Fish Scale Collagen Peptides Protect against CoCl2/TNF- α-Induced Cytotoxicity and Inflammation via Inhibition of ROS, MAPK, and NF- κ B Pathways in HaCaT Cells

Fazli Subhan, Hae Yeong Kang, Yeseon Lim, Muhammad Ikram, Sun-Yong Baek, Songwan Jin, Young Hun Jeong, Jong Young Kwak, Sik Yoon, Fazli Subhan, Hae Yeong Kang, Yeseon Lim, Muhammad Ikram, Sun-Yong Baek, Songwan Jin, Young Hun Jeong, Jong Young Kwak, Sik Yoon

Abstract

Skin diseases associated with inflammation or oxidative stress represent the most common problem in dermatology. The present study demonstrates that fish scale collagen peptides (FSCP) protect against CoCl2-induced cytotoxicity and TNF-α-induced inflammatory responses in human HaCaT keratinocyte cells. Our study is the first to report that FSCP increase cell viability and ameliorate oxidative injury in HaCaT cells through mechanisms mediated by the downregulation of key proinflammatory cytokines, namely, TNF-α, IL-1β, IL-8, and iNOS. FSCP also prevent cell apoptosis by repressing Bax expression, caspase-3 activity, and cytochrome c release and by upregulating Bcl-2 protein levels in CoCl2- or TNF-α-stimulated HaCaT cells. In addition, the inhibitory effects of FSCP on cytotoxicity and the induction of proinflammatory cytokine expression were found to be associated with suppression of the ROS, MAPK (p38/MAPK, ERK, and JNK), and NF-κB signaling pathways. Taken together, our data suggest that FSCP are useful as immunomodulatory agents in inflammatory or immune-mediated skin diseases. Furthermore, our results provide new insights into the potential therapeutic use of FSCP in the prevention and treatment of various oxidative- or inflammatory stress-related inflammation and injuries.

Figures

Figure 1
Figure 1
Stimulatory effects of FSCP on proliferation and viability of HaCaT cells. Treatment of HaCaT cells with FSCP for 24 h significantly enhanced cell proliferation (a) and attenuated the CoCl2-induced (500 μM CoCl2) decrease in cell viability (b). Results are presented as the means ± SD of three independent experiments. #p < 0.05 versus the control and ∗∗p < 0.01 versus the CoCl2-treated group.
Figure 2
Figure 2
Inhibitory effects of FSCP on CoCl2/TNF-α-induced ROS generation in HaCaT cells. Intracellular ROS levels were determined via fluorescence microscopy (a, c) or flow cytometry (b, d) using DCFH-DA. The increased ROS levels induced by 100 μM CoCl2 treatment was significantly attenuated by treatment with 1 mg/mL FSCP for 24 h or NAC for 2 h (a, b). Elevated ROS levels were induced by treatment with 20 ng/mL TNF-α but were reversed by treatment with 1 mg/mL FSCP for 12 or 24 h (c, d). Staining intensities were measured using ImageJ software (a, c). Blue histograms represent ROS negative cells, red histograms represent ROS positive cells, and the median fluorescence intensity (MFI) of ROS expression is plotted along the y-axes (b, d). Results are presented as the means ± SD of three independent experiments. #p < 0.05 versus the control and ∗∗p < 0.01 and ∗∗∗p < 0.001 versus the CoCl2/TNF-α-treated group.
Figure 3
Figure 3
Inhibitory effects of FSCP on CoCl2/TNF-α-induced proinflammatory cytokine expression. The expression of the key proinflammatory cytokines, namely, TNF-α, IL-1β, IL-8, and iNOS, was analyzed via RT-PCR. Elevated levels of TNF-α and iNOS mRNA in CoCl2-treated HaCaT cells were attenuated by treatment with FSCP for 24 h (a, b). Upregulation of IL-1β and IL-8 mRNA in TNF-α-treated HaCaT cells was decreased by treatment with FSCP for 24 h (c, d). Results are presented as the means ± SD of three independent experiments. #p < 0.05 versus the control and ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus the CoCl2/TNF-α-treated group.
Figure 4
Figure 4
Effects of FSCP on CoCl2-induced altered expression of apoptosis-related proteins. The expression of apoptosis-related proteins was analyzed by western blotting. Bcl-2 downregulation and Bax upregulation in CoCl2-treated HaCaT cells were reversed by treatment with FSCP and NAC (a, b). Potentiated levels of caspase-3 activity and cytochrome c release in CoCl2-treated HaCaT cells were attenuated by treatment with FSCP and NAC (c). Results are presented as the means ± SD of three independent experiments. Cl-caspase-3: cleaved-caspase-3. #p < 0.05 versus the control and ∗∗p < 0.01 and ∗∗∗p < 0.001 versus the CoCl2-treated group.
Figure 5
Figure 5
Inhibitory effects of FSCP on CoCl2-induced activation of p38/MAPK signaling pathway. P-p38/MAPK levels were increased in CoCl2-treated HaCaT cells (a). Treatment of HaCaT cells with FSCP and NAC (a) and SB203580 (SB) (b) blocked CoCl2-induced phosphorylation of p38/MAPK. Treatment of HaCaT cells with FSCP and SB203580 (SB) blocked CoCl2-induced intracellular ROS activity (c), and staining intensities were measured using ImageJ software (d). Treatment with FSCP blocked CoCl2-induced cytotoxicity in HaCaT cells (e). Western blot analysis showed that the treatment of HaCaT cells with FSCP and SB203580 (SB) suppressed CoCl2-induced elevated expression of iNOS and Bax (f). Results are presented as the means ± SD of three independent experiments. #p < 0.05 versus the control and ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus the CoCl2-treated group.
Figure 6
Figure 6
Inhibitory effects of FSCP on TNF-α-induced activation of the MAPK signaling pathway. Treatment of HaCaT cells with FSCP did not significantly inhibit TNF-α-induced increase in p38/MAPK phosphorylation (a). Treatment of HaCaT cells with FSCP suppressed TNF-α-induced ERK (b) and JNK (c) activation. Results are presented as the means ± SD of three independent experiments. #p < 0.05 versus the control and ∗∗p < 0.01 versus the TNF-α-treated group.
Figure 7
Figure 7
Inhibitory effects of FSCP on TNF-α-induced activation of the NF-κB signaling pathway. Western blot analysis showed NF-κB p65 levels were decreased in the cytoplasm and increased in the nucleus in TNF-α-treated HaCaT cells (a). Immunofluorescence microscopic assay revealed that FSCP prevented the translocation of NF-κB p65 to the nucleus in TNF-α-treated HaCaT cells (b). Results are presented as the means ± SD of three independent experiments. CE: cytoplasmic extracts; NE: nuclear extract. #p < 0.05 versus the control and ∗∗p < 0.01 and ∗∗∗p < 0.001 versus the TNF-α-treated group.
Figure 8
Figure 8
Schematic representation of the regulatory mechanism of FSCP on CoCl2-/TNF-α-induced oxidative and inflammatory stress in HaCaT cells.

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