Chinese Herbal Formula, Huayu Tongbi Fang, Attenuates Inflammatory Proliferation of Rat Synoviocytes Induced by IL-1β by Regulating Proliferation and Differentiation of T Lymphocytes

The inflammatory proliferation of fibroblast-like synoviocytes (FLSs) and functional imbalances in T lymphocytes play critical roles in the pathogenesis of rheumatoid arthritis (RA). The clinical efficacy of Huayu Tongbi Fang (HYTB, a traditional herbal formula) in RA treatment has been validated. In this study, we aimed to explore the regulatory mechanisms of HYTB on the proliferation and differentiation of T lymphocytes, and the inhibitory effect of HYTB on inflammatory proliferation of FLSs. The RCS-364 (Rat FLSs) cells were cocultured with rat splenic lymphocytes that were induced by interleukin-1β in Transwell chambers. After freeze-dried HYTB powder treatment, the percentage of T-cell subset and apoptosis rates of FLSs were measured using flow cytometry. Furthermore, protein expression of key molecules of NF-κB and JAK/STAT signaling pathways was quantified using Western blot. The granulocyte-macrophage colony-stimulating factor (GM-CSF) was measured using enzyme-linked immunosorbent assay. The results showed that HYTB could inhibit the inflammatory proliferation of FLSs through inducing cell apoptosis. Additionally, HYTB treatment could intervene in the proliferation and differentiation of T lymphocytes and regulate protein expression of key molecules in NF-κB and JAK/STAT cell signaling pathways. Moreover, it could inhibit FLS activation by suppressing GM-CSF production by T cells and FLSs. Therefore, the HYTB formula should be used as a traditional medicine against RA in modern complementary and alternative therapies.


Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects 0.5%-1.0% of adults worldwide. RA joint inflammation is associated with immune cell infiltration, synovial inflammatory hyperplasia, and excessive proinflammatory mediator production, eventually resulting in articular cartilage injury [1,2]. Although the detailed etiology and pathogenesis of RA remain uncertain, recent studies have demonstrated that inflammatory proliferation and activation of fibroblast-like synoviocytes (FLSs) and abnormal T-cell subset differentiation play critical roles in RA pathogenesis [3,4]. T-cell activation also contributes to RA pathogenesis. Abnormal balance of T helper cells, 1/ 2, and Treg/ 17 induces the inflammatory immune response and FLS activation. FLSs produce proinflammatory cytokines and chemokines, recruit effective T cells to synovial tissue in the joint, and initiate autoimmune arthritis. e proinflammatory cytokine secretion, including granulocyte-macrophage colony-stimulating factor (GM-CSF), initiates and augments autoimmune arthritis [5][6][7][8]. Effective cells and activating FLSs participate in autoimmune responses and aggravate bone and cartilage degradation [9,10]. e nuclear factor-κB (NF-κB) signaling pathway is related to several pathophysiological changes including inflammation, cell survival, proliferation, and differentiation. Activation of this pathway regulates proinflammatory cytokine production and accelerates the RA pathological progression. Moreover, inflammatory cytokines activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway (also known as IL-6 signal pathway) and elevate the matrix metalloproteinase gene expression, resulting in apoptosis resistance in FLSs [11]. e proinflammatory cytokines activate 17 and FLSs to secrete GM-CSF. GM-CSF stimulates monocytes, dendritic cells (DCs), and 17 cells and augments innate and adaptive immune cell activation and the synovitis in joints [10].
Huayu Tongbi Fang (HYTB), a Chinese herbal formula, is considered to improve the RA pathological microenvironment by activating blood circulation, dissipating blood stasis, and dredging meridians and collaterals. Its curative effect and drug safety have been established through long clinical practice in RA treatment. Despite the good curative effect of HYTB in RA treatment [12], the mechanisms for inhibiting abnormal T-cell differentiation and inflammatory proliferation of FLSs are not clear. In this study, RSC-364 cells (Rat FLSs cell line) [4,13] cocultured with rat lymphocytes that were induced by IL-1β in Transwell chambers were used as cell models. Following freeze-dried HYTB powder treatment, the rates of T-cell subsets and FLSs apoptosis were measured. e expression of key molecules in NF-κB and JAK/STAT signaling pathways and GM-CSF level were also studied. We wanted to explore the possible mechanisms by which HYTB regulates T-cell proliferation and differentiation, thus inhibiting the FLSs activation and inflammatory proliferation.

