Protection by collagen peptides from walleye pollock skin on bone formation via inhibition of oxidative stress

2.1 Cell culture and treatment

Human osteoblastic cell line Saos-2 was purchased from the Chinese Academy of Sciences. The cells were cultured in α-MEM with 10% fetal bovine serum, 100 U/ml of penicillin and 100 μg/ml of streptomycin in an incubator at 37 °C with 5% CO₂.

Saos-2 cells were pretreated with or without CP (Qingdao Fusheng Food Co., Ltd., China) for 48 h before exposing to 300 μM H₂O₂ (Sigma-Aldrich) for 6 h·H₂O₂ was used as exogenous generator of ROS whereas N-acetyl-l-cysteine (NAC, 1 mM) was the positive control in the study.

2.2 Assays of cell viability

The MTT assay was used to determine cell viability. Briefly, the cells were incubated with 100 μl of medium supplemented with 10 μl of MTT solution (5 mg/ml, Sigma-Aldrich) per well at 37 °C for 4 h after washing the cells twice with phosphate buffered saline. Next, DMSO was added and the plates were shaken for 10 min to dissolve the formazan products. The absorbance of each well was determined by a microplate reader at 570 nm. The cell viability of the control group was defined as 100%.

2.3 Assays of malondialdehyde (MDA), superoxide dismutase (SOD) and alkaline phosphatase (ALP)

MDA, SOD and ALP levels were measured by detection kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. Total protein concentrations were determined using BCA protein assay (Beyotime Biotechnology, China). The absorbance of each sample was normalized against the protein concentration.

2.4 Intracellular reactive oxygen species (ROS) measurement

The intracellular ROS expression level was determined using the ROS assay kit (AAT Bioquest Inc., USA). Briefly, 100 μl of Amplite^TM ROS Green working solution was added and the cells were incubated for 1 h at 37 °C with 5% CO₂. All treatment groups except for the normal control group were treated with or without CP (800 μg/ml) and H₂O₂. The fluorescence was detected using a multi-detection microplate reader at 490 nm excitation and 525 nm emission.

2.5 Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using TRIzol reagent (Invitrogen). The single-stranded cDNA was synthesized using the Fast Quant RT kit (TIANGEN Biotechnology, China). The qRT-PCR was performed using Superreal PreMix Plus (SYBR Green) with the 7300 Real-time PCR System (Applied Biosystems). The expression of target genes FBXW7, p100 and NF-κB were normalized to the reference gene GAPDH. The 2^−ΔΔCt method was applied to calculate the relative gene expression. The primers used in the study were as follows: FBXW7, 5′-CCGTGTTTGGGATGTG GAGA-3′ (sense) and 3′-CCCCCACTCTCCAATGTGAC-5′ (antisense); p100, 5′-CTACTCGACTACGGCGTCAC-3′ (sense) and 3′-GAGTCTCCATGCCGATCC AG-5′ (antisense); NF-κB, 5′-CCAGACCAACAACAACCCCT-3′ (sense) and 3′-GCCTGGTCCCGTGAAATACA-5′ (antisense); GAPDH, 5′-GAGAAGGCTGG GGCTCAT TT-3′ (sense) and 3′-AGTGTGGC ATGGACTGTGG-5′ (antisense).

2.6 Western blotting

The western blotting procedure was performed as previously described with slight modifications (Liu et al., 2016). Briefly, the proteins were separated by SDS-PAGE electrophoresis, transferred onto polyvinylidene fluoride membranes and incubated with the antibodies: anti-FBXW7 (Abcam), anti-p100 (Thermo), anti-NF-κB (Abcam), and anti-β-actin (Abcam). The protein bands were visualized with the ECL Western Blotting kit (Shenggong Bioengineering, China) and quantified using Quantity One analysis software (Bio-Rad, UK).

2.7 Construction and transfection of plasmids

FBXW7 overexpression plasmids, p100 interference plasmids, NF-κB interference plasmids, and their respective negative control plasmids were all constructed by Shanghai Biotech Engineering Co., Ltd. (China). Plasmids were transfected using Lipofectamine 2000 (Invitrogen). Expression of FBXW7, p100, and NF-κB mRNA and protein levels were analyzed by qRT-PCR and western blotting, respectively.

