Aqueous extract of Kan-Lu-Hsiao-Tu-Tan ameliorates collagen-induced arthritis in mice by regulating immune and inammatory responses

Abstract: Background: Kan-Lu-Hsiao-Tu-Tan (KLHTT) exhibits anti-psoriatic effects through anti-inammatory activity in mice. However, the therapeutic effects of KLHTT on rheumatoid arthritis (RA), another signicant autoimmune inammatory disorder, are not elucidated. Herein, we explored the antiarthritic effects of KLHTT on collagen-induced arthritis (CIA) in mice. Methods: KLHTT was extracted by boiling water and subjected to spectroscopic analysis. Chicken collagen type II (CII) with complete Freund’s adjuvant was intradermally injected to induce CIA in DBA/1J mice. Anti-CII antibody, cytokines, malondialdehyde (MDA), and hydrogen peroxide (H 2 O 2 ) were measured using ELISA, thiobarbituric acid reactive substances, and hydrogen peroxide assay kit. Splenocyte proliferation was tested using thymidine incorporation. Th1 and Th17 cells were analyzed by ow cytometry. Results: Oral KLHTT treatment (50 and 100 mg/kg) ameliorated mouse CIA by decreasing the levels of interleukin (IL)-1β, IL-6, IL-17A, and tumour necrosis factor-α in the paw homogenates and serum. KLHTT also suppressed anti-CII antibody formation, splenocyte proliferation, and splenic Th1 and Th17 cell numbers. Additionally, KLHTT showed antioxidant activity by reducing the concentrations of MDA and H 2 O 2 in paw tissues. Conclusions: The therapeutic effects of KLHTT in CIA mice were through regulating oxidative stress and inammatory responses. Our results suggest that KLHTT has potential to treat RA.


Introduction
Rheumatoid arthritis (RA) is an autoimmune disease affecting approximately 1 % of the global population, which is characterised by synovitis, cartilage damage, and bone resorption in the joint [1].
Moreover, RA is associated with fatigue [2], cervical spine disease, carpal tunnel syndrome [3], interstitial lung disease [4], cardiovascular disease [5], depression [6], and sleep disorders [7]. RA can cause personal and emotional problems, and impose a signi cant socio-economic burden [8,9]. The medical treatment of RA includes biological disease-modifying anti-rheumatic drugs, conventional disease-modifying antirheumatic drugs, and analgesics. However, these available therapies cannot treat the disease completely and exert signi cant side effects. Therefore, the development of new therapeutics for RA is needed [10,11].
RA is characterised by synovitis accompanied by the in ltration of immune cells [12], including T cells, B cells, dendritic cells [13], neutrophils [14], and macrophages [15]. Studies have indicated that anticitrullinated protein antibodies (ACPAs) and in ammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-17, and tumour necrosis factor-alpha (TNF-α), are pivotal mediators in RA [16]. ACPA is also a speci c diagnostic biomarker for RA [17]. Furthermore, oxidative stress caused by reactive oxygen species (ROS) is crucial in joint in ammation, and RA patients exhibit high level of ROS in serum [18,19]. Hence, antioxidant drugs may be effective in treating RA [20]. Collagen-induced arthritis (CIA) in mice recapitulates the clinical and pathogenic characteristics of human RA, and are widely used to study RA [21].
Kan-Lu-Hsiao-Tu-Tan (KLHTT), a Chinese medicine (CM), has been used to treat in ammatory conditions such as RA, systemic lupus erythematosus, dermatomyositis [22], sinusitis, gingivitis, gastritis, hepatitis [23], and dermatitis [24]. Our previous study demonstrated that KLHTT exerts ROS scavenging ability and anti-in ammatory activity in human neutrophils and exhibits anti-psoriatic activity in mice [25]. However, the pharmacologic effects of KLHTT on RA, another signi cant autoimmune in ammatory disorder, are not yet elucidated. Herein, in this study, we aimed to investigate the anti-arthritic effects of KLHTT in CIA mice and evaluate its value in the treatment of RA. (stem and leaf plot), and 1.67 g Belamcanda chinensis (L.) DC (rhizome)) was extracted by boiling water (12 times the weight of the herbs) for 1 h, and then concentrated to a voucher specimen (CGU_KLHTT-01) by the freeze dryer (LABCONCO, USA) [25]. The voucher specimen complied with Chang Gung University guidelines.

