Tak 242

The human colon cancer cell lines SW480 and Caco-2 were grown in DMEM high glucose, supplemented with 10% fetal bovine serum (FBS, Bovogen), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Beyotime, china) in a 37°C, 5% CO₂ controlled incubator. A stock solution of the TLR4 antagonist TAK-242 (MedChemexpress, USA) was prepared in DMSO, and then further diluted with DMEM to yield a final concentration of 1 μM. TLR4 was inhibited with 1 μM TAK-242 for 1 hour prior to Fn or LPS stimulation. PAK1 was inhibited by IPA-3 (MedChemexpress, USA) (10 μM final concentration in DMEM) for 1 hour prior to stimulation.

Pomegranate fruits were obtained from Lintong, Shaanxi province of China, and the extraction and purification of PPPs were performed in our laboratory (20 (link), 21 (link)). Detailed methods and results have been presented in the previous article (28 (link)). The polyphenol content of PPPs was 57.09%. The main polyphenol compounds were gallic acid, Punicalagin (Punicalagin-α and Punicalagin-β), catechin, chlorogenic acid, epicatechin, and ellagic acid. The yielding of PC, which is a major component of PPPs, was 464.48 mg/g, and the yielding of EA was 71.50 mg/g. The concentration of the other components of PPPs – catechin, gallic acid, epicatechin, and chlorogenic acid – was 45.14, 38.24, 35.28, and 8.85 mg/g, respectively.
Punicalagin, ellagic acid, and LPS (Escherichia coli 055:B5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture reagents and fetal bovine serum were purchased from Gibco BRL (Rockville, MD, USA). TAK-242 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Bay11-7082, MG-132, ROS, and NO assay kits were purchased from Beyotime Biotechnology (Beijing, China). TLR4, P65, p-IκBα, IκBα, histone 1, and β-actin primary antibodies for Western blot analysis were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA).

TAK242, BMS-345541, BAY 11-7082, and SecinH3 were acquired from MedChemExpress (Shanghai, China). LPS (L2880) from Escherichia coli 055:B5 was obtained from Sigma (St. Louis, USA). Recombinant human ARF6 was purchased from Proteintech (Wuhan, China).

Six-week-old C57BL/6 background ApoE^−/− deficit mice purchased from Vital River (Beijing, China) were employed to establish the T2DM AS model according to previous descriptions with several modifications [13 (link), 14 (link)]. Animals were maintained in independent polypropylene cages in controlled ambient conditions and were free to water and standard chow. Mice were weaned for 2 weeks. Intraperitoneal injections of streptozotocin (STZ, 55 mg/kg bodyweight, Sigma-Aldrich) were administrated to mice for 5 consecutive days. Then, the animals were fed Western diet (21% milk fat and 0.15% total cholesterol, TD.88137, Harlan Teklad) for 16 weeks. Control rats received intraperitoneal injections of vehicle buffer and fed Western diet. An automatic blood glucose analyzer (Roche) was used to determine fasting blood glucose (FBP) in blood sampled from the tail vein 6 weeks after STZ injections. The TLR4 inhibitor was administrated to animals according to previous descriptions with modification [15 (link)]. DM AS animals were pretreated with TAK-242 (MedChemExpress, Shanghai, China) and twice a week for 16 weeks after modeling by intraperitoneal injection (dissolved in DMSO, 3 mg/kg bodyweight) prior to STZ injections. Animal experimental protocols were reviewed and approved by Medical Research Ethics Committee of Shaanxi Provincial People's Hospital.

SEA of S. japonicum and S. japonicum cercariae were obtained from Jiangsu Institute of Parasitic Diseases (China). SEA was sterile-filtered and endotoxin was removed with Polymyxin B agarose beads (Sigma, USA). Limulus amebocyte lysate assay kit (Lonza, Switzerland) was used to confirm the removal of endotoxins from the SEA as previously described29 (link). Primary antibodies for α-SMA, p53, p21, p16, Toll-like receptor (TLR) 4, SOCS3 and STAT3 were purchased from Santa Cruz Biotechnology (USA). Primary antibody for collagen I was purchased from Abcam. Primary antibodies for phospho-p53 (Ser15) and phospho-STAT3 (Tyr-705) were purchased from Cell Signaling Technology (USA). All of the secondary antibodies were obtained from Santa Cruz Biotechnology (USA). TAK-242, a small-molecule-specific inhibitor of TLR4 signaling, was purchased from Medchemexpress.

The aerial parts of Tetrastigma hemsleyanum Diels et Gilg were obtained from Hangzhou China Agrotime Agri-Tech Co., Ltd. SYQP was prepared and characterized in our laboratory as previously reported (Bingqi Zhu et al., 2020 (link)).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (#M2128-100 MG), dimethyl sulfoxide (DMSO) (#V900090), LPS (#l2880-100 mg), dexamethasone (DEX) (#D4902-100 MG) and Pam3CSK4 (P3C) (#tlrl-pms) were purchased from Sigma Chemical Co. (MO, United States). TAK-242 (#HY-11109-10 mg) and C29 (C₁₆H₁₅NO₄) (#HY-100461-5 mg) were purchased from MedChemExpress (MCE) (NJ, United States). Peroxidase-conjugated goat anti-mouse IgG (#115–035–003) and goat anti-rabbit IgG (#111–035–003) were purchased from Jackson ImmunoResearch (PA, United States). RNA-Quick Purification Kit (#RN001) was purchased from ESscience Biotech (Shanghai, China). A BeyoRT™ II First Strand cDNA Synthesis Kit (#D7170M) was purchased from Beyotime Biotechnology (Shanghai, China). PowerUp™ SYBR™ Green Master Mix (#A25742) was purchased from Thermo Fisher Scientific (MA, United States), ROS Assay Kit (2′,7′-dichlorofluorescin diacetate (DCFH-DA) (#S0033S) was purchased from Beyotime Biotechnology (Shanghai, China).