Disease-Modifying Anti-rheumatic Drug Prescription Baihu-Guizhi Decoction Attenuates Rheumatoid Arthritis via Suppressing Toll-Like Receptor 4-mediated NLRP3 Inflammasome Activation

As a traditional Chinese medicine-originated disease-modifying anti-rheumatic drug prescription, Baihu-Guizhi decoction (BHGZD) is extensively used for the treatment of rheumatoid arthritis (RA) with a satisfying therapeutic efficacy. Mechanically, our previous data indicated that BHGZD may ameliorate RA partially by restoring the balance of the “inflammation-immune” system through regulating the TLR4-c-Fos-IL2-TNF-alpha axis. Toll-like receptor 4 (TLR4) has been revealed to be involved in the activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome complex. Thus, the aim of the current study was to determine the regulatory effects of BHGZD on the TLR4–mediated inflammasome activation during RA progression based on the modified adjuvant-induced arthritis model (AIA-M) and the lipopolysaccharide/adenosine triphosphate (LPS/ATP)–induced pyroptosis cellular models. As a result, oral administration of BHGZD exhibited prominent improvement in the disease severity of AIA-M rats, such as reducing the redness and swelling of joints, arthritis incidence, arthritic scores, and diameter of the limb and increasing pain thresholds. In line with the in vivo findings, BHGZD treatment effectively inhibited the LPS/ATP–induced pyroptosis of both Raw264.7 macrophage and MH7A cells in vitro by reducing pyroptotic cell death morphology (swollen cells) and decreasing propidium iodide–positive and terminal deoxynucleotidyl transferase–mediated dUTP-fluorescein nick end labeling (TUNEL)–positive cells. Notably, the increased expression levels of TLR4, NLRP3, interleukin 1β, and interleukin 18 proteins and the elevated activities of caspase-1 and lactic dehydrogenase in in vivo and in vitro disease models were markedly reversed by the treatment with BHGZD. In conclusion, the above findings proved the immunomodulatory and anti-inflammatory activities of BHGZD, especially in pyroptosis, which may be attributed to the activation of TLR4–mediated NLRP3 inflammasome signaling.


