MB is a more potent ROS scavenger than NAC, MitoQ, and mTEM

To evaluate the effectiveness of MB as an antioxidant, we first compared the effects of MB treatment with the effects of three other popular ROS scavengers, specifically, N-Acetyl-L-cysteine (NAC), MitoQ, and MitoTEMPO (mTEM). (Supplemental Tables 1 and 2). NAC is a widely used, general ROS scavenger that acts as a precursor of glutathione synthesis and stimulates certain enzymes involved in glutathione regeneration15. MitoQ is a modified coenzyme Q10 with a selective accumulation in mitochondria16. mTEM is a mitochondrial-targeting superoxide dismutase mimetic that possesses superoxide and alkyl radical scavenging properties17, 18. In order to evaluate the effects of each antioxidant, primary skin fibroblasts from a middle-aged normal individual and an HGPS patient were treated for 4 weeks. Mitochondrial ROS (indicated by MitoSOX), the main resource of the total cellular ROS, was then measured through FACS analysis. HGPS cells were used as an accelerated model for normal aging since they share many features in common with physiological aging19 (Supplemental Tables 1 and 2).

NAC was supplemented in the culture medium at a concentration of 100 μM according to a previous publication20. In contrast to the anti-aging effects of MB, long-term treatment with NAC did not reduce mitochondrial ROS level and appeared to delay cell proliferation in both normal and HGPS cells (Fig. 1A and B). To our surprise, treatment with MitoQ at 100 nM, as suggested by previous studies16, 21, did not reduce but drastically increased mitochondrial ROS level (Fig. 1C). Additionally, MitoQ treatment did not promote but inhibited cell proliferation in both normal and HGPS cells (Fig. 1D). Treatment of mTEM at 100 nM showed moderate ROS scavenging effects on HGPS cells (Fig. 1E). It also moderately promoted normal cell proliferation but failed to stimulate HGPS cells (Fig. 1F). Amongst all four tested anti-oxidants, MB was the most effective in reducing mitochondrial ROS and promoting skin cell proliferation (Fig. 1A–F, Supplemental Table 2).

Figure 1 MB is a more potent ROS scavenger than NAC, MitoQ and mTEM. (A) Comparison of mitochondrial specific superoxide (MitoSOX) levels in normal and HGPS fibroblasts treated with vehicle, 100 nM MB or 100 μM NAC for four weeks. (B) Growth curves of normal and HGPS fibroblasts during the four-week treatment with vehicle, 100 nM MB or 100 μM NAC. (C) Comparison of MitoSox levels in normal and HGPS fibroblasts treated with vehicle, 100 nM MB or 100 nM MitoQ for four weeks. (D) Growth curves of normal and HGPS fibroblasts during the four-week treatment with vehicle, 100 nM MB or 100 nM MitoQ. (E) Comparison of MitoSox levels in normal and HGPS fibroblasts treated with vehicle, 100 nM MB or 100 nM mTEM for four weeks. (F) Growth curves of normal and HGPS fibroblasts during the four-week treatment with vehicle, 100 nM MB or 100 nM mTEM. (*p < 0.05, **p < 0.01). Full size image

MB reduces aging signs in old skin cells

Next, we asked whether MB treatment could delay or reverse aging phenotypes from skin cells derived from old individuals. Two old dermal fibroblast lines from individuals over 80 years old (3-OM & 4-OF) and two young skin fibroblast lines from individuals below 30 years old (1-YM & 2-YF) were selected for MB treatment (Supplemental Table 1). The old fibroblasts, particularly the 4-OF cells, showed severe senescence phenotypes. At the molecular level, 3-OM and 4-OF lines demonstrated increased SA-β-gal signals and p16 expression, two widely used senescence biomarkers, in comparison to those in the young cells (1-YM & 2-YF) (Fig. 2A and B). FACS analysis revealed much higher levels of mitochondrial ROS in 3-OM and 4-OF than control cells 1-YM and 2-YF (Fig. 2C). Furthermore, the old cells proliferated much more slowly than the young cells (Fig. 2D, solid lines). Particularly, the 4-OF cells stopped growing towards the end of the experiment at passage 18 (Fig. 2D).

