The present disclosure relates to the field of biotechnology, and in particular, to a use of α-ketoglutarate in the manufacture of a medicament.
Osteoarthritis (OA) is a common chronic joint disease. The prevalence of osteoarthritis is high, ranking it as the sixth most disabling disease in the world. By 2020, osteoarthritis is expected to be the fourth most disabling disease. The onset of the disease is mostly seen in middle-aged and elderly people, more women than men. Osteoarthritis mostly occurs in a heavily loaded knee, ankle, spine, and frequently moving finger joints. The main pathological changes in osteoarthritis include degeneration of the articular cartilage. Clinical symptoms of osteoarthritis usually include joint pain, pressing pain, stiffness and functional limitation, with synovitis and secondary osteophyte formation. Osteoarthritis leads to destruction of articular cartilage, as well as subchondral bone sclerosis. These pathological changes are closely related to the painful symptoms of the patients' joints and cause joint mobility impairment and joint deformity, which severely affects the patients' quality of life, causes or exacerbates other comorbidities, and reduces life expectancy. In advanced stages of this disease, the articular cartilage will be extensively stripped and the subchondral bone will be directly stressed, resulting in loss of joint function and eventual disability. The cause of osteoarthritis is not fully understood; its development is a long-term, chronic, and progressive pathological process, with age, obesity, mechanical injury, and genetics being the main risk factors. Each year, more than 200 million people seek medical attention for symptoms associated with osteoarthritis. With the rapidly increasing elderly population, the socioeconomic impact of osteoarthritis will become more significant.
Most clinical treatments such as pharmacological pain relief, physical therapy and joint cavity injection can only relieve the symptoms of osteoarthritis but do not prevent the destruction of articular cartilage and the formation and development of secondary osteophyte. Articular cartilage is composed of chondrocytes and cartilage matrix. Chondrocytes are the only cells present in articular cartilage tissue and play an important role in maintaining, remodeling and repairing cartilage, a non-vascular tissue. Chondrocytes integrate and respond to signals from different types and intensities of biomechanical stimuli (shear force, stress and pressure), and remodel the extracellular matrix to adapt to mechanical stimuli through the secretion of synthetic and catabolic factors. In this process, chondrocytes achieve a dynamic balance in the cartilage matrix by balancing synthetic (production of type II collagen and proteoglycan) and catabolic processes (production of various enzymes that degrade matrix components). In summary, the imbalance between synthetic and catabolic processes in cartilage tissue leads to the development of osteoarthritis.
Alpha-ketoglutarate (α-KG) is an intermediate product of action in the tricarboxylic acid cycle and a downstream metabolite for the synthesis of various amino acids and proteins, such as glutamine (Gln). It has been shown that Gln-derived α-ketoglutarate regulates macrophage differentiation to the M2 phenotype and inhibits macrophage differentiation to the M1 phenotype via the histone demethylase JMJD3. Also, α-KG inhibits the formation of p-IKKα/β and suppresses the expression of induced inflammatory factors.
Currently, there are no reports of α-KG in the treatment of arthritis; therefore, providing a medicament containing α-KG has great potential application.
The present disclosure provides a use of alpha-KG in the manufacture of a medicament, the medicament being used for: 1) treating osteoarthritis and related diseases; and/or, 2) inhibiting the catabolic phenotype of chondrocytes; and/or, 3) promoting the synthetic phenotype of chondrocytes; and/or, 4) promoting the regeneration of skin follicles.
The present disclosure further provides a pharmaceutical composition.
A first aspect of the present disclosure provides a use of alpha-KG in the manufacture of a medicament, the medicament being used for: 1) treating osteoarthritis and related diseases; and/or, 2) inhibiting the catabolic phenotype of chondrocytes; and/or, 3) promoting the synthetic phenotype of chondrocytes; and/or, 4) promoting the regeneration of skin follicles.
Optionally, the α-KG is used to inhibit the activation and/or transduction of the NF-κB signaling pathway in osteocytes.
Optionally, the α-KG regulates the catabolic phenotype and/or synthetic phenotype of chondrocytes in an interleukin-1β (IL-1β) stimulated environment.
Optionally, the IL-1β is used to stimulate chondrocytes.
Optionally, the concentration of IL-1β is 1-15 ng/ml.
Optionally, the form of α-ketoglutarate in the medicament is dimethyl-α-ketoglutarate.
Optionally, the medicament is administered via intra-articular injection.
