Baicalein Induces Apoptosis of Rheumatoid Arthritis Synovial Fibroblasts through Inactivation of the PI3K/Akt/mTOR Pathway

Purpose Rheumatoid arthritis (RA) shows abnormal proliferation, apoptosis, and invasion in fibroblast-like synoviocytes (FLSs). Baicalein (BAI), extracted from Scutellaria baicalensis, is used as an anticancer drug through inducing cancer cells apoptosis. However, the mechanism of BAI in RA progression still remains unknown. Here, we demonstrated that BAI inhibited FLS proliferation and migration, whereas it enhanced apoptosis via the PI3K/Akt/mTOR pathway in vitro. Methods Cell viability and colony formation were analyzed by MTT and plate colony formation assays in SW982 cells, respectively. Apoptosis was detected by flow cytometry and western blotting. Epithelial-mesenchymal transition (EMT), MMP family proteins (MMP2/9), and the PI3K/Akt/mTOR pathway were detected by western blot. Cell migration was detected by scratch healing assay under BAI treatment in SW982 cells. Results BAI dose-dependently inhibited cell viability and colony forming in SW982 cells. BAI upregulated apoptotic proteins and downregulated EMT-related proteins, resulting in enhanced cell apoptosis and inhibited cell migration in SW982 cells. BAI also dose-dependently inhibited the phosphorylation of PI3K, Akt, and mTOR. Conclusions These results indicated that BAI inhibited FLSs proliferation and EMT, whereas induced cell apoptosis through blocking the PI3K/Akt/mTOR pathway, supporting clinical application for RA progression.


Introduction
Rheumatoid arthritis (RA) is an autoimmune and systemic inflammatory disease that affects 0.5%-1% of the global population [1]. e basic pathological manifestations of rheumatoid arthritis are synovial inflammation, pannus formation, and progressive destruction of articular cartilage and bone, resulting in joint deformity and loss of function. RA shows abnormal proliferation, apoptosis and invasion in Fibroblast-like synoviocytes (FLSs). FSL activation induced chemokine secretion and immune cell recruitment to destroy bone and cartilage in the inflammatory environment of the synovium tissues [2][3][4]. To date, nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids and disease-modifying antirheumatic drugs (DMARDs) are commonly used for RA treatment [5]. However, most of the drugs for RA patients' treatment have gastrointestinal side effect or are very difficult to treat for a long time because of their high cost.
erefore, development of a novel therapy for RA which has low cost, is effective, and has less side effects is very important [6]. New studies and reports have confirmed that extracts or active components of flavonoids, alkaloids, and triterpenoids of traditional Chinese herbal medicine have anti-RA effect [7].
Baicalein (BAI) (5,6,7-trihydroxyflavone) ( Figure S1(a)) [8,9], a traditional Chinese herbal medicine, extracted from Scutellaria baicalensis, is widely used as an anti-inflammatory or anticancer drug. Scutellaria baicalensis has been applied in the treatment of respiratory infection, diarrhea, jaundice, and hepatitis in traditional Chinese medicine [10]. In previous studies, we have demonstrated that BAI could inhibit gastric cancer cell proliferation and migration through FAK interaction via downregulation in AKT/ mTOR signaling [11]. BAI also inhibited cell proliferation via blocking of the mTOR/p70S6K pathway and suppressed EMT pathway-associated migration through the TGFβ pathway in cervical cancer HeLa cells [12]. According to network protein analysis of molecular docking system ( Figure S1(b)), recent reports also found that BAI highly binding affinity to AKT1, MAPK1, MMP2, and MMP9 which is involved in cell proliferation apoptosis and migration [13].
Recently, studies have also shown that BAI can treat osteoarthritis (OA) by inhibiting chondrocyte apoptosis, reducing cartilage damage, and reducing synovitis, suggesting that BAI has the potential to slow the pathological progression of OA [14]. BAI significantly suppressed TNF-α, IL-6, and IL-1β mRNA expression in FLSs, demonstrating a potential therapeutic effect for RA through anti-inflammatory action [15]. Clinical investigation reveals a strong association RA progression with increased FLSs proliferation and migration, whereas inhibited FLSs apoptosis. However, the mechanism of BAI in RA progression still remains unknown. In this study, we found BAI inhibits FLS proliferation and EMT, whereas it induces cell apoptosis through blocking the PI3K/Akt/mTOR pathway.

