CY and LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA). CY was dissolved in DMSO at concentrations of 1 mol/l. And it was stored at −20°C. The additional proportion of DMSO in the culture medium was less than 0.05%. LPS was dissolved in PBS at concentrations of 1 mg/ml and was stored at 4°C. The P-ERK, ERK, P-JNK, JNK, P-P38, P38 and GAPDH antibody and secondary antibody were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Collagen II and aggrecan were purchased from Abcam (Cambridge, UK). The IHC secondary antibody was MaxVision TM HRP-Polymer kit from Maixin Bio (Fuzhou, China).

The present study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. NP cells were isolated from the lumbar spines of Sprague Dawley rats (6–8 weeks old, mixed male and female), using standard enzymatic digestion and culture in complete media (high-glucose DMEM with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin) up to passage 2–3 at 37°C in a humidifed atmosphere with 5% CO₂.

A cell counting kit-8 (CCK8) (Dojindo, Kumamoto, Japan) was used to test the viability of NP cells after CY treatment for 1 day according to previously reported methods (24). Approximately 5×10³ NP cells were seeded on each film and transferred to 96-well plates. incubated with various concentrations of CY for 24 h. The conditioned culture medium was removed before CCK8 examination. Subsequently, 100 µl of DMEM and 10 µl of CCK8 solution were added to each well, followed by CCK8 incubation at 37°C for 2.5 h. The optical density (OD) at 450 nm was determined using a microplate reader (BioTek, Winooski, VT, USA). Cell viability was calculated as follows: Cell viability to control (%)=(OD_{drug-treated group}-OD_blank)/(OD_{control group}-OD_blank).

Cell apoptosis was measured by flow cytometry using Annexin V/propidium iodide (PI) double-immunofluorescent staining according to previously reported methods (25). NP cells were cultured with 200 µM CY and/or 1 µg/ml LPS for 24 h. Then the cells were washed with cold PBS and resuspended with 1X Annexin-binding buffer. After that, all cells were stained with Annexin V-FITC and Propidium iodide according to the manufacturer's protocols. The apoptosis rate was measured by flow cytometry (FCM). Apoptotic events were indicated as a combination of fluorescein isothiocyanate (FITC)+/PI- (early apoptotic) and FITC+/PI+ (late apoptotic or dead) events. The final results are expressed as the percentage of early, late and total apoptotis.

NP cells were incubated with different concentrations of CY and 1 µg/ml LPS for 24 h. LPS could induce inflammation and matrix degradation in IVD. Total RNA was isolated by the AxyPrep™ Multisource Total RNA Miniprep kit (Axygen Biosciences, Union City, CA, USA). Then 1 µg RNA was converted into complementary DNA (cDNA) with PrimeScript™ RT reagent kit (Takara Bio, Inc., Otsu, Japan). Quantitative PCR was performed using an ABI 7500 Sequencing Detection System and SYBR® Premix Ex Taq™ (Takara Bio, Inc.). Cycling conditions were as follows: 40 cycles at 95°C for 5 sec and 60°C for 34 sec. The primers were used to amplify target genes are listed in Table I. The primers were designed and selected using blast in pubmed, and GAPDH was used as the internal control.

For signaling pathway protein assay, the cells were treated with various concentrations of CY and/or 1 µg/ml LPS for 24 h. For aggrecan and collagen II protein assay, the cells were treated with various concentrations of CY and/or 1 µg/ml LPS for 5 or 8 days. Cell total proteins were extracted using NE-PER® Nuclear and Cytoplasmic Extraction Reagents according to the manufacturer's instructions. 20 µg protein (each sample) was loaded into gel, and separated by 7.5–12.5% SDS-PAGE, then transferred to 0.22-µm PVDF membranes (Millipore Corp., Billerica, MA, USA). The membranes were blocked with 5% fat-free milk at room temperature for 1 h and subsequently incubated with primary antibodies overnight at 4°C (1:1,000 dilution, Cell Signaling Technology). After three washes in TBST, the membranes were probed with the corresponding secondary antibody for 1 h at room temperature. The membranes were washed again in TBST, and the protein bands were visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Positive immunoreactive bands were densitometrically quantified and normalized to GAPDH.

