Baicalein alleviates osteoarthritis by protecting subchondral bone, inhibiting angiogenesis and synovial proliferation

Abstract

Osteoarthritis (OA) is one of the most frequent chronic joint diseases with the increasing life expectancy. The main characteristics of the disease are loss of articular cartilage, subchondral bone sclerosis and synovium inflammation. Physical measures, drug therapy and surgery are the mainstay of treatments for OA, whereas drug therapies are mainly limited to analgesics, glucocorticoids, hyaluronic acids and some alternative therapies because of single therapeutic target of OA joints. Baicalein, a traditional Chinese medicine extracted from Scutellaria baicalensis Georgi, has been widely used in anti-inflammatory therapies. Previous studies revealed that baicalein could alleviate cartilage degeneration effectively by acting on articular chondrocytes. However, the mechanisms involved in baicalein-mediated protection of the OA are not completely understood in consideration of integrality of arthrosis. In this study, we found that intra-articular injection of baicalein ameliorated subchondral bone remodelling. Further studies showed that baicalein could decrease the number of differentiated osteoblasts by inhibiting pre-osteoblasts proliferation and promoting pre-osteoblasts apoptosis. In addition, baicalein impaired angiogenesis of endothelial cells and inhibited proliferation of synovial cells. Taken together, these results implicated that baicalein might be an effective medicine for treating OA by regulating multiple targets.

Keywords: angiogenesis; baicalein; osteoarthritis; subchondral bone; synovitis.

FIGURE 1

Intra‐articular injection of baicalein ameliorates subchondral bone remodelling in OA. A, Representative H&E staining and safranin O staining of the articular cartilage and subchondral bone after treating osteoarthritis employing baicalein. B, The effect of baicalein on the structure of tibial subchondral bone analysed by micro‐CT. C‐F, Baicalein reduced tibial subchondral bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th) and increased trabecular separation(Tb.Sp) compared to DMM surgery groups (**P < .01, *P < .05)

FIGURE 2

Baicalein decreases the differentiation of pre‐osteoblasts in the subchondral bone of OA. A, Immunofluorescence staining (Runx2, Osterix) visualised baicalein obviously reduced osteogenic ability of pre‐osteoblasts in subchondral bone compared to DMM surgery group. B,C, Quantification of the intensity of Runx2 and Osterix signal in subchondral bone of the tibia, n = 5, ***P < .001, **P < .01. D‐G, The qRT‐PCR analyses indicated the mRNA expression of ALP, Runx2 and OCN, n = 3, ***P < .001, **P < .01, *P < .05. G, Western blotting analysis of ALP, Runx2 and OCN expression, β‐actin was used as reference gene. H, Immunofluorescence assay results showing the expression level of Runx2. I, ALP staining and alizarin red staining indicated that baicalein decreases pre‐osteoblasts differentiation

FIGURE 3

Baicalein induces apoptosis of pre‐osteoblasts in vivo and in vitro. A,B, TUNEL staining and quantification intensity of joints longitudinal sections were performed to identify apoptotic cells in vivo after injected baicalein, n = 5, **P < .01. C, Flow cytometry assay showed apoptotic cell percentage markedly increased in baicalein group than control group. D, The results of flow cytometric analysis are expressed as percentages of positive mean values ± SD, n = 3, ***P < .001, **P < .01, *P < .05. E,F, TUNEL staining and positive cell rates of pre‐osteoblasts were applied to show apoptotic cells in vitro. G, Caspase‐3 activity of pre‐osteoblasts after treated with baicalein in different concentrations, n = 3, **P < .01, *P < .05. H, Western blotting results of caspase‐3, bcl‐2 and bax

FIGURE 4

Baicalein decreases proliferation of pre‐osteoblasts in vivo and in vitro. A,B, EdU assay and corresponding statistical data at different baicalein concentrations demonstrated that baicalein decreased pre‐osteoblast proliferation, n = 3, ***P < .001, **P < .01, *P < .05. C, CCK‐8 assay revealed that baicalein induced a notable inhibition of cell viability compared with scramble control, n = 3

FIGURE 5

Baicalein impairs angiogenesis of subchondral bone in vivo and in vitro. A,B, Immunofluorescence staining (CD31) and quantification intensity of joints longitudinal sections visualized baicalein obviously reduced angiogenesis in subchondral bone compared to DMM surgery group, n = 5, *P < .05. C, Tube formation assay evaluated angiogenesis effects of baicalein in vitro. D,E, Matrigel plug formation and HE staining of Matrigel sections show baicalein can impair angiogenesis obviously

FIGURE 6

Baicalein inhibits proliferation of synovial cells. A,B, EdU assay and corresponding statistical data demonstrated that baicalein decreased proliferation n = 3, **P < .01, *P < .05. C, CCK‐8 assay revealed that baicalein induced an inhibition of FLSs compared with control, n = 3