Application of irisin in resisting ossification of yellow ligament

Abstract

The invention provides an application of tectoridin in resisting ossification of yellow ligament, and particularly provides an application of tectoridin, or a prodrug thereof, or a degradation product thereof, or a fusion protein thereof, and the tectoridin, or the prodrug thereof, or the degradation product thereof, or the fusion protein thereof is used for preparing a pharmaceutical composition, and the application of the tectoridin in resisting ossification of yellow ligament in resisting ossification of yellow ligament in resisting ossification of yellow ligament in resisting ossification of yellow ligament in resisting ossification of yellow ligament. The pharmaceutical composition is used for preventing and / or treating yellow ligament ossification (OLF).

Description

Technical Field

[0001] This invention relates to the fields of life sciences and pharmaceutical technology, and more specifically, to the application of irisin in the treatment of ossification of the ligamentum flavum. Background Technology

[0002] Irisin is a myogenic factor secreted by skeletal muscle during exercise. It is a polypeptide fragment cleaved from fibronectin type III domain-containing protein 5 (FNDC5). It can enter the bloodstream or be secreted paracrinely to facilitate interactions between muscles and other systems in the body. [1-3] As an endogenous substance that can be produced by exercise and distributed throughout the body via multiple pathways, irisin has been reported to alleviate disease progression and promote tissue repair in multiple systems, including the nervous, endocrine, skeletal, and cardiovascular systems, through mechanisms such as inflammation regulation, inhibition of ferroptosis, and activation of autophagy. [4-8] In recent years, irisin has often been associated with exercise therapy and plays an important role in degenerative diseases.

[0003] Ossification of the ligamentum flavum (OLF) is a specific disease characterized by heterotopic ossification of the ligamentum flavum in the spine. [9-12] OLF has an insidious onset, with a prevalence rate of approximately 3.6-36% in the general population. It is more common in middle-aged and elderly Asian populations, with no significant difference in incidence between genders. [9,12-17] Although ossified ligamentum flavum (OLF) can occur anywhere in the spine where it is present, it is most common in the thoracic spine and is one of the leading causes of thoracic spinal stenosis. Importantly, the ossified ligamentum flavum often compresses the spinal cord, leading to severe spinal cord injury, causing paralysis and bowel and bladder dysfunction. [18-19] OLF worsens as the disease progresses, and currently there is a lack of effective conservative treatments. Therefore, for patients with symptomatic spinal OLF, surgical treatment is currently the most effective treatment method.

[0004] Surgical treatment of ossification of the ligamentum flavum (OLF) is challenging because it commonly occurs in the thoracic spine. However, the thoracic spinal cord has poor blood supply, and ectopic ossification often adheres to surrounding tissues (e.g., the dural sac). Therefore, literature confirms that OLF surgery not only easily leads to surgery-related complications such as cerebrospinal fluid leakage and postoperative infection, but approximately 10-20% of patients may also experience ischemia-reperfusion injury. [9] These factors can lead to transient or even permanent worsening of motor and sensory function after surgery, causing serious harm to the patient's physical and mental health, and also placing a heavy economic burden on society and family.

[0005] Therefore, finding a feasible non-surgical treatment to prevent and treat ossification of the ligamentum flavum is of great significance.

[0006] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of the present invention, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to explore the mechanism of ossification of the ligamentum flavum, find possible non-surgical treatment methods, delay the occurrence and development of ossification of the ligamentum flavum, and achieve the goal of early intervention or even prevention.

[0008] In a first aspect of the invention, there is provided the use of irisin, or a prodrug thereof, or a degradation product thereof, or a fusion protein thereof, for the preparation of a pharmaceutical composition for the prevention and / or treatment of ossification of the ligamentum flavum (OLF).

[0009] In another preferred embodiment, the ossification of the ligamentum flavum is ossification of the ligamentum flavum caused by mechanical stimulation.

[0010] In another preferred embodiment, the pharmaceutical composition is also used to regulate calcium signaling.

[0011] In another preferred embodiment, the pharmaceutical composition is also used to limit the influx of extracellular calcium ions and stabilize the intracellular calcium ion concentration.

[0012] In another preferred embodiment, the intensity of the Piezo1 channel's response to mechanical stimulation is reduced.

[0013] In another preferred embodiment, the pharmaceutical composition is also used to treat cell proliferation and apoptosis caused by mechanical stress.

[0014] In another preferred embodiment, the pharmaceutical composition is also used to stabilize cell state.

[0015] In another preferred embodiment, the pharmaceutical composition is also used to reduce intracellular ROS levels and / or alleviate cellular oxidative stress, such as in ligamentum flavum cells.

[0016] In another preferred embodiment, the pharmaceutical composition is also used to protect mitochondrial homeostasis.

[0017] In another preferred embodiment, the pharmaceutical composition is also used to maintain calcium homeostasis in mitochondria.

[0018] In another preferred embodiment, the pharmaceutical composition is also used to downregulate the expression of osteogenic-related markers in mammalian ligamentum flavum cells.

[0019] In another preferred embodiment, the mammals include humans and non-human mammals.

[0020] In another preferred embodiment, the non-human mammals include mice and rats.

[0021] In another preferred embodiment, the pharmaceutical composition is also used to inhibit osteogenic differentiation of ligamentum flavum cells.

[0022] In another preferred embodiment, the osteogenic differentiation of the ligamentum flavum cells is induced by the activation of the Piezo1 channel.

[0023] In another preferred embodiment, the pharmaceutical composition is also used to inhibit osteogenic differentiation of ligamentum flavum cells induced by mechanical stimulation.

[0024] In a second aspect of the invention, a method for preventing and / or treating ossification of the ligamentum flavum (OLF) is provided, comprising the steps of administering a safe and effective amount of irisin, or a prodrug thereof, or a degradation product thereof, or a fusion protein thereof, to a subject in need.

