Materials and reagents
The Kadsura heteroclita dried stems were collected from Shimen City, Hunan Province, China. The collection of Kadsura heteroclita dried stems has been approved by the Agriculture and Rural Bureau of Shimen County, Changde City, Hunan Province and Hunan University of Chinese Medicine. The permissions of collection of Kadsura heteroclita dried stems were obtained from Agriculture and Rural Bureau of Shimen County, Changde City, Hunan Province and Hunan University of Chinese Medicine. The collection process of Kadsura heteroclita stem complies with the Plant Protection Law of the People's Republic of China, conforms to the standards of agricultural and rural plant collection of Shimen County. And the Experiment complied with relevant institutional, national, and international guidelines and legislation. The plant materials of Kadsura heteroclita are stored in TCM and Ethnomedicine Innovation & Development International Laboratory, Innovative Materia Medica Research Institute, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, 410208, China. The specimen number is 2020081501. The plant identification and the standard of XTS was conducted by Prof. Wang Wei. The Cell Counting Kit-8 (CCK-8) kit, Apoptosis assay kit and protein extraction kit were purchased from SEVEN Co., Ltd (Beijing, China). The antibodies used, including β-actin (AF7018), MMP-9 (AF5228), Bax (AF0120), Bcl-2 (AF6139), Caspase-3 (AF7022), and HMGB-1 (AF7020), were obtained from Affinity Co., Ltd (Jiangsu, China).
Extraction and isolation
It has been reported that 1.08 g XTS can be extracted and isolated from 3 kg Xuetong stems[8]. The optimized the extraction process of XTS in this study as follows: The air-dried Xuetong stem (13.2 kg) was extracted thrice using 90% ethyl alcohol (EtOH), each time for 1.5 hours, with the assistance of ultrasound. Subsequently, the extracted solvents were evaporated under reduced pressure to yield a crude EtOH extract (3.2 kg). The crude extract was then suspended in water and sequentially partitioned with dichloromethane (DCM) and ethyl acetate (EtOAc), resulting in DCM-soluble and EtOAc-soluble fractions. The DCM fraction was further purified using silica gel column chromatography eluted with a PE-EtOAc gradient (from 1:0 to 0:1), producing six fractions (Fr. A-Fr. F). Subfraction Fr. B underwent further separation using silica gel column chromatography, Sephadex LH-20 gel chromatography, and recrystallization to yield XTS (26.22 g).
Structure characterization of XTS
The XTS spectra, including 1H, 13C, 1H-1H COSY, HSQC, and HMBC, were acquired using a Bruker AV-600 spectrometer equipped with a single NMR probe operating at 600 MHz for 1H and 150 MHz for 13C in CDCl3. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) data were collected on a Waters UHPLC-H-CLASS/XEVO G2-XS Q-TOF mass spectrometer. UV-visible (UV-vis) spectral analysis was conducted using a SHIMADZU UV-vis spectrophotometer.
Qualitative and quantitative analysis
To perform a qualitative and quantitative assessment of the enriched XTS, we employed high-performance liquid chromatography (HPLC) to extract, identify, and quantify XTS. The analysis was carried out utilizing an Agilent 1260 system featuring a diode array detector. A suitable Agilent TC-C18 column (5 µm, 150 mm × 4.6 mm) was selected and operated with a mobile phase consisting of an aqueous solution containing 0.1% phosphoric acid and 100% acetonitrile. A gradient elution protocol was followed: over 0–30 minutes, the acetonitrile concentration was adjusted from 65–85%. The optimized parameters consisted of a flow rate of 1 mL/min, a column temperature of 25°C, an injection volume of 5 µL, and detection at a wavelength of 210 nm. The content of XTS was quantified employing the external standard method based on the standard concentration.
Cell culture
RAFLS and HFLS cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). RAW264.7 cells and NIH-3T3 cells were obtained from Procell Life Science & Technology Co., Ltd (Wuhan, China). These cells were cultured in DMEM/F-12 = 1:1 medium (RAFLS and HFLS cells) or DMEM medium (RAW264.7 cells and NIH-3T3 cells), supplemented with 10% FBS and 1% penicillin/streptomycin.
