We collected the fruits of C. speciosa from Bozhou, Anhui Province, China, in October 2014 (authenticated by Prof. Hanming Zhang). A voucher specimen (No. CS20141008) for C. speciosa fruit was submitted to the authors’ laboratory. We performed hot water extraction of the fruits of C. speciosa (100 kg; 1:15), followed by deproteinization using 15% trichloroacetic acid, and precipitation with 70% ethanol. After centrifugation, the precipitate was collected to obtain the crude polysaccharide. The total carbohydrate content of the polysaccharides was determined by the phenol-sulfuric acid method with glucose as standard, and determined the content was 52.97%.

Mannose (B25303), rhamnose (YY91163), galacturonic acid (B21894), glucose (B21882), galactose (YY91011), and arabinose (B25845) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. PMP (1-phenyl-3-methyl-5-pyrazolone) pre-column derivatization combined with high performance liquid chromatography (HPLC) were used to determine the monosaccharide compositions of CSP (Cao et al., 2019). The polysaccharide (1.0 mg/ml) was hydrolyzed using 4 M trifluoroacetic acid (TFA) at 110°C for 6 h. The residual TFA was obtained by evaporating the residue under reduced pressure in methanol (2 ml). The hydrolysate and the hybrid standard monosaccharide solution (100 μl, glucose, D-mannose, D-galactose, xylose, maltose, L-arabinose, all 1 mg/ml) were dissolved in 200 μl water; the supernatant was mixed with PMP and 0.3 M NaOH solutions. The reaction was carried out at 70°C for 45 min before being stopped using an HCl solution. The reaction product was removed thrice with chloroform, and the upper layer was centrifuged, and filtered using a 0.22 μM membrane, followed by HPLC analysis. We used the Agilent Eclipse XDB-C18 chromatographic column (30°C), equipped with a UV-detector. The mobile phase was PBS (pH 6.8) mixed with acetonitrile (87: 13) and eluted isocratically for 60 min.

We purchased the Sprague-Dawley (SD) (Male, weight: 120–140 g) rats from the Laboratory Animal Research Center at the Second Military Medical University (SCXK (hu) 2017-0010). The animal study was reviewed and approved by the Animal Ethic Committee of Second Military Medical University (No.CZ201705126) in the accordance with the National Institutes of Health guidelines for animal care. These animals were freely access to food/water and were hosted in rat cages at (24 ± 2)°C, 12 h:12 h light/dark cycle, 40–70% humidity.

Our experimental protocols followed the guidelines of the Animal Guidelines of Second Military Medical University. We followed a previously described method to establish the CFA-induced arthritis model in SD rats (Gupta et al., 2020). First, we measured the volume of the foot (reference), and then intradermally injected CFA (0.1 ml) into the foot pad and recorded as day 0. On day 7, the immunized rats were divided into five group, and then the five groups were administrated with saline (model group), 12.5 mg/kg CSP (low-dose group), 25.0 mg/kg CSP (middle-dose group), 50.0 mg/kg CSP (high-dose group), and 20.0 mg/kg imrecoxib (positive control group) via oral gavage, respectively from day 8. Another 10 non-immunized rats were treated with saline (“the control group”). Each group had 10 rats who were treated daily for 3 weeks.

After treatment initiation, foot volume was measured every 3 days and each time the joints were marked based on the initial measurement. The swelling degree and swelling inhibition were calculated based on the swelling volume as follows:

Swelling degree (ΔmL) = paw volume after inflammation—paw volume before inflammation.

Swelling inhibition rate (%) = (paw volume in model group—paw volume in the treatment group)/paw volume in model group × 100%

The pro-inflammatory cytokines in serum, including TNF-α, COX-2, and IL-1β were measured by ELISA kits (Jiancheng Bioengineering, Nanjing, China) according to the manufacturer’s instructions.

On day 29, we used isoflurane to sacrifice the rats. The legs below the knee joints (together with the feet) were simultaneously excised, fixed with 10% formalin, and treated with 5% formic acid. The paraffin-embedded tissues were sectioned, stained with H&E stain, and analyzed histopathologically.

The KM mice (male, weight 23 ± 0.5 g) were purchased from the Laboratory Animal Research Center at the Second Military Medical University and hosted under identical conditions as the SD rats.

The KM mice were divided into five groups (n = 10 each): model, aspirin (positive group, 200 mg/kg), low-dose (25 mg/kg CSP), medium-dose (50 mg/kg CSP), and high-dose (100 mg/kg CSP). The animals were administered respective treatments daily for 5 days, and 1 h post-administration, each group was intraperitoneally injected with HAc (10 mg/ml) and observed and recorded for 15 min (Tadiwos et al., 2017).

