Aqueous Extract of Kan-Lu-Hsiao-Tu-Tan Ameliorates Collagen-Induced Arthritis in Mice by Inhibiting Oxidative Stress and Inflammatory Responses

Background: Kan-Lu-Hsiao-Tu-Tan (KLHTT) exhibits anti-psoriatic effects through anti-inflammatory activity in mice. However, the therapeutic effects of KLHTT on rheumatoid arthritis (RA), another significant autoimmune inflammatory disorder, have not been elucidated. Herein, we explored the anti-arthritic 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 (H2O2) were measured using ELISA, thiobarbituric acid reactive substances, and a hydrogen peroxide assay kit. Splenocyte proliferation was tested using thymidine incorporation. Th1 and Th17 cells were analyzed by flow 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 H2O2 in paw tissues. Conclusions: The therapeutic effects of KLHTT in CIA mice were through regulating oxidative stress and inflammatory responses. Our results suggest that KLHTT has potential to treat RA.

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 profiling 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 fixed 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 specific 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 (approval code: 109-115).

CIA Model Establishment
CIA was induced by active immunisation with chicken CII [26]. Briefly, 2 mg/mL CII was dissolved in 10 mM acetic acid solution and emulsified 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 on day 21 after the first 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 a positive control. Mice were euthanized with CO 2 exposure (100% CO 2 for 5 min) by experienced experimenters humanely on day 42. The arthritis severity score was recorded every 3 days after the treatment of drugs. The biochemical and histological assays were performed 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 a plethysmometer 37,140 (Ugo Basile SRL, Comerio, VA, Italy).

Assessment of Histological Arthritis Severity
After the mice were humanely sacrificed, the hind limbs were fixed in 10% buffered formalin, decalcified in 15% EDTA, and embedded in paraffin. Serial paraffin sections (5 µm) were stained with haematoxylin and eosin. The severity of histopathological lesions was scored [26] as follows: 0, normal appearance; 1, mild infiltration of inflammatory cells, mild pannus front, and minimal cartilage damage; 2, moderate infiltration of inflammatory cells, erosive pannus front, and moderate cartilage damage; 3, diffuse infiltration of inflammatory cells, severe cartilage damage and bone resorption.

Measurement of Pro-Inflammatory 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].

Statistical Analysis
Data are presented as mean ± SD. Statistical analyses were performed using one-or two-way ANOVA followed by Tukey's honest significant difference test. A p-value < 0.05 was considered statistically significant.

Identification of Flavone Derivatives in KLHTT Extract
In this study, qualitative analysis of flavonoid-derived constituents was performed by UPLC-MS/MS under 330 nm. The most significant components of the KLHTT extract were flavonoid derivatives ( Figure 1A). It has been reported that flavone glycosides are the major constituents of the KLHTT aqueous extract [27]. Moreover, some of the flavonoids and their glycosides such as baicalin and baicalein were found to display significant anti-inflammatory properties [25]. Therefore, we conducted tandem mass (MS/MS) spectrometry experiments to identify the constituents of KLHTT for flavonoids and their glycosides specifically. The specific mass fragmentations were compared with previous references [28][29][30][31][32][33][34], and eight flavonoids were identified: chrysin 6-C-arabinoside-8-C-glucoside (1), (6), wogonoside (7), and baicalein (8) ( Figure 1B) (Table 1). Table 1. UV and multi-stage mass spectrometry data for the identification of the constituents of Kan-Lu-Hsiao-Tu-Tan extract.

