Use of hsa-let-7f-5p in the preparation of a drug for treating muscle steatosis

Abstract

The present application relates to the microRNA drug technical field, specifically to the application of hsa-let-7f-5p in the preparation of a drug for treating muscle steatosis, and discloses that hsa-let-7f-5p can inhibit muscle fat infiltration by inhibiting fibroblast / adipogenic progenitor cell adipogenic differentiation, and hsa-let-7f-5p can be used as a microRNA drug in the treatment of diseases such as muscle fat infiltration and muscle steatosis, and diseases related to fat infiltration, for example, muscle atrophy, muscle injury, obesity, diabetes, etc. The relevant nucleic acid fragments are wrapped in liposomes, so that the drug stability and drug efficiency can be improved. The technical scheme can solve the technical problem that there is no drug capable of effectively inhibiting muscle steatosis and treating muscle injury in the prior art, and has an ideal application and promotion prospect in the fields of sports medicine and rehabilitation treatment.

Description

Technical Field

[0001] This invention relates to the field of microRNA drug technology, specifically to the application of hsa-let-7f-5p in the preparation of drugs for treating myofascitis. Background Technology

[0002] Fatty infiltration refers to the presence of fat cells in the interstitium of tissues or organs that normally contain little or no fat cells. This is particularly common in tissues such as the heart, pancreas, and skeletal muscle. It is noteworthy that fatty infiltration in skeletal muscle is an underappreciated health risk factor. It not only leads to fat deposition within muscles, eventually causing muscle fatty degeneration (a condition characterized by abnormal fat accumulation in muscle tissue), but it can also be caused by genetic factors, metabolic disorders, malnutrition, lack of exercise, or certain diseases. Furthermore, it is closely related to the development and progression of various diseases.

[0003] Specifically, fat deposition can be divided into two forms: intramuscular and intramuscular. The former refers to the deposition of fat cells in the available space between skeletal muscles; the latter encompasses all fat cells embedded between active skeletal muscle fibers. Muscle steatosis is closely related to skeletal muscle diseases. Regarding muscle atrophy, as the disease progresses, the number and volume of muscle fibers decrease, while extensive fat cell infiltration and fibrosis occur. This not only hinders muscle regeneration but also accelerates the process of muscle atrophy. When skeletal muscle is injured, muscle fibers are destroyed or reduced in number, accompanied by an inflammatory response. During this repair process, the infiltration of a large number of fat cells and the occurrence of fibrosis further impede muscle regeneration, thus affecting the recovery effect. Therefore, inhibiting skeletal muscle steatosis is of great significance for promoting recovery after muscle injury.

[0004] Furthermore, studies have shown that intramuscular fat deposition (whether intramuscular or intermuscular) is closely associated with conditions such as obesity, diabetes, and metabolic syndrome (Uezumi A, Fukada S, Yamamoto N, Takeda S, Tsuchida K. Mesenchymal progenitors distinct from satellite cells contribute to ectopicfat cell formation in skeletal muscle. Nat Cell Biol. 2010; 12:143-52.). Intramuscular adipose tissue, as one of the early changes in muscle structure, appears before strength and functional abnormalities manifest, and it is a precursor to metabolic changes associated with obesity and diabetes. The combined effect of fat accumulation and myofibril degeneration in muscles significantly reduces muscle mass and is positively correlated with morbidity and mortality (Zamboni, M., Gattazzo, S. & Rossi, AP Myosteatosis: a relevant, yet poorly explored element of sarcopenia. Eur Geriatr Med 10, 5–6 (2019); Hausman GJ, Basu U, Du M, Fernyhough-Culver M, Dodson MV. Intermuscular and intramuscular adipose tissues: Bad vs. good adipose tissues. Adipocyte. 2014 Dec 10; 3(4): 242-55.).

