Pharmaceutical composition for prevention or treatment of rotatory muscle tear

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of rotator cuff tear, comprising a high-density lipoprotein.

Description

Pharmaceutical composition for the prevention or treatment of rotator cuff tears

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of rotator cuff tears.

[0002] A rotator cuff tear refers to a condition in which the tendons of the rotator cuff, which play a crucial role in maintaining the stability of the shoulder joint and enabling movement in various directions, are damaged. The rotator cuff consists of four major muscles (supraspinatus, infraspinatus, subscapularis, and teres minor) and the tendons connected to them. Rotator cuff tears primarily occur due to causes such as aging, overuse, and trauma, resulting in symptoms such as shoulder pain, limited range of motion, and weakness.

[0003] Treatment for rotator cuff tears typically involves non-surgical methods such as physical therapy, medication, and injections, as well as surgical methods in severe cases. However, these treatments do not guarantee complete recovery, and damage to rotator cuff cells and functional decline can be accelerated, particularly in hypoxic environments. A hypoxic environment can further aggravate rotator cuff damage by inducing an increase in intracellular reactive oxygen species (ROS), activation of inflammatory responses, and apoptosis.

[0004] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of rotator cuff tears.

[0005]

[0006] 1. A pharmaceutical composition for the prevention or treatment of rotator cuff tears comprising high-density lipoprotein.

[0007] 2. A pharmaceutical composition for the prevention or treatment of a rotator cuff tear, wherein the rotator cuff tear is induced by hypoxia, in accordance with 1 above.

[0008]

[0009] The pharmaceutical composition of the present invention can exhibit excellent pharmacological effects against rotator cuff tears.

[0010]

[0011] Fig. 1. Western blot analysis to identify rotator cuff fibroblasts (RCFs). (A, B) The cultured cells studied were characterized using specific cell markers, including the fibroblast marker AS02 and tendon cell-specific markers such as Sklaxis, Tenasin-C, Mohawk, and Tenomodulin. The cultured cells expressed these cell markers, suggesting that the cultured cells were human RCFs. Data are expressed as mean ± SD (n=3).

[0012] Fig. 2. Fluorescence microscopy analysis of RCFs exposed to CoCl2. (A, B) Exposure of human RCFs to 1000 μM CoCl2 significantly increases the production of reactive oxygen species (ROS). Magnification: 4X. Scale bar: 2500 μm. Data are expressed as mean ± SD (n=3). Each data point represents the mean of technical replicas of independent experiments. *** :p<0.001 was compared to normal oxygen.

[0013] Fig. 3. Western blot analysis of human RCFs exposed to CoCl2. Exposure of human RCFs to CoCl2 increases the expression of hypoxia-inducible factor-1α (HIF1-α), heme oxygenase-1 (HO-1), and Bcl-2 / E1B-19kDa interacting protein 3 (BNIP3), which serve as markers of hypoxia. (A) HIF-1α expression was significantly higher in all CoCl2 study groups than in the normal hypoxia group (p ≤ 0.011). (B) HO-1 expression was significantly higher in the 1,000 μmol / L CoCl2 study groups than in the normal hypoxia group (p = 0.044). (C) BNIP3 expression was significantly higher in the 500 and 1,000 μmol / L CoCl2 study groups than in the normal hypoxia group (p ≤ 0.033). Data are expressed as mean ± SD (n=3). *:p< 0.05, **:p< 0.01, ***:p< 0.001 is compared to normal oxygen saturation.

[0014] Fig. 4. Effect of CoCl2 on cell viability. (A) The MTT assay shows that CoCl2 significantly induces apoptosis (p < 0.010). (B) The live and dead assays show that CoCl2 significantly increases apoptosis. Magnification: 10x, Scale bar: 500 μm, Data are presented as mean ± SD (n=4). One data point represents the mean of technical replications of independent experiments. * :p< 0.05, ** :p< 0.01 and *** :p < 0.001 is compared to normal oxygen.

