Application of fingolimod hydrochloride in preparation of medicine for promoting skeletal muscle injury repair

Abstract

The invention discloses an application of fingolimod hydrochloride in preparation of a medicine for promoting skeletal muscle injury repair. According to the prepared medicine, the phenotypic transformation promoting effect, the immunoregulation effect, the myoblast proliferation and differentiation promoting effect and the fibrosis reaction inhibiting effect of fingolimod hydrochloride on macrophages at the skeletal muscle injury part are utilized, so that the local inflammatory response at the skeletal muscle injury part is relieved, myoblast differentiation is promoted, myotube atrophy is reduced, and the skeletal muscle injury part can be cured. Meanwhile, the fibrosis reaction of a skeletal muscle injury part is inhibited, so that remarkable skeletal muscle defect repair is realized. The new application can effectively promote repair and regeneration of muscles at skeletal muscle injury parts, can significantly improve the morphology and quality of the injured muscles, promotes regeneration of new muscle fibers and reduces muscle fibrosis, is suitable for treatment of traumatic or non-traumatic large-volume skeletal muscle defects, and has a wide application prospect. A new theoretical basis can be provided for research and development of skeletal muscle defect repairing injections, and the skeletal muscle defect repairing injections have great scientific significance and clinical transformation value.

Description

Technical Field

[0001] The present invention belongs to the technical field of regenerative medicine, and particularly relates to the application of fingolimod hydrochloride (FTY720) in the preparation of a drug for promoting the repair of skeletal muscle injury. Background Art

[0002] Skeletal muscle injury is a common clinical problem. Muscle tissue has a certain self-repair ability. However, when the muscle mass loss caused by severe injury exceeds 20%, it is called volumetric muscle loss (VML). VML exceeds the endogenous repair ability of muscle, leading to persistent inflammatory reaction at the injury site, irreversible fibrosis and muscle atrophy, thus affecting muscle function and the quality of life of patients. At present, the clinical treatment strategy for VML is limited to free functional muscle transfer. However, autologous muscle transplantation has many complications and poor prognosis. Therefore, there is still a lack of effective clinical treatment strategies for VML.

[0003] The immune response plays a "double-edged sword" role in the repair process after skeletal muscle injury. An appropriate immune response can promote muscle repair by clearing necrotic tissue and providing repair signals. However, an excessive immune response at the VML injury site will lead to persistent inflammation, cause muscle atrophy, inhibit muscle regeneration, and promote the occurrence of fibrosis. In recent years, more and more studies have begun to focus on the importance of immune regulation in muscle injury repair. By regulating the immune response, excessive inflammatory response can be avoided, the formation of fibrosis can be reduced, and muscle regeneration and functional recovery can be promoted.

[0004] Fingolimod hydrochloride (FTY720) is an S1P receptor modulator, which was first used to treat multiple sclerosis. FTY720 binds to the S1P1 receptor, inhibits the peripheral migration of T cells and other immune cells, thereby reducing the accumulation of immune cells at the injury site and alleviating the inflammatory response. The immune regulatory effect of FTY720 and its potential promoting effect on muscle regeneration make it a promising new drug for treating skeletal muscle injury. At present, the research on FTY720 in skeletal muscle injury repair is still in its initial stage, and there are still few studies on its specific mechanism, optimal dose, administration method and long-term treatment effect. Therefore, how to effectively apply FTY720 to the clinical treatment of skeletal muscle injury and achieve more precise local treatment is still the focus of current research. Summary of the Invention

[0005] Aiming at the defects and deficiencies existing in the current skeletal muscle injury repair technology, the present invention provides the application of fingolimod hydrochloride (FTY720) in the preparation of a drug for promoting the repair of skeletal muscle injury, and utilizes the effects of FTY720 in immune regulation, cell proliferation and differentiation, fibrosis inhibition, etc., to improve the repair effect of skeletal muscle.

[0006] To solve the above technical problems, the present invention is achieved through the following technical solutions:

[0007] Use of fingolimod hydrochloride (FTY720) in the preparation of a drug for promoting the repair of skeletal muscle injury.

