Application of Guaiacol in treating osteoarthritis and promoting articular cartilage repair

Abstract

The invention discloses application of Guaiacol in treating osteoarthritis and promoting articular cartilage repair, it is found for the first time and proved through experiments that Guaiacol can be used for effective treatment of osteoarthritis, Guaiacol is a natural compound and has the advantages of being low in toxic and side effect, high in safety, high in effectiveness and the like, in addition, Guaiacol is rich in raw material resource and low in cost, and the Guaiacol can be applied to treatment of osteoarthritis and promotion of articular cartilage repair. The invention provides a brand new thought and strategy for the research and development of osteoarthritis treatment medicines, has a wide application prospect in the technical field of osteoarthritis treatment, and has important conversion significance.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular, relates to the application of Guaiacol in treating osteoarthritis and promoting the repair of articular cartilage. Background Art

[0002] Osteoarthritis (OA) is a common joint disease with a high incidence rate worldwide. With the aging of the population, its prevalence rate is increasing year by year. According to statistics, the incidence rate of osteoarthritis is about 50% in people over 60 years old, and this proportion is as high as 80% in people over 75 years old. The main symptoms of osteoarthritis are joint pain, swelling and stiffness, which will gradually worsen as the disease progresses. Pain can seriously affect the patient's sleep quality and daily living activities, causing the patient to have difficulty in moving and even lose the ability to work. Osteoarthritis patients need long-term treatment and rehabilitation care, which imposes a huge economic burden on the patient's family and society.

[0003] At present, the treatment of osteoarthritis mainly focuses on relieving pain and improving symptoms, and there is a lack of effective drugs that can delay disease progression or promote cartilage repair. Commonly used analgesics and anti-inflammatory drugs have gastrointestinal, cardiovascular and other side effects. For patients with advanced osteoarthritis, joint replacement surgery is the ultimate treatment option, but the operation is high-risk, expensive, and there are complications such as prosthesis loosening and infection. Due to the many limitations of existing treatment methods, they cannot meet the treatment needs of osteoarthritis patients. Therefore, this field urgently needs to develop new therapeutic drugs that can promote the repair and regeneration of articular cartilage, relieve the pain symptoms of osteoarthritis patients, and improve the quality of life of osteoarthritis patients.

[0004] Guaiacol, also known as o-methoxyphenol, is an organic compound with important application value. Guaiacol exists in a variety of natural substances, such as guaiacol resin, pine tar, etc. It can be extracted, and trace amounts of guaiacol are also found in the components of some plants such as tobacco and coffee. In addition, guaiacol can be synthesized industrially by a variety of methods. The common method is to use o-nitrochlorobenzene as the raw material and prepare it through a series of reactions such as methoxylation, reduction and diazotization hydrolysis. Guaiacol is one of the commonly used raw materials in the spice industry. So far, there has been no research or report on the application of guaiacol in the treatment of osteoarthritis and the promotion of articular cartilage repair. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide the use of Guaiacol in treating osteoarthritis and promoting the repair of articular cartilage, in order to provide a new low-toxic and highly effective osteoarthritis therapeutic drug for the art.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0007] The first aspect of the present invention provides the use of Guaiacol or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing osteoarthritis or promoting the repair of articular cartilage.

[0008] Furthermore, the structural formula of the Guaiacol is shown in formula (I):

[0009]

[0010] Formula (I).

[0011] Furthermore, the medicament comprises an effective amount of Guaiacol or a pharmaceutically acceptable salt thereof.

[0012] Furthermore, the Guaiacol or a pharmaceutically acceptable salt thereof inhibits chondrocyte apoptosis and extracellular matrix degradation by activating the MAPK pathway and reducing the expression of ADAMTS4.

[0013] Furthermore, the Guaiacol or a pharmaceutically acceptable salt thereof reduces the expression of ADAMTS4, BAX, COX2, MMP13, IL-6, and MMP9, and effectively protects cartilage and subchondral bone.