[0015] Fig. 5. Effect of CoCl2 on apoptosis. (A) Annexin V-PI double staining analysis showed that the apoptosis rates in the 100, 500, and 1,000 μmol / L CoCl2 study groups were significantly higher than in the normal oxygen administration group (p≤ 0.005). (B, C) Western blot analysis showed that 1,000 μmol / L CoCl2 induced increased expression of cleaved caspase-3 (p= 0.023) and cleaved PARP-1 (p= 0.002). Data are expressed as mean ± SD (n=3). * : p<0.05, ** : p<0.01 and *** : p<0.001 is compared to normal oxygen saturation.

[0016] Fig. 6. Changes in vascular endothelial growth factor (VEGF) and matrix metalloprotease-2 (MMP-2) expression by CoCl2. (A) VEGF expression was significantly higher in the 1,000 μmol / L CoCl2 treatment group than in the normal hypoxia group (p<0.001). (B) MMP-2 expression was significantly higher in the 1,000 μmol / L CoCl2 treatment group than in the normal hypoxia group (p<0.030). Data are expressed as mean ± SD (n=3). * : p < 0.05 and ***: p < 0.001 is compared to normal oxygen saturation.

[0017] Fig. 7. Changes in collagen I and III expression due to CoCl2. CoCl2 induces a decrease in the expression of collagen I and III. (A) Collagen I expression was significantly lower in the 1,000 μmol / L CoCl2 study group than in the normoxia group. (B) Collagen III expression was significantly lower in all CoCl2 study groups than in the normoxia group (p≤ 0.034). Data are expressed as mean ± SD (n=3). * :p < 0.05 is compared to normal oxygen levels.

[0018] Fig. 8. DPPH radical scavenging activity assay. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay demonstrates significant antioxidant capacity compared to the lowest concentrations of N-acetylcysteine ​​(NAC), Vitamin C (Vit C), reconstituted HDL (rHDL), and high-density lipoprotein (HDL). These results demonstrate that the studied rHDL exhibits significant oxidative radical scavenging capacity similar to that of NAC, Vitamin C, and HDL. Data are presented as mean ± SD (n=3). Each data point represents the mean of technical replications from independent experiments. * :p< 0.05, ** :p< 0.01 and *** :p < 0.001 is compared to the control group.

[0019] Fig. 9. rHDL inhibits CoCl2-induced intracellular reactive oxygen species (ROS) production. (A, B) Fluorescence microscopy analysis showed that reconstituted HDL (rHDL) and other antioxidants significantly inhibited CoCl2-induced intracellular ROS production (p < 0.001). Magnification: 4x. Scale: 2500 μm. Data are expressed as mean ± standard deviation (n = 4), and each data point represents the descriptive replicate mean of independent experiments. ***: p < 0.001 when compared to normal oxygen concentration. ††† : p< 0.001 when compared to the hypoxia group. ns : Indicates that there is no significant difference from normal oxygen concentration.

[0020] Fig. 10. Inhibitory effect of rHDL on the expression of CoCl2-induced hypoxia-inducible factor-1α (HIF-1α), heme oxygenase-1 (HO-1), and Bcl-2 / E1B-19kDa interacting protein 3 (BNIP3). (A) HIF-1α expression was significantly increased in the hypoxia group compared to the normal oxygen concentration group (p < 0.001). Similar to the hypoxia groups pretreated with NAC, Vitamin C (Vit C), and HDL, the hypoxia group pretreated with rHDL showed significantly lower HIF-1α expression than the hypoxia group (p ≤ 0.034). (B) HO-1 expression was significantly increased in the hypoxia group compared to the normal oxygen concentration group (p < 0.001). Similar to the hypoxia groups pretreated with NAC, vitamin C, and HDL, the hypoxia group pretreated with rHDL showed significantly lower HO-1 expression than the hypoxia group (p ≤ 0.013). (C) BNIP3 expression was significantly increased in the hypoxia group compared to the normal oxygen concentration group (p < 0.001). The hypoxia groups pretreated with rHDL and HDL showed significantly lower BNIP3 expression than the hypoxia group (p ≤ 0.007). Data were expressed as mean ± standard deviation (n = 3), * :p< 0.05, ** :p< 0.01, *** :p < 0.001 when compared to normal oxygen concentration, † :p< 0.05, †† :p < 0.01 is compared to the hypoxia group. ns : Indicates that there is no significant difference from normal oxygen concentration, NS : Indicates no significant difference from the hypoxia group.

