No detection kit

Parthenolide (PAR), complete Freund’s adjuvant (CFA), lipopolysaccharides (LPS) and naloxone were purchased from Sigma-Aldrich (St. Louis, MO) (+)-Bicuculline (BIC) was purchased from Topscience (Shanghai, China). 3-mercaptoethanol (3-MP) was purchased from Meryer (Shanghai, China). Morphine was obtained from Northeast Pharmaceutical Group (Shenyang, China). NO detection kit was obtained from Beyotime Institute of Biotechnology (Jiangsu, China). The ReverTra Ace qPCR RT kit was purchased from TOYOBO Co., Ltd (Osaka, Japan). IL-6 ELISA kit and IL-1β ELISA kit were obtained from Thermofisher Co., Ltd (MA, United States) and TNF-α ELISA kit was purchased from Neobioscience (Shenzhen, China). Antibodies were purchased as following: Anti-ERK, JNK, p38, iNOS, p65, AKT, and phosphor-ERK, -JNK, -p38, -p65, -AKT were purchased from Cell Signaling Technology (Danvers, MA, United States). Anti-β-actin was purchased from Bioss (Beijing, China). Anti-GABA_A Receptor alpha 2 (anti-GABA_ARα2) was purchased from Abcam (Cambridge, United Kingdom). GAPDH was obtained from Proteintech (Wuhan, China). IRDye®800CW secondary antibodies were purchased from LI-COR (Lincoln, NE, United States).

Nitrite (NO₂⁻) was measured as an indicator of NO synthesis and was estimated using the Griess reagent [62 (link)]. A NO detection kit (Beyotime, Shanghai, China) was used according to the manufacturer’s instructions. In brief, 50 μL samples were mixed with an equal volume of Griess reagents I and II at room temperature. The absorbance was determined at 540 nm and calibrated with a nitrite standard curve to determine the nitrate concentration in samples.

Strains (2.2 × 10⁸ cells/mL) were inoculated in TYA medium containing 0, 0.77 or 1.03 M NaCl, respectively. The bacteria were collected by centrifugation at 2, 4, and 8 h, respectively. After ultrasonic cell-break at 4 °C, the supernatant (as crude enzyme solution) was collected by centrifugation at 12,000 r/min for 20 min. CAT and POD activities were determined according to the method described in Liang et al. [33 (link)]. One unit of CAT activity is defined as the amount of enzyme that reduces A₂₄₀ 0.01 per min. One unit of POD activity is defined as the amount of enzyme that increases A₄₇₀ 0.01 per min. MDA content was determined using thiobarbituric acid method according to the method described in Liang et al. [33 (link)]. MDA content (μM) = 6.45 × (A₅₃₂ − A₆₀₀) − 0.56 × A₄₅₀. NO content was determined using NO detection kit (Beyotime Biotechnology Company, Shanghai, China) according to the method from the manufacturer. A₆₀₀ value of the cell cultures was also determined simultaneously. The enzyme activity and MDA and NO content were calculated according to the number of cells (10⁹ cells).

The wild type Ds-26-16 was derived from our previous work [19 (link)]. p21-ORF is the plasmid that inserted the open reading frame of Ds-26-16 (159 bp) into the SalI/HindIII site of pET-21b(+). dITP were obtained from Thermo Fisher Scientific. Inc. (Waltham, MA, USA). rTaq DNA polymerase were obtained from Dalian TaKaRa Biotech. Co., Ltd. (Dalian, China). NO detection kit was provided by Beyotime Biotechnology Inc. (Shanghai, China). E. coli DH5α-FT and BL21(DE3) were kept in our laboratory.
To simplify the description, LB medium containing a final concentration of 1 mM Isopropyl-β-d-thiogalactopyranside (IPTG) and 0.1 mg/mL Amp was named as LBA. 2× TY medium containing a final concentration of 1 mM IPTG and 0.1 mg/mL Amp was named as TYA. BL21(DE3) containing pET-21b(+) plasmid or p21-ORF plasmid were named as pET-21b(+) strain or Ds-26-16 strain, respectively. BL21(DE3) containing pET-21b(+) that carried the evolved gene of Ds-26-16 was named after its evolved gene.

Raspberries (cultivar name: Autumn Britain) were obtained from Qinghai Raspberry Agriculture and Forestry Industrialization Ltd. (Qinghai, China), located in Huangyuan county of Qinghai province (latitude of 101.256, longitude of 36.682 and elevation of 2650 m).
RAW264.7 macrophages were supplied by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S), dextran standards, and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The Cell Counting Kit-8 (CCK-8) and NO detection kit were purchased from Beyotime Biotechnology (Shanghai, China). The enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-6, and IL-1β were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). The monosaccharide standards, including rhamnose (Rha), fucose (Fuc), arabinose (Ara), xylose (Xyl), mannose (Man), glucose (Glu), etc., were obtained from The National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). The DEAE Sepharose Fast Flow gel and Sephadex G-200 gel were purchased from Yangzhou BoRui Saccharide Biotech Co. Ltd. (Jiangsu, China). All chemical reagents were of analytical reagent grade.

Recombinant human/mouse/rat activin A was provided by R&D systems (Minneapolis, MN, USA). Recombinant murine TNF-α and recombinant murine SDF-1α (CXCL12) were obtained by PeproTech (Rocky Hill, NJ, USA). Cell Counting Kit-8 (CCK-8) was bought from GlpBio Biotechnology Co. (Shanghai, China). NO detection kit was purchased Beyotime Biotechnology Co. (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kit for TGF-β1 was obtained from eBioscience (San Diego, USA); ELISA kit for IL-6 was provided by R&D systems (Minneapolis, MN, USA). Reverse transcription-PCR (RT-PCR) kit was purchased from Takara Biotechnology Co. (Dalian, China). The antibodies used for Western blotting were as follow: ActRIIA (Absin), Smad3 (Immunoway), p-Smad3 (Abcam), ERK1/2 and p-ERK1/2 (Cell Signaling), MMP-2 (Absin), MMP-9 (Absin) and GAPDH (Absin).