3 methyladenine 3 ma

d-Gal and GlcN were procured from Sigma-Aldrich (Darmstadt, Germany). 3-Methyladenine (3-MA) was purchased from Selleckchem (Houston, TX, USA). ELISA kits for cytokines IL-1β and IL-6 were procured from Boster (Wuhan, Hubei, China). Malonaldehyde (MDA) and superoxide dismutase (SOD) assay kit was purchased from Jiancheng Bioengineering (Nanjing, Jiangsu, China).The cDNA was synthesized by reverse transcription using a cDNA synthesis kit (Takara, Japan), and was analyzed with SYBR Green Real-time PCR kit (Novizan Biotechnology, Nanjing, Jiangsu, China).The antibodies included LC3, Beclin-1, P62, RUNX2, OCN, BMP2, Bcl-2, BAX and Cleaved Caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA), and the antibodies to Ki-67 and p16 were all obtained from Beyotime (Shanghai, China). The antibody to β-actin and the second antibody conjugated HRP (goat-anti-mouse and goat-anti-rabbit) were purchased from Proteintech (Chicago, IL, USA). The Alkaline phosphatase (ALP) staining kit, Goldner trichrome staining kit and Tartrate-resistant acid phosphatase (TRAP) staining kit were purchased from Solarbio (Beijing, China).

We cultured THP-1 cells (American Type Culture Collection, ATCC, Manassas, VA, USA) in RPMI-1640 complete medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) at 37 °C in a humidified 5% CO₂ and 95% air atmosphere. For further experiments, THP-1 cells were incubated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA, AdipoGen, San Diego, CA, USA) for 24 h to obtain M0 macrophages. For the autophagy state regulation, M0 macrophages were pretreated with RAPA (RAPA, Selleck Chemicals, Houston, TX, USA) or 3-methyladenine (3-MA, Selleck Chemicals, Houston, TX, USA) for 4 h, and polarized into M1 macrophages with 100 ng/mL lipopolysaccharide (LPS, InvivoGen, San Diego, CA, USA) + 20 ng/mL interferon-γ (IFN-γ, Cyagen, Santa Clara, CA, USA) stimulation for 24 h. Human umbilical vein endothelial cells (HUVECs) from the ATCC were cultured in Dulbecco’s modified eagle medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% FBS, 50 ng/mL gentamicin, and 50 ng/mL amphotericin B at 37 °C in an atmosphere of 95% O₂ and 5% CO₂.

Baicalein (≥ 99%, Yousi Scientific Co., Ltd, Shanghai, China) was dissolved in DMSO at concentration of 200 mM and stored at -20 ℃. Mdivi-1, an inhibitor of Drp1, was purchased from Selleck (Huston, TX, USA). 3-Methyladenine (3-MA), an inhibitor of autophagosomes, and Bafilomycin A1 (Baf-A1), an inhibitor of H⁺-ATPase, were purchased from Selleck. Antibodies against PARP (#9542), Drp1 (#5391), AMPKα (#5831), p-AMPKα (Thr172) (#2535), LC3 (#12741), Bak (#6947) and β-actin (#3700) were obtained from Cell Signaling Technology (Boston, MA, USA). Antibodies against Caspase 3 (#19677-1-AP), Caspase 9 (#10380-1-AP), Bcl2 (#12789-1-AP), Bcl-xl (#10783-1-AP), Bax (#50599-1-AP), Cytochrome c (#10993-1-AP), Aif (#17984-1-AP), Cox IV (#11242-1-AP), Fis1 (#10956-1-AP), Opa1 (#27733-1-AP), Mfn1 (#13798-1-AP), Ndufs1 (#12444-1-AP), Sdha (#14865-1-AP), Uqcrc1 (#21705-1-AP), Atp5a1 (#14676-1-AP), p62 (#18420-1-AP), and Beclin1 (#11306-1-AP) were obtained from Proteintech (Wuhan, China). Antibody against p-Drp1 (Ser616) (#12749) was obtained from Signalway Antibody (College Park, MD, USA). Secondary goat anti-rabbit or rabbit anti-mouse antibodies were purchased from Proteintech. Fluorescent-labeled antibody Annexin V-FITC, Annexin V-APC, PI, 7-AAD and 10 × binding buffer were obtained from BD (Franklin Lakes, New Jersey, USA).