Lipopolysaccharide lps

Murine RAW264.7 (ATCC, United States) was incubated in a complete medium (DMEM; Gibco, Grand Island, United States) supplemented with streptomycin/penicillin (1%, Hyclone, United States) and fetal bovine serum (10%, FBS; Gibco, Grand Island, United States). In each well of a six-well plate, 1 × 10⁶ cells were plated for each group. Macrophages were incubated with 100 ng/ml Lipopolysaccharide (LPS, Solarbio, China) and 20 ng/ml INF-γ (Beyotime, China) to induce M1 polarization, while 20 ng/ml IL-4 (Peprotech, United States) induced M2 polarization (Zhang et al., 2017 (link); Xia et al., 2020 (link)). As a control, M0 macrophages were incubated in a complete culture medium. The cells were washed three times in PBS followed by incubation with a complete medium after 24 h. The medium supernatants derived from macrophage were centrifuged after collection for 20 min at 1950 g after another 24 h of culturing. The conditioned medium (CM) comprising M0, M1, and M2 cell cultures were given the respective names CM0, CM1, and CM2. As a control, the entire medium (Norm) was used. Quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry, and fluorescence staining and imaging were used to identify the phenotypes of polarized cells (as activated by IL-4 or LPS plus INF-γ) and unpolarized cells (complete culture medium).

The source of human rheumatoid arthritis fibroblast-like synovial cells (RA-FLS) in this study was from the European Collection of Authenticated Cell Cultures (ECACC), and human synovial tissues were collected from donors with RA. RA-FLS were purchased from Jennio Biotechnology Co., Ltd. Under an inverted phase-contrast microscope, RA-FLS have certain characteristics that distinguish them. FLS are triangular and spindle-shaped, with an oval nucleus, a clear nucleolus in the center of the cell, with an accumulation of secretions surrounding the cells. RA-FLS were cultured in DMEM-H complete medium (Jennio, Guangzhou, China) supplemented with 2% GermClean, L-glutamine, sodium bicarbonate and 15% heat-inactivated FBS (Gibco, Carlsbad, California, USA). Before stimulation, the cells were seeded in a six-well plate and treated with DMEM-H medium containing 10 μmol/L β-caryophyllene oxide (TargetMol, China) and myrtenal (yuanye, Shanghai, China) for 24 h. Cells were stimulated with 10 ng/ml lipopolysaccharide (LPS) (Solarbio Life Science, Beijing, China) for 6 h. Cell lysates and supernatants were collected and used for cytokine detection and Western blot analysis.

Anoectochilus formosanus was obtained from Fujian province and verified by Prof. Boyun Yang from the Life Science Center of Nanchang University. It was then dried at 55 °C for 48 h before preparation. The monosaccharide standards used, D-Mannose, L-Arabinose, D-Ribose, etc., were purchased from Merck Co. (Darmstadt, Germany). Levamisole hydrochloride and cyclophosphamide were purchased from Aladdin Industrial Inc. (Shanghai, China). Cytokine (IgA, IgG, SIgA, IL-2, IL-6, IFN-γ, TNF-α) detecting ELISA kits were purchased from Biosharp biological technology Co., Ltd. (Shanghai, China). Lipopolysaccharide (LPS) and PBS buffer powder were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Commercially available analytical grade reagents were used in this study.

After incubation with PBS, OA-RD17 (1 nM), lipopolysaccharide (LPS, 1 μg/mL) (Solarbio, China), specific TLR4 inhibitor (1 μg/mL), miR-632 mimic (50 nM), or miR-632 inhibitor (100 nM) for 24 h, respectively, cell lysates (RIPA: PMSF: phosphatase inhibitor = 100:1:1; RIPA and PMSF, Meilun Biotechnology, Dalian, China; phosphatase inhibitor, Roche, Shanghai, China) were used to extract total protein in keratinocytes and macrophages. Moreover, cell lysates were also used to extract total protein in wound tissues from SD rats. The extracted proteins were quantified using the Bradford method (BCA protein analysis kit, Meilun, Dalian, China). The protein samples were then separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electro-imprinted on polyvinylidene fluoride membranes, and recorded and analyzed quantitatively using the Bole exposure software system. Primary antibodies, including GAPDH, Lamin B1, P38, P-P38, ERK, P-ERK, JNK, P-JNK, IκB, P-IκB, P65, P-P65 (Affinity, China), GSK3β, β-catenin, Cyclin D1, c-MYC, and Vimentin (ZEN BIO, China) were used following the provided instructions.