Application of chlorella in preparation of medicine for treating osteoarthritis

Abstract

The invention discloses application of chlorella in preparation of a medicine for treating osteoarthritis, in-vivo experiments and in-vitro experiments prove that chlorella can maintain a chondrocyte metabolism steady state, inhibit expression of chondrocyte extracellular matrix catabolism-related genes and proteins, and inhibit osteoarthritis. The expression of genes and proteins related to chondrocyte extracellular matrix anabolism is promoted, and by injecting a chlorella solution into a knee joint cavity, the effects of relieving and improving OA pain are achieved; furthermore, polydopamine is used for modifying the chlorella to obtain the polydopamine-coated chlorella Ch (at) P with a photothermal effect, and the Ch (at) P not only maintains the biological activity of the chlorella, but also has the effect of delaying cartilage degeneration and OA process due to the photothermal effect under near infrared (NIR) irradiation.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application of Chlorella vulgaris in preparing a drug for treating osteoarthritis. Background Art

[0002] Osteoarthritis (OA) is a chronic degenerative disease characterized by joint pain, stiffness, and dysfunction, severely impacting patients' physical and mental health and quality of life. With the aging population, the prevalence of OA is expected to increase annually. Its high incidence and disability rates place a heavy burden on patients, their families, and society. Unfortunately, current treatment options for OA are very limited, and there are no drugs that can alter or reverse the progression of OA.

[0003] Currently, the main clinical treatments for OA include medication and surgery. Medication primarily involves pain-relieving drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids, while surgery primarily includes arthroscopic surgery, osteotomy, and total joint replacement. Aside from total joint replacement surgery for patients with end-stage OA, there is no definitive and effective treatment. Commonly used anti-inflammatory and analgesic drugs only alleviate OA pain symptoms and have significant side effects. Their effectiveness needs to be improved, and they are prone to recurrent attacks and are unable to truly slow the progression of OA. Although total joint replacement surgery is the most effective treatment, it is only suitable for patients with end-stage OA and carries certain surgical risks, such as infection, aseptic loosening, and periprosthetic fractures. Compared to total joint replacement surgery for end-stage OA, intervening in the early stages of OA to prevent or slow its progression would be a more effective treatment strategy. If OA could be addressed through medication, thereby avoiding surgery, it would be a significant boon for OA patients. Summary of the Invention

[0004] The purpose of the present invention is to provide a drug that can prevent or delay the progression of osteoarthritis.

[0005] In order to achieve the above object, the present invention provides a use of Chlorella in preparing a drug for treating osteoarthritis.

[0006] Optionally, the characteristic size of the Chlorella is 2 μm to 10 μm.

[0007] Optionally, the drug is a chlorella solution.

[0008] Optionally, the concentration of the Chlorella solution is 1*10 5 cells / mL~1*10 8 cells / mL.

[0009] Optionally, the Chlorella is Chlorella vulgaris ZF strain GY-D25 Chlorella vulgaris.

[0010] Optionally, the drug is polydopamine-coated Chlorella.

[0011] Optionally, the concentration of the polydopamine-coated Chlorella is 1*10 5 cells / mL~1*10 8 cells / mL.

[0012] Optionally, the preparation method of the polydopamine-coated Chlorella comprises: centrifuging Chlorella from a Chlorella culture medium to obtain a precipitate, washing the precipitate with PBS, dispersing the washed precipitate in a dopamine hydrochloride solution, stirring at room temperature in the dark, washing with PBS after the stirring, and resuspending the precipitate by centrifugation to obtain the polydopamine-coated Chlorella.

[0013] Optionally, the polydopamine-coated Chlorella has a photothermal effect under irradiation with 808 nm near-infrared light.

[0014] The present invention also provides a medicine for treating osteoarthritis, which contains Chlorella.

[0015] Compared with the prior art, the beneficial effects of the present invention include at least:

[0016] The present invention demonstrates that Chlorella vulgaris can maintain chondrocyte metabolic homeostasis, inhibit the catabolism of cartilage extracellular matrix (ECM), and promote the anabolism of cartilage extracellular matrix. Injecting a Chlorella solution into the knee joint cavity alleviates osteoarthritis pain, improves gait, delays articular cartilage degeneration, and reduces osteophyte proliferation and synovial inflammation. Furthermore, the present invention uses polydopamine to modify Chlorella vulgaris to obtain polydopamine-coated Chlorella at photothermal effect. Ch@P not only maintains the biological activity of Chlorella vulgaris, but also, due to the photothermal effect under near-infrared (NIR) irradiation, has the effect of delaying cartilage degeneration and the progression of OA. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The following are characterization images of Chlorella vulgaris according to the present invention. A is an optical micrograph of Chlorella vulgaris, and B is a SEM image of Chlorella vulgaris.

