Taxifolin alleviates FCA-induced arthritis via regulating the Th1/Th2 balance, and down-regulates NLRP3 inammasome axis activation in Jurkat T cells

Rheumatoid arthritis (RA) is a chronic inammatory joint disease mediated by T cells. In traditional Chinese medicine, Smilacis Glabrae Rhizoma is commonly used to treat deoxidation, dampness and ease joint movement. One of its active components, a avonoid called taxifolin, has been the focus of several studies in recent years. However, the pharmacological action of taxifolin in the development of RA remains unknown. Here, we investigated the therapeutic effects of taxifolin on Freund's complete adjuvant (FCA)-induced arthritis model, and then veried the underlying immunoregulatory mechanisms of taxifolin on activated Jurkat T cells. Taxifolin ameliorated the physical signs including paw volume (PV), arthritis index (AI) and body weight (BW) and reduced the organ coecients (spleen and thymus) in FCA-induced rats, as well as the inammatory responses in the left hind paw and plasma. The results also showed that taxifolin greatly improved the imbalance of T helper (Th)1/Th2 status in the plasma and spleen. Further, the Th1/Th2 imbalance status and NLR family pyrin domain containing 3 (NLRP3) inammasome activation in the activated Jurkat T cells was inhibited signicantly by taxifolin. In conclusion, these results suggested that taxifolin potentially targeted the Th1/Th2 status and NLRP3 inammasome axis in T cells, contributing valuable insights to elucidating the mechanism of action of taxifolin for future studies on RA therapeutics.


Introduction
Pathologically, rheumatoid arthritis (RA) is one of the most prevalent chronic and refractory immunemediated diseases that involve several degrees of in ammation in multiple joints, severe pain, and loss of joint functions [22]. In clinical practice, traditional synthetic disease-modifying antirheumatic drugs (DMARDs), nonsteroidal anti-in ammatory drugs (NSAIDs) and corticosteroids are the basis for RA treatment and are approved by both domestic and foreign authorities as rst-line treatments, and biologicals usually used for alternative inhibition of the speci c immune system molecules. However, existing drugs have been shown to have signi cant adverse effects, such as increased risk of gastrointestinal reactions, infection and cancer [8]. Due to numerous patients who do not respond or continue to respond to existing RA therapies, both conventional and biologic, the development of drugs for RA is of great interest to researchers. Therefore, it is urgent to develop new effective therapies, especially herb drugs and natural medicinal components to meet the clinical needs for RA patients.
Previous studies in humans and rats proved the vital roles of activated CD4 + T cells in the manifestations of RA, demonstrating that the in ammatory milieu in the whole body is regulated by a complex network of cytokines and chemokines [29,19]. T-helper (Th) cells are considerable subtypes of CD4 + T cells that play a central role in autoimmune pathophysiology and participate in systemic immunity and antigen recognition in the joints [21]. Th1 / Th2 imbalance is considered to be the driving factor of organ pathology in RA and other autoimmune diseases [13]. Studies have shown that Th1 cells and their marker cytokines are involved in the in ammatory development of joint. In clinical, an effective treatment for early RA patients is to suppress Th1cells reaction, and Th2 cells play a protective role in RA [28].
In this study, we examined the therapeutic effects of taxifolin and two extract from Smilacis Glabrae Rhizoma on Freund's complete adjuvant (FCA)-induced arthritis model in vivo, and veri ed the underlying mechanisms in activated Jurkat T cells in vitro. Our ndings may provide new insights to promote a reasonable and effective application of taxifolin for the RA therapeutics.

Chemicals
The taxifolin (purity > 98%) used for cell culture was purchased from Yuanye Bio-Technology Co., Ltd.
(Shanghai, China). Prednisone acetate was purchased from Shanghai Pharmaceuticals Holding Co., Ltd.

