Tgf β1

To investigate the effects of Mg²⁺-doped CaSO₄/β-TCP on the secretion of cytokines and chemokines in macrophage, Mg²⁺-doped CaSO₄/β-TCP scaffolds were inoculated with THP-1 cells, and after 3 days, the supernatant was collected. The levels of the cytokines (Interleukin-1β (IL-1β) and Interleukin-10 (IL-10) and chemokines (Transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein (BMP-2) in the supernatant were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (BMP-2 [Elab science, E-EL-H0011c]; IL-1β [ml058059]; IL-10 [ml064299]; TGF-β1 [Elabscience, E-EL-0162c]; AKP [Nanjing Jiancheng Institute of Biological Engineering, A059-2]) according to the manufacturer’s instructions. The absorbance was detected at 450 nm using a microplate reader (SPECTCA MAX190).

An ELISA was performed on the serum samples to measure the levels of TGFβ1(Elabscience), TGFβ2(Elabscience) and TGFβ3(Elabscience) according to the manufacturer's instructions. Human serum samples were derived from autopsy cases. All procedures concerning human samples conformed to the principles outlined in the Declaration of Helsinki and were approved by Dalian Medical University. Material care was reviewed and approved by the Institutional Ethics Committee.

NT-PCP-III (Elabscience Biotechnology, China), TGF-β1 (Elabscience Biotechnology, China), SOD activity (COIBO Biotechnology, China), GSH (COIBO Biotechnology, China), GSSG (COIBO Biotechnology, China) and MDA (COIBO Biotechnology, China) levels in BALF were determined by double antibody-coated tube radioimmunoassay method, according to the instructions of the kit. Briefly, BALF was added to specific antibody-coated microtiter plate wells. Then horseradish peroxidase-labeled detection antibody was added and incubated to form antibody–antigen–enzyme–antibody complex. After washing, 3,3′,5,5′-tetramethylbenzidine solution was added to react with horseradish peroxidase for color development, and the optical density was measured at a wavelength of 450 nm with spectrophotometer. Finally, the concentration of biomarkers in the BALF was calculated by standard curve. The assay was repeated three times for each sample and the average of these three independent experiments was used as the final measured concentration.

In addition to IHC, enzyme linked immunosorbent assay (ELISA) kits were used following the manufacturer’s protocol to measure cardiac Tnfα, Il1β, cTn-I (Abcam; Cambridge, MA), and transforming growth factor beta1 or Tgfβ1 (Elabscience; Houston, TX).

The DSS (Cat no.160110) was purchased from the MP Biomedicals (Aurora, USA). The HSPA5 inhibitor HA15 (batch no. 1609402-14-3) was purchased from the MedChemExpress (New Jersey, USA). Dexamethasone sodium phosphate injection was obtained from the Western Pharmacy of Wuhan Union Hospital. ELISA kits, IL-10 (batch no. JYM0005Mo) and IL-6 (batch no. JYM0012Mo), were purchased from the ColorfulGene Biological Technology Company, Wuhan; TGF-β1 (batch no. E-EL-0162c) was purchased from the Elabscience (Wuhan, Hubei province). The anti-HSPA5 antibody (batch no. 11587-1-AP) was purchased from the Proteintech Group (Chicago, IL). The anti-HSPA1A antibody (batch no. PA534772) was purchased from the Thermo Company (Wyman Street, Waltham, MA), and the anti-CHIP antibody (batch no. 2080S) was purchased from the CST Company (Danvers, MA). FVS 510 (batch no. 564406), PE-Cy7-anti-CD4 (batch no. 552775), APC-Cy7-anti-CD45 (batch no. 557659), and PE-anti-FOXP3 (batch no. 563101) were purchased from the BD Biosciences (San Jose, CA).