[0018] Figure 2Figure 1 shows how Chlorella vulgaris promotes the expression of genes related to ECM anabolism and inhibits the expression of genes related to ECM catabolism in chondrocytes. A shows how Chlorella vulgaris promotes the expression of genes related to ECM anabolism and inhibits the expression of genes related to ECM catabolism in chondrocytes. B shows how Chlorella vulgaris promotes the expression of proteins related to ECM anabolism and inhibits the expression of proteins related to ECM catabolism in chondrocytes. C shows the quantitative analysis of protein expression.

[0019] Figure 3 The figures are the results of toluidine blue and Alcian blue staining of the present invention. Among them, A is the toluidine and Alcian blue staining of chondrocytes, B is the quantitative analysis of toluidine staining, and C is the quantitative analysis of Alcian blue staining.

[0020] Figure 4 Figure 1 shows the experimental results of the present invention's Chlorella treatment to alleviate pain and improve gait in OA mice. Figure A shows Chlorella reducing mechanical pain sensitivity in OA mice, Figure B shows Chlorella reducing acute thermal pain sensitivity in OA mice, and Figures C and D show Chlorella improving gait in OA mice.

[0021] Figure 5 This figure shows the results of the Chlorella vulgaris algae in reducing cartilage degeneration and synovial inflammation in OA mice. A shows Chlorella vulgaris reducing cartilage degeneration and synovial inflammation in OA mice, and B and B show the synovial inflammation score, OARSI score, and cartilage area and thickness score, respectively.

[0022] Figure 6 The following is a diagram showing the photothermal effect of polydopamine-coated Chlorella vulgaris. Ch@P-1 represents polydopamine-coated Chlorella vulgaris (with an initial dopamine reaction concentration of 5 mM), and Ch@P-2 represents polydopamine-coated Chlorella vulgaris (with an initial dopamine reaction concentration of 10 mM). A shows infrared photothermal images of Ch@P-1 and Ch@P-2 under 808 nm near-infrared laser irradiation; B shows the photothermal temperature rise curves of Ch@P-1 and Ch@P-2 under 808 nm near-infrared laser irradiation. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0024] Articular cartilage degeneration is one of the main pathological features of osteoarthritis. Cartilage tissue is composed of chondrocytes and cartilage matrix. As the only cell type within articular cartilage, chondrocytes are responsible for the synthesis and secretion of the extracellular matrix, maintaining the integrity and balance of the cartilage matrix. Abnormal chondrocyte metabolism can disrupt cartilage homeostasis, leading to the occurrence and development of OA. In the chronic inflammatory microenvironment of OA, inflammatory factors can activate cartilage extracellular matrix catabolic factors (MMPs, ADMTS, etc.) and inhibit the expression of anabolic factors (Sox9, Col2a1, ACAN, etc.), causing an imbalance in cartilage extracellular matrix metabolism, leading to cartilage extracellular matrix degradation and cartilage degeneration. The development of new therapeutic drugs that can regulate chondrocyte metabolism and promote articular cartilage repair and regeneration is of great significance for the treatment of OA and the research of new treatment strategies.

[0025] Chlorella, a spherical, single-celled green microalgae found in eutrophic freshwater, is a natural, environmentally friendly, and renewable biomaterial with a wide range of applications in food, bioenergy, health supplements, aquaculture, and environmental protection. It contains a variety of functional compounds, including polysaccharides, proteins, and lipids, with potential antioxidant, anti-inflammatory, and immunomodulatory effects. Chlorella or Chlorella extracts have been shown to modulate immune responses, inhibit bacteria, and exhibit significant anticancer effects against various cancer types. However, to date, there have been no reports on the use of Chlorella for the treatment of osteoarthritis.

[0026] The chlorella used in the present invention is the common chlorella ZF strain GY-D25 Chlorella vulgaris, which was purchased from Shanghai Guangyu Biotechnology Co., Ltd.

[0027] 1. Characterization of Chlorella and Preparation of Chlorella Solution

[0028] Optical microscope, scanning electron microscope and other microscopes were used to collect images of Chlorella to observe the characteristics of Chlorella. Figure 1 The results show that Chlorella is a spherical, single-celled freshwater algae that exhibits good dispersion in water. Scanning electron microscopy (SEM) further confirmed the typical spherical single-cell morphology of natural Chlorella.