[0025] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the invention. Attached Figure Description

[0026] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention. It is obvious that the drawings described below are merely some embodiments of the invention, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0027] Figure 1A and Figure 1B The comparison of stress on the ligamentum flavum in each segment is shown, including:

[0028] Figure 1A The average stress intensity of the ligamentum flavum in each segment is shown under resting gravity load and under dynamic loads of different directions and intensities.

[0029] Figure 1B The maximum stress on each segment of the ligamentum flavum is shown under resting gravity load and dynamic loads of different directions and intensities.

[0030] Figure 2 The distribution of ossification frequency and stress intensity in the lower thoracic ligamentum flavum region is shown. (A) Fitting map of the stress distribution (ventral view) under a 10000 N / mm rotational load on T10 / 11 and the ossification area in a representative T10 / 11 single-segment ligamentum flavum ossification patient. (B) Comparison of the ossification area distribution map in a single-segment ligamentum flavum ossification patient in the lower thoracic region and the stress distribution (ventral view) under a 10000 N / mm rotational load on T10 / 11.

[0031] Figure 3 The expression of osteogenic markers in ligamentum flavum cells was detected after time-gradient tensile stress intervention. ***P<0.001; **P<0.01; *P<0.05, n=3.

[0032] Figure 4A , Figure 4B and Figure 4C The transcriptome sequencing analysis of human ligamentum flavum tissue is shown (3 vs. 3), in which:

[0033] Figure 4A Heatmap of differential gene expression in the transcriptome between the ossified and non-ossified groups;

[0034] Figure 4B Volcano plot of differential gene expression in transcriptomes between ossified and non-ossified groups;

[0035] Figure 4C Heatmap showing the difference in expression of key ion channel genes in the transcriptome between the ossified and non-ossified groups.

[0036] Figure 5A , Figure 5B , Figure 5C , Figure 5D , Figure 5E , Figure 5F and Figure 5G The ossification phenotype of human ligamentum flavum cells after in vitro intervention with Piezo1 is shown. ***P<0.001; **P<0.01; *P<0.05, n=3, where:

[0037] Figure 5A qRT-PCR detection of osteogenic markers in human ligamentum flavum cells;

[0038] Figure 5B Western blotting was used to detect the expression of various osteogenic markers. GAPDH, glyceraldehyde-3-phosphate dehydrogenase, served as an internal control between experimental groups. Sh.NC represented the group transfected with the blank vector plasmid as a control. Sh.Piezo1 represented the group transfected with a Piezo1-silencing plasmid. Yoda1 represented the group that received the specific Piezo1 agonist Yoda1 (drug name).

[0039] Figure 5C Western blot analysis was performed to detect the expression of various osteogenic markers.

[0040] Figure 5DAlizarin Red staining analysis of osteogenic-induced human ligamentum flavum cells. Control group was the blank control group; Yoda1 group received the specific agonist Yoda1 (drug name); Sh.Piezo1 group was transfected with a Piezo1-silencing plasmid; and Sh.Piezo1+Yoda1 group was transfected with both the Piezo1-silencing plasmid and the Piezo1 agonist Yoda1.

[0041] Figure 5E Alizarin Red staining analysis of osteogenic-inducing culture of human ligamentum flavum cells;

[0042] Figure 5F ALP staining analysis of osteogenic induction culture of human ligamentum flavum cells;

[0043] Figure 5G ALP staining analysis of osteogenic induction culture of human ligamentum flavum cells.

[0044] Figure 6 This study demonstrates the effect of in vivo regulation of Piezo1 on ossification of the ligamentum flavum. (A) Micro-CT images of the thoracic and lumbar spine in mice. (B) Statistical analysis of ligamentum flavum thickness in the thoracic and lumbar spine of mice. (C) IHC staining and analysis of osteogenic markers in the thoracic and lumbar spine of mice. ***P<0.001; **P<0.01; *P<0.05, n=6. The Piezo1CKO+ standing group refers to the group using mice with Piezo1 specifically knocked out in ligament tissue for standing experiments.

[0045] Figure 7 The function of Piezo1 channels in human ligamentum flavum cells is shown, where OLF represents the ossified group and non-OLF represents the non-ossified group. (A) Comparison of intracellular calcium fluorescence fluo-4 imaging before and after the addition of 5 μmol / L Yoda1 (200x). (B) Confocal imaging of the relative intensity change curve of intracellular calcium fluorescence fluo-4 in ligamentum flavum cells before and after Yoda1 concentration gradient intervention. (C) Intracellular calcium fluorescence fluo-4 imaging in ligamentum flavum cells 48 hours after Yoda1 concentration gradient intervention. (D) Patch-clamp current curve when ligamentum flavum cells are stimulated by a mechanical force gradient. (E) Mechanical stimulation intensity-current relationship curve of ligamentum flavum cells. n=3. Wherein, OLFSh.NC refers to the group of ligamentum flavum cells derived from ossified patients transfected with a blank vector plasmid, non-OLF Sh.NC refers to the group of ligamentum flavum cells derived from non-ossified patients transfected with a blank vector plasmid, and OLF Sh.Piezo1 refers to the group of ligamentum flavum cells derived from ossified patients transfected with a Piezo1-silencing plasmid.

[0046] Figure 8Pseudo-time analysis of single-cell sequencing in tissues (3 vs. 3). (A) Pseudo-time cell trajectory analysis. (B) Trends in key genes and gene clustering heatmap (Top 100) in pseudo-time analysis. Component 1 is the first principal component, representing the dimension with the greatest cellular variation in the pseudo-time analysis; component 2 is the second principal component, representing the dimension with the second greatest cellular variation in the pseudo-time analysis; cell_type refers to cell type; and Pseudotime refers to the pseudo-time period.

