Interleukin 1β il 1β

The mice were killed, and theircollagenase I-treated tendon tissue were fixed with 4% paraformaldehyde, and stained with hematoxylin & eosin to evaluate the change of collagen alignment. Based on our previous study described in detail [22 (link)], a modified semiquantitative 4-point scoring method for each factor was used. Tendinopathy severity, based on the sum of the scores, was graded as 0–3 (0, ≤ 2, 3–4, ≥5 points). After scoring, the tendon tissue were subjected to undergo immunohistochemistry and TUNEL assay. The tendon tissue from normal and overiectomy rats (under static and stretch conditions) were immunohistochemically analyzed. The processed tissuewere snap-frozen and embedded in paraffin. The sections were deparaffinized in xylene, dehydrated in alcohol, treated with proteinase K, washed with H₂O₂ in PBS, and stained with antibodies against ER-β (Abcam), collagen type I (Abcam), cleaved caspase 3 (Cell Signaling Technology), interleukin-1β (IL)-1β (Cell Signaling), and matrix metalloproteinase-9 (MMP)-9 (Abcam), in combination with the chromogen 3-amino-9-ethylcarbazole (Zymed). The signal intensity was further quantitated using Image J 1.42q (National Institutes of Health) in three randomly chosen fields.

Total protein was extracted from tissues, resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and transferred to a polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Danvers, MA, USA). After blocking, the membranes were incubated overnight with antibodies against α-tubulin (GeneTex, Irvine, CA, USA), type I collagen (COLA-1; ABclonal, Woburn, MA, USA), type III collagen (COLA-3; ABclonal), fibronectin (GeneTex), NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3; Proteintech, Rosemont, IL, USA), caspase-1 (Proteintech), interleukin-1β (IL-1β; Cell Signaling Technology, Danvers, MA, USA), interleukin-18 (IL-18; Proteintech), and gasdermin D (GSDMD; Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (EMD Millipore) for 1 h. Protein bands were visualized with enhanced chemiluminescence (ECL) reagents (EMD Millipore) and quantified using densitometry with Image J software (v1.46). The band intensities of the proteins of interest were normalized by that of α-tubulin or the uncleaved band.