Cell Apoptosis Assay.
e percentage of apoptotic cells were determined using FITC Annexin V Apoptosis Detection kit II (BD Bioscience, USA) and all the experiment steps were performed according to manufacturer's instructions. SW982 cells (1 × 10 6 cells/well) were incubated with different concentrations of BAI for 48 hours and collected in the 15 ml conical tube and then washed twice with cold PBS. Cells were then resuspended in a 1X binding buffer (0.1 M HEPES/NaOH, pH7.4, 1.4 M NaCl, 25 mM CaCl 2 ) and 1 × 10 5 cells were transferred to 5 ml flow cytometry (FACS) tubes. FITC Annexin V (5 μl) and propidium iodide (5 μl) were added and the cells were incubated for 15 minutes at room temperature under a dark condition. e cells were analyzed by FACS advantage equipment (BD Bioscience, USA).

Wound
Healing Assay. SW982 cells were plated at a density of 5 × 10 4 cells/well on 24 well plate and were incubated for 24 hours upon reaching a confluent monolayer. Artificial wounds were formed by scratching with a 200 μl pipette tip. e scratch-wounded cells were washed with 1 X PBS to remove cell fragments and were incubated in a fresh media with various concentrations of BAI. ree independent images of scratch-wounded region of interest (ROI) were captured by using a digital camera (Olympus, Japan) at 0, 24, 48 and 72 hours after BAI treatment.

Western
Blotting. SW982 cells were cultured with different concentrations of BAI for 48 hours. Cell was lysed with RIPA buffer (50 mM Tris-HCl, pH7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM Na 3 VO 4 , and a protease inhibitor cocktail; Sigma-Aldrich). Cell lysates were measured Bradford protein assay kit (Bio-Rad) and were loaded on 8% or 10% polyacrylamide gels and electrophoresed in SDS-PAGE. Proteins were transferred to a PVDF membrane (Millipore, Eschborn, Germany) and then was blocked with 5% skim milk and immunoblotted with a primary antibody (Cell Signaling Technology, USA). Proteins were detected by enzyme-linked chemiluminescence (ECL) and quantified with a chemiluminescence and fluorescence imaging system (Bio-Rad, USA).

Statistical Analysis.
All data analyses were performed using SPSS 26.0 software (SPSS, Chicago, IL) and GraphPad Prism 9.1.2 software (GraphPad Software, USA). e least significant difference (LSD) t test was used to compare two groups and one-way ANOVA was used for multiple comparisons. e value of P < 0.05 was considered statistically significant. Average values of at least 3 independent experiments were used for analysis.

BAI Inhibited the Proliferation and Colony
Formation of SW982 Cells. BAI inhibited the proliferation and growth of human osteosarcoma cells [16]; however, the effect on the proliferation of FSLs was not elucidated. MTT assay was used to detect the effects of BAI on SW982 cell viability and proliferation (Figures 1(a) and 1(b)). BAI dose-dependently and time-dependently inhibited SW982 cells proliferation at the concentration of 25 μM, 50 μM, 75 μM, and 100 μM. In addition, we also carried out plate cloning forming assay to detect effect of BAI on SW982 cells viability (Figures 1(c) and 1(d)). BAI significantly inhibited colony formation ability of SW982 cells at the concentration of 25 μM, 50 μM, 75 μM, and 100 μM. ese results suggested that BAI could significantly inhibit cell proliferation and viability in human synovial sarcoma SW982 cells.

BAI Induced Apoptosis of SW982 Cells.
Enhanced proliferation and inhibited apoptosis in FLS are generally considered as a major pathological reason for RA development. However, rheumatoid arthritis FLSs (RA-FLSs) are characterized by apoptosis resistance, increased invasiveness, and the production of inflammatory mediators. BAI-treated groups at 15 μM, 25 μM, and 50 μM promoted apoptosis of SW982 cells in a dose-dependent manner compared to the control group by FITC/PI double staining flow cytometry (Figures 2(a) and 2(b)). Expression of cleaved-caspase3, caspase3, and Bax expression were dose-dependently increased while the Bcl-2 expression was dose-dependently decreased at BAI treatment in the SW982 cells (Figures 2(c) and 2(d)). ese results indicated BAI could induce SW982 cells apoptosis.