[0029] Preparation of Chlorella solution: After the purchased Chlorella was expanded and centrifuged, it was dispersed into PBS solution. In the subsequent in vitro cell experiments, the Chlorella was diluted with cell culture medium. The culture medium was any one of F / 2 concentrated culture medium, BG11 culture medium or SE culture medium, all purchased from Shanghai Guangyu Biotechnology Co., Ltd. In the subsequent co-culture with cells, after the cells attached to the wall, the Chlorella solution was added, and about 3*10 cells per well were added. 5 cells, and cultured for 24 h.

[0030] 2. Chlorella alleviates cartilage extracellular matrix degradation

[0031] Experimental methods:

[0032] (1) Cell culture

[0033] Primary chondrocytes were isolated and cultured from the articular cartilage of newborn mice. The articular cartilage was cut into small pieces and digested with trypsin at 37°C for 30 minutes, followed by digestion with 0.2% type II collagenase for 6 hours. DMEM / F12 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin solution) was added and placed in an incubator to expand and culture the primary chondrocytes.

[0034] (2) Fluorescence quantitative PCR

[0035] Chondrocytes were cultured at 2×10 5 / well were inoculated in a six-well plate, and then a concentration of 2*10 6 Cells / mL of Chlorella solution were used for treatment. Total RNA was extracted from chondrocytes using a total RNA kit, and RNA concentration was measured using a NanoDrop spectrophotometer. RNA was reverse transcribed into cDNA using a qScript cDNA synthesis kit, and fluorescent quantitative PCR experiments were performed using a SYBR Premix Ex Taq kit.

[0036] (3) Western blotting

[0037] Chondrocytes were cultured at 2×10 5 / well were inoculated in a six-well plate, and then a concentration of 2*10 6 Cells were treated with a 100 cells / mL solution of Chlorella vulgaris. The cells were gently washed three times with pre-chilled PBS and lysed on ice for 30 minutes using RIPA buffer containing 1% protease and phosphatase inhibitors. The cell lysate supernatant was collected after centrifugation at 12,000 g for 15 minutes at 4°C. Protein concentration was measured using a BCA protein quantification kit. After adding the buffer, the cells were boiled at 95°C for 15 minutes. Proteins were separated using 4%-20% polyacrylamide gel electrophoresis and electrotransferred onto a PVDF membrane. Subsequently, the membrane was blocked with 3% BSA solution for 1 hour at room temperature and incubated with a primary antibody overnight at 4°C. The membrane was washed with Tris buffer containing Tween-20 and incubated with a secondary antibody solution on a shaker in the dark for 1 hour. Images were acquired using an Odyssey dual-color infrared fluorescence imager.

[0038] (4) High-density culture and staining of chondrocytes

[0039] Use trypsin to digest chondrocytes, centrifuge at 300g for 5min, resuspend the cells and count them. According to the cell counting results, the cell density is adjusted to 1.5*107 / ml. Add 10 μl / well of cell suspension to the center of a 24-well plate. Incubate in an incubator for 2 hours, then add 1 ml of cell culture medium. Change the cell culture medium every other day. Stain with Alcian blue and toluidine blue after approximately 7 days of culture. Aspirate the cell culture medium, wash three times with PBS, fix with 4% paraformaldehyde at room temperature for 30 minutes, and wash three times with PBS. Stain with Alcian blue or toluidine blue staining solution at room temperature for 30 minutes, wash three times with PBS, and capture images.

[0040] Experimental results:

[0041] Articular cartilage degeneration is the main hallmark of osteoarthritis. Chondrocytes, as the only cell type in articular cartilage, regulate the synthesis and secretion of ECM and maintain the homeostasis of articular cartilage. This application studies whether natural Chlorella vulgaris can effectively alleviate the degradation of chondrocyte ECM under inflammatory stimulation. Figure 2 As shown, PCR and WB results showed that after stimulation with the inflammatory factor IL-1β, the expression of chondrocyte ECM anabolic genes and proteins (Sox9, Col2a1) decreased, while the expression of ECM catabolism genes and proteins (MMP3, MMP13) increased. Treatment with Chlorella promoted the expression of ECM anabolic genes and proteins and inhibited the expression of ECM catabolism genes and proteins. High-density chondrocyte culture results showed that after stimulation with the inflammatory factor IL-1β, chondrocyte ECM degradation occurred, while treatment with Chlorella delayed chondrocyte ECM degradation. Furthermore, immunofluorescence staining results showed that the addition of Chlorella increased the proportion of Col2a1- and Sox9-positive chondrocytes, further confirming that Chlorella can promote chondrocyte ECM synthesis. These results indicate that Chlorella can maintain chondrocyte homeostasis and alleviate chondrocyte ECM degradation in the inflammatory OA microenvironment.

