A two-herb formula inhibits hyperproliferation of rheumatoid arthritis fibroblast-like synoviocytes

Fibroblast-like synoviocytes (FLS) play a pathogenic role in rheumatoid arthritis (RA). STAT3 signaling is activated in FLS of RA patients (RA-FLS), which in turn causes RA-FLS hyperproliferation. RL is a traditional remedy for treating inflammatory diseases in China. It comprises Rosae Multiflorae Fructus and Lonicerae Japonicae Flos. A standardized ethanolic extract of RL (RLE) has been shown to exert anti-arthritic effects in collagen-induced arthritis (CIA) rats. Some constituents of RLE were reported to inhibit JAK2/STAT3 signaling in rat FLS. Here, we determined whether RLE inhibits FLS hyperproliferation, and explored the involvement of STAT3 signaling in this inhibition. In joints of CIA rats, RLE increased apoptotic FLS. In IL-6/sIL-6R-stimulated RA-FLS, RLE reduced cell viability and evoked cell apoptosis. In synovial tissues of CIA rats, RLE lowered the protein level of phospho-STAT3. In IL-6/sIL-6R-stimulated RA-FLS, RLE inhibited activation/phosphorylation of STAT3 and JAK2, decreased the nuclear localization of STAT3, and downregulated protein levels of Bcl-2 and Mcl-1. Over-activation of STAT3 diminished RLE’s anti-proliferative effects in IL-6/sIL-6R-stimulated RA-FLS. In summary, RLE inhibits hyperproliferation of FLS in rat and cell models, and suppression of STAT3 signaling contributes to the underlying mechanisms. This study provides further pharmacological groundwork for developing RLE as a modern anti-arthritic drug.

www.nature.com/scientificreports/ Pharmacological studies showed that RMF 17 and LJF-containing formulas 18,19 can attenuate collagen-induced arthritis (CIA). We previously found that a standardized ethanolic extract of RL (RLE for short) has anti-RA effects in CIA rats 20 . Chlorogenic acid, a compound used for controlling RLE's quality 20 , has been shown to be able to inhibit the proliferation of IL-6-stimulated rat FLS and suppress JAK2/STAT3 signaling 21 . In this study, we investigated the effects of RLE on apoptosis of FLS in joints of CIA rats, and on proliferation of, and apoptosis in, interleukin-6/soluble interleukin-6 receptor (IL-6/sIL-6R)-stimulated RA-FLS. We also investigated the involvement of STAT3 signaling in these effects.

Results
RLE increases apoptotic FLS in joints of CIA rats. RA-FLS are resistant to apoptosis 2 . We examined the effect of RLE on apoptotic rate of FLS in joints of CIA rats using the TUNEL assay and immunohistochemistry (IHC) staining of cleaved caspase-3. DNA fragmentation resulting from apoptotic signaling cascades can be detected by TUNEL assays. Cleaved caspase-3 is an apoptosis marker. In normal and model groups, TUNELpositive FLS (Fig. 1A) and cleaved caspase-3-positive FLS (Fig. 1B) in synovia of rat joints were very few, and there was no difference between the two groups. TUNEL assays showed that RLE dose-dependently and significantly induced apoptosis (Fig. 1A). In IHC staining assays, although RLE was not able to significantly increase apoptotic FLS at 330 mg/kg, it exerted significant effect at 660 mg/kg (Fig. 1B). The positive control indomethacin significantly increased TUNEL-positive FLS (Fig. 1A) and cleaved caspase-3-positive FLS (Fig. 1B) as well.
These findings indicate that RLE promotes apoptosis of FLS in joints of CIA rats in a dose-dependent manner.
RLE inhibits proliferation of, and induces apoptosis in, IL-6/sIL-6R-stimulated RA-FLS.  signaling activates STAT3 in RA-FLS, which has been implicated in hyperproliferation and resistance to apoptosis of RA-FLS 3 . To determine the effects of RLE on proliferation of patient-derived synoviocytes, CCK-8 and , rat joints were collected from a previous study (n = 6; randomly selected 6 from 8 rats in each group). *P < 0.05, **P < 0.01 vs. model group. www.nature.com/scientificreports/ crystal violet staining assays were performed. RLE dose-and time-dependently reduced viability of IL-6/sIL-6R-stimulated synoviocytes in CCK-8 assays ( Fig. 2A). The anti-proliferative effects of 450 μg/mL of RLE were comparable to that of 100 μM of indomethacin ( Fig. 2A). Crystal violet staining visualized RLE's inhibitory effects on RA-FLS proliferation (Fig. 2B). To determine the effects of RLE on the proliferation of normal cells, mouse fibroblast L929 and human normal liver-derived MIHA cells were used. In the presence of IL-6/sIL-6R, the anti-proliferative effects of RLE in both L929 and MIHA lines were weak (Fig. 2C,D) and less potent than in RA-FLS ( Fig. 2A,B). To determine the effects of RLE on apoptosis of synoviocytes, Annexin V-FITC/PI double staining assays were conducted. RLE dose-dependently induced apoptosis in IL-6/sIL-6R-stimulated synoviocytes (Fig. 3A). RLE's apoptotic effects were verified by its ability to dose-dependently cleave caspase-3 and caspase-9 (Fig. 3B). As compared with the solvent treatment, IL-6/sIL-6R stimulation did not significantly affect synoviocyte apoptotsis (data not shown). These findings indicate that RLE inhibits proliferation of, and induces apoptosis in, IL-6/sIL-6R-stimulated RA-FLS.