BAI Inhibited the PI3K/Akt/mTOR Signaling Pathway.
e PI3K/Akt/mTOR signaling pathway is an important signaling pathway in the cell proliferation and apoptosis. To elucidate the molecular mechanism of BAI-induced apoptosis, we confirmed the PI3K/Akt/mTOR signaling pathway. Interestingly, BAI inhibited the expression of p-PI3K, p-Akt, and p-mTOR, while the expression of PI3K, Akt and  mTOR did not change significantly (Figures 3(a) and 3(b)). ese results indicated BAI could dose-dependently inactivate of PI3K/Akt/mTOR signaling pathway to regulate SW982 cells apoptosis.

BAI Inhibited Migration of SW982 Cells.
RA-FLSs secrete inflammatory cytokines, chemokines, and MMPs and induce cell migration and invasion to destroy joints. erefore, the regulation of RA-FLS migration may be a new therapeutic strategy for the pathologic progression of RA [17]. Due to migration of FLSs involved in the RA development and progression, we further examined the effect of BAI on SW982 cell migration through a scratch healing experiment. Lateral migration of SW982 cells was significantly reduced in the BAI-treated group (25 μM and 50 μM) at 24, 48, 72, and 96 hours (Figures 4(a)-4(b)). BAI treatment significantly upregulated epithelial markers of E-cadherin in a dose-dependent manner compared with controls, whereas it dose-dependently downregulated epithelial markers of vimentin, snail, MMP2, and MMP9 (Figures 4(c)-4(e)).
ese results suggested that BAI significantly inhibited the lateral and vertical migration of SW982 cells via regulation of epithelial-mesenchymal transformation (EMT)-related proteins and MMP family proteins expression.