[0042] 3. Chlorella delays articular cartilage degeneration and OA progression

[0043] Experimental methods:

[0044] (1) Establishment of the OA model in mice with destabilized medial meniscus (DMM)

[0045] Eighteen 3-month-old male C57BL / 6 mice were randomly divided into three groups: (1) Sham group, (2) DMM group, and (3) DMM + Chlorella group, with 6 mice in each group. The experimental animals were housed under standard conditions, with 12-h alternating light and dark lighting, temperature (22-24°C), relative humidity (40-60%), and adequate water and food.

[0046] Medial meniscotibial ligament amputation (DMM): Mice were anesthetized, the surgical site was prepared and disinfected, and a scalpel blade was used to incise the skin and medial aspect of the patellar ligament to expose the knee joint cavity. The intercondylar fat pad was bluntly dissected to expose the medial meniscotibial ligament, which was then cut with microscissors and the knee joint capsule and skin were sutured layer by layer. In the sham group, only the medial meniscotibial ligament was exposed without amputation, and samples were collected 8 weeks later.

[0047] The Chlorella solution was injected into the knee joint cavity of mice using a microsyringe, 10 μL per mouse, once a week for 8 weeks.

[0048] (2) Behavioral experiments

[0049] Mechanical pain sensitivity in mice was measured using a Von Frey analgesia. Mice were placed on a grid platform and allowed to acclimate to the experimental environment to minimize stress. A fiber was gently touched perpendicularly to the sole of the hind paw for several seconds to observe for a rapid paw withdrawal response. The intensity of each stimulus and the mouse's response were recorded, and the mean mechanical pain threshold was calculated.

[0050] The hot plate test measures acute thermal pain sensitivity in mice. Heat a hot plate to a constant temperature of 55°C, place the mouse on the hot plate, and start a timer. Closely observe and record the time (latency) until the mouse first displays a pain response, such as licking its paw or jumping. After the test, immediately remove the mouse and return it to its cage, and record the reaction time.

[0051] The CatWalk gait analysis system was used to assess gait changes in mice. Mice were placed on a glass-floored platform in the gait analyzer and acclimated to the experimental environment to minimize stress. The mice were then recorded as they walked freely from one end to the other. Gait analysis, including footprint area, maximum contact area, average contact force, and percentage of stance phase, was performed using CatWalk software.

[0052] (3) Histological staining

[0053] Safranin Fast Green Staining: Place the paraffin sections of the mouse knee joint in environmentally friendly dewaxing and clearing solution I (20 min) - dewaxing and clearing solution II (20 min) - anhydrous ethanol I (5 min) - anhydrous ethanol II (5 min) - 75% alcohol (5 min) in sequence, and finally wash with tap water. Place the sections in fast green staining solution and stain for 1-5 minutes, use tap water to wash off excess staining solution until the cartilage becomes colorless, soak in 1% hydrochloric acid alcohol for 10 seconds, and rinse slightly with tap water. Place the sections in safranin staining solution for 1-5 seconds, then quickly dehydrate in four cylinders of anhydrous ethanol, and examine under a microscope after the fourth dehydration. Use clean xylene to make it transparent for 5 minutes, and then seal the sections with neutral gum. Perform microscopic examination under a microscope, and collect and analyze images.

[0054] HE staining: Place the sections in hematoxylin solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash again with tap water, then use bluing solution to bluish, and rinse with running water. Dehydrate the sections in 85% and 95% graded alcohol for 5 minutes each, then place in eosin solution for 5 minutes. Transparent the sections in anhydrous ethanol I (5 minutes) - anhydrous ethanol II (5 minutes) - anhydrous ethanol III (5 minutes) - xylene I (5 minutes) - xylene II (5 minutes), and seal with neutral gum. Examination was performed under a microscope, and images were collected and analyzed.

