ACE Inhibitory Peptide from Skin Collagen Hydrolysate of Takifugu bimaculatus as Potential for Protecting HUVECs Injury

Abstract

Angiotensin-I-converting enzyme (ACE) is a crucial enzyme or receptor that catalyzes the generation of potent vasopressor angiotensin II (Ang II). ACE inhibitory peptides from fish showed effective ACE inhibitory activity. In this study, we reported an ACE inhibitory peptide from Takifugu bimaculatus (T. bimaculatus), which was obtained by molecular docking with acid-soluble collagen (ASC) hydrolysate of T. bimaculatus. The antihypertensive effects and potential mechanism were conducted using Ang-II-induced human umbilical vein endothelial cells (HUVECs) as a model. The results showed that FNLRMQ alleviated the viability and facilitated apoptosis of Ang-II-induced HUVECs. Further research suggested that FNLRMQ may protect Ang-II-induced endothelial injury by regulating Nrf2/HO-1 and PI3K/Akt/eNOS signaling pathways. This study, herein, reveals that collagen peptide FNLRMQ could be used as a potential candidate compound for antihypertensive treatment, and could provide scientific evidence for the high-value utilization of marine resources including T. bimaculatus.

Keywords: HUVECs; Takifugu bimaculatus; antihypertension; skin collagen.

Figure 1

FNLRMQ enhanced cell viability in Ang-II-induced HUVECs. (a) Structure of FNLRMQ. (b) Evaluation of the effect of FNLRMQ against the HUVECs. HUVECs were treated with FNLRMQ (1–40 µM) without Ang II treatment for 24 h. (c) Evaluation of the effect of Cap against the HUVECs. HUVECs were treated with Cap (1–500 nM) without Ang II for 24 h. (d) Effect of FNLRMQ (1–40 µM) on the viability of Ang-II-induced HUVECs. Cap as the positive control. HUVECs’ viability was measured using a CCK-8 assay. The values are expressed as the means ± SD, n = 3. ** p < 0.01, ^## p < 0.01. The figure marked ** was compared with the normal control group and the figure marked ## was compared with the Ang-II-induced-model group.

Figure 2

FNLRMQ ameliorated Ang-II-induced HUVECs apoptosis. HUVECs were incubated with Ang II for 24 h in the absence or presence of FNLRMQ (5–20 µM). (a) The flow cytometry assay was adopted to detect the effect of FNLRMQ on the apoptosis of Ang-II-induced HUVECs. (b) The percentage rate of apoptotic cells (mean ± SD, n = 3). (c) Expression of cytochrome C and anti-apoptotic protein Bcl-2 and pro-apoptosis proteins caspase-3 and Bax was examined by Western blotting analysis in HUVECs with different treatments. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. (d,e) The quantification of the immunoreactive bands. The values are expressed as the means ± SD, n = 3. ** p < 0.01, ^# p < 0.05, ^## p < 0.01. The figure marked ** was compared with the normal control group and the figure marked ## was compared with the Ang-II-induced-model group.

Figure 3

Protective effects of FNLRMQ on oxidative stress in Ang-II-induced HUVECs. HUVECs were incubated with Ang II for 24 h in the absence or presence of FNLRMQ (5–20 µM). (a) The fluorescent probe, DCFH-DA was adopted to measure total intracellular ROS level in HUVECs, and photos were taken by fluorescence inverted microscopy. (b) Mean fluorescence intensity was analyzed by ImageJ software. The values are expressed as the means ± SD, n = 3. (c,d) HUVECs’ oxidative damage was assessed by MDA and LDH detection. (e) T-AOC was determined using an assay kit. The values are expressed as the means ± SD, n = 3. ** p < 0.01, * p < 0.05, ^## p < 0.01, ^# p < 0.05. The figure marked ** was compared with the normal control group and the figure marked ## was compared with the Ang-II-induced-model group.

Figure 4

FNLRMQ catalyzed the Nrf2 nuclear translocation and activation of the Nrf2/HO-1 signaling pathway. HUVECs were incubated with Ang II for 24 h in the absence or presence of FNLRMQ (5–20 µM). (a) Effect of FNLRMQ on protein expression of Nrf2 in the nucleus and cytoplasm. Histone H3 and GAPDH were used as the internal control. (b,c) The quantification of the immunoreactive bands of Nrf2 in the nucleus and cytoplasm, respectively. (d) IF staining of HUVECs with anti-Nrf-2 antibody (green) and DAPI (blue). (e) Mean fluorescence intensity of IF. The values are expressed as the means ± SD, n = 3. (f) Effect of FNLRMQ on the expression of antioxidant enzyme in HUVECs. Protein expression of the HO-1 and Keap-1 was measured by Western blotting analysis. GAPDH functioned as the control. (g) The quantification of the immunoreactive bands. (h,i) The effects of FNLRMQ on the activities of antioxidant enzymes SOD and GSH were determined by kit assays. The values are expressed as the means ± SD, n = 3. ** p < 0.01, * p < 0.05, ^## p < 0.01, ^# p < 0.05. The figure marked ** was compared with the normal control group and the figure marked ## was compared with the Ang-II-induced-model group.

Figure 5

The effects of FNLRMQ on the production of NO and NOS and the role of the eNOS in response to Ang-II-induced oxidative stress. (a) The effects of FNLRMQ on the production of NO. HUVECs cultured with Ang II (1 µM) were treated with FNLRMQ (5–20 µM) for 24 h. Subsequently, the level of NO was determined with the fluorescent probe DAF-FM DA, following detection by a microplate reader. (b) NOS was detected by assay kits. (c) Western blotting analysis was performed to evaluate the effect of FNLRMQ on the expression of eNOS and p-eNOS. (d) The quantification of the immunoreactive bands. (e) HUVECs were initially exposed to the eNOS inhibitor L-NAME (100 μM) for 30 min, then treatment with Ang II and FNLRMQ for 24 h. Finally, the production of NO was detected. The values are expressed as the means ± SD, n = 3. ** p < 0.01, * p < 0.05, ^## p < 0.01, ^# p < 0.05. The figure marked ** was compared with the normal control group and the figure marked ## was compared with the Ang-II-induced-model group.

Figure 6

FNLRMQ attenuated Ang-II-induced HUVECs dysfunction through the PI3K/Akt pathway. (a) HUVECs treated with Ang II (1 µM) were incubated with FNLRMQ (5-20 µM) and Cap (0.1 μM) for 24 h, then the protein expression of PI3K, Akt, and p-Akt was detected by Western blotting analysis. (b)The quantification of the immunoreactive bands. (c) HUVECs were exposed to LY294002 (50 μM) for 30 min before being treated with Ang II for 24 h in the absence or presence of FNLRMQ. Subsequently, the expression of Nrf2 in the cytoplasm and nucleus was assessed by Western blotting analysis. (d,e) The quantification of the immunoreactive bands of Nrf2 in the nucleus and cytoplasm, respectively. (f,g) HUVECs were exposed to LY294002 (50 μM) for 30 min before being treated with Ang II for 24 h in the absence or presence of FNLRMQ (20 μM). Subsequently, the levels of ROS and NO were measured. The values are expressed as the means ± SD, n = 3. ** p < 0.01, ^## p < 0.01. The figure marked ** was compared with the normal control group and the figure marked ## was compared with the Ang-II-induced-model group.