Tnf α

The following ELISA kits were used to analyze rat serum samples, according to the manufacturers' protocols: Interleukin (IL)-1β (SEA563Ra; Uscn Life Sciences, Inc., Wuhan, China), IL-8 (H008; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), tumor necrosis factor α (TNF-α; SEA133Ra; Uscn Life Sciences, Inc.), IL-10 (SEA056Ra; Uscn Life Sciences, Inc.), malondialdehyde (MDA; A003-2; Nanjing Jiancheng Bioengineering Institute), superoxide dismutase (SOD; A001-3; Nanjing Jiancheng Bioengineering Institute) and nitric oxide (NO; A013-2; Nanjing Jiancheng Bioengineering Institute). Blank wells, standard wells and detected wells were used. Blank wells contained 100 µl PBS; the other wells contained 100 µl standard preparation (provided with the kits) or rat serum samples. All were incubated at 37°C for 2 h. Following removal of the liquid, the plate was washed three times with the reagent provided by the kits. HRP-conjugated fluid (100 µl) was added to each well at 37°C for 1 h. Following removal of the liquid, sections were dried, and 100 µl substrate solution was added to each well and incubated at 37°C in the dark for ~15 min. Stop buffer (50 µl) was subsequently added to each well. Optical density values were measured in each well at 450 nm using a microplate reader.

The samples were washed with PBS, fixed with 4 % paraformaldehyde for 30 min, according to experimental requirements, were incubated with anti-Pdpn (proteintech, 1:200), anti-Grem1 (CST, 1:50), anti-Cd200 (proteintech, 1:200), anti-Ctsk (Abcam, 1:200), anti-Ptgs2 (proteintech, 1:200), anti-vinculin (proteintech, 1:200) or anti-YAP (CST, 1:200) at 4 °C for 8–12 h. After that, samples incubated with goat anti-rabbit IgG (Servicebio, Alexa Fluor 488, 1:300), goat anti-rabbit IgG (Servicebio, Cy3, 1:300) or goat anti-mouse IgG (Servicebio, Alexa Fluor 488, 1:200) for 1 h at room temperature. Nuclear staining was conducted with DAPI (Sigma), and F-actin stained by rhodamine phalloidin (Beyotime). Tissue samples were fixed in 4 % paraformaldehyde. After decalcification, all samples were embedded in paraffin and sectioned at 6-μm slices. The tissue sections were immunofluorescently stained with primary antibodies targeting Sry (Santa Cruz, 1:200), CD11b (Servicebio, 1:500), TNF-α (Uscn Life Science Inc., 1:1000), IL-1β (Servicebio, 1:1200) and IL-10 (Servicebio, 1:1000), and the goat anti-rabbit IgG (Servicebio, Alexa Fluor 488, 1:400) and goat anti-rabbit IgG (Servicebio, Cy3, 1:500). DAPI was used to stain the nuclei. The images were observed by a fluorescence microscope (Olympus CKX53) or Nikon confocal microscope (Nikon).

Commercial ELISA kits used for measuring serum levels of IL-6 (cat no SEA079Hu), IL-8 (cat no SEA080Hu), TNF-α (cat no SEA133Hu), and PICP (cat no SEA570Hu) were purchased from Uscn Life Science lnc (Wuhan, China). The commercial competitive radioimmunoassay kits were used to measure serum levels of ICTP (cat no 06099; UniQ, Orion Diagnostica, Espoo, Finland). These procedures were performed according to the manufacturer’s instructions.

BALF was collected and centrifuged at 600 × g for 5 min at 25°C, for quantification of total protein content in the BALF supernatant standard BALF collection was performed, as described previously (25 (link)). The protein concentration was measured using a BCA Protein Assay kit (Beyotime Institute of Biotechnology) following the manufacturer's instructions. BALF cytokine levels were examined using IL-10 (cat. no. SEA056Ra, Wuhan USCN Business Co., Ltd.) and TNF-α (cat. no. E-EL-R0019c, Elabscience Biotechnology, Inc.) ELISA kits.

ELISA kits were used to detect the inflammatory factors interleukin (IL)-1β (cat. no. CSB-E08055r; CUSABIO Technology), IL-6 (cat. no. SEA079Ra; Wuhan USCN Business Co., Ltd.), TNF-α (cat. no. SEA133Si; Wuhan USCN Business Co., Ltd.) and IL-10 (cat. no. SEA056Ra; Wuhan USCN Business Co., Ltd.) in the serum from the rats. In addition, the oxidative stress indicators superoxidase dismutase (SOD; cat. no. SES134Hu), malondialdehyde (MDA; cat. no. CEA597Ge), nitric oxide (NO; cat. no. IS100) and the intestinal injury markers D-lactic acid in serum (cat. no. CEV643Ge), diamine oxidase (DAO; cat. no. SEJ298Hu) and intestinal fatty acid-binding protein (I-FABP; cat. no. SEA559Ra) were detected in the serum using kits from Wuhan USCN Business Co., Ltd. The tests were performed according to the manufacturer's instructions.

Protein expression changes of markers of myocardial injury [lactase dehydrogenase (LDH; cat. no. SEB864Ra; Uscn Life Sciences, Inc., Wuhan, China), troponin I (cTnI; cat. no. SEA478Ra; Uscn Life Sciences, Inc.), creatine kinase (CK; cat. no. SEA109Ra; Uscn Life Sciences, Inc.) and heart type fatty acid binding protein (hFABP; cat. no. SEB243Ra; Uscn Life Sciences, Inc.)], inflammatory cytokines [interleukin (IL) 1, 6 (cat. nos. SEA563Ra and SEA079Ra, respectfully; Uscn Life Sciences, Inc.) and tumor necrosis factor (TNF)-α (cat. no. SEA133Ra; Uscn Life Sciences, Inc.)], and oxidative stress factors [malondialdehyde (MDA; cat. no. CEA597Ge; Uscn Life Sciences, Inc.) and sorbitol dehydrogenase (SDH; cat. no. SEB495Ra; Uscn Life Sciences, Inc.)] were detected by ELISA kits. The optical density (OD) at 450 nm was measured with microplate reader.