[0021] Fig. 11. Inhibitory effect of rHDL on CoCl2-induced cytotoxicity. (A) Cell viability in the hypoxia group pretreated with rHDL was significantly higher than in the hypoxia group pretreated with NAC, Vitamin C (Vit C), and HDL (p ≤ 0.013). (B) Live and dead analysis results showed that, similar to the hypoxia groups pretreated with NAC, Vitamin C, and HDL, the proportion of living cells was significantly higher and the proportion of dead cells (red) was significantly lower in the hypoxia group pretreated with rHDL. Arrows indicate dead cells marked in red. Zoom: 10x, Scale: 500 μm. Data are expressed as mean ± standard deviation (n = 4), and each data point represents the descriptive replicate mean of independent experiments. *** : p < 0.001 when compared to normal oxygen concentration. † : p< 0.05 when compared to CoCl2, ††† : p < 0.001 when compared to CoCl2.

[0022] Fig. 12. Inhibitory effect of rHDL on CoCl2-induced apoptosis. (A) FACS analysis using Annexin V / PI double staining showed that the apoptosis rate in the hypoxia group was significantly higher than in the normal oxygen concentration group (p < 0.001). The apoptosis rate in the hypoxia group pretreated with rHDL and the hypoxia group pretreated with NAC, vitamin C, and HDL was significantly lower than in the hypoxia group (p < 0.001). (B) The expression of cleaved caspase-3 was significantly higher in the hypoxia group than in the normal oxygen concentration group (p < 0.001). The expression of cleaved caspase-3 in the hypoxia group pretreated with rHDL and the hypoxia group pretreated with NAC, vitamin C, and HDL was significantly lower than in the hypoxia group (p ≤ 0.002). (C) Expression of cleaved PARP-1 was significantly higher in the hypoxia group than in the normoxia group (p = 0.007). Expression of cleaved PARP-1 in the hypoxia group pretreated with rHDL and the hypoxia group pretreated with NAC, vitamin C, and HDL was significantly lower than in the hypoxia group (p ≤ 0.034). (D) TUNEL analysis showed that apoptotic nuclei (green) were significantly more numerous in the hypoxia group than in the normoxia group. Apoptotic nuclei were significantly fewer in the hypoxia group pretreated with rHDL and the hypoxia group pretreated with NAC, vitamin C, and HDL than in the hypoxia group. Arrows indicate TUNEL-FITC positive cells marked in green. Data are expressed as mean ± standard deviation (n = 3), ** : p < 0.01 when compared to normal oxygen concentration, *** : p < 0.001 when compared to normal oxygen concentration, † : p< 0.05 when compared to the hypoxia group, †† : p< 0.01 when compared to the hypoxia group, ††† : p< 0.001 when compared to the hypoxia group, §§ : p < 0.01 when compared to normal oxygen concentration,§§§ : p < 0.001 when compared to normal oxygen concentration, † : p< 0.05 when compared to the hypoxia group, †† : p< 0.01 when compared to the hypoxia group, ns : Indicates that there is no significant difference from normal oxygen concentration, NS : Indicates no significant difference from the hypoxia group.