INTRODUCTION
Rheumatoid arthritis (RA), a common systemic autoimmune disease involving multiple organs, is characterized by persistent synovitis, systemic inflammation, and progressive destruction of the cartilage, joint, and bone (Scott, Wolfe, & Huizinga, 2010). RA affects 0.5-1.0% of adults worldwide annually (Gabriel & Michaud, 2009). RA at the active stage (active RA) is characterized by hyperactive immune response and excessive inflammatory cytokines (Mackiewicz, Schooltink, Heinrich, & Rose-John, 1992;;Wang et al., 2012), including interleukin 1 (IL-1), interleukin 6 (IL-6), and interleukin 17 (IL-17) exerting their influences on both osteoclast differentiation and osteoblasts and active joint inflammation (Kawanaka et al., 2002;Dharmapatni et al., 2009;Karmakar et al., 2010). The key therapeutic agents, including disease-modifying antirheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and biological response modifiers, reduce synovitis and systemic inflammation and improve their function (Kirwan, 1995). Among them, methotrexate (MTX), one of the DMARDs, is indicated for severe active RA. MTX has been used in the treatment of RA since the 1980s and is commonly used as the first-line medication for RA treatment . However, poor patient's response, infection, and high costs often restrict the prescription of this drug (Urushibara et al., 2004;Andersson et al., 2008;Aletaha et al., 2017). Therefore, it is an urgent necessity to explore and identify alternative therapeutic strategies for RA treatment.
Traditional Chinese medicine (TCM) has its unique advantages in treating complex and chronic diseases, especially in the clinical treatment of active RA (Zhang et al., 2010). Baihu-Guizhi decoction (BHGZD), a TCM-originated DMARD prescription, is recorded by a Chinese medical sage Zhang Zhongjing in Jin Kui Yao Lue and consists of Gypsum (sulfates; Gypsum Fibrosum), Anemarrhena asphodeloides Bge. (Liliaceae; Anemarrhenae Rhizoma), Cinnamomum cassia Presl (Lauraceae; Cinnamomi Ramulus), Oryza sativa L. (Gramineae; Oryza Semen), and Glycyrrhiza uralensis Fisch (Leguminosae; Glycyrrhizae Radix et Rhizoma). Among them, Oryza sativa L. and Glycyrrhiza uralensis Fisch are renowned for their homology of medicine and food superiority in traditional and alternative medicine, having a high research value.
Accumulated clinical trials have revealed that BHGZD achieves satisfactory therapeutic response in the treatment of active RA, alleviating symptoms and signs of the disease such as pain, morning stiffness, joint tenderness, swelling, and deformity, as well as levels of rheumatoid factor (RF), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) especially in RA patients with wind-damp-heat stimulation with a clinical efficiency as high as 90% (Yuan et al., 2019;Wu, 2021). In addition, the mean DAS28 score of RA patients decreases after BHGZD treatment (DAS28 score: 5.49 to 2.93) (Yuan, et al., 2019). BHGZD significantly ameliorates infectious mononucleosis via decreasing the levels of CD3 + , CD8 + , aspartate aminotransferase (AST), lactic dehydrogenase (LDH), creatine (9CK), and isoenzyme (CK-MB) and increasing the levels of CD4 + , CD4+/CD8+, and IgA and IgG . Previously, our group, combining the chemical and transcriptomic profiling, target prediction, network calculation, and experimental validations, identified the chemical constituents contained in BHGZD and revealed that BHGZD may ameliorate RA partially by restoring the balance of the "inflammation-immune" system and subsequently reversing the pathological events during RA progression through regulating the TLR4-c-Fos-IL2-TNF-alpha axis , which was in line with the findings of other research groups Fang, 2018).
Pyroptosis is a highly regulated process of cell death, which is essential for physiological processes such as organ development, cell renewal, and differentiation (Shi et al., 2017). The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome composed of NLRP3 proteins, apoptosis-associated speck-like proteins containing a caspase recruitment domain (ASC), and caspase-1 have been extensively studied (Martinon et al., 2002;Ting et al., 2008;Zeng et al., 2019). Increasing evidence showed that NLRP3 inflammasome may be involved in the pathogenesis of RA (Guo et al., 2018;Kolly et al., 2010). NLRP3 may be activated by TLR4 signaling (Chi et al., 2015;Martin-Rodriguez et al., 2015), resulting in the formation of intracellular inflammasome complexes with the adaptor protein ASC and caspase-1-dependent cleavage and secretion of interleukin 1 beta (IL-1β) (Netea et al., 2008;Kate and jurg, 2010;Strowig et al., 2012;, which is a well-characterized outcome of TLR and inflammasome cooperation (Chi et al., 2015). In the current study, we investigated the regulatory effects of BHGZD on TLR4-mediated NLRP3 inflammasome activation during RA progression based on the

Ethics Statement
The study was approved by the Research Ethics Committee of the Institute of Basic Theory of Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, China (SYXK 2016-0021, Beijing, China). All animal studies were treated in accordance with the guidelines and regulations for the use and care of animals of the Center for Laboratory Animal Care, China Academy of Chinese Medical Sciences.
(15 g), Cinnamomum cassia Presl (10 g), Oryza sativa L. (30 g), and Glycyrrhiza uralensis Fisch (5 g) were purchased from Beijing Tongrentang Co., Ltd. (Beijing, China). BHGZD was prepared according to the original composition of the formula recorded in the Chinese Pharmacopoeia 2020 edition and obtained based on our previous study (Supplementary Material Section 1). The filtrate was combined and concentrated under reduced pressure and dried out in an oven at 70°C overnight to obtain the BHGZD powder.

