Promitotic Action Of Oenothera Biennis On Senescent Human Dermal Fibroblasts Part 1

Abstract: Accumulation of senescent dermal fibroblasts drives skin aging. The reactivation of proliferation is one strategy to modulate cell senescence. Recently, we reported the exact chemical composition of the hydrophilic extract of Oenothera biennis cell cultures (ObHEx) and we showed its skin anti-aging properties. This work aims to assess its biological effect specifically on cell senescence. ObHEx action has been evaluated on normal human dermal fibroblasts subjected to stress-induced premature senescence (SIPS) through an ultra-deep proteomic analysis, leading to the most global senescence-associated proteome so far. Mass spectrometry data show that the treatment with ObHEx re-establishes levels of crucial mitotic proteins, strongly downregulated in senescent cells. To validate our proteomics findings, we proved that ObHEx can, in part, restore the activity of ‘senescence-associated-ß-galactosidase’, the most common hallmark of senescent cells. Furthermore, to assess if the upregulation of mitotic protein levels translates into a cell cycle re-entry, FACS experiments have been carried out, demonstrating a small but significant reactivation of senescent cell proliferation by ObHEx. In conclusion, the deep senescence-associated global proteome profiling published here provides a panel of hundreds of proteins deregulated by SIPS that can be used by the community to further understand senescence and the effect of new potential modulators. Moreover, proteomics analysis pointed to a specific pro-mitotic effect of ObHEx on senescent cells. Thus, we suggest ObHEx as a powerful adjuvant against senescence associated with skin aging.

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Keywords: Oenothera biennis hydrophilic extract; senescence; fibroblasts; proteomics; mass  spectrometry; diaPASEF; mitosis; skin aging

1. Introduction

Senescence is the main cause of aging and age-related diseases. Senescent cells undergo proliferation arrest as a response to physiological telomere erosion (replicative senescence) or to stress triggers including DNA damage, oxidative stress, organelle strain, and oncogene activation (stress-induced premature senescence, SIPS) [1]. Despite DNA Damage Response (DDR) induced growth arrest, senescent cells display a high metabolic state and lysosomal activity, crucial for their increased secretory activity, known as senescence-associated secretory phenotype (SASP) [2,3]. SASP factors include several families of soluble (interleukins, chemokines, growth factors, secreted proteases) and insoluble (secreted insoluble proteins and extracellular matrix components) proteins that negatively affect the surrounding tissue, generating a chronic low-grade inflammatory state.

The accumulation of senescent fibroblasts in the derma drives skin aging, impairing the integrity of the extracellular matrix (ECM) and the function of neighboring microvascular endothelial cells, epidermal melanocytes, and keratinocytes [4–6]. The three main strategies to counteract senescence include (i) the clearance of senescent cells by the induction of apoptosis or immune activation; (ii) the modulation of the SASP and (iii) the reactivation of cell proliferation. Indeed, although senescence has been originally described as an irreversible cell cycle arrest, current evidence suggests that proliferation can be restored [7–9].

In this scenario, ingredients able to prevent and/or modulate cellular senescence are of great interest in the cosmetic field as valuable weapons to counteract skin aging. In particular, extracts derived from the species Oenothera biennis (O. biennis, Evening Primrose),  belonging to the family Onagraceae, are traditionally used for cosmetic applications due to their anti-inflammatory and antioxidant activities [10]. Methanolic extracts prepared from aerial parts and aqueous leaf extracts mainly contain phenolic acids and flavonoids,  whereas methanolic extracts obtained from roots mostly contain sterols and triterpenes [11]. Although the biological activities of alcoholic and hydroalcoholic aerial part extracts have been demonstrated [12–14], the most frequently used extracts of O. biennis are the oils obtained from seeds, which are extremely rich in fatty acids. Indeed, since their high content of linoleic acid (LA) and γ-linolenic acid (GLA), O. biennis seed oils improve the proper functioning of the skin and, in particular, they have shown beneficial effects in patients with chronic dermatitis [11,15].