[0014] Furthermore, the medicine also contains pharmaceutically acceptable adjuvants and / or excipients.

[0015] In the present invention, the Guaiacol refers to guaiacol, also known as o-methoxyphenol, which is a natural organic substance with a molecular formula of C6H4(OH)(OCH3). It can be prepared from guaiacol resin, pine oil, etc. Common guaiacol presents a dark color due to exposure to air or light. Guaiacol is widely used in industry. Guaiacol is commonly used to produce various spices, such as eugenol, vanillin and artificial musk. Guaiacol is also used in medicine. It can be used to synthesize guaiacol benzenesulfonate (potassium guaiacolsulfonate), used as a local anesthetic or preservative. Guaiacol can also be used as a dye, organic synthesis raw material and standard substance for analytical determination. At present, there is no relevant research or report on the application of guaiacol in treating osteoarthritis and promoting articular cartilage repair, and the present invention is the first report.

[0016] Furthermore, ADAMTS4 refers to A disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS4) with platelet thrombospondin-like motifs. The family of Adisintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) is the main protease that causes the degradation of the extracellular matrix (ECM) of osteoarthritis cartilage. Among them, ADAMTS1, ADAMTS4, ADAMTS5, ADAMTS8, ADAMTS9, ADAMTS15 and other ADAMTS mainly cause the degradation of proteoglycans in the ECM of articular cartilage. Proteoglycans can make articular cartilage have good elasticity and compression resistance. The loss and degradation of proteoglycans cause the imbalance of ECM anabolism and catabolism, which is considered to play an important initiating role in the early lesions of osteoarthritis. Among the numerous ADAMTS, ADAMTS4 and ADAMTS5 are the main proteoglycanases that cause proteoglycan degradation. It has been proven that the expression of ADAMTS4 in the cartilage of osteoarthritis patients is significantly higher than that in normal people. ADAMTS4 is an important enzyme that causes proteoglycan degradation.

[0017] Further, the BAX refers to Bcl-2 associated X protein, which is a member of the Bcl-2 protein family and is mainly located on the mitochondrial membrane. BAX is a pro-apoptotic protein that plays a key role in the regulation of cell apoptosis. Under normal circumstances, the expression of BAX in cells is in a relatively balanced state to maintain the normal survival and death regulation of cells. In osteoarthritis, the expression of BAX is usually increased. Under the influence of factors such as inflammatory stimulation, mechanical damage, and oxidative stress, the apoptotic signaling pathway in articular chondrocytes is activated. This will lead to upregulation of BAX expression, conformational changes in BAX protein and aggregation on the outer membrane of mitochondria to form pores, resulting in a decrease in mitochondrial membrane potential, and the release of apoptosis-related substances such as cytochrome C into the cytoplasm, thereby activating the caspase cascade reaction, and ultimately inducing chondrocyte apoptosis. Massive apoptosis of chondrocytes will destroy the cell structure and function of cartilage tissue and accelerate the development of osteoarthritis.

[0018] Furthermore, the COX2 refers to cyclooxygenase-2, which is an inducible enzyme. Under normal physiological conditions, its expression level is low. However, in the inflammatory environment of osteoarthritis, inflammatory factors (such as interleukin-1β, tumor necrosis factor-α, etc.) stimulate synovial cells, chondrocytes, etc. to express a large amount of COX2, that is, COX2 is highly expressed in osteoarthritis. COX2 can catalyze the conversion of arachidonic acid into inflammatory mediators such as prostaglandins, which can lead to joint pain, swelling and increased inflammatory response. In addition, the high expression of COX2 is also involved in the apoptosis of chondrocytes and matrix degradation, further promoting the development of osteoarthritis.