Preparation of the Freeze-Dried HYTB Powder.
e modified HYTB formula was composed of six medicinal herbs. e constitution ratio of the six herbs was composed of Ligusticum chuanxiong Hort [4], Dioscorea nipponica Makino [4], Radix Aconiti Lateralis Preparata [2], Astragalus membranaceus [4], Radix Paeoniae Alba [3], and Corydalis yanhusuo [3]. e herbal mixture was soaked in deionized water for 20 min and extracted with boiling water at 10 : 1 (v/ w) for 2.5 h. e extracted liquid was collected, and the herb residue was then added with deionized water and boiled for another 1 h. e extracted liquid was collected and combined with the first one. After the water extract was concentrated by heating the mixture at 75°C for 2.5 h. e herbal extract was filtered using a standard test sieve of 150 μm, freeze-dried, and maintained in desiccators at 4°C until use.

High-Performance Liquid Chromatography-Electrospray Ionization/Mass Spectrometer (HPLC-ESI/MS n ) Analysis.
e constituents of the freeze-dried HYTB powder were measured using HPLC-ESI/MS n . e specific measuring procedures were based on our previously described method [14]. e PeakView ™ 2.2 software was used to analyze the data including retention time, accurate mass, and MS/MS spectrum comparison.

Cell Viability
Assay. Cell viability assay was performed using the cell counting kit-8 (CCK8) method. In brief, RSC-364 cells were seeded into a 96-well plate at a density of 4,000 cells/well for 24 h at 37°C and 5% CO 2 in a cell incubator. Following treatment with various HYTB concentrations (0, 10, 50, 100, 250, 500, 750, and 1000 µg/mL, six repetitions) for 24 h at 37°C, cells were added to wells with 10 µL CCK8 (Beyotime, Nanjing, China) per well and incubated for 4 h. Subsequently, optical density (OD) was measured at 450 nm using a Varioskan Flash microplate reader ( ermo Fisher Scientific, Inc.). According to the experimental results, we used the 100 and 250 µg/mL concentrations as cell stimulation concentrations in this study ( Figure S1).

Preparation of Rat
Lymphocytes. Sprague Dawley rats (weighing 180-220 g) were obtained from the Beijing Vital River Laboratory Animal Technology Co. Ltd, Beijing, China (certificate number SCXK: 2012-0001). is study was approved by the Institutional Ethics Committee of the Beijing University of Chinese Medicine (No. 17-02, Beijing, China), and all animal protocols complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (revised 2011). After intraperitoneal injection (50 mg/kg) of pentobarbital sodium anesthesia, the spleen was isolated under sterile conditions. e rats were sacrificed by intravenous pentobarbital sodium anesthesia (150 mg/kg) administration, and the death of rats after cardiac arrest was confirmed for 30 min. Single-cell suspensions from rat spleens were collected with gentle MACS Dissociator (Macs Miltenyi, Teterow, Germany). e spleens were placed in a homogenate pipe with phosphate-buffered saline (PBS). e suspension was filtered through a 70 μm nylon cell strainer. After centrifugation (1000 rpm, 5 min), cells were resuspended in 5 mL of Red Blood Cell Lysis Buffer for 5 min and centrifuged at 2000 rpm for 5 minutes. e splenic lymphocytes were cultured in 2 mL RPMI-1640 medium supplemented with 3% fetal bovine serum (FBS).

Cell
Culture and Treatment. RSC-364 cells were collected and seeded into six-well culture plates (1 × 10 6 cells/ per well). After synchronization, cells were washed three times with PBS. Transwell inserts were placed onto culture plates. e lymphocytes (1 × 10 6 cells/per well) were then seeded into the upper chambers. e freeze-dried HYTB powder was dissolved in RPMI-1640 medium. Afterward, cells were stimulated for 12 h and 24 h in RPMI-1640 medium containing 5%. FBS, with 25 ng/mL IL-β alone or together with 25 ng/mL IL-1 receptor antagonist (IL-1RA) of treatment, cells were harvested, washed three times with PBS, and incubated with 5 μL Annexin V-FITC and 10 μL PI for 20 min at room temperature. To measure the 1, 2, or 17 cell percentages, lymphocytes were harvested and stimulated with phorbol 12-myristate 13-acetate (PMA) (50 ng/mL) and ionomycin (Ion) (1 μg/mL) (Sigma, San Francisco, CA, USA) in the presence of GolgiPlug (BD Bioscience) for 5 h at 37°C and 5% CO 2 in a cell incubator. After being surface-labeled with anti-rat CD4 PE-Cya-nine5 antibody (eBioscience), lymphocytes were blocked, fixed, permeabilized using Fixation/Permeabilization kit (BD Bioscience), and stained with anti-rat IFNc PE, IL-4 PE-Cyanine7, or IL-17A FITC antibodies (BD Bioscience). e stained apoptotic cells and cells were measured using a FACS Calibur cytometer, and data were analyzed using CellQuest software (Beckman Coulter, Brea, CA, USA).