[0047] Figure 9A , Figure 9B , Figure 9C and Figure 9D The study showed decreased irisin levels in patients with ossification of the ligamentum flavum. ***P<0.001; **P<0.01; *P<0.05, where:

[0048] Figure 9A Volcano plot of differential gene expression in transcriptomes between ossified and non-ossified groups;

[0049] Figure 9B UMAP diagram of FNDC5 expression distribution in ligament cells;

[0050] Figure 9C Serum irisin levels in patients with ossification and non-ossification were measured by ELISA (10v10).

[0051] Figure 9D ELISA detection of irisin content in paraspinal muscles of patients in the ossification group and the non-ossification group (10v10).

[0052] Figure 10 The regulatory effect of irisin on calcium signaling in human ligamentum flavum cells. (A) Comparison of intracellular calcium fluorescence fluo-4 imaging before and after the addition of 5 μmol / L Yoda1 (200x). (B) Confocal imaging of the relative intensity changes of intracellular calcium fluorescence fluo-4 in ligamentum flavum cells before and after Yoda1 intervention at different concentration gradients. In this model, OLF Sh.NC represents the ossified group (Sh.NC), non-OLF Sh.NC represents the non-ossified group (Sh.NC), and OLF Sh.NC Irisin represents the ossified group (Sh.NC + irisin). (C) Mitochondrial calcium ion staining ( -Rhod2 co-staining, 200x). (D) Analysis of mitochondrial calcium content. (E) Patch-clamp current curves of ligamentum flavum cells stimulated by mechanical force gradient. (F) Mechanical stimulation intensity-current relationship curves of ligamentum flavum cells. ***P<0.001; **P<0.01; *P<0.05, n=3.

[0053] Figure 11Regulation of mitochondrial homeostasis in human ligamentum flavum cells by irisin. (A) EdU staining (200x) after irisin intervention. (B) JC-1 staining (200x) after irisin intervention. (C) ROS staining (200x) after irisin intervention, where DCFH-DA is a staining reagent that reflects the level of reactive oxygen species in cells. (D) Quantitative analysis of EDU levels. (E) RNA-seq analysis. (F) Quantitative analysis of ROS levels. (G) Electron microscopy and analysis of mitochondria after irisin intervention. ***P<0.001; **P<0.01; *P<0.05, n=3.

[0054] Figure 12 Regulation of osteogenic differentiation of human ligamentum flavum cells by irisin. (A) qRT-PCR detection of osteogenic markers in human ligamentum flavum cells. (B) Alizarin red staining analysis of human ligamentum flavum cell osteogenic induction culture. (C) ALP staining analysis of human ligamentum flavum cell osteogenic induction culture. ***P<0.001; **P<0.01; *P<0.05, n=3.

[0055] Figure 13 The in vivo regulation of ligamentum flavum ossification by irisin is shown. (A) Micro-CT images of the thoracic and lumbar spine in mice. (B) Statistical analysis of ligamentum flavum thickness in the thoracic and lumbar spine of mice. (C) IHC staining of osteogenic markers in the thoracic and lumbar spine of mice. ***P<0.001; **P<0.01; *P<0.05, n=6.

[0056] Figure 14 A schematic diagram of the mechanism of this invention. Detailed Implementation

[0057] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, they are provided so that the invention will be thorough and complete, and the concept of the exemplary embodiments will be fully conveyed to those skilled in the art.

[0058] Unless otherwise defined, all terms and phrases used herein include their meanings as they have in the art, unless explicitly stated otherwise or clearly indicated from the context of their use. While any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, specific methods and materials are now described.

[0059] Through extensive and in-depth research, the inventors have unexpectedly discovered for the first time that irisin can limit the function of Piezo1 in response to mechanical stimulation, thereby limiting the influx of extracellular calcium ions, stabilizing intracellular calcium signals, maintaining calcium homeostasis in mitochondria, and thus playing a protective role for mitochondria, thereby alleviating osteogenic differentiation of ligamentum flavum cells regulated by Piezo1 activation.

[0060] Specifically, in this invention, in order to find a treatment method for ligamentum flavum ossification (OLF) with minimal impact on physiological state, the inventors, based on previous research findings, identified a myofactor secreted by exercise stimulation: irisin. After observing differences in clinical samples, the inventors verified the regulatory effects of irisin on calcium signaling, mitochondria, and ossification phenotype. Based on the pathogenic mechanism of mechanical stimulation leading to OLF ossification, the invention verified that irisin, as an endogenous substance, can delay the development of OLF ossification, providing important theoretical support for the development of drug therapy, physical therapy, and exercise therapy for OLF ossification. Irisin is an endogenous myofactor secreted by human skeletal muscle after exercise. Numerous studies have confirmed that exercise and electrical muscle stimulation can increase irisin levels in the body, achieving a therapeutic effect on the disease. This research also provides new ideas for the development of drug and physical therapies for OLF in clinical practice. Based on this, this invention was completed.

[0061] the term

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0063] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0064] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0065] Unless otherwise defined, the terms used in the specification and claims have the meanings commonly understood by one of ordinary skill in the art. Unless otherwise stated, all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety.

[0066] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this application, unless specifically stated otherwise, the singular is used to include the plural. It must be noted that unless clearly stated otherwise, the singular form used in this specification and claims includes the plural form of the referred to. It should also be noted that unless otherwise stated, “or” or “or” means “and / or”. Furthermore, the terms “containing” or “comprising” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “made of”.

[0067] As used herein, "active ingredient" and "active ingredient of the present invention" are used interchangeably and both refer to irisin, or its prodrug, or its degradation product, or its fusion protein.

[0068] Piezo1

[0069] Piezo1 is a family of mechanically gated cation channels. This mechanosensor can sense changes in the mechanical forces of the cell membrane, thereby inducing calcium ion influx, realizing the conversion of mechanical signals into bioelectrical signals, and participating in physiological activities such as angiogenesis, erythrocyte volume regulation, and blood pressure homeostasis.