[0005] Despite the growing importance of skeletal myofibril degeneration, effective drug treatments for this condition remain lacking in clinical practice. Particularly in treating muscle injury or atrophy, there are no specific drugs that can promote muscle repair by inhibiting muscle fat deposition. This area requires further research and exploration to find more effective treatments. Summary of the Invention

[0006] The present invention aims to provide the application of hsa-let-7f-5p in the preparation of a drug for treating myofascial degeneration, in order to solve the technical problem of the lack of drugs in the prior art that can effectively inhibit myofascial degeneration and treat muscle damage.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The application of hsa-let-7f-5p in the preparation of drugs for treating myofascitis, the sequence of which is shown in SEQ ID NO.1. RNA.

[0009] Furthermore, hsa-let-7f-5p is generated from double-stranded RNA; the sense strand of the double-stranded RNA is shown in SEQ ID NO.2, and the antisense strand is shown in SEQ ID NO.3.

[0010] Furthermore, double-stranded RNA or hsa-let-7f-5p is an inhibitor of adipogenic differentiation from fibroblasts / adipogenic progenitor cells.

[0011] Furthermore, double-stranded RNA or hsa-let-7f-5p is an inhibitor of Tgfbr3 gene expression.

[0012] Furthermore, muscle fat infiltration is caused by muscle damage.

[0013] This technical solution also provides the application of double-stranded RNA in the preparation of drugs for treating muscle injury, wherein the sense strand is shown in SEQ ID NO.2 and the antisense strand is shown in SEQ ID NO.3; the double-stranded RNA is used to form the sequence hsa-let-7f-5p as shown in SEQ ID NO.1.

[0014] Furthermore, double-stranded RNA or hsa-let-7f-5p is used to promote regeneration and repair after muscle injury, and to inhibit fat deposition after muscle injury.

[0015] Furthermore, the hsa-let-7f-5p is enriched in exosomes derived from platelet-rich plasma.

[0016] This technical solution also provides a liposome vesicle encapsulating double-stranded RNA.

[0017] Furthermore, the raw materials for liposome vesicles include SPC, DOTAP, cholesterol, and DSPE-PEG-2k;

[0018] Liposomes encapsulating double-stranded RNA were prepared by the following method: SPC, DOTAP, cholesterol and DSPE-PEG-2k were dissolved in anhydrous ethanol to form a lipid mixture; citrate buffer containing dissolved double-stranded RNA and ethanol was added to the lipid mixture; and after sonication, extrusion using a liposome extruder and dialysis, liposomes encapsulating double-stranded RNA were obtained.

[0019] The principle and beneficial effects of this technical solution are as follows:

[0020] Through in-depth research on the role of platelet-rich plasma (PRP) in inhibiting fatty infiltration and adipogenesis in muscle tissue, the inventors discovered that PRP-derived exosomes have significant effects. The inventors first removed DNA, RNA, or protein from PRP exosomes and then intervened in vitro on adipogenic fibroblast / adipogenic progenitor cells. Experimental results showed that after RNA removal, the inhibitory effect of PRP-derived exosomes on the adipogenic differentiation ability of fibroblast / adipogenic progenitor cells was significantly weakened. This indicates that RNA is a key component of PRP-derived exosomes influencing the adipogenic differentiation of these progenitor cells. To identify the specific active components, the inventors used high-throughput sequencing technology to comprehensively analyze the miRNA composition of exosomes. The study found that hsa-let-7f-5p was highly enriched in exosomes, suggesting that this miRNA may be one of the key factors playing a crucial role.

[0021] Based on the above findings, the inventors synthesized double-stranded RNA mimics (mimics) matching the mature sequence of hsa-let-7f-5p, used to form hsa-let-7f-5p in cells and tissues, and specifically studied their role in inhibiting adipogenic differentiation of fibroblasts / adipogenic progenitor cells. The results showed that hsa-let-7f-5p mimics significantly inhibited the adipogenic differentiation ability of fibroblasts / adipogenic progenitor cells, demonstrating the important role of this miRNA in this process. In addition, the study found that the target of hsa-let-7f-5p is the Tgfbr3 gene.