[0019] Furthermore, the MMP13 refers to matrix metalloproteinase 13, which is a zinc-dependent proteolytic enzyme mainly produced by chondrocytes, synoviocytes and osteoclasts. MMP13 is highly expressed in osteoarthritis. In the pathological process of osteoarthritis, the massive release of inflammatory factors (such as interleukin-1β, tumor necrosis factor-α, etc.) stimulates cells to synthesize and secrete MMP13. MMP13 has a strong ability to degrade the extracellular matrix. MMP13 can specifically cut the triple helix structure of type II collagen, leading to the degradation and destruction of the cartilage matrix, thereby promoting the occurrence and development of osteoarthritis.

[0020] Furthermore, the IL-6 refers to interleukin 6, which is significantly expressed in osteoarthritis. During the onset of osteoarthritis, various cells in the joints (such as synovial cells, chondrocytes, macrophages, etc.) will produce IL-6. IL-6 can participate in the pathological process of osteoarthritis through various pathways. IL-6 can promote the amplification of the inflammatory response and induce the production of other inflammatory factors (such as interleukin-1β, tumor necrosis factor-α, etc.), forming an inflammatory cascade reaction. At the same time, IL-6 can also regulate the activity of osteoclasts, promote bone resorption, and lead to the destruction of subchondral bone in the joints. In addition, IL-6 can inhibit the synthesis of extracellular matrix by chondrocytes, promote the apoptosis of chondrocytes, and accelerate the degeneration of cartilage.

[0021] Furthermore, the MMP9 refers to matrix metalloproteinase 9, which is a zinc-dependent proteolytic enzyme. Under normal physiological conditions, MMP9 participates in tissue remodeling, repair, and renewal of extracellular matrix. However, in normal joints, the expression level of MMP9 is low and strictly regulated. In osteoarthritis, MMP9 is highly expressed. The inflammatory microenvironment in the joint stimulates synovial cells, chondrocytes, macrophages and other cells to synthesize and secrete MMP9. Inflammatory cytokines (such as IL-1β, TNF-α, etc.) can promote the transcription and expression of MMP9 gene by activating intracellular signaling pathways. MMP9 has a wide range of substrate specificity and can degrade a variety of extracellular matrix components, including COL2, gelatin, fibronectin, etc. Its high expression will lead to excessive degradation of cartilage matrix, destroy the normal structure and function of cartilage, and may also participate in the inflammatory response and angiogenesis of periarticular tissues, further aggravating the pathological process of osteoarthritis.

[0022] In some embodiments, in addition to the Guaiacol or its pharmaceutically acceptable salt, the related uses of the Guaiacol or its hydrates, enantiomers, diastereomers, solvates or crystalline forms in the preparation of drugs for treating and / or preventing osteoarthritis or promoting articular cartilage repair also fall within the protection scope of the present invention.

[0023] In the present invention, the pharmaceutically acceptable salt refers to the pharmaceutically acceptable salt of the Guaiacol, which means those carboxylates, amino acid addition salts, etc. of the compound of the present invention (the Guaiacol), which are suitable for contact with patients within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic reaction, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, including (where possible) the zwitterionic form of the compound of the present invention (the Guaiacol).

[0024] In some embodiments, examples of pharmaceutically acceptable salts of Guaiacol include, but are not limited to, those having (as counter ions) alkali metal ions such as Li + 、Na + or K + or salts with alkaline earth metal ions such as Mg 2+ or Ca 2+ or any other pharmaceutically acceptable metal ion such as Zn 2+ or Al 3+ or a pharmaceutically acceptable salt formed with an organic base such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine or tromethamine.

[0025] In some embodiments, the pharmaceutically acceptable adjuvants and / or excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995). These substances are used to help the stability of the drug or to help improve the activity of the active ingredient (i.e., the Guaiacol or its pharmaceutically acceptable salt as described above in the present invention), and the substances include but are not limited to: diluents, surfactants, wetting agents, adhesives, fillers, disintegrants, adsorption carriers, lubricants, stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents, and the pharmaceutical composition thus prepared can be administered by any appropriate administration method known to those skilled in the art as needed.