A second aspect of the present disclosure provides a pharmaceutical composition, which includes α-KG.
Optionally, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
As described above, the present disclosure provides a use of α-KG in the manufacture of a medicament, and a pharmaceutical composition. According to the use of the present disclosure, the medicament is used for: 1) treating osteoarthritis and related diseases; and/or, 2) inhibiting the catabolic phenotype of chondrocytes; and/or, 3) promoting the synthetic phenotype of chondrocytes; and/or, 4) promoting the regeneration of skin hair follicles. The present disclosure also discloses the regulation of α-KG on the phenotypes of chondrocytes under the stimulation of IL-1β. In addition, the present disclosure also established a model simulating osteoarthritis in vivo, which proved that the administration of α-KG via intra-articular injection could significantly inhibit the progression of osteoarthritis. Therefore, the pharmaceutical composition of the present disclosure can protect cartilage tissue and impede the progression of osteoarthritis, and has great potential application for the clinical treatment and prevention of osteoarthritis. Further, the medicament provided by the present disclosure can significantly increase the number of skin hair follicles, which also has great potential application value for the treatment and improvement of hair loss. Other features, advantages and effects can be referred to the content disclosed within the claims and specification of the present disclosure.
The embodiments of the present disclosure will be described below. Those skilled in the art can easily understand other advantages and effects of the present disclosure according to contents disclosed by the specification. The present disclosure may also be implemented or applied through other different specific implementation modes. Various modifications or changes may be made to all details in the specification based on different points of view and applications without departing from the spirit of the present disclosure.
Before further describing the specific embodiments of the present disclosure, it should be understood that the scope of protection of the present disclosure is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present disclosure are just for describing the specific embodiments instead of limiting the scope of the present disclosure. In the present specification and claims, the singular forms “a”, “an” and “the” include the plural forms, unless specifically stated otherwise.
When the numerical values are given by the embodiments, it is to be understood that the two endpoints of each numerical range and any one between the two may be selected unless otherwise stated. Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as commonly understood by one skill in the art. In addition to the specific method, equipment and material used in the embodiments, any method, equipment and material in the existing technology similar or equivalent to the method, equipment and material mentioned in the embodiments of the present disclosure may be used to realize the invention according to the grasp of the existing technology and the record of the invention by those skilled in the art.
A first aspect of the present disclosure provides a use of α-KG in the manufacture of a medicament, the medicament being used for: 1) treating osteoarthritis and related diseases; and/or, 2) inhibiting the catabolic phenotype of chondrocytes; and/or, 3) promoting the synthetic phenotype of chondrocytes; and/or, 4) promoting the regeneration of skin follicles.
In a specific embodiment of the present disclosure, the α-KG is used, for example, to inhibit the activation and/or transduction of the NF-κB signaling pathway in osteocytes. Further, the osteocytes may be, for example, chondrocytes in cartilage tissues. The concentration of the α-KG may be, for example, 3 to 20 mM. Further, to improve the effective inhibition to osteoarthritis, the concentration of the α-KG may be 3 to 10 mM, for example, 3 mM, 5 mM, 7 mM, or 8 mM. Further, the form of α-KG in the medicament is dimethyl-α-ketoglutarate, which enters the cell for regulatory effect.
In a specific embodiment of the present disclosure, the α-KG regulates the catabolic phenotype and/or synthetic phenotype of chondrocytes in an IL-1β-stimulated environment. Further, the IL-1β is used to stimulate chondrocytes, and the concentration of IL-1β is 1-15 ng/ml, such as 1 ng/ml, 5 ng/ml, or 10 ng/ml.
A second aspect of the present disclosure provides a pharmaceutical composition, which includes α-KG.
The pharmaceutical composition may further include a pharmaceutically acceptable carrier. The carriers may include various excipients and diluents that are not themselves essential active ingredients and are not unduly toxic upon application. The dosage to be considered in the administration of the pharmaceutical composition should depend on the frequency and pattern of administration, the age, gender, weight, and general condition of the subject being treated, the condition and severity of the disease, the route of administration, any accompanying diseases to be treated, and other factors apparent to those skilled in the art. Also, depending on the condition of the subject being treated and other pathological conditions, the pharmaceutical composition of the present disclosure may be administered or applied in combination with one or more other therapeutically active compounds or substances. In a specific embodiment of the present disclosure, the medicament or pharmaceutical composition may be administered via intra-articular injection.