[0070] Pharmaceutical Compositions and Applications

[0071] The present invention also provides the use of one or more of irisin, or a prodrug thereof, or a degradation product thereof, or a fusion protein thereof as an active ingredient in the preparation of a medicament for the prevention and / or treatment of ossification of the ligamentum flavum (OLF) and related diseases.

[0072] The pharmaceutical composition provided by the present invention preferably contains 0.001-99 wt% of an active ingredient, preferably irisin as the active ingredient accounting for 0.1 wt% to 90 wt% of the total weight, with the remainder being a pharmaceutically acceptable carrier, diluent, solution, or salt solution.

[0073] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the aforementioned irisin, or a prodrug thereof, or a degradation product thereof, or a fusion protein thereof, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated.

[0074] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): oral, respiratory, intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition of the present invention is preferably administered by injection or oral administration. Injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions.

[0075] The pharmaceutical composition described in this invention is a pharmaceutical composition for the prevention and / or treatment of diseases associated with ossification of the ligamentum flavum (OLF).

[0076] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described irisin of the present invention, or its prodrug, or its degradation product, or its fusion protein, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 10 mg / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0077] In this invention, preferably, the pharmaceutical composition further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01–99.99% of the above-mentioned polypeptide and 0.01–99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0078] In this invention, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the conventional range.

[0079] When using a pharmaceutical composition, a safe and effective amount of the active ingredient is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 8 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 1 milligram per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0080] The advantages of this invention include:

[0081] (1) Based on the pathogenic mechanism of mechanical stimulation leading to ossification of the ligamentum flavum, the study verified that irisin, as an endogenous substance, can delay the occurrence and development of ossification of the ligamentum flavum, providing important theoretical support for the development of drug therapy, physical therapy and exercise therapy for ossification of the ligamentum flavum.

[0082] (2) Iris extract can be used for the early prevention and treatment of ossification of the ligamentum flavum, and a non-invasive and preventive intervention method for ossification of the ligamentum flavum can be developed to avoid irreversible nerve damage and surgical complications.

[0083] The present invention will be further illustrated below with reference to specific embodiments.

[0084] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Guide* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the manufacturer's recommendations (e.g., product instructions). Unless otherwise stated, percentages and parts are by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available or can be prepared according to literature methods.

[0085] Materials and methods

[0086] Table 1: Main reagents and consumables used in the experiment

[0087]

[0088]

[0089]

[0090]

[0091] Table 2: Main Experimental Instruments

[0092]

[0093]

[0094] The solution of the present invention

[0095] Here, the inventors conduct a preliminary investigation into the therapeutic effect of irisin on ossification of the ligamentum flavum. First, the inventors verified the effect of mechanical stimulation on ossification of the ligamentum flavum through three-dimensional finite element analysis and cell experiments. Single-cell sequencing analysis confirmed that ossified ligamentum flavum is transformed from normal ligamentum flavum; therefore, the essence of treating this disease is to treat the osteogenic differentiation of ligamentum flavum cells. Subsequently, the inventors performed differential analysis on tissue transcriptome and single-cell sequencing results and collected clinical samples to detect irisin content, verifying the differences in disease groups. Next, cell experiments were conducted to verify its regulatory effect on intracellular calcium homeostasis and mitochondrial function. Finally, through in vivo and in vitro experiments, the therapeutic effect of irisin on osteogenic differentiation of the ligamentum flavum was clarified, aiming to provide new insights for the future treatment of ligamentum flavum ossification. The mechanism of this invention is described in [link to invention]. Figure 14 The details are as follows:

[0096] Example 1: Mechanical stimulation is an important factor in the ossification of the ligamentum flavum.

[0097] A retrospective study was conducted on patients with ligamentum flavum ossification. The spatial distribution of ossified segments was analyzed, dividing the thoracic spine into upper thoracic (T1-T4), middle thoracic (T4-T9), and lower thoracic (T9-T12) segments. Of the 151 patients, ossification was confined to a single segment in 95 cases (upper:middle:lower = 11:0:84) (Table 3), while ossification involved multiple segments in 56 cases, indicating a clear localized tendency in the development of ligamentum flavum ossification in the thoracic spine. Further analysis of the frequency of ossification in different segments confirmed that the frequency of ligamentum flavum ossification in the lower thoracic segment was significantly higher than in the other two regions (Table 3). Analysis of the most severely ossified parts of the ligamentum flavum showed that the probability of the most severe ossification was significantly higher in the lower thoracic segment than in the other two regions (Table 3). Furthermore, the results showed that T2 / 3, T6 / 7, and T10 / 11 were the sites with the highest incidence of ossification in the upper, middle, and lower thoracic segments, respectively. Therefore, these segments were used as representatives for subsequent three-dimensional finite element analysis.

[0098] Table 3 Clinical Case Analysis of Thoracic OLF

[0099]

[0100] Three-dimensional finite element models of the ligamentum flavum were constructed using CT and MRI images of healthy volunteers, covering the upper thoracic vertebrae 2-3 (T2 / 3), middle thoracic vertebrae 6-7 (T6 / 7), lower thoracic vertebrae 10-11 (T10 / 11), cervical vertebrae 5-6 (C5 / 6), and lumbar vertebrae 4-5 (L4 / 5). The stress distribution of the ligamentum flavum was analyzed using software such as Mimics 21.0, Geomagic 2021, SolidWorks 2022, and Ansys 23.0. The results showed that ossification of the ligamentum flavum was most common in the lower thoracic segment, and the stress on the ligamentum flavum in the lower thoracic segment was also the highest. Figure 1A , Figure 1B Furthermore, comparing the frequency distribution of ossification areas in 84 patients with single-region OLF in the lower thoracic spine with the stress distribution of the ligamentum flavum analyzed by three-dimensional finite element method, it was found that the stress concentration area of ​​the lower thoracic ligamentum flavum coincided with the high incidence area of ​​ossification of the lower thoracic ligamentum flavum, suggesting a close relationship between tensile stimulation and the occurrence of heterotopic ossification of the ligamentum flavum. Figure 2 This confirms that mechanical stimulation is an important pathogenic factor in ossification of the ligamentum flavum.