[0008] Furthermore, the skeletal muscle injury is a large - volume skeletal muscle injury with a defective muscle mass exceeding 20% of the total mass of the original normal muscle.

[0009] Furthermore, the skeletal muscle injury includes traumatic skeletal muscle injury or non - traumatic skeletal muscle injury.

[0010] Among them,

[0011] The etiologies of the traumatic skeletal muscle injury mainly include: war injuries, severe traumas, surgical lesion resections, etc.;

[0012] The etiologies of the non - traumatic skeletal muscle injury mainly include: degenerative diseases such as sarcopenia, muscle atrophy, muscular dystrophy, muscle denervation, etc.

[0013] Furthermore, the drug is an injection containing FTY720, which can directly act on the skeletal muscle injury site by local injection to promote the repair and regeneration of the skeletal muscle injury site.

[0014] The effects of promoting the repair and regeneration of the skeletal muscle injury site include: significantly improving the morphology and quality of the injured muscle, promoting the regeneration of new muscle fibers while reducing muscle fibrosis.

[0015] The optimized injection regimen of the injection containing FTY720 is to inject 0.5 mg of FTY720 per kilogram of mammalian body weight, with a dosing frequency of once a week and a treatment cycle of 6 weeks.

[0016] Furthermore, the drug utilizes the immunomodulatory effect, myoblast proliferation and differentiation promoting effect, and fibrosis reaction inhibitory effect of FTY720 on the skeletal muscle injury site to reduce the local inflammatory reaction at the skeletal muscle injury site, promote myogenic differentiation, reduce myotube atrophy, and at the same time inhibit the fibrosis reaction at the skeletal muscle injury site, thereby achieving the repair of skeletal muscle defects.

[0017] Furthermore, the immunomodulatory effect is specifically that FTY720 in the drug reduces the local inflammatory reaction at the skeletal muscle injury site by regulating the phenotypic transformation of macrophages at the skeletal muscle injury site, creating a favorable immune microenvironment for the repair of skeletal muscle in the early stage of injury.

[0018] Furthermore, the specific regulation of macrophage phenotypic transformation at the skeletal muscle injury site is that the drug uses FTY720 to promote the transformation of macrophages at the skeletal muscle injury site from pro-inflammatory M1 type (decrease in iNOS + macrophages) to pro-regenerative M2 type (increase in CD206 + macrophages).

[0019] Furthermore, the specific promotion of myoblast proliferation and differentiation is that after FTY720 in the drug reduces the local inflammatory response through immunomodulation, it then protects the cell viability of myoblasts under inflammatory conditions stimulated by LPS, enhances the differentiation ability of myoblasts, increases the average diameter and fusion index of mature myotubes, promotes the expression of myogenic differentiation-related genes, thereby promoting myogenic differentiation at the skeletal muscle injury site and reducing myotube atrophy at the skeletal muscle injury site.

[0020] The optimal concentration of FTY720 in the above in vitro experiment is 10 μM.

[0021] The above in vitro cell culture inflammatory conditions are simulated by adding LPS to the culture medium.

[0022] Furthermore, the myogenic differentiation-related genes include MyoD, MyoG, and MHC.

[0023] Furthermore, the specific inhibition of the fibrotic response is that the drug uses FTY720 to reduce the deposition of collagen in the skeletal muscle injury site, avoid the formation of scar tissue, improve the muscle tissue structure, thereby inhibiting the fibrotic response at the skeletal muscle injury site.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present invention first proves the mechanism of action of fingolimod hydrochloride (FTY720) in regulating the inflammatory response in the muscle injury area, promoting the transformation of the inflammatory immune microenvironment into a regenerative immune microenvironment, inhibiting the fibrotic response, promoting the proliferation and differentiation of myoblasts under inflammatory conditions, and promoting myotube fusion and maturation. These findings can provide a new theoretical basis for the research and development of therapeutic drugs related to muscle injury repair, and have great scientific significance and clinical translation value.