2.8 Animals and experimental design

One hundred 8-week-old Sprague-Dawley female rats were obtained from the Experimental Animal Center of the Lu Kang Pharmaceutical Co., Ltd. (Shandong, China). The rats were housed in a well-ventilated room at 24 ℃ with a 12 h/12 h light/dark cycle and were given free access to food and water. The animals were randomly divided into five groups: sham surgery group (SHAM), ovariectomized group (OVX), CP pre-protection group (CP-P), low concentration CP group (CP-L) and high concentration CP group (CP-H).

The rats underwent sham surgery or bilateral ovariectomy at the age of 12 weeks via abdominal incision under general anesthesia. From the age of 18 weeks, rats in the CP-L and CP-H groups were daily intragastrically administered with CP at a concentration of 0.5 or 1.0 g/kg/d for 17 weeks while the OVX and SHAM groups were given an equivalent volume of saline. Unlike other groups, the rats in the CP-P group were administered intragastrically with CP at a dose of 1 g/kg/d from the age of 9 weeks for 26 weeks, except for the 3 days after ovariectomy.

2.9 Histomorphometric analyses

The right proximal tibia of each animal was fixed with 4% paraformaldehyde, processed for decalcification in 10% EDTA-2Na and embedded in paraffin following standard procedures. Sections were stained with hematoxylin-eosin (H&E). The images were captured using Olympus BX60 light microscope (Japan) and analyzed using ImageJ 1.48v software (National Institutes of Health, USA).

2.10 Statistical analyses

Statistical analyses were performed using SPSS version 17.0 (SPSS Inc., USA). Data were described as means ± standard deviation (SD). All data were analyzed using a one-way analysis of variance (ANOVA) with a Tamhame’s multiple comparison post hoc test. Statistical significance was defined as p < 0.05.

3.1 Protective effects of CP on H₂O₂-induced cytotoxicity in Saos-2 cells

The concentration-dependent effects of CP on Saos-2 cell viability and H₂O₂-induced cell death were first examined. As shown in Fig. 1A, CP did not exhibit cytotoxic effect at concentration ≤1000 μg/mL under normal condition. Instead, it significantly increased Saos-2 cell viability at concentrations range from 200 to 1000 μg/mL. With H₂O₂ treatment, the cell viability was significantly decreased (p < 0.01, Fig. 1B). Treatment with CP significantly protected Saos-2 cells against H₂O₂-induced cell injury at concentrations of 400 and 800 μg/mL but not 200 μg/mL (Fig. 1B). These results suggest that CP promotes cell viability and suppresses H₂O₂-induced cell death.

Close

Fig. 1

Effect of CP on cell viability, ALP activity and antioxidant levels in H₂O₂-induced oxidative stress in Saos-2 cells. (A) Effects of CP on the cell viability (B, C, D) Effects of H₂O₂, CP, overexpressed FBXW7, p100 and NF-κB silencing on cell viability, ALP activity, and ROS levels in Saos-2 cells. (E, F) Effects of H₂O₂, and CP on MDA productions and SOD activity in Saos-2 cells. NAC is the positive control. Data shown represent the means ± SD. *p < 0.05 and **p < 0.01 vs. control group, ^#p < 0.05 and ^##p < 0.01 vs·H₂O₂ group, and ^&p < 0.05 and ^&&p < 0.01 vs. CP (800 μg/ml) group.

Export to PPT

3.2 CP reversed H₂O₂-induced inhibition of ALP activity in Saos-2 cells

ALP is a marker at the early stage of osteoblast differentiation and osteogenic activity. As shown in Fig. 1C, H₂O₂ markedly decreased ALP activity whereas CP attenuated the loss of ALP activity induced by H₂O₂ in a concentration-dependent manner (p < 0.05, CP200 versus H₂O₂; p < 0.01, CP400/CP800 versus H₂O₂), suggesting that CP promotes osteogenic differentiation.

3.3 CP scavenged H₂O₂-induced ROS and MDA productions, and promoted SOD activity in Saos-2 cells

Assessment of the antioxidant activity of CP showed that H₂O₂ significantly increased the production of ROS (Fig. 1D) and MDA (Fig. 1E) while inhibiting the activity of SOD (Fig. 1F). In cells pre-incubated with CP, the levels of ROS (Fig. 1D) and MDA (Fig. 1E) were suppressed in a concentration-dependent manner, whereas SOD (Fig. 1F) activity significantly increased as compared with the H₂O₂-treated group. These data revealed that CP could attenuate oxidative stress injury induced by H₂O₂.

