Il 1β

Complete Freund’s adjuvant (CFA) was obtained from Chondrex, Inc. (USA). D-luciferin was purchased from Abcam (UK). Rabbit anti-BMP3, anti-TNF-a, and anti-TIMP metallopeptidase inhibitor 1 (TIMP-1) antibodies were obtained from Abcam (UK). Rabbit anti-MMP-3 and anti-MMP-9 antibodies were acquired from Merck Millipore (USA). Rabbit anti-IL-6, anti-IL-1β, and anti-IL-17A were purchased from Bioworld (USA). The rat anti-β-actin antibody was purchased from CST (USA). A horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin (IgG) was procured from Zhongshan Biotechnology Corporation (China). BMP3, MMP-3, MMP-9, TIMP-1, TNF-a, IL-6, IL-1β, IL-17A, CCL-2, CCL-3, VCAM-1, and β-actin primers were synthesized by the Shanghai Sangon Biological and Technological Company (China).

The supernatant was separated from cell lysate via centrifugation at 12,000×g for 15 min at 4°C. Protein samples were separated by12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (PVDF, Millipore, Bedford, MA USA). Membranes were blocked in TBST buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 0.1% Tween-20) with 5% nonfat dry milk for 1 h. The blots were then incubated with primary antibodies anti-TLR4, NF-κB, COX2, IL-1β, TNF-α, β-actin, phospho-TLR4, and phospho-NF-κB p65 (Ser529) (Sangon, Shanghai, China) overnight at 4°C. The blots were rinsed with TBST buffer and incubated with HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (at 1 : 5000 dilution, Sangon, Shanghai, China). Target proteins were visualized using chemiluminescence horseradish peroxidase (Millipore, Bedford, MA USA) and analyzed by densitometry using ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA).

The strain of GL1 was isolated by the traditional streaking separation method from a traditional region of Tibet in Naqu, China, and it was recognized by 16S rRNA sequence analysis. A fragment 16S rDNA of GL1 was amplified by the 16S rDNA primer (Supplementary Table S1). The amplification was performed in 50 μL (final volume) of the reaction mixture containing 25 μL Rapid Taq Master Mix, 2 uL of each primer, 8 to 10 ng of purified genomic DNA 2 μL, and 19 μL deionized water. The PCR parameters were 94 °C for 2 min, 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s, and a final extension at 72 °C for 10 min. Then, the sequences were determined by the BLAST program of the GenBank database, and GL1 was identified as L. paracasei. The RAW264.7 murine macrophage cell line was purchased from Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. MD34 dialysis bags were purchased from Biosharp (Beijing Labgic Technology Co. Ltd., Beijing, China). The nitric oxide (NO) kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The primers of TNF-α, IL-1β, iNOS, and ꞵ-actin were synthesized by the Sangon Bioengineering Institute (Shanghai, China).

PTER (SP9570, purity ≥98%) was purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). LPS (L2880, purity >97%) was purchased from the Sigma Chemical Co. (St. Louis, MO, United States). MPO (A044-1–1), SOD (A001-3–2), CAT (A007-1–1), GSH-Px (A005-1–2) and MDA (A003-1–2) Assay Kit and were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TNF-α, IL-6, IL-1β, COX-2 and iNOS primers were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). p-p65 rabbit polyclonal antibody (A13599) was acquired from Boster Biological Technology co.Itd. p65 rabbit polyclonal antibody (BS9879M), p-IκB rabbit polyclonal antibody (BS4105), IκB rabbit polyclonal antibody (BS3601), Nrf2 rabbit polyclonal antibody (BS1258) and HO-1 rabbit polyclonal antibody (BS6626) were acquired from Bioworld Technology, Inc (Minnesota, United States). Actin-β polyclonal antibody (YT0099) was acquired from ImmunoWay Biotechnology Company (Plano, TX, United States). All other chemicals were at the reagent level.