Baicalin Promotes Chondrocyte Viability and the Synthesis of Extracellular Matrix through TGF-β/Smad3 Pathway in Mouse Chondrocytes

Background: Osteoarthritis (OA) is epidemic in the elderly people as a common chronic joint disease. By now, drug and surgical treatments are two main therapies for OA worldwidely. Baicalin (BA) is a avonoid monomer extracted from Scutellaria baicalensis Georgi and is reported that BA has anti-inammatory, anti-deformation and anti-bacterial effects. Methods: Micromass culture, alcian blue and Safran O (SO)/fast green staining were used to investigate chondrocyte viability and ECM synthesis in chondrocytes of all groups. The expression of SOX9 and Smad3 in chondrocytes of all groups were detected by western blot and RT-qPCR, the expression of aggrecan (AGG), type II collagen (Col2α), MMP9/13 and ADAMTS5 were detected by RT-qPCR. In current study, we demonstrated that BA neutralized the down-regulation of chondrocyte viability and extracellular matrix (ECM) secretion, including AGG and Col2α, induced by IL-1β. As the key regulators of ECM, the down-regulation of SOX9, and the up-regulation of MMP9/13 and ADAMTS5 induced by IL-1β were abolished by BA. Moreover, BA increased the nuclear translocation and phosphorylation of Smad3, one key mediator of TGF-β/Smads pathway. Interestingly, the addition of Smad3 inhibitor SIS3 reversed the promotions of BA on chondrocyte viability, ECM secretion, SOX9 expression, Smad3 nuclear translocation and Smad3 phosphorylation, and the down-regulation of BA on the expressions of MMP9/13 and ADAMTS5. Conclusions: These results imply that BA can protect chondrocytes against IL-1β-induced inammatory injury through the acceleration of Smad3 phosphorylation and nuclear translocation in chondrocytes. This study demonstrates that BA may be a potential drug for OA clinical treatment.


Background
Osteoarthritis (OA) is epidemic in the elderly people as a common chronic joint disease [1]. OA has been characterized as joint pain, limb stiffness and others, as well as di culty in movement which seriously affects the daily travel of patients [2]. The rapid growth of the epidemic brings a great burden on individuals and society [3]. By now, the treatments of OA in the world mainly include drug treatment and surgical treatment [4]. The surgical treatment with high cost is usually used in the advanced stage of OA patients [5], while the drug treatment of OA is trapped in the inhibition the in ammation. The drugs have an advantage in relieve in ammation and pain symptoms, but cannot prevent or delay the progress of OA completely [6]. So, it is urgent and necessary to seek new drugs for OA treatments.
Baicalin (BA) is a avonoid monomer extracted from Scutellaria baicalensis Georgi. BA has antiin ammatory, anti-bacterial and diuretic effects [7]. It has been reported that BA has a signi cantly inhibition on the in ammatory response and the expression of IL-1β in chondrocytes [8]. IL-1β is a proin ammatory factor and has shown to inhibit SOX9 expression and extracellular matrix (ECM) synthesis and to induce the productions of MMPs and ADAMTS5 [9]. BA can increase the transcription activity of SOX9, the synthesis of aggrecan (AGG) and type II collagen (Col2α), two main components of cartilage ECM [10] and inhibit the expressions of MMPs [11]. SOX9 is a key transcription factor in the development and differentiation of chondrocytes. The decreased expression of SOX9 leads to arrest differentiation of prechondrocytes and serious skeletal deformities [12]. MMPs are proteases and can degrade the ECM [13]. SOX9 is involved in several pathways including TGF-β/Smads pathway [14]. TGF-β/Smads pathway plays a key role in regulating SOX9 expression during cartilage formation [15]. Recent studies have shown that TGF-β/Smads could activate SOX9 expression [16]. As a key component of TGF-β/Smads pathway, Smad3 was con rmed to involve in chondrocyte differentiation during cartilage development [17]. TGF-β/Smads signaling pathway that results in accelerated chondrocyte maturation is inactivated by de phosphorylation of Smad3 [18]. Since BA can protect chondrocytes by up-regulating the expression of SOX9, but it remains unclear that whether BA protects chondrocytes through the activation of TGFβ/Samd3 by phosphorylating Smad3. Therefore, a chondrocyte in ammatory model in vitro is established by IL-1β in this study. Cellular and molecular biology methods are used to investigate whether BA protects chondrocyte viability and ECM expression from in ammation through TGF-β/Smad3 pathway in mouse chondrocytes.

