochondria were extracted from co-cultured cardiomyocytes and stained again with MitoTracker Red. Fluorescence intensity was analyzed by flow cytometry to quantitatively assess mitochondrial number. Intracellular reactive oxygen species (ROS) levels were detected using the fluorescent probe DCFH-DA to assess oxidative stress and verify the effect of improved mitochondrial function. Mitochondrial function and energy metabolism levels were examined using an oxygen consumption rate (OCR) assay. Example 2

[0046] Preparation of small molecule chemical complexes: Based on serum-free mesenchymal stem cell culture medium, a drug combination was added. The final concentrations were: metformin 2 mM, β-nicotinamide mononucleotide 0.8 mM, nicotinamide 0.3 mM, ergothioneine 0.8 mM, and pyrroloquinoline quinone 0.05 μM. The compounds were stirred to dissolve, ensuring homogeneous mixing.

[0047] Mesenchymal stem cell culture: Collected bone marrow-derived mesenchymal stem cells were cultured in GMP-grade serum-free mesenchymal stem cell medium at 2×10⁻⁶ ppm. 4 Cells were seeded at a density of 6 cells / cm² in 6-well culture plates and incubated at 37°C in a 5% CO2 incubator.

[0048] Mesenchymal stem cell treatment: The collected mesenchymal stem cells were processed at a rate of 2 × 10⁻⁶. 4 Cells were seeded into culture dishes or flasks at a density of 1 / cm². After the cells adhered, the prepared compound culture medium was added in two stages. In the first stage (0-24 h), metformin, β-nicotinamide mononucleotide, and nicotinamide were added. In the second stage (24-48 h), ergothionein and pyrroloquinoline quinone were added.

[0049] Mesenchymal stem cell co-culture with cardiomyocytes: A direct co-culture method was used, in which treated mesenchymal stem cells and cultured cardiomyocytes were cultured in serum-free medium at 1×10⁻⁶. 4Cells were seeded at a density of 1:1 in 6-well culture plates, and the plates were gently shaken to ensure even cell distribution. The plates were then incubated at 37°C in a 5% CO2 incubator for 48 hours. During incubation, cell morphology changes were observed using an inverted microscope, and cell growth status, adhesion, and signs of contamination were recorded to ensure the stability of the co-culture system and the reliability of the experimental results. The cultured cells can be used for subsequent assays.

[0050] Mitochondrial-related assays: Mitochondria of co-cultured cardiomyocytes were stained with the fluorescent probe MitoTracker Red and photographed using a fluorescence microscope to indicate changes in mitochondrial number. Mitochondria were extracted from co-cultured cardiomyocytes and stained again with MitoTracker Red. Fluorescence intensity was analyzed by flow cytometry to quantitatively assess mitochondrial number. Intracellular reactive oxygen species (ROS) levels were detected using the fluorescent probe DCFH-DA to assess oxidative stress and verify the effect of improved mitochondrial function. Mitochondrial function and energy metabolism levels were examined using an oxygen consumption rate (OCR) assay. Example 3

[0051] Preparation of small molecule chemical complexes: Based on serum-free mesenchymal stem cell culture medium, a drug combination was added. The final concentrations were: metformin 4 mM, β-nicotinamide mononucleotide 0.3 mM, nicotinamide 0.2 mM, ergothioneine 0.4 mM, and pyrroloquinoline quinone 0.2 μM. The compounds were stirred to dissolve, ensuring homogeneous mixing.

[0052] Mesenchymal stem cell culture: Collected adipose-derived mesenchymal stem cells were cultured in GMP-grade serum-free mesenchymal stem cell medium at 1×10⁻⁶ ppm. 4 Cells were seeded at a density of 6 cells / cm² in 6-well culture plates and incubated at 37°C in a 5% CO2 incubator.