[0022] In a muscle injury model established using glycerol treatment, the inventors further verified the function of hsa-let-7f-5p. The results showed that hsa-let-7f-5p effectively prevented fatty infiltration of muscle tissue caused by muscle injury, thereby promoting the muscle repair process. Therefore, hsa-let-7f-5p can not only be used alone to inhibit adipogenic differentiation of fibroblasts / adipogenic progenitors, but its application in the treatment of muscle injuries with symptoms of fatty infiltration can also achieve relatively ideal results.

[0023] To improve the stability of RNA drugs, this technical approach also explored the fabrication of liposomes encapsulating hsa-let-7f-5p. The inherent instability of RNA molecules and the challenges they face during in vivo delivery (such as enzymatic degradation, immunogenicity, and poor targeting specificity) limit their widespread application. Liposomes, as carriers of RNA drugs, can effectively overcome these problems. Liposomes can form a physical barrier, protecting RNA from degradation by nucleases in both in vivo and in vitro environments. Optimizing the composition of liposomes can further enhance the chemical stability of RNA drugs. Liposomes also exhibit better compatibility with receptor cell membranes, promoting cellular uptake of RNA drugs.

[0024] In summary, this study reveals the importance of hsa-let-7f-5p, abundant in PRP-derived exosomes, in inhibiting adipogenic differentiation of fibroblasts / adipogenic progenitors and promoting the repair of muscle injuries associated with fat infiltration. These findings not only deepen our understanding of exosome-mediated muscle repair mechanisms but also provide a theoretical basis for developing new therapeutic strategies. In particular, hsa-let-7f-5p shows promise for the prevention and treatment of muscle injuries and related fat infiltration problems. hsa-let-7f-5p helps maintain the normal structure and function of muscle tissue and prevents inappropriate fat infiltration. In a muscle injury and fat infiltration model established by glycerol treatment, hsa-let-7f-5p effectively prevented fat infiltration in muscle tissue, which is of great significance for the prevention and treatment of pathological changes caused by muscle injury, especially in the fields of sports medicine, rehabilitation therapy, and age-related muscle degeneration.

[0025] In exploring the role of platelet-rich plasma (PRP) in inhibiting muscle adipogenesis and fat accumulation, researchers discovered that PRP-derived exosomes exhibited significant effects. To identify their active components, researchers first removed DNA, RNA, or proteins from PRP exosomes and conducted in vitro intervention experiments on fibroblast / adipogenic progenitor cells. The results showed that removing RNA significantly reduced the inhibitory effect of PRP-derived exosomes on adipogenic differentiation, indicating that RNA is a key component.

[0026] To further identify the specific active ingredients, researchers used high-throughput sequencing to analyze the miRNA composition in exosomes, finding that hsa-let-7f-5p was highly enriched in exosomes, suggesting it may be one of the key factors playing a major role. Based on this finding, researchers synthesized double-stranded RNA mimics (mimics) matching the mature sequence of hsa-let-7f-5p, used to form hsa-let-7f-5p in cells and tissues, and investigated their effects on inhibiting adipogenic differentiation. Experimental results showed that hsa-let-7f-5p mimics significantly reduced the likelihood of adipogenic differentiation, demonstrating the important role this miRNA plays in this process. Furthermore, the study identified the Tgfbr3 gene as a target of hsa-let-7f-5p.

[0027] In a glycerol-induced muscle injury model, researchers validated the function of hsa-let-7f-5p, finding that it effectively prevented fatty infiltration caused by muscle injury and promoted muscle tissue repair. This indicates that hsa-let-7f-5p not only has the potential to inhibit adipogenic differentiation alone, but also shows promise in treating muscle injuries accompanied by fatty infiltration.

[0028] Considering the challenges encountered during in vivo delivery of RNA drugs, such as enzymatic degradation, immunogenicity, and poor targeting specificity, researchers attempted to encapsulate hsa-let-7f-5p in liposome vesicles to improve its stability. Liposomes, as carriers, not only form a physical barrier to protect RNA from nuclease damage but also enhance chemical stability and promote cellular uptake by optimizing their composition.

