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.

of OA. [4][5][6] However, recent researches revealed that targeting AC alone may not be sufficient to halt the disease progression and the 'arthrosis is an organ' concept has begun to be recognized as a fundamental basic science as our understanding of the pathophysiology of OA grows. 3,7,8 Growing evidence suggested that both subchondral bone and synovium were also actively involved in OA development and often preceded AC damage. 9,10 Treatment approaches focussed on AC degeneration, subchondral bone abnormalities and synovitis together have been promised to be new choices for OA. 11,12 Subchondral bone is a bone area below the AC and formed by the subchondral plate, which is a layer of highly vascular cortical bone. 13 Subchondral bone sclerosis and osteophyte formation were associated with the abnormal phenotype and increased anabolism of the osteoblasts. [14][15][16] Additionally, studies have shown that osteogenesis accompanied with abnormal subchondral bone angiogenesis may contribute to the development of subchondral bone remodelling. Thus, osteoblasts and vascular endothelial cells can act as key targets for OA treatment. 17 Baicalein is a flavonoid extracted from Scutellaria baicalensis Georgi (HuangQin in Chinese), a Chinese medical plant used in anti-inflammatory and anti-allergic therapies frequently. 18,19 Simultaneously, baicalein has been revealed to alleviate the OA disease effectively by ameliorating apoptotic and catabolic phenotype of chondrocytes in AC. 20,21 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 human umbilical vein endothelial cells (HUVECs) and inhibited proliferation of fibroblast-like synovial cells (FLSs).
Taken together, these results implicated that baicalein might be an effective medicine for treating OA through by regulating multiple targets.

| Animal experiments
Adult male Sprague-Dawley rats were used to induce OA model by destabilized medial meniscus (DMM) surgery (three groups and in each group n = 5; eight weeks old; mean body weight = 220 g).
Briefly, after anaesthetisation, a medial articular incision approach was made to expose the right knee joint. Then, the medial meniscus ligament was transected, and the medial meniscus was dissociated gently. Finally, the medial capsular incision was sutured, and the skin was closed. A sham operation was performed by only opening the joint cavity. The skin wound healed 1 week after DMM surgery, and a total of 1 mg baicalein (Cayman Chemicals) per knee were intra-articular injected once weekly for 10 consecutive weeks, whereas injected saline was used as control. The usage of baicalein is in line with the product instruction. First, baicalein was dissolved in DMSO at storage concentrations (20 mg/ mL). Baicalein is sparingly soluble in aqueous; therefore, we apply Tween-80 as co-solubiliser. The detailed proportion is as follows: baicalein (100 μL, 20 mg/mL) + normal saline (800 μL) + Tween-80 (100 μL). Injected dose is 50 μL solution per knee. All animal experiments were performed according to the protocol approved by the Shanghai Jiao Tong University (SJTU) Animal Care and Use Committee.

| Radiological evaluation
Knees of OA rats were received to radiographical evaluation at 12 week after surgery. The intact knee joints were obtained and fixed in 70% ethanol for 24 hours after killing by excessive anaesthesia. Samples were scanned using SkyScan1172 high-resolution micro-CT (Bruker) as previously described, 20 with some modifications that set the parameters as follows: 100 kVp, 100 μA and 10.0 μm per pixel. The data were reconstructed and three-dimensional modelled for further analysis.

| Histomorphometry and immunofluorescence
Samples were fixed in 4% paraformaldehyde overnight, followed by decalcification in EDTA-buffered saline solution (pH 7.4, 0.25 mol/L) for 21 days. Tissue sections were then cut longitudinally to obtain 10 μm sections. Histological changes and subchondral bone content were observed by HE and Safranin-O/Fast Green staining.

| Osteoblast culture
Osteoblasts were isolated from the calvaria of neonatal Sprague-Dawley rats by collagenase digestion following the previous protocols. 21 Briefly, dissected calvarium from neonatal rats and removed periosteum and blood vessels to reduce other cells.