Micromass culture and Alcian blue & Safranin O staining
Twenty microliters of chondrocyte suspension (1 × 10 6 cells) was added to 12-well plates and incubated for 3 hours at 37°C for cell attachment. One milliliter of fresh DMEM medium containing various concentrations of drugs were added to each well. The experiments were divided into four groups, namely control group, IL-1β group, IL-1β + BA group and IL-1β + BA + SIS3 group. Every three days, medium was changed. After 2 weeks, aggrecan (AGG) was checked by Alcian blue & Safranin O staining. The staining procedure was as below: wells were washed once with PBS (phosphate buffer saline) and then xed for 20 min with 1.0 mL of 4% (v/v) paraformaldehyde (PFA). Subsequently, 0.5 ml of 1.0 % (w/v) Alcian blue (Sigma) (v/v) and 0.5 mL Safranin O solutions were added to each well and incubated for 20 min at room temperature. After 1 wash with 70% (v/v) ethanol and three washes with PBS, photomicrographs of the stained cell mass were obtained by a scanner (EPSON, V550, Japan).
After 24 hours of culture, total RNA was extracted using TRIzol reagent (Thermo Fisher Scienti c). cDNA was synthesized with a PrimeScriptTM Master Mix reagent kit (Takara Bio, Inc. Japan). qRT-PCR was performed with SYBR Premix ExTaq (Takara Bio, Inc.) using the qTOWER version 3.0 PCR system (Jena Industries, Inc.). The mRNA relative expressions of all target genes were analyzed using a formula (2 -△△T ). GAPDH was used as the internal control. The primer sequences of all genes were shown in Table 1. Table 1 Sequences of primers used for gene ampli cation.

Genes
Forward Reverse Immunohistochemistry Chondrocytes (1 × 10 5 /mL) were seeded on the slides placed on 6-well plates. After 24 hours, the chondrocytes were xed with PFA for 10 min at room temperature, and then were incubated with the primary antibody diluent of rabbit anti-p-Smad3 (# BM4033;

Statistical analysis
All data (n=3) were expressed as mean ± standard deviation and were calculated by SPSS 22.0 software. Student's t-test and One-way ANOVA were used for comparison between two groups and among many groups respectively.

BA promotes the viability of ADTC5 chondrocytes
In order to con rm the optimal concentration of BA on chondrocytes viability, the chondrocytes were cultured with increasing concentrations of BA (0, 10, 20 and 40 μmol/L) for 24 hours. The chondrocytes viability was analyzed by MTS method. Various concentrations of BA (10-40 μmol/L) increased the cell viability (p < 0.05) (Fig. 1b). The most signi cant promoting effect on chondrocyte activity was found at 20 μmol/L of BA. As shown in Fig. 1c, cell viability was signi cantly decreased in 10 ng/mL of IL-1β group compared with control group (p < 0.05).
Combing MTS experiment results and literature [19], 20 μmol/L of BA, 10 ng/mL of IL-1β and 10 μmol/L of SIS3 were used in subsequent experiments. The experimental groups were divided into 4 groups, namely control group (without any drugs), IL-1β group, IL-1β + BA group and IL-1β + BA + SIS3 group. After 24 hours of culture, cell viability was analyzed by MTS method. As shown in Fig. 1d, the downregulation of chondrocyte viability induced by IL-1β was signi cantly abolished by BA treatment (p < 0.05). The addition of SIS3 signi cantly inhibited the protective effect of BA on chondrocyte viability.

BA promotes ECM synthesis in ADTC5 chondrocytes
Alcian blue and Safranin O staining are classic methods to detect ECM key component AGG [20]. As shown in Fig. 2a, IL-1β treatment obviously down-regulated the expression of AGG in chondrocytes compared with control groups. On the contrary, the down-regulation of AGG induced by IL-β was obviously inhibited by BA. Moreover, the addition of SIS3 obviously reversed the protective effect of BA on the expression trend of AGG. The staining results indicated that BA promoted the secretion of AGG in chondrocytes and Smad3 inhibitor SIS3 disturbed the promotion of BA on the secretion of AGG.
By Alcian blue and Safranin O staining, we con rmed that BA promoted the expression of AGG in chondrocytes. Since AGG and Col2α are two key components of ECM, we checked the expression of AGG and Col2α in chondrocytes by qRT-PCR. Compared with control group, the expression of AGG and Col2α in IL-1β group was signi cantly down-regulated (p < 0.05, Fig. 2b & 2c). BA abolished the inhibition of IL-1β on AGG and Col2α expressions (p < 0.05). The addition of SIS3 reversed the inhibitory effect of BA on IL-1β-induced down-regulation of AGG and Col2α (p < 0.05). These results indicated that BA promoted ECM secretion in chondrocytes and Smad3 inhibitor SIS3 disturbed the promotion of BA on ECM secretion.

