Application of oleanolic acid in promoting chondrogenic differentiation of chondrocytes

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of oleanolic acid in promoting chondrogenic differentiation of chondrocytes. This invention discovers that oleanolic acid promotes the expression of proteoglycans and calcium deposition in ATDC5 cells undergoing chondrogenic differentiation, and also promotes the expression of ATDC5 cell chondrogenic differentiation markers. Therefore, it has the potential to be formulated into a drug that can promote chondrogenic differentiation of chondrocytes, thereby promoting cartilage repair and regeneration for the treatment of osteoarthritis and rheumatoid arthritis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of oleanolic acid in promoting chondrogenic differentiation of chondrocytes. Background Technology

[0002] Osteoarthritis (OA) is one of the most common rheumatic diseases, predominantly affecting middle-aged and elderly individuals, with its incidence increasing with age. OA initially affects the cartilage. Due to the limited repair capacity of cartilage, it cannot heal after damage. The lesions then invade the subchondral bone plate and synovium, leading to focal, erosive cartilage destruction, subchondral sclerosis, cystic changes, and compensatory osteophyte formation. Chronic progressive cartilage destruction is one of the main pathological features of OA. Furthermore, chondrocytes are terminally differentiated cells with limited repair and regeneration capabilities. Therefore, targeting the differentiation of chondrocyte precursor cells to promote cartilage repair and regeneration is a potential treatment strategy for OA.

[0003] The ATDC5 cell line was first isolated from the differentiated teratoma stem cell line AT805 and is commonly used as a model for in vitro chondrocyte research. The ATDC5 cell line exhibits a high degree of similarity in differentiation to chondrocytes, maintaining an undifferentiated state and possessing strong proliferative capacity under normal in vitro culture conditions. Therefore, it is considered a reliable cell model for simulating cartilage formation. Consequently, an increasing number of researchers are using ATDC5 cells to study cell morphology, cell viability, growth status, and the mechanisms of chondrocyte formation.

[0004] Osteocyte formation begins with the aggregation of mesenchymal cells. These cells undergo a series of changes, including aggregation, migration, proliferation, and fusion, eventually differentiating into chondrocyte progenitors. These progenitors then differentiate into bone through four phases: resting phase, proliferative phase, pre-hypertrophic phase, and hypertrophic phase. During bone formation, chondrocytes undergo changes in gene and protein expression, cell morphology, cell surface markers, and metabolic activity. Protein and gene expression during chondrocyte proliferation and differentiation involves the regulation of numerous cell signaling pathways and cytokines. For example, some transcription factors (nuclear factor-cl, Runx2, Sox9, activating transcription factor-4, and AP-1) can interact to regulate the expression of chondrocyte markers. Other important regulatory pathways include the Wnt / β-catenin pathway, transforming growth factor-β (TGF-β), and Ihh, which can influence columnar chondrocyte differentiation by acting on downstream target factors.

[0005] Chondrocyte differentiation is essential for subsequent stages of bone formation, namely the deposition of cartilage matrix proteins such as Co12, type IX collagen, and Aggrecan. A hallmark factor expressed early in chondrogenesis is the nuclear transcription factor Sox9. Before the deposition of cartilage matrix proteins, Sox9 is the most important transcription factor for the expression of cartilage matrix proteins such as Col2al, Col11a2, and CD-RAP. Two other members of the Sox family, L-Sox5 and Sox6, are co-transcribed with Sox9 during chondrocyte proliferation and differentiation. Except within the HMG cassette, Sox9 shares high sequence identity with L-Sox5 and Sox6. L-Sox5 forms homodimers or heterodimers with Sox6, allowing for more efficient binding to the HMG cassette dual site, regulating the expression of Col9al, Aggrecan, FN1, and Col2al during chondrocyte differentiation. During chondrocyte aggregation, Sox protein expression depends on the bone morphogenetic protein (BMP) signaling pathway involving two signaling factors (BMPR1A and BMPR1B), which are not expressed in the perichondrium. The BMP signaling pathway is primarily transduced via heteropolymeric complexes (type I and type II receptors with serine-threonine kinase activity). Upon receptor binding, BMPRII phosphorylates type I receptors, ALK-2, BMPRIA / ALK-3, and BMPRIB / ALK-6. Other BMP-mediated transcription factors include members of the JunB, JunB, JunD, ID, and DLX families. BMPs can transmit signals by activating protein kinase 1 via TGF-β, which in turn activates p38 and JNK through a cascade of interaction with MEKK1. BMPs can also send signals by activating the Ras / ERK1 / 2 / RhoA / ROCK signaling pathway. The p38 signaling pathway facilitates chondrocyte aggregation, and the interaction between activated ERK1 / 2 and the BMP-2-induced signaling pathway can regulate chondrogenesis.