The following formulas were used to calculate the number of writhing turnovers of the mice in the group, and the writhing inhibition rate:

Writhing Inhibition Rate (%) = (N₁−N₂)/N₁ × 100%

N₁: Number of writhing in model group; N₂: Number of writhing in CSP group.

We cultured NR8383 murine macrophage cells (Institute of Biochemistry and Cell Biology, Shanghai) in high-glucose DMEM with 10% FBS and 1% antibiotics at 37°C in 5% CO₂. The CCK-8 assay was used to determine the cytotoxicity of CSP (25.0, 50.0, 100.0, and 200.0 μg/ml) (Yu et al., 2020).

The Total RNA Kit was used to isolate total RNA from the spleen tissues, followed by quantification via the µ Drop Plate (Thermo scientific, Finland). A Revertaid First Strand cDNA Synthesis Kit was used to synthesize cDNA, followed by qRT-PCR using a SYBR Green Master Mix using corresponding primers for cytokines. The primer sequences were as follows: TNF-α: (F) 5′- GGG​GCC​ACC​ACG​CTC​TTC​TGT​CTA-3′; (R) 5′- CCT​CCG​CTT​GGT​GGT​TTG​CTA​CG -3′), IL-1β (F) 5′- CCT​CTG​TGA​CTC​GTG​GGA​TGA​TG-3′; (R) 5′- CAG​GGA​TTT​TGT​CGT​TGC​TTG​TCT -3′), COX-2 (F) 5′- CTG​GTG​CCG​GGT​CTG​ATG​ATG​TA-3′; (R) 5′- AGC​AGG​TGT​GGG​TCG​AAC​TTG​AG -3′), β-actin (F) 5′- CCT​AAG​GCC​AAC​CGT​GAA​AAG​ATG-3′; (R) 5′- GTC​CCG​GCC​AGC​CAG​GTC​CAG -3′). The following PCR conditions were used: pre-denaturation for 5 min at 95°C, then for 10 s at 95°C, and annealing for 20 s at 60°C for 40 cycles. The data was normalized to β-actin to estimate the relative expression of these genes.

NR8383 cells (1 × 10⁵ cells/well) were cultured in 96-well plates for 24 h, followed by pretreatment with CSP (25, 50, 100, and 200.0 μg/ml) for 12 h, and p38 inhibitor (SB 203580, 10 µM) and JNK inhibitor (SP600125, 10 µM) for 5 h, followed by LPS stimulation (1 μg/ml) for another 30 min. Next, the concentrations of IL-1β, TNF-α, and COX-2 were determined in the supernatant using respective ELISA kits.

The samples for western blot were prepared following a previously described protocol (Xiong et al., 2019). Briefly, NR8383 cells (1 × 10⁵ cells/well) were cultured in 96-well plates for 24 h, followed by pretreatment with CSP (25, 50, 100, and 200.0 μg/ml) for 12 h, and p38 inhibitor (SB 203580, 10 µM) and JNK inhibitor (SP600125, 10 µM) for 5 h, followed by LPS stimulation (1 μg/ml) for another 30 min. Next, the cells were washed with cold PBS and lysed in ice-cold lysis buffer containing the Mammalian Protein Extraction Reagent. The cell lysates were centrifuged (4°C; 10,000 g; 5 min) and the supernatant was used to measure the expression of MAPKs (p-p38, p38, ERK 1/2, JNK, p-JNK) and GAPDH (Santa Cruz, United States) was used as the internal control. The BCA method was used for protein quantification. After separating the protein specimens on SDS-PAGE, they were transferred to a PVDF membrane, which was treated overnight with GAPDH (1:1,000), ERK1/2 (1:600), p-ERK1/2 (1:400), p38 (1:800), p-p38 (1:600), p-JNK (1:500), JNK (1:500) primary antibodies at 4°C. Next, the membranes were treated with HRP-conjugated secondary antibody. The protein bands were visualized using Pierce^® ECL Substrate.

All experiments were repeated at least thrice and data were represented as means ± SEM. Statistical differences were examined via GraphPad Prism 7.0 (GraphPad, La Jolla, CA, United States) by one-way ANOVA, followed by Dunnett’s test.

Monosaccharide content of CSP was evaluated post-PMP derivatization. HPLC chromatogram showed the presence of six peaks (19.12, 27.95, 37.98, 44.01, 51.02, and 56.89 min) (Figure 2B), and were identified as mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose, according to the references (Figure 2A), which were found to be present in a ratio of 1.66: 2.92: 4.72: 4.25: 9.42: 77.02.