[0029] In summary, this study reveals the importance of hsa-let-7f-5p in PRP-derived exosomes in inhibiting adipogenic differentiation and promoting muscle damage repair. These findings not only enhance our understanding of exosome-mediated muscle repair mechanisms but also provide a theoretical basis for developing new treatment methods. In particular, hsa-let-7f-5p shows great promise in sports medicine, rehabilitation therapy, and age-related muscle degeneration, helping to prevent and treat fatty infiltration caused by muscle damage and maintain the normal structure and function of muscle tissue. Attached Figure Description

[0030] Figure 1 This is the result of an in vitro study on the inhibition of adipogenic differentiation of fibroblasts / adipogenic progenitor cells by hsa-let-7f-5p in Example 1 of the present invention.

[0031] Figure 2 This invention relates to the effect of hsa-let-7f-5p on adipogenic differentiation under Tgfbr3 overexpression in Example 2 of this invention.

[0032] Figure 3 This is the experimental result of hsa-let-7f-5p inhibiting muscle fat formation after muscle damage in vivo, as described in Example 3 of this invention.

[0033] Figure 4 The results of exosome morphology and biomarker identification in Example 4 of this invention, as well as the effect of PRP-derived exosomes on the adipogenic ability of adipogenic fibroblasts / adipogenic progenitor cells detected by Oil Red staining, are presented.

[0034] Figure 5 The Oil Red staining assay in Example 4 of this invention was used to detect the effect of removing DNA, RNA, or protein from PRP exosomes on the adipogenic ability of fibroblasts / adipogenic progenitor cells undergoing adipogenic differentiation.

[0035] Figure 6 The results of high-throughput sequencing of miRNAs in Example 4 of this invention are shown (the number and percentage of short sequence reads of different miRNAs in PRP exosome samples from young and elderly individuals).

[0036] Figure 7Microscopic images of liposomes encapsulating hsa-let-7f-5p and experimental results of their therapeutic effects in Example 5 of this invention. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; and the materials, reagents, etc. used are all commercially available.

[0038] Example 1: In vitro study of hsa-let-7f-5p inhibiting adipogenic differentiation of fibroblasts / adipogenic progenitor cells

[0039] The mature sequence of hsa-let-7f-5p is: 5'-UGAGGUAGUAGAUUGUAUAGUU-3' (SEQ ID NO.1).

[0040] Double-stranded RNA (hsa-let-7f-5p) mimics, consisting of a sense strand and an antisense strand, were synthesized based on the mature sequence of hsa-let-7f-5p.

[0041] Chain of Justice: 5'-UGAGGUAGUAGAUUGUAUAGUU-3'(SEQ ID NO.2);

[0042] Antisense chain: 5'-CUAUACAAUCUACUACCUCAUU-3' (SEQ ID NO.3).

[0043] In the sequence listing, the U in SEQ ID NO.1-SEQ ID NO.3 above is replaced with T. Simultaneously, a negative control scRNA is synthesized for use in subsequent experiments.

[0044] The experiment was conducted as follows:

[0045] Before transfection with the above-mentioned hsa-let-7f-5p mimic and scRNA, prepare a stock solution of the lyophilized hsa-let-7f-5p mimic and scRNA powders. Transfect cells with hsa-let-7f-5p mimic and scRNA at a final concentration of 80 μM using INVDNA RNA Transfection Reagent (Invigentech, a standard transfection reagent). Primary muscle fibroblast / adipogenic progenitor cells were sorted using flow cytometry (FACS) and cultured in vitro using standard techniques. The fibroblast / adipogenic progenitor cells were cultured at a density of 3 × 10⁶ cells / year. 5After seeding at a rate of [number] cells / mL and culturing in DMEM medium for 72 h, fibroblast / adipogenic progenitor cells were transfected using the aforementioned RNA-INVIDNA RNA Transfection Reagent complex according to the kit description. After 24 h, the medium was replaced with medium containing neither RNA nor INVIDNA RNA Transfection Reagent, and the cells were cultured for 96 h. Relevant transcriptional expression was then detected. The transfected fibroblast / adipogenic progenitor cells from different experimental groups underwent the same routine adipogenic differentiation induction, using adipogenic differentiation induction medium to induce adipogenic differentiation in fibroblast / adipogenic progenitor cells (i.e., to establish a cell adipogenic differentiation model). Oil Red staining was performed at the same time point to detect adipocyte formation capacity.

