Cysteine hydrochloride

After disc organ culture, the central NP tissue was isolated as described above to measure the glycosaminoglycan (GAG) and hydroxyproline (HYP) contents. Briefly, one part of NP samples was lyophilized for 24 h, and digested at 60°C for 24 h in 1 ml water containing 5 mg/ml papain, 0.2 mol/l NaCl, 0.01 mol/l cysteine hydrochloride, 0.1 mol/l CH₃COONa, and 0.05 mol/l Na₂-EDTA (Sangon, Biotech Co., Ltd., China). Then, the GAG content was calculated using a dimethyl methylene blue (DMMB) assay [21 (link)]. Another part of NP samples was weighed to determine their wet weights, and then the HYP content was determined using an HYP quantitation kit (Nanjing Jiancheng, China) according to the manufacturer’s instructions.

Material used in this experiment were lipoic acid (Huayu Pharmaceutical Co., Ltd., Wuxi China); cysteine hydrochloride, sodium cholate, methanol and docetaxel (Sangon Biotech, Shanghai, China); PLGA (50:50, Mw35000, Lack Siomaterials, US); chlorpromazine hydrochloride and amiloride hydrochloride (Sigma-Aldrich, (St. Louis, MO, US); 4′,6-diamidino-2-phenylindole (DAPI) and Filipin III (Cayman Chemical, Michigan, US); 1,1-Dioctadecyl −3,3,30,30 – tetramethyl indotricarbocyanineiodide (DiR) (Biotium, CA, US); Apoptosis Assay Kit and Cell Cycle and Annexin V-FIT Kit (Becton Dickinson and Company, US); zeocin and puromycin (Invitrogen, Carlsbad, CA, US); RPMI-1640, foetalbovine serum (FBS), PBS and trypsin (Life Technologies, Grand Island, US).
Female nude mice aged 5 weeks and weighing about 16 g were purchased from the Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). All animals were raised at the Second Military Medical University (Shanghai, China).
All animal experiments were performed in accordance with the protocols evaluated and approved by the ethics committee of the Second Military Medical University.