Discussion
RA is a chronic inflammatory disease characterized by autoimmunity, infiltration of activated inflammatory cells into the synovial membrane of the joint, synovial proliferation, new angiogenesis, and progressive destruction of cartilage and bone; the main pathological change is chronic inflammation of synovial tissue [18,19]. e synovial environment of RA is conducive to the survival of FLSs and inhibits its apoptosis, thereby participating in preventing its elimination. e imbalance of FLS proliferation and apoptosis has been considered as an initiation factor in RA development [20,21]. erefore, the development of a novel therapy for RA which inhibits antiproliferation and induces apoptosis is a very important approach. BAI inhibits cell proliferation via blocking of the mTOR/p70S6K pathway and suppresses EMT pathway-associated migration through the TGFβ pathway in cervical cancer HeLa cells [12]; however, the effect on the mechanism of BAI on RA  development and progression still remained unknown. e human synovial sarcoma cell line SW982 was one of the well-known models for synovitis in RA [22]; therefore, we used human synovial sarcoma cell line SW982 to determine mechanism of BAI in RA development and progression.
Chinese herbal medicine is considered as a valuable resource for novel drug candidates with low toxicity. A large number of studies have confirmed that Chinese herbs and their components have proapoptotic activities on FLSs. Zhang et al. reviewed these Chinese herbs and pointed out that inducing apoptosis of FLSs is an important molecular mechanism of the active components of Chinese herbs in the treatment of rheumatoid arthritis [6].
BAI is a traditional Chinese herbal medicine with antiinflammatory and antioxidant activities [23,24]. However, the therapeutic effect of BAI on RA is not fully understood [25]. Tang et al. found that BAI may be a candidate drug for treatment of RA and the biological function of the grouping network related to the pathogenesis of RA is clarified. Including some viral infections and cancers, involving related signaling pathways such as the PI3K-Akt signaling pathway [26], Hu et al. found that BAI inhibited cervical cancer cell growth and cell cycle whereas enhanced cell apoptosis [27]. Recent reports demonstrated that BAI regulated Bax, Bcl-2, and caspase3 apoptosis signaling pathways to protect OA development [14].
Synovial hyperplasia and infiltration of immune cells lead to excessive expansion and destruction of articular cartilage in RA [28]. Activated FLSs are resistant to cell apoptosis. Indeed, activated FLSs induce chemokine secretion to promote inflammation, and neovascularization ultimately accelerate cartilage degradation [29,30]. Small molecule inflammatory mediators and proteolytic enzymes produced by FLSs can degrade the extracellular matrix in RA [31]. However, simple TNF-α blockade could not directly kill inflammatory FLSs under the microenvironment of RA patients. Direct targeting of inflammatory FLSs to enhance apoptosis could be an effective therapeutic strategy for RA treatment [32]. In this study, we found that BAI enhanced the apoptosis of SW982 cells while inhibited the proliferation of SW982 cells, which might provide a new basis for the treatment of RA. e PI3K/Akt/mTOR pathway regulates cell proliferation and migration; therefore, its role in RA synovial hypertrophy and immune cell infiltration has been extensively explored. e PI3K/Akt/mTOR signaling pathway is activated in a variety of RA cell lines [33][34][35]. In synovial cells of RA patients, the signaling pathway is continuously abnormally activated, resulting in a high expression of antiapoptotic genes [36]. e PI3K/Akt/mTOR signaling pathway was significantly activated in RA synovial cells and attenuated RA development by modulating the PI3K/Akt/ mTOR signaling pathway [37]. us, inhibition of the PI3K/ Akt signaling pathway mediated an antiapoptotic action, which is the main therapeutic target for RA treatment [38]. Recent relevant studies reported that inactivation of the PI3K/Akt/mTOR signaling pathway enhanced autophagy, inhibited the proliferation and inflammatory response of synovial cells, and thus alleviated local symptoms of RA [39]. In addition, studies have also shown that galangin inhibited the development of RA through the PI3K/AKT signaling pathway, which also revealed a new mechanism which might be a feasible method for the treatment of RA [40]. Recent studies showed that BAI not only inhibited nasopharyngeal carcinoma (NPC) cells proliferation and induced NPC cells apoptosis but also blocked the PI3K/AKT and p53 signaling pathways in CNE2 cells [41]. Our studies want to clarify the molecular mechanism of BAI-induced apoptosis, and the relation with the PI3K/Akt/mTOR signaling pathway in RA-FLSs ( Figure 5). We confirmed BAI inhibited cell  Evidence-Based Complementary and Alternative Medicine invasiveness and resistance to apoptosis [42,43]. Snail induces EMT through the suppression of many epithelial markers, including E-cadherin and claudins [44,45]. E-cadherin is one of the molecular markers of the epithelial phenotype, downregulation or loss of E-cadherin leads to the breakdown of intercellular connections and promotes cell metastasis, which is the most significant event during EMT [46]. Ma et al. reported BAI significantly downregulated snail and vimentin protein, upregulated E-cadherin protein in a dose-and time-dependent manner on breast cancer [47]. Similarly, in our study, BAI was also proved to inhibit the occurrence of EMT by downregulated snail and vimentin proteins, upregulated the E-cadherin protein thus inhibiting the migration of SW982 cells. e migration and invasion of RA-FLSs play an important role in synovitis and bone destruction [48]. Activated RA-FLSs not only migrate and invade adjacent unaffected joints but also migrate to the sublayer of synovial lining, promoting angiogenesis, and exacerbating synovial hyperplasia and bone destruction [49]. Recently, some scholars confirmed that miR-653-5p inhibited the cell migration and invasion in RA-FLS cells and revealed that regulating the migration and invasion of RA-FLSs might be a new strategy for RA treatment [50]. It is well known that MMPs, mainly produced by FLSs in RA, are proteases involved in extracellular matrix remodeling and play an important role in FLSs migration and progressive joint destruction in RA. Rui et al. found that BAI significantly inhibited the invasive and metastatic ability of colorectal cancer cells by regulating the expression levels of MMP2/9 via inhibition of the AKT signaling pathway [51]. In this paper, we further confirmed that BAI could inhibit the migration of SW982 cells by a cell scratch healing experiment, and its migration mechanism might be related to the downregulation of the MMP2/9 expression, EMT-related proteins, and PI3K/Akt/mTOR signaling pathway.

Conclusion
In summary, BAI inhibited FLS proliferation and EMT, whereas induced cell apoptosis through blocking the PI3K/ Akt/mTOR pathway, supporting clinical application for RA progression. e abovementioned studies indicated the possibility of BAI as a candidate drug for the treatment of RA in the future and provided a solid data and theoretical basis for its application in clinical treatment. Of course, more research could be needed to confirm this, and this will be the focus of our future research.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.