[0053] Mesenchymal stem cell treatment: The collected mesenchymal stem cells were processed at a rate of 2 × 10⁻⁶. 4 Cells were seeded into culture dishes or flasks at a density of 1 / cm². After the cells adhered, the prepared compound culture medium was added in two stages. In the first stage (0-24 h), metformin, β-nicotinamide mononucleotide, and nicotinamide were added. In the second stage (24-48 h), ergothionein and pyrroloquinoline quinone were added.

[0054] Co-culture of mesenchymal stem cells and skeletal muscle cells: An indirect co-culture method was used, in which treated mesenchymal stem cells were cultured in serum-free medium at 1×10⁻⁶ mg / L. 4Cells were precisely seeded at a density of 10 cells / cm² in the upper chamber of a Transwell culture plate. The plate was gently shaken to ensure even distribution of cells, and then incubated for 5 minutes to promote uniform cell dispersion. Simultaneously, skeletal muscle cells in the logarithmic growth phase were cultured in a specialized medium at 1×10⁻⁶. 4 cells / cm 2 Cells were evenly seeded at a uniform density in the lower chamber of a 6-well culture plate to ensure uniform cell distribution. The plate was then incubated at 37°C in a 5% CO2 incubator for 48 hours. During incubation, cell morphology changes could be observed using an inverted microscope, and cell growth status, adhesion, and signs of contamination could be recorded to ensure the stability of the co-culture system and the reliability of the experimental results. The cultured cells can then be used for subsequent assays.

[0055] Mitochondrial-related assays: Mitochondria of co-cultured cardiomyocytes were stained with the fluorescent probe MitoTracker Red and photographed using a fluorescence microscope to indicate changes in mitochondrial number. Mitochondria were extracted from co-cultured cardiomyocytes and stained again with MitoTracker Red. Fluorescence intensity was analyzed by flow cytometry to quantitatively assess mitochondrial number. Intracellular reactive oxygen species (ROS) levels were detected using the fluorescent probe DCFH-DA to assess oxidative stress and verify the effect of improved mitochondrial function. Mitochondrial function and energy metabolism levels were examined using an oxygen consumption rate (OCR) assay.

[0056] Comparative Example 1: The comparative example of this application provides a mesenchymal stem cell culture medium without the addition of any additional compounds, which is used for the normal culture of mesenchymal stem cells.

[0057] Mesenchymal stem cell culture: Collected umbilical cord-derived mesenchymal stem cells were cultured in GMP-grade serum-free mesenchymal stem cell medium at 1×10⁻⁶ ppm. 4 Cells were seeded at a density of 6 cells / cm² in 6-well culture plates and incubated at 37°C in a 5% CO2 incubator.

[0058] Mesenchymal stem cells co-cultured with cardiomyocytes: An indirect co-culture method was used, in which the conventionally cultured mesenchymal stem cells were cultured in serum-free medium at 2×10⁻⁶ mg / L. 4 Cells were precisely seeded at a density of 10 cells / cm² in the upper chamber of a Transwell culture plate. The plate was gently shaken to ensure even distribution of cells, and then incubated for 10 minutes to promote uniform cell dispersion. Simultaneously, cardiomyocytes in the logarithmic growth phase were cultured in a specialized medium at 1×10⁻⁶... 4 cells / cm 2Cells were evenly seeded at a uniform density in the lower chamber of a 6-well culture plate to ensure uniform cell distribution. The plate was then incubated at 37°C in a 5% CO2 incubator for 48 hours. During incubation, cell morphology changes could be observed using an inverted microscope, and cell growth status, adhesion, and signs of contamination could be recorded to ensure the stability of the co-culture system and the reliability of the experimental results. The cultured cells can then be used for subsequent assays.

[0059] Mitochondrial-related assays: Mitochondria of co-cultured cardiomyocytes were stained with the fluorescent probe MitoTracker Red and photographed using a fluorescence microscope to indicate changes in mitochondrial number. Mitochondria were extracted from co-cultured cardiomyocytes and stained again with MitoTracker Red. Fluorescence intensity was analyzed by flow cytometry to quantitatively assess mitochondrial number. Intracellular reactive oxygen species (ROS) levels were detected using the fluorescent probe DCFH-DA to assess oxidative stress and verify the effect of improved mitochondrial function. Mitochondrial function and energy metabolism levels were examined using an oxygen consumption rate (OCR) assay.