[0023] Fig. 13. Effects of rHDL on CoCl2-induced vascular endothelial growth factor (VEGF) and matrix metalloprotein-2 (MMP-2). (A) VEGF expression was significantly higher in the hypoxia group than in the normoxia group (p = 0.008). VEGF expression in the hypoxia group pretreated with rHDL and the hypoxia group pretreated with NAC, vitamin C, and HDL was significantly lower than in the hypoxia group (p ≤ 0.038). (B) MMP-2 expression was significantly higher in the hypoxia group than in the normoxia group (p = 0.015). MMP-2 expression in the hypoxia group pretreated with rHDL and the hypoxia group pretreated with NAC, vitamin C, and HDL was significantly lower than in the hypoxia group (p ≤ 0.044). Data are expressed as mean ± standard deviation (n = 3), * : p< 0.05 when compared to normal oxygen concentration, ** : p < 0.01 when compared to normal oxygen concentration, † : p< 0.05 when compared to the hypoxia group, †† : p< 0.01 when compared to the hypoxia group, ns : Indicates that there is no significant difference from normal oxygen concentration.

[0024] Fig. 14. Effects of rHDL on CoCl2-induced collagen I and III. (A) Collagen I expression was significantly lower in the hypoxia group than in the normoxia group (p = 0.006). Collagen I expression in the hypoxia group pretreated with rHDL and the hypoxia group pretreated with HDL was significantly higher than in the hypoxia group (p ≤ 0.038). (B) Collagen III expression was significantly lower in the hypoxia group than in the normoxia group (p = 0.039). Collagen III expression in the hypoxia group pretreated with rHDL, the hypoxia group pretreated with NAC, and the hypoxia group pretreated with HDL was significantly higher than in the hypoxia group (p ≤ 0.046). Data are expressed as mean ± standard deviation (n = 3), * : p< 0.05 when compared to normal oxygen concentration, ** : p < 0.01 when compared to normal oxygen concentration, † : p< 0.05 when compared to the hypoxia group, ns : Indicates that there is no significant difference from normal oxygen concentration, NS : Indicates no significant difference from the hypoxia group.

[0025] Fig. 15. Cell proliferation effect of rHDL on CoCl2-induced hypoxia. (A,B) Ki-67 staining analysis revealed that the antioxidants under study, including rHDL, exhibited a cell proliferation effect on CoCl2-induced hypoxia. Ki-67 is indicated in green, actin in red, and DAPI in blue, with both displayed as overlaid. The hypoxia group showed a significant decrease in the number of Ki-67-positive cells, indicating reduced cell proliferation. Conversely, the groups pretreated with rHDL and other antioxidants showed a significant increase in the number of Ki-67-positive cells, indicating cell proliferation. Data are expressed as mean ± standard deviation (n = 3). * : p< 0.05 when compared to normal oxygen concentration, **: p < 0.01 when compared to normal oxygen concentration, *** : p < 0.001 when compared to normal oxygen concentration, † : p< 0.05 when compared to the hypoxia group, ††† : p< 0.001 when compared to the hypoxia group, ns : Indicates that there is no significant difference from normal oxygen concentration, NS : Indicates no significant difference from the hypoxia group.

[0026]

[0027] The present invention will be described in detail below.

[0028] The present invention relates to a pharmaceutical composition for the prevention or treatment of rotator cuff tears.

[0029] The pharmaceutical composition of the present invention comprises high-density lipoprotein (HDL).

[0030] High-density lipoprotein may be of the same species as the subject being taken.

[0031] High-density lipoproteins may be obtained from living organisms or produced by recombination.

[0032] Recombinant human high-density lipoprotein may be used, for example, from Aivasystem Bio, but is not limited thereto.

[0033] The rotator cuff is a collection of important muscles and tendons that stabilize the shoulder joint and enable movement in various directions. The rotator cuff consists of four major muscles (supraspinatus, infraspinatus, subscapularis, and teres minor) and the tendons connected to them.

[0034] The rotator cuff tear that is the target of treatment by the pharmaceutical composition of the present invention is a condition in which the rotator cuff is damaged, and the cause of the damage is not limited. For example, it may be degenerative changes, overuse, trauma, hypoxic conditions, etc., and specifically, it may be a rotator cuff tear induced by hypoxia.

[0035] The pharmaceutical composition of the present invention is not limited to these but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. For injectable preparations, it may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., in combination; and for topical administration, a base, excipients, lubricants, preservatives, etc. may be used. The formulations of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injectables, it can be manufactured in the form of unit dosing ampoules or multiple dosing ampoules. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained-release formulation, etc.