The present disclosure also conducted experiments in which IL-1β stimulation inhibits chondrocytes, to study the metabolism of Gln within chondrocytes. In an embodiment of the present disclosure, the stimulation of chondrocytes by IL-1β inhibits the metabolism of Gln within chondrocytes and reduces the amount of Gln in chondrocytes, thus establishing that osteoarthritis is associated with the level of Gln content in chondrocytes.
The present disclosure also conducted experiments in which II-1β stimulation inhibits chondrocytes, to study the metabolism of the downstream metabolite α-KG of Gln in chondrocytes. In a specific embodiment of the present disclosure, IL-1β stimulates chondrocytes, and inhibits the expression of genes that produce α-KG in chondrocytes. The amount of α-KG in chondrocytes becomes less, thus providing insight that supplementation/complementation of α-KG can inhibit the progression of osteoarthritis.
The present disclosure also performs experiments on the effect of α-KG on the catabolic and synthetic phenotypes of chondrocytes, and on NF-κB signaling. In a specific embodiment of the present disclosure, by means of whole-genome sequencing, it was detected that α-KG significantly promoted the synthetic phenotype of chondrocytes and inhibited the catabolic phenotype of cartilage after α-KG was complemented. This result was verified by q-PCR and western blot. Furthermore, whole-genome sequencing also detected that upon IL-1β stimulation conditions, the expression of many genes in the NF-κB signaling pathway, such as P65 in the nucleus of the NF-κB signaling pathway, was suppressed after α-KG was complemented. Therefore, it was thus established that α-KG could inhibit the production of inflammation and could regulate the phenotype of chondrocytes by suppressing the NF-κB signaling pathway.
The present disclosure also conducted experiments in which an osteoarthritis model was established. In an embodiment of the present disclosure, α-KG was injected into the articular cavity, OARSI quantitative scoring was performed, and it was found that α-KG significantly inhibited the progression of osteoarthritis.
The present disclosure also conducted experiments in which the regeneration level of skin hair follicles was studied. In an embodiment of the present disclosure, α-KG was injected by intradermal injection, and the results were observed by HE staining. The results indicated that α-KG could significantly increase the number of skin hair follicles and can be used to alleviate and/or treat hair loss.
The present disclosure will be described in more detail in the following by means of specific embodiments. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present disclosure all employ conventional techniques of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology in the technical field and related fields. These techniques are well described in the prior literature.
The experimental methods without specific conditions noted in the following embodiments are based on conventional methods and conditions, or selected according to the products' instructions. The α-KG used in the present disclosure is dimethyl-α-KG. The dimethyl-α-KG enters cells to play a regulatory role. The dimethyl-α-KG is commercially available, and the rest of the reagents and raw materials used in the present disclosure are also commercially available.
Mouse chondrocytes were obtained from 7-day-old C57BL/6 mice (Shanghai Slac laboratory animal Co., Ltd.), and the procedure was as follows: under aseptic surgical conditions, bilateral knee cartilages of C57 mice were carefully stripped and digested with 0.2% neutral collagenase (Serva, Germany) for 8 h; cell precipitates were obtained by centrifugation, and the cells were resuspended in modified Eagle medium (DMEM) (Gibco, USA) containing 10% FBS, inoculated in culture dishes, and cultured with high-sugar DMEM medium (containing 10% FBS, 100 U/mL penicillin and 100 mg/L streptomycin) at 37° C. in a 5% CO2 constant temperature incubator. The medium was changed for the first time at 48 h. When the cell fusion rate was observed to reach 80%-90%, the cells were digested with trypsin (Gibco, USA) at a concentration of 0.25% and then subcultured. The cells were collected by centrifugation and resuspended, and the concentration was adjusted for inoculation in culture dishes. The cells were subcultured by the above method, and the obtained cells were labeled as P1, P2, P3 generations, etc.
Nineteen amino acid standard substances were weighed accurately and prepared with water into a mixture so that the final concentrations of the nineteen amino acids in the mixture were 0.05 μg/mL, 0.2 μg/mL, 0.5 μg/mL, 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL, and 100 μg/mL (eleven gradients).