Post-immunization, CFA mice were orally administered CSP (12.5, 25, and 50 mg/kg) from days 8 to 29 to examine the inhibitory effects of CSP (Figure 3A). The swelling volume of the injured paws was used to evaluate CFA-induced disease progression. The rats in the CFA group started showing substantially enhanced paw swelling from day 7 post-injection. Pain swelling was inhibited (p < 0.01) after treatment with iremcoxib from day 20 (Figures 3B–C). In the high-dose and medium-dose groups, CSP considerably improved paw swelling from day 22 post-injection of CFA (p < 0.01, Figures 3B–C), which improved with time. Additionally, the swelling inhibition rate of the high-dose group was similar to that of the positive control (imrecoxib) from day 22 (Figure 3D).

The results of histopathological examination showed that the model group showed more severe pathological changes compared with normal rats not induced by CFA (Figure4). All rats exhibited various degrees of pathological changes in the ankle joints and feet, mainly due to the proliferation of ankle synovial cells, inflammatory cell infiltration; the articular cartilage and bone tissue in the joint cavity were severely damaged, there was fibrous connective tissue proliferation on the cartilage surface, part of the periosteal fibrosis, and even joint cavity stenosis; obvious soft tissue around the joint inflammation, etc., which suggested the successful establishment of the rheumatoid arthritis model in rats. There was a substantial reduction in the pathological changes in the mouse ankle joint and foot tissue. There was relatively less infiltration of inflammatory cells and proliferation of synovial cells in the ankle joint of rats. The fibrosis of articular cartilage and bone tissue was not severe, and there was no obvious articular surface stenosis. Also, adhesions and inflammation of the surrounding soft tissues were not severe. Additionally, the pathological changes in the 25.0 and 12.5 mg/kg CSP groups were slighter than that in the model group. Thus, CSP could effectively reduce the inflammatory response of the joint synovium and prevent the destruction of cartilage tissue, bone tissue, and tissues around the joint.

Additionally, the degree of pathological changes was quantified (Table 1). The pathological changes in the synovial membrane, articular cartilage, and skin ulcer were divided into four levels, including slight (±), mild (+), moderate (++), and severe (+++). In this study, the number of pathological changes (moderate and severe) in the CSP (50 mg kg−¹) group were reduced by 33 and 67% in the synovial membrane and skin ulcer, respectively, than the model group. We observed no significant change in the articular cartilage in the CSP (50 mg kg−¹) group after 3-weeks administration, suggesting that CSP could alleviate pathological changes in CFA-induced rats.

Figure 5 exhibits the production of various pro-inflammatory cytokines in the serum of CFA-induced rats. When compared to the normal group, the model group had higher contents of TNF-α, COX-2, IL-6, and IL-1β in the serum (p < 0.01), suggesting that an apparent inflammation existed in the model group (Figures 5A–C). However, the pro-inflammatory cytokines production decreased in the CSP group compared with the model group, especially for the high group with significant difference (p < 0.05). These findings suggested that CSP has a significant anti-inflammatory activity in CFA-induced rats.

The results of HAc-induced writhing method showed that the extract showed substantial analgesic activity. In the HAc-induced writhing method, we found that CSP dose-dependently promoted a reduction in writhing movement in mice compared with the model group; especially for the high-dose group (100 mg/kg), which showed a significant difference (p < 0.01) (Figure 6A). In addition, 200 mg/kg Aspirin showed significant difference in reduction in writhing movement (p < 0.01), compared with the model group. Moreover, the writhing inhibition rate of CSP (100 mg/kg) was around 43.2%, which is higher than the positive control group (Aspirin 200 mg/kg).

We performed western blot to test evaluate the potential mechanism involved in CSP-induced attenuation of arthritis. In order to explain whether the improvement of inflammation by CSP is related to MAPK signaling pathway, the key proteins including extracellular signal regulated kinase (ERK), p-ERK, p38 mitogen activated protein kinase (p38), p-p38, c-Jun N-terminal kinase (JNK) and p-JNK were measured (Figure 8). The results of this study demonstrated that the protein expression levels of p-ERK, p-JNK were significantly elevated in the model group. Furthermore, CSP degraded the protein expression levels of p-ERK and p-JNK, while p-p38 showed no significance after CSP treatment. Moreover, when NR8383 cells were pretreated with SP600125, a specific JNK inhibitor, this increase was markedly inhibited, which was similar to the effects of CSP treatment. Similar results were obtained when the inhibitor was replaced by SB203580, which is a broad-spectrum inhibitor of p38 (Figure 8).