Papain

Assay for inhibitory activity against protease was performed as previously described with modification (23 (link)–27 (link)). Trypsin, α-chymotyrpsin, pronase, subtilisin, thrombin were purchased from Sigma-Aldrich (St. Louis, USA), and proteinase K and papain were purchased from Sangon Biotech (Shanghai, China). SPINK7 (100 μg) was preincubated with 10 μg protease in 100 μL Fluoro assay buffer (100 mM Tris-HCl and 20 mM CaCl₂, pH 7.5) for 30 min at 37°C. Then, 100 μL FITC-casein substrate buffer (G-Biosciences, USA) was added and incubated at 37°C in the dark for 1 h. The fluorescence was measured at 485/535 nm (excitation/emission). Protease inhibition by SPINK7 was assessed using the following formula: % residual activity = (residual enzyme activity/enzyme activity without inhibitor) × 100. Three independent replicates were performed for each experiment. Student’s t test was used to evaluate statistical significance. These data were analyzed by GraphPad Prism 5 software.

The 1,9-dimethylmethyleneblue (DMMB) assay was applied to measure the glycosaminoglycan (GAG) content of the cultured NP cells as described in a previous report [26 (link)]. Briefly, the NP cells were collected and digested by 5 mg/ml papain (Sangon, Biotech Co., Ltd., China) for 6 h at 60°C. Then, the GAG content in the digested solution was calculated according to the absorbance value at 525 nm. Additionally, the shark cartilage chondroitin sulfate (Sigma, USA) was used as a standard in the DMMB assay.

To quantify the GAG content synthesized by the NP cells, a DMMB assay was performed according to a previous method25 (link). Briefly, after NP cells (seeded in a 6-well plate, 1 × 10⁴ cells/well) were incubated with different test compounds for 1 and 3 days, NP cells were collected and suspended in PBS containing 5 mg/mL papain (Sangon, Biotech Co., Ltd., China) for 6–8 hours at 60 °C. Then, the GAG content in the digested sample was measured using the 1,9-dimethyl methylene blue (DMMB) assay, in which shark cartilage chondroitin sulfate (Sigma, USA) was used as a standard, and the absorbance at 525 nm was measured.

To evaluate the sensitivity of antibacterial substances to catalase and protease, neutralised CFS from each strain was incubated for 2 h at 37 °C with the following: catalase (3000 U/mg, Sangon, Shanghai, China), pepsin (15,000 U/mg, Sangon, Shanghai, China), trypsin (250 U/mg, Sangon, Shanghai, China) and papain (6000 U/mg, Sangon, Shanghai, China). The enzymes were used at a final concentration of 1 mg/mL in 50 mmol sodium phosphate buffer (pH 7.00), except for pepsin (pH 2.00) [22 (link),23 (link)]. Subsequently, the enzymes were inactivated by boiling at 100 °C for 10 min, after which the pH of each CFS was re-adjusted to 6.00. The antibacterial ability was determined according to the procedure described in Section 2.1.
To evaluate the sensitivity of antibacterial substances to heat, CFS from each strain was incubated under the following conditions: 60 °C for 10 min, 60 °C for 30 min, 60 °C for 1 h, 90 °C for 10 min, 90 °C for 30 min and 121 °C for 15 min. Subsequently, the pH of each CFS was re-adjusted to 6.00. The antibacterial ability was determined according to the procedure described in Section 2.1.

The entire jelly-like NP was isolated from each level discs at 6 months after surgery (n = 6). The amount of proteoglycan content, mainly s-GAG, was quantified using the 1,9-dimethylmethylene blue (DMMB) method [38 (link), 39 (link)]. Briefly, each lyophilized sample was digested with 125 μg/mL papain (Sangon Inc, ShangHai; PRC) in sterile PBS, 5 mM EDTA, and 5 mM cysteine · HCl at pH 6.8 and 60 °C overnight. After complete digestion, 20 μL papain digest were added to 200 μL of DMMB solution, with absorbance detected at 520 nm. Total sGAG in the disc for each group was normalized according to the tested DNA amount, and then the sGAG/DNA ratio was measured and reported.

The DMMB assay was performed to measure GAG synthesized by NP cells [41 (link)]. Briefly, after DBM samples were suspended in 1 mL of PBS containing 5 mg/mL papain (Sangon, Biotech Co., Ltd., Shanghai, China) for 6–8 hours at 60 °C. Then, the GAG content in the digested sample was measured at an absorbance value at 525 nm via the 1,9-dimethyl methylene blue (DMMB) assay, in which shark cartilage chondroitin sulphate (Sigma-Aldrich) was used as a standard.

The brain tissue was cut and digested in HEPES (12,360,038, Gibco™) containing 30 U/ml papain (A501612, Sangon Biotech) and 40 μg/ml DNase I for 60 min as described above. At the end of digestion, 30% bovine serum albumin (A8010, Solarbio) was added to the PBS. The resulting cell suspension was centrifuged at 250 × g for 5 min, and the cells were resuspended in endothelial cell culture medium (CC-3202, Lonza) and plated in culture dishes precoated with 0.02% collagen type I (C8061, Solarbio). After 1 day of culture in a 37 °C cell incubator at 5% CO2, the medium was changed. The endothelial cells were passaged 3 times to purify the endothelial cells, and the cells were used for subsequent cell experiments after the purity of the cells was > 99%, as determined by immunofluorescence staining with CD 31 (1:100, NB100-2284, NOVUS).