[0026] On this basis, the present invention first proposes to apply fingolimod hydrochloride (FTY720) to the repair of skeletal muscle injury. It not only utilizes the immunomodulatory effect of FTY720, but also combines mechanisms such as regulating macrophage phenotypic transformation in the muscle injury area, promoting the proliferation and differentiation of myoblasts under inflammatory conditions, promoting the regeneration of new muscle fibers while inhibiting the fibrotic response, providing a new and effective treatment method for the repair of large-volume muscle defects.

[0027] By formulating FTY720 into an injection and directly applying it to the skeletal muscle injury site in a local injection manner with appropriate concentration and frequency, it is ensured that the drug acts more directly and efficiently on the injury site, reducing systemic side effects while enhancing the effect of promoting muscle injury repair and regeneration.

[0028] The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the invention and implement it according to the content of the specification, the following details the preferred embodiments of the present invention in conjunction with the accompanying drawings. The specific implementation manners of the present invention are given in detail by the following embodiments and their accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings described herein are used to provide a further understanding of the present invention and form a part of this application. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0030] Figure 1 It is a graph showing the experimental results of the repair of large - volume skeletal muscle defects in the tibialis anterior muscle of rats by local injection of FTY720 at different injection doses and frequencies in Example 1 of the present invention.

[0031] Figure 2 It is a graph showing the quantitative analysis results of the muscle mass of the tibialis anterior muscle, the quantitative analysis results of the number of neonatal myofibers, and the quantitative analysis results of the fibrotic area in Example 1 of the present invention (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0032] Figure 3 It is a graph showing the results of immunofluorescence staining of iNOS and CD206 and the quantitative analysis results of the number of iNOS macrophages and CD206 macrophages in the defect area at 2 weeks after VML in Example 2 of the present invention (**p < 0.01). + macrophages and CD206 + It is a graph showing the quantitative analysis results of the number of macrophages (**p < 0.01).

[0033] Figure 4 It is a graph showing the CCK - 8 detection results of culturing C2C12 myoblasts in a medium supplemented with different concentrations of FTY720 for 1 day and 3 days in Example 3 of the present invention and the CCK - 8 detection results of culturing C2C12 myoblasts in a medium supplemented with 10 μM FTY720 for 3 days under inflammatory conditions stimulated by LPS (ns: no significant difference; *p < 0.05; **p < 0.01).

[0034] Figure 5This is the MHC immunofluorescence staining image of myotubes formed after culturing C2C12 myoblasts in the myogenic differentiation medium supplemented with 10 μM FTY720 under LPS-stimulated inflammatory conditions for 6 days in Example 4 of the present invention, as well as the quantitative analysis results of the average myotube diameter and myotube fusion index in each group (*p<0.05; **p<0.01).

[0035] Figure 6 This is the analysis result graph of the relative mRNA expression levels of MyoD, MyoG, and MHC in myotubes formed after culturing C2C12 myoblasts in the myogenic differentiation medium supplemented with 10 μM FTY720 under LPS-stimulated inflammatory conditions for 6 days in Example 5 of the present invention (*p<0.05). Detailed implementation manners

[0036] The following will describe the preferred embodiments of the present invention in detail with reference to the accompanying drawings, so as to more clearly understand the purpose, features, and advantages of the invention. It should be understood that the embodiments shown in the drawings are not limitations on the scope of the present invention, but only to illustrate the essential spirit of the technical solution of the present invention.

[0037] In the following description, certain specific details are set forth for purposes of explaining various disclosed embodiments to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other instances, well-known devices, structures, and techniques associated with the present application may not be shown or described in detail so as not to unnecessarily obscure the description of the embodiments.

[0038] Unless the context requires otherwise, throughout the specification and claims, the words "comprise" and its variations, such as "comprising" and "having", should be understood in an open, inclusive sense, i.e., construed as "including, but not limited to".

[0039] References to "an embodiment" or "one embodiment" in the specification throughout represent that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment. Thus, the appearances of "in an embodiment" or "in one embodiment" in various places throughout the specification are not necessarily all referring to the same embodiment. Additionally, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0040] As used in this specification and the appended claims, the singular forms "a" and "the" include plural referents unless the context clearly dictates otherwise. It should be noted that the term "or" is generally used in its inclusive sense of "and / or" unless the context clearly dictates otherwise.