Animals
Male Lewis rats (n 51, 6∼8-week-old, 200 ± 20 g in weight) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (production license no. SCXK 2016-0006, Beijing, China). All animals were maintained under specific pathogen-free conditions in a temperature-controlled room at a constant temperature of 24 ± 1°C with a 12°h light/dark cycle and had free access to the standard rodent diet and water ad libitum.
Induction of the AIA-M was performed as previously reported Mao et al., 2020), and AIA-M rats were established to simulate the pathological changes and characteristics of active RA after AIA-M induction. In brief, male Lewis rats were injected intradermally through the base of the tail with 10 mg/mL M tuberculosis H37 Ra (Difco, BD company, New Jersey, United States) suspended in liquid paraffin (Freund's complete adjuvant (CFA)). From the day of primary immunization, the male Lewis rats were kept in an artificial climate box (production license no. RXZ-380A-LED) for 2 h daily with certain wind velocity (6 m/s), temperature (37°C), and humidity (90%) for a period of 15 days.
In the AIA-M-BHGZD treatment group, the dosage selection for BHGZD was 21.4 g/kg, nearly equivalent to two times of the daily dosage of RA patients in clinics, which has been proved to exert the most prominent therapeutic efficacy as in our previous study . The dosage for MTX was 0.2 mg/kg. All treatments were performed for 30 days via oral administration from the day of primary immunization.

Assessment of Arthritis Severity
The severity of arthritis was evaluated by arthritis accidence, arthritis score, diameter of the limb, and body weight as per our previous studies Guo et al., 2016;Li W. et al., 2020;). Detailed information has been given in Supplementary Material Section 2. The temperature of the articular surface was detected as described in Supplementary Material Section 3.

Measurement of Mechanically, Acetone-, and Thermally Induced Hyperalgesia
The pain threshold was evaluated by mechanically, acetone-, and thermally induced hyperalgesia as described previously Guo et al., 2016;Li W. et al., 2020;. Detailed information on the protocol is provided in Supplementary Material Sections 4-6.

Thymus and Spleen Indexes
The weight ratios of the thymus, spleen, liver, and kidney relative to the total brain weight were calculated, as described in Supplementary Material Section 7.

Cell Culture
Mouse macrophage cell lines (Raw264.7) and immortalized cell lines of synovial fibroblasts from the articular cavity in RA patients (MH7A cells) were used for experiment validation in vitro. Raw264.7 macrophage cells were maintained in DMEM (Hyclone, Logan, UT, United States), supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, United States), 100 U/mL penicillin, 100 μg/ml streptomycin, and 2 mML glutamine (TBD, Tianjin, China) in a humidified 5% CO 2 incubator at the temperature of 37°C. The Raw264.7 macrophage cells of low passage number were used in the current experiment validations.
MH7A cells were cultured in a sterile synoviocyte growth medium (Cell Applications, Inc., San Diego, CA) added with 10% synoviocyte growth supplement (Cell Applications, Inc., San Diego, CA), 100 U/ mL penicillin, 100 μg/ml streptomycin, and 2 mML glutamine (TBD, Tianjin, China) in a humidified 5% CO 2 incubator at the temperature of 37°C. MH7A cells of passage numbers four to eight were used in the current experiment validations.

Enzyme-Linked Immunosorbent Assay
On the 31st day, all rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). The rats were secured in the supine position, and the blood was taken from the abdominal aorta using one-time anticoagulant negative pressure blood collection tubes. The blood was placed at room temperature for 20 min and centrifuged at 12,000 rpm at low temperature (4°C) for 15 min. The supernatant was collected, vortexed, and centrifuged again at high speed for 15 min. The cells were centrifuged, and supernatants were collected.

Statistical Analyses
Statistical analyses were performed using GraphPad Prism 8.0 Software (San Diego, CA, United States). Data are expressed as the mean ± S.D. and were analyzed by one-way ANOVA with Bonferroni's or Dunnett's post hoc test for comparison of multiple columns. Differences were considered statistically significant when the p value was less than 0.05.