Ultra-performance liquid chromatography-tandem mass spectrometry
The chemical pro le of KLHTT extract was obtained using ultra-performance liquid chromatographytandem mass spectrometry (UPLC-MS/MS) comprising a LC-30AD pump, SIL-30AC auto-sampler, CTO-20AC column, and SPD-M20A Photodiode Array Detector (Nexera X2, Shimazu, Kyoto, Japan). Prior to being loaded onto the UPLC column, 1 mg of KLHTT extract was rst dissolved in 1 mL of methanol and ltered through a 0.45 μm membrane. Sample injections of 1 μL were then performed automatically. Liquid chromatography was performed using a CORTECS UPLC C18 column (90Å, 1. Multiple reaction monitoring (MRM) experiments (in negative) were carried out using Shimazu LCMS-8045 triple quadrupole mass spectrometry to identify the constituents of KLHTT extract. The precursor ion settings of the corresponding pro ling peaks were determined using the full scan experiment (50-1000 amu). The product ions were settled according to previously reported data. The dwell time was xed at 100 ms and the collision energy was set at 25-45 eV. All MS data were acquired and processed using LCMS LabSolutions software Version 5.93 (Shimazu, Kyoto, Japan).

Experimental animals
DBA/1J mice (male, six-to eight-week old, weight 20-22 g) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and maintained at 20-25 °C with half day light/dark cycle under a speci c pathogen-free condition. All mice were treated according to the guidelines of the Institutional Animal Care and Use Committee of National Chung Hsing University (NCHU). The study protocol was approved by NCHU ethics committee.

CIA model establishment
CIA was induced by active immunisation with chicken CII [26]. Brie y, 2 mg/ml CII was dissolved in 10 mM acetic acid solution and emulsi ed with an equal volume of complete Freund's adjuvant containing Mycobacterium tuberculosis H37RA (250 μg/mouse). The mixture (200 μL/mouse) was intradermally injected at the base of the tail. Incomplete Freund's adjuvant and CII were administered as booster injections to the mice 21 days after the rst immunisation. ddH 2 O, KLHTT, or MTX was administered orally once a day from day 21 to 42. Mice were divided into four groups (n = 6/group) randomly as follows: Group I, Normal; Group II, Vehicle (ddH 2 O) + CII; Group III, KLHTT (50 mg/kg) + CII; Group IV, KLHTT (100 mg/kg) + CII. MTX (0.5 mg/kg) was used as positive control. Mice were euthanized with CO 2 exposure (100 % CO 2 for 5 min) by experienced experimenters humanely on day 42.

Assessment of clinical arthritis severity
The body weight and arthritis severity score were obtained [26]. The arthritis severity score was evaluated as: 0, no swelling nor redness; 1, mild swelling and redness restricted to the tarsals or the ankle joint; 2, mild swelling and redness from the tarsals to the ankle; 3, moderate swelling and redness extending to the metatarsal joints; 4. severe swelling and redness from the ankle to the foot and the digits, or limb ankyloses. In addition, paw volume was measured using Plethysmometer 37140 (Ugo Basile SRL, Comerio, VA, Italy).

Assessment of histological arthritis severity
After the mice were humanly sacri ced, the hind limbs were xed in 10 % buffered formalin, decalci ed in 15 % EDTA, and embedded in para n. Serial para n sections (5 μm) were stained with haematoxylin and eosin. The severity of histopathological lesions was scored [26] as follows: 0, normal appearance; 1, mild in ltration of in ammatory cells, mild pannus front, and minimal cartilage damage; 2, moderate in ltration of in ammatory cells, erosive pannus front, and moderate cartilage damage; 3: diffuse in ltration of in ammatory cells, severe cartilage damage and bone resorption.

