Semi-synthetic chondroitin sulfate CS-semi5 upregulates miR-122-5p, conferring a therapeutic effect on osteoarthritis via the p38/MMP13 pathway

Abstract

Osteoarthritis (OA) is an aging-associated disease characterized by joint stiffness pain and destroyed articular cartilage. Traditional treatments for OA are limited to alleviating various OA symptoms. There is a lack of drugs available in clinical practice that can truly repair cartilage damage. Here, we developed the chondroitin sulfate analog CS-semi5, semi-synthesized from chondroitin sulfate A. In vivo, CS-semi5 alleviated inflammation, provided analgesic effects, and protected cartilage in the modified Hulth OA rat model and papain-induced OA rat model. A bioinformatics analysis was performed on samples from OA patients and an exosome analysis on papain-induced OA rats, revealing miR-122-5p as the key regulator associated with CS-semi5 in OA treatment. Binding prediction revealed that miR-122-5p acted on the 3'-untranslated region of p38 mitogen-activated protein kinase, which was related to MMP13 regulation. Subsequent in vitro experiments revealed that CS-semi5 effectively reduced cartilage degeneration and maintained matrix homeostasis by inhibiting matrix breakdown through the miR-122-5p/p38/MMP13 axis, which was further validated in the articular cartilage of OA rats. This is the first study to investigate the semi-synthesized chondroitin sulfate CS-semi5, revealing its cartilage-protecting, anti-inflammatory, and analgesic properties that show promising therapeutic effects in OA via the miR-122-5p/p38/MMP13 pathway.

Keywords: Cartilage; Chondroitin sulfate; Extracellular matrix; Inflammation; MMP13; Osteoarthritis; miRNA; p38.

Graphical abstract

Figure 1

The structure–activity relationship and preliminary activity of CS-semi5. (A) Different types of CS. (B) Synthesis of CS-semi compounds. (C) The characteristics of various CS-semi compounds. (D) CS-semi5 had the best anti-inflammatory activity among the CS-semi series compounds (n = 8). (E) CS-semi5 had a better anti-inflammatory effect than CS-A and D-Glu (n = 8). (F) CS-semi5 inhibited inflammation in a dose-dependent manner (n = 8). (G) CS-semi5 exhibited analgesic activity in the hot plate test (n = 8). (H) CS-semi5 effectively inhibited acetic acid-induced pain (n = 10); ∗P < 0.05 and ∗∗P < 0.01 compared with the Model group. (I) CS-semi5 protected primary chondrocytes from complement-induced death (scale bar: 100 μm). The black box represents the enlarged part of the original image. (J) Quantitative results of the complement-induced death experiment (n = 3); ∗P < 0.05 and ∗∗P < 0.01 compared with NHS group. (K) CS-semi5 improved primary chondrocytes extracellular matrix from complement-induced death (scale bar: 100 μm). The black box represents the enlarged part of the original image. (L) Quantitative results of the Alcian blue staining of extracellular sulfated GAGs (n = 3); ∗P < 0.05 and ∗∗P < 0.01 compared with NHS group.

Figure 2

CS-semi5 decreased MMP13 expression in primary chondrocytes. (A) CS-semi5 had no toxic effects on primary chondrocytes (n = 3). (B) ELISA demonstrated that CS-semi5 reduced MMP13 secretion in a dose-dependent manner (n = 3). Western blotting with quantitative results (C–E) and immunofluorescence analysis (F–H) indicated that CS-semi5 exerted cartilage-protective effects on MMP13, extracellular matrix components ACAN and COL Ⅱ in IL-1β (10 ng/mL)-stimulated primary chondrocytes (n = 3); ∗P < 0.05 and ∗∗P < 0.01 compared with the IL-1β group; scale bar: 25 μm.

Figure 3

CS-semi5 alleviated surgeon-induced OA in Hulth model rats (n = 6). (A) Schematic of the experimental process. (B) Weight changes of rats in all groups. (C) The width of the operated knee joint at the end of the experiment. (D) View of knee joint structure after operation and administration. (E) T1 MRI of operated joints. (F) Sagittal and coronal views of reconstructed joints. The quantitative outcomes of bone volume/total volume (G) and bone surface area/bone volume (H) measurements. ∗∗P < 0.05 and ∗P < 0.01 compared with the modified Hulth model group.

Figure 4

CS-semi5 alleviated papain-induced OA in rats (n = 6). (A) Schematic of the induction and treatment process. (B, C) show the weight changes and changes in the width of the left knee joint in rats during the experimental process. (D) Number of shocks in 2 min in the running test. (E) HE staining (40 × ); scale bar: 500 μm. (F) MRI of the junction between the femur and tibia in groups of control, papain-model, and CS-semi5 200 mg/kg. The T2 time (G) and density values (H) of MR images indicate joint water content. (I) The cross view of subchondral bone reconstruction on micro-CT with sagittal and coronal views of three-dimensional images of the knee joints. ∗P < 0.05 and ∗∗P < 0.01 compared with the Model group.

Figure 5

Preliminary safety evaluation of CS-semi5 (100 and 500 mg/kg) (n = 5). (A) The effect of CS-semi5 on the body weight of rats. (B) The effect of CS-semi5 on the main organ coefficient of rats. (C) HE staining of main organs, including the heart, liver, spleen, lung, kidney, and testicles; scale bar: 1 mm. (D) Hematological, (E) biochemical, and (F) coagulation function analyses of rat blood after 4 weeks of treatment with CS-semi5 at 100 and 500 mg/kg. N.S., no significance between control and treatment groups.

Figure 6

The molecular mechanism by which CS-semi5 treats OA (n = 3). (A) Heatmap showing the different microRNA levels in the control, papain model, and CS-semi5 treatment groups. (B) Venn diagram showing differentially expressed genes in the different groups. (C) CS-semi5 increased miR-122-5p expression in IL-1β induced chondrocytes, as demonstrated by qPCR. ∗∗P < 0.01 compared with the IL-1β-induced model group. (D) The interaction network of target genes for MMP13 and predicted potential binding site of Mapk14 3′UTR mRNA. (E) Luciferase activity in 293T cells transfected with a dual luciferase reporter gene with the miR-122-5p mimic. ∗∗P < 0.01 compared with the group added miR-122-5p mimic. (F) Relative Mmp13 expression level by qPCR in IL-1β induced chondrocytes treated with CS-semi5 treatment with or without the miR-122-5p inhibitor. Immunofluorescence (G) and protein blotting (H) of p-p38 in IL-1β induced chondrocytes with CS-semi5 treatment; scale bar: 25 μm. Western blotting (I) of MMP13 (upper) and quantification (lower) in IL-1β induced chondrocytes treated with CS-semi5 with or without p38 inhibitor SB230580. ∗∗P < 0.01 compared with the IL-1β-induced model group, ^##P < 0.01 compared with the CS-semi5 treated model group. (J) Immunofluorescence of COL II expression in IL-1β induced chondrocytes treated with CS-semi5 with or without the miR-122-5p inhibitor; scale bar: 25 μm.

Figure 7

CS-semi5 inhibits the p38–MMP13 axis in the articular cartilage of OA rats. Immunohistochemical images of papain-induced OA rats show that CS-semi5 reduced p-p38 and MMP13 levels and increased COL II levels in the cartilage vs. those observed in the model groups, scale bar: 100 μm in safranine-O fast green staining images, 20 μm in immunohistochemical images.