Il 1β elisa kit

The culture media were collected from the transfected rASTROs and centrifuged at 4000 rpm for 10 min at 4 °C. The levels of cytokines, neurotrophic factors, and dopamine in the supernatants were then determined using ELISA. The following commercially available ELISA kits were employed: Rat brain-derived neurotrophic factor (BDNF) ELISA Kit PicoKine (EK0308, Boster Biological Technology, Pleasanton, CA, USA), Rat glial cell line-derived neurotrophic factor (GDNF) ELISA Kit PicoKine (EK0363, Boster Biological Technology), Rat Nerve Growth Factor (NGF) ELISA Kit (MBS261790. MyBioSource, Inc., San Diego, CA, USA), Rat tumor necrosis factor-alpha (TNFα) DuoSet ELISA (DY5100-05; R&D System, Minneapolis, MN, USA), Rat Interleukin 1β (IL-1β) ELISA Kit (CSB-E08055r; CUSABIO, Houston, TX, USA), and Dopamine ELISA kit (KA3838, Abnova, Taipei City, Taiwan). All analyses were conducted according to the manufacturers’ instructions. The concentration of each protein was estimated using the standards provided in the kit.

Jatrorrhizine hydrochloride (Purity ≥ 98%) was purchased from Nanjing Zelang Medical Technology Co. Ltd (Nanjing, China) (Figure 1A). MTX was obtained from Shanghai Sine Pharmaceutical Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Gibco BRL, Grand Island, NY, USA). Recombinant tumor necrosis factor alpha (TNFα) was purchased from Peprotech (PeproTech, Inc., Rocky Hill, NJ, USA). MTS reagents were obtained from Sigma-Aldrich (St. Louis, MI, USA). Trizol reagent was obtained from Invitrogen (Carlsbad, CA, USA). Primary antibodies targeting phosphorylated (p) ERK, p-p38, p-JNK, ERK, p38, JNK, IκBα, and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, CA, USA). Anti-p65, anti-MMP2, and anti-MMP3 antibodies were purchased from CST (Cell Signaling Technology Inc., Beverly, MA, USA). Commercial kits for ALT and AST measurement were from Jiancheng Institute of Biotechnology (Nanjing, China). TNFα and IL-1β ELISA kits were purchased from CUSABIO BIOTECH CO., Ltd (Wuhan, China). Lyophilized native chicken type II collagen (CII) was purchased from Sigma Chemical Company (St. Louis, MO, USA).

E. ulmoides polysaccharide (EUP, content: 60%, batch number TR20180607, extracted from E. ulmoides leaves) was obtained from Xi'an Tianrui Bio-Tech Co., Ltd. (Xi'an, China). The lipid peroxidation malondialdehyde (MDA) assay kit, dihydroethidium (DHE), and CCK-8 reagent were acquired from Beyotime (Shanghai, China), and the superoxide dismutase (SOD) assay kit was acquired from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). Primary antibodies against Toll-like receptor 4 (TLR4), high-mobility group protein B1 (HMGB1), myeloid differentiation factor 88 (MyD88), NF-κB p65, IKB-α, interferon regulatory factor 1 (IRF-1), β-tubulin, β-actin, and horseradish peroxidase- (HRP-) conjugated secondary antibody were purchased from Proteintech (Wuhan, China). Primary antibodies against tumor necrosis factor-receptor-associated factor 6 (TRAF6), P-p65, and P-IKB-α were purchased from Affinity Biosciences (Cincinnati, OH, USA). HMGB1, TNF-α, and IL-1β ELISA kits were obtained from CUSABIO (Wuhan, China). Fetal bovine serum (FBS), RPMI-1640 medium, penicillin, and streptomycin were purchased from Gibco (Rockville, MD, USA). TLR-4 overexpression plasmid, empty plasmid, and polybrene were purchased from Hanbio Biotechnology Co., Ltd. (Shanghai, China).

The serum was collected and centrifuged at 3,000 rpm for 5 min. The levels of creatine kinase (CK) and CK-MB in serum were determined by Beckman LX-20 Fully Automated Biochemistry Analyzer (Beckman, California, United States) according to the manufacturer’s instructions. IL-1β ELISA kits (Cusabio Biotech Co., Ltd., Hubei, China; Item No. 011614MO) and IL-18 ELISA kits (Cusabio Biotech Co., Ltd., Hubei, China; Item No. E04609m) were used to determine IL-1β and IL-18 levels in serum and cell supernatants following manufacturer’s instructions. SIRT1 activity assay kit (Abcam, Cambridge, MA, United States; Item No. 156065) and caspase-1 activity assay kit (Beyotime, Nan Jing, China, Item No. C1101) were used to determine SIRT1 and caspase-1 activity in myocardial tissue or cell extracts.