[0046] For detailed experimental results, please refer to Figure 1 (n=3, the percentage of Oil Red staining area in the total field of view) shows the Oil Red staining status of different experimental groups. Increasing the amount of hsa-let-7f-5p in cells (transfection with hsa-let-7f-5p mimics) will reduce the area of ​​adipocytes, that is, reduce the adipogenic differentiation of fibroblasts / adipogenic progenitor cells.

[0047] Example 2: hsa-let-7f-5p regulates the adipogenic differentiation capacity of fibroblasts / adipogenic progenitor cells by targeting Tgfbr3.

[0048] The Tgfbr3 (Transforming Growth Factor Beta Receptor III) gene encodes transforming growth factor beta receptor III (TGF-β Receptor III), also known as beta-glycan. This receptor is expressed on the cell surface and is an important component of the TGF-β signaling pathway. Overexpressing the Tgfbr3 gene in normal cells and further inducing adipogenic differentiation using adipogenic differentiation-inducing medium can obtain an enhanced adipogenic differentiation model, which can be used to study the effects of drugs on adipogenic differentiation. Extensive adipogenic differentiation of fibroblasts / adipogenic progenitor cells in muscle leads to muscle fatty infiltration and degeneration.

[0049] Experiments demonstrated that hsa-let-7f-5p can inhibit adipogenic differentiation of fibroblast / adipogenic progenitor cells overexpressing the Tgfbr3 gene. In vitro transfection with a Tgfbr3 overexpression plasmid (TGFBR OE) or an empty vector was performed. In cells transfected with the Tgfbr3 overexpression plasmid (TGFBR OE), hsa-let-7f-5p (the aforementioned double-stranded hsa-let-7f-5p mimics) or scRNA was then transfected. Adipogenic differentiation of fibroblast / adipogenic progenitor cells was induced using adipogenic differentiation induction medium, and adipocyte formation capacity was detected by Oil Red staining. Specifically, in fibroblast / adipogenic progenitor cells transfected with a Tgfbr3 overexpression plasmid or an empty vector, hsa-let-7f-5p or scRNA was transfected according to the aforementioned method, and then adipocyte formation capacity was detected using Oil Red staining. Detailed experimental results can be found in [link to experimental results]. Figure 2 (n=3, the percentage of Oil Red staining area in the total field of view) demonstrates the regulatory effect of Tgfbr3 overexpression on the inhibition of adipogenic differentiation of fibroblasts / adipogenic progenitor cells by hsa-let-7f-5p, as detected by Oil Red staining. In fibroblasts / adipogenic progenitor cells overexpressing the Tgfbr3 gene, overexpression of the Tgfbr3 gene leads to reduced adipogenic differentiation of fibroblasts / adipogenic progenitor cells. Simultaneously, increasing the amount of hsa-let-7f-5p in these cells reduces the area of ​​adipocytes, thus reducing adipogenic differentiation of fibroblasts / adipogenic progenitor cells.

[0050] Although existing technologies have reported that one of the targets of hsa-let-7f-5p is the Tgfbr1 gene, which can be applied to the treatment of osteoporosis, there are significant differences between the Tgfbr1 and Tgfbr3 genes. For example, their structural characteristics differ (the former possesses serine / threonine kinase activity, while the latter lacks intrinsic kinase activity). They also differ in ligand binding patterns, signal transduction functions, tissue distribution, and biological effects. Therefore, the Tgfbr3 gene is a newly discovered target of hsa-let-7f-5p in this protocol. By targeting this gene, adipogenic differentiation of fibroblasts / adipogenic progenitors is inhibited, thereby treating myofascial degeneration and muscle damage associated with it.