Measurement of pro-in ammatory cytokine levels
Hind paw was dissected and homogenised in ice-cold saline using a tissue homogeniser. After being centrifuged at 3000 rpm (4 °C, 10 min, twice), the hind paw homogenates were harvested. Blood was collected from the heart. The levels of cytokines in hind paw homogenates and serum were measured by ELISA [16].

Measurement of the concentrations of oxidative markers
Malondialdehyde (MDA) concentration was determined by thiobarbituric acid reactive substances assay at 532 nm. The standard curve was obtained using 1,1,3,3-tetramethoxypropane. Hydrogen peroxide (H 2 O 2 ) concentration was measured using a colorimetric OxiSelect™ hydrogen peroxide assay kit at 560 nm [16].
The level of IgG1 and IgG2a was measured at 450 nm by an ELISA reader (Sunrise, Tecan Inc., Switzerland) [16].

Statistical analysis
Data are presented as mean ± SD. Statistical analyses were performed using one-or two-way ANOVA followed by Tukey's honestly signi cant difference test. A P-value < 0.05 was considered statistically signi cant.

KLHTT exerts anti-arthritic effects in CIA mice
The CIA mouse model is a well-established and commonly used model mimicking the clinical symptoms and immunopathogenesis of human RA [35]. Mice immunised with CII induced increases in clinical arthritis scores, paw volume, and histopathological damage. The normal group exhibited no gross or histological changes. KLHTT (50 and 100 mg/kg) showed inhibitory effects on arthritis severity ( Fig.   2A) and paw erythema and swelling ( Fig. 2B and 2C). The body weight loss in CIA mice was also restored by KLHTT (Fig. 2D). Histopathological analysis of the CIA mice revealed in ammatory cell in ltration into articular tissues, exudates within the synovial space, synovial hyperplasia, and cartilage erosion. KLHTT-treated mice demonstrated well-preserved joint spaces with minimal in ammatory exudates, normal cartilage structure, and clear synovial spaces, along with improved histological arthritis severity scores (Fig. 3). MTX (0.5 mg/kg) was used as a positive control and showed comparable inhibitory effects with KLHTT in CIA mice ( Fig. 2 and 3).

KLHTT inhibits cytokine production in CIA mice
The pathogenesis of RA involves activated T cells promoting macrophages to release pro-in ammatory cytokines [35]. Therefore, the levels of TNF-α, IL-1β, IL-6, and IL-17 in hind paw homogenates and serum samples were measured by sandwich ELISA. KLHTT (50 and 100 mg/kg) treatment inhibited the levels of IL-1β, IL-6, IL-17, and TNF-α in paw homogenates (Fig. 4A) and serum samples (Fig. 4B) in CIA mice. These results indicated that KLHTT effectively attenuates in ammation in CIA mice.

KLHTT reduces oxidative stress in CIA mice
RA patients exhibit high level of oxidative stress, which correlates with joint in ammation and may contribute to the chronicity of RA [36]. Signi cantly elevated levels of MDA and H 2 O 2 were noted in the hind paw homogenates of CIA mice. KLHTT (50 and 100 mg/kg) signi cantly reduced the levels of MDA (Fig. 5A) and H 2 O 2 (Fig. 5B). These data showed that KLHTT protects mice from oxidative damage, which may have contributed to the amelioration of CIA. KLHTT, Kan-Lu-Hsiao-Tu-Tan; MDA, malondialdehyde.

KLHTT inhibits CII-speci c antibody production and splenocyte proliferation in CIA mice
Autoantibodies targeting IgG play a major role in RA. Similarly, elevated levels of IgG1 and IgG2a antibodies were detected in serum samples from CIA mice. KLHTT signi cantly suppressed IgG1 and IgG2a antibody production (Fig. 6A). Furthermore, KLHTT signi cantly inhibited the proliferation of CIIinduced splenocytes (Fig. 6B).

KLHTT reduces the levels of splenic Th1 and Th17 cells in CIA mice
The pro-in ammatory Th1 and Th17 cell axes play crucial roles in RA. The levels of splenic Th1 and Th17 cells were higher after CII induction. KLHTT signi cantly decreased the numbers of CD4 + IFNγ + Th1 and CD4 + IL17A + Th17 cells (Fig. 7). KLHTT also mitigated the levels of pro-in ammatory cytokines in CIA mice (Fig. 4). These results indicated that KLHTT decreases splenic pro-in ammatory Th1 and Th17 cells in CIA mice.