Statistical Analysis.
All data are presented as mean-± standard deviation (SD). e SPSS version 13.0 was used for statistical analyses. A P < 0.05 was considered statistically significant.

Identification of Chemical Constituents in HYTB by HPLC-ESI/MS n .
Representative liquid chromatographymass spectrometry chromatograms are shown in Figure 1. Negative (Figure 1(a)) and positive ( Figure 1(b)) modes were operated in the HPLC-ESI/MS n experiment. Twenty-five constituents were identified by comparing the retention time with the IDA method. e identified compounds are shown in Table 1.

HYTB Regulated the Differentiation of and Treg Cells of Lymphocytes in the Coculture System Induced by IL-1β.
e percentages of 1, 2, 17, and Treg cells in lymphocytes were measured by flow cytometry. e 1 (IFN + CD4 + , Figure 2(a)) and 17 (IL-17 + CD4 + , Figure 3(a)) cell percentages were significantly increased compared with those of the model group after 12 and 24 h of IL-1β induction. After 12 or 24 h of HYTB treatment, the abnormal differentiation of 1 and 17 cells was suppressed. However, the percentages of 2 (IL-4 + CD4 + ,        Figure 4(a)) cells were significantly decreased by IL-1β. After 12 h of HYTB treatment, the number of 2 cells was significantly increased. e percentages of Treg (CD4 + CD25 + , Figure 5) cells in the HYTB group (250 μg/mL) were significantly increased compared to those induced by IL-1β alone at these two-time nodes of treatment. ese results indicated that HYTB treatment could interfere with the proliferation and differentiation of and Treg cells induced by IL-1β, and the regulatory effects were time-and dosedependent.
To explore HYTB treatment mechanisms on intervening in the proliferation and differentiation of cells, the protein expression of specific transcription factors in cells was measured using Western blot analysis. As shown in Figures 2(b), 3(b), and 4(b), the T-bet protein levels (the specific transcription factor of 1) and RORct (that of 17) were significantly decreased, and GATA-3 (that of 2) was remarkably increased in lymphocytes after HYTB treatment at two-time nodes. e regulatory effects of HYTB treatment, especially of 250 μg/mL HYTB treatment groups, were equal or superior to those of IL-1RA groups.

HYTB Inhibited the Activation of the NF-κB Signaling
Pathway in Lymphocytes Induced by IL-1β. IL-1β can activate the NF-κB signaling pathway and contribute to the inflammatory immune response [15]. We measured the protein levels of key molecules in the NF-κB signaling pathway of lymphocytes after HYTB treatment. As shown in Figures 6 and 7, the protein levels of TRAF2, IKKα/β, and NF-κB p50 were significantly downregulated after 12 and 24 h of HYTB treatment. Phospho-IKKα/β also significantly decreased. e HYTB inhibitory effects (250 μg/mL) of the treatment groups were better than those of the other groups after 24 h of treatment.

HYTB Suppressed the Inflammatory Proliferation of FLSs by Inducing Cell Apoptosis.
e HE staining analysis showed that RSC-364 cells appeared regular with large and spindleshaped nuclei in normal group, and cells induced by IL-1β exhibited irregular spindle shape and various degrees of damage in the cytoplasm. e inflammatory proliferation of RSC-364 cells also appeared. HYTB treatment significantly  inhibited FLSs proliferation and repaired the cell damage (Figure 8(a)).
In addition, we also measured apoptotic cell percentage in RSC-364 cells after treatment. As shown in Figure 8(b), the apoptotic cell percentage in the HYTB treatment groups significantly increased compared with that induced by IL-1β alone after 12 h of treatment. e inducing effect of HYTB treatment was superior to that of IL-1RA.  Figure 6: Effect of HYTB on inhibiting the NF-κB signaling pathway activation in lymphocytes after 12 h of treatment. e expression levels of TRAF2, IKKα/β, p-KKα/β, and NF-κB p50 in lymphocytes were measured using the Western blot analysis. e quantified results are presented in bar charts. GAPDH is used as an internal control. Data are presented as the mean ± SD (n � 3). * P < 0.05 and * * P < 0.01.