[0101] Finally, the extracted ligamentum flavum cells were cultured in vitro using the FlexCell system under tensile stress. A deformation rate of 10%, a frequency of 1 Hz, a dynamic sinusoidal waveform, and time gradients of 2, 4, 6, 8, 12, 24, and 48 hours were used. Bone markers, including bone morphogenetic protein type 2 (BMP2), osteocalcin (OCN), osteopontin (OPN), collagen type I alpha1 (Col1α1), and Runt-related transcription factor 2 (RUNX2), were detected using quantitative real-time polymerase chain reaction (qRT-PCR). This demonstrated that tensile stimulation induced osteogenic activity in ligamentum flavum cells on the cellular level. Figure 3 ).

[0102] Example 2: Mechanical stimulation regulates ossification of the ligamentum flavum by activating Piezo1.

[0103] Transcriptome sequencing was performed on three ossified ligamentum flavum tissue samples from patients with ossified ligamentum flavum and three unossified ligamentum flavum tissue samples from control patients. The results showed that the gene level of the mechanosensitive ion channel Piezo1 was significantly elevated in the ossified ligamentum flavum tissue. Figure 4A , Figure 4B and Figure 4C Using non-ossified ligamentum flavum cells, Piezo1 was activated using the specific activator Yoda1, and Piezo1 knockout plasmids were used to downregulate Piezo1 function to regulate Piezo1 channel function. After 48 hours of intervention with 5 μmol / L Yoda1, qRT-PCR and Western blotting revealed that when Piezo1 channels were specifically activated by Yoda1, the expression levels of osteogenic markers BMP2, OCN, OPN, Col1α1, and RUNX2 increased at both the transcriptomic and protein levels. However, transfection with Sh. Piezo1 to reduce intracellular Piezo1 expression inhibited the osteogenic effect of Yoda1. Figure 5A , Figure 5B , Figure 5C In addition, osteogenic induction was performed on ligamentum flavum cells under the same conditions to regulate Piezo1 channel activity. Alizarin red staining and alkaline phosphatase (ALP) staining were performed on day 21 of osteogenic induction culture. The results showed that after Yoda1 activated the Piezo1 channel, calcium nodule deposition increased and ALP activity rose, suggesting enhanced osteogenic differentiation. However, after Piezo1 knockdown, there was no significant calcium nodule deposition, and ALP activity was significantly lower than in the control group, and could not be restored by Yoda1, suggesting decreased osteogenic differentiation. Figure 5D , Figure 5E , Figure 5F , Figure 5G The results show that the mechanosensitive ion channel Piezo1, as a key element for ligamentum flavum cells to sense mechanical stimulation signals and convert them into biological signals, plays an indispensable role in the osteogenic differentiation of ligamentum flavum cells by activation.

[0104] Furthermore, tamoxifen-induced Piezo1flox / flox-Scx-CreERT2 mice were used as Piezo1-cKO mice to specifically knock out Piezo1 in the ligamentum flavum tissue of mice. Utilizing the hydrophobia-induced standing characteristic of mice, the stress on the ligamentum flavum was increased to construct a mouse model of ligamentum flavum ossification. Mice stood for 6 hours / day (3 hours of standing, followed by 1 hour of rest in their cages), 5 days a week, for a total of 16 weeks. Mice were euthanized on the first day after the end of standing for tissue collection. The spine from the thoracolumbar region down was completely removed, fixed with 4% paraformaldehyde, and subjected to micro-CT imaging. The control group showed generally normal imaging with no obvious ectopic ossification, while the standing group showed ectopic ossification in some segments of the ligamentum flavum (circled in red). When Piezo1 was knocked out in the ligament, the ligamentum flavum ossification caused by the increased stress on the ligamentum flavum due to standing did not occur. Figure 6 (See section A). Next, serial pathological sections were prepared from the specimens, and osteogenic markers were selected for IHC staining. Firstly, the sections revealed that: in the control group, the ligamentum flavum was smooth, with a regular arrangement of ligament fibers and ligament cells distributed parallel to the long axis of the ligament; in the ossification group, the ligamentum flavum showed localized thickening, disordered arrangement of ligament fibers, and the formation of hypertrophic chondrocytes and ossification foci within the ligamentum flavum tissue (see section A). Figure 6 (Red arrow in C) The surrounding ligament cells are scattered; the ligamentum flavum in the Piezo1-cKO group has a slender structure with a large amount of vacuolated fatty tissue in the fibrous structure, and the density of ligament cell nuclei is significantly lower than that in other groups. Figure 6 (B and C). Further analysis of the expression levels of BMP2, OCN, OPN, and Col1α1 in the ligamentum flavum revealed that osteogenic-related markers were upregulated in the ligamentum flavum tissue of mice after 16 weeks of standing. However, knocking out Piezo1 in the ligament did not stimulate the upregulation of osteogenic-related markers; instead, they were downregulated compared to the control group. Figure 6 (C). Based on the above results, it can be inferred that Piezo1 not only mediates stress-induced regulation of ligamentum flavum ossification, but may also participate in the normal growth and development of the ligamentum flavum.