3.4 CP attenuated H₂O₂-induced oxidative stress injury by inhibiting FBXW7/p100/NF-κB pathway

As shown in Fig. 2A, H₂O₂ increased the mRNA expression of FBXW7, suggesting that H₂O₂ induces oxidative stress damage through FBXW7. The elevated expression of FBXW7 was significantly reversed by CP. In addition, CP treatment at concentrations 400 and 800 μg/mL showed a significant reversal effect on the mRNA expression of NF-κB (CP400/CP800 versus H₂O₂, p < 0.01, Fig. 2B) and p100 (CP400/CP800 versus H₂O₂, p < 0.01, Fig. 2C) induced by H₂O₂.

Close

Fig. 2

qRT-PCR analysis of mRNA expression of (A) FBXW7 (B) NF-κB (C) p100 in Saos-2 cells treated with and without overexpressed FBXW7, p100 silencing, H₂O₂ and CP. Data shown represent the means ± SD. *p < 0.05 and **p < 0.01 vs. control group, ^#p < 0.05 and ^##p < 0.01 vs. H₂O₂ group, and ^&p < 0.05 and ^&&p < 0.01 vs. CP (800 μg/ml) group.

Export to PPT

To further explore the mechanism of CP, Saos-2 cell lines overexpressing FBXW7, silencing NF-κB or silencing p100 were established. Their proteins and mRNAs expression levels were examined by western blotting (Fig. 3A) and qRT-PCR (Fig. 3B), respectively. Overexpression of FBXW7 down-regulated the expression of p100 and up-regulated the expression of NF-κB. Interestingly, CP significantly suppressed FBXW7 and NF-κB and promoted p100 expression in Saos-2 cells, thereby reversing the effect of H₂O₂ and overexpressed FBXW7 in cells (Fig. 3C). In addition, silencing of p100 upregulate the expression of NF-κB (Fig. 3D). These data strongly suggest that a mutual regulatory relationship between FBXW7, p100, and NF-κB and that FBXW7 is an upstream regulatory factor of p100 and NF-κB while p100 is a negatively regulator of NF-κB.

Close

Fig. 3

The mRNA and protein expression levels of FBXW7, p100 and NF-κB were detected by qRT-PCR and Western Blot, respectively. (A, B) Efficiency of overexpressing and silencing of the genes. (C–E) FBXW7 regulates expression levels of p100 and NF-κB. p100 negatively regulate NF-κB protein expression. The values shown represent the means ± SD. *p < 0.05 and **p < 0.01 vs. vector control.

Export to PPT

Further examination showed that the effects of CP on cell viability, ALP and ROS were attenuated by overexpressing FBXW7 or silencing p100 (Fig. 1B, C, D). These results further demonstrated that the effect of CP via FBXW7/p100/NF-κB axis and that FBXW7 is a key factor in this pathway.

3.5 CP improved osteoporosis in ovariectomized rats over time

To evaluate the effect CP on oxidative stress injury in vivo, ovariectomized rats as a model of osteoporosis was examined. Six weeks after ovariectomy, we observed decreased in trabecular number (Tb.N) and trabecular thickness (Tb.Th), increased trabecular separation (Tb.Sp), and interrupted continuity of trabecular meshwork in the OVX group as compared to the SHAM group (data not shown), indicating that the pathological model of osteoporosis was successfully established. Twenty-three weeks after ovariectomy (35-week-old rats), the bone tissue microstructure of the OVX rats were severely damaged, the number of trabeculae and the thickness of the trabeculae were further reduced, and the trabecular meshwork disappeared. Compared with the OVX group, all CP-treated groups significantly increased Tb.N, decreased Tb.Sp, and improved the integrity of the trabecular meshwork connection integrity to different extents (p < 0.05, Fig. 4).

Close

Fig. 4

Representative images of H&E staining (4× magnification) and histomorphometric analyses of the right tibias of 35-week-old ovariectomized rats treated with low concentration CP (CP-L), high concentration CP (CP-H) or pretreated with CP (CP-P). The experiments were repeated three times. Data are expressed as means ± SD. *p < 0.05, vs SHAM group, ^#p < 0.05, vs. OVX group.

Export to PPT