HYTB Treatment Inhibited GM-CSF Production in the Coculture System of Lymphocytes and FLSs Induced by IL-1β.
IL-1β signaling drives 17 cells and FLSs to produce GM-CSF, active DCs, and macrophages, contributing to perpetuate autoimmune inflammation [16,17]. We measured the GM-CSF levels in the supernatant of the coculture solution after treatment by ELISA. As shown in Figure 9, the GM-CSF levels in a coculture system of lymphocytes and FLSs significantly increased after IL-1β stimulation. HYTB treatment downregulated the GM-CSF secretion. e HYTB   treatment inhibitory effect at 250 μg/mL concentration was equal or superior to that of IL-RA.

Inhibitory Effects of HYTB on the Activation of NF-κB and JAK/STAT Signaling Pathways of RSC-364 Cells in the
Transwell System. Activation of NF-κB and JAK/STAT signaling pathways plays key roles in the RA pathogenesis [18]. For these reasons, we measured the protein levels of key molecules in NF-κB and JAK/STAT signaling pathways in RSC-364 cells after treatment. As shown in Figures 10 and  11, the protein expression of MyD88, TRAF2, IKKα/β, and NF-κB p50 in RSC-364 cells of HYTB treatment groups was significantly lower than those induced by IL-1β alone at two time nodes. Furthermore, IKKα/β protein phosphorylation was inhibited by HYTB treatment. e phospho-STAT3 and JAK1 protein levels in RSC-364 cells also significantly decreased after HYTB treatment (Figures 12 and 13). e inhibitory effects of 250 μg/mL HYTB were better than those of IL-1RA groups.