[0041] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0042] To make the technical solutions of the present invention more clearly understandable, the feasibility and effectiveness of the application of FTY720 in the repair of skeletal muscle defects are verified through experimental examples below.

[0043] Experimental Example 1: Application of FTY720 as an injection in the repair of volumetric muscle loss (VML) of the tibialis anterior (TA) muscle in rats by local injection, and optimization of the local injection regimen of FTY720.

[0044] (1) Experimental animals: Twelve-week-old male Sprague-Dawley rats, weighing approximately 300 g, a total of 25 rats were selected.

[0045] (2) Establishment of a model of large-volume skeletal muscle defect in the tibialis anterior muscle: SD rats were anesthetized with pentobarbital, weighed, and the mass of the tibialis anterior muscle was calculated according to the formula: tibialis anterior muscle weight = 0.0017 × rat body weight - 0.0716. The right tibialis anterior muscle was exposed, and a muscle volume of approximately 10×7×3 mm 3 was excised, weighing while cutting until the excised mass reached 25% of the total mass, and the fascia and skin were sutured. After the operation, the rats were given a standard breeding environment. An FTY720 treatment group and a control group were set up, with 5 SD rats in each group after the above operation. In the FTY720 treatment group, FTY720 was dissolved in physiological saline and injected into the skeletal muscle defect site by local injection. By comparing different doses (0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, once a week) and injection frequencies (0.5 mg / kg, once every two weeks), the local injection regimen of FTY720 was optimized, and the treatment period was 6 weeks. The control group was injected with the same volume of physiological saline.

[0046] (3) After 6 weeks, the rats were euthanized, and the bilateral TA muscles were carefully dissected, taking care to keep the Achilles tendon intact and completely removing the fascia and adipose tissue on the muscle surface. After taking pictures of the appearance of the TA muscles with a camera, the mass (g) of the bilateral TA muscles was weighed. To exclude individual differences, the percentage of the mass of the damaged TA muscle to the mass of the contralateral normal muscle was used to evaluate the mass recovery of the damaged skeletal muscle. Then, the repair situation was observed by histological section staining.

[0047] See Figure 1As shown, it can be seen that all VML injuries were established on the right TA muscle during the experiment, and the uninjured left TA muscle was used as the normal control. At 6 weeks after surgery, there were no signs of infection or toxic reactions in the appearance of the TA muscle after local injection of FTY720, demonstrating the safety and reliability of local injection of FTY720. The TA muscles on the VML injury side and the normal TA muscles on the contralateral side in each group had unique "teardrop-shaped" morphological characteristics, and the length of the muscle in the vertical direction was approximately twice the width in the horizontal direction. The surface of the muscle in the control group was uneven and filled with fibrous scar-like tissue, while the muscle in the FTY720 local injection group was relatively plump, and the defect was filled with muscle-like tissue.

[0048] First, the percentage of the TA muscle mass on the VML injury side to the mass of the normal muscle on the contralateral side was compared to evaluate the muscle repair situation. Compared with the mass of the normal contralateral muscle, the muscle mass in each group decreased at 6 weeks after VML injury. See Figure 2 As shown, it can be seen that compared with the control group, there was a tendency for an increase in the percentage of muscle mass in the FTY720 local injection group. Among them, there was a statistical difference between local injection of FTY720 (0.5 mg / kg, once a week) and the control group. The results showed that the muscle could not repair itself after VML injury, leading to muscle atrophy, and local injection of FTY720 could effectively promote the recovery of muscle morphology and mass after VML injury.

[0049] Next, histological sections of rat muscle tissue were analyzed using H&E staining and Masson staining. In the control group, there were no signs of muscle regeneration in the surgically resected area, and fibroblasts infiltrated the defect area to form fibrous scars, and obvious depressions were seen in the defect area under the microscope. In the FTY720 local injection group, new myofibers were observed to grow into the defect area. The nuclei of the new myofibers were located in the center, and the diameters were uneven, which was a manifestation of immature myofibers.