[0026] In some embodiments, the medicament may additionally contain liquids such as water, saline, glycerol, and ethanol.

[0027] In some embodiments, the amount of Guaiacol or a pharmaceutically acceptable salt thereof is 0.01-100 mg / kg.

[0028] In some embodiments, the amount of Guaiacol or a pharmaceutically acceptable salt thereof is 5-50 mg / kg.

[0029] A second aspect of the present invention provides a pharmaceutical composition.

[0030] Furthermore, the pharmaceutical composition comprises the Guaiacol or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention.

[0031] In some embodiments, the main active ingredient of the pharmaceutical composition is Guaiacol or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention. In addition to Guaiacol or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention, the pharmaceutical composition may further comprise other drugs that can be used for the treatment and / or prevention, or for the auxiliary treatment and / or prevention of osteoarthritis.

[0032] A third aspect of the present invention provides a pharmaceutical preparation.

[0033] Furthermore, the pharmaceutical preparation comprises the pharmaceutical composition described in the second aspect of the present invention.

[0034] Furthermore, the dosage form of the pharmaceutical preparation is a solution, a sustained-release agent, a suspension, a granule, a tablet, a capsule, a powder, an emulsion, a syrup or a drop.

[0035] In some embodiments, the dosage form of the pharmaceutical preparation is a dosage form for enteral administration or a dosage form for parenteral administration.

[0036] Furthermore, the dosage form for administration through the gastrointestinal tract is a solution, tablet, capsule, suspension, granule, powder, sustained-release agent, emulsion, syrup, effervescent agent, drops or chewable agent; the dosage form for administration through the parenteral tract is an injection dosage form, a respiratory tract dosage form, a cavity dosage form, a mucosal dosage form or a skin dosage form.

[0037] In some embodiments, the pharmaceutical composition or pharmaceutical preparation described in the present invention can be prepared into a dosage form suitable for oral administration, parenteral administration or topical administration by conventional methods currently known in the art, such as by conventional mixing, granulation, coating, solvent or lyophilization methods. The dosage form includes but is not limited to: oral solution, oral emulsion, oral suspension, tablet, capsule, granule, powder for injection, sustained-release tablet, injection, injection emulsion, gel, ointment, suppository, controlled-release tablet, etc.

[0038] Those skilled in the art will appreciate that, although the pharmaceutical composition or pharmaceutical preparation mentioned above of the present invention may further comprise pharmaceutically acceptable adjuvants and / or excipients, when the Guaiacol or its pharmaceutically acceptable salt is used as a drug for humans or animals, they may also be administered in their own form, i.e., the present invention may be achieved without adding any of the above-mentioned pharmaceutically acceptable adjuvants and / or excipients.

[0039] In some embodiments, suitable administration methods for the pharmaceutical compositions or pharmaceutical formulations described herein include any of the various methods and delivery systems known to those skilled in the art to physically introduce the pharmaceutical compositions or pharmaceutical formulations described herein into the subject, including but not limited to oral administration, parenteral administration, topical administration, administration by inhalation spray, nasal administration, rectal administration, buccal administration, or administration by an implanted reservoir.

[0040] In the present invention, the effective amount refers to an amount having a therapeutic and / or preventive effect or an amount required to produce a therapeutic and / or preventive effect in a subject to be treated and / or prevented. For example, a therapeutically or pharmaceutically effective amount refers to the amount of a drug required to produce the desired therapeutic and / or preventive effect, and the therapeutic and / or preventive effect can be reflected by the results of clinical trials, model animal studies and / or in vitro studies. The pharmaceutically effective amount depends on several factors, including (but not limited to): characteristic factors of the subject to be treated (such as the subject's height, weight, gender, age and medication history), and the severity of the disease.

