Application of L-histidine in treatment of osteoarthritis

Abstract

The invention discloses application of L-histidine in treatment of osteoarthritis, and belongs to the technical field of medicine application. Through verification of cell experiments and animal experiments, it is found that L-histidine can improve osteophyte hyperplasia, meanwhile, expression of matrix metalloproteinases MMP13 and MMP3 and expression of proteoglycan ADAMTS5 can be reduced, expression of SOX9, Aggrecan and COL2A1 in extracellular matrixes can be effectively increased, and therefore the effect of treating osteoarthritis is achieved; the invention clarifies the influence and action mechanism of L-histidine on osteoarthritis, and provides a new thought for clinical treatment of osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the technical field of drug application, in particular to the application of L-histidine in treating osteoarthritis. Background Art

[0002] Osteoarthritis (OA) is the most common degenerative musculoskeletal disease. In an aging society, it has become a major social health problem that cannot be ignored by the elderly. OA can affect various synovial joints, such as the most common knee, lumbar spine, cervical spine, ankle joints, etc. Although the pathological process of OA formation varies in different types of joints, it is usually characterized by progressive degeneration of articular cartilage, synovitis and bone remodeling.

[0003] In recent years, an increasing number of studies have shown that metabolic disorders are closely related to the development of OA. In vivo and in vitro studies have shown a potential link between OA and metabolic disorders. Several recent metabolomics studies have reported many circulating biomarkers in humans and animal models, including amino acids, carbohydrates, and lipids. Although considerable efforts have been made to understand the nature of OA, the mechanisms and risk factors of OA remain elusive.

[0004] Functional amino acids play an important role in improving the health, survival, growth and development of organisms by regulating key metabolic pathways. In bones, functional amino acids are not only important components of the bone matrix, but also regulators of bone growth, maturation and homeostasis. Metabolomics analysis has been used to describe the metabolic pathways of various functional amino acids, as well as the regulatory role of major metabolic intermediates and enzymes in bone homeostasis. As a type of functional amino acid, small amino acid molecules have shown significant advantages in the construction of nanopharmaceutical preparations due to their diversity, clear structure, easy modification and low immunogenicity. For example, selenomethionine (SeMet) can act as an antioxidant and GPX-like redox catalyst, and can also supplement selenium levels, which may have a beneficial effect on inhibiting the progression of OA.

[0005] L-Histidine is a component of protein with high safety. It plays a unique role in proton buffering, metal ion chelation, active oxygen and nitrogen species scavenging, erythropoiesis and histaminergic system. Histidine metabolism is physiologically involved in key pathways such as satiety, recognition memory, skin, neuroprotection and inflammatory diseases. Metabolomics analysis has previously found that a variety of substances including L-histidine play an important role in the occurrence and development of osteoarthritis. The present invention uses L-histidine to treat osteoarthritis, studies the phenotypic changes during the treatment process, and explains the internal mechanism to prove the efficacy of the amino acid, which is of great significance for the treatment of osteoarthritis. Summary of the invention

[0006] The purpose of the present invention is to provide an application of L-histidine in the treatment of osteoarthritis to solve the problems existing in the above-mentioned prior art. L-histidine can improve osteophyte hyperplasia and play a role in treating osteoarthritis. The present invention provides a new idea for the clinical treatment of osteoarthritis.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides application of L-histidine in preparing medicine for treating osteoarthritis.

[0009] Optionally, the L-histidine plays a role in treating osteoarthritis by improving bone hyperplasia;

[0010] The osteoarthritis includes osteoarthritis caused by inflammation or trauma.

[0011] Optionally, the L-histidine plays a role in treating osteoarthritis by inhibiting the expression of MMP13, MMP3 and ADAMTS5.

[0012] Optionally, the L-histidine plays a role in treating osteoarthritis by upregulating the expression of SOX9, Aggrecan and COL2A1.

[0013] The present invention also provides a drug for treating osteoarthritis, wherein the active ingredient of the drug is L-histidine;

[0014] The drug further comprises a pharmaceutically acceptable excipient;

[0015] The auxiliary materials include at least one of diluents, fillers, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

[0016] Optionally, the drug plays a role in treating osteoarthritis by improving bone hyperplasia.