Treatment conditions
Based on our previous experimental results, we determined the appropriate dosage of taxifolin in experimental SD rats, which were randomly divided into 8 treatment groups (n = 5 each): control (0.2 mL 0.5% CMC-Na), model (0.2 mL 0.5% CMC-Na), PDN (20 mg/kg prednisone), aqueous extract (a-EXT; 8 mg/kg aqueous extract of Smilacis Glabrae Rhizoma), ethanol extract (e-EXT; 9 mg/kg ethanol extract of Smilacis Glabrae Rhizoma), L-taxifolin (20 mg/kg taxifolin), M-taxifolin (40 mg/kg taxifolin), and Htaxifolin (80 mg/kg taxifolin). Development of the arthritis model was induced by injecting (sub-plantar) 0.2 mL FCA mixed with heat-killed BCG in a 5:1(w/v) ratio into the left hind paw of the rat (intra-dermally) for 15 d. The paw volume (PV), arthritis index (AI) and body weight (BW) levels were measured to check for arthritis. Then, the chemicals were administered via intragastric gavage daily for 14 d. All rats were sacri ced after the treatments, and biochemical examinations were performed.

PV, AI and BW evaluations
The paw volume (PV), arthritis index (AI) and body weight (BW) levels were measured to check for arthritis [14].The left hind PV of all rats were measured before FCA injection on day 0 and subsequently at different time intervals until day 30 using a plethysmometer (YLS-7B; Equipment Station of the Shandong Academy of Medical Sciences, Shandong, China).

Samples collection
On day 30, heparinized blood was withdrawn from the abdominal aorta in the enterocoelia of rats by arterial blood collection method. The blood was stored at -80°C for subsequent enzyme-linked immunosorbent assay (ELISA) analysis. In addition, the spleen and thymus were obtained at the end of the experiment and weighted.

Pathological examination
The xed left hind paws were dehydrated with gradient alcohol, embedded with para n, sectioned, stained with hematoxylin/eosin (HE), and examined under a DM2700M RL/TL light microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany).

Cell treatment and viability assay
Jurkat T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were seeded into 6-, 24-, and 96-well plates at a suitable density, respectively. After overnight incubation, taxifolin at different concentrations (10, 20, and 40 µM) or NAC (5 µM) was added to the medium for 30 min prior to PHA (5 µg/mL) treatment. Additionally, the controls received no treatment, while the models were treated with PHA only. Then, the cells were cultured for 24 h.
And the cell viability was determined using the CCK-8 assay kit.

Flow cytometry
After 24 h treatment, the cells were harvested and then labeled with 5 µL anti-CD25-PE antibody in the dark for 30 min. The speci c uorescence intensity was measured by ow cytometry (BD Biosciences, San Jose, CA, USA) according to the manufacturer's guidelines.

Detection of intracellular reactive oxygen species (ROS)
The ROS assay kit (Beyotime, Shanghai, China), according to manufacturer's instructions, was used to measure the intensity of intracellular ROS.

Quantitative reverse transcription PCR (qRT-PCR)
The total RNA of cells and tissues was extracted according to previous methods [10] by using TRIzol™ reagent (Invitrogen, Carlsbad, California, USA). The RNA expression levels (as shown in supplementary data 1) were analyzed by qRT-PCR according to the manufacturer's protocol of the manufactures. RNA samples (2 µg) were reverse-transcribed using Transcriptor First Strand cDNA Synthesis Kit. The qRT-PCR mix was prepared using FastStart Universal SYBR Green Master (ROX) (Roche, Basel, Switzerland).
Relative gene expression levels were calculated by second derivative maximum analysis using the 2 −∆∆Ct equation, with the data standardized to the endogenous reference mRNA levels of β-actin based on Eq. (2): where, Ct1 represents the cycles of the tested gene in the control samples when the ampli cation curve reached the threshold value of the uorescence signal, Ct2 indicates the cycles of β-actin in the same group that the ampli cation curve reached the threshold value of the uorescence signal, Ct1' represents the cycles of the tested gene in the treatment groups when the ampli cation curve reached the threshold value of the uorescence signal, and Ct2' represents the cycles of β-actin in the parallel group samples when the ampli cation curve achieved to the threshold value of the uorescence signal. In the abovementioned calculations, the gene expression levels in the control samples (without treatment) were set as the default (numerical values were 1).

