DL-3-N-Butylphthalide Promotes Cartilage Extracellular Matrix Synthesis and Inhibits Osteoarthritis Development by Regulating FoxO3a

Abstract

Osteoarthritis (OA) has been reported as a progressive disease in the elderly, primarily characterized by degenerated articular cartilage. There has been no satisfactory drug for the treatment of OA. DL-3-n-butylphthalide (NBP), a small molecule compound extracted from celery seeds, may have antiapoptotic, antioxidant, and anti-inflammatory activities in numerous studies. However, the effects of NBP on OA and its mechanisms have been rarely reported. In this study, the effect of NBP on OA in vitro and in vivo and its possible mechanism were investigated. The results showed that NBP injection into the knee joint inhibited osteoarthritis development in a rat model of osteoarthritis induced by DMM+ACLT. NBP could increase the expressions of extracellular matrix-related components (such as type II collagen, aggrecan, proteoglycan 4, and SRY-box 9) in human osteoarthritic chondrocytes and cartilage explants. Moreover, NBP promoted the expressions of SOD and CAT. NBP upregulated the expression of FoxO3a by inhibiting the PI3K/AKT pathway, which subsequently inhibited the apoptosis of human OA chondrocytes. In conclusion, NBP promotes cartilage extracellular matrix synthesis and inhibits osteoarthritis development and the underlying mechanism related to the activation of FoxO3a.

Figure 1

NBP was identified as a chondrogenic inducer that increased ECM anabolism of human OA chondrocytes in vitro. (a) Chemical structure of NBP. (b) Alcian blue staining (scale bar, 100 μm). (c) Immunofluorescence staining of COLII (scale bar, 100 μm). (d) The CCK8 assay measured the cytotoxicity of NBP on chondrocytes at different concentrations for 24 h. (e, f) The protein expressions of COL2A1 and ACAN were detection by Western blot analysis after chondrocytes were treated with NBP for 24 h. (g) The mRNA levels of COL2A1, ACAN, PRG4, and SOX9 were detected using qRT-PCR. (h, i) The expressions of COLII and ACAN were detected by the immunofluorescence assay (scale bar, 100 μm). All data are expressed as the mean ± standard deviation (SD). ^∗P < 0.05,  ^∗∗P < 0.01, and^∗∗∗P < 0.001.

Figure 2

NBP promoted ECM anabolism in OA cartilage explants. (a) Proteoglycan content was measured by the safranin O-fast green staining after incubation with NBP for 2 w (scale bar, 100 μm). (b) COLII content was measured by immunohistochemistry staining (scale bar, 100 μm). (c) Average optical density values of proteoglycan content determined by safranin O-fast green staining. (d, e) GAG and DNA content per wet weight were assessed after NBP (0.1 μM) treatment for 2 w. (f–i) The mRNA levels of COL2A1, ACAN, PRG4, and SOX9 were detected using qRT-PCR after NBP (0.1 μM) treatment for 2 w. All data are expressed as the mean ± standard deviation (SD). ^∗P < 0.05,  ^∗∗P < 0.01, and^∗∗∗P < 0.001.

Figure 3

NBP inhibited the development of knee OA after intra-articular injection of NBP in a rat model of DMM+ACLT for 8 w. (a) Representative images of HE for cartilage destruction, safranin O-fast green for cartilage regeneration, and immunohistochemistry staining for COLII expression, respectively. (b) OARSI scores in different treatment groups (n = 6). (c) Pain response time when rats were placed onto a 55°C hot plate after the surgery (n = 6). (d) The differences in two hind limb weights after the surgery in rats (n = 6). All data are expressed as the mean ± standard deviation (SD). ^∗P < 0.05,  ^∗∗P < 0.01, and^∗∗∗P < 0.001.

Figure 4

NBP inhibited apoptosis and oxidative stress of human OA chondrocytes. (a, c) Apoptosis was analyzed by flow cytometry and apoptosis rates are expressed in the respective group. (b, d) Apoptotic assay by TUNEL staining was observed under a microscope, and TUNEL-positive cells were calculated (n = 3, scale bar, 100 μm). (e, f) The mRNA levels of Bcl-2 and Bax were measured by qRT-PCR. (g) The protein expressions of CAT, SOD2, Bcl-2, Bax, and Caspase 3 were determined after treatment with NBP (0-10 μM) for 24 h by Western blot analysis. (h, i) The expression levels of SOD and GSH in chondrocytes treated with NBP (0-10 μM) for 24 h. (j, k) The mRNA levels of SOD2 and CAT were measured by qRT-PCR. All data are expressed as the mean ± standard deviation (SD). ^∗P < 0.05,  ^∗∗P < 0.01, and^∗∗∗P < 0.001.

Figure 5

Bioinformatics analysis based on network pharmacology and transcriptome sequencing result after treated with NBP. (a–c) Network pharmacology. (a) Venn diagram of NBP-OA. (b) Bubble plot of the enrichment analysis results: cell components. (c) Signaling pathways. (d–f) Transcriptome sequencing. (d) Volcano plots of differentially expressed genes in the transcriptome. (e) Gene Ontology (GO) analysis of the transcriptome of chondrocytes administrated with NBP. (f) Pathway analysis from the chondrocyte transcriptome.

Figure 6

NBP regulated the PI3K/AKT/FoxO3a pathway in human OA chondrocytes. (a, b) The protein expressions of FoxO1, FoxO3a, p-FoxO3a, FoxO4, PI3K, p-PI3K, AKT, and p-AKT were determined by Western blot assay. (c–e) The mRNA levels of FoxO1, FoxO3a, and FoxO4 were measured using qRT-PCR. (f) Immunofluorescence staining of FoxO3a and ACTIN (scale bar, 20 μm). All data are expressed as the mean ± standard deviation (SD). ^∗P < 0.05,  ^∗∗P < 0.01, and^∗∗∗P < 0.001.

Figure 7

NBP increases ECM anabolism in human OA chondrocytes by regulating FoxO3a. (a, b) The protein and mRNA levels of FoxO3a were evaluated by Western blot and qRT-PCR after treated with and/or Lv-FoxO3a for 24 h. (c) Proteins expressions of ACAN and COL2A1 were detected by Western blot. (d) Immunofluorescence staining for COLII (scale bar, 50 μm). (e–h) The mRNA levels of COL2A1, ACAN, SOX9, and PRG4 were determined by qRT-PCR. All data are expressed as the mean ± standard deviation (SD). ^∗P < 0.05,  ^∗∗P < 0.01, and^∗∗∗P < 0.001.

Figure 8

NBP inhibited apoptosis of human OA chondrocytes by upregulating FoxO3a. (a) Cluster analysis of mRNA levels of downstream genes in the FoxO pathway. (b, c) The mRNA levels Bcl-2 and Bax were determined. (d) The protein expressions of Bcl-2, Bax, and Caspase 3 were determined. All data are expressed as the mean ± standard deviation (SD). ^∗P < 0.05,  ^∗∗P < 0.01, and^∗∗∗P < 0.001.

Figure 9

Molecular mechanism of NBP on inhibiting OA development. NBP possesses protective effect on human OA chondrocytes from apoptosis by regulating PI3K/AKT/FoxO3a pathway (created with https://biorender.com/).