Combination of Autophagy and Stem Cell Enhancing Properties of Natural Product Extracts in Human Dermal Papilla Stem Cells

Abstract

Background/Aim

Dermal papilla (DP) stem cells are known for their remarkable regenerative capacity, making them a valuable model for assessing the effects of natural products on cellular processes, including stemness, and autophagy.

Materials and Methods

Autophagy and stemness characteristics were assessed using real-time RT-PCR to analyze mRNA levels, along with immunofluorescence and western blot techniques for protein level evaluation.

Results

Butterfly Pea, Emblica Fruits, Kaffir Lime, and Thunbergia Laurifolia extracts induced autophagy in DP cells. Kaffir Lime-treated cells exhibited increase in the OCT4, NANOG, and SOX2 mRNA (6-, 5, and 5.5-fold, respectively), and protein levels (4-, 3-, and 1.5-fold, respectively). All extracts activated the survival protein kinase B (Akt) in DP cells.

Conclusion

Natural products are a promising source for  promoting hair growth by rejuvenating hair stem cells.

Recent advancements in stem cell and tissue engineering research offer promising avenues for utilizing stem cell techniques to regenerate hair and prevent hair loss. Specifically, dermal papilla (DP) cells, hair mesenchymal stem cells, have shown potential for hair regeneration and induction (1,2). Damage, depletion, or inactivity of DP stem cells can lead to hair loss, particularly in conditions like androgenetic alopecia, where DP cells show reduced activity, resulting in shorter and thinner hair production (3). Studies have demonstrated that the decrease in the stemness of DP cells directly correlates with a decline in their ability to regenerate hair (4). Autophagy, the process of cellular self-digestion, is observed in both keratinocytes and mesenchymal cells surrounding hair follicles (5). Recent research highlights the active involvement of autophagy in hair growth, with its inhibition leading to follicle regression (6). Autophagy supports stem cell preservation, essential for self-renewal (7). In addition, the survival PI3K/Akt signaling pathway has been long shown to control cell proliferation and induce stem-like characteristics in human cells (8). In addition, activation of Akt signaling has been shown to be essential for the de novo hair follicle regeneration (9).

Research on plant-derived compounds and extracts has shown promising effects on DP cells, aiding in prolonging the anagen phase and stimulating hair growth (10). In our research, Butterfly Pea, Emblica Fruits, Kaffir lime peels, Soybean, and Thunbergia Laurifolia extracts were selected to study the potential to induce stemness as well as autophagy in human DP cells.

Materials and Methods

Methods for extraction of natural products. Herbal extracts were prepared by maceration and solvent extraction methods. The desired part of plants was dried in hot air oven at 50˚C for 10-12 h. The dry plants (batches of 200 g) were grounded slightly and soaked in ethanol: water solution for 24 h. The contents were then filtered through Whatman filter paper no.1. The filtrates were then evaporated using a rotary evaporator (Büchi R-210, Flawil, Switzerland) under 100 mbar pressure at 40˚C.

Cells. Human primary DP cells were obtained from Applied Biological Materials Inc. (Richmond, BC, Canada) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 2 mM glutamine, and 1% Anti-Anti (Gibco). The cells were maintained at 37˚C with 5% CO₂.

Cell viability assay. Viability was determined at 24 h and 48 h using the MTT reagent (4 mg/ml in PBS) and incubated for 3 h. The MTT reagent was removed and DMSO (100 μl) was added to solubilize the formazan crystals. The density of the resulting purple color was measured at 570 nm using a microplate reader (CLARIOstar, BMG Labtech, Ortenberg, Germany).

Monodansylcadaverine staining. DP cells were treated with various concentration of extracts (0, 10, 20, 100 μg/ml) for 6 h. The cells were stained with mono dansyl cadaverine (0.1 mM) at 37˚C for 30 min. The fluorescence intensity was captured using a fluorescence microscope (Olympus IX 51 with DP70, Olympus America Inc., Center Valley, PA, USA).

Western blot analysis. The cells were incubated with lysis buffer for 30 min on ice. Cell lysates were collected. The protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After gel separation, the proteins were transferred onto nitrocellulose membrane and incubated with 5% nonfat dry milk in TBST for 1 h and subsequently incubated with appropriated antibodies. A chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA) was used to detect proteins and the signal was quantified using densitometry.

Real time polymerase chain reaction (RT-PCR). The RNA was extracted using the GENEzol reagent. SuperScript III reverse transcriptase was utilized to synthesize cDNA from total RNA. After the cDNA synthesis, 100 ng of the cDNA was employed in RT-PCR using Luna Universal qPCR Master Mix (NEB, Ipswich, UK) according to manufacturer’s instruction. The reaction was conducted using the CFX 96 Real-time PCR system (Bio-Rad, Hercules, CA, USA). The relative mRNA gene expression level for each gene was determined based on the comparative Cq values.

Immunofluorescence. The cells were fixed with 4% paraformaldehyde for 15 min. The cells were permeabilized with 0.5% triton-X for 5 min and non-specific protein binding was blocked following incubation with 10% FBS in 0.1% Triton-X PBS for 1 h at room temperature. The cells were incubated with 1:400 of primary antibodies (OCT4, NANOG, SOX2) at 4˚C for overnight. The images were captured using a fluorescence microscope (Olympus IX 51 with DP70, Olympus America Inc.).