The samples were vortexed for 30 s, fast-frozen in liquid nitrogen; the quenched cell solution was dissolved at room temperature, vortexed for 30 s, and centrifuged at 800 g for 1 min; the supernatant was transferred into a centrifuge tube and placed on dry ice; the cells were reset with 500 μL methanol (−80° C), fast-frozen in liquid nitrogen, and subjected to repeated vortex and centrifugation operations; the supernatant was combined, blown dry with liquid nitrogen, added with 1000 μL of alanine-d4 isotope internal standard at a concentration of 1 μg/mL, vortexed for mixing well, and subjected to short centrifugation; 100 μL of the samples were added with 60 μL of concentrated hydrochloric acid: n-butanol (1:3), mixed well before short centrifugation (for shaking off the liquid droplets from the lid), placed at a constant temperature of 65° C. for 15 min for derivatization, subjected to short centrifugation, and volatilized at a temperature below 45° C.; 100 μL of 80% acetonitrile in water was added for redissolving, and the mixed solution was directly loaded into LC-MS for analysis.
Chromatographic conditions: Column: ACQUITY UPLC® BEH C18 column (2.1×100 mm, 1.7 μm, Waters Corporation, USA); injection volume: 5 μL, column temperature: 40° C.; mobile phase A—acetonitrile containing 0.1% formic acid and 0.1% heptafluorobutyric acid, mobile phase B-0.1% formic acid in water; flow rate: 0.2 mL/min; gradient elution procedure: 0˜1.5 min, 5% A; 1.5˜2 min, 5˜20% A; 2˜7 min, 20˜30% A; 7˜8.5 min, 30˜98% A; 8.5˜10.5 min, 98% A; 10.5˜11min, 98˜5% A; 11˜12.5min, 5% A.
MS conditions: electrospray ionization (ESI) source, positive ionization mode. Ion source voltage: 3200 V, solvent temperature: 380° C., cone voltage: 20 V. Scanning was performed using multiple reaction monitoring (MRM). The ion pairs for quantitative analysis are shown in Table 1 below.
The linearity of the standard solutions was investigated by performing LC-MS assays on the concentration series of the standards, respectively, with the concentration of the standards as the horizontal coordinate and the peak area ratio as the vertical coordinate. The linear regression equation obtained for each amino acid is shown in Table 2. Correlation coefficient >0.99.
The P1 generation mouse chondrocytes cultured in Embodiment 1 were used in this Embodiment. After the P1 generation chondrocytes were grown adherent, they were cultured in serum-free DMEM culture medium containing 10 ng/ml IL-1β. For chondrocytes to be treated without glutamine, the chondrocytes were first changed to glutamamine-free medium 12 h in advance, and then treated with IL-1β.
(2) Real-Time Fluorescent Quantitative PCR (Real Time q-PCR) Analysis
The supernatant was carefully discarded, 1 ml of 75% ethanol was slowly added along the wall of the centrifuge tube (do not touch the precipitate), the wall of the centrifuge tube was washed by gently turning the centrifuge tube upside down, the centrifuge tube was centrifuged at 12000 g under 4° C. for 5 minutes, and then the ethanol was carefully discarded (to better control the salt ion content in RNA, ethanol should be removed as much as possible).
The precipitate was dried at room temperature for 2˜5 minutes, an appropriate amount of RNase-free water was added to dissolve the precipitate, and if necessary, a pipette may be used to gently pipet the precipitate. After the RNA precipitate was completely dissolved, the concentration was immediately measured, and reverse transcription was carried out. The remaining RNA was stored at −80° C.
The RNA samples obtained in (4) were measured for determining concentrations by Nanodrop 2000 instrument.
The RNA samples obtained in (5) were subjected to a reverse transcription reaction, in which the reaction system and reaction conditions are shown in Table 3.
On ice, SYBR Premix Ex Taq, upstream and downstream primers of catabolic and synthetic genes of chondrocytes, ROX Reference Dye, cDNA template and dH2O were added into a test tube. After being mixed evenly, the mixture was evenly divided into PCR tubes. The PCR tubes were placed in a Q-PCR instrument to detect the product. The reaction system and reaction conditions for real-time PCR are shown in Table 4. Real-time PCR primer sequences are shown in Table 5.
P1 generation mouse chondrocytes cultured in Embodiment 1 were subjected to western blot analysis through operations including the following:
P1 generation mouse chondrocytes cultured in Embodiment 1 were used to extract nuclear proteins for observing changes of P65 in the NF-κB signaling pathway by a method including the following:
Mouse chondrocytes of P1 generation obtained in Embodiment 1 were cultured in blank DMEM culture medium, DMEM culture medium containing 10 ng/ml IL-1β, and DMEM culture medium containing 10 ng/ml IL-1β and 7 mM α-KG, respectively, to obtain the corresponding Ctrl group, IL-1β group, and IL-1β+αKG group.