[0017] Optionally, the dosage form of the drug includes a liquid dosage form.

[0018] Optionally, the drug may be administered orally or parenterally.

[0019] The present invention discloses the following technical effects:

[0020] The present invention has been verified through cell experiments and animal experiments, and found that L-histidine can improve osteophyte hyperplasia, and can also down-regulate the expression of matrix metalloproteinases MMP13, MMP3 and proteoglycanase ADAMTS5, and effectively up-regulate the expression of SOX9, Aggrecan and COL2A1 in the extracellular matrix, thereby playing a role in treating osteoarthritis. The present invention clarifies the influence and mechanism of action of L-histidine on osteoarthritis, and provides a new idea for the clinical treatment of osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The metabolomics test results in Example 1;

[0023] Figure 2 The effect of L-histidine on each group after LPS stimulation in Example 2; A: the expression level of each protein in chondrocytes of each group; B: the expression level of each gene in chondrocytes of each group; C: the upper figure is the immunofluorescence image of MMP-13 and COL2A1, and the lower figure is the quantitative statistical graph;

[0024] Figure 3 The effect of L-histidine on each group after IL-1β stimulation and TNF-α stimulation in Example 2; A: the expression level of each protein in each group of chondrocytes after IL-1β stimulation; B: the expression level of each gene in each group of chondrocytes after IL-1β stimulation; C: the expression level of each protein in each group of chondrocytes after TNF-α stimulation; D: the expression level of each gene in each group of chondrocytes after TNF-α stimulation;

[0025] Figure 4 The figures are the results of animal experiments in Example 3; A: C57BL / 6 mouse modeling, drug administration and in vivo detection and evaluation flow chart; B: The left side is the SafraninO / FastGreen staining result, and the right side is the OARSI grading result; C: The left side is the immunohistochemical staining image, and the right side is the quantitative result; D: The left side is the micro-CT scan image; The right side is the statistical graph of the number of osteophytes. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1 Metabolomics Analysis of Human Joint Fluid

[0032] Metabolomics and PCA analysis were performed on 20 pairs of synovial fluid from normal subjects and osteoarthritis patients. The results showed that compared with the normal group (Control), the L-histidine metabolites in the synovial fluid of osteoarthritis patients (Model) were significantly downregulated (see Figure 1 ).

[0033] Example 2 Cell experiment

[0034] 1. Western Blot

[0035] Chondrocytes in the logarithmic growth phase were divided into four groups: control group (NC), model group (LPS), 10mM L-histidine treatment group (LPS+L-Histidine 10mM) and 20mM L-histidine treatment group (LPS+L-Histidine 20mM). Chondrocytes were stimulated with lipopolysaccharide to construct a cell inflammation model. After the modeling was completed, the L-histidine treatment group was given the corresponding concentration of L-histidine.

[0036] After 48 h, chondrocytes were lysed using RIPA lysis buffer (Beyotime, Shanghai, China). The cell lysate was first subjected to polyacrylamide gel electrophoresis, and then the protein bands were transferred to polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, St. Louis, MO, USA) by transfer. Then, the membrane bands were blocked with 5% BSA (Beyotime, Shanghai, China) for one hour, and then the membrane bands were incubated with the corresponding primary antibodies at 4 ° C overnight. Then wash and incubate with the secondary antibody at room temperature for one hour. Protein bands were detected using Fude ECL luminescent solution (Fudebio, Hangzhou, China), and protein images were taken using a chemiluminescence system (Bio-Rad, USA). The signal intensity was quantified using Image J software. The following antibodies were used: β-actin antibody (1:1000, 4970s, Cell Signaling Technology), MMP3 antibody (1:1000, ab52915, Abcam), MMP13 antibody (1:1000, ab51072, Abcam), ADAMTS5 antibody (1:1000, ab41037, Abcam), SOX9 antibody (1:1000, ab185966, Abcam), Aggrecan antibody (1:1000, ab3778, Abcam), COL2A1 antibody (1:1000, ab34712, Abcam). Figure 2 As shown in A, the results of Western Blot immunoblotting experiments showed that compared with the LPS group, L-histidine treatment in chondrocytes could down-regulate the expression of MMP13 (matrix metalloproteinase), MMP3 (matrix metalloproteinase) and ADAMTS5 (proteoglycanase), and up-regulate the expression of SOX9, Aggrecan and COL2A1 in the extracellular matrix, indicating that L-histidine can inhibit the synthesis of degradases and promote the production of synthases, and there is a concentration gradient effect.

