Reagents

The H&E staining kit was purchased from BaSO (Zhuhai, China). Carrageenan was brought from Solarbio (Beijing, China).

Preparation of PRE

PRE was generously donated by Dr Huang from the School of Pharmacy, Cheng du University of traditional Chinese medicine. The details were as follows: Paridis rhizoma was ground and sifted through a 65-mesh (250 μm) sieve. The resulting powder was weighed to approximately 0.5 g and placed in a conical bottle with a cork. Then, 25 mL of 70% ethanol was added to the bottle. The contents were heated and refluxed at a specific temperature for 30 min. After cooling, the bottle was weighed again, and the lost weight was compensated by adding 70% ethanol. The mixture was shaken thoroughly and centrifuged at 12,000 × g·min-1 for 15 min. The resulting supernatant was filtered through a 0.22 μm microporous filter membrane to obtain the PRE.

Cell culture

RAW264.7 cells were purchased from Procell Life Science and Technology Co., Ltd. (Wuhan, China) and incubated at 37 °C in 5% CO2 humidified air in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with a mixture of 1% penicillin/streptomycin and 10% fetal bovine serum (FBS).

Cell viability assay

The Cell Counting Kit (CCK)-8 assay (Biosharp, China) was used to evaluate the cytotoxic effects of PRE on Raw264.7 cells. Briefly, the cells were seeded into 96-well plates at a density of 4 × 103 /ml and cultured for 24 h at 37 °C in an atmosphere of 5% CO2.The cells were then treated with different concentrations of PRE (0, 10, 25, 50, 100, 200, 400, 800 ng/ml) for 24 h. The viability of cells after the treatments was evaluated by addition of 10μL of CCK-8 solution (Beyotime, Shanghai, China) to each well and incubated at 37 °C for 2 h. The final absorbance was read at a wavelength of 450 nm using a microplate reader (Molecular Device, Shanghai, China).

Western blotting

Protein was isolated from cells using a radioimmunoprecipitation assay buffer and quantified using a Bicinchoninic Acid Protein Assay Kit (Beyotime, Jiangsu, China). The proteins were separated using 10% sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk in Tris-buffered saline for 1 h and were incubated with anti-phospho-p65 (Affinity, USA), anti-p65 (Affinity, USA), anti-phospho-IKB alpha (Affinity, USA), anti-IKB alpha (Affinity, USA), anti-IL-6 (Proteintech), anti-TNF alpha (Proteintech), and anti-β-actin (Proteintech) at 4 °C overnight. They were then washed with Tris-buffered saline Tween-20, and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Proteins blots were detected with enhanced chemiluminescence reagent (Beyotime, Jiangsu, China).

Quantitative real-time reverse transcriptase polymerase chain reaction

Total RNA was isolated from cells using TRIzol™ (Solarbio, Beijing, China) reagent according to the manufacturer's instructions. The cDNA was synthesized using reverse transcription kit of the first strand using the SynScript® III cDNA Synthesis Mix. The mRNA levels were analysed by qRT-PCR (Bio-ER) in a total volume of 20μL using 2 × TSINGKE® Master qPCR Mix (SYBR Green I, Tsingke, China). Relative mRNA expression was calculated using the 2‑ΔΔct method after normalization to the expression level of GAPDH expression. The gene-specific primers used are listed in the Table 1.

Table 1 Primers used for qPCRMeasurement of reactive oxygen species

RAW264.7 cells were cultured overnight in 6-well plates at a density of 1 × 106 cells/well and then exposed to different concentrations of PRE and LPS (200 ng/ml) alone or in combination. The intracellular ROS level in Raw264.7 cells was determined using 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA; Yeasen). Simply, Raw264.7 cells were incubated with 10 µM DCFH-DA in DMEM without FBS at 37 °C for 20 min and washed three times with DMEM. The 2′,7′-dichlorofluorescein (DCF) fluorescence was observed using an inverted fluorescence microscope. The average fluorescence intensity was assessed using ImageJ.

Animals

Adult male Sprague–Dawley rats (8–10 weeks old, 180–200 g) were purchased from SPF Laboratory Animal Technology Co., Ltd. (Beijing, China) and acclimatized for 1 week before experimentation. All animal procedures were performed in compliance with the National Institutes of Health Guidelines for Care and Use of Laboratory Animals. The protocol for this study was approved by the Bioethics Committee of Chengdu University of Traditional Chinese Medicine (2022–65).

Carrageenan-induced rat paw edema model

According to Winter et al. [21], the paw edema was induced by carrageenan. Rats were randomly divided into 6 groups each comprising 10 rats. They were pretreated orally for a week with the vehicle (0.9% saline, control group, n = 10), model (untreated group, n = 10), PRE low dosage (PRE-LD) (low dose, n = 10), PRE medium dosage (PRE-MD) (medium dose, n = 10), PRE high dose (PRE-HD) (high dose, n = 10) and indomethacin (INDO) (5 mg/kg, po) [22]. Rats in the low-, medium-, and high-dose PRE groups received 0.315 g, 0.63 g, and 1.26 g/kg PRE daily, respectively. Edema was induced 30 min after the last treatment, by injection of 0.1 mL of carrageenan (100 µg/paw) in saline into the right hind paw while the control received 0.9% saline (0.1 ml). Inflammation was quantified by measuring the volume (mL) displaced by the paw using plethysmometer (Tai Meng Software Co., Ltd, Chengdu, China) at 0, 0.5, 1, 1.5, and 2 h after carrageenan injection. All rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (50 mg/kg) [23].

Histopathological examination

The paws were fixed overnight in 4% paraformaldehyde containing in 0.1 M PBS and embedded in paraffin. Slides with a 5-μm thickness were prepared, deparaffinized and stained with Haematoxylin/eosin (H&E).

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of tumor necrosis factor-α (TNF-α) (Jianglai Biotechnology, JL13202), prostaglandin E2 (PGE2) (Jianglai Biotechnology, JL12636), cyclooxygenase-2 (COX-2) (Jianglai Biotechnology, JL21044) and interleukin-6 (IL-6) (Jianglai Biotechnology, JL20896) in sera were detected using ELISA following the manufacturer’s instructions.

Statistical analysis

The statistical analyses were performed with GraphPad prism 8.0. Data were presented as means ± standard error of the mean (SEM). Comparisons among multiple groups were performed by a one-way analysis of variance (ANOVA). If one-way ANOVA yielded a significant F ratio, Bonferroni post-hoc analysis or Tamhane's T2 was conducted between the groups. P values < 0.05 were considered statistically significant.