[0041] In some embodiments, when the pharmaceutical composition or pharmaceutical preparation as described above is actually used, its administration regimen and dosage regimen can be selected according to a variety of factors, including the type, species, age, weight, sex and type of disease to be treated of the subject; the severity of the disease to be treated; the route of administration; the patient's renal and liver function; and the specific compound or salt thereof used. A dosing and / or dosage regimen can be used, for example, to prevent a disease, inhibit (completely or partially inhibit) a disease, or stop the development of the disease.

[0042] In the present invention, the subject refers to any animal, including humans and non-human animals. The non-human animals include all vertebrates, for example, mammals, such as non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc. In a specific embodiment of the present invention, the subject is preferably a human.

[0043] The fourth aspect of the present invention provides use of the Guaiacol or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention as an active ingredient in the preparation of a reagent.

[0044] Further, the reagent is used in any one or more of the following aspects:

[0045] (1) Reduce the expression of ADAMTS4, BAX, COX2, MMP13, IL-6 or MMP9 in vitro;

[0046] (2) Inhibit chondrocyte apoptosis in vitro;

[0047] (3) Inhibit the degradation of chondrocyte extracellular matrix in vitro.

[0048] In a specific embodiment of the present invention, the present invention proves through in vivo and in vitro experiments that the Guaiacol has a significant therapeutic effect on osteoarthritis, can effectively protect cartilage and subchondral bone, effectively inhibit chondrocyte apoptosis, and effectively inhibit the degradation of chondrocyte extracellular matrix. In addition, it can be used to inhibit chondrocyte apoptosis in vitro, inhibit the degradation of chondrocyte extracellular matrix in vitro, and reduce the expression of ADAMTS4, BAX, COX2, MMP13, IL-6 or MMP9 in vitro.

[0049] The fifth aspect of the present invention provides a method for non-therapeutic in vitro inhibition of chondrocyte apoptosis, inhibition of chondrocyte extracellular matrix degradation, or reduction of ADAMTS4, BAX, COX2, MMP13, IL-6 or MMP9 expression.

[0050] Further, the method comprises the following steps: contacting the Guaiacol or a pharmaceutically acceptable salt thereof described in the first aspect of the present invention with a cell system in need thereof, thereby inhibiting chondrocyte apoptosis, inhibiting degradation of chondrocyte extracellular matrix, or reducing the expression of ADAMTS4, BAX, COX2, MMP13, IL-6 or MMP9.

[0051] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0052] The present invention discovers for the first time and confirms through experiments that Guaiacol can be used for the effective treatment of osteoarthritis. Guaiacol is a natural compound with the advantages of low toxicity, high safety, high effectiveness, etc. In addition, Guaiacol has abundant raw material resources and good social and economic benefits. The present invention provides a new idea and strategy for the research and development of drugs for the treatment of osteoarthritis, has broad application prospects in the technical field of osteoarthritis treatment, and has important transformation significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The diagrams are the experimental animal grouping and experimental steps, the radiographic examination of the left knee joint of mice, and the bone parameter indexes of the subchondral bone of the left tibial plateau of mice;

[0054] Figure 2 Guaiacol inhibits cartilage destruction and increases anabolism in mouse knee joints;

[0055] Figure 3 It is a measure of the viability of human primary chondrocytes;

[0056] Figure 4 Guaiacol inhibits extracellular matrix degradation and promotes matrix synthesis;

[0057] Figure 5 Guaiacol inhibits apoptosis;

[0058] Figure 6 This is the differentially expressed genes, GO biological function enrichment analysis diagram and PPI analysis diagram between the IL-1β treated group and the untreated group. DETAILED DESCRIPTION

[0059] The present invention is further described below in conjunction with specific embodiments. The following specific embodiments are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0060] The reagents, experimental consumables and raw materials used in the present invention are easily obtained by those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0061] Example 1 Study on the therapeutic effect of Guaiacol on anterior cruciate ligament resection (ACLT) osteoarthritis mouse model

[0062] 1. Experimental Materials

[0063] Guaiacol was purchased from Shanghai Taoshu Biochemical Technology Co., Ltd., and mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. They were C57BL / 6J mice, 6 weeks old, male.

