Heptapeptide Isolated from Isochrysis zhanjiangensis Exhibited Anti-Photoaging Potential via MAPK/AP-1/MMP Pathway and Anti-Apoptosis in UVB-Irradiated HaCaT Cells

Abstract

Marine microalgae can be used as sustainable protein sources in many fields with positive effects on human and animal health. DAPTMGY is a heptapeptide isolated from Isochrysis zhanjiangensis which is a microalga. In this study, we evaluated its anti-photoaging properties and mechanism of action in human immortalized keratinocytes cells (HaCaT). The results showed that DAPTMGY scavenged reactive oxygen species (ROS) and increase the level of endogenous antioxidants. In addition, through the exploration of its mechanism, it was determined that DAPIMGY exerted anti-photoaging effects. Specifically, the heptapeptide inhibits UVB-induced apoptosis through down-regulation of p53, caspase-8, caspase-3 and Bax and up-regulation of Bcl-2. Thus, DAPTMGY, isolated from I. zhanjiangensis, exhibits protective effects against UVB-induced damage.

Keywords: MMPs; UVB; apoptosis; heptapeptide; photoaging.

Figure 1

Effect of DAPTMGY on cell viability of HaCaT cells. (a) the viability of various doses of DAPTMGY (0, 10, 20, 50, 100, 150, and 200 µM) on HaCaT cells; (b) protective effects of DAPTMGY (0, 20, 50 and 100 µM) on UVB -induced (40 mJ/cm²) HaCaT cell injury. Data are shown as mean ± S.D (n = 3). (###) p < 0.001, as compared to the blank group (untreated cells). ** p < 0.01 and *** p < 0.001, respectively, as compared to the control group.

Figure 2

Effect of DAPTMGY on intercellular ROS generation and on the contents of Total superoxide dismutase (T-SOD), catalase (CAT) and glutathione peroxidase 1 (GPX1) in UVB-stimulated (40 mJ/cm²) HaCaT cells. Cells were cultured with DAPTMGY (20, 50, and 100 µM) for 2 h and then cultured for another 24 h after UVB irradiation. (a) Intercellular ROS production was measured in intact HaCaT cells using DCFH-DA, and photos were captured under fluorescence inverted microscopy. (b) The relative DCF fluorescence intensity analysis was conducted using Image J (fluorescence experiments were performed four times; n = 3 per group). (c–e) The levels of T-SOD, CAT, and GPX1 were measured using biochemical assay kits and ELISA kits. Results are shown as the mean ± SD (n = 3). ^## p < 0.01 and ^### p < 0.001 compared to the blank group (untreated cells); * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control group.

Figure 3

The influence of DAPTMGY on the levels of related apoptosis proteins in UVB-stimulated HaCaT cells. (a) Western blotting bands of p53, Bcl-2, Bax, caspase-3 and caspase-8. (b,d,e) The proteins expression of p53, caspase-3 and caspase-8, respectively. (c) The ratios of Bcl-2/Bax. (f) HaCaT cells stained with DAPI were observed using an inverted fluorescence microscope. (g) Nuclear translocation of p53 was observed by immunofluorescence through an overlay of green p53 staining, blue DAPI staining and Actin red. Data are shown as mean ± S.D (n = 3). (#, ## and ###) p < 0.05, p < 0.01 and p < 0.001, as compared to the blank group (untreated cells). * p < 0.05, ** p < 0.01 and *** p < 0.001, respectively, as compared to the control group. β-actin was used as an internal control.

Figure 3

The influence of DAPTMGY on the levels of related apoptosis proteins in UVB-stimulated HaCaT cells. (a) Western blotting bands of p53, Bcl-2, Bax, caspase-3 and caspase-8. (b,d,e) The proteins expression of p53, caspase-3 and caspase-8, respectively. (c) The ratios of Bcl-2/Bax. (f) HaCaT cells stained with DAPI were observed using an inverted fluorescence microscope. (g) Nuclear translocation of p53 was observed by immunofluorescence through an overlay of green p53 staining, blue DAPI staining and Actin red. Data are shown as mean ± S.D (n = 3). (#, ## and ###) p < 0.05, p < 0.01 and p < 0.001, as compared to the blank group (untreated cells). * p < 0.05, ** p < 0.01 and *** p < 0.001, respectively, as compared to the control group. β-actin was used as an internal control.

Figure 4

DAPTMGY blocked phosphorylation of ERK, p-38 and c-Jun in UVB-stimulated HaCaT cells. (a) Western blotting bands of MAPK pathway and AP-1. (b) The ratios of p-ERK/ERK, (c) The ratios of p-JNK/JNK, (d) The ratios of p-p38/p38 and (e) The ratios of p-c-Jun/c-Jun are calculated respectively. Data are shown as mean ± S.D (n = 3). (# and ##) p < 0.05 and p < 0.01, as compared to the blank group (untreated cells). * p < 0.05, ** p < 0.01 and *** p < 0.001, respectively, as compared to the control group. β-actin functioned as the internal control.

Figure 5

DAPTMGY suppressed the UVB-stimulated NF-κB signal pathway in HaCaT. (a) Western blotting bands of expression of NF-κB pathway. (b–d) The ratios of p-NF-κB p65/NF-κB p65, p-IκB/IκB and p-p50/p50 are calculated respectively. β-actin functioned as the control and all of the experiments were performed in at least triplicate. Data are shown as mean ± S.D (n = 3). (##) p < 0.01, as compared to the blank group (untreated cells). * p < 0.05, ** p < 0.01 and *** p < 0.001, respectively, as compared to the control group. β-actin functioned as the internal control.

Figure 6

DAPTMGY suppresses MMP-1 and MMP-3 secretion and increase procollagen I. (a) The secretion in condition media of MMP-1, (b) The secretion in condition media of MMP-3, and (c) The secretion in condition media of procollagen I was assessed by ELISA kit. Data are shown as mean ± S.D (n = 3). (#, ## and ###) p < 0.005, p < 0.01 and p < 0.001, as compared to the blank group (untreated cells). * p < 0.005, ** p < 0.01 and *** p < 0.001, respectively, as compared to the control group.

Figure 7

DAPTMGY interacted with MMP-1 (a) and MMP-3 (b). The optimal 3D poses structure and two-dimensional diagram of DAPTMGY and MMP-1 and MMP-3. Sticks stand for DAPTMGY and dotted lines is represent bond formation.

Figure 8

The amino acid sequence of DAPTMGY.

Figure 9

Mechanism summary of DAPTMGY against UVB-irradiated HaCaT cells oxidative and apoptosis.