(2) Real-Time Fluorescent Quantitative PCR (Real Time q-PCR) Analysis
The Ctrl group, IL-1β group and IL-1β+αKG group were subjected to q-PCR assay under the same experimental conditions as in Embodiment 3, respectively.
The Ctrl group, IL-1β group and IL-1β+αKG group were subjected to western blot assay under the same experimental conditions as in Embodiment 3, respectively.
Male C57BL/6 mice (n=30) were purchased from Shanghai Slac laboratory animal Co., Ltd. and reared to 8 weeks of age. The animal experimental operation method was reviewed and approved by the Ethics Committee of Tongji University School of Medicine.
Male C57BL/6 mice reared to 8 weeks of age were anaesthetized, and a longitudinal incision was made in the medial side of the knee joint. The articular cavity was opened along the medial side of the patellar ligament, the fat pad in the intercondylar area was bluntly separated, and the meniscus tibial ligament connecting the medial meniscus was found and transected. The incision was closed after hemostasis by compression, and the mice were continued to be fed. The mice were anesthetized and sacrificed at 4, 8 and 12 weeks after surgery (n=8 for each group), and the knee joints were isolated for histological examination, forming the treatment group.
Male C57BL/6 mice reared to 8 weeks of age were anaesthetized, and a longitudinal incision was made in the medial side of the knee joint. The articular cavity was opened along the medial side of the patellar ligament, the fat pad in the intercondylar area was bluntly separated, and the meniscus tibial ligament connecting the medial meniscus was found, exposed, but not cut. The incision was closed after hemostasis by compression, and the mice were continued to be fed. The mice were anesthetized and sacrificed at 4, 8 and 12 weeks after surgery (n=8 for each group), and the knee joints were isolated for histological examination, forming the sham group.
(2) Intra-Articular Injection of α-KG into the Knee Joint of Mice for Histological Staining
Three weeks after DMM surgery, intra-articular injections were given to the mice in the treatment group and sham group, once a week, 5 times in total. After the mice were anesthetized, the skin at the knee joint was wiped with alcohol for disinfection, and the articular cavity of the treatment group was injected with 10 μL of PBS-diluted α-KG (1 μl dimethyl-α-KG dissolved in 9 μl PBS) using an insulin needle. A sham operation was performed and PBS was injected as a control. After 5 injections, i.e., 8 weeks after DMM surgery, samples were collected, and histological staining was performed to observe the changes of articular cartilage.
3 weeks after the DMM surgery, mice in sham group was injected with PBS, mice in a DMM group was injected with PBS, and mice in a DMM+α-KG group was injected with α-ketoglutarate. The corresponding mouse knee joints were taken, and treated by the following methods to form specimens:
Safranin-O/fast green staining includes the following procedures:
The damage degree of articular cartilage was observed by Safranin-O/ fast green staining and quantitatively evaluated by OARSI score. The evaluation criteria are shown in Table 6 below.
The scores of sham group, DMM group, and DMM+αKG group were tested by unpaired samples t-test. The statistical software used was SPSS11.0, and P<0.05 was considered statistically different.
The skin on the back of posterior limbs of 12-month-old aged mice was taken, and the treatment group was intradermally injected with PBS-diluted α-KG (1 μl of dimethyl-α-KG dissolved in 4 μl of PBS). A sham operation was performed and PBS was injected as a control. Histological staining was performed to observe the changes of skin hair follicles at 4 weeks after injection.
In summary, the present disclosure effectively overcomes various shortcomings and has high industrial utilization value. The above-mentioned embodiments are just used for exemplarily describing the principle and effects of the present disclosure instead of limiting the present disclosure. Modifications or variations of the above-described embodiments may be made by those skilled in the art without departing from the spirit and scope of the present disclosure. Therefore, all equivalent modifications or changes made by those who have common knowledge in the art without departing from the spirit and technical concept disclosed by the present disclosure shall be still covered by the claims of the present disclosure.
Number | Date | Country | Kind |
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201911353216.5 | Dec 2019 | CN | national |
Filing Document | Filing Date | Country | Kind |
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PCT/CN2020/138554 | 12/23/2020 | WO |