[0036] Examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

[0037] The routes of administration of the pharmaceutical composition of the present invention are not limited to but include oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal. Oral or parenteral administration is preferred. The parenteral administration includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques.

[0038] The pharmaceutical composition of the present invention may vary depending on various factors including age, body weight, general health, gender, diet, time of administration, route of administration, release rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may be appropriately selected by a person skilled in the art, depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, and may be administered at a dose of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be administered once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, coated tablet, capsule, liquid, gel, syrup, slurry, or suspension.

[0039]

[0040] The present invention will be explained in more detail with reference to the following examples.

[0041]

[0042] Examples

[0043] Materials and Methods

[0044] Clinical sample collection

[0045] Primary RCF was obtained from a patient who underwent arthroscopic rotator cuff reconstruction with the approval of the Institutional Review Board of Gyeongsang National University (GNU-170918-R0043). The tissue was washed twice with PBS (Lonza, Walkersville, Maryland, USA), chopped into small pieces with a sterile scalpel, and cultured in DMEM (Thermo Fisher Scientific, Waltham, Massachusetts, USA) in 6-well tissue culture plates (Thermo Fisher Scientific, Waltham, Massachusetts, USA). 20% FBS (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 1% antibiotic-antifungal agent (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were added, and the cells were cultured at 37°C under a 5% CO2 atmosphere. After 2 weeks, the cells reached 90% confluence. The cells were then treated with TrypLE TM Trypsin treatment was performed for 5 minutes using Express (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 0.02% EDTA (Lonza, Walkersville, Maryland, USA), centrifuged at 1300 rpm for 3 minutes, and expanded during the second culture step. The cells were TrypLE TM Cells were collected using Express (Thermo Fisher Scientific, Waltham, Massachusetts, USA), frozen, and then thawed for use in 3 to 6 culture cycles. The cultured cells were characterized as tendon fibroblasts using markers specific to fibroblasts and tendon cells (Fig. 1).

[0046]

[0047] reagent

[0048] Reagents containing CoCl2 (Sigma-Aldrich, St. Louis, Missouri, USA), a complex of human apolypoprotein AI and 1-palmitoyl-2-oleoyl phosphatidylcholine (molar ratio of 1 to 100), rHDL (Aivasystem bio, San Diego, California, USA), and HDL were used, as well as Vitamin C (Vit C) and NAC (all derived from Sigma-Aldrich, St. Louis, Missouri, USA).

[0049]

[0050] Research Design

[0051] Approval was obtained from the Institutional Review Board of Gyeongsang National University (IRB: GNU-170918-R0043). Human RCFs were divided into study groups for normoxia, hypoxia, NAC-hypoxia, Vitamin C-hypoxia, rHDL-hypoxia, and HDL-hypoxia. Hypoxia was induced using 1000 μM CoCl2, a well-known chemohypoxia based on prior studies. This study evaluated the expression of HIF-1α, heme oxygenase-1 (HO-1), and BNIP3, cell viability, intracellular reactive oxygen species (ROS) production, and apoptosis, as well as cleaved caspase-3, cleaved poly-ADP-ribose polymerase-1 (PARP-1), vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), collagen I and III production, and cell proliferative capacity (Figs. 2-7). Cells were exposed to CoCl2 for 24 hours, excluding HIF-1α (4 hours), VEGF (1 hour), and ROS (1 minute). Antioxidants 10 mM NAC, 2 mM Vitamin C, 200 μM rHDL, and 200 μM HDL were administered for 1 hour prior to exposure to CoCl2. The concentrations of the antioxidants were selected based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (Fig. 8).

[0052]

[0053] Measurement of intracellular ROS production

[0054] Intracellular ROS production in each study subset was qualitatively evaluated using fluorescence microscopy. 1 x 10 4 Human RCFs containing RCF were inoculated into plates and cultured for 24 hours. After washing with PBS and adding serum-free medium, the cells were incubated with 5 μmol / L DCF-DA solution at 37°C for 15 minutes. Finally, intracellular ROS production was analyzed using a fluorescence microscope (Nikon, Ti2-UFL, Tokyo, Japan).