[0105] Subsequently, the inventors identified the function of the Piezo1 channel in human ligamentum flavum cells. First, they activated the Piezo1 channel specifically using Yoda1, and then used a cell membrane-permeable calcium ion fluorescent probe (Fluo-4, AM, Cell Permeant) to detect intracellular calcium ion concentration. Fluo-4 is a non-fluorescent substance, but it produces strong fluorescence when it binds to intracellular calcium ions, which increases with increasing calcium ion concentration. Ligamentum flavum cells from both patients with and without ligamentum flavum ossification were selected and stimulated with 5 μmol / L Yoda1. Video recordings were performed using confocal microscopy to observe the intracellular fluorescence intensity after drug administration, and fluorescence intensity-time curves were plotted to reflect the changes in intracellular calcium ion concentration after Yoda1 activation of the Piezo1 channel. To clarify that the change in intracellular calcium ion concentration was caused by Piezo1 channel activation, ossified cells were transfected with a Piezo1 knockout plasmid, and other ossified and non-ossified ligamentum flavum cells were transfected with an empty vector plasmid. The inventors discovered that ligamentum flavum cells showed a significant increase in intracellular calcium ion concentration after Piezo1 channel activation, and the ossified group cells responded more strongly to the same concentration of Yoda1 stimulation than the non-ossified group. After a short period of peak fluorescence intensity, the calcium ion concentration slowly decreased and gradually stabilized at a level higher than the initial concentration, while the stable level of the ossified group cells was higher than that of the non-ossified cells. When Piezo1 was knocked out, the intracellular calcium concentration did not fluctuate due to Yoda1 stimulation. Figure 7 (A and B). The inventors also stimulated non-ossified ligamentum flavum cells with different concentration gradients of Yoda1 for 48 hours, followed by incubation with Fluo-4 probes. Fluorescence intensity was detected using fluorescence microscopy, confirming that the increased intracellular calcium ion concentration after Yoda1 activation of the Piezo1 channel could be maintained for a relatively long time. Figure 7 (C). Furthermore, the inventors prepared a special detection solution and external solution for the Piezo1 channel. Using mechanical stress to load whole-cell patch clamps, they directly detected the current in ligamentum flavum cells after being subjected to gradient-set mechanical stimulation. Simultaneously, a Piezo1 plasmid knockout group was established, confirming that the current on the cell membrane was mainly caused by the response of the Piezo1 channel to mechanical stimulation. The results showed that mechanical stimulation of ligamentum flavum cells induced a significant inward current, and the current intensity changed positively with the stimulation intensity. Similarly, the ossified group cells responded more strongly to mechanical stimulation than the non-ossified group, and after Piezo1 knockout, this inward current generated with mechanical stimulation almost disappeared. Figure 7(D and E in the text). These results indicate that the Piezo1 channel is a key mechanoreceptor on human ligamentum flavum cells. Its activation primarily induces calcium ion translocation into the cell, increasing intracellular calcium ion concentration. The Piezo1 channel converts mechanotransmission into calcium transduction, potentially representing a crucial upstream link in the osteogenic differentiation mechanism of ligamentum flavum cells. Furthermore, the excessively high sensitivity of Piezo1 to mechanotransmission in ligamentum flavum cells from ossified patients is a significant contributing factor to abnormal osteogenic differentiation in these cells.

[0106] Example 3: Ossified ligamentum flavum originates from the differentiation of normal ligamentum flavum tissue.

[0107] Nine ossified ligamentum flavum tissue samples from patients with ossification and nine unossified ligamentum flavum tissue samples from control patients were collected (intra-group pooling, three samples per group) for single-cell sequencing. Pseudo-time series analysis was used to infer the differentiation trajectories of each cell subset. Results confirmed that the fibrous-like cell subset (F01-F04) was concentrated near the differentiation initiation point, while the ossification-like cell subset (C01-C03) was mostly near the differentiation endpoint, especially C02 and C03, which occupied the majority of cells in the endpoint region. Figure 8 (A). Simultaneously, it was found that at the time endpoint, there was a high expression of osteogenic differentiation-related markers (BMP2, CHAD, CRTAC1, BMP6, etc.) and low expression of fibrous tissue-related markers (FBLN1, etc.), which confirms that ligament cells can differentiate into osteoblast-like cells starting from fibroblast-like cells. Figure 8 (B) The pseudo-time series analysis results show that there are clear evolutionary trajectories among the various cell subpopulations within the ligamentum flavum. During the occurrence and development of ligamentum flavum ossification, the ossified ligamentum flavum cells are differentiated from normal ligamentum flavum cells, rather than evolving from other cell types.

[0108] Example 4: Decreased irisin levels in patients with ossification of the ligamentum flavum

[0109] The precursor protein of irisin is encoded by the FNDC5 gene. First, FNDC5 expression was investigated using tissue transcriptome sequencing results. It was found that FNDC5 expression was downregulated in the osteotomy group, but the difference was not statistically significant. Figure 9A Meanwhile, single-cell sequencing results also showed that FNDC5 expression was higher in fibroblast-like cells than in osteoblast-like cells. Figure 9B These results suggest that irisin secretion may be reduced in the ligamentum flavum of ossified lesions. Furthermore, blood samples and surgically removed paraspinal muscle tissue were collected from some patients, and the irisin content was detected using ELISA. The results showed that the irisin content in muscle tissue was higher than in serum. The irisin content in serum and paraspinal muscle tissue of patients in the ossified group was significantly downregulated compared to patients in the non-ossified group, and the downregulation was more significant in muscle tissue. Figure 9C and Figure 9D This indicates that, for adjacent tissues, the direct paracrine effect of irisin through muscle tissue may be stronger than its endocrine effect via the bloodstream. Furthermore, in patients with ossification, the ligamentum flavum may experience a weaker effect from irisin compared to the non-ossified group. These results validate the differences in the occurrence and development of ligamentum flavum ossification with irisin. The regulatory role of irisin in the pathogenesis of ligamentum flavum ossification will be explored further in the future.

