Lipopolysaccharide lps

Antibodies against α-SMA (1 : 1000), TGF-β1 (1 : 1000), EGBB2 (1 : 1000), MMP2 (1 : 1000), MMP9 (1 : 1000), PTGS2 (1 : 1000), APP (1 : 500), EGFR (1 : 1000), and β-actin (1 : 1000), as well as HRP goat anti-mouse antibody (1 : 10000) and FITC goat anti-mouse antibody (1 : 1000), were obtained from BD Pharmingen. Sanhua cigarette was provided by Hunan Tobacco (Changsha, China). LPS (lipopolysaccharide) was purchased from Sigma Chemical Co. (St. Louis, MO, United States). Reactive Oxygen Species Assay Kit (ROS, DCFH-DA) and DAPI were obtained from Beijing Solarbio Technology Co., Ltd. (Beijing, China). Mice quantitative ELISA kits, IL-6 (M6000 B) and TGF-β(MB100 B), were obtained from R&D Systems (Minnesota, USA).
Luteolin (purity > 98% via HPLC, batch number: L107328) was purchased from Lianshuo Biotechnology Co., Ltd. (Shanghai, China). Dexamethasone Tablets (75 mg/tablet, batch number: 191067) were purchased from Zhangzhongjing Pharmacy (Zhengzhou, China). Ix53 inverted fluorescence microscope and Cx31 upright microscope were purchased from OLYMPUS company, Japan. G: BOX multifunctional gel imaging system was purchased from Syngene, UK. The animal respiratory metabolic measurement system was obtained from Sable Systems International, United States.

To assure that most stomata were open before experiments were initiated, plants were kept under light (100 mEm⁻²s⁻¹) for at least 3 h [69 (link)]. The epidermis was peeled from fully expanded leaves and immediately immersed in MES buffer (25 mM MES-KOH (pH 6.15) and 10 mM KCl), 5 mM flg22 peptide (Flagellin Fragment, Anaspec, Fremont, CA, USA), or 100 ng/μL LPS (lipopolysaccharide, Sigma). Flg22 was dissolved in MES buffer. LPS was dissolved in MES buffer solution containing 0.25 mM MgCl₂ and 0.1 mM CaCl₂ [70 (link),71 (link)]. At 1 and 3 hpi, samples were placed on glass slides and observed under a microscope. The width and length of the stomatal aperture was measured using the Image-Pro (Olympus Corporation, Tokyo, Japan). For the callose experiment, solution (Mgcl₂ (10 mM), flg22 (5 mM) or LPS (100 ng/μL)) was injected into rosette leaves using 1 mL needleless syringes, respectively. Leaves were stained with aniline blue (dissolved in 150 mM k₂HPO₄ (pH 9.5)) at 18 hpi to detect callose deposition.

To detect NO release, evaluated as (NO₂⁻), HUVEC and SC cells (1 × 10⁴) were seeded on 96-well plates and sodium mesoglycan or Prisma^® Skin 0.3 mg/mL were administrated for 24 h in presence or not of LPS (lipopolysaccharide) 10 µg/mL (Sigma Aldrich). N^ω-Nitro-l-Arginine Methyl Ester hydrochloride (L-NAME) 100 µM (Abcam, Cambrige, UK) was used as technical control as potent NOS inhibitor. NO generation was evaluated in the culture medium by Griess reaction. The amount of NO₂⁻, evaluated as µM concentration, in the samples was calculated by a sodium NO₂⁻ standard curve.

Peritoneal macrophages were harvested by 5 peritoneal washes with 1 ml of PBS containing 3 U/ml heparin. The obtained cell suspension was centrifuged at 1200 rpm for 5 minutes. Cells were suspended in the RPMI 1640 culture medium containing 10% FBS and cultured (37°C, 5% CO₂) for 8 hours. Then, the nonadherent cells were removed. The anchorage-dependent cells were detected by flow cytometry or activated and induced by the CM. The grouping of macrophages is detailed in Table 1. Cells were cultured (37°C, 5% CO2) in DMEM plus 10% FBS with reagents mentioned above; LPS (lipopolysaccharide), IL-4, and IL-13 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

ASP (4-Di-1-ASP (4-(4-(Dimethylamino)styryl)-N-Methylpyridinium Iodide)) was obtained from Invitrogen (Carlsbad, CA, USA). LPS (Lipopolysaccharide) and Fluoxetine were purchased from Sigma-Aldrich (St Louis, MO, USA). Stock solutions were prepared in dimethyl sulfoxide (DMSO) and stored at -20°C. ASP and LPS were diluted in fresh medium before each experiment, and the final concentration of DMSO was <0.1%.

The Buffy coats from healthy voluteers were used to isolate human monocytes. Ficoll-Hypaque (Pharmacia Corporation) was utilized to isolate peripheral blood monocytes followed by density gradient centrifugation for 50 min at 400 g. Cells were seeded into 24-well plates at a density of 2 × 10⁶ cells/ml in RPMI 1640 medium plus 10% heat-inactivated human AB serum, 50 U penicillin and streptomycin/ml, 2 mM L-glutamine, and 100 ng/ml human macrophage colony-stimulating factor, which was aimed at stimulating the differentiation of macrophages. Warm medium was used to wash away the cells that did not adhere gently and repeatedly after 6 days of culture. CD14⁺ macrophages constituted over 95% of the adherent cells using this approach. For the activation of M1 macrophages in vitro, 2 × 10⁶ cells/l of the isolated cells (as described above) were treated for 1 day with 25 μg/ml LPS (lipopolysaccharide; Sigma) to the above-isolated cells, while for the activation of M2-polarized macrophages, 45 ng/ml recombinant human interleukin-4 (IL-4, R&D) was used. Flow cytometry was conducted to check the differentiated macrophages from monocytes. Conditioned media for the next round of in vitro experiments was obtained from PBS washing of cells followed by incubation in medium minus supplements for another 24 h.