BA adjusts the expressions of ECM regulating genes in ADTC5 chondrocytes
As we know, SOX9, MMPs and ADMTS are key ECM regulators. SOX9 promotes ECM synthesis, and MMPs and ADMTS restrain ECM synthesis. By qRT-PCR and/or western blot, we checked their expressions in chondrocytes. Compared with control groups, the protein expression of SOX9 in IL-1β group was signi cantly down-regulated (p < 0.05, Fig. 3a & 3b). BA abolished the inhibition of IL-1β on protein expression of SOX9 (p < 0.05). The addition of SIS3 reversed the inhibitory effect of BA on IL-1βinduced down-regulation in protein expression of SOX9 (p < 0.05). The change in mRNA expression of SOX9 was same as SOX9 protein (Fig. 3c). As shown in Fig. 3d-3f, the mRNA expressions of MMP9, MMP13 and ADAMTS5 in IL-1β group were signi cantly up-regulated compared with control group, (p < 0.05). However, the inhibition of IL-1β on the expressions of MMP9, MMP13 and ADAMTS5 was obviously abolished by BA treatment (p < 0.05). The addition of SIS3 reversed the protective effect of BA on chondrocytes treated by IL-1β (p < 0.05). These results suggested that BA increased the expressions of ECM synthesizing genes and decreased the expressions of ECM degrading genes and Smad3 inhibitor SIS3 disturbed the adjusting effect of BA on the ECM regulating genes in chondrocytes.

BA activates Smad3 in ADTC5 chondrocytes
Since Smad3 inhibitor SIS3 disturbed the promoting effect of BA on the secretion of ECM, it is reasonable to believe that BA may increase the activity of Smad3 which is related with Smad3 phosphorylation and nuclear localization and Smad3 activation may be one of the protective mechanisms of BA on chondrocyte viability and ECM synthesis. The effect of BA on the activation of Smad3 treated by IL-1β was detected by immuno uorescence and western blot. As shown in Fig. 4a, Smad3 was expressed both in the cytoplasm and nucleus of chondrocytes in the control group, while Smad3 was mainly expressed in the cytoplasm of chondrocytes in the IL-1β group. Immuno uorescence results showed that the Smad3 in the nuclear of chondrocytes in IL-1β-induced groups was obviously less than that of BA + IL-1β groups.
Oppositely, the addition of SIS3 obviously reversed the nuclear translocation trend of Smad3. On the other side, as shown in Fig. 4b& 4c, the ratio of p-Smad3/Smad3 was down-regulated by IL-1β treatment (p < 0.05). But BA treatment neutralized the decrease of p-Smad3/Smad3 induced by IL-1β treatment (p < 0.05). Additionally, in the presence of IL-1β and BA simultaneously, the addition of SIS3 obviously inhibited the protective effect of BA on chondrocytes. These results revealed that BA activates Smad3 in chondrocytes.

Discussion
OA is epidemic in the elderly people as a common chronic joint disease [1]. By now, drug and surgical treatments are two main therapies for OA worldwidely [4]. Comparing with the high cost of surgical treatment, drug treatment of OA is more acceptable [5]. These drugs have an advantage in relieve in ammation and pain symptoms. It is reported that BA has anti-in ammatory, anti-deformation, antibacterial and diuretic effects [7,8]. In current study, we demonstrate that BA increases the viability of chondrocytes and neutralized the in ammation induced by IL-1β through TGF-β/Smad3 pathway.
IL-1β is an in ammatory factor and is related to the progress of OA [20]. It has been shown that IL-1β inhibits the secretion of Col2α and AGG [21]. In addition, IL-1β stimulates the releases of MMP9/13 and ADAMTS5 in chondrocytes which are responsible for the degradation of ECM [9,22]. IL-1β has been also shown to inhibit ECM synthesis through decreasing the expression of SOX9 [9]. By being treated with 10 ng/mL of IL-1β, we successfully established the in ammatory and injury model of chondrocytes. After treated by IL-1β, cell viability and the secretion of Col2α and AGG were reduced; SOX9, one of ECM synthesis promoter was inhibited; on the contrast, MMP9, MMP13 and ADAMTS5, the ECM degradation promoters, were induced. However, BA signi cantly reversed the inhibition of IL-1β on the secretion of Col2α and AGG and the expression of SOX9 which mainly regulates the synthesis of Col2α and AGG [23][24][25], and decreased the induction of IL-1β on MMP9, MMP13 and ADAMTS5. The promoting effect of BA on the expression of SOX9 and ECM components Col2α and AGG is consistent with previous report [26].
The transcriptional activations of SOX9 and TGF-β are necessary for prechondrocyte differentiation [27,28]. It is reported that TGF-β regulates the transcription of SOX9 in human chondrocytes via TGFβ/Smad2/3 signaling pathway [29]. It is suggested that the overexpression of Smad3 can signi cantly induce the formation of primary cartilage originated from human mesenchymal stem cell [23]. In addition, Smad3 enhances the transcription activity of SOX9 and Col2α expression, while Smad3 silence inhibits the expression of SOX9 [30]. According to the literature [31], Smad2/3 is associated with SOX9 in TGF-β dependent manner and forms a transcription complex with SOX9 in the enhancer region of Col2α. The TGF-β/Smad3 pathway plays a key role in SOX9-dependent cartilage formation through up-regulating Smad3 phosphorylation [32].