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After treatment, primary astrocytes were lysed in RIPA lysis buffer (150 mmol/L NaCl, 25 mmol/L Tris–HCl, pH 7.6, 1% sodium deoxycholate, and 1% NP-40) with a phosphatase inhibitor and a protease inhibitor (Roche, Switzerland). Proteins were kept on ice and mixed with loading buffer. About 20–40 μg cell lysate was isolated by SDS-PAGE (8% or 12.5% gel) and transferred to a PVDF membrane (Millipore). PVDF membrane was blocked with 5% milk powder or 5% BSA in TBS containing 0.1% Tween-20 and incubated for 1 h at room temperature. Blots were incubated with the primary antibody at 4°C overnight. The primary antibodies used were: LRRK2 (ab133474, 1:8000, Abcam, Cambridge, UK), NLRP3 (AG-20B-0014-C100, 1:2000, AdipoGen, San Diego, CA, USA), P-IKK (2697S, 1:500, Cell Signaling Technology, Danvers, MA, USA), P-P65 (AB19870, 1:1000, Abcam, Cambridge, MA, USA), and IKB (4812S, 1:1000, Cell Signaling Technology) and incubated with the corresponding secondary antibody for 2 h. The proteins were visualized using ECL detection kits (Vazyme, China) with chemiluminescence (6000EXP, TOUCH, China).

Rabbit polyclonal antibody specific for phosphate-p85, a heterodimer of phosphatidylinositol 3 kinase (PI3K), p-Akt, p-IKK were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibodies specific for PI3K, Akt, IKK, NF-κB, β-actin, and mouse polyclonal antibodies specific for OSM were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The recombinant human adiponectin was purchased from PeproTech (Rocky Hill, NJ, USA). PI3K inhibitors (Wortmannin and Ly294002), Akt inhibitor, and NF-κB inhibitors pyrrolidine dithiocarbamate (PDTC) and L-1-tosylamido-2-phenylenylethyl chloromethyl ketone (TPCK) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Small-interfering RNAs (siRNAs) against p85, Akt, and p65 were purchased from Dharmacon Research (Lafayette, CO, USA). OSM ELISA kit was purchased from R&D Systems (Minneapolis, MN, USA). DMEM, fetal bovine serum (FBS), and all the other cell culture reagents were purchased from Gibco life technologies (Grand Island, NY, USA).

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.

Equal amounts of protein were separated by SDS-PAGE on 10% gel. Proteins were transferred onto a polyvinylidene difluoride membrane. The membrane was incubated overnight with primary antibodies against p-IKK, IKK, p-p38, p38, p-ERK1/2, ERK1/2, p-JNK, JNK, p-Smad3, Smad3 (all from Cell Signaling Technology) and, fibronectin (Santa Cruz Biotechnology), incubated for 1 h with HRP-conjugated secondary antibodies (PIERCE). Protein bands were detected using the enhanced chemiluminescent plus system. Bands intensities were analyzed by NIH Image J software.

Hispolon was acquired from BJYM Pharmaceutical. & Chemical Co., Ltd. (Beijing, China). The purity of hispolon was higher than 95% (Figure 1A). LPS, dexamethasone (DEX) and other chemicals, solvents, and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits for the determination of cytokine secretion were acquired from BioLegend Inc. (San Diego, CA, USA). Anti-PI3k and Anti-p-AKT were obtained from Merck Millipore (Merck KGaA, Darmstadt, Germany). The antibodies against TLR4, AKT, p-JNK, ATF6, p-CaMKK2, p-LKB1, catalase, GPx, SOD, Keap1, COX-2, caspase 12, IRE1, GRP78, PERK, CHOP, Beclin 1 and LC3 I/II were obtained from GeneTex (Irvine, CA, USA). Antibodies against IKK, p-IKK, JNK, p-ERK, ERK, p-p38, mTOR and p-mTOR were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-iNOS, anti-HO-1, anti-Nrf-2, anti-PPARγ, anti-IκBα, anti-NF-κB, anti-p38, and anti-β-actin were purchased from Abcam (Cambridge, UK). Determination of protein concentration using a Bio-Rad protein assay kit (Bio-Rad Laboratories Ltd., Watford, UK).