Statistical analysis
Data were expressed as mean ± standard error of mean (SEM) and analyzed by independent samples ttest for comparisons using SPSS 15.0 software (SPSS, Inc. Chicago, IL, USA). Data with P-values < 0.05 were considered statistically signi cant.

Taxifolin alleviated the physical signs and reduced the organ injury in FCA-treated rats
Compared to the model group, a signi cant decrease in PV was observed in all treatment groups on days 19, 23, 27 and 30 (Fig. 1A). Furthermore, the AI scores in the M-and H-taxifolin groups on the 30th day signi cantly decreased to 61 and 60%, respectively (Fig. 1B). The BW results also showed in Fig. 1C.
Additionally, the spleen and thymus are the key immune organs of the animal physiological body and the organ coe cients of both organs are regarded as a common and important index to evaluate the immune function of the body [9]. We found that the spleen and thymus coe cients of the PDN and Htaxifolin groups were all signi cantly reduced by 36 and 32% and by 36 and 34%, respectively (Fig. 1D), indicating that 80 mg/kg taxifolin can suppress the coe cients of spleen and thymus in FCA-treated rats. These results suggested that taxifolin alleviated the physical symptoms of RA and reduced the organ injury in FCA-treated rats.

Taxifolin relieved the in ammation in FCA-treated rats
After treatment, all treatment groups decreased different degree brous tissue hyperplasia, cartilage and bone destruction, granulation tissue formation, and in ammatory cell in ltration (Fig. 1E and F). When compared to the model group, MCP-1, TNF-α, IL-12 and IL-17 expression levels were signi cantly decreased with the increasing dosage of taxifolin (Fig. 2). Interesting, taxifolin treatment caused signi cantly increased IL-8 expression level (Fig. 2B). These results indicated that taxifolin could improve the in ammatory response in FCA-induced RA rats.
3.3. Taxifolin regulated the Th1/Th2 balance in the plasma and spleen of FCA-treated rats Previous studies have reported that Th1/Th2 imbalance is a key factor in the pathogenesis of in ammatory diseases [3]. The results showed that taxifolin treatment caused signi cantly decreased IFNγ and IL-4 levels in the plasma compared to models (Fig. 3A, B). What's more, compared the control group, the IFN-γ/ IL-4 ratio in the plasma of rats in the model group re ected that the Th1/ Th2 status polarized to Th1 status; while the IFN-γ/ IL-4 ratios decreased to 45 and 23% after the 40 and 80 mg/kg taxifolin treatment, respectively (Fig. 3C). Further, the relative expression level of T-bet, GATA-3, IFN-γ and IL-4 were upregulated in the spleen, particularly in the H-taxifolin group ( Fig. 3D and E, G and H). Similarly, the ratio of T-bet/GATA-3 and IFN-γ/ IL-4 in the spleen showed that the Th1/ Th2 status polarized to Th2 status. By contrast, the 20, 40 and 80 mg/kg taxifolin treatment displayed a correction effect, with about 5.10-and 2.95-, 5.24-and 4.08-, 9.05-and 4.18-fold higher in the ratio of T-bet/GATA-3 and IFN-γ/IL-4, respectively (Fig. 3F, I), indicating that taxifolin can modulate the Th1/ Th2 imbalance in the spleen of FCA-treated rats. Taken together, our ndings suggested that taxifolin can relieve the polarization to Th1 cells in the plasma while relieve the polarization to Th2 cells in the spleen. However, no signi cant difference was observed in the thymus (Supplementary Data 2).