[0050] See Figure 2 As shown, quantitative statistics showed that there was a statistically significant increase in the number of new myofibers in the FTY720 local injection group (0.5 mg / kg, once a week) compared with the other groups. See Figure 1 As shown, the histological analysis results of H&E staining indicated that local injection of FTY720 (0.5 mg / kg, once a week) significantly promoted myofiber regeneration at the VML injury site. See Figure 1 As shown, by analyzing the blue area in Masson staining, collagen deposition could be detected at the defect site in each group. The blue area was more obvious in the control group and the FTY720 local injection group (0.1 mg / kg, once a week), indicating a large amount of fibrosis. Figure 2As shown, the quantitative statistical results of the percentage of fibrotic area in the defect area showed that, compared with the other groups, the fibrotic area in the FTY720 local injection group (0.5 mg / kg, once a week) was significantly reduced.

[0051] The above results indicate that local injection of FTY720 can significantly improve the morphology and quality of VML-injured muscles, promote myofiber regeneration while inhibiting the formation of fibrotic tissue. Moreover, the optimal dose and administration frequency are 0.5 mg / kg, once a week.

[0052] Experimental Example 2: The effect of FTY720 as an injection on regulating the immune microenvironment of the inflammatory response at the VML defect site by local injection.

[0053] To study the regulatory effect of FTY720 on the early acute inflammatory response of VML injury. A rat anterior tibial muscle VML model was established according to the experimental method in Example 1. In the experimental group, FTY720 (0.5 mg / kg, once a week) was injected at the defect, while the control group was injected with the same volume of normal saline. At 2 weeks after surgery, immunofluorescence staining of the pro-inflammatory marker iNOS and the anti-inflammatory marker CD206 was performed on TA muscle tissue sections. The brief steps are as follows: citrate antigen repair, draw a circle with an immunohistochemical pen, punch holes with 0.1% Triton X-100, block with 2% BSA, incubate with iNOS or CD206 antibody, incubate with the corresponding fluorescent secondary antibody, add DAPI staining solution to label the cell nucleus, and mount the slides. After observing and photographing under a fluorescence microscope, the number of iNOS + (red fluorescence) and CD206 + (green fluorescence) macrophages in the VML injury area was counted using ImageJ software.

[0054] At 2 weeks after surgery, iNOS (one of the markers for M1 macrophages) and CD206 (one of the markers for M2 macrophages) were used to characterize the phenotypic distribution of macrophages in the injury area. As shown in Figure 3 A, the white dotted line is the boundary between the defect area and the remaining muscle below. It can be seen that there are a large number of macrophage infiltrations in the defect areas of both groups under high magnification. In the control group, iNOS + macrophages are the main type, distributed both in the defect area and at the interface between the defect area and the remaining muscle below. The CD206 + macrophages in the control group are concentrated in the defect area, and there are fewer CD206 + macrophages. In contrast, the FTY720 group is mainly composed of CD206 + macrophages, which are distributed both in the defect area and above and below the interface. As shown in Figure 3 B, the quantitative statistical results show that the total amount of macrophages in the two groups is similar. The control group shows more iNOS+ macrophages, and CD206 + There were fewer macrophages. However, in the FTY720 group, iNOS was counted + decreased macrophages and a large number of CD206 + macrophages, which showed a statistically significant difference compared with the control group, indicating that the VML injury site in the FTY720 group exhibited a pro-regenerative immune microenvironment dominated by M2 macrophages.

[0055] These data indicate that FTY720 regulates macrophages from the M1 type (decreased iNOS + macrophages) to the M2 type (increased CD206 + macrophages), reduces the inflammatory response, and creates a favorable immune microenvironment for skeletal muscle repair in the early stage of injury.

[0056] Experimental Example 3: Effects of FTY720 on the activity of myoblasts under normal culture conditions and inflammatory stimulation conditions.