[0051] More specifically, Tgfbr3 is expressed on the cell surface and is an important component of the TGF-β signaling pathway. The Tgfbr3 gene is closely related to muscle injury repair and adipogenic differentiation of fibroblasts / adipogenic progenitors. In particular, regarding its role in muscle injury repair, the Tgfbr3 gene participates in the TGF-β signaling pathway, which regulates various cellular activities during muscle injury repair. For example, this pathway is related to the formation of new muscle fibers, the synthesis and deposition of extracellular matrix, and the regulation of inflammatory responses during muscle injury. Subsequent experiments using a glycerol-induced muscle injury model demonstrated that treatment of muscle injury with hsa-let-7f-5p mimics can effectively and comprehensively promote muscle injury repair. The inventors analyzed that the reason is that hsa-let-7f-5p can exert a comprehensive positive regulatory effect on aspects such as myofiber formation, extracellular matrix synthesis and deposition, inflammatory responses, and fat deposition during muscle injury repair by acting on the target gene Tgfbr3.

[0052] Example 3: Experimental study on the in vivo inhibition of fat infiltration after muscle damage by hsa-let-7f-5p

[0053] Animal model construction: An acute injury model of muscle fatty infiltration was induced by local injection of 50 μL of 50% (v / v) glycerol solution into the tibialis anterior muscle of mice. Glycerol, as a solvent, can induce muscle fiber dissolution and inflammatory response when injected locally into muscle at high concentrations, thus simulating muscle injury. One hour after glycerol injection, 4 μg of hsa-let-7f-5p (the aforementioned double-stranded hsa-let-7f-5p mimics) or NC (scRNA) was injected locally. Samples were collected on day 14 after model establishment for subsequent testing. Glycerol treatment of muscle tissue is a method used to establish a muscle injury model, which leads to muscle fatty infiltration and fatty degeneration, thus serving as a model for muscle fatty infiltration (muscle fatty degeneration). This model can be used to study skeletal muscle regeneration, repair, fatty infiltration (muscle fatty degeneration) mechanisms, and to test potential treatment strategies. Necrotic injury of muscle tissue is induced by local injection of high-concentration glycerol; after injury, fat deposition occurs in the skeletal muscle, affecting muscle regeneration and repair.

[0054] Tibial anterior muscle specimens were collected and fixed in 4% paraformaldehyde for 12 hours, then dehydrated overnight with 30% sucrose solution. The dehydrated samples were then prepared into frozen sections of 8-10 μm thickness using a cryostat. Fluorescent immunohistochemistry was performed on the samples to determine the extent of fatty infiltration; detailed results can be found in [link to results]. Figure 3 (n=4, detect perilipin) + (Percentage of positive area to total slide area). Figure 3 The green portion is labeled with Perilipin, used to show adipocyte infiltration; the red portion is labeled with Phalloidine, showing the tissue cytoskeleton; and the blue portion is labeled with Hoechst 33342, showing the cell nucleus. Figure 3 The chart on the right is for Perilipin + Statistics on the area of ​​the marked region. (From...) Figure 3 The experimental results show that treatment with hsa-let-7f-5p can reduce fatty infiltration in muscle tissue and inhibit muscle fat formation.

[0055] In addition, the hsa-let-7f-5p treatment in this protocol not only inhibits adipogenesis in fibroblasts / adipocyte progenitors but also promotes regeneration and repair processes after skeletal muscle injury. Phalloidine is a toxin commonly used to label and visualize actin filaments (F-actin). It has a high affinity for F-actin, and fluorescently labeled phalloidin can be used to observe actin structures in the cytoskeleton. When studying the degree of muscle injury recovery, phalloidin staining can help assess the integrity of myofibril structure and its repair effectiveness. Figure 3 It can be seen that, compared with the negative control, the Phalloidine-labeled myotubes in the hsa-let-7f-5p group had a larger average cross-sectional area and were more neatly arranged, indicating that the recovery of muscle damage was more ideal after injection of double-chain hsa-let-7f-5p mimics.