Taxifolin decreased T cell activation and regulated the cytokine levels in Jurkat T cells
To further investigate the effects of taxifolin treatment on the regulation of T cells, we screened the optimum concentrations of taxifolin in Jurkat T cells, a well-known model for studying T cell signaling. Previous studies usually used PHA to stimulate lymphocyte function in the cell [11]. The test for relative cell viability revealed that a low dose of taxifolin had no signi cant effect on normal cell proliferation (as shown in Supplementary Data 3).
CD25 expression is a known biomarker of CD4 + T cell activation [17]. After PHA (5 µg/mL) stimulation, the positive rate of CD25 signi cantly increased by 24.4-fold in Jurkat T cells compared with untreated cells, which suggesting T cells have activated into CD4 + cells; compared to the PHA-stimulated cells, CD25 expression levels in the successfully activated Jurkat T cells decreased to 88, 83 and 70% with the increased dosage of taxifolin at 10, 20, and 40 µM, respectively (Fig. 4A, B). This result suggests that taxifolin can signi cantly inhibit T cell activation in Jurkat T cells.
We also assessed whether taxifolin can exert anti-in ammatory effect in activated Jurkat T cells.
Compared to cells treated with PHA only, the IL-8 levels in cells treated with 10, 20 and 40 µM taxifolin were approximately 1.4-, 2.6-, 4.5-fold higher, respectively (Fig. 4C). By contrast, the TNF-α expression levels decreased signi cantly in the cells treated with 10, 20, and 40 µM taxifolin, separately (Fig. 4D). These results indicated that taxifolin could regulate the in ammatory response in the activated T cells.

Taxifolin attenuated Th1 and Th2 imbalance in Jurkat T cells
Research has shown that all of the Th1, Th2 cells are differentiated from naive CD4 T cells when they are activated through T-cell receptor (TCR)-mediated signaling [34]. We further investigated whether taxifolin can regulate the Th1/Th2 status in activated Jurkat T cells. Results showed that taxifolin downregulated the levels of T-bet and IFN-γ ( Fig. 5A and D), but upregulated the expression of GATA-3 and IL-4 ( Fig. 5B  and E). Likewise, the Th1/Th2 ratios of the studied transcription factors and cytokines were signi cantly reduced after taxifolin treatment (Fig. 5C and F). These results suggested that taxifolin could correct the Th1/ Th2 imbalanced status in activated Jurkat T cells.
3.6. Taxifolin-downregulates the NLRP3 in ammasome expression in Jurkat T cells NLR family pyrin domain containing 3 (NLRP3) triggers a sequence of in ammatory responses by activating in ammatory caspase to produce IL-1β and IL-18 [23]. In the present study, PHA successfully triggered the assembly of NLRP3 in ammasomes in activated Jurkat T cells. In cells co-treated with PHA and taxifolin, we observed that the mRNA and protein expression levels of NLRP3, Caspase-1 and ASC were signi cantly downregulated with increasing dosage of taxifolin ( Fig. 6A-C, G), indicating that taxifolin can inhibit the NLRP3, ASC and Caspase-1 expression levels.
Besides, previous studies have shown that ROS generation can cause in ammasome activation [6]. To verify whether these alterations are related to ROS activity, the Jurkat T cells were subsequently co-treated with NAC (5 µM) and PHA for 24 h. NAC is a strong oxidant that can e ciently scavenge ROS [15]. The results showed that IL-1β and IL-18 expression levels and ROS intensity were signi cantly downregulated with the increasing dosage of taxifolin ( Fig. 6D-F). In all, our ndings con rmed that taxifolin could downregulate NLRP3 in ammasome expression and reduce intracellular oxidative stress in Jurkat T cells.