RLE lowers the protein level of p-STAT3 in synovia of CIA rats.
In the mechanistic studies, we found that protein level of phospho-STAT3 (Tyr 705) in synovial tissues was higher in CIA rats than in normal rats, and RLE treatments significantly and dose-dependently down-regulated the level of this protein (Fig. 4).
Indomethacin also inhibited phosphorylation of STAT3 in synovia of CIA rats (Fig. 4), which is in line with a previous report 22 . These data indicate that RLE inhibits STAT3 activation in synovia of CIA rats.
These results indicate that RLE inhibits STAT3 signaling in RA-FLS.

Discussion
In a previous study, we found that RLE reduces paw swelling and arthritis clinical scores, and alleviates bone destruction in CIA rats without observable adverse reactions 20 . Moreover, RLE improves body weight gain of CIA rats 20 . In the present study, we found that RLE promotes apoptosis of pathogenic FLS in CIA rats (Fig. 1A,B), and inhibits proliferation ( Fig. 2A,B) of, and induces apoptosis (Fig. 3A) in, IL-6/sIL-6R-stimulated RA-FLS. These findings of our previous and the present studies indicate that RLE has potential to be developed as an effective and safe modern drug for treating RA. We previously observed that RLE inhibits diverse toll-like receptor 4 (TLR4) signaling components in LPSstimulated RAW264.7 and THP-1 cells 24 ; and that RLE lowers the protein level of TLR4 and inhibits phosphorylation/activation of TLR4 downstream transcription factors, NF-κB p65 (nuclear factor-κB p65), AP-1 (activator protein-1) and IRF3 (interferon regulatory factor 3) in joints of CIA rats 20 . We also observed that RLE reduces production of pro-inflammatory cytokines IL-6, TNF-α and IL-1β which are regulated by transcription factors NF-κB, AP-1 and IRF3 in joint tissues and sera of CIA rats 20 . These findings indicate that inhibiting TLR4 signaling is one of the mechanisms of the anti-RA effects of RLE. Pro-inflammatory cytokines IL-6, TNF-α and IL-1β are also regulated by the transcription factor STAT3. These cytokines can be produced by RA-FLS 25 . It is reported that chlorogenic acid, a quality control marker of RLE 20 , can suppress JAK2/STAT3 signaling in rat FLS 21 . Thus, in this study, we investigated the involvement of STAT3 signaling in RLE's inhibitory effects on FLS proliferation. We found that RLE inhibits STAT3 activation in synovia of CIA rats (Fig. 4) and suppresses STAT3 signaling in cell models (Fig. 5B-D). Over-activation of STAT3 attenuates the anti-proliferative effects of RLE in IL-6/sIL-6R-stimulated synoviocytes (Fig. 6B). Results of the present study indicate that inhibiting STAT3 signaling to suppress RA-FLS hyperproliferation is another anti-RA mechanism of RLE (Fig. 7).
RA-FLS play a key role in RA-associated bone erosion 26 . In RA-FLS, IL-6/sIL-6R induces expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which is crucial for osteoclastogenesis 27 . In a previous study, we found that RLE alleviates bone erosion in CIA rats 20 . Further studies are warranted to determine whether RLE inhibits RANKL expression in IL-6/sIL-6R-stimulated RA-FLS by inhibiting the STAT3 pathway.
In summary, we for the first time found that RLE has pro-apoptotic effect in FLS of CIA rats, and has antiproliferative and pro-apoptotic effects in IL-6/sIL-6R-stimulated RA-FLS; and that inhibition of the STAT3 signaling pathway contributes to the underlying mechanisms (Fig. 7). This study provides further pharmacological justifications for the traditional use of RL in treating inflammatory diseases, and provides additional pharmacological groundwork for developing RLE as a modern anti-arthritic agent.