2×10⁴/ml cells were seeded in 24-well plates, and these NP cells were cultured with diverse CY concentrations and/or 1 µg/ml LPS for 5 or 8 days. Cells were fixed with 4% paraformaldehyde before making cells slides. After fixation, NP cells were treated with 0.1% Triton X-100 for 15 min. Then the cells were blocked with 2% bovine serum albumin (Sigma-Aldrich) for 1 h. Then, cells slides were incubated with the corresponding antibody, including anti-collagen II and anti-aggrecan antibody (1:200 dilution; Abcam) overnight at 4°C. For immunohistochemistry, the secondary antibody was used for 15 min at room temprature. The DAB (Maixin Bio) solution was used as the chromogen. We used an inverted microscope microscopy (Olympus, Tokyo, Japan) to acquire the images. The integral optical density (IOD) of every photo was measured using the Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

The statistical package for the social sciences (SPSS) version 19.0 was used to analyze the data. The significance of differences between experimental groups and controls were assessed using the Student's t-test or one-way analysis of variance (ANOVA) as appropriate. The data are expressed as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

To study the potential cytotoxicity of CY, we measured the cell viability of NP cells. After incubating with different concentrations of CY for 24 h, we used CCK-8 to assay the cell viability of NP cells. As shown in Fig. 1, CY did not inhibit proliferation of NP cells at concentration of 50, 100 and 200 µM.

To verify the cytotoxicity of CY, we examined the apoptosis rate of NP cells after treating with CY for 24 h. CY could decrease the early apoptosis rate at 200 µM with 1 µg/ml LPS (2.47% in 1 µg/ml LPS vs. 1.93% which were treated with CY of 200 µM and 1 µg/ml LPS, P<0.05), and CY could increase the late apoptosis rate at 200 µM with 1 µg/ml LPS (0.58% in 1 µg/ml LPS vs. 1.36% which were treated with CY of 200 µM and 1 µg/ml LPS, P<0.05), but the total apoptosis has no difference between two groups (Fig. 2A). And quantitative data of early, late and total apoptosis rate was consistant with this phenomenon (Fig. 2B-D).

NP cells were incubated with 1 µg/ml LPS in the various concentration of CY (100 or 200 µM) for 5 or 8 days. After that, we detected extracellular matrix content by cell immunohistochemistry. Our results indicated that the expression of collagen II and aggrecan increased over time in NP cells, while LPS strikingly decreased the aggrecan and collagen II amount (Fig. 3A). Immunohistochemistry staining showed CY significantly increased the content of aggrecan and collagen II compared to the LPS groups (Fig. 3A). The quantification of IOD also indicated that the CY group gained more aggrecan and collagen II staining (Fig. 3B and C).

After incubated with LPS and various concentration of CY for 5 or 8 days, we also used western blotting to measure the collagen II and aggrecan protein content. The result was similar with immunohistochemistry staining. As shown in Fig. 4, LPS induced collagen II and aggrecan protein down-expression, and CY significantly attenuated the collagen II and aggrecan loss at day 5 and 8. This effect was more obvious at day 8 (Fig. 4).

NP cells were stimulated with 1 µg/ml LPS and 0, 100 or 200 µM CY, followed by quantitative PCR assay. PCR results showed that LPS significantly up-regulated the gene expression of proinflammatory cytokines TNF-α (Fig. 5A). LPS also down-regulated the gene expression of collagen II and aggrecan in NP cells, which was reversed by CY (Fig. 5B and C). And CY also inhibited multiple matrix-degrading enzymes (MMP-3, ADAMTS-4 and ADAMTS-5) gene overexpression induced by LPS in NP cells (Fig. 5D-F).

We measured the activation of the MAPKs pathway related proteins by western blotting to evaluate the potential mechanisms of effects of CY on LPS induced NP cells. Cells were pretreated with various concentrations of CY for 2 h, then treated with or without 1 µg/ml LPS for 30 min. The results showed that 1 µg/ml LPS significantly activated the MAPKs pathways by promoting JNK, ERK and P38 protein phosphorylation (Fig. 6). And CY could inhibit the phosphorylation of JNK, ERK and P38 in a dose dependent manner (Fig. 6). This result indicated the MAPKs pathway was involved while CY exerted anti-inflammatory effect in NP cells.