Tnf α

PHI was prepared by our laboratory (purity > 98.0%). DSS (molecular weight of 36,000–50,000 Da) was obtained from MP Biochemicals (Santa Ana, CA, USA). The enzyme-linked immunosorbent assay (ELISA) kits (such as TNF-α, IL-1β, IL-6, and IL-10) and TUNEL assay kit were purchased from Boster Biological Technology (Wuhan, China). The activity assay kits of MPO, SOD, and MDA were purchased from Jiancheng Bioengineering Institute (Nanjing, China). The NO assay kit purchased from Beyotime Biotechnology (Shanghai, China). The antibodies such as p-Src (ab185617), p-p38 (ab195049), JNK (ab179461), p-JNK (ab124956), ERK1/2 (ab184699), p-ERK1/2 (ab201015), p65 (ab32536), p-p65 (ab76302), IκBα (ab76429), p-IκBα (ab133462), occludin (ab216327), E-cadherin (ab231303), and ZO-1 (ab190085) were purchased from Abcam (Cambridge, UK). The other antibodies of iNOS (A3200), TLR4 (A5258), and Src (A0324) were purchased from ABclonal Technology (Wuhan, China). The antibody p38 (D155224) and the MightyScript Plus First Strand cDNA Synthesis Master Mix were obtained from Sangon Biotechnology (Wuhan, China). SYBR green mix was purchased from Mei5 Biotechnology (Beijing, China). Triol reagent was obtained from Takara (Shiga, Japan).

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.

Serum was collected and cytokine levels were measured using an ELISA kit to determine the expression of alanine aminotransferase (ALT) (Servicebio), aspartate aminotransferase (AST) (Servicebio), PGE2 (Mlbio, Shanghai, China), TNF-α (Boster Biological Technology, Pleasanton, CA, USA), and IL-1β (Mlbio) according to the manufacturer’s instructions. After incubation, absorbance was finally read at 450 nm on a SpectraMax Absorbance Reader (Molecular Devices, Sunnyvale, CA, USA).

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).

Using commercially available cytokine ELISA kits, the protein concentrations of pro inflammatory cytokines, IL6 and TNF-α (BosterBio, USA), were determined in spleen and lung tissue samples of both infected and uninfected control mice euthanized at each time points. The assays were carried out following manufacturer’s instructions. Briefly, 100 μl of lyophilized recombinant Mouse cytokine standard and test samples (including infected and control groups) were added to the duplicate wells pre-coated with anti-mouse TNF-α or IL-6 antibodies, followed by 100 μl of biotinylated anti-Mouse IL6/TNF-α antibody and incubated for 2 h. Following three washes, 100 μl of Avidin-Biotin-Peroxidase Complex was added to each well and incubated for 30 min. Following color development with 90 μl of TMB substrate, the reaction was stopped and absorbance was measured at 450 nm. From the standard curves derived individually for TNF-α or IL-6 by plotting absorbance against the known concentrations of the recombinant cytokines, the level of these cytokines in tissue samples were estimated and the significant difference was calculated using Tukey’s HSD in JMP 12.0.

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.