Materials and methods
Preparation of RLE. Collection and authentication of RMF and LJF were the same as in a previous report 9 .
Voucher specimens of the two herbs (RMF: YS6, LJF: JYH) were deposited at our School. As originally recorded in Tang dynasty of China, patients with inflammatory diseases were asked to drink rice wine in which the two ingredient herbs of RL had been soaked 10 . This suggests that active constituents in the formula are lipophilic. For use in this work, we extracted RL with different concentrations of ethanol and compared the effects of the extracts on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophages. Results showed that among the extracts, RLE exhibited the most potent inhibitory effects (Fig. S1). RLE was extracted with absolute ethanol 20 ; its yield was 15.2%. Contents of chlorogenic acid and gallic acid in RLE were quantified using an HPLC method (Fig. S2). www.nature.com/scientificreports/ TUNEL assays. Knee joints of CIA and normal rats that had been dosed with drugs or vehicle in a previous study 20 were collected. All care and handling of animals were performed in accordance with the recommendations of the Committee on the Use of Human & Animal Subjects of the Hong Kong Baptist University. The protocol was approved by the Department of Health, Hong Kong [Reference number: (19)(20)(21) in DH/SHS/8/2/6 Pt. 3]. The ARRIVE guidelines for reporting animal research were fully implemented. In that study, there were three groups of RLE intragastrically (i.g.)-dosed rats; of those three groups, results from only two (330 mg/kg group and 660 mg/kg group) were used in this study. The two groups exhibited stronger anti-arthritic effects than the 165 mg/kg group 20 . We also included the normal control (0.5% carboxymethyl cellulose-Na, i.g.), model (0.5% carboxymethyl cellulose-Na, i.g.) and positive control (2.5 mg/kg of indomethacin, i.g.) groups in the present study. There were 8 rats in each group, for a total of 48 rats. The joints were fixed and sectioned as described previously 28 . The sections were processed for TUNEL assays (In Situ Apoptosis Detection Kit, USA) following the manufacturer's instructions. Cryosection imaging was performed as described 29

IHC staining
Knee joints of rats were fixed and sectioned. IHC staining was performed as described 29 . The primary cleaved caspase-3 antibody was bought from Cell Signaling Technology (#9579; USA) and the secondary antibody was bought from Abcam (#ab205719; Cambridge, UK). Cryosection imaging was performed as described in the TUNEL assay section. IHC staining results were quantified by counting the DAPI and cleaved caspase-3-positive cells in three individual fields using ImageJ software. Percentages of cleaved caspase-3-positive cells in synovia of rat joints were calculated using the following equation Apoptosis assay. RLE's apoptotic effects on RA-FLS were measured by Annexin V-FITC/PI double staining 32 using an Apoptosis Detection Kit (#ab14085; Abcam). Synoviocytes seeded in 6-well plates (1 × 10 5 cells/well) were pre-treated with RLE (0, 150, 300 μg/mL) for 1 h and then treated with IL-6/sIL-6R for 24 h. Flow cytometric analysis was performed using a BD Accuri C6 flow cytometer (BD Biosciences, USA).
Western blot analysis. Proteins were prepared from ankle synovial tissues of rats and cultured cells.