[0056] Example 4: Discovery of hsa-let-7f-5p

[0057] Studies have found that platelet-rich plasma (PRP) has the function of inhibiting muscle fat infiltration, and this technical protocol further investigated its mechanism of action. By extracting exosomes from PRP, it was found that exosomes can significantly inhibit muscle fat infiltration by suppressing the adipogenic differentiation ability of fibroblasts / adipogenic progenitor cells. Through screening for effective components, RNA was found to be a key component mediating the inhibition of adipogenesis by PRP-derived exosomes. Using high-throughput miRNA sequencing, hsa-let-7f-5p was identified as a possible key molecule in the inhibition of adipogenesis by PRP-derived exosomes.

[0058] Exosomes were extracted from PRP using conventional ultracentrifugation methods for identification. Detailed experimental results can be found in [link to experimental results]. Figure 4 Electron microscopy was used to examine the morphology of exosomes. Figure 4 (Top left) and Western blot detection of typical exosome marker expression ( Figure 4Top right), Oil Red staining to detect the effect of PRP-derived exosomes on the adipogenic capacity of fibroblasts / adipogenic progenitors undergoing adipogenic differentiation ( Figure 4 Next, n=4, detect perilipin + The percentage of positive area in the slide specimen indicates that the use of PRP-derived exosomes can effectively inhibit adipogenic differentiation of fibroblasts / adipocyte progenitors.

[0059] DNA, RNA, or protein were removed from PRP exosomes, and then these were used to intervene in adipogenic differentiation of fibroblasts / adipogenic progenitor cells in vitro. The results showed that RNA removal significantly weakened the inhibitory effect of PRP-derived exosomes on the adipogenic differentiation of fibroblasts / adipogenic progenitor cells, suggesting that RNA is a key component of PRP-derived exosomes influencing the adipogenic differentiation of fibroblasts / adipogenic progenitor cells. The experimental results were detected by Oil Red staining. See [link to experimental details]. Figure 5 (n=5, detect perilipin) + (Percentage of positive area to total slide area).

[0060] High-throughput miRNA sequencing was used to detect miRNA-rich components in PRP-derived exosomes, and hsa-let-7f-5p was found to be highly enriched in these exosomes. Figure 6 In PRP-derived exosomes, sequencing revealed a high number and proportion of short reads of hsa-let-7f-5p. Therefore, this technical approach selected double-stranded mimics based on the mature hsa-let-7f-5p sequence to study their effect on inhibiting muscle fat accumulation, thereby discovering this potential RNA drug for the treatment of related diseases.

[0061] Example 5: Liposome vesicles encapsulating hsa-let-7f-5p

[0062] Let-7f was encapsulated in liposomes and then locally injected to intervene in a glycerol-induced tibialis anterior muscle injury model. Muscle samples were collected on day 21 for frozen sectioning, and the percentage of Perilipin expression area to the total area of ​​the sample sections was detected by immunofluorescence staining. The experimental method is the same as in Example 3. The injection volume of liposomes encapsulating hsa-let-7f-5p (lipo-miRNAs) and liposomes encapsulating scRNA (lipo-scRNA) was 60 μL. The liposomes encapsulating hsa-let-7f-5p (double-stranded hsa-let-7f-5pmimics) or scRNA (lipo-miRNAs and lipo-scRNA) were prepared using conventional liposome preparation methods, that is, RNA is encapsulated in lipid-formed vesicles. The general procedure is as follows: SPC (phosphatidylcholine), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), cholesterol, and DSPE-PEG-2k (1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]) are dissolved in 2 mL of anhydrous ethanol and transferred to a round-bottom flask to form a lipid mixture. The ratio of the above raw materials can be determined according to conventional methods, for example: SPC:DOTAP:cholesterol:DSPE-PEG-2k = 40-60 mg: 2-3 mg: 2-3 mg: 2-3 mg: 2 mL. RNA (i.e., RNA drug, double-stranded hsa-let-7f-5pmimics or scRNA) was dissolved in citrate buffer (50 mM citrate, pH = 4) containing 25% ethanol. This solution was slowly added to the aforementioned lipid mixture, mixed thoroughly, and incubated for 20 minutes. The mixture was then subjected to sonication and a liposome extruder (200 nm filter membrane). Dialysis was then performed using a nanodialysis device (polycarbonate membrane, 50 nm pore size) to remove unloaded RNA. Finally, sterile water was added to bring the volume to 10 mL.