[0057] (1) To study the effect of FTY720 concentration on the activity of myoblasts, C2C12 myoblasts were cultured in 96-well plates (3000 cells / well). After the cells were completely adherent, the medium was changed, and after rinsing 3 times with PBS, the experimental groups were respectively added with high-glucose DMEM complete medium containing 1 μM, 5 μM, 10 μM, and 15 μM FTY720, and the control group was added with normal high-glucose DMEM complete medium. After continuing to culture for 1 day and 3 days, the medium was replaced with CCK-8 solution. To avoid the influence of bubbles and impurities on the results, after incubating for 2 h, 100 μL was aspirated from each well and transferred to another new 96-well plate, and the absorbance value of the solution was measured at a wavelength of 450 nm. There were five replicates in each group, and the experiment was independently repeated three times.

[0058] (2) 100 ng / ml LPS was added to the medium to study the effect of FTY720 on the biological activity of myoblasts under inflammatory stimulation conditions. LPS (final concentration 100 ng / ml) was added to the DMEM complete medium containing 10 μM FTY720 and normal high-glucose DMEM complete medium, and the high-glucose DMEM complete medium without LPS stimulation was used as the control group, and the remaining experimental steps were the same as above.

[0059] See Figure 4As shown in Figure A, the results showed that after culturing C2C12 myoblasts in the medium containing different concentrations of FTY720 for 1 day, there was no significant difference in cell viability among the groups with different concentrations compared with the control group. After culturing for 3 days, there was no significant difference in the viability of C2C12 myoblasts in the groups with FTY720 concentration less than 10 μM compared with the control group, but it was significantly decreased compared with the control group after the FTY720 concentration was higher than 15 μM. Therefore, we selected 10 μM FTY720 for subsequent research.

[0060] Then, the effect of FTY720 on the viability of C2C12 myoblasts under inflammatory stimulation conditions was further studied. See Figure 4 As shown in Figure B, the results showed that after culturing for 3 days, the viability of C2C12 myoblasts in the medium group supplemented with LPS was significantly decreased compared with the normal control group, while the cell viability in the LPS + FTY720 group was significantly higher than that in the LPS group. It indicated that the inflammatory stimulation caused by LPS significantly decreased the cell viability of C2C12 myoblasts, while FTY720 had a protective effect on the cell viability of C2C12 under inflammatory conditions.

[0061] Experimental Example 4: Effect of FTY720 on the morphology of myotubes differentiated from myoblasts under inflammatory stimulation conditions.

[0062] When C2C12 myoblasts were cultured in a 24-well plate (5×10 4 cells / well) until the cell confluence reached about 95%, the medium was changed. After rinsing 3 times with PBS, myogenic differentiation medium containing LPS (100 ng / mL) or LPS + FTY720 (10 μM) was added, and the ordinary DMEM medium containing 2% HS was used as the control group. The medium was changed every other day and continued to be cultured for 6 days until mature myotubes were formed. Immunofluorescence staining was performed on the myotube-specific marker MHC. The experimental steps were as follows: Myotube cells were fixed with 4% paraformaldehyde at 4 °C for 25 min, the paraformaldehyde was washed away with PBS, permeabilized with 0.1% Triton-X100 for 15 min, and 500 μL of 1.5% BSA was added to each well and blocked at 37 °C for 1 h. The MHC antibody was added and incubated overnight at 4 °C. The primary antibody was washed away with PBS, and the fluorescent secondary antibody was added and incubated in the dark. After rinsing 3 times with PBS, the nuclei were labeled with DAPI staining solution. The stained myotubes were observed with an inverted fluorescence microscope, and the fusion index in 5 random fields of view in each group was statistically analyzed using Image J software according to the following formula. Fusion index = the number of nuclei in MHC-positive myotubes / the total number of nuclei in the field of view × 100%.

[0063] See Figure 5As shown in A, the results of MHC immunofluorescence staining of mature myotubes showed that after the addition of LPS, inflammatory stimulation caused the myotubes in the LPS group to become significantly thinner and atrophy, and the number of myotubes in the field of view was also significantly sparser. In contrast, the myotubes in the LPS+FTY720 group were in good shape, with only a very small number of myotubes becoming thinner, and most of the myotubes were similar in shape to those in the normal control group. See Figure 5 As shown in B, the quantitative statistical results showed that the myotube diameter in the LPS+FTY720 group was comparable to that in the normal control group. Compared with these two groups, the myotube diameter in the LPS group was significantly reduced, and the myotubes were significantly atrophied. The trend of the fusion index results of myotubes in each group was the same as that of the myotube diameter results. Among them, the fusion index in the LPS group was the lowest. The addition of FTY720 significantly reversed the decrease in the fusion index caused by inflammatory stimulation.