Baihu-Guizhi Decoction Treatment Alleviates Disease Severity of Arthritis in Adjuvant-Induced Arthritis Model Rats
To determine the pharmacological effects of BHGZD against AIA-M, we successfully established AIA-M rats simulating pathological changes and characteristics of RA with severe redness and swelling ( Figure 1A). The final incidence (incidence on the 25th day after the first immunization), mean arthritis score, diameter of the limb, and articular temperature were approximately 100%, 43.56, 10.95 cm, and 33.65°C, respectively ( Figure 1D∼F). BHGZD treatment strikingly improved the severity of arthritis, including the reduced arthritis incidence (p < 0.05, Figure 1D), arthritis scores (p < 0.001, Figure 1E), diameter of the limb (p < 0.05, Figure 1F), and articular temperature (p < 0.05, Figure 1G), which were consistent with the macroscopic evidence of arthritis ( Figure 1A).
In terms of the response to inflammation, the thymus and spleen indexes in different groups were determined ( Figure 1H∼I). BHGZD treatment decreased the thymus and spleen indexes of AIA-M rats significantly.
Simultaneously, the body weight ( Figure 1C) and pain thresholds (mechanically, acetone-, and thermally induced hyperalgesia) in AIA-M rats were significantly elevated when treated with BHGZD (all p < 0.05, Figure 1J∼K). The pharmacological effects of BHGZD were similar to that of the positive drug MTX. No hepatic or renal toxicities were observed after BHGZD treatment (Supplementary Material Figure S1).

Baihu-Guizhi Decoction Suppresses TLR4-Mediated NLRP3 Inflammasome Activation in Adjuvant-Induced Arthritis Model Rats
Consistent with our previous study , the expression of TLR4 proteins was significantly higher in both sera and the knee joint of AIA-M rats, which was reduced by the treatment with BHGZD (all p < 0.001, Figure 2).
To address the regulation of BHGZD on NLRP3 inflammasome activation, the expression levels of the NLRP3 inflammasome pathway were detected by ELISA and Western blotting, respectively. The protein expression levels of NLRP3, ASC, and caspase-1 p20 (the active form of caspase-1) were markedly increased, which were decreased by the treatment with BHGZD (all p < 0.001, Figure 2). In addition, the activity of caspase-1 in sera was detected by ELISA, which demonstrated the reduced effect of BHGZD on the activity of caspase-1. Notably, we found that the protein expression levels of GSDMD and inflammatory cytokines (IL-1β and IL-18) were markedly increased, which were reduced by the treatment with BHGZD (p < 0.001, Figure 2). Moreover, the LDH activity was used to assess pore formation and release of intracellular soluble components. The enhancing activity of LDH in the sera of AIA-M rats was observed, which was decreased by BHGZD treatment (p < 0.05, Figure 2A).

Baihu-Guizhi Decoction Suppresses
Lipopolysaccharide/Adenosine Triphosphate-Induced Pyroptosis in Raw264.7 Macrophage and MH7A Cells To verify our in vivo findings based on AIA-M rats, an established method (LPS plus ATP) was applied to induce NLRP3 inflammasome activation in both Raw264.7 macrophage and MH7A cells. We initially evaluated the cytotoxicity of BHGZD on the growth of Raw264.7 macrophage cells using flow cytometry analysis, exhibiting no cell toxicity under BHGZD treatments of 7.14, 14.27, and 28.54 μg/ml, as shown in Supplementary Material Figure S2. Thus, the low, middle, and high doses of BHGZD treatment were chosen in the following assays.
The aggravated pyroptotic cell death morphology in cultured Raw264.7 macrophage and MH7A cells (membrane swell, Figure 3D) was observed, which was reversed by the treatment with BHGZD in different doses (7.14, 14.27, and 28.54 μg/ml). Then, flow cytometry analysis revealed that BHGZD treatment prominently reduced the PI-positive cell rate [a marker of cells that stains necrotic, dead, and Frontiers in Pharmacology | www.frontiersin.org October 2021 | Volume 12 | Article 743086 membrane-compromised cells ], indicating the amelioration of cell membrane pore formation induced by LPS/ATP ( Figure 3A∼B). In addition, the co-staining with FAM-FLICA caspase-1 and PI was performed to visualize pyroptosis. Importantly, activation of caspase-1 was observed in LPS/ ATP-induced Raw264.7 macrophage cells exposed to radiation by FAM-FLICA caspase-1 staining, which was suppressed by the treatment with BHGZD ( Figure 3C). Moreover, BHGZD apparently decreased TUNEL-positive cells in LPS/ ATP-induced MH7A cells ( Figure 3E).