[0055]

[0056] Western blot analysis

[0057] Human RCF (3 x 10) in various research reagents depending on the subset of the study 5After exposure to ), the studied cells were washed with cold PBS and scraped in 100 μL of RIPA buffer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) to prepare total cell lysates. The lysed cells were then sonicated and centrifuged at 13,000 rpm for 20 minutes at 4°C to remove insoluble impurities. The samples were lysed in an 8–12% SDS-polyacrylamide gel and then transferred to a PVDF membrane by electrophoresis using a wet technique. Then, the membrane was blocked with 5% skim milk powder in TBS-T buffer (IBS-BT008, iNtRon, Seongnam, South Korea) for 1 hour and incubated with primary antibodies against HIF-1α (1:10,000, A300-286A, BETHYL Laboratories, Montgomery, Texas, USA), HO-1, cleavage Caspase-3, cleavage PARP-1, MMP-2 (1:1000, #43966, #9662, #9542, #40994, Cell Signaling Technology, Danvers, Massachusetts, USA), BNIP3, Collagen I, Collagen III (1:1000, ab1093, ab138492, ab184993, abcam, Cambridge, UK), VEGF (1:100, sc-7269 (Santa Cruz, California, USA), β-actin (1:10,000, MA1-744, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added to TBS-T buffer containing 5% skim milk (Biopure, Seoul, South Korea). Specific antibody binding was detected by the horseradish peroxidase-conjugated secondary antibody (anti-rabbit and anti-mouse, 1:5000; 1460, 31430, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and visualized using an enhanced chemiluminescence detection reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

[0058]

[0059] Cell viability analysis

[0060] To evaluate cell viability, the MTT assay (Sigma-Aldrich, St. Louis, Missouri, USA) and the Live / dead viability / cytotoxicity kit (Invitrogen, Carlsbad, California, USA) were used.

[0061] For MTT analysis, human RCF(2 x 10 4 ) were seeded in a 24-well plate and exposed to various agents for 24 hours, after which MTT solution was added. Absorbance was measured at 570 nm to confirm cell viability. Cell viability was evaluated using MTT. Human RCF (2 × 10⁶ 4 ) was seeded into each well of a 24-well plate. Cells were maintained for 24 hours in a 5% CO2, 37°C incubator. Each study group was exposed to culture medium, NAC, vitamin C, rHDL or HDL, and CoCl2 according to the study subset. 500 μL of MTT solution (0.5 mg / mL in glass medium) was briefly added to each well of the 24-well plate. Then, the plates were incubated for 2 hours. Afterward, the cell supernatant was removed, and 200 μL of DMSO (Merck, Darmstadt, Germany) was added to each vessel. The absorbance of the plates was measured at 570 nm using a microplate reader. Cell viability was expressed as the percentage of living cells compared to a control set at 100%.

[0062] Cell viability was also evaluated using the Live / dead viability / cytotoxicity kit (Invitrogen, Carlsbad, California, USA). Human RCFs (1 x 10⁻¹⁰ 5) was seeded on a 35 mm confocal plate. Cells were maintained in an incubator at 5% CO2, 37°C for 24 hours. Depending on the study area, various study substances were applied to the cells. Briefly, the Live / dead kit solution (5X dye) was added to the refractometer. After the refractometer was incubated at room temperature for 10 minutes, the cells were evaluated using a fluorescence microscope (Nikon, Ti2-U FL, Tokyo, Japan), and digital photographs were taken at 100x magnification.

[0063]

[0064] Cell death rate analysis

[0065] FACS analysis was performed using Annexin V / PI double staining to analyze the apoptosis rate. Human RCF (1 x 10 5 ) was seeded into each well of a 6-well plate. After 24 hours of incubation, the study groups were exposed to various study drugs depending on the study portion. Human RCFs were collected after trypsin treatment and then centrifuged. These cells were washed with PBS and then stained using the FITC Annexin V / PI kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. Cell viability was measured using a flow cytometer (Cytomics FC500, Beckman Coulter, Brea, California, USA) as follows: living cells were labeled with both stains, early apoptotic cells were labeled with Annexin V only, necrotic cells were labeled with PI only, and apoptotic cells were labeled with both Annexin V and PI.