[0060] Comparative Example 2: The comparative example of this application provides a mesenchymal stem cell culture medium without the addition of any additional compounds, which is used for the normal culture of mesenchymal stem cells.

[0061] Mesenchymal stem cell culture: Collected bone marrow-derived mesenchymal stem cells were cultured in GMP-grade serum-free mesenchymal stem cell medium at 2×10⁻⁶ ppm. 4 Cells were seeded at a density of 6 cells / cm² in 6-well culture plates and incubated at 37°C in a 5% CO2 incubator.

[0062] Mesenchymal stem cell treatment: The collected mesenchymal stem cells were processed at a rate of 2 × 10⁻⁶. 4 Cells were seeded into culture dishes or flasks at a density of 1 / cm². After the cells adhered, the prepared compound culture medium was added in two stages. In the first stage (0-24 h), metformin, β-nicotinamide mononucleotide, and nicotinamide were added. In the second stage (24-48 h), ergothionein and pyrroloquinoline quinone were added.

[0063] Mesenchymal stem cell co-culture with cardiomyocytes: A direct co-culture method was used, in which treated mesenchymal stem cells and cultured cardiomyocytes were cultured in serum-free medium at 1×10⁻⁶. 4Cells were seeded at a density of 1:1 in 6-well culture plates, and the plates were gently shaken to ensure even cell distribution. The plates were then incubated at 37°C in a 5% CO2 incubator for 48 hours. During incubation, cell morphology changes were observed using an inverted microscope, and cell growth status, adhesion, and signs of contamination were recorded to ensure the stability of the co-culture system and the reliability of the experimental results. The cultured cells can be used for subsequent assays.

[0064] Mitochondrial-related assays: Mitochondria of co-cultured cardiomyocytes were stained with the fluorescent probe MitoTracker Red and photographed using a fluorescence microscope to indicate changes in mitochondrial number. Mitochondria were extracted from co-cultured cardiomyocytes and stained again with MitoTracker Red. Fluorescence intensity was analyzed by flow cytometry to quantitatively assess mitochondrial number. Intracellular reactive oxygen species (ROS) levels were detected using the fluorescent probe DCFH-DA to assess oxidative stress and verify the effect of improved mitochondrial function. Mitochondrial function and energy metabolism levels were examined using an oxygen consumption rate (OCR) assay.