[0110] Example 5: Regulation of intracellular calcium signaling in human ligamentum flavum cells by irisin

[0111] First, the effect of irisin on Piezo1 activation-related calcium signaling was investigated. Osteoplastic ligamentum flavum cells were pre-incubated with irisin (200 ng / ml, 48 hours). Intracellular calcium ion concentration changes were monitored using a Fluo-4 fluorescent probe combined with confocal microscopy. It was found that the addition of irisin did not completely inhibit the calcium ion influx induced by Piezo1 channel activation, but it weakened the Piezo1 channel response intensity in ossified ligamentum flavum cells, making the peak and stable calcium ion concentrations comparable to those in non-ossified cells after Piezo1 channel activation. Figure 10 (A and B). Further, to investigate changes in mitochondrial calcium homeostasis, the inventors selected another calcium ion fluorescent probe, Rhod-2, to stain ossified ligamentum flavum cells. Compared to Fluo-4, Rhod-2 is more sensitive to changes in calcium ion levels, is localized to mitochondria, and is a long-wavelength calcium ion probe. It was then compared with a mitochondrial green fluorescent probe (…). Co-staining with Green FM was used to precisely locate calcium ions in mitochondria, and changes in their concentration were observed one hour after drug administration. The results showed that yoda1 (5 μmol / L) activation of the Piezo1 channel led to an upregulation of calcium ion concentration in mitochondria, and the addition of irisin partially reversed this phenomenon. Figure 10 (C and D). Tracing the source of changes in calcium ion concentration, the inventors used a whole-cell patch-clamp system under mechanical stress to detect the inward current mediated by the Piezo1 channel. They found that irisin could attenuate the Piezo1 channel's response to mechanical stimulation, but did not completely inhibit Piezo1 channel function. Figure 10 (E and F in the middle). In summary, irisin can inhibit the excessive response of Piezo1 channels to stimuli, stabilize intracellular calcium ion concentration, and protect mitochondrial calcium homeostasis.

[0112] Example 6: Regulation of mitochondrial homeostasis in human ligamentum flavum cells by irisin

[0113] After clarifying that irisin regulates intracellular calcium signaling, the inventors tested indicators reflecting mitochondrial functional morphology. Non-ossified ligamentum flavum cells were selected and stained with EdU and JC-1. The results showed that irisin (200 ng / ml, 48 hours) could treat the increased cell proliferation and apoptosis induced by Yoda1 (5 μmol / L, 48 hours), downregulate the positive rate of EdU in cells, partially restore RNA-seq, and stabilize the cell state. Figure 11 (A, B, D, and E). DCFH-DA staining showed that irisin could reduce ROS levels induced by yoda1 addition and alleviate cellular oxidative stress. Figure 11 (C and F). Furthermore, transmission electron microscopy of the cells revealed that, compared to Yoda1 stimulation alone, the addition of irisin reduced mitochondrial division and fusion, resulting in morphology closer to normal and restoration of damaged cells. Figure 11 (G). This part of the results confirms that irisin has a protective effect on mitochondrial homeostasis.

[0114] Example 7: The therapeutic effect of irisin on ossification of the ligamentum flavum

[0115] Finally, the inventors conducted preliminary verification of the effect of irisin in treating ossification of the ligamentum flavum. In the in vitro experiment, ligamentum flavum cells from the non-ossified group were used. After intervention with Yoda1 (5 μmol / L, 48 hours) and irisin (200 ng / ml, 48 hours), osteogenic markers were detected by qRT-PCR. The results showed that irisin could improve the upregulation of osteogenic markers in ligamentum flavum cells induced by Piezo1 activation. Figure 12 (A). Furthermore, 21 days after osteogenic induction and drug stimulation, the area of ​​positive regions for Alizarin Red and ALP was significantly reduced in the irisin treatment group, suggesting that irisin weakened the effect of Piezo1 activation stimulating osteogenic differentiation of ligamentum flavum cells. Figure 12 (Figures B and C). In the in vivo experimental section, the mouse hydrophobia standing model was used to establish the ossification of the ligamentum flavum. Simultaneously, one group of mice was intraperitoneally injected with physiological saline solution of irisin (400 μg / kg, twice a week for 16 weeks). Micro-CT results of the thoracic and lumbar spine showed that no obvious ossification signal was observed in the ligamentum flavum of the mice in the irisin treatment group. Figure 13 (A). Subsequently, the specimens were sectioned and IHC stained for osteogenic markers. The sections showed that the ligamentum flavum morphology in the irisin-treated group was normal, and the thickness of the ligamentum flavum in the control group was comparable. The expression levels of most osteogenic markers were also downregulated compared to the simple standing group, and were close to those in the control group. Figure 13 (B and C). These results indicate that irisin can treat and inhibit abnormal osteogenic activity of ligamentum flavum cells caused by mechanical stimulation without disrupting the normal structure and physiological function of the tissue.

[0116] discuss

[0117] Based on the fact that the therapeutic mechanism of irisin aligns with the pathogenic mechanism of ligamentum flavum ossification related to mechanical stimulation verified by the inventors, the inventors hypothesize that irisin may have a therapeutic effect on ligamentum flavum ossification. Given that irisin is an endogenous substance that can be naturally produced through exercise, it may be a less physiologically disruptive intervention, thus providing a basis for exercise therapy. Based on preliminary research, the inventors speculate that irisin may have a positive effect on the prevention and treatment of ligamentum flavum ossification. If its therapeutic effect on ligamentum flavum ossification can be verified and used for its early prevention and treatment, a non-invasive, preventative intervention for ligamentum flavum ossification could be developed, avoiding irreversible nerve damage and surgical complications.

[0118] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the appended claims.

[0119] References

[0120] [1]Maak S,Norheim F,Drevon CA,et al.Progress and Challenges in theBiology of FNDC5and Irisin[J].Endocr Rev,2021,42(4):436-56.