Figure 2 MB reduces aging signs in old skin cells. (A) The images of senescence-associated β-galactosidase (SA-β-gal) staining in two young (1-YM & 2-YF) and two old (3-OM & 4-OF) human skin fibroblast lines that were treated with vehicle or 100 nM MB for four weeks. Scale bar = 200 μm. (B) Western blotting analysis with an anti-p16 antibody in the two young (1-YM & 2-YF) and two old (3-OM & 4-OF) cells after four weeks of treatment with vehicle or MB at 100 nM. (C) Relative fold change of mitochondrial superoxide (MitoSOX) levels in the two young and two old fibroblasts after four weeks of treatment with vehicle or MB 100 nM. (D) Growth curves for vehicle- (solid lines) or MB- (dashed lines) treated young and old fibroblasts during the four-week treatment of each drug. (*p < 0.05, **p < 0.01). Full size image

After growing cells in culture medium supplementing 100 nM MB for four weeks, it was evident that the aging-related phenotypes were significantly reduced in the old cell lines 3-OM and 4-OF. MB treatment effectively reduced SA-β-gal signals and decreased the expression of p16 in 3-OM and 4-OF cells (Fig. 2A and B). In addition, MB treatment decreased the elevated MitoSOX in old cell lines, especially in the 3-OM line, to a level comparable to that in young cells (Fig. 2C). Furthermore, growth curve analysis indicated that all cell lines (both young and old) proliferated better in a cell medium supplemented with MB (dotted lines, Fig. 2D). Together, these results indicated that MB treatment is capable of reducing and/or reversing aging phenotypes in old skin fibroblasts.

MB upregulates the expression of Nrf2 and its downstream antioxidant genes

The nuclear factor erythroid 2-related factor 2 (Nrf2) is known as an essential regulator of antioxidant defense system by inducing the expression of an array of antioxidant response element (ARE)-containing genes, thereby decreasing overall cellular ROS22. A recent study has implicated the Nrf2 antioxidant pathway as a driver mechanism in HGPS23. Importantly, MB has been shown to upregulate Nrf2 in neurons24.

We speculate that MB may activate the Nrf2-mediated antioxidative response, thereby simulating ROS quenching in the skin fibroblasts. To test this idea, we first examined Nrf2 expression in all six lines of human dermal fibroblasts used in the studies of Figs 1 and 2. Western blotting analysis confirmed the increased Nrf2 protein amounts in most cell lines treated with MB compared to vehicle control (Fig. 3A and B). The old cell 3-OM did not show an obvious increase of Nrf2 protein upon MB treatment probably due to its extreme senescent cell stage thus limited cells for the analysis. Next, we investigated the mRNA levels of Nrf2 downstream ARE-containing genes in HGPS fibroblast line, where the most significant increase of Nrf2 protein upon MB treatment was observed. Quantitative RT-PCR analysis revealed a significant increase in the mRNA expression of a subset of ARE-containing genes, including GCLC, GSR, GPX7, GSTM1, and TBP (Fig. 3C). Together, these analyses support the idea that MB regulates cellular ROS levels at least partially through activating the Nrf2-mediated antioxidative response.

Figure 3 MB upregulates the expression of Nrf2 and its downstream ARE-response genes in human fibroblasts. (A) Western blotting analysis showing the changes of Nrf2 protein amounts in normal and HGPS fibroblasts upon four weeks of MB treatment at 100 nM. (**p < 0.01) (B) Western blotting analysis showing the changes of Nrf2 protein amounts in the two young and two old fibroblasts after four weeks of treatment upon four weeks of MB treatment at 100 nM. (*p < 0.05) (C) Quantitative RT-PCR analysis showing mRNA levels of Nrf2 and its targeting ARE genes in HGPS fibroblasts after four weeks of MB treatment at 100 nM. (*p < 0.05; **p < 0.01). Full size image

MB increases tissue viability and shows no signs of irritation on in vitro reconstructed 3D human skin

Based on MB’s potential as a powerful antioxidant in 2D fibroblast lines, we then explored the effects of MB on 3D reconstructed human skin epidermis. We used two available skin models: the EpiDerm skin model EPI-200 (Fig. 4A, upper panel) and the EpiDerm Full Thickness skin model EFT-412 (Fig. 4A, lower panel). These in vitro skin models consist of normal human-derived epidermal keratinocytes and fibroblasts cultured at the air-liquid interface on a semi-permeable tissue culture insert (Fig. 4B, details described in Material and Methods), which mimic human normal skin epidermis and are used as approved replacements of Draize rabbits for the in vitro skin irritation test (SIT)18.