| Fibroblast-like synovial cells culture
The FLSs were isolated from synovial tissues from 12 weeks rat joints following the previous protocols. 24 foetal bovine serum (FBS, Gibco Laboratories). Next, the culture flask was placed upright with 5% CO 2 at 37°C for tissue adhesion.
After 4 hours, cell culture flasks were carefully laid flat. FLSs migrated out from tissue explants and grown into a monolayer of 95% confluency within three weeks. The culture medium was renewed every 2-3 days. The FLSs from passage 3-6 were used for further analysis. The total number of osteoblasts was calculated using DAPI staining. Finally, the cells and sections were observed with a confocal microscopy.

| CCK-8 assay
The effects of baicalein on osteoblasts and synovial cells viability were determined by a CCK-8 assay (Dojindo Laboratories).

Briefly, osteoblasts and synovial cells were inoculated into 96-well
plates.100 µL of WST-8 was added into each well of the plates at 37°C for 1 hour. The absorbance of each sample was measured on microplate reader (Infinite F50, Tecan) at a wavelength of 450 nm.

| 5-Ethynil-2′-deoxyuridine (EdU) proliferation assay
Osteoblasts and synovial cells were inoculated into 24-well plates and incubated with 50 μmol/L EdU (Sigma-Aldrich) for 2 and 12 hours, respectively. Next, cells were fixed and permeabilised at room temperature. After washing the cells three times with PBS, 100 μL of 1X Apollo reaction mixture was added to each well for 30 minutes. At last, the cells were stained with Hoechst 33258. The

EdU rates were calculated as the ratio of EdU-positive cells (red cells)
to total Hoechst 33342-positive cells (blue cells).

| Western blotting analysis
Western blotting analysis was accomplished according to a previous report. 26 Total protein from each group was fractionated by 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane followed by blocking with 5% non-fat milk and incubated with primary antibod-

| Matrigel plug assay
Matrigel plug angiogenesis was applied as previously described. 27 HUVECs treated with 0, 10, 20 and 50 μmol/L baicalein resuspended with 300 μL Matrigel and subcutaneously injected into four-weekold male nude mice. After seven days, Matrigel pellets were acquired, partially fixed with 4% formalin, embedded in paraffin, then processed HE staining.

| Statistical analysis
The composite data are expressed as mean ± SD Statistical analysis was performed with two-sided Student's t test and one-way analysis to show the difference between groups. Differences were considered be significant at P < .05.

| Intra-articular injection of baicalein ameliorates subchondral bone remodelling in OA
H&E and safranin O staining showed the cartilage integrity and subchondral bone histological patterns in sham, OA and baicaleininjected OA group. Baicalein could ameliorate the progression of OA induced by DMM surgery effectively ( Figure 1A). Furthermore, the effect of baicalein on the structure of tibial subchondral bone was analysed by micro-CT ( Figure 1B). As shown in Figure 1C

| Baicalein decreases the differentiation of preosteoblasts in the subchondral bone of OA
To investigate whether baicalein is functional for the develop-  Figure 2H). In addition, ALP staining and alizarin red staining ( Figure 2I) indicated that baicalein could significantly decrease pre-osteoblasts osteogenesis. All results above consistently revealed that baicalein at high concentrations effectively inhibited the differentiation of pre-osteoblasts. cytometry assay showed that the apoptotic cell percentage was markedly increased in the baicalein-treated group than in the control group ( Figure 3C,D). Similar results were obtained by TUNEL staining (Figure 3E,F). Additionally, as presented in Figure 3G,H, baicalein resulted in a remarkable enhancement of caspase-3 activity. Consistently, compared with negative control, baicalein-treated F I G U R E 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 F I G U R E 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 3H). In summary, these results suggested that baicalein induced apoptosis in pre-osteoblasts.

