Cleaved caspase 3

Decalcified bone sections were deparaffinized with xylene, and endogenous peroxidase activity was quenched with 3% H₂O₂ for 15 min., followed by antigen retrieval with trypsinization for 15 min. Then, sections were blocked with normal goat serum for 30 min. and incubated at 4°C overnight with primary antibody followed by the biotinylated secondary antibody and horseradish peroxidase‐conjugated streptavidin–biotin staining. Immunoreactivity was visualized with a 3, 3′‐diaminobenzidine tetrahydrochloride kit (ZSGB‐BIO, Beijing, China) followed by counterstaining with methyl green. Primary antibodies against the following proteins were used: collagen II (1:400; Chondrex, Redmond, WA, USA), collagen X (1:200; Abcam, Cambridge, MA, USA), Aggrecan (1:200 Abcam), MMP13 (1:200; PeproTech, Chicago, IL, USA), ADAMTS5 (1:200; Abcam), cleaved caspase‐3 (1:100; Boster, China) and phosphor‐P65 (1:200; CST, Danvers, MA, USA). The number of immunoreactive cells in sections was counted using Image‐Pro Plus 5.1 (Media Cybernetics, Rockville, MD, USA).

SD rats (male, 4–6 weeks old, 200 ± 20 g) were purchased from Zhejiang Chinese Medical University Animal Center (Grade SPF). All rats had free access to food and water and were tested in the SPF barrier laboratory of Zhejiang Chinese Medical University Animal Center. The whole protocol was approved by the Animal Ethics Committee of Zhejiang Chinese Medical University. Every effort was made to minimize the suffering of the animals during the experiment.
The main formula of YQJPR is as follows: Huang Qi, 30 g; Dang Shen, 20 g; Bai Zhu, 12 g; Fu Lin, 15 g; Zhu Li Ban Xia, 12 g; Chen Pi, 9 g; Qian Hu, 12 g; Dan Pi, 12 g; and Gan Cao 3 g. All herbs were provided by the TCM Pharmacy of the first affiliated hospital of Zhejiang Chinese Medical University (Hangzhou, China), and one dose was finally decocted to 125 ml of liquid (1 g/ml).
Hongtashan cigarettes (84-mm flue-cured cigarettes, smoke nicotine content: 0.9 mg, tar content: 10 mg, smoke carbon monoxide content: 11 mg) were provided by Hongta Tobacco Co., Ltd.
Antibody types are as follows: primary antibodies [Phospho-Akt (CST, 9271), Phospho-p44/42 MAPK (p-ERK1/2) (CST, 4370), c-Myc (CST, 5605), cleaved caspase-3 (CST, 9664), Phospho-Stat3 (CST, 9134), and GAPDH (BOSTER, BM1623)], and secondary antibodies [anti-rabbit IgG, HRP-linked antibody (CST, 7074) and anti-mouse IgG, HRP-linked Antibody (CST, 7076)].