Baihu-Guizhi Decoction Suppresses the Activation of the NLRP3 Inflammasome in Raw264.7 Macrophage and MH7A Cells
To further explore the mechanism of BHGZD against pyroptosis, the expression levels of the proteins related to the NLRP3 inflammasome pathway were detected by immunofluorescence staining, ELISA, and Western blotting. The immunofluorescence staining result showed that the co-expression level of NLRP3 and ASC was increased, which was significantly reduced by the treatment with BHGZD in both Raw264.7 macrophage and MH7A cells, similar to the effects of MCC950 ( Figure 4A and Figure 5A). As shown in Figure 4B∼D, BHGZD treatment decreased the expression levels of IL-1β proteins in both cultured cells and supernatants and LDH release in LPS/ATP-induced Raw264.7 macrophage cells (all p < 0.05 in 28.54 μg/ml BHGZD). Consistent with the data, BHGZD treatment decreased the expression levels of IL-1β proteins in both cultured cells and supernatants, as well as the level of IL-18 and LDH release in MH7A cell supernatants (all p < 0.05 in 14.27 and 28.54 μg/ml BHGZD treatment, Figure 5B∼D). As the maturation of IL-1β results from NLRP3 inflammasome activation, which was characterized by decreased protein expression levels of NLRP3, ASC, and caspase-1 when treated with BHGZD in LPS/ATP-induced Raw264.7 macrophage and MH7A cells ( Figure 4C∼D and Figure 5C∼D). In addition, LPS/ATP induction profoundly increased the expression of TLR4 proteins, while BHGZD treatment significantly reduced the TLR4 protein expression (all p < 0.05, Figure 4C∼D and Figure 5C∼D), revealing that BHGZD suppressed toll-like signaling activation. Moreover, Western blotting results showed that GSDMD occurred in the LPS/ ATP-induced group and was decreased by the treatment with BHGZD (all p < 0.001, Figure 4C∼D and Figure 5C∼D).