Additionally, EdU assay visualised by fluorescence microscope and
corresponding statistical data further demonstrated that baicalein decreased pre-osteoblast proliferation in a concentration-dependent manner ( Figure 4A,B). Moreover, CCK-8 assay revealed that baicalein induced a notable inhibition of cell viability compared with scramble control ( Figure 4C). Taken together, these results indicated that baicalein could hamper pre-osteoblast proliferation effectively.

| Baicalein impairs angiogenesis of subchondral bone in vivo and in vitro
In OA, angiogenesis plays an important role in osteophyte development, subchondral bone remodelling and cartilage mineralization.
We then detected angiogenesis in subchondral bone of OA rats. The number and percentage area of new blood vessels (CD31+) were increased in subchondral bone/bone marrow in OA group, whereas the neo-vessel formation in baicalein-injected group was decreased in osteoarthritic joints ( Figure 5A,B). Effects of baicalein on angiogenesis in vitro were evaluated by tube formation assay. HUVECs were treated with baicalein at different concentrations and cultured in 48-well plates with 100 μL matrigel in each well. The results showed that baicalein obviously reduced tube formation in number and length in a concentration-dependent manner ( Figure 5C). We also analysed matrigel plug formation following subcutaneous implantation in vivo. After implanted seven days, matrigel pellets were obtained. Matrigel mixed with baicalein-treated HUVECs decreased blood vessel growth ( Figure 5D) and HE staining of matrigel sections showed the similar results ( Figure 5E). In summary, baicalein can obviously impair angiogenesis of subchondral bone in OA.

| Baicalein inhibits proliferation of FLSs
There is increasing recognition that hyperplasia of the synovium plays an important role in the pathogenesis of OA. Thus, we next observed the effect of baicalein on FLSs. FLSs were treated with 0, 2.5, 5, 10, 20 and 50 μmol/L baicalein for 24 hours, followed by

F I G U R E 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 the measurement of cell proliferative rate. EdU assay showed that baicalein decreased FLSs proliferation in a concentration-dependent manner by fluorescence microscope (Figure 6A,B). In addition, CCK-8 assay revealed that baicalein induced inhibition of cell proliferation compared with control group ( Figure 6C). Taken together, these results indicated that baicalein could inhibit synovial cells proliferation.

| D ISCUSS I ON
Although some progress has been made in the pathogenesis and therapeutic options of OA, OA is still a major obstacle of human health with high morbidity. Previous studies showed that baicalein Abnormal vascular formation in subchondral bone is also one of the characteristic pathological features of OA. 30 OA is thought to aggravate by osteochondral angiogenesis where blood vessels grow into the tidemark at the osteochondral junction. 31 Consistently, studies have shown that aberrant subchondral bone angiogenesis accompanied with osteogenesis may contribute to the development of subchondral bone sclerosis and osteophyte formation. 32 Hence, subchondral bone angiogenesis emerged in sight as a fresh therapeutic target to delay the progress of OA. Baicalein has been revealed reducing angiogenesis in different circumstances, such as inflammatory microenvironment and tumour tissues. 33,34 In this study, the effect of baicalein on vascularisation of HUVECs was tested in vivo and in vitro and the results showed that baicalein could effectively suppress angiogenesis. Thus, our study provides a new mechanism that baicalein ameliorates subchondral bone remodelling through inhibiting abnormal vascular formation and generates an outstanding approach in managing subchondral bone aberrant angiogenesis.
Vascular growth is also increased in the synovium of OA joints and associated with synovitis. 35 Therefore, the inhibition of vascularization by baicalein is beneficial to synovium inflammation simultaneously. Furthermore, FLSs are the major effector cells that lead to synovitis. 36 The imbalance of FLSs proliferation and apoptosis has long been considered as initiating factor for rheumatoid arthritis (RA). 37 Taken together, these results indicated that baicalein might be a hypothetical candidate for the treatment of OA and inspire a new approach to develop potential therapeutic agents treating degenerative joints as a whole organ.

ACK N OWLED G EM ENTS
This study was sponsored by the National Natural Science Foundation of China (81870617, 81672202), the Science and Technology Commission of Shanghai Municipality (15411950900).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.