Discussions
In this study, we have performed that the avonoid component taxifolin modulated in ammatory response in FCA-induced RA rats. The changes in PV, AI score, and BW have been classically used for evaluating the anti-in ammatory and anti-arthritic effects of certain treatments on RA [7]. We found that taxifolin ameliorated the pathological symptoms of FCA-induced RA rats. Similarly, the spleen and thymus coe cients signi cantly decreased in taxifolin groups. And the rats in each treatment group showed alleviation of bone destruction and in ammatory cell in ltration in the left hind paw after treatment. The MCP-1, TNF-α, IL-12 and IL-17 expression levels all signi cantly decreased as the taxifolin dosage increased. IL-8 is a classical prototypical chemokine and responsible for inducing chemotaxis, which directly migrates cells to the in ammatory site [20]. In this study, our results showed that because in ammatory responses were gradually improved in all treatment groups, the IL-8 expression level in plasma returned to normal state.
RA is a T cell-dependent disease; thus, increasing efforts have focused on understanding the phenotype and function of CD4 + T cells in rheumatoid in ammation. Th1 and Th2 cells, two important subtypes of CD4 + Th cells, are associated with RA [29]. Research shows that Th1 effector cells initiate cellular immune responses through proin ammatory cytokines such as IFN-γ, while Th2 cells can produce several types cytokines (e.g., IL-4, IL-10, IL-13 etc.) that are required for humoral immunity [5]. Furthermore, Th1 and Th2 cytokine gene expression is regulated by T-bet and GATA-3, two major Thspeci c transcription factors that play a central role in Th cell differentiation [24]. Our results demonstrated that the mechanism of action of taxifolin in the plasma of FCA-induced rats may involve humoral immunity. By contrast, the mode of action in the spleen tissue may involve cellular immune response. Further, the spleen may play a regulatory function on Th1/Th2 cells, but not thymus tissue. Therefore, the spleen plays a crucial role in this study. Taken together, taxifolin exhibited immunoregulatory effect of Th1/Th2 in a dose-dependent manner in vivo.
In the successfully activated Jurkat T cells, the cells mostly polarized into Th1 status; however, this extreme pathological shift was reversed by taxifolin as the dosage increased, suggesting that taxifolin can correct Th1 cell polarization in vitro. These results were in good agreement with previous conclusion of Th1/Th2 phenomenon in the plasma. Combining these in vivo and in vitro results, taxifolin had potential immunomodulatory effects.
The activated sensor protein NLRP3, which triggered ASC through the pyrin domain structure (PYD), and then the CARD domain in ASC recruited the CARD domain in pro-caspase-1 to form the NLRP3-ASCcaspase-1 complex, subsequently secreting IL-1β and IL-18, which plays a malignant role in the development of in ammatory and immune responses [1]. For example, a previous study on a spontaneous arthritis mouse model demonstrated that the pathology of arthritis may be associated with the NLRP3 in ammasome/IL1β axis [26]. In this study, the NLRP3, ASC, caspase-1 expression levels all dramatically increased after the inducement of PHA, and suggest that this NLRP3 in ammasome were activated in vitro. However, taxifolin reduced NLRP3, ASC, caspase-1 expression levels, revealing that taxifolin may exhibited anti-in ammation effect through NLRP3 in ammasome. Simultaneously, ROS accumulation, which is also involved with the NLRP3 in ammasome [6,1], was also investigated. Our results showed that taxifolin and NAC both reduced the IL-1β and IL-18 expression levels and ROS activity in vitro. These ndings demonstrated that the anti-in ammation mechanism of action of taxifolin involved the NLRP3 in ammasome in vitro.
In conclusion, our study demonstrated the Th1/Th2 status plays a signi cant role in the pharmacologic activity of taxifolin in the FCA-induced RA rats. Further, this study con rmed the role of taxifolin in correcting the activated of NLRP3 in ammasome axis in the classical Jurkat T cell model in vitro. Our ndings also corroborate previously published data and validate that taxifolin has both antioxidant and immunomodulatory effects, providing the basis for the development of new drugs for RA treatment with traditional Chinese medicine. Naturally, the speci c mechanism of taxifolin improving NLRP3 remains to be further studied and clari ed.

Declarations
Ethics approval and consent to participate All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Tianjin Institute of Pharmaceutical Research (TIPR) new drug assessment. Written informed consent for publication was obtained from all participants.

Consent for publication
Not applicable.