Ex527

To investigate the effect of SIRT1 on propagation of N. caninum, caprine EECs were incubated with an activator resveratrol (RSV) (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) or an inhibitor Ex 527 (Beyotime Biotechnology, Shanghai, China) of SIRT1 for 1 h and then infected with N. caninum tachyzoites at a MOI of 3:1 (parasite:cell) for 48 h. The numbers of N. caninum tachyzoites per parasitophorous vacuole were calculated by using inverted optical microscopy (Olympus Co., Tokyo, Japan), and a total of 100 vacuoles were counted. In addition, the cytotoxicities of RSV and Ex 527 were analyzed using a cell counting kit (CCK-8; Zeta life, California, USA), and both 50 μM RSV and 20 μM Ex 527 had no significant cytotoxicities for caprine EECs (Additional file 2: Fig. S1).

LO2 cells were obtained from Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in high-glucose dulbecco’s modified eagle medium (DMEM) (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) medium supplemented with 10% fetal bovine serum (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin antibiotics (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) in a humidity atmosphere of 5% CO₂ at 37 °C. The cells were seeded at an appropriate density on 96-well plates, 24-well plates, 12-well plates, and 6-well plates, and then treated with 0.5 mM palmitic acid (PA; Sigma-Aldrich, St. Louis, MO, USA) for 24 h to mimic a nonalcoholic steatohepatitis model. The liquefied PA was diluted with 5% bovine serum albumin (BSA, Biofroxx, Guangzhou, China) solution gradually and sterilized by ultraviolet light to prepare a PA stock solution required by the study on the basis of a previous study [12 (link)]. Different concentrations of empagliflozin (Empa; 5.5, 11 and 22 μM; BioChemPartner, Shanghai, China), EX527 (10 μM; Beyotime, Shanghai, China), and mitoquinone (mitoQ; 500 nΜ; MedChemExpress, Shanghai, China) were added to investigate the protective effects on PA-induced LO2 cells.

OVA was purchased from Sangon Biotech Co., Ltd. (Shanghai, China; BC Grade). Aluminum hydroxide was purchased from Sigma-Aldrich (St. Louis, MO, United States). Bergenin was purchased from Aladdin Chemistry (Shanghai, China; >98% purity) for cell experiments and Solarbio (Beijing, China; >98% purity) for animal experiments. EX-527, an Ultrasensitive ECL Chemiluminescence Kit, and IL-1β and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Beyotime Biotechnology (Shanghai, China). IL-5 ELISA kit was purchased from NeoBioscience Technology Co, Ltd (Shenzhen, China). Antibodies used in western blotting and immunofluorescence are listed in Table 1.

Cell culture media and additives were obtained from the HyClone Company (Beijing, China). Antibodies for mTOR (Cat. No. 32028), p-mTOR (Cat. No. 109268), p-S6K1 (Cat. No. 131436), and p-4EBP1 (Cat. No. 75767) were purchased from Abcam (Cambridge, MA, USA). PARP-1 (Cat. No. 9542S) and SIRT1 (Cat. No. 9475 T) antibodies were purchased from Cell Signaling Technology (Shanghai, China). pADPr (PAR) (Cat. No. 56198) and AIF (Cat. No. 9416) antibodies were acquired from Santa Cruz Biotechnology (Beijing, China). The β-ACTIN (Cat. No. 21800) and LaminB1 (Cat. No. 40413) antibodies, in addition to secondary antibodies, were obtained from Signalway Technology (St. Louis, MO, USA). Hoechst/PI and EX527 (a SIRT1 inhibitor) were purchased from Beyotime Biotechnology (Shanghai, China). Rapamycin (an mTOR inhibitor) and 3-Aminobenzamide (3AB, a PARP-1 inhibitor) were obtained from Abmole (Beijing, China). All other reagents were acquired from Sigma-Aldrich (Shanghai, China).

The experiment was approved by the Animal Ethics Committee in the Tianjin Nankai Hospital (Approval No. NKYY-DWLL-2019-012, Tianjin, China). Six-week-old (20 ∼ 25 g) male C57BL/6 mice were supplied by Experimental Animal Technical Co. Ltd. (Beijing, China; animal license number: SYXK (Jing)2017-0024) and maintained in a temperature-controlled facility (humidity: 40%–60%, temperature: 22–25 °C) with a 12-h light/dark cycle. The animals were randomly assigned into seven groups (n = 8): control, LPS, LPS + NMN, NMN, LPS + NMN + EX-527, LPS + EX-527, and EX-527. To establish the endotoxin-related ALI model, lipopolysaccharide (15 mg/kg, Sigma, USA) was injected intraperitoneally as reported previously (Shi et al. 2021 (link)). Mice were pretreated intraperitoneally with NMN (500 mg/kg, Sigma, USA) and/or SIRT1 inhibitor EX-527 (5 mg/kg, Beyotime, China) 1 h before LPS injection as previously described (Caton et al. 2011 (link); Xu et al. 2016 (link)). Additionally, an equal volume of sterile saline served as a vehicle control. The mice were euthanized under deep anaesthesia via cervical dislocation after 12 h, and care was taken to reduce animal suffering. Serum was obtained by eyeball extirpation with centrifugation at 3000 rpm for 10 min for further analysis. Lung tissues were fixed in 4% paraformaldehyde or stored at −80 °C for further analyses.