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]. Immunisation of mice 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 ( Figure 2A) and paw erythema and swelling ( Figure 2B,C). The body weight loss in CIA mice was also restored by KLHTT ( Figure 2D).
Histopathological analysis of the CIA mice revealed inflammatory cell infiltration into articular tissues, exudates within the synovial space, synovial hyperplasia, and cartilage erosion. KLHTT-treated mice demonstrated well-preserved joint spaces with minimal inflammatory exudates, normal cartilage structure, and clear synovial spaces, along with improved histological arthritis severity scores (Figure 3). MTX (0.5 mg/kg) was used as a positive control and showed comparable inhibitory effects with KLHTT in CIA mice (Figures 2 and 3). 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 (Figure 2A) and paw erythema and swelling ( Figure 2B,C). The body weight loss in CIA mice was also restored by KLHTT ( Figure 2D). Histopathological analysis of the CIA mice revealed inflammatory cell infiltration into articular tissues, exudates within the synovial space, synovial hyperplasia, and cartilage erosion. KLHTTtreated mice demonstrated well-preserved joint spaces with minimal inflammatory exudates, normal cartilage structure, and clear synovial spaces, along with improved histological arthritis severity scores ( Figure 3). MTX (0.5 mg/kg) was used as a positive control and showed comparable inhibitory effects with KLHTT in CIA mice (Figures 2 and 3).

KLHTT Inhibits Cytokine Production in CIA Mice
The pathogenesis of RA involves activated T cells promoting macrophages to release pro-inflammatory 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 ( Figure 4A) and serum samples ( Figure 4B) in CIA mice. These results indicated that KLHTT effectively attenuates inflammation in CIA mice.

KLHTT Reduces Oxidative Stress in CIA Mice
RA patients exhibit high level of oxidative stress, which correlates with joint inflammation and may contribute to the chronicity of RA [36]. Significantly elevated levels of MDA and H2O2 were noted in the hind paw homogenates of CIA mice. KLHTT (50 and 100 mg/kg) significantly reduced . KLHTT inhibits pro-inflammatory cytokine production in CIA mice. CIA was induced by active immunisation with chicken CII in DBA/1J mice. Drugs were administered orally once a day from day 21 to 42. The levels of cytokines in hind paw homogenates (A) and serum (B) from CIA mice were measured on day 42 by enzyme-linked immunosorbent assay. Data are expressed as mean ± SD (n = 6). * p < 0.05, ** p < 0.01, and *** p < 0.001 versus vehicle-treated CIA control mice; one-way ANOVA. CIA, collagen-induced arthritis; CII, collagen type II; IL, interleukin; KLHTT, Kan-Lu-Hsiao-Tu-Tan; TNF-α, tumour necrosis factor-α.

KLHTT Reduces Oxidative Stress in CIA Mice
RA patients exhibit high level of oxidative stress, which correlates with joint inflammation and may contribute to the chronicity of RA [36]. Significantly 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) significantly reduced the levels of MDA ( Figure 5A) and H 2 O 2 ( Figure 5B). These data show that KLHTT protects mice from oxidative damage, which may have contributed to the amelioration of CIA.
Life 2020, 10, x FOR PEER REVIEW 11 of 18 the levels of MDA ( Figure 5A) and H2O2 ( Figure 5B). These data show that KLHTT protects mice from oxidative damage, which may have contributed to the amelioration of CIA.

KLHTT Inhibits CII-Specific 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 significantly suppressed (an ROS marker) were determined on day 42 by the thiobarbituric acid reactive substances assay and the hydrogen peroxide assay kit, respectively. Data are expressed as mean ± SD (n = 6). * p < 0.05, ** p < 0.01, and *** p < 0.001 versus vehicle-treated CIA control mice; one-way ANOVA. CIA, collagen-induced arthritis; CII, collagen type II; H 2 O 2 , hydrogen peroxide; KLHTT, Kan-Lu-Hsiao-Tu-Tan; MDA, malondialdehyde; ROS, reactive oxygen species.