[0006] Oleanolic acid, a pentacyclic triterpenoid compound, also known as angelic acid, is widely distributed in privet fruit and possesses strong anti-inflammatory activity. The anti-inflammatory effects of oleanolic acid have been confirmed by numerous studies, demonstrating its ability to alleviate pain and inflammation in osteoarthritis. In our previous studies, we found that oleanolic acid can inhibit IL-1β-induced inflammatory responses in human synovial cells (SW982) by mediating the MAPK, PI3K / Akt, and NF-κB signaling pathways. However, there are currently no reports on oleanolic acid promoting chondrocyte differentiation. Summary of the Invention

[0007] In response to the shortcomings of existing technologies and practical needs, this invention provides the application of oleanolic acid in promoting chondrogenic differentiation of chondrocytes.

[0008] In a first aspect, the present invention provides the application of oleanolic acid in promoting chondrogenic differentiation of chondrocytes in vitro, wherein the CAS No. of oleanolic acid is 508-02-1.

[0009] Secondly, the present invention provides a chondrogenic differentiation induction culture medium, the culture medium comprising a basal culture medium and oleanolic acid added to the basal culture medium.

[0010] Furthermore, the concentration of oleanolic acid in the basal culture medium is 1 nmol-10 μmol.

[0011] Thirdly, the present invention provides a method for inducing chondrocyte differentiation into chondrocytes in vitro, the method comprising culturing chondrocytes using the aforementioned culture medium.

[0012] Furthermore, the culture time is 3-7 days.

[0013] Fourthly, the present invention provides the use of the oleanolic acid in the preparation of a medicament for treating arthritis, including osteoarthritis and rheumatoid arthritis.

[0014] Furthermore, the drug achieves its therapeutic purpose by promoting cartilage repair and regeneration, which is achieved by promoting chondrogenic differentiation of chondrocytes.

[0015] Furthermore, the drug comprises a pharmaceutically acceptable carrier.

[0016] The present invention has the following beneficial effects:

[0017] This invention is the first to discover that oleanolic acid can promote the chondrogenic differentiation of chondrocytes in vitro. Therefore, it has the potential to be prepared into a drug that can promote the chondrogenic differentiation of chondrocytes and thus promote cartilage repair and regeneration to treat osteoarthritis and rheumatoid arthritis. Attached Figure Description

[0018] Figure 1 This is the chemical structural formula of oleanolic acid.

[0019] Figure 2 The effect of different concentrations of oleanolic acid on the viability of ATDC5 cells.

[0020] Figure 3 Alcian blue staining (A) and Alizarin red staining (B) are images of ATDC5 cells cultured in medium containing 10 nM oleanolic acid for 0, 3, and 7 days, respectively.

[0021] Figure 4 The protein expression levels (A), quantitative analysis results (B), and mRNA expression levels (C) of ATDC5 cells cultured for 0, 3, and 7 days with 10 nM oleanolic acid were measured in 10 nM oleanolic acid medium.

