Il 1β

Supernatants from peritoneal macrophage culture assayed for IL‐6, IL‐1β, and TNF‐α (BOSTER, Wuhan, China) according to the manufacturer's instructions. In the screening assay, peritoneal macrophages were seeded in a 96‐well plate at 2 × 10⁴ per well. The PMs were pre‐treated with compounds at a concentration of 10 µm for 1 h and then stimulated with LPS for another 24 h. Cell supernatants were analyzed by the ELISA assay. For in vivo analysis, after appropriate treatment, BALF of mice and cells supernatants were collected for ELISA analysis. The absorbance of each sample was measured by a microplate reader (BioTek, CA, USA) at 450 nm.

Before feeding the LNCaP cells with the collected CM, TNFα (Invitrogen, Carlsbad, US), interleukin-6 (IL6) and interleukin-1beta (IL1β) (Boster Biological Technology Co., US) levels were examined using an ELISA according to the manufacturer's instructions. Because exposure is a major component in our inflammation model, the time (0, 2, 4, 6, 12 and 24 h) and the dose (62.5, 125, 250 or 500 pg/ml TNFα-containing CM) for the courses of CM treatments were optimized separately. Ultimately, TNFα was chosen as a measure of CM exposure.

Using proteins extracted from plasma and colon tissue, IL-1β, IL-6, IL-10, TNF-α, (BOSTER, Wuhan, China), IL-8, IgA (NEOBIOSCIENCE, Shenzhen, China) levels were measured according to the kit instructions.

CQMUH-011 (purity > 95%) was provided by the Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology (Chongqing, China). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), and penicillin and streptomycin were obtained from GIBCO (Life Technologies, Grand Island, NY, USA). Trypsin-EDTA was purchased from Thermo Scientific (Waltham, MA, USA). TNF-α, IL-1β, and ELISA kits were purchased from Boster (Wuhan, China). LPS (Escherichia coli, 055:B5) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, USA). Antibodies against extracellular signal-regulated kinase (ERK), phospho-ERK, phospho-nuclear factor-kappa (NF-κB) p65 (P-p65), inhibitory kappa B-alpha (IκBα), phospho-IκBα, p38 mitogen-activated protein kinases (MAPK), and phospho-p38 (MAPK) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against proliferating cell nuclear antigen (PCNA), Cyclin D1, Bax, Bcl-2, IBA1, OX-42, Toll-like receptor 4 (TLR4), and myeloid differentiation factor 88 (MyD88) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against β-actin and horseradish peroxide (HRP)–conjugated goat anti-rabbit IgG (H + L) were purchased from Proteintech Biotechnology (Proteintech, Wuhan, China).

Monoclone antibodies IR, PI3K P85, p-NF-κB p65, IKK, BACE-1, APP, α7nAChR and polyclone antibody Aβ were purchased from Abcam (Cambridge, MA, USA). Monoclone antibody p-Akt (Ser473), AKT, NF-κB, p-IKK, p-IRS-1(Ser307), and IRS-1 were purchased from Cell Signaling Technology (Boston, MA, USA). Insulin ELISA kit (EZRMI-13) and PVDF membrane (0.45 µm) were obtained from Millipore (Billerica, MA, USA). The cytokines of IL-1β, IL-18 and TNF-α were purchased from BOSTER (Wuhan, China) and the ACh kits (A105-1: tissue, A105-2: Serum) and the AChE kits (A024) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The ladder marker was obtained from Thermo Scientific (Waltham, MA, USA). Finally, the GLU kit was purchased from Shanghai Mind Bioengineering Co., Ltd. (Shanghai, China). Berberine was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (99% pure, Shanghai, China). All other reagents purchased from located market were of analytical grade.