IC50 assay and Cell viability assay
RAFLS cells were seeded in a 96-well plate at a density of 1.0 × 105 cells/well and and allowed to proliferate logarithmically. The cells were cultured in above medium supplemented with 1% FBS and 1% penicillin/streptomycin and treated with varying concentrations of XTS, sinomenine (SIN), indomethacin (Indo), and methotrexate (MTX) (0, 2.5, 5, 7.5, 10, 15, 20 µM) for 48 hours. Following that, the aforementioned medium was substituted with a medium supplemented with 10% CCK-8 solution. After a 30-minute incubation period, the optical density (OD) at 450 nm for each well was quantified using a microplate reader (Thermo Multiskan Sky, Waltham, MA, United States). Cell viability was determined using the following calculation:
Cell Viability = OD450 nm/sample/ OD450 nm/control× 100%
RAFLS cells were treated with XTS at concentrations (0, 4.5, 9, 18 µM) or Indo (10 µM) for 48 hours. Cell viability was assessed using the previously described method.
The cytotoxic effects of XTS were further evaluated by examining its impact on the viability of HFLS, NIH-3T3, and RAW264.7 cell lines. These cells were cultured with 1% FBS and 1% penicillin/streptomycin in corresponding medium, treated with different concentrations of XTS (0, 4.5, 9, 18 µM) or Indo (5.0 µM), MTX (5.0 µM), and SIN (5.0 µM) for 48 hours. Cell viability was determined following the protocol mentioned above.
Wound healing assay
After treatment with various concentrations of XTS (0, 4.5, 9, 18 µM) for 48 hours, a sterile plastic scraper was used to create a wound in the center of the RAFLS cell monolayer. The migratory capacity of RAFLS cells was assessed by measuring the wound area at 0, 12, 24, and 48 h post-injury. The percentage of the wound closure was determined by comparing the healed area at each time point to the initial wound area. The rate of cell migration was calculated using the formula:
Cell migration rate (%) = (Original wound distance-Residual wound distance) / Original wound distance × 100.
Apoptosis assay
Following a 48-hour treatment of RAFLS cells with various concentrations of XTS (0, 4.5, 9, 18 µM), the cells were collected into 1.5 mL Eppendorf (EP) tubes. Subsequently, they underwent three washes with pre-chilled phosphate-buffered saline (PBS), with each wash involving centrifugation at 800 rpm (approximately 100 g) for 5 minutes at 4°C. After the final wash, the cells were resuspended in 500 µL of 1× Binding Buffer. To each cell suspension, 5 µL of Annexin V-FITC and 5 µL of Propidium Iodide (PI) were added, and the mixture was incubated in the dark for 10 minutes. Flow cytometry was then employed to detect apoptotic cells, and the proportions (%) were measured using FlowJo 7.6 software.
Cell protein analysis by Western blot
Following 48-hour treatment with various concentrations of XTS (0, 4.5, 9, 18 µM), RAFLS cells were lysed in protein extract buffer on ice. The cells, comprising both the supernatant and adherent fractions, were collected through centrifugation at 800 rpm for 5 minutes. The protein concentrations were determined using the BCA assay. Subsequently, the proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were then blocked with rapid sealing fluid at 25°C for 10 minutes. After blocking, the membranes were incubated overnight at 4°C with antibodies against Bcl-2, Bax, Caspase-3, MMP-9, and HMGB-1, which were diluted in TBST at a 1:1600 ratio. Following the incubation with secondary antibodies, the protein bands were visualized using enhanced chemiluminescence and quantified using Image J software.
Animals
Male SD rats weighing 70–90 g (Hunan SJA Animal Co., Ltd) were maintained under SPF conditions. All experiments were carried out in compliance with the ARRIVE guidelines. All methods were performed in accordance with the relevant guidelines and regulations. The animals were accommodated in a pathogen-free facility, with ambient temperature maintained at 23 ± 2°C, and provided with an libitum access to feed and water. The animal study was granted ethical approval by the ethics committee of Hunan University of Chinese Medicine (permit number: LL202103160001).