Statistical analysis. The results, presented as mean±standard deviation (SD), were derived from at least three independent experiments. Statistical analyses involved one-way ANOVA with post hoc tests using GraphPad Prism 9 software (La Jolla, CA, USA). A p-value <0.05 was considered statistically significant for group differences.

Results

Cytotoxicity of natural product extracts on DP cells. The cytotoxicity of natural product extracts, including Butterfly Pea, Emblica Fruit, Kaffir Lime, Soybean, and Thunbergia Laurifolia, was assessed in human DP cells using the MTT assay. Results indicated significant cytotoxic effects of Emblica Fruit and Kaffir Lime at 1,000 μg/ml after 24 h, while Butterfly Pea, Soybean, and Thunbergia Laurifolia showed no toxicity at that concentration within the same timeframe (Figure 1A). Similar trends were observed after 48 h, although with different IC₅₀ values (Figure 1B). The IC₅₀ values for Butterfly Pea, Soybean, and Thunbergia Laurifolia exceeded 1,000 μg/ml at both 24 and 48 h. However, for Emblica Fruit and Kaffir Lime, the IC₅₀ values were 998 μg/ml and 640.1 μg/ml at 24 h, respectively, and 540 μg/ml and 353.3 μg/ml at 48 h, respectively (Figure 1C and D).

Figure 1. Cytotoxicity of natural products on human dermal papilla (DP) cells. (A, B) The effect of extracts from Butterfly Pea, Emblica Fruit, Kaffir Lime Peel, Soybean, Thunbergia Laurifolia on DP cell viability at 24 and 48 h, respectively was evaluated using MTT assays. (C, D) IC₅₀ values calculated for different extracts in DP cells treated for 24 h and 48 h, respectively. Data are presented as mean±SD (n=3). Significant compared to the control group, *p<0.05, ***p<0.001 versus untreated control cells.

Natural product extracts induced autophagy. Treatment of DP cells with various concentrations of extracts (0-100 μg/ml) for 6 h resulted in the formation of cytoplasmic vacuoles. Staining with monodansylcadaverine (0.1 mM) confirmed the presence of autophagosomes in the cells treated with extracts compared to untreated cells, with higher number of green fluorescence vesicles indicative of autophagic vacuoles (Figure 2A). Additionally, western blot analysis confirmed the increased conversion of LC3B-I to LC3B-II in most extracts except Soybean. Butterfly Pea (3.2 fold), Emblica Fruit (3 fold), Kaffir Lime Peel (2.8 fold), and Thunbergia Laurifolia (2.8 fold) treated DP cells exhibited significantly elevated levels of LC3B-II, indicating enhanced autophagy (Figure 2B). Overall, these extracts induced the highest levels of autophagy activity in DP cells.

Figure 2. Effect of natural product extracts on autophagy and autophagy-related proteins in dermal papilla (DP) cells. (A) DP cells were treated with natural product extracts (0-100 μg/ml) for 6 h and stained with monodansylcadaverine (0.1 mM). Images were visualized using fluorescence microscopy. Fluorescence intensity was measured using image J software. (B) DP cells were treated with natural product extracts (20 μg/ml) for 14 h. LC3B-II protein levels were determined using western blot. The blot was reprobed with β-actin to confirm equal loading of protein. Data are presented as mean±SD (n=3). Significant compared to the control group, *p<0.05, **p<0.01, ***p<0.001 versus untreated.

Natural product extracts induced high expression of stemness transcription factors mediated by the Akt-dependent mechanism in DP cells. To investigate the effect of extracts on stemness, DP cells were treated with extracts at 0-20 μg/ml for 14 h. Subsequently, mRNA levels of stem cell transcription factors were assessed. Overall treatment with extracts led to up to 6-fold increase in OCT4 mRNA expression in DP cells, particularly with Kaffir Lime treatment (Figure 3A). Immunofluorescence analysis further confirmed higher OCT4 protein expression in DP cells treated with Kaffir Lime (Figure 3C).

Figure 3. The effect of natural products on the expression of stemness transcription factors in dermal papilla (DP) cells was mediated by a mechanism dependent on Akt. (A) DP cells were treated with natural product extracts (0-20 μg/ml) for 14 h, and the mRNA expression levels of stemness transcription factors, OCT4, NANOG, and SOX2 were determined using RT-PCR. The mRNA level was normalized using the housekeeping mRNA GAPDH. The relative mRNA expression was calculated by using comparative Ct cycles. (B) The expression levels of pAkt and (C, D, E) stemness transcription factors, OCT4, NANOG, and SOX2, respectively, were captured using immunofluorescence and the fluorescence intensity was determined using Image J software. (F) The protein expression levels of stemness transcription factors, OCT4, and Akt and its activated form, p-Akt (Ser 473) were determined using western blot. The blots were reprobed with β-actin to confirm equal loading of protein and band intensity was determined using Image J software. Data are presented as mean±SD (n=3). Significant compared to the control group, *p<0.05, **p<0.01, ***p<0.001 versus untreated control cells.