Discussion
In traditional Chinese medicine, multiple agents contained in one formula will synergistically work as medical treatment. Twenty-five chemical constituents of HYTB formula were identified by HPLC-ESI/MS n analysis. Pharmacological research demonstrated that paeoniflorin can inhibit lipopolysaccharide-induced production of tumor necrosis factor-α (TNF-α) and IL-1β, thereby increasing IL-10 production [19]. Coptisine can inhibit IL-1β-induced inflammatory response by suppressing NF-κB signaling pathway [20]. Moreover, isoschaftoside and jatrorrhizine have been shown to inhibit proliferation, migration, and production of inflammatory mediators [21,22]. Epiberberine, coptisine, and p-coumaric acid have antibacterial, anti-inflammatory, and antioxidative effects [23][24][25][26]. Pharmacological studies have also shown that calycosin-7-O-glucoside could reduce the expression of platelet-derived growth factor, fibroblast growth factor, and toll-like receptor [27]. Tanshinone IIA could upregulate lncRNA GAS5 and block cell cycle in the G2/M phase, inducing FLSs apoptosis [28,29]. erefore, we suggest that these HYTB constituents could be contributed by interacting with multiple targets and exerting synergistic therapeutic effects on regulating proliferation and differentiation of and Treg cells, thereby inhibiting inflammatory FLSs proliferation by inducing apoptosis, and attenuating proinflammatory cytokine production for the suppression and resolution of inflammation in RA.
T-cell infiltration and inflammatory cytokine imbalance in the joint lumen could induce an abnormal anti-immune response and initiate inflammatory proliferation of synovial tissue and the autoimmune arthritis, resulting in articular cartilage injury [30]. e imbalances in the differentiation and function of 1 and 2/ 17 cells influence RA pathogenesis [3,31,32]. Naïve CD4 + T cells differentiate into either type of cell subset depending on the stimulation of specific transcription factors.
1 cells play a predominant role in the RA pathology. Synovium-infiltrating 1 cells secrete IFN-c and activate macrophages and TNF production [36]. However, several studies have demonstrated that 1 phenotype does not explain all mechanisms involved in RA. IL-17-producing 17 cells were found in peripheral blood monocytes in a large proportion of RA patients. e 17 cell proportion is related to disease activity during RA progression [36]. IL-17 secreted by 17 cells activates osteoblasts and FLSs and induces osteoclastogenesis [37]. IL-17 also induces production of TNF and IL-6 from FLSs and macrophages and augments recruitment of inflammatory cells into the synovial tissue. CD4 + T cells overexpressing RORct induce the CCR6 production and promote the CD4 + T-cell migration into inflamed joints [38]. 2 cells coordinating with IL-4 and IL-10 suppress 1 cell differentiation. In addition, IL-4R controls IL-17 production. GATA-3 overexpression reduces 17 cell differentiation [39]. Treg cells have regulatory roles in the development of autoimmune arthritis. Depletion of CD25 + T cells augments the severity of collagen-induced arthritis in a murine model. CD4 + CD25 + Treg cell transplantation suppresses the progression of arthritis [40]. In this study, our results indicated that HYTB treatment has a regulatory effect on cells differentiation.  Figure 10: e key molecules protein levels of the NF-κB signaling pathway in RSC-364 cells after 12 h of treatment. TRAF2, MyD88, IKKα/ β, p-IKKα/β, and NF-κB p50 were quantified using Western blot analysis. e quantified results are presented in bar charts. GAPDH is used as an internal control. Data are presented as the mean ± SD (n � 3). * P < 0.05 and * * P < 0.01.
Evidence-Based Complementary and Alternative Medicine 13 stimulation. One possible mechanism is interfering with protein expression of specific transcription factors. Synovial hyperplasia and infiltration of immune cells lead to excessive expansion and destruction of articular cartilage in RA [41]. Activated FLSs have resistance to cell apoptosis. Small molecule inflammatory mediators and proteolytic enzymes produced by FLSs can degrade the extracellular matrix in RA [42]. Our results showed that HYTB treatment could inhibit FLSs inflammatory proliferation induced by IL-1β by promoting apoptosis.
GM-CSF, a key proinflammatory cytokine, activates dendritic cells and macrophages. 17 cells induced by IL-1 and IL-23 signaling secrete GM-CSF and initiate autoimmune inflammation [43]. FLSs are important effector cells that produce large amounts of inflammatory cytokines, contributing to cartilage and bone degradation in  Figure 11: e key molecules protein levels of the NF-κB signaling pathway in RSC-364 cells after 24 h of treatment. TRAF2, MyD88, IKKα/ β, p-IKKα/β, and NF-κB p50 were detected using Western blot analysis. e quantified results are presented in bar charts. GAPDH was used as an internal control. Data are presented as the mean ± SD (n � 3). * P < 0.05 and * * P < 0.01.
RA. IL-17 combined with IL-1 increases GM-CSF production by FLSs. Loss of GM-CSF production by 17 cells and FLSs could inhibit progression of autoimmune arthritis [44]. HYTB treatment decreased GM-CSF production in the coculture system of lymphocytes and FLSs induced by IL-1β. e NF-κB signaling pathway plays a key role in the pathogenesis of RA. NF-κB signaling pathway activation stimulates proinflammatory cytokines expression, leading to the inflammatory response of RA [45]. It can also induce expression of antiapoptotic cytokines, inhibit apoptosis of FLSs, and contribute to the synoviocyte hyperplasia stimulated by proinflammatory cytokines [46]. e JAK/ STAT3 signaling pathway also plays a key role in the RA pathological progression. e cell cycle and apoptosis of FLSs in RA are regulated by activation of JAK/STAT3 signaling pathway [47]. e results of present study showed that HYTB treatment could intervene in the protein expression of key molecules of NF-κB and JAK/ STAT3 signaling pathways, inhibit activation of these pathways, induce FLSs apoptosis, and contribute toward inhibiting inflammatory proliferation of FLSs induced by proinflammatory cytokines.
Our study demonstrated that the HYTB formula could regulate proliferation and differentiation of cells, suppress NF-κB and JAK/STAT signaling pathways activation, induce apoptosis in FLSs, and decrease GM-CSF production, resulting in the suppression of inflammatory proliferation of FLSs stimulated by proinflammatory cytokines. HYTB can restore homeostasis in the tissue microenvironment of RA. ese in vitro experimental results and preliminary clinical observation results provide supporting supplements for HYTB formula in the treatment of autoimmune arthritis, including RA. However, in vivo experiments and clinical trials are necessary to confirm the anti-inflammatory activities of HYTB. is is also a key point in our follow-up research work. Taken together, this Chinese medical formula, HYTB, should be used as a complementary or alternative traditional medicine for RA treatment. Data are presented as the mean ± SD (n � 3). * P < 0.05 and * * P < 0.01.

Data Availability
e data used to support the findings of this study are included within the article and the Supplementary Materials.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper. Data are presented as the mean ± SD (n � 3). * P < 0.05 and * * P < 0.01. 16 Evidence-Based Complementary and Alternative Medicine