[0065] Cardiomyocytes co-cultured with mesenchymal stem cells in Comparative Example 1 and Example 1 (defined as CM-MSC and CM-CiMSC, respectively) were photographed under a bright-field microscope, and the appendages could be observed. Figure 1 The mesenchymal stem cells cultured in Comparative Example 1 and Example 1 showed no significant difference in cell morphology, indicating that the mesenchymal stem cells treated with the combined compounds in this example had no significant effect on the morphology of the co-cultured cardiomyocytes. MitoTracker Red staining was performed on the cardiomyocytes co-cultured with the mesenchymal stem cells from Comparative Example 1 and Example 1, and the images were taken under a fluorescence microscope, revealing the presence of... Figure 2 The expression of mitochondria in cardiomyocytes indirectly co-cultured with mesenchymal stem cells in Comparative Example 1 and Example 1 is shown. The number of mitochondria in Example 1 is significantly greater than that in Comparative Example 1, indicating that the mesenchymal stem cells treated by the method of this invention can indirectly transfer more mitochondria to cardiomyocytes. Mitochondria were extracted from cardiomyocytes co-cultured with the same number of cells in Comparative Example 1 and Example 1, stained with MitoTracker Red, and the number of mitochondria was quantified by analyzing the fluorescence intensity using flow cytometry, as shown in the attached figure. Figure 3 As shown, the number of mitochondria in Example 1 was significantly greater than that in Comparative Example 1. *** P<0.001), indicating that the method of the present invention can significantly promote the transfer of mitochondria from mesenchymal stem cells to cardiomyocytes. To further visualize the mitochondrial transfer, the mesenchymal stem cells obtained in Comparative Example 2 and Example 2 were used to label the mitochondria of cells with dsRed (dsRed-MSCs), and human cardiomyocytes AC16 were labeled with DiO (DiO-AC16). The mesenchymal stem cells and cardiomyocytes were co-cultured, and the transfer of mitochondria was observed under a fluorescence microscope. Figure 4 In Comparative Example 2, a small amount of mitochondria from mesenchymal stem cells migrated to cardiomyocytes, while the appendages... Figure 5 In Example 2, the mesenchymal stem cells showed increased mitochondrial translocation to cardiomyocytes (as indicated by the white arrows), demonstrating that the mesenchymal stem cells treated by the method of this invention can also transfer more mitochondria to cardiomyocytes through direct contact. To further investigate the effect of mesenchymal stem cells treated by this method on cardiomyocyte ROS, a dexamethasone (DEX)-induced cardiomyocyte oxidative stress model was constructed, as shown in the attached figure. Figure 6 As shown, compared with the blank control group (Control), the ROS expression of cardiomyocytes in the DEX group was significantly increased. The ROS expression in cardiomyocytes co-cultured in Example 1 was significantly lower than that in the DEX group, while the ROS expression in cardiomyocytes co-cultured in Example 1 was the lowest. This suggests that the mesenchymal stem cells with increased mitochondrial numbers obtained by this method can significantly inhibit ROS expression in cardiomyocytes. Mitochondrial function and energy metabolism levels were examined using an oxygen consumption rate (OCR) experiment, as shown in the attached figure. Figure 7 As shown, the basal oxygen consumption rate, especially the maximum respiratory rate, of the cardiomyocytes co-cultured in Example 1 was significantly higher than that of the cardiomyocytes co-cultured in Comparative Example 1, indicating that the mesenchymal stem cells obtained by this method can further improve the mitochondrial function and energy metabolism level of cardiomyocytes.

[0066] In summary, the innovativeness of this invention can be summarized as follows: Multi-target synergistic mechanism: The small chemical molecule complex achieves comprehensive regulation of mitochondrial function in mesenchymal stem cells through multiple pathways, including simultaneous activation of the AMPK signaling pathway, enhancement of NAD+ levels, ROS scavenging, and promotion of mitochondrial biogenesis. This multi-target synergistic mechanism is key to increasing the quantity and quality of mitochondria and enhancing their delivery capacity. For example, AMPK activation not only promotes mitochondrial biosynthesis but also regulates cellular energy metabolism, placing the cell in a state conducive to mitochondrial synthesis and release. Simultaneously, increased NAD+ levels provide the necessary conditions for the normal operation of the mitochondrial respiratory chain, while ROS scavenging protects newly formed mitochondria from oxidative damage, ensuring their functional integrity. These effects synergistically form a positive feedback loop, promoting the abundant generation and efficient delivery of mitochondria.

[0067] A phased treatment strategy: Adding small molecule chemical complexes in stages effectively avoids drug interactions and ensures that each key regulatory step functions optimally. In the first stage, metformin, NMN, and nicotinamide synergistically activate AMPK and increase NAD+ levels, laying the foundation for mitochondrial biogenesis. Adding antioxidants at this stage might interfere with AMPK activation-related redox signaling, potentially reducing mitochondrial biogenesis efficiency. In the second stage, after the number of mitochondria begins to increase, the addition of ergothioneine and pyrrolidinequinoline quinone more precisely scavenges ROS and directly promotes mitochondrial synthesis, maximizing both mitochondrial quality and quantity. This phased treatment strategy represents a refined manipulation of mesenchymal stem cell mitochondrial function and is a crucial technological step in achieving efficient mitochondrial delivery.