[0064] 2. Construction of anterior cruciate ligament transection (ACLT) osteoarthritis mouse model

[0065] Mice were anesthetized with intramuscular injection of dexmedetomidine (0.25 mg / kg) and ketamine (25 mg / kg), and the left knee joint was routinely prepared and disinfected. The medial parapatellar incision was made to expose the knee joint. After the patella was exposed, the patella was dislocated laterally and the knee joint was flexed as much as possible to expose the anterior cruciate ligament (ACLT). The anterior cruciate ligament was cut under direct vision, and the bleeding was completely stopped. The patella was repositioned and the joint cavity was closed layer by layer with No. 1 silk thread. In the sham operation group, only the left knee joint was incised and sutured, and the anterior cruciate ligament was not cut.

[0066] 3. Study on the therapeutic effect of Guaiacol on the ACLT mouse model of osteoarthritis

[0067] Six-week-old male C57BL / 6J mice (n=30) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. After 2 weeks of acclimatization, the mice were randomly divided into 6 groups, with 5 mice in each group: ① normal group, no intraperitoneal injection; ② sham operation group, no intraperitoneal injection; ③ ACLT model group, intraperitoneal injection of solvent; ④ low-dose ACLT model group, intraperitoneal injection of guaiacol (5 mg / kg) after ACLT; ⑤ medium-dose ACLT model group, intraperitoneal injection of guaiacol (10 mg / kg) after ACLT; ⑥ high-dose ACLT model group, intraperitoneal injection of guaiacol (50 mg / kg) after ACLT ( Figure 1 A).

[0068] 4. micro-CT, HE and Safranin Fast Green Staining

[0069] After 8 weeks of medication, the left knee joint specimens were taken, the surrounding tissues were removed, and the specimens were placed in a Micro-CT (SkyScan1172, Belgium) instrument for scanning after moisturizing. The scanning method was 180° rotation, 0.4° step length, 21 μm scanning resolution, and 70 kV tube voltage. After scanning, more than 1,000 cross-sectional images of the specimens were obtained using the equipment's built-in reconstruction software NRecon (V1.6.9.4). The complete knee joint bone tissue of the specimen was used as the region of interest (ROI), and the three-dimensional image was displayed using CTVol (v.2.2) software ( Figure 1 B), the image was binarized (threshold 80-255) and analyzed using CTan (V1.14.4) software ( Figure 1 BC).

[0070] The knee joint specimens fixed with 4% paraformaldehyde were rinsed with saline for 3 times, decalcified in EDTA solution, dehydrated with ethanol, and embedded in paraffin. 5 μm sections were routinely dewaxed to water and stained with hematoxylin-eosin (HE) and safranin O-fast green ( Figure 2 A).

[0071] 5. Immunohistochemical staining

[0072] Routine sections were dewaxed and hydrated, antigen repaired for 10 minutes, incubated with 3% H2O2 at room temperature for 10 minutes, blocked with goat serum at room temperature for 15 minutes, and then added with primary antibody (1:200) at 4°C overnight, HRP-labeled secondary antibody (working concentration 1:200) at room temperature for 15 minutes, DAB color development, hematoxylin counterstaining, dehydrated with gradient ethanol, transparentized with xylene, and sealed with neutral resin to detect the expression of COL2, Acan, MMP13, and ADAMTS4 molecules ( Figure 2 ).

[0073] 6. Experimental results

[0074] The results showed that within a certain concentration, Guaiacol had no significant effect on the body weight of mice, and ACLT could cause the destruction of the tibial plateau articular cartilage and subchondral bone in mice ( Figure 1 BC), while Guaiacol effectively protects cartilage and subchondral bone ( Figure 2 ). This showed that Guaiacol had a significant therapeutic effect on the ACLT mouse model of osteoarthritis.