[0121] [2]Bao JF,She QY,Hu PP,et al.Irisin,a fascinating field in our times[J].Trends EndocrinolMetab,2022,33(9):601-13.

[0122] [3] P,Wu J,Jedrychowski MP,et al.A PGC1-α-dependent myokinethat drives brown-fat-like development of white fat and thermogenesis[J].Nature,2012,481(7382):463-8.

[0123] [4]Kim H,Wrann C D,Jedrychowski M,et al.Irisin Mediates Effects onBone and Fat viaαVIntegrin Receptors[J].Cell,2018,175(7):1756-68.e17.

[0124] [5]Wang J,Zhu Q,Wang Y,et al.Irisin protects against sepsis-associated encephalopathy bysuppressing ferroptosis via activation of theNrf2 / GPX4 signal axis[J].Free Radic BiolMed,2022,187:171-84.

[0125] [6]Slate-Romano J J,Yano N,Zhao T C.Irisin reduces inflammatorysignaling pathways ininflammation-mediated metabolic syndrome[J].Mol CellEndocrinol,2022,552:111676.

[0126] [7]Zhou W,Shi Y,Wang H,et al.Exercise-induced FNDC5 / irisin protectsnucleus pulposuscells against senescence and apoptosis by activatingautophagy[J].Exp Mol Med,2022,54(7):1038-48.

[0127] [8]Perakakis N,Triantafyllou G A,Fernández-Real J M,et al.Physiologyand role of irisin inglucose homeostasis[J].Nat Rev Endocrinol,2017,13(6):324-37.

[0128] [9]Guo J J,Luk K D,Karppinen J,et al.Prevalence,distribution,andmorphology ofossification of the ligamentum flavum:a population study of onethousand seven hundredthirty-six magnetic resonance imaging scans[J].Spine(Phila Pa 1976),2010,35(1):51-6.

[0129]

[10] Hur H,Lee J K,Lee J H,et al.Thoracic myelopathy caused byossification of theligamentum flavum[J].J Korean Neurosurg Soc,2009,46(3):189-94.

[0130]

[11] Daniels A H,Mcdonald C L,Basques B A,et al.Ossified LigamentumFlavum:Epidemiology,Treatment,and Outcomes[J].J Am Acad Orthop Surg,2022,30(12):e842-e51.

[0131]

[12] Liang H,Liu G,Lu S,et al.Epidemiology of ossification of thespinal ligaments andassociated factors in the Chinese population:a cross-sectional study of 2000consecutiveindividuals[J].BMC Musculoskelet Disord,2019,20(1):253.

[0132]

[13] Fujimori T,Watabe T,Iwamoto Y,et al.Prevalence,Concomitance,andDistribution ofOssification of the Spinal Ligaments:Results of Whole Spine CTScans in 1500JapanesePatients[J].Spine(Phila Pa 1976),2016,41(21):1668-76.

[0133]

[14] Mori K,Kasahara T,Mimura T,et al.Prevalence,distribution,andmorphology of thoracicossification of the yellow ligament in Japanese:resultsof CT-based cross-sectional study[J].Spine(Phila Pa 1976),2013,38(19):E1216-22.

[0134]

[15] Kim S I,Ha K Y,Lee J W,et al.Prevalence and related clinicalfactors of thoracicossification of the ligamentum flavum-a computedtomography-based cross-sectional study[J].Spine J,2018,18(4):551-7.

[0135]

[16] Moon B J,Kuh S U,Kim S,et al.Prevalence,Distribution,andSignificance of IncidentalThoracic Ossification of the Ligamentum Flavum inKorean Patients with Back or LegPain:MR-Based Cross Sectional Study[J].JKorean Neurosurg Soc,2015,58(2):112-8.

[0136]

[17] Aizawa T,Sato T,Tanaka Y,et al.Thoracic myelopathy in Japan:epidemiologicalretrospective study in Miyagi Prefecture during 15 years[J].Tohoku J Exp Med,2006,210(3):199-208.

[0137]

[18] Wang T,Pan M,Yin C Q,et al.Spinal Cord Kinking in ThoracicMyelopathy Caused byOssification of the Ligamentum Flavum[J].Chin Med J(Engl),2015,128(19):2595-8.

[0138]

[19] Feng F B,Sun C G,Chen Z Q.Progress on clinical characteristicsand identification oflocation of thoracic ossification of the ligamentumflavum[J].Orthop Surg,2015,7(2):87-96.

Claims

1. The use of irisin, or its prodrug, or its degradation product, or its fusion protein, characterized in that, Used to prepare a pharmaceutical composition for the prevention and / or treatment of ossification of the ligamentum flavum.

2. The use as described in claim 1, characterized in that, The ossification of the ligamentum flavum mentioned above is an ossification of the ligamentum flavum caused by mechanical stimulation.

3. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to regulate calcium signaling.

4. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to reduce intracellular ROS levels and / or alleviate cellular oxidative stress.

5. The use as described in claim 1, characterized in that, The pharmaceutical composition described above is also used to protect mitochondrial homeostasis.

6. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to downregulate the expression of osteogenic-related markers in mammalian ligamentum flavum cells.

7. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to inhibit osteogenic differentiation of ligamentum flavum cells.

8. The use as described in claim 7, characterized in that, The osteogenic differentiation of the ligamentum flavum cells is caused by the activation of the Piezo1 channel.

9. The use as described in claim 1, characterized in that, The pharmaceutical composition is also used to inhibit osteogenic differentiation of ligamentum flavum cells induced by mechanical stimulation.

10. A method for preventing and / or treating ossification of the ligamentum flavum, characterized in that, The steps include: administering a safe and effective amount of irisin, or its prodrug, or its degradation product, or its fusion protein to the desired subject.

Images