The NANOG mRNA was found to be 5-fold higher in DP cells treated with Emblica Fruit, Kaffir Lime, and Thunbergia Laurifolia extracts (Figure 3A). Furthermore, the fluorescence signal of NANOG was notably increased by 3-fold in DP cells upon treatment with these natural product extracts (Figure 3D). SOX2 mRNA showed a significant increase, approximately 5.5-fold, in DP cells treated with extracts from Emblica Fruits, Kaffir Lime, and Soybean (Figure 3A). Notably, the protein expression level of the SOX2 marker increased by 1.5-fold in DP cells treated with extracts from Emblica Fruits, Kaffir Lime, and Soybean (Figure 3E). Overall, expression of OCT4, NANOG, and SOX2 transcription factors increased in all DP cells treated with natural product extracts.

In addition, the effect of extracts on Akt signal, particularly its activated form pAkt (Ser473), was evaluated in DP cells using immunofluorescence and western blot analysis. DP cells treated with extracts from Emblica Fruits, Kaffir Lime, Soybean, and Thunbergia Laurifolia showed a 1.5-fold increase in pAkt fluorescence signal (Figure 3B) and a notable over 3-fold increase in pAkt protein expression compared to controls (Figure 3F).

According to our research, the tested extracts induced activities of autophagy, Akt signaling, and stemness in DP cells (Figure 4).

Figure 4. The effects of natural product extracts revealed that autophagy, and stem cells properties are interconnected and are important for the maintenance of hair follicles in dermal papilla (DP) cells. HS: Hair shaft; B: bulge; APM: arrector pili muscles; SG: sebaceous gland; HF: hair follicle.

Discussion

Hair follicles undergo a unique life cycle, continuously renewing and regenerating throughout one’s life. Stem cells within them engage in constant self-renewal and differentiation, crucial for regulating hair growth and skin balance. The DP acts as a crucial signaling center, maintaining hair-inducing activity and supporting hair follicle regeneration and development (2,11).

Autophagy is crucial for hair follicle maintenance (12). Our research indicates that Butterfly Pea, Emblica Fruits, Kaffir Lime, and Thunbergia Laurifolia extracts effectively induce autophagy in DP cells (Figure 2). Increasing autophagy can facilitate entry into the active growth phase (anagen) of the natural hair follicle cycle (5). Autophagy also maintains the self-renewal and stemness properties of hair follicles. Stem cells within hair follicles play key roles in wound healing and follicle regeneration, possessing the ability to undergo reprogramming (13).

Our findings also highlight that Kaffir Lime extracts significantly upregulate pluripotent stem cell markers, including OCT4, NANOG, and SOX2 (Figure 3A, C-E). These markers play a crucial role in regulating the self-renewal processes necessary for hair follicle formation (2). OCT4 acts as upstream in the signaling pathway governing stem cell self-renewal capacity (14). NANOG expression is vital for sustaining cell self-renewal, regulated by OCT4 and SOX2 (15). NANOG and OCT4 accumulation in DP cells can prolong self-renewal capacity and preserve stemness, preventing cell differentiation (16). In addition, Kaffir Lime extracts could induce Akt activation (Figure 3B and F). Akt activation occurs in regions like the epidermis, hair infundibulum, bulge, and hair bulb during hair follicle stem cell proliferation, promoting the Wnt/β-catenin pathway for hair regeneration (10). Our findings indicate that extracts from Emblica Fruit, Soybean, and Thunbergia Laurifolia activate the Akt signaling pathway (Figure 3B and F). This activation correlates with increased expression of pluripotent stem cell markers, such as OCT4, NANOG, and SOX2.

The current research findings reveal that in a cell-based model, extracts obtained from Butterfly Pea, Emblica Fruits, Kaffir Lime, Soybean, and Thunbergia Laurifolia demonstrate the ability to enhance autophagy and induce stemness characteristics in DP cells. However, we believe that utilizing animal models and conducting clinical investigations should be recommended to explore such activities for future evaluations.

In conclusion, we observed a positive correlation between autophagy, activated Akt, and cell stemness in DP cells, as illustrated in Figure 4. This interplay is crucial for hair maintenance and regeneration. Information on extracts enhancing the stemness as well as its potentiating factors would benefit the development of novel hair regenerative approaches.

Funding

This research was funded by Thailand Science research and Innovation Fund Chulalongkorn University.

Conflicts of Interest

All Authors declare that there are no conflicts of interest in relation to this study.

Authors’ Contributions

Conceptualization, P.C.; methodology, P.C. and V.C.; validation, P.C. and V.C.; formal analysis, Z.Z.E., V.C. and P.C.; investigation, Z.Z.E. and P.C.; resources, S.K.; P.H. and P.C.; writing-original draft preparation, Z.Z.E.; writing-review and editing, P.C.; supervision, P.C. All Authors have read and agreed to the published version of the manuscript.

Acknowledgements

The Author (Z.Z.E.) is grateful to the Second Century Fund (C2F) for the postdoctoral fellowship, Chulalongkorn University.