Hemolysis assay
Blood samples were obtained from the orbital venous plexus of rats, and thereafter, red blood cells (RBCs) were isolated and washed with PBS buffer. A 40 µL aliquot of 2.5% RBCs suspended in PBS was mixed with 460 µL of XTS solutions at various concentrations (0.21, 0.42, 0.84, 1.68, 3.36 mg/mL), followed by incubation at 37°C for 4 hours. Concurrently, 40 µL of 2.5% RBCs were diluted in 460 µL of ultra-pure water to serve as a positive control. After incubation, samples were centrifuged at 3000 rpm for 5 minutes at 4°C. A UV-vis spectrophotometer was used to check the absorbance of the supernatant 540 nm. Microscopic examination was conducted to assess the morphological alterations in RBCs. The percentage of hemolysis was determined using the following formula:
Hemolysis (%) = (I / I0) × 100%.
In the formula, "I" represents the absorbance of the supernatant after the addition of XTS at different concentrations to the red blood cell suspension, while "I0" represents the absorbance of complete hemolysis in pure water.
Induction of RA models and drug administration
To induce arthritis in SD rats, an adjuvant-induced arthritis (AIA) model was established by injecting 150 µL of complete Freund's adjuvant (CFA) emulsion subcutaneously at the base of the tail. The CFA emulsion contained 2 mg/mL of heat-inactivated Mycobacterium tuberculosis (Mtb). The rats selected for this model weighed between 80–100 g. Following the induction of AIA, the rats were divided into several groups, which included a normal group, a model group (Model), an Indo (1 mg/kg) treatment group, and three groups receiving varying doses of XTS (0.21 mg/kg, 0.42 mg/kg, 0.84 mg/kg). From days 10 to 30 post-AIA immunization, all groups except the normal control were administered their respective treatments intravenously every 2 days. The XTS-treated groups received varying doses of XTS (0.21 mg/kg, 0.42 mg/kg, 0.84 mg/kg), the positive group received 1.0 mg/kg of Indo, while the model group injected an equivalent volume of saline.
Evaluation of arthritis index and radiologic analysis
After the AIA immunization, the body weight and hind paw swelling of each rat were recorded every three days using an electronic balance and a Vernier caliper, respectively. The degree of paw swelling was evaluated and scored based on the observed extent of swelling during these measurements. After a treatment period of 30 days, all rats were deep anesthesia by using 3% isoflurane and euthanized. Then their hind paws were severed using bone scissors for analysis. The excised paws were subjected to Micro-CT analysis (PerkinElmer-Caliper LS Quantum FX Demo, USA) to assess bone destruction.
Histopathological analysis
After 30 days with XTS treatment, the hind paws of rats were fixed in 4% paraformaldehyde for one week. Subsequently, decalcification was carried out using 10% EDTA for a duration of 1–3 months. The decalcified hind paws were then embedded in hot melted paraffin wax, allowed to cool and solidify, and subsequently sectioned into thin slices. To visualize the tissue morphology, the sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope.
Western blot analysis
Paw tissues were snap-frozen in liquid nitrogen to ensure their integrity before being pulverized. Proteins were then extracted using a lysis buffer supplemented with a protease inhibitor. The concentration of protein in each lysate was assessed using the BCA protein assay kit. Equal quantities of protein were loaded into the wells of an SDS-PAGE gel for electrophoresis. Following electrophoresis, the separated proteins were transferred onto a PVDF membrane. The PVDF membrane was blocked with rapid sealing fluid at 25°C for 10 minutes, and then incubated overnight at 4°C with the target antibody. The washed membrane was incubated with a secondary antibody for 1.5 hours. The protein bands were visualized using an imaging system.
Statistical analysis
The data are expressed as mean ± standard deviation (SD) and are derived from a minimum of three independent experiments. Statistical analyses were performed using SPSS version 22.0. Student's t-test was employed for single comparisons, while one-way ANOVA was used for multiple comparisons. Statistical significance was defined as P < 0.05.