[0068] Optimized formulation of small molecule chemical complexes: This invention optimized the concentration range and formulation of each component in the small molecule chemical complex through extensive experiments. Metformin (1-5 mM), β-nicotinamide mononucleotide (0.1-1 mM), nicotinamide (0.1-1 mM), ergothioneine (0.1-1 mM), and pyrroloquinoline quinone (0.01-0.1 μM) achieved optimal synergistic effects within this formulation range, significantly improving the efficiency of mitochondrial biogeneration and delivery in mesenchymal stem cells. This optimized formulation is a core element for achieving efficient mitochondrial delivery and functional recovery.

[0069] Composition and synergistic mechanism of the small chemical molecule complex: The core innovation of this invention lies in the combination of specific small chemical molecules (metformin, β-nicotinamide mononucleotide, nicotinamide, ergothioneine, and pyrroloquinoline quinone) and their synergistic mechanism. These components, formulated in specific concentration ranges, synergistically enhance mitochondrial biogenesis and function in mesenchymal stem cells through multiple pathways, including activating the AMPK signaling pathway, increasing NAD+ levels, scavenging ROS, and promoting mitochondrial biogenesis.

[0070] The phased treatment time division and drug addition sequence: The phased treatment strategy, including the time division of each stage and the drug addition sequence, is an important technical means for improving mitochondrial delivery efficiency in this invention. Metformin, NMN, and nicotinamide are added in the first stage (0-24 h), and ergothioneine and pyrrolidinequinoline quinone are added in the second stage (24-48 h). This specific timing arrangement ensures the orderly progress of mitochondrial biogenesis, avoids interference between drugs, and maximizes the effect of each component.

[0071] Optimized formulation of small molecule chemical complexes: This invention experimentally determined the optimal concentration range and formulation of each component in the small molecule chemical complexes. A specific formulation of metformin (1-5 mM), β-nicotinamide mononucleotide (0.1-1 mM), nicotinamide (0.1-1 mM), ergothioneine (0.1-1 mM), and pyrroloquinoline quinone (0.01-0.1 μM) achieved optimal synergistic effects, significantly improving the efficiency of mitochondrial biogeneration and delivery in mesenchymal stem cells. This optimized formulation is a crucial component of the technical solution of this invention.

[0072] Definitions: Mesenchymal stem cells: a type of adult stem cell with self-renewal and multi-directional differentiation potential.

[0073] Muscle cells: including skeletal muscle cells, cardiac muscle cells, and smooth muscle cells, have contractile function and are the basic units that make up muscle tissue.

[0074] Mitochondria are important organelles within cells, often referred to as the cell's "energy factories." The main process of aerobic respiration takes place in mitochondria, producing the energy needed for cellular life activities. Mitochondria also participate in various physiological processes such as apoptosis and signal transduction.

Claims

1. A small chemical molecule complex that promotes the delivery of mitochondria from mesenchymal stem cells to muscle cells, characterized in that... It consists of the following components: metformin, β-nicotinamide mononucleotide, nicotinamide, ergothionein, and pyrroloquinoline quinone.

2. The small molecule complex according to claim 1, characterized in that... The metformin concentration is 1-5 mM, used to activate the AMPK signaling pathway and promote mitochondrial biogenesis.

3. The small molecule complex according to claim 1, characterized in that... The β-nicotinamide mononucleotide is at a concentration of 0.1-1 mM and is used to increase NAD+ levels.

4. The small molecule complex according to claim 1, characterized in that... The nicotinamide concentration is 0.1-1 mM, used to assist the NAD+ cycle. It can stabilize NAD+ metabolism, prevent its degradation, and work synergistically with β-nicotinamide mononucleotide to further improve the availability of NAD+ in mitochondria.

5. The small molecule complex according to claim 1, characterized in that... The ergothioneine concentration is 0.1-1 mM, and it is used as a mitochondrial-targeted antioxidant to scavenge ROS.