[0066] To detect apoptotic cells, a TUNEL assay was performed using the TUNEL kit (Roche Applied Science, Indianapolis, Indiana, USA) according to the manufacturer's protocol. Briefly, human RCFs (1 x 10 4) was seeded in confocal dishes. After 24 hours of incubation, the study groups were exposed to various study drugs depending on the part of the study. Cells were stained with DAPI (4',6-damidino-2-phenylindole, Sigma-Aldrich, St. Louis, Missouri, USA). Then, the cells were evaluated at 200x magnification using a fluorescence microscope (Nikon, Ti2-U FL, Tokyo, Japan). The percentage of apoptotic cells was calculated as the ratio of TUNEL-positive cells to DAPI-stained cells.

[0067]

[0068] Cell proliferation analysis

[0069] Cell proliferation was evaluated using Ki-67 staining. Human RCF (1 x 10⁻¹⁰ 5) was seeded in 35 mm confocal dishes. After 24 hours of incubation, the study groups were exposed to various target substances depending on the study portion. A fixation solution in cold methanol was added to each vessel, and the cells were subsequently incubated at 4°C for 20 minutes. 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, Missouri, USA) was added to each vessel to infiltrate the cells, and they were subsequently incubated at room temperature for 20 minutes. Then, the cells were incubated at room temperature for 1 hour with 1% bovine serum albumin in PBS (Amresco, Solon, Ohio, USA). After that, primary antibodies of Ki-67 (ab15580, Abcam, Cambridge, Massachusetts, USA) and β-actin (Thermo Fisher Scientific, Waltham, Massachusetts, USA) diluted 1:200 were added, and the cells were incubated at room temperature for 2 hours. A secondary antibody (ab150119, Goat Anti-Mouse IgG H&L Alexa Fluor® 647 / ab150081, Goat-Anti-Rabbit IgG H&L Alexa Fluor® 488, abcam, Cambridge, Massachusetts, USA) diluted 1:200 was used for 1 hour at room temperature, and cells were stained with 1 μg / mL of DAPI (4',6-Damidino-2-phenylindole, Thermo Fisher Scientific, Waltham, Massachusetts, USA). Then, cells were evaluated using a fluorescence microscope (Nikon, Ti2-U FL, Tokyo, Japan), and digital photographs were taken at 200x magnification.

[0070]

[0071] Statistical analysis

[0072] Data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) compared the mean levels of the explored parameters, followed by multiple comparisons using Tukey's method. Statistical significance was set at p < 0.05, indicating a statistically significant difference. All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, California, USA).

[0073]

[0074] result

[0075] Intracellular ROS production rate

[0076] The intracellular ROS production rate was significantly higher in the hypoxia group than in the normal oxygen concentration group (p < 0.001). However, the intracellular ROS production rate in the hypoxia groups pretreated with NAC, vitamin C, rHDL, or HDL was significantly lower compared to the hypoxia group (p < 0.001) (Fig. 9).

[0077]

[0078] Analysis of HIF-1α, HO-1, and BNIP3 expression

[0079] The expression of HIF-1α, HO-1, and BNIP3 was significantly higher in the hypoxia group than in the normal oxygen concentration group (p < 0.001). When the hypoxia group was pretreated with rHDL, NAC, vitamin C, or HDL, the expression of HIF-1α was significantly lower (p ≤ 0.034). Similarly, when the hypoxia group was pretreated with rHDL, NAC, vitamin C, or HDL, the expression of HO-1 was significantly lower (p ≤ 0.013). When the hypoxia group was pretreated with rHDL and HDL, the expression of BNIP3 was significantly lower (p ≤ 0.007) (Figs. 10A-C).