[0055] Experimental results:

[0056] like Figures 3 to 5 Von Frey and hot plate tests showed that Chlorella treatment alleviated lower limb pain in OA mice 4 and 8 weeks after DMM surgery. Gait analysis showed that Chlorella treatment improved gait in OA mice. Safranin fast green and HE staining revealed that DMM mice had severe cartilage degeneration and significant synovial inflammation, and Chlorella treatment alleviated these conditions.

[0057] 4. Preparation method of polydopamine-coated Chlorella and its photothermal effect

[0058] Experimental methods:

[0059] (1) Preparation method of polydopamine-coated Chlorella (Ch@P)

[0060] Chlorella (10 mL) was centrifuged and washed twice with sterile PBS, then dispersed in dopamine hydrochloride solution (5 mM or 10 mM Tris-HCl solution, 10 mL, pH 8.0) and gently stirred for 2 hours (300 rpm) at room temperature in the dark, and finally washed three times with sterile PBS, centrifuged and resuspended.

[0061] (2) Photothermal effect of Ch@P

[0062] Ch@P with initial dopamine concentrations of 5 mM and 10 mM were dispersed in PBS (Ch@P-1, Ch@P-2). The 808 nm near-infrared laser was used at different powers (0, 1.0, and 1.5 W / cm 2 ) for 10 minutes. The temperature was recorded every 60 seconds using a thermal imaging camera.

[0063] Experimental results:

[0064] like Figure 6As shown, optical microscopy and laser confocal microscopy observations found that the Ch@P solution appeared gray, and dopamine coating did not change the fluorescence characteristics of Chlorella. Transmission electron microscopy results showed that Chlorella had a good morphology, and dopamine was obviously aggregated around Chlorella. The elemental mapping results showed that the C, O and N elements of dopamine were evenly distributed on the surface of Chlorella. Compared with natural Chlorella, the C, O and N elements of Ch@P were significantly increased. Ultraviolet-visible absorption spectrum (UV-Vis) showed that the absorption peak of Ch@P near 690nm was well maintained. Thermal infrared image and photothermal curve results showed that under 808nm NIR laser (power density of 1.0W / cm 2 ), Ch@P exhibited significant photothermal conversion capabilities under irradiation, indicating that polydopamine-coated Chlorella vulgaris has the effect of delaying cartilage degeneration and OA progression.

[0065] In summary, the present invention confirms that Chlorella can maintain the metabolic homeostasis of chondrocytes, inhibit the catabolism of cartilage extracellular matrix, and promote the anabolism of cartilage extracellular matrix. By injecting Chlorella solution into the knee joint cavity, it has the effects of alleviating osteoarthritis pain, improving gait, delaying articular cartilage degeneration, and reducing osteophyte hyperplasia and synovial inflammation. Furthermore, the present invention uses polydopamine to transform and modify Chlorella to obtain polydopamine-coated Chlorella Ch@P with photothermal effect. Ch@P not only maintains the biological activity of Chlorella, but also has the effect of delaying cartilage degeneration and OA progression due to the photothermal effect under near-infrared (NIR) irradiation.

[0066] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. Use of Chlorella vulgaris in the preparation of a drug for treating osteoarthritis.

2. The use according to claim 1, characterized in that The characteristic size of the Chlorella is 2 μm to 10 μm.

3. The use according to claim 1, characterized in that The medicine is chlorella solution.

4. The use according to claim 1, wherein The concentration of the chlorella solution is 1*10 5 cells / mL~1*10 8 cells / mL.

5. The use according to claim 1, characterized in that The chlorella is the common chlorella ZF strain GY-D25 Chlorella vulgaris.

6. The use according to claim 1, wherein The drug is polydopamine-coated Chlorella.

7. The use according to claim 6, characterized in that In the polydopamine-coated chlorella, the concentration of chlorella is 1*10 5 cells / mL~1*10 8 cells / mL.

8. The use according to claim 6, characterized in that The preparation method of the polydopamine-coated chlorella comprises the following steps: centrifugally separating the chlorella from the chlorella culture medium to obtain a precipitate; washing the precipitate with PBS; dispersing the washed precipitate in a dopamine hydrochloride solution; stirring the precipitate at room temperature in the dark; washing the precipitate with PBS after the stirring is completed; and resuspending the precipitate by centrifugation to obtain the polydopamine-coated chlorella.

9. The use according to claim 6, characterized in that The polydopamine-coated chlorella has a photothermal effect under irradiation of 808nm near-infrared light.

10. A drug for treating osteoarthritis, characterized in that: The medicine contains chlorella.

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