DISCUSSION
Increasing clinical evidence shows that BHGZD may be highly applied to most patients with RA during the active phase representing the damp-heat impeding syndrome (Wang et al., 2012), featured by severe immune response and inflammatory reaction (Latz et al., 2013;;Wang et al., 2012). Previously, we indicated that BHGZD reversed the imbalance of the "inflammation-immune" system by suppressing TLR4 activation . Accumulated studies have demonstrated that TLR4 initiates NLRP3 inflammasome-mediated pyroptosis (Chi et al., 2015;Martin-Rodriguez et al., 2015;Yin et al., 2020;Zhang et al., 2020), which is a type of programmed cell death (Hersoug et al., 2018), largely involved in the pathogenesis of cell death, liver fibrosis (Wree et al., 2014), and chronic and acute inflammation (So et al., 2013;Zamyatina & Heine, 2020), such as RA (Guo et al., 2018;Li Z. et al., 2020;Pope & Tschopp, 2007;Shen et al., 2018;Wu et al., 2020). Especially, high expression levels of NLRP3 mRNA and NLRP3 inflammasome protein were observed in monocytes/ macrophages, fibroblast-like synoviocytes (FLS), dendritic cells, and neutrophils from RA patients (Choulaki et al., 2015;Kim et al., 2017;Ruscitti et al., 2015;;Yang Z. et al., 2016). In the current study, our data based on the in vivo and in vitro experiments demonstrated for the first time the regulatory effects of BHGZD on the TLR4-mediated pyroptosis, which may underlie the main pathological changes during active RA .
In the current study, in vivo experimental data showed that BHGZD treatment significantly ameliorated the disease severity of arthritis in AIA-M rats, including the prominent improvement of redness and swelling of joints, arthritis scores, diameter of the limb, articular temperature, body weight, and pain thresholds. In addition, the thymus and spleen indexes of AIA-M rats were increased by the treatment with BHGZD. The spleen and thymus, central immune organs, are the main places for immune cell development, maturation, differentiation, and recruitment (Res & Spits, 1999). The values of spleen and thymus indexes reflect and affect the immune function in the body. The above data suggest that BHGZD may regulate the immune reaction in the progression of active RA, in line with our previous findings that BHGZD treatment typically preserved the articular cartilage matrix integrity in markedly inflamed joints . In addition, our previous study verified that the antiinflammatory and immunomodulatory effects of BHGZD were achieved through suppressing TLR4 activation . Accordingly, we focused on determining the regulatory effects of BHGZD on the TLR4-mediated pyroptosis using the LPS/ATP-induced pyroptosis cellular models. As a result, BHGZD treatment significantly improved pyroptotic cell death morphology (swollen cells), decreased PI-and TUNEL-positive cells, and suppressed caspase-1 activation. RA involves erosions in the marginal bone along with the articular cartilage, leading to joint destruction. The process of pyroptosis is linked with the destruction of the plasma membrane, which releases cytokines, and excessive release of other mediators (Chadha et al., 2020;Wu et al., 2020). Active caspase-1 formation is associated with pyroptosis and inflammasome activity, and active caspase-1 takes on the role of a key housekeeping enzyme in its conversion of pro-IL-1β proteins into the active IL-1β cytokine and is measured via irreversible binding of a probe composed of an inhibitor-peptide sequence containing a fluorescent tag, referred to as FAM-FLICA (Fernandez et al., 2016). Pyroptosis is a type of programmed cell death with several features including DNA damage detected by the TUNEL assay (Miao et al., 2011). Similar to apoptotic cells, the pyroptotic cells were PI-or TUNEL-positive (Labbe & Saleh, 2008). These results clearly indicated that BHGZD significantly inhibited pyroptotic death through suppressing TLR4-mediated NLRP3 inflammasome activation.
It is well known that chronic inflammatory response may be the mainly protracted course of RA disease. We further confirmed that BHGZD inhibited the TLR4-mediated inflammasome activation by decreasing the expression levels of TLR4 proteins, NLRP3 inflammasome components (NLRP3, ASC, and caspase-1), GSDMD proteins, pro-inflammatory cytokines, such as IL-1β and IL-18, and LDH release. GSDMD, as a key executor in inflammatory caspase-induced pyroptosis (Kesavardhana & Kanneganti, 2017;Zhao Y. et al., 2018), is cleaved by caspase-1 at a specific site (the N-terminal domain of GSDMD), which binds to membrane lipid and lyses cells through pores with an inner diameter on the membrane, subsequently causing cell lysis and IL-1β release (DiPeso et al., 2017;Evavold et al., 2018;;Shi et al., 2015). A number of studies showed that NLRP3 inflammasome may play a vital role in the pathogenesis of active RA, including high expression of NLRP3 in the synovial tissue of collagen-induced arthritis (CIA) mice  and the peripheral blood cells of patients with active RA (Choulaki et al., 2015). Furthermore, the treatment with the NLRP3 selective inhibitor MCC950 exerted demonstrable pharmacodynamic effects on suppressing the NLRP3 inflammasome activation and amelioration of arthritic symptoms and cartilage destruction in CIA (Fan et al., 2016). Consistent with these previous studies, our results herein demonstrated that BHGZD may suppress the TLR4-mediated pyroptosis both in vivo and in vitro ( Figure 6).

CONCLUSION
The current study offers the experimental evidence that BHGZD may reverse the imbalance of the inflammation-immune system in the development and progression of RA via suppressing the TLR4-mediated NLRP3 inflammasome activation. These findings provide an in-depth understanding on the pharmacological mechanisms of BHGZD against RA, which may be of notable significance to the popularization of the application of TCM in the treatment of RA.

DATA AVAILABILITY STATEMENT
Publicly available datasets were analyzed in this study. These data can be found here: GSE136098 (https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc GSE136098).