[0037] 2. Real-time fluorescence quantitative PCR

[0038] Real-time fluorescence quantitative PCR (qRT-PCR) was performed on chondrocytes in each group. According to the instructions of the reagent manufacturer, TRIzol reagent (Invitrogen, USA) was used to extract total RNA of chondrocytes. 1g of total RNA was reverse transcribed into cDNA using PrimeScript RT kit (Accurate Biology, China). In order to evaluate the expression level of candidate genes, qRT-PCR experiments were performed using SYBR Green PCR kit (Yeasen Biotechnology, China) and evaluated in ABI 7900HT system (Applied Biosystems, USA). 2 -△△CT The relative expression level of mRNA was calculated by the method and the expression level of β-actin was normalized. Figure 2 As shown in B, the results of qRT-PCR experiments showed that compared with the LPS group, L-histidine treatment in chondrocytes could inhibit the synthesis of degradative enzymes, promote the increase of synthetic enzyme production, and downregulate the levels of inflammatory factors (IL-6 and TNF-α), and there was a concentration gradient effect.

[0039] 3. Immunofluorescence

[0040] Each group of cells was fixed on a glass slide with 4% paraformaldehyde for 30 minutes, and then washed three times with PBS for 5 minutes each time. The cells were incubated with PBS containing 0.5% Triton X-100 for 15 minutes and blocked with 5% BSA for 1 hour at room temperature. The following antibodies were then diluted with PBS at a dilution of 1:100: MMP13 antibody (18165-1-AP, Proteintech, Wuhan, China) and COL2A1 antibody (ab34712, Abcam, Cambridge, UK). The cells were incubated overnight at 4°C with the above antibodies. The cells were then washed three times with PBS, incubated with goat anti-rabbit IgG (H&L) at room temperature for 1 hour, and then washed. The cell nuclei were stained with DAPI for 5 minutes. The fluorescence brightness of MMP13 and COL2A1 proteins was observed using a fluorescence microscope (Carl Zeiss, USA). The results of chondrocyte MMP13 and COL2A1 immunofluorescence are shown in Figure 2. Figure 2 As shown in C, compared with the LPS group, L-histidine treatment in chondrocytes can inhibit the expression of MMP13 and promote the increased production of COL2A1, and there is a concentration gradient effect.

[0041] IL-1β and TNF-α were used to replace lipopolysaccharide to construct a cellular inflammation model, and the above-mentioned "1. Protein immunoblotting" and "2. Real-time fluorescence quantitative PCR" tests were performed. The results showed that the cellular inflammation model constructed by treating IL-1β and TNF-α with L-histidine showed the same trend as the above experimental results ( Figure 3 ).

[0042] Example 3 Animal Experiment

[0043] 1. Mouse model construction and drug administration

[0044] Grouping: 8-week-old male C57BL / 6 mice (n=27) were used for in vivo experiments. The flowchart of mouse modeling, drug administration and in vivo testing and evaluation is shown in Figure 4 Middle A. Mice were randomly divided into three groups (n=9 / group): NC group (negative control group), model group (DMM), and L-histidine treatment group (DMM+L-Histidine); the DMM group used medial meniscus destabilization (DMM) surgery to construct post-traumatic osteoarthritis as a positive control; the sham operation group was the negative control group (NC); osteoarthritis model mice were injected with L-histidine, which was the DMM+L-Histidine group.

[0045] Modeling: After C57BL / 6 mice were anesthetized with pentobarbital, the medial joint capsule of the knee joint was incised, and then the medial meniscus ligament (MMTL) was cut with microsurgical scissors to destroy the stability of the medial meniscus. The mice were then rinsed with sterile saline and the incision was closed with sutures to construct the DMM model.