[0064] Experimental Example 5: Effect of FTY720 on the expression of genes related to myoblast differentiation under inflammatory stimulation.

[0065] To further clarify the effect of the inflammatory microenvironment on the myogenic differentiation ability of myoblasts and explore the effect of FTY720 on myogenic differentiation under inflammatory stimulation. When C2C12 myoblasts were cultured in a 6-well plate (10 5 cells / well) until the cell confluence reached about 95%, the medium was changed. After rinsing 3 times with PBS, myogenic differentiation medium containing LPS (100 ng / ml) or LPS+FTY720 (10 μM) was added, and ordinary DMEM medium containing 2% HS was used as the control group. The medium was changed every other day and continued to be cultured for 6 days until mature myotubes were formed. The myogenic differentiation genes: myogenic regulatory factor (MyoD), myogenin (MyoG), and myosin heavy chain (MHC) were detected by PCR. The main steps were as follows: Rinse 2 times with PBS, and collect the cell suspension lysed by Trizol into a 1.5 ml EP tube. Add chloroform to make the RNA enter the aqueous phase, vortex and mix it thoroughly, and collect the colorless and transparent liquid on the top layer after centrifugation. Add an equal volume of isopropanol to precipitate the RNA, add 75% ethanol to wash the RNA precipitate, and repeat 2 times. After drying, add about 10 μL of DEPC water to each tube and heat it in a 58 °C metal pot for 8 min. After measuring the purity and concentration of RNA, reverse transcription was performed to obtain cDNA. The cDNA was prepared into a 10 μL system of PCR reaction solution, loaded, and the relative expression levels of genes related to myogenic differentiation were calculated after detection on the machine.

[0066] See Figure 6As shown, the PCR results of specific genes (MyoD, MyoG, and MHC) after myogenic differentiation showed that under inflammatory stimulation conditions, the mRNA levels of MyoD, MyoG, and MHC in the LPS group were significantly lower than those in the normal control group. In the LPS+FTY720 group, the expression of MyoD, MyoG, and MHC genes was higher than that in the LPS group, and the differences in the results of MyoG and MHC were statistically significant. The mRNA expression levels of MyoG and MHC reached similar levels to those in the normal control group.

[0067] According to the experimental results of the above 5 experimental examples, it can be proved that FTY720 can be fully applied to promote the repair of skeletal muscle injury, and can specifically be applied in the preparation of drugs for promoting the repair of skeletal muscle injury. This drug utilizes the immunomodulatory effect of FTY720 on the skeletal muscle injury site, the promoting effect on the proliferation and differentiation of myoblasts, and the inhibitory effect on the fibrotic reaction to reduce the local inflammatory reaction at the skeletal muscle injury site, promote myogenic differentiation, reduce myotube atrophy, and at the same time inhibit the fibrotic reaction at the skeletal muscle injury site, thereby achieving the repair of skeletal muscle defects.

[0068] The specific immunomodulatory effect is that FTY720 in the drug promotes the transformation of macrophages at the skeletal muscle injury site from pro-inflammatory M1 type (iNOS + macrophages decreased) to pro-regenerative M2 type (CD206 + macrophages increased), thereby reducing the local inflammatory reaction at the skeletal muscle injury site and creating a favorable immune microenvironment for the repair of skeletal muscle in the early stage of injury.

[0069] The specific promoting effect on the proliferation and differentiation of myoblasts is that after FTY720 in the drug reduces the local inflammatory reaction through the immunomodulatory effect, it further protects the cell viability of myoblasts under inflammatory conditions stimulated by LPS, enhances the differentiation ability of myoblasts, increases the average diameter and fusion index of mature myotubes, and promotes the expression of myogenic differentiation-related genes MyoD, MyoG, and MHC, thereby promoting myogenic differentiation at the skeletal muscle injury site and reducing myotube atrophy at the skeletal muscle injury site.

