Voghera Sweet Pepper Regulates Cell Death Pathways in an Aging In Vitro Model

Abstract

Background/Objectives: Aging and its related disorders are important issues nowadays, and ROS overproduction is one of the primary contributors to this physio-pathological condition. In this regard, ascorbic acid is a strong antioxidant molecule and its anti-aging proprieties are well known. Our previous data demonstrated that Voghera sweet pepper (VP), a peculiar type of pepper cultivated in Italy, is particularly rich in ascorbic acid and displayed a potential anti-aging effect in both young and aged in vitro models, regulating oxidative stress and senescence/proliferation. Based on these data, the anti-aging effect mediated by the extract of the edible part of VP, in terms of regulation of specific cell death mechanisms, was evaluated in an in vitro model of both young and old Normal Human Dermal Fibroblasts (NHDF). Methods: Immunofluorescence analyses were performed to assess the expression levels of specific markers related to autophagy (p62, LC3b) and mitophagy (Pink1, Parkin), as well as the apoptotic marker caspase-3. In addition, transmission electron microscopy (TEM) was used to analyze cellular ultrastructure and to provide further morphological evidence of the extract's impact. Results: Immunofluorescence analyses revealed that VP extract led to modulated expression levels of p62, LC3b, Pink1, and Parkin, along with a reduction in caspase-3 activity, indicating decreased apoptosis. TEM ultrastructural analysis supported these findings, showing morphological changes consistent with the modulatory effects of VP extract during aging. Conclusions: Based on these results, we may suppose that Voghera pepper (VP) is able to modulate different mechanisms of regulated cell death (RCD) in our in vitro aging model.

Keywords: NHDF; Nutraceutics; Voghera sweet pepper; aging; antioxidant; cell death pathways.

Figure 1

Immunocytochemical detection of p62 (red signal) and beta-actin (green signal) by fluorescence microscopy in Control (CTR) (A–C and J–L), Carmagnola Pepper (CP) (D,E and M–O) and Voghera Pepper (VP) (F–H and P–R) treatment, in both young and old NHDF (A–I and J–R, respectively). DNA counterstaining with Hoechst 33258 (blue fluorescence). Histograms depicting the quantitative measurement of young (Y-) (left) and old (O-) (right) p62 (S) and beta-actin (T) mean fluorescence intensity. Statistically significant data: * p < 0.05. Magnification: 60×. Scale bars: 30 µm.

Figure 2

Immunocytochemical detection of LC3b (green signal) and alpha-tubulin (red signal) by fluorescence microscopy in Control (CTR) (A–C and J–L), Carmagnola Pepper (CP) (D–F and M–O) and Voghera Pepper (VP) (G–I and P–R) treatment, in both young and old NHDF (A–I and J–R, respectively). DNA counterstaining with Hoechst 33258 (blue fluorescence). Histograms depicting the quantitative measurement of young (Y-) (left) and old (O-) (right) LC3b (S) and alpha-tubulin (T) mean fluorescence intensity. Statistically significant data: *** p < 0.001. Magnification: 60×. Scale bars: 30 µm.

Figure 3

Immunocytochemical detection of lysosomes (red signal) by fluorescence microscopy in Control (CTR) (A–C and J–L), Carmagnola Pepper (CP) (D–F and M–O) and Voghera Pepper (VP) (G–I and P–R) treatment, in both young and old NHDF (A–I and J–R, respectively). DNA counterstaining with Hoechst 33258 (blue fluorescence). Histograms depicting the quantitative measurement of young (Y-) (S) and old (O-) (T) lysosomes mean fluorescence intensity. Statistically significant data: * p < 0.05, ** p < 0.01, *** p < 0.001. Magnification: 40×. Scale bars: 50 µm.

Figure 4

Immunocytochemical detection of Pink1 (green signal) and mitochondria (red signal) by fluorescence microscopy in Control (CTR) (A–C and J–L), Carmagnola Pepper (CP) (D–F and M–O) and Voghera Pepper (VP) (G–I and P–R) treatment, in both young and old NHDF (A–I and J–R, respectively). DNA counterstaining with Hoechst 33258 (blue fluorescence). Histograms depicting the quantitative measurement of young (Y-) (left) and old (O-) (right) Pink1 (S) and mitochondria (T) mean fluorescence intensity. Statistically significant data: ** p < 0.01, *** p < 0.001. Magnification: 60×. Scale bars: 30 µm.

Figure 5

Immunocytochemical detection of parkin (red signal) and mitochondria (green signal) by fluorescence microscopy in Control (CTR) (A–C and J–L), Carmagnola Pepper (CP) (D–F and M–O) and Voghera Pepper (VP) (G–I and P–R) treatment, in both young and old NHDF (A–I and J–R, respectively). DNA counterstaining with Hoechst 33258 (blue fluorescence). Histograms depicting the quantitative measurement of young (Y-) (left) and old (O-) (right) of Parkin (S) and mitochondria (T) mean fluorescence intensity. Statistically significant data: * p < 0.05, ** p < 0.01, *** p < 0.001. Magnification: 60×. Scale bars: 30 µm.

Figure 6

Immunocytochemical detection of caspase3 (green signal) and mitochondria (red signal) by fluorescence microscopy in Control (CTR) (A–C and J–L), Carmagnola Pepper (CP) (D–F and M–O) and Voghera Pepper (VP) (G–I and P–R) treatment, in both young and old NHDF (A–I and J–R, respectively). DNA counterstaining with Hoechst 33258 (blue fluorescence). Histograms depicting the quantitative measurement of young (Y-) (left) and old (O-) (right) cleaved caspase3 (S) and alpha-tubulin (T) mean fluorescence intensity per cell. Statistically significant data: *** p < 0.001. Magnification: 40×. Scale bars: 30 µm.

Figure 7

TEM ultrastructural analysis of Control (CTR) (A–C), Carmagnola Pepper (CP) (D–F) and Voghera Pepper (VP) (G–I) treatment in young (Y-) NHDF. Histogram (J) depicting the quantitative measurement of mitochondria area in young NHDF. Arrowheads indicate rough endoplasmic reticulum, while arrows point to mitochondria. Statistically significant data: * p < 0.05, ** p < 0.01. Magnification: 12,000× (A); 20,000× (D); 30,000× (G); 40,000× (E,F,H); 50,000× (B,C,I).

Figure 8

TEM ultrastructural analysis of Control (CTR) (A–C), Carmagnola Pepper (CP) (D–F) and Voghera Pepper (VP) (G–I) treatment in old (O-) NHDF. Histogram (J) depicting the quantitative measurement of mitochondria area in old NHDF. Arrowheads indicate rough endoplasmic reticulum, while arrows point to mitochondria. Statistically significant data: ** p < 0.01. Magnification: 10,000× (A); 20,000× (B,D); 25,000× (G); 30,000× (E,H); 40,000× (C); 50,000× (F,I).