No kit

After one week adaptive training, systolic blood pressure (SBP) was measured via tail-cuff method weekly during waking hours (BP-2010A, Softron Beijing Biotechnology Co., Ltd, Beijing, China). SBP was measured three times under rest state, and then average values were obtained. Blood samples were collected 8 weeks later. Within 4 hours, whole blood viscosity was detected. Vascular contractility was preliminarily measured by relevant vasoactive substances in serum. ABC-ELISA kits (Westang Biotechnology Co., Ltd, Shanghai, China) were used to detect angiotensin II (Ang II) and epinephrine (EPI) levels in serum. In addition, vascular relaxant factor, nitric oxide (NO) in serum, was measured by NO kit (Beyotime Biotechnology, China) with Griess reagent. All of the experimental procedures were followed in accordance with the rules designated by the manufacturer.

Specific optical rotation was measured on a PerkinElmer 341 instrument at 25°C. Cary 5000 spectrophotometer was used to record UV spectra in MeOH. ECD data were obtained by Model 420SF CD spectrometer (Aviv Biomedical Inc). In KBr discs, IR spectra were obtained using Fourier infrared IS50 spectrometer. All NMR experiments were performed at room temperature on a Bruker AVANCE 500 spectrometer using the signals of residual solvent protons (CDCl₃: δ_H 7.26; CD₃OD: δ_H 3.31) and carbons (CDCl₃: δ_C 77.1; CD₃OD: δ_C 49.2). HRESIMS spectra were tested by Waters TQ-XS mass spectrometer. Column chromatography (CC) was conducted by silica gel (200–300 mesh, Yantai Huiyou Silica gel company) and Sephadex LH-20 (CHCl₂/MeOH, v/v 1:1) (Pharmacia Sweden). On silica gel plates, thin layer chromatography (TLC) was conducted (GF 254 Silica gel Thin Layer Plate Yantai Huiyou Silica company). The Typical Culture Preservation Committee Cell Bank, China provided RAW264.7 cells; the fetal bovine serum (FBS) was obtained by Gibco; ProCell provided Dulbecco's modified Eagle's medium (DMEM); Sigma supplied LPS and _L-NMMA; Shanghai Beyotime Biotechnology supplied the NO kit. Thermo Fisher Scientific (Shanghai, China) provided the primers for iNOS. Cell Signaling (Beverly, MA, USA) supplied all the antibodies.

Free total rhubarb anthraquinones were purchased from the Research Institute (Huaian, Jiangsu, China, batch number: 20130211) and dissolved in normal saline (NS). Sodium taurocholate (STC) was purchased from Sigma–Aldrich Co. (St. Louis, MO, United States). Rat TNF-α and IL-1β ELISA Kits were purchased from United States Systems R&D (Long Beach, CA, United States). NO Kit was purchased from Beyotime Biotechnology Research Institute (Shanghai, China). MPO kit was purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, Jiangsu, China). Fixative solution, permeabilization wash buffer, Foxp3 fixative permeabilization wash solution, monensin, APC anti-rats CD4, FITC anti-rats IFN-γ, PE anti-rat IL-4, PE anti-rat CD25, Alexa fluor 488 anti-rat Foxp3 and goat anti-rat SIgA were purchased from Biolegend Company (San Diego, CA, United States). Rabbit anti-rat NLRP3, goat anti-rat ASC, mouse anti-rat caspase-1 were purchased from Santa Company (Santa Cruz, CA, United States). Triton, bovine serum albumin (BSA) DAB color liquid, goat super sensitive two-step reagent Kit, mouse one-step kit, TRITC labeled rabbit ant- goat secondary antibodies, fluorescein (FITC) labeled sheep anti-rabbit secondary antibodies were purchased from ZSGB Biological Company (Beijing, China).

Nitric oxide (NO) levels were measured according to the Griess method by detecting NO₃⁻ and NO₂⁻ with a NO Kit (S0021, Beyotime, China). Standard samples were diluted into different concentrations (0, 1, 2, 5, 10, 20, 40, 60, and 100 μM) to plot the standard curve. After the above treatment, 50 μL of the culture supernatant fluid was collected and 50 μL (per well) Griess Reagent I and 50 μL (per well) Griess Reagent II were added. Then, NO production was measured using OD value readings with a microplate spectrophotometer (Tecan, Crailsheim, Germany) at an absorbance wavelength of 540 nm. The concentrations were calculated from the standard curve.

The intracellular NO content was detected using a NO kit (Beyotime). Fluorescent probes for Dihydroethidium (DHE, 10 µM), mitochondrial membrane potential [a probe of mitochondrial membrane potential (JC-1), 1x], ROS [2ʹ,7ʹ-Dichlorodihydrofluorescein diacetate (DCFH-DA), 10 µM], and a probe to enable mitochondria visualization (Mitotracker Red CMXRos, 200 nM) were used to detect the corresponding intracellular indicator. All probes were incubated for 20 min at 37 °C. RAW264.7 cells were washed with PBS buffer twice and visualised under a fluorescence microscope. Image processing was performed using ImageJ analysis software. Analyses were performed using GraphPad Prism 6.0 software.