More recently, extracts obtained from O. biennis cell cultures have been proposed to fully substitute extracts from spontaneous plants as they are bio-sustainable, contaminant-free, and standardized secondary metabolites mixtures [16]. It is precisely the combination of the compounds with their synergistic effect that is responsible for the overall activity of botanical extracts [17].

Here, we have focused on the hydrophilic extract of O. biennis cell cultures (ObHEx),  rich in lignans and triterpenes, as it showed skin anti-aging properties when tested in in vitro and ex vivo models [18]. Mass spectrometry-based chemical characterization of this extract revealed that it contains several classes of interesting secondary metabolites. They belong to lignans (salvadoraside and liriodendron) and triterpenes (muriatic acid,  arjunolic acid, aspartic acid, and hederagenin), some of which have previously been associated with pro-collagen I production in human fibroblasts [19,20]. In particular, we have previously proved that ObHEx promotes matrix collagen contraction, actin polymerization, and the production of ECM proteins, thus improving skin firmness and elasticity, via an increase in Myosin light chain kinase (MYLK) gene expression.

To date, the molecular mechanisms of ObHEx effects on skin aging and cellular senescence, in particular, have not been investigated. This work aims to explore ObHEx activity on senescent human dermal fibroblasts. We used a data-independent mass spectrometry ultra-deep proteomic approach to obtain hints of the mechanism of action and bio-orthogonal methods to validate and investigate it (Figure 1).

2. Results 

Stress-induced premature senescence (SIPS) was performed in normal human dermal fibroblasts (NHDF) by treatment with hydrogen peroxide (H2O2). Oxidative stress-induced senescent cells are an excellent in vitro model for aging research and H2O2 is widely used to achieve it [21,22].

To understand the biological pathways altered by ObHEx on senescent NHDF cells,  we used an ultra-deep proteomic approach. SIPS and control cells were treated or not for 48 h with the extract at 0.01% (p/v) since, from the MTT assay reported in [18], this results in the highest concentration that does not cause any cell toxicity after 48 h incubation.

Cell lysates were then subjected to tryptic digestion and nano-UPLC MS/MS analysis. To gain the optimal depth of the proteome, mass spectrometry data were acquired in Data Independent Acquisition (DIA) mode, which allowed the robust quantification of 9650 proteins (Table S1), with data completeness of 98%. Our study reports the most complete proteome analysis of senescent cells to date.

2.1. Stress-Induced Premature Senescence (SIPS) Evaluation

First of all, we verified if the senescence induction in NHDF was successful, analyzing the global proteome of H2O2-treated NHDF by mass spectrometry (MS) and examining their morphology by flow cytometry.

2.1.1. SIPS Evaluation by Mass Spectrometry (MS)

To evaluate SIPS induction, we compared the total proteome of H2O2-treated NHDF  cells versus untreated ones. Among the 3256 proteins that were deregulated, several biomarkers of senescence were found to be significantly altered (Figure 2A, B). Firstly,  we observed the upregulation of the most widely used biomarker of senescent cells, the ‘senescence-associated-ß-galactosidase’ (SA-β-gal, GLB1). The increased levels of the lysosomal enzymes GLB1 and Tissue alpha-L-fucosidase (FUCA1), which have also been found upregulated by MS, reflect the characteristic increment in the lysosomal content of senescent cells [23,24]. Indeed, lysosomes favor catabolic processes, which provide energy and raw materials required by senescence-associated secretion [25].

Secondly, the protein level of serine/threonine-protein kinase ATR and serine-protein kinase ATM, both involved in DDR signaling, was also found to be increased [26]. DDR is activated by nuclear DNA damage, generally associated with the establishment of cellular senescence, and it converges into the activation of tumor suppressor p53. In addition to this, the upregulation of the histone 2A variants MACROH2A1 and MACRO2A2 has also been observed. These proteins participate in the ATR-dependent formation of Senescence Associated Heterochromatin Foci (SAHFs), dense and repressive chromatin structures characteristic of senescent cells [1,2,27].