[0075] Example 2 Study on the Effect of Guaiacol on Human Chondrocytes Stimulated by IL-1β

[0076] 1. Experimental Materials

[0077] Human primary chondrocytes and complete culture medium were purchased from Beijing Beina Chuanglian Biotechnology Research Institute, RNA extract (TRIzol) was purchased from Invitrogen, and IL-1β was purchased from Sigma.

[0078] 2. Experimental methods

[0079] (1) Detecting cell activity using CCK8

[0080] Human primary chondrocytes were cultured at 2×10 5 Cells were seeded in 96-well plates at a density of 1 mL / well and cultured for 12 hours. Different concentrations of Guaiacol (0-1000 μM) were added to complete culture medium and cultured for 24 hours. 10 μL / well CCK-8 solution was added and incubated at 37°C for 2 hours. The absorbance at a wavelength of 450 nm was measured using a multifunctional microplate reader. The cell viability of the control group was set as 100% ( Figure 3 D). In addition, we also cultured primary human chondrocytes (2×10 5 Cells / mL) and different concentrations of Guaiacol (0, 50, 100, 200, 500, 1000 μM) were cultured for 3 h, and then CCCP (10 μM) was added for 24 h. Then, 10 μL / well CCK-8 solution was added and incubated at 37°C for 2 h. The absorbance was detected as above ( Figure 3 BE). CCCP refers to carbonyl cyanide m-chlorophenylhydrazone. By treating primary human chondrocytes with CCCP, the damaged mitochondrial function of chondrocytes in osteoarthritis was simulated. Mitochondrial dysfunction will further induce intracellular oxidative stress and inflammatory response, which is also an important link in the development of osteoarthritis.

[0081] (2) Fluorescence quantitative PCR and protein immunoblotting to detect the expression of COL2, MMP13, MMP9, ADAMTS4, Bax and other factors

[0082] Human primary chondrocytes were cultured at 2×10 5Cells were seeded at a density of 100 μg / well in 6-well plates and cultured for 12 hours. Cells were stimulated with complete medium containing different concentrations of Guaiacol (0, 50, 100, 200, 500, 1000 μM), and 0 or 10 ng / mL of IL-1β was added to the complete medium, and the cells were cultured for another 24 hours. After washing twice with PBS, RNA was extracted according to the TRIzol extraction solution operation sequence. Reverse transcription was performed using a reverse transcription kit (Promega, USA), and the reverse transcription product cDNA was used as a PCR amplification template. The target genes were TNFα, IL-6, COL2, MMP13, MMP9, ADAMTS4 ( Figure 4 ). After washing twice with PBS, the total protein was extracted according to the operating sequence of the total protein extraction kit (Thermo Fisher, USA), and the protein concentration was detected by Bradford method. After 20 μg of protein was electrophoresed in 4-12% acrylamide gel, the protein was transferred to PVDF membrane (Bio-Rad, USA) and blocked with 5% skim milk powder for 60 minutes. The primary antibodies used monoclonal antibodies ADAMTS-4 (abcam, USA), MMP-13 (abcam, USA), MMP-9 (abcam, USA), COL II (proteintech, USA), IL-6 (abcam, USA) and actin (abcam, USA) at 4°C overnight. After washing the membrane with TBST, the secondary antibody was labeled with mouse or rabbit IgG with horseradish peroxidase and incubated at room temperature for 1 hour. Imaging was performed using a fully automatic chemiluminescence imaging analysis system (Tanon 5800, China) ( Figure 4 ).