[0070] The specific inhibitory effect on the fibrotic reaction is that the drug utilizes FTY720 to reduce the deposition of collagen in the skeletal muscle injury site to avoid the formation of scar tissue, improve the muscle tissue structure, and thereby inhibit the fibrotic reaction at the skeletal muscle injury site.

[0071] The new application proposed by the present invention is applicable to the repair of large - volume skeletal muscle injuries with muscle mass defects exceeding 20% of the total original normal muscle mass. Skeletal muscle injuries can be traumatic skeletal muscle injuries caused by factors such as war injuries, severe traumas, surgical lesion resections, etc., or non - traumatic skeletal muscle injuries caused by degenerative diseases such as sarcopenia, muscle atrophy, muscular dystrophy, muscle denervation, etc.

[0072] For the new application proposed by the present invention, FTY720 is preferably prepared into an injection. By means of local injection, FTY720 is directly applied to the skeletal muscle injury site to promote the repair and regeneration of the skeletal muscle injury site. The main effects of repair and regeneration include: significantly improving the morphology and quality of the injured muscle, promoting the regeneration of new muscle fibers while reducing muscle fibrosis. The optimized injection plan is: injecting 0.5 mg of FTY720 per kilogram of mammalian body weight, with the administration frequency of once a week and the treatment cycle of 6 weeks.

[0073] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of fingolimod hydrochloride in the preparation of drugs for promoting the repair of skeletal muscle damage.

2. The use according to claim 1, characterized in that: The medicine is an injection containing fingolimod hydrochloride.

3. The use according to claim 1, characterized in that: The drug utilizes the immunomodulatory effects of fingolimod hydrochloride on the site of skeletal muscle injury, the promoting effects on myoblast proliferation and differentiation, and the inhibitory effects on fibrosis response to reduce the local inflammatory response at the site of skeletal muscle injury, promote myogenesis differentiation, reduce myotube atrophy, and inhibit the fibrosis response at the site of skeletal muscle injury, thereby achieving skeletal muscle defect repair.

4. The use according to any one of claims 1 to 3, characterized in that: The skeletal muscle injury is a large-volume skeletal muscle injury in which the defective muscle mass exceeds the original normal muscle total mass by more than 20%.

5. The use according to claim 3, characterized in that: The immunomodulatory effect is specifically that fingolimod hydrochloride in the drug regulates the phenotypic transformation of macrophages at the site of skeletal muscle injury, thereby alleviating the local inflammatory response at the site of skeletal muscle injury and creating a favorable immune microenvironment for skeletal muscle repair in the early stages of injury.

6. The use according to claim 5, characterized in that: The method of regulating the phenotypic change of macrophages at the site of skeletal muscle injury is specifically that the drug uses fingolimod hydrochloride to promote the change of macrophages at the site of skeletal muscle injury from the pro-inflammatory M1 type to the pro-regenerative M2 type, wherein the pro-inflammatory M1 type represents iNOS + Macrophages decreased, the pro-regenerative M2 phenotype expressed CD206 + Increased macrophages.

7. The use according to claim 3, characterized in that: The myoblast proliferation and differentiation promoting effect is specifically that fingolimod hydrochloride in the drug, after alleviating the local inflammatory response through immunomodulatory effects, further protects the cell viability of myoblasts under LPS-stimulated inflammatory conditions, enhances the differentiation ability of myoblasts, increases the average diameter and fusion index of mature myotubes, and promotes the expression of myoblast differentiation-related genes, thereby promoting myoblast differentiation at the site of skeletal muscle injury and reducing myotube atrophy at the site of skeletal muscle injury.

8. The use according to claim 7, characterized in that: The myogenic differentiation-related genes include MyoD, MyoG and MHC.

9. The use according to claim 3, characterized in that: The fibrosis reaction inhibitory effect is specifically that the drug uses fingolimod hydrochloride to reduce the deposition of collagen at the site of skeletal muscle injury to avoid the formation of scar tissue, improve muscle tissue structure, and thus inhibit the fibrosis reaction at the site of skeletal muscle injury.

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