[0046] One week after surgery, 2.5 mg of L-Histidine was dissolved in 0.2 mL of PBS and injected into the abdominal cavity of each mouse in the third group (DMM+Histidine) once every 2 days, and 0.2 mL of PBS was injected into the NC mice in the control group once every 2 days. Two months after treatment, the mice were killed, and the bilateral knee cartilage was collected, and a part of it was taken to make paraffin sections for subsequent analysis.

[0047] 2. SafraninO / FastGreen staining

[0048] After paraffin sections were dewaxed to water, they were stained with 0.1% SafraninO stain and 0.001% FastGreen stain (Sigma-Aldrich, St. Louis, MO, USA) for 5 minutes, respectively, and then dehydrated to wax seal. Images of SafraninO / FastGreen stained sections were obtained using a microscope (CX33TRF, Olympus, Tokyo, Japan). The staining results are shown in Figure 4 As shown in the left figure of middle B, intraperitoneal injection of L-histidine can rescue the cartilage matrix degradation caused by DMM. The severity of osteoarthritis in the slice specimens was assessed using the OARSI grading system. The OARSI score is as follows Figure 4 As shown in the right figure of middle B, the grading results showed that the DMM group had obvious pathological degeneration, and L-histidine improved the pathological degeneration.

[0049] 3. Immunofluorescence

[0050] The paraffin sections were fixed on glass slides with 4% paraformaldehyde for 30 minutes, and then rinsed three times with PBS buffer for five minutes each time. The paraffin sections were then incubated with PBS buffer containing 0.5% TritonX-100 for 15 minutes, and then blocked with 5% BSA for 1 hour at room temperature. The following antibodies were diluted with PBS at a dilution of 1:100: MMP13 antibody, COL2A1 antibody, and the sections were incubated with the above antibodies at 4°C overnight. Then they were washed three times with PBS, and the paraffin sections were incubated with goat anti-rabbit IgG (H&L) at room temperature for 1 hour, and then washed. The cell nuclei were stained with DAPI for 5 minutes. The fluorescence brightness of the target protein was observed by fluorescence microscopy (CarlZeiss, USA). Tissue immunofluorescence staining results of MMP13 and COL2A1 Figure 4 As shown in Figure C, the results showed that compared with the DMM group, the DMM+L-Histidine group could effectively inhibit the production of MMP13 and increase COL2A1.

[0051] 4. Micro-CT analysis

[0052] Knee joint specimens fixed in paraformaldehyde were scanned using high-resolution micro-CT (Skyscan 1072; Skyscan, Aartselaar, Belgium) with a scanning tube volume of 11 mm 3 , current is 180mA, voltage is 55kV, scanning time is 115 minutes, the scanning results are as follows Figure 4 The data were analyzed using Skyscan software (VG Studio MAX; Volume Graphics, Heidelberg, Germany). Figure 4 As shown in the right picture of D. Figure 4 The results in Figure 3 showed that the knee joints of the NC group were normal, while the knee joints of the DMM group had obvious osteophyte hyperplasia. Compared with the DMM group, the amount of osteophyte hyperplasia in the DMM+L-Histidine group was significantly reduced.

[0053] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of L-histidine in the preparation of drugs for treating osteoarthritis.

2. The use according to claim 1, characterized in that The L-histidine plays a role in treating osteoarthritis by improving bone hyperplasia; The osteoarthritis includes osteoarthritis caused by inflammation or trauma.

3. The use according to claim 1, characterized in that The L-histidine plays a role in treating osteoarthritis by inhibiting the expression of MMP13, MMP3 and ADAMTS5.

4. The use according to claim 1, characterized in that The L-histidine plays a role in treating osteoarthritis by upregulating the expressions of SOX9, Aggrecan and COL2A1.

5. A drug for treating osteoarthritis, characterized in that: The active ingredient of the drug is L-histidine; The drug further comprises a pharmaceutically acceptable excipient; The auxiliary materials include at least one of diluents, fillers, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

6. The drug according to claim 5, characterized in that The drug plays a role in treating osteoarthritis by improving osteophyte hyperplasia.

7. The drug according to claim 5, characterized in that The dosage form of the drug includes a liquid dosage form.

8. The drug according to claim 5, characterized in that The administration method of the drug includes oral or parenteral administration.

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