KLHTT Inhibits CII-Specific 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 significantly suppressed IgG1 and IgG2a antibody production ( Figure 6A). Furthermore, KLHTT significantly inhibited the proliferation of CII-induced splenocytes ( Figure 6B).
Life 2020, 10, x FOR PEER REVIEW 12 of 18 IgG1 and IgG2a antibody production ( Figure 6A). Furthermore, KLHTT significantly inhibited the proliferation of CII-induced splenocytes ( Figure 6B). Figure 6. KLHTT inhibits anti-IgG CII antibody production and splenocyte proliferation in CIA mice. CIA was induced by active immunisation with chicken CII in DBA/1J mice. Drugs were administered orally once a day from day 21 to 42. (A) The levels of anti-CII IgG1 and IgG2a antibodies were detected on day 42 using enzyme-linked immunosorbent assay. (B) Splenocytes were cultured with CII for 40 h, and then cell proliferation was measured by incorporation of ( 3 H)-thymidine. Data are expressed as mean ± SD (n = 6). ** p < 0.01, and *** p < 0.001 versus vehicle-treated CIA control mice; one-way ANOVA. CIA, collagen-induced arthritis; CII, collagen type II; KLHTT, Kan-Lu-Hsiao-Tu-Tan.

KLHTT Reduces the Levels of Splenic Th1 and Th17 Cells in CIA Mice
The pro-inflammatory Th1 and Th17 cell axes play crucial roles in RA. The levels of splenic Th1 and Th17 cells were higher after CII induction. KLHTT significantly decreased the numbers of CD4 + IFNγ + Th1 and CD4 + IL17A + Th17 cells (Figure 7). KLHTT also mitigated the levels of pro-inflammatory cytokines in CIA mice (Figure 4). These results indicated that KLHTT decreases splenic proinflammatory Th1 and Th17 cells in CIA mice. Figure 6. KLHTT inhibits anti-IgG CII antibody production and splenocyte proliferation in CIA mice. CIA was induced by active immunisation with chicken CII in DBA/1J mice. Drugs were administered orally once a day from day 21 to 42. (A) The levels of anti-CII IgG1 and IgG2a antibodies were detected on day 42 using enzyme-linked immunosorbent assay. (B) Splenocytes were cultured with CII for 40 h, and then cell proliferation was measured by incorporation of ( 3 H)-thymidine. Data are expressed as mean ± SD (n = 6). ** p < 0.01, and *** p < 0.001 versus vehicle-treated CIA control mice; one-way ANOVA. CIA, collagen-induced arthritis; CII, collagen type II; KLHTT, Kan-Lu-Hsiao-Tu-Tan.

KLHTT Reduces the Levels of Splenic Th1 and Th17 Cells in CIA Mice
The pro-inflammatory Th1 and Th17 cell axes play crucial roles in RA. The levels of splenic Th1 and Th17 cells were higher after CII induction. KLHTT significantly decreased the numbers of CD4 + IFNγ + Th1 and CD4 + IL17A + Th17 cells (Figure 7). KLHTT also mitigated the levels of pro-inflammatory cytokines in CIA mice (Figure 4). These results indicated that KLHTT decreases splenic pro-inflammatory Th1 and Th17 cells in CIA mice.

Discussion
RA is a chronic autoimmune inflammatory disease [1]. Patients with RA have systemic inflammatory comorbidities. The therapeutic armamentarium for RA has expanded from analgesics and nonsteroidal anti-inflammatory drugs to biological disease-modifying anti-rheumatic drugs and conventional disease-modifying anti-rheumatic drugs; however, these available therapies may cause Life 2020, 10, 313 13 of 17 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 inflammatory 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 inflammatory cell infiltration. Both in CIA and human RA, pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-17 trigger autoimmune reactions and enhance chronic inflammation in synovial tissues [38,39]. These pro-inflammatory cytokines activate synovial fibroblasts and chondrocytes to produce enzymes which degrade collagen and proteoglycans, thus damaging adjacent joint tissues [40]. KLHTT significantly 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 anti-inflammatory 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]. Additionally, 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 significantly decreased the levels of H 2 O 2 (an 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 infiltration in the synovium [46,47]. Infliximab, 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 decreased the levels of splenic Th1 and Th17 cells. The levels of pro-inflammatory 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.