Lastly, the deregulated levels of Cyclin-Dependent Kinase Inhibitor 1A or p21 (CDK1NA)  and proliferation marker protein Ki-67 (MKI67) reflect senescent cell cycle arrest at the G2 phase [28,29]. Indeed, p21 increases levels, promoted by a DNA damage-induced p53  pathway, and inhibits the CDK2-cyclin E complex. This causes the dephosphorylation of retinoblastoma protein (RB), which in its hypo-phosphorylated state sequesters E2F, a  transcriptional factor that favors the progression of the cell cycle [2].

Beyond these already-known markers of senescence, a broader pathway enrichment analysis of the most upregulated proteins in H2O2 treated versus control cells points to  p53/p21 pathway activation induced by DNA damage (Figure 2C, Table S2). Instead,  proteins associated with proliferation and mitosis are downregulated, pointing to the consequent proliferation arrest (Figure 2D, Table S3).

2.1.2. SIPS Evaluation by Flow Cytometry

SIPS has also been confirmed by flow cytometry analysis. We observed an increase in cytoplasmic granularity of H2O2-treated cells (Figure S1A, B), due to the augmentation in number and size of the lysosomes in senescent cells. This is the result of the balance between the gradual accumulation of dysfunctional lysosomes and the production of new ones [25]. Moreover, we also detected an intra-lysosomal accumulation of lipofuscin aggresomes: this growth, associated with an increment of autofluorescence in the 525/50 nm bandpass channel of the flow cytometer, is another marker of senescence related to a lysosomal malfunction (Figure S1C, D) [30].

2.2. ObHEx Partially Restored Mitotic Proteins Downregulated in Senescent NHDF Cells

ObHEx treatment was able to partially restore the levels of 71 proteins deregulated in the senescent condition (Two-way ANOVA test q-value < 0.05). More precisely, 46 proteins were downregulated by the extract and 25 were upregulated (Figure 3A, Tables S4 and S5).

Reactome enrichment analysis of the 46 proteins whose levels were decreased by ObHEx suggests that the extract can reduce p53 pathway activation and affect phosphatidylinositol metabolism and Hedgehog signaling, whose alterations were found in aging [31,32] (Figure 3B, Table S6).

Regarding the 25 upregulated proteins, the enrichment analysis points to mitotic cell cycle pathway reactivation and, in particular, to DNA unwinding and replication,  chromosome condensation, and the resolution of sister chromatids (Figure 3C, Table S7).

An in-depth analysis shows that 18 of the 25 restored proteins cluster strongly together (Figure 4A). In this cluster, it is possible to discern CDK1, all the subunits of the condensin I complex (SMC2, SMC4, NCAPD2, NCAPH, and NCAPG), three kinetochore-related proteins (KNTC1, NUF2, and TRIP13), all the six subunits of the replicative minichromosome maintenance (MCM) complex (MCM2-7) together with IQGAP3, PBK, and DHFR.

The ability of ObHEx to partially restore the levels of these proteins is shown well in the profile plots in Figure 4B: protein expression levels were mostly unaltered in the proliferating cells treated or not with the extract, dropped in the senescent cells, and partially increased after ObHEx treatment.

2.3. Biological Assays Confifirmed the ObHEx Promitotic Mechanism of Action

The most common hallmark of senescent cells is the increased SA-β-gal activity that can be evaluated by a staining assay. Our results show that H2O2 treatment induced a  significant increase in the number of SA-β-gal positive cells (blue), from 4.7% to 22.9% (Figure 5A, B). ObHEx incubation significantly reduced SA-β-gal activity with a decrease of 38.4% (p < 0.01).

Moreover, to assess if the restoration of mitotic protein expression by ObHEx translates to the reactivation of the cell cycle, the control, and senescent cells were treated or not with the extract for 72 h and Fluorescence Activated Cell Sorting (FACS) experiments were performed. The senescent cell results showed a block in the G2/M phases of the cell cycle (36.8%), while only a much lower fraction of the control cells (7.2%) were in these phases since they reached confluence, accordingly to the literature [33] (Figure 5C, D). The treatment of senescent cells with ObHEx was able to reduce the G2/M population by 4.3% (p < 0.05). Of notice, when related to the senescence cell portion after SIPS (22.9%), the G2/M escape could be estimated to be 18.9%. The same ObHEx treatment for 48 h has been carried out, but the results were not statistically significant.

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