Protegrin-1

In mammals, oxidative stress-induced apoptosis of granulosa cells is one of the major causes of follicular atresia, affecting ovarian physiological function. Protegrin-1 (PG-1) is an antimicrobial peptide with effective antimicrobial activity, immunomodulatory function, and porcine growth-promoting effects. PG-1 has been detected in porcine ovaries follicles. This study aimed to investigate the effect of PG-1 on oxidative stress-induced apoptosis of porcine ovarian granulosa cells and the underlying molecular mechanism. Granulosa cells were obtained from porcine follicles and treated with H2O2 to establish the oxidative stress model, and then treated with or without PG-1 (10 μg/mL). PG-1 significantly suppressed H2O2-induced apoptosis in granulosa cells after 24 h of treatment. Furthermore, these results revealed that PG-1 increased the mRNA and protein expression of anti-apoptotic B cell lymphoma/leukemia 2 (BCL2) and the BCL2/Bcl-2-associated X protein (BAX) ratio while decreasing the expression of pro-apoptotic BAX and active caspase-3. Using Western blot analysis, it was found that PG-1 decreased the phosphorylation of RNA-like endoplasmic reticulum kinase (PERK) and the α-subunit of eukaryotic initiation factor 2 (eIF2α) as well as the protein expression level of CCAAT enhancer-binding protein homologous protein (CHOP), all of which were increased by H2O2. Moreover, inhibitors against PERK and phospho-eIF2ɑ both suppressed the H2O2-induced granulosa cells apoptosis and enhanced the anti-apoptosis effect of PG-1. Taken together, our findings demonstrated that PG-1 inhibited porcine ovarian granulosa cell apoptosis from oxidative stress via the PERK/eIF2α/CHOP signaling pathway in vitro, which suggests the novel regulatory function of the antimicrobial peptide in the ovary.

Antimicrobial peptides (AMPs) are traditionally known to be essential components in host defense via their broad activities against bacteria, fungi, viruses, and protozoa. Their immunomodulatory properties have also recently received considerable attention in mammalian somatic tissues of various species. However, little is known regarding the role of AMPs in the development and maturation of ovarian follicles. Protegrin-1 (PG-1) is an antimicrobial peptide which is known to have potent antimicrobial activity against both gram positive and negative bacteria. Here we report that the PG-1 is present in the porcine ovarian follicular fluid. Treatment of granulosa cell with PG-1 enhanced granulosa cell proliferation in a dose-dependent manner. This is accompanied by increased expression of cell-cycle progression-related genes such as cyclin D1(CCND1), cyclin D2 (CCND2), and cyclin B1(CCNB1). Additionally, Western blot analysis showed that PG-1 increased phosphorylated epidermal growth factor receptor (EGFR), and the phosphorylated-/total extracellular signal-regulated kinase (ERK)1/2 ratio. Pretreatment with either U0126, a specific ERK1/2 phosphorylation inhibitor, or EGFR kinase inhibitor, AG1478, blocked the PG-1 induced proliferation. Moreover, luciferase reporter assay revealed that ETS domain-containing protein-1 (Elk1) C/EBP homologous protein (CHOP), and the transcription activators downstream of the MAPK pathway, were activated by PG-1. These data collectively suggest that PG-1 may regulate pig granulosa cell proliferation via EGFR-MAPK pathway., Hence, our finding offers insights into the role of antimicrobial peptides on follicular development regulation.

Porcine protegrin-1 (PG-1) is a broad-spectrum antimicrobial peptide (AMP) with potent antimicrobial activities. We produced recombinant PG-1 and evaluated its cytotoxicity toward various types of mammalian cell lines, including embryonic fibroblasts, retinal cells, embryonic kidney cells, neuroblastoma cells, alveolar macrophage cells, and neutrophils. The sensitivity of the different mammalian cells to cytotoxic damage induced by PG-1 differed significantly among the cell types, with retinal neuron cells and neutrophils being the most significantly affected. A circular dichroism analysis showed there was a precise correlation between conformational changes in PG-1 and the magnitude of cytotoxicity among the various cell type. Subsequently, a green fluorescent protein (GFP) penetration assay using positively charged GFPs indicated there was a close correlation between the degree of penetration of charged GFP into cells and the magnitude of PG-1 cytotoxicity. Furthermore, we also showed that inhibition of the synthesis of anionic sulphated proteoglycans on the cell surface decreases the cytotoxic damage induced by PG-1 treatment. Taken together, the observed cytotoxicity of PG-1 towards different membrane surfaces is highly driven by the membrane's anionic properties. Our results reveal a possible mechanism underlying cell-type dependent differences in cytotoxicity of AMPs, such as PG-1, toward mammalian cells.