6. The small molecule complex according to claim 1, characterized in that... The pyrroloquinoline quinone is at a concentration of 0.01-0.1 μM and is used to promote mitochondrial biogenesis.

7. A method for staged treatment of mesenchymal stem cells using a small chemical molecule complex, comprising the following steps: Step 1, First Stage, 0-24h: Metformin, β-nicotinamide mononucleotide, and nicotinamide were added to the culture medium to achieve a metformin concentration of 1-5 mM, a β-nicotinamide mononucleotide concentration of 0.1-1 mM, and a nicotinamide concentration of 0.1-1 mM. The main purpose of this stage was to activate AMPK and increase NAD+ levels, thereby initiating mitochondrial biogenesis. After AMPK activation, it upregulates the expression of genes related to mitochondrial biosynthesis by phosphorylating downstream signaling molecules; at the same time, β-nicotinamide mononucleotide and nicotinamide work synergistically to increase intracellular NAD+ concentration, providing necessary coenzymes for the normal operation of the mitochondrial respiratory chain and promoting the initial recovery of mitochondrial function. Step 2, Second Stage, 24-48 hours: Ergothioneine and pyrroloquinoline quinone were added to the culture medium to make the ergothioneine concentration 0.1-1 mM and the pyrroloquinoline quinone concentration 0.01-0.1 μM; the focus of this stage is to clear ROS and directly promote mitochondrial synthesis. After entering the cell, ergothione selectively accumulates in the mitochondrial matrix and reacts specifically with ROS molecules, reducing them to harmless water and alcohols. This clears excess ROS in the mitochondria and reduces oxidative stress damage to the mitochondria. Meanwhile, pyrroloquinoline quinone, as a highly efficient redox agent, can stimulate mitochondrial DNA replication and mitochondrial protein synthesis, accelerate mitochondrial biogenesis, and further increase the number and function of mitochondria in the cell. Step 3: Co-culture of mesenchymal stem cells and muscle cells: Two methods were employed: direct contact co-culture and indirect co-culture, such as using Transwell. Direct contact co-culture can simulate direct interactions between cells in vivo, promoting direct mitochondrial delivery through the intercellular junction pathway. In indirect co-culture, mesenchymal stem cells were seeded in the upper chamber of the Transwell insert, and muscle cells were seeded in the lower chamber. The two cell layers shared nutrients and signaling molecules through the culture medium, avoiding direct cell-cell contact, which is beneficial for investigating the mitochondrial delivery mechanism through the paracrine pathway. The processed mesenchymal stem cells and muscle cells (skeletal muscle cells, cardiomyocytes, and smooth muscle cells) were seeded in a co-culture system at a certain ratio. For direct contact co-culture, mesenchymal stem cells and muscle cells are seeded separately in a culture plate so that they can come into direct contact in the same culture medium; For indirect co-culture, mesenchymal stem cells were seeded in the upper chamber of the Transwell insert, and muscle cells were seeded in the lower chamber, ensuring that the culture medium could flow between the two cell layers. Co-culture was carried out in an incubator at 37°C and 5% CO2 for 24-48 hours. During co-culture, the culture medium was changed regularly to maintain the cell growth environment and the effective concentration of small chemical molecule complexes. At the same time, the cell growth status and mitochondrial distribution were observed using a fluorescence microscope to preliminarily determine the mitochondrial delivery efficiency. Step 4, Mitochondrial-related detection: Mitochondria from treated mesenchymal stem cells or co-cultured cells were stained with the fluorescent probe MitoTracker Red and photographed using a fluorescence microscope to indicate changes in mitochondrial number. Mitochondria were extracted from mesenchymal stem cells and muscle cells, stained with MitoTracker Red again, and their fluorescence intensity was analyzed by flow cytometry to quantitatively assess mitochondrial number. Intracellular reactive oxygen species (ROS) levels were detected using the fluorescent probe DCFH-DA to assess oxidative stress and verify the effect of improved mitochondrial function. Mitochondria function and energy metabolism levels were examined using an oxygen consumption rate (OCR) assay.

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