[0022] Figure 5 The protein expression levels (A), quantitative analysis results (B), and mRNA expression levels (C) of ATDC5 cells cultured for 1 day in medium containing 5 nM and 10 nM oleanolic acid, which are markers of chondrogenesis. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0024] Example 1: Chemical structure of oleanolic acid and its effect on cell viability

[0025] The chemical structure of oleanolic acid is as follows: Figure 1 As shown, ATDC5 cells were seeded in 96-well plates. When the cell density reached 70%-80%, different concentrations of oleanolic acid (1 nM, 10 nM, 100 nM, 1 μM, 10 μM) were added and cultured for another 24 h. The effect on the viability of ATDC5 cells was then detected using a CCK8 assay.

[0026] The results are as follows Figure 2 As shown, compared with the control group (ctl, without oleanolic acid), oleanolic acid (≤10uM) had no toxic effects and no impact on cell viability, and can be used for subsequent experiments.

[0027] Example 2: Oleanolic acid promotes the expression of proteoglycans and calcium deposition in ATDC5 cells undergoing chondrogenic differentiation.

[0028] ATDC5 cells in good growth condition were seeded into 35 mm dishes and replaced with complete medium containing 10 nM oleanolic acid after 24 h. The culture was then continued for 0, 3, and 7 days. Alcian blue and Mordant Red staining were used to detect the effects of oleanolic acid on proteoglycan expression and calcium deposition in ATDC5 cells.

[0029] Alcian blue staining diagram ( Figure 3 A) and quantitative results showed that, compared with the control group, proteoglycan expression increased significantly from day 3 to the highest level at day 7, as indicated by Alizarin Red staining ( Figure 3B) and the quantitative results showed a similar trend in calcium deposition. These results indicate that oleanolic acid can affect chondrogenic differentiation of ATDC cells.

[0030] Example 3: Oleanolic acid promotes the expression of chondrogenic differentiation markers in ATDC5 cells

[0031] ATDC5 cells in good growth condition were seeded into 35 mm dishes and cultured for 24 hours. The culture medium was then replaced with complete medium containing 10 nM oleanolic acid. Cells were cultured for 0, 3, and 7 days. Total protein and total RNA were then extracted. Western blotting was used to detect and quantify the protein expression levels of chondrocyte differentiation markers Sox9 and Collagen II. qPCR was used to detect the mRNA expression levels of Sox9 and Coll2a. Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA (n = 3); x ± s. *p < 0.1, ** < 0.01, ***p < 0.001, compared with the control group.

[0032] Western blot results ( Figure 4 A) and grayscale analysis ( Figure 4 B) showed that, compared to day 0, the cartilage markers Sox9 and Coll2 significantly increased from day 3, reaching their peak expression level at day 7. qRT-PCR also showed a similar trend for Sox9 and Coll2 proteins. Figure 4 C). These results suggest that oleanolic acid can promote chondrogenic differentiation of ATDC5 cells, and the degree of effect is directly proportional to the duration of oleanolic acid treatment.

[0033] ATDC5 cells in good growth condition were seeded into 35 mm dishes. After 24 h, the culture medium was replaced with complete medium containing 5 nM and 10 nM oleanolic acid, respectively. After 1 day, total protein and total RNA were extracted from the cells and the control group, respectively. Western blotting was used to detect and quantify the protein expression levels of chondrocyte differentiation markers Sox9 and Collagen II. qPCR was used to detect the mRNA expression levels of Sox9 and Coll2a.

[0034] Western blot results ( Figure 5 A) and grayscale analysis ( Figure 5 B) showed that, compared with the control group, the expression levels of cartilage markers Sox9 and Coll2 increased with increasing treatment concentration. qRT-PCR also showed a similar trend for Sox9 and Coll2 proteins. Figure 5 C). These results suggest that oleanolic acid can promote chondrogenic differentiation of ATDC5 cells, and the effect is proportional to the concentration of oleanolic acid.

[0035] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0036] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of oleanolic acid for promoting chondrogenic differentiation of chondrocytes in vitro, characterized in that, The application specifically is culturing chondrocytes with chondrogenic differentiation induction medium, the medium includes base medium and oleanolic acid added in the base medium, the concentration of the oleanolic acid in the base medium is 10nM, and the culturing time is 3-7 days.

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