[0063] Figure 7 A is an electron micrograph of liposome vesicles encapsulating hsa-let-7f-5p. Figure 7 B involves performing fluorescence immunohistochemical detection on the samples to determine the extent of fatty infiltration (n=4, detecting perilipin). + (Percentage of positive area to total slide area). Figure 7 In B, the green portion is labeled with Perilipin, used to show adipocyte infiltration; the red portion is labeled with Phalloidine, showing the tissue cytoskeleton; and the blue portion is labeled with Hoechst 33342, showing the cell nucleus. Figure 7 The statistics chart for C is for Perilipin. + Statistics on the area of ​​the marked region. (From...) Figure 7 The experimental results show that treatment with liposomes encapsulating hsa-let-7f-5p can reduce fatty infiltration in muscle tissue and inhibit muscle fat formation. Furthermore, compared to the negative control (lipo-scRNA group), the lipo-miRNA group showed that the myotubes labeled with phalloidine had a larger average cross-sectional area and were arranged more regularly and densely, indicating that the recovery of muscle damage was more ideal after injection of liposomes encapsulating hsa-let-7f-5p.

[0064] RNA drugs, including small interfering RNA (siRNA), microRNA (miRNA), and messenger RNA (mRNA), have shown great potential in treating a variety of diseases. However, the inherent instability of RNA molecules and the challenges they face during in vivo delivery (such as enzymatic degradation, immunogenicity, and poor targeting specificity) limit their widespread application. Liposome vesicles, as carriers of RNA drugs, can effectively overcome these problems, and this protocol attempts to encapsulate RNA drugs in liposome vesicles.

[0065] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. The use of hsa-let-7f-5p in the preparation of medicaments for treating muscle injuries with muscle fat infiltration, characterized in that: Its sequence is shown in SEQ ID NO.1; hsa-let-7f-5p is used to inhibit the adipation of fibroblasts / adipocyte progenitors and to promote regeneration and repair after muscle injury.

2. The use of hsa-let-7f-5p according to claim 1 in the preparation of a medicament for treating muscle injury with muscle fat infiltration, characterized in that: The hsa-let-7f-5p has the effect of inhibiting muscle fat degeneration.

3. The use of hsa-let-7f-5p according to claim 1 in the preparation of a medicament for treating muscle injury with muscle fat infiltration, characterized in that: It is produced by double-stranded RNA; the sense strand of the double-stranded RNA is shown in SEQ ID NO.2, and the antisense strand is shown in SEQ ID NO.

3.

4. The use of hsa-let-7f-5p according to claim 3 in the preparation of a medicament for treating muscle injury with muscle fat infiltration, characterized in that: Double-stranded RNA or hsa-let-7f-5p are inhibitors of Tgfbr3 gene expression.

5. The use of hsa-let-7f-5p according to claim 1 in the preparation of a medicament for treating muscle injury with muscle fat infiltration, characterized in that: Muscle fat infiltration is caused by muscle damage.

6. The use of hsa-let-7f-5p according to claim 1 in the preparation of a medicament for treating muscle injury with muscle fat infiltration, characterized in that: The hsa-let-7f-5p is enriched in exosomes derived from platelet-rich plasma.

7. The use of hsa-let-7f-5p according to claim 3 in the preparation of a medicament for treating muscle injury with muscle fat infiltration, characterized in that: The double-stranded RNA is encapsulated in liposome vesicles, and the raw materials of the liposome vesicles include SPC, DOTAP, cholesterol, and DSPE-PEG-2k. Liposomes encapsulating double-stranded RNA were prepared by the following method: SPC, DOTAP, cholesterol and DSPE-PEG-2k were dissolved in anhydrous ethanol to form a lipid mixture; citrate buffer containing dissolved double-stranded RNA and ethanol was added to the lipid mixture; and after sonication, extrusion using a liposome extruder and dialysis, liposomes encapsulating double-stranded RNA were obtained.

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