[0080]

[0081] Cell viability analysis

[0082] Cell viability was significantly lower in the hypoxia group than in the normal oxygen concentration group (p < 0.001). In the hypoxia groups pretreated with NAC, vitamin C, rHDL, or HDL, cell viability was significantly higher than in the hypoxia group (p ≤ 0.013) (Fig. 11A). In the live and dead analysis as well, the number of dead cells (red) in the hypoxia group was significantly higher than in the normal oxygen concentration group. In the hypoxia groups pretreated with NAC, vitamin C, rHDL, or HDL, the number of dead cells was significantly lower than in the hypoxia group (Fig. 11B).

[0083]

[0084] Cell death analysis

[0085] The apoptosis rate was significantly higher in the hypoxia group than in the normal oxygen concentration group (p < 0.001). In the hypoxia group pretreated with NAC, vitamin C, rHDL, or HDL, the apoptosis rate was significantly lower than in the hypoxia group (p < 0.001) (Fig. 12A). The expression of cleaved caspase-3 was significantly higher in the hypoxia group than in the normal oxygen concentration group (p < 0.001). However, in the hypoxia group pretreated with NAC, vitamin C, rHDL, or HDL, the expression of cleaved caspase-3 was significantly lower than in the hypoxia group (p ≤ 0.002) (Fig. 12B). The expression of cleaved PARP-1 was significantly higher in the hypoxia group than in the normal oxygen concentration group (p = 0.007). However, in the hypoxia groups pretreated with NAC, vitamin C, rHDL, or HDL, the expression of cleaved PARP-1 was significantly lower than in the hypoxia group (p≤ 0.034) (Fig. 12C). In TUNEL analysis, the number of apoptotic nuclei (green) in the hypoxia group was significantly higher than in the normal oxygen concentration group, but in the hypoxia groups pretreated with NAC, vitamin C, rHDL, or HDL, the number of apoptotic nuclei was significantly lower than in the hypoxia group (Fig. 12D).

[0086]

[0087] VEGF and MMP-2 Expression Analysis

[0088] VEGF expression was significantly higher in the hypoxia group than in the normal oxygen concentration group (p = 0.008). In the hypoxia group pretreated with NAC, vitamin C, rHDL, or HDL, VEGF expression was significantly lower than in the hypoxia group (p ≤ 0.038) (Fig. 13A). MMP-2 expression was significantly higher in the hypoxia group than in the normal oxygen concentration group (p = 0.015). However, in the hypoxia group pretreated with NAC, vitamin C, rHDL, or HDL, MMP-2 expression was significantly lower than in the hypoxia group (p ≤ 0.044) (Fig. 13B).

[0089]

[0090] Collagen I and III production analysis

[0091] Type 1 collagen production was significantly lower in the hypoxia group than in the normal oxygen concentration group (p = 0.006). Additionally, in the hypoxia group pretreated with rHDL and the hypoxia group pretreated with HDL, Type 1 collagen production was significantly higher than in the hypoxia group (p ≤ 0.038) (Fig. 14A). Type 3 collagen production was also significantly lower in the hypoxia group than in the normal oxygen concentration group (p = 0.039). In the hypoxia groups pretreated with rHDL, NAC, or HDL, Type 3 collagen production was significantly higher than in the hypoxia group (p ≤ 0.046) (Fig. 14B).

[0092]

[0093] Analysis of cell proliferation capacity

[0094] Cell proliferation activity, represented by Ki-67 positive cells, was significantly lower in the hypoxia group than in the normal oxygen concentration group (p < 0.001). However, in the hypoxia groups pretreated with rHDL, vitamin C, or HDL, the number of Ki-67 positive cells was significantly higher than in the hypoxia group (p ≤ 0.049) (Fig. 15).

Claims

1. A pharmaceutical composition for the prevention or treatment of rotator cuff tears comprising high-density lipoprotein.

2. A pharmaceutical composition for the prevention or treatment of a rotator cuff tear according to claim 1, wherein the rotator cuff tear is induced by hypoxia.

Images