Abstract
Dermal fibroblast replicative senescence that often occurs in aging skin is characterized by loss of cell proliferative capacity, cell cycle arrest, decreased cell elongation, and decreased synthesis of dermal extracellular matrix (ECM) components. Although Panax notoginseng is known for its effectiveness in alleviating many age-related degenerative diseases, few studies have evaluated P. notoginseng components for efficacy or mechanisms of action in delaying cell replicative senescence. In this study, P. notoginseng oligosaccharides (PNO) were isolated using a stepwise purification procedure involving water extraction and alcohol precipitation followed by DEAE Sepharose Fast Flow column chromatography, preparative high performance liquid chromatography, and size-exclusion chromatography. Monosaccharides detected in PNO constituents included mannose, galactose, and sorbitose in relative molar proportions of 14.2:12.3:1, respectively, aligning with PNO absorption spectrum results resembling typical known spectra for sugars. In vitro, PNO treatment of replicative senescent NIH-3T3 fibroblasts significantly promoted cell vitality, inhibited SA-β-galactosidase (SA-β-Gal) activity, and reduced p16 and p21 protein-level expression. Moreover, PNO treatment of senescent fibroblasts led to a lower proportion of G1 phase cells and higher proportion of S phase cells, while also inducing aging NIH-3T3 cells to migrate and synthesize collagen-I (CoL-I). Mechanistically, PNO treatment up-regulated expression of proliferating cell nuclear antigen (PCNA), cyclin E, cyclin D1, and cyclin-dependent kinase 4 (CDK4) proteins and promoted phosphorylation of MEK, p38, and ERK1/2 to trigger cell cycle progression. Additionally, PNO treatment also up-regulated protein-level expression of TGF-β1 and levels of p-Smad2/3, p-FAK, and p-Pax to trigger CoL-I synthesis and cell migration. Taken together, these findings demonstrate that oligosaccharides purified from P. notoginseng could reverse fibroblast replicative senescence by promoting fibroblast cell proliferation, migration, and CoL-I production.
Highlights
As people get older, skin undergoes endogenous aging that mainly manifests itself as thinning of skin leading to atrophic appearance, reduced elasticity, fine wrinkles, loss of underlying fat, dryness, and increased sensitivity (Gu et al, 2020)
Mouse monoclonal antibodies against p16, p21, type I collagen (CoL-I), cell proliferation antigen (PCNA), cyclin D1, cyclin E, cycle-dependent kinases 4 (CDK4), focal adhesion kinase (FAK)/p-FAK, Pax/p-Pax, MEK/p-MEK, ERK/p-ERK, p38/ p-p38, transforming growth factor-β1 (TGF-β1), Smad 2/3/p-Smad 2/3, GAPDH, and β-actin were purchased from Abcam
A gel column was used to separate components according to particle size, resulting in collection of peak I associated with material exhibiting target activity that counteracted dermal fibroblast replicative senescence, P. notoginseng oligosaccharide (PNO) (Figure 1C)
Summary
Skin undergoes endogenous aging that mainly manifests itself as thinning of skin leading to atrophic appearance, reduced elasticity, fine wrinkles, loss of underlying fat, dryness, and increased sensitivity (Gu et al, 2020). Major pathological changes associated with aging skin include epidermal layer thinning accompanied by epidermal rete ridge loss, dermal papillae attenuation, extracellular matrix atrophy, and decreases in numbers of fibroblasts, collagen fibers, and elastic fibers (Russell-Goldman and Murphy, 2020). Prominent age-related dermal fibroblast characteristics include reduced cell size, decreased cell elongation, and decreased cell ability to synthesize ECM (Kanaki et al, 2016). These changes appear to be related to a fibroblast-aging phenomenon known as replicative senescence. Replicative senescence has been linked to telomere shortening and imbalanced oxidative stress responses (Mohamad Kamal et al, 2020; Sadowska-Bartosz and Bartosz, 2020)
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