Discussion
RA is a chronic autoimmune in ammatory disease [1]. Patients with RA have systemic in ammatory comorbidities. The therapeutic armamentarium for RA has expanded from analgesics and nonsteroidal anti-in ammatory drugs to biological disease-modifying anti-rheumatic drugs and conventional diseasemodifying anti-rheumatic drugs; however, these available therapies may cause adverse reactions and fail to achieve long-term remission [37]. Therefore, the development of new drugs is required to improve the treatment of RA.
KLHTT is a well-known CM and has been used to treat in ammatory diseases [22]. In this study, we investigated the anti-arthritic effect of KLHTT in CIA mice. Mice actively immunised with CII develop CIA, which closely resembles human RA. CIA mice showed paw erythema and swelling, synovitis, cartilage damage, and bone erosion [35]. KLHTT reduced arthritis severity scores and paw swelling, and restored body weight in CIA mice. KLHTT also decreased in ammatory cell in ltration. Both in CIA and human RA, pro-in ammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-17 trigger autoimmune reactions and enhance chronic in ammation in synovial tissues [38,39]. These pro-in ammatory cytokines activate synovial broblasts and chondrocytes to produce enzymes which degrade collagen and proteoglycans, thus damaging adjacent joint tissues [40]. KLHTT signi cantly reduced the levels of TNF-α, IL-1β, IL-6, and IL-17 in serum samples and joint homogenates. Therefore, KLHTT exerts local and systemic antiin ammatory effects, which may explain its anti-arthritic activity.
Autoantibodies such as rheumatoid factor and ACPAs can be detected in 50-80 % of RA patients.
Increased levels of anti-CII IgG correlate with elevated TNF-α and IL-6 in RA patients [41]. In CIA mice, anti-CII antibodies initiate arthritis and CII-reactive T cells promote the progression of the disease [42]. In this study, KLHTT reduced the levels of anti-CII IgG1 and IgG2a in serum samples from CIA mice. Anti-CII IgG2 autoantibodies are the predominant subclass of autoantibodies in CIA mice [35]. Also, KLHTT inhibited CII-induced splenocyte proliferation and reduced the levels of splenic Th1 and Th17 cells. These results indicate that KLHTT has immunomodulatory effects in CIA.
Oxidative stress is involved in the pathogenesis of RA [36,43]. In this study, KLHTT signi cantly decreased the levels of H 2 O 2 (a ROS marker) and MDA (a lipid peroxidation marker) in joint homogenates from CIA mice. MDA-related reactions are highly immunogenic. MDA levels correlate with RA severity and can be used to predict RA severity [43]. In addition, autoreactive T cells such as Th1 and Th17 cells are crucial in the pathogenesis of RA [44]. The newly diagnosed RA patients have higher levels of serum Th1 and Th17 cells [45]. RA patients show increased Th17 cell in ltration in the synovium [46,47]. In iximab, an anti-TNF-α antibody, promotes Th1 cell apoptosis in RA patients, thus impeding RA progression [48]. Adalimumab, another anti-TNF-α drug, mitigates the homing of Th17 cells to the synovium, consequently improving joint damage [49]. In this study, KLHTT showed a decrease effect in the levels of splenic Th1 and Th17 cells. The levels of pro-in ammatory cytokines, such as IL-1β, IL-6, IL-17, and TNF-α, were also inhibited by KLHTT. Therefore, we suggest that the regulation of Th1 and Th17 cells is also involved in the anti-arthritic effects of KLHTT.

Conclusion
In summary, our results indicate that KLHTT, a CM formulation, shows signi cantly anti-in ammatory effects, antioxidant activities, and immunomodulatory functions in CIA mice. The present study also demonstrates that KLHTT has potential to treat RA. Availability of data and materials Upon request, data used to support the ndings of this study are available from the corresponding authors. Table   Table 1. UV and multi-stage mass spectrometry data for the identification of the constituents of Kan-Lu-Hsiao-Tu-Tan extract.