High fat diet (hfd)

Male C57BL/6N mice aged 6 weeks were purchased from Charles River Laboratories and were randomly divided into three groups: (1) a control group (Con) (n = 12); (2) a HFD + L-NAME-treated group (HFD + L-NAME) (n = 12); and (3) an ADRQβ-004 vaccine group (ADRQβ-004) (n = 12). Two mice were maintained in each cage on a 12-hour light/dark cycle from 6 AM to 6 PM and had unrestricted access to food and water. The control group mice received standard diet and water, while the HFD + L-NAME group mice and the ADRQβ-004 vaccine group mice received HFD (D12492; Research Diet, Inc, New Brunswick, USA (60% of kilocalories from fat (lard))) and L-NAME (1 g/L; TCI, Tokyo, Japan) in drinking water after adjusting the pH to 7.4. HFD food and the L-NAME drinking water were supplied for 18 weeks. Mice in the ADRQβ-004 vaccine group were immunized subcutaneously with the ADRQβ-004 vaccine at weeks 0, 2, 4, and 10 at 200 μg per mouse, while mice in the control and HFD + L-NAME groups were treated with 200 μL of PBS per mouse in the same way. The body weight (BW) of all groups was measured every week. All mice were sacrificed at week 18.

MED was isolated from the fermentation broth of D. sp. HLY-1, as previously described [19 (link)]. The identity of MED was confirmed by HRMS and ¹H and ¹³C NMR analysis, and the purity of MED exceeded 95.7 % according to HPLC analysis. MED was dissolved in dimethylsulfoxide (DMSO) and stored at −20 °C. DMSO, MTT, oil red O, and anti-β-actin antibody were obtained from Sigma-Aldrich (Sigma, St. Louis, MO, USA); IL-1β ELISA kit, IL-6 ELISA kit, and TNF-α ELISA kit were obtained from eBioscience (eBioscience, SanDiego, CA, USA); DMEM was purchased from Gibco (Gibco, Grand Island,NY, USA); fetal bovine serum (FBS) was obtained from Hyclone (Thermo Scientific, IL, USA); Ox-LDL was purchased from Unionbiol (Beijing, China); anti-IκB-α antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against p65 and PARP were purchased from Cell Signaling Technology (Danvers, MA, USA); antibody against LOX-1 was obtained from R&D systems (Minneapolis, MN, USA); high-fat diet was produced by Vital River Laboratories (Beijing, China) according to the formula from Harlan (TD.88137).