Figure 4 MB increases tissue viability and shows no signs of irritation on the in vitro reconstructed 3D human skin. (A) H&E staining images showing two kinds of engineered human skin tissues (obtained from MatTek, Ashland, USA). Upper Panel: EpiDerm EPI-200 consists of normal human-derived epidermal keratinocytes (NHEK) and the outer-most stratum corneum layer. This model was only used for the in vitro skin irritation test in 3 C. Lower Panel: EpiDerm Full Thickness EFT-412 consists of normal human dermal fibroblasts (NHFB), NHEK and stratum corneum, which was used for most of the 3D skin tissue experiment described in this study. (B) Schematic illustrations of top (Left Panel) and side (Right Panel) views of the engineered 3D skin tissue cultured on a microporous membrane insert. (C) The short term skin irritation test. MTT assay was conducted on EPI-200 tissues after topical applications of MB in serial doses for 60 minutes. 5% SDS was used as a positive control (strong irritation) and PBS was used as a negative control (no irritation) in this experiment. (D) Long term cell viability test. MTT assay was conducted on EpiDerm Full-Thickness (EFT-412) skin tissues that had been treated with MB for two weeks at the indicated concentrations (*p < 0.05). Full size image

First, we evaluated MB’s safety by conducting the SIT on the model EPI-200. Skin irritation is characterized by a reversible local inflammatory reaction, and MTT cell viability assay is used to estimate the damage caused by the testing irritant. A reduction of MTT over 50% was indicative of skin irritation, as shown by the positive control (5% SDS, Fig. 4C). MB was tested over a wide range of concentrations from 0.2 μM to 500 μM. None of these dosages significantly affected cell viabilities upon a 60-minute topical exposure (Fig. 4C).

To further test the potential irritations of MB after long-term application on the skin, we supplemented MB at different concentrations in culture medium and incubated the full thickness skin model EFT-412 in these media for a total of two weeks. On day 14, the MTT assay was performed. During this two-week incubation period, we noticed the skin tissues treated with high concentrations of MB (5.0 μM and above) started to appear blue after 3 days, suggesting that MB dosage needs to be limited to avoid its colorant side effect on skin appearance. The tissues treated with lower concentrations of MB (from 0.1 μM to 2.5 μM) did not show any tissue coloring. Consistent with the ability of MB to stimulate cell proliferation, we noticed that, at the dosage of 0.5 μM, MB significantly increased cell viability in comparison to the PBS control. In addition, the tissues treated with higher concentrations of MB (5.0 μM and 10.0 μM) showed a reduction in cell viability (Fig. 4D). Based on these results, it can be concluded that low concentrations of MB (less than 2.5 μM) neither irritate nor color skin and are therefore safe for long-term use. As a result, we performed the follow-up studies with MB at concentrations below 2.5 μM.

MB increases skin thickness and hydration

Human skin thickness decreases at an averaged rate of 6% per decade25. The gradually thinning skin with age mainly includes a thinner epidermis and dermis, which results in a lowered resistance to shearing forces and higher susceptibility to wounds after trauma25, 26. To study the effect of MB treatment on skin thickness, we conducted H&E staining on the EFT-412 skin tissues that had been incubated in culture medium containing 0.1 μM, 0.5 μM or 2.5 μM MB for two weeks, with a fresh replacement of each medium daily. Cross sections of the dermis were then measured after H&E staining (Fig. 5A). We noticed that MB treated skin tissues showed thicker dermis layers than the control PBS-treated dermis (Fig. 5A and B). Quantitative analysis further revealed that the greatest increase in dermis thickness occurred at 0.5 μM MB (Fig. 5B). We also attempted to analyze the thickness of stratum corneum and epidermis layers in those H&E stained skin tissues but found that the thicknesses of these layers were intrinsically highly variable, likely due to tissue preparation and experimental handling.

Figure 5 MB increases skin thickness and hydration. (A) Representative cross-section images from H&E staining of Epiderm EFT-412 tissues after MB treatment at various concentrations for two weeks. The three skin layers are pointed by colored arrows. Red: the stratum corneum layer, Blue: the keratinocyte layer and black: the dermis fibroblast layer. Scale bar = 200 μm. (B) Histogram plot comparing the thickness of fibroblast layer (black arrows in A) under different concentrations of MB treatment. The thickness of fibroblast layer was quantified using ImageJ on six consecutive slides for each skin tissue and the average thickness was shown. (C) Skin hydration assessed by DPM9003 device showing significantly escalated levels of hydration on the Epiderm EFT-412 skin tissues after two weeks of MB treatment at 0.5 and 2.5 μM. (*p < 0.05, **p < 0.01). Full size image