[0083] (3) Detection of chondrocyte apoptosis using ROS and TUNEL reagents

[0084] Human primary chondrocytes were cultured at 2×10 5 Cells were seeded in 6-well plates at a density of 100 μM and cultured for 12 hours. Guaiacol at a concentration of 100 μM was co-cultured with human primary chondrocytes for 3 hours, and CCCP (10 μM) was added for co-culture for 24 hours. After 24 hours, the cells were washed 2-3 times with PBS, and then fixed with 4% paraformaldehyde. TUNEL staining was performed according to the instructions of the ROS and TUNEL apoptosis kits, and the cell nuclei were counterstained with DAPI staining solution ( Figure 5 A, B).

[0085] (4) Research on the action pathway of Guaiacol

[0086] Human primary chondrocytes were cultured at 2×10 5The cells were seeded at a density of 1.5 cells / mL in a 6-well plate (3.5 cm in diameter) and cultured for 12 hours. The cells were cultured for 24 hours in complete medium containing different concentrations of Guaiacol (50, 100, 200 μM). At the same time, 0 or 10 ng / mL of IL-1 was added to the complete medium to stimulate the cells for 24 hours. RNA was extracted using TRIzol RNA extraction solution. The RNA chip detection steps include: ① total RNA sample preparation and quality control, ② library construction and quality control, ③ hybridization washing and staining scanning, and ④ information analysis. Gene chip technology was provided by Beijing Zhongkangbo Biotechnology Co., Ltd. ( Figure 6 ).

[0087] 3. Experimental results

[0088] The results showed that Guaiacol inhibited cell apoptosis and extracellular matrix degradation by activating the MAPK pathway ( Figure 6 AF), which further demonstrates that Guaiacol can effectively prevent and treat osteoarthritis.

Claims

1. Use of Guaiacol or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing osteoarthritis or promoting the repair of articular cartilage, characterized in that: The structural formula of the Guaiacol is shown in formula (I): Formula (I).

2. The use according to claim 1, characterized in that: The medicament comprises an effective amount of Guaiacol or a pharmaceutically acceptable salt thereof.

3. The use according to claim 1, characterized in that: The Guaiacol or a pharmaceutically acceptable salt thereof inhibits chondrocyte apoptosis and extracellular matrix degradation by activating the MAPK pathway and reducing the expression of ADAMTS4.

4. The use according to claim 1, characterized in that: The Guaiacol or a pharmaceutically acceptable salt thereof reduces the expression of ADAMTS4, BAX, COX2, MMP13, IL-6, and MMP9, and effectively protects cartilage and subchondral bone.

5. The use according to claim 1, characterized in that: The medicament further comprises pharmaceutically acceptable adjuvants and / or excipients.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Guaiacol or a pharmaceutically acceptable salt thereof as claimed in claim 1.

7. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the pharmaceutical composition according to claim 6.

8. The pharmaceutical preparation according to claim 7, characterized in that The dosage form of the pharmaceutical preparation is solution, sustained-release agent, suspension, granule, tablet, capsule, powder, emulsion, syrup or drops.

9. Use of Guaiacol or a pharmaceutically acceptable salt thereof as claimed in claim 1 as an active ingredient in the preparation of a reagent, characterized in that: The reagent is used in any one or more of the following aspects: (1) Reduce the expression of ADAMTS4, BAX, COX2, MMP13, IL-6 or MMP9 in vitro; (2) Inhibit chondrocyte apoptosis in vitro; (3) Inhibit the degradation of chondrocyte extracellular matrix in vitro.

10. A method for non-therapeutic in vitro inhibition of chondrocyte apoptosis, inhibition of chondrocyte extracellular matrix degradation, or reduction of ADAMTS4, BAX, COX2, MMP13, IL-6 or MMP9 expression, characterized in that: The method comprises the following steps: contacting the Guaiacol or a pharmaceutically acceptable salt thereof described in claim 1 with a cell system in need thereof, thereby inhibiting chondrocyte apoptosis, inhibiting degradation of chondrocyte extracellular matrix, or reducing the expression of ADAMTS4, BAX, COX2, MMP13, IL-6 or MMP9.

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