Human skin retains water mostly through the outermost stratum corneum layer. Loss of hydration in aged skin, due to a decline in function of the stratum corneum, results in a sagging and wrinkling appearance27, 28. To study the effect of MB on the stratum corneum layer, we evaluated the water content of the EFT-412 skin tissues that had been incubated in culture medium supplemented with MB for two weeks. In this experiment, the electrical impedances of skin tissues were determined and used as indicators of water content. Consistent with the dermis thickness measurement, we found that skin hydration levels were significantly higher in EFT-412 tissues treated with MB at 0.5 μM and 2.5 μM compared to the PBS control (Fig. 5C). Together, these experiments revealed that MB treatment increases skin dermis thickness and improves skin hydration.

MB treatment alters the expression of a subset of ECM proteins, including upregulation of elastin and collagen 2A1

Elastin, one of the most abundant ECM components in skin dermis, plays an important role in maintaining skin elasticity and resilience. It is synthesized and secreted by dermal fibroblasts and organizes with other ECM proteins into high-order structures2, 3. During physiological aging, the elastin production remains relatively stable up to 30~40 years of age then drastically declines afterward3, 29. Our previous study indicated that the elastin mRNA level is upregulated by at least two folds in MB-treated normal fibroblasts compared to mock-treated control cells13. To test whether this result is transferrable from 2D fibroblast culture to 3D human skin models, we extracted RNA from EFT-412 skin tissues following two-week MB treatment at 0.1, 0.5 and 2.5 μM concentrations. Quantitative RT-PCR analysis revealed that the elastin mRNA levels were significantly increased in the skin tissues treated with MB at all three dosages (Fig. 6A). Western blotting analysis further confirmed the increased elastin protein in MB treated-skin tissues compared to vehicle control (Fig. 6B). Immunohistochemistry with an anti-elastin antibody on the EFT-412 tissue cross sections revealed significantly more elastin fibers in the dermis in 0.5 μM or 2.5 μM MB-treated skin, and a moderate increase of elastin fibers in 0.1 μM MB-treated tissue samples (Fig. 6C).

Figure 6 MB upregulates elastin expression and alters other ECM genes expression on 3D skin tissue. (A) Quantitative PCR analysis showing significantly upregulated mRNA levels of elastin in EFT-412 skin tissue after two weeks of MB treatment at 0.1, 0.5, or 2.5 μM. (**p < 0.01) (B) Western blotting analysis showing increased elastin protein amounts in EFT-412 skin tissues upon two-week treatment of MB at 0.1, 0.5, or 2.5 μM. (**p < 0.01) (C) Representative IHC images showing the signals from an anti-elastin antibody (green) on the paraffin slides of 3D skin tissues. Two-week treatment with MB raised elastin signals compared to PBS treatment. Scale bar = 100 μm. (D) qPCR analysis showing the significantly up- or down-regulated ECM genes in EFT-412 skin tissues in response to MB treatment compared to PBS treatment. Full size image

To further explore whether MB regulates additional ECM components besides elastin, we screened ECM genes using the Bio-Rad PCR array, which contains 30 genes known to be involved in human ECM remodeling. Of these 30 genes, five genes, including COL2A1, IGF1, KLK3, AC002094.1, and PLG, were upregulated by MB and two genes, MMP9 and LAMC2, were downregulated by MB in EFT-412 tissues. Notably, most of these genes showed a dose-dependent response to the concentration of MB (Fig. 6D).

MB promotes wound healing in dermal fibroblasts

Cutaneous wound healing processes include epidermal keratinocyte migration, dermal fibroblast migration, and the interactions of these cells with the ECM30. The skin repair capabilities decline with age due to structural and functional changes, such as reduced proliferation and migration of fibroblasts and degraded collagen and elastin in the ECM31. Based on the results from Figs 1–6, we speculate that MB treatment will promote the wound healing of the skin.

To test this hypothesis, we performed an in vitro wound assay, which mimics the cutaneous wound healing process30, 32. Fibroblast monolayers were wounded with a scratch and images of cell movement in the scratched area were captured at 0 and 24 hours post wounding. Two normal skin fibroblast lines, one derived from a middle-aged individual and the other from an 84-year old individual, were investigated. As expected, fibroblasts from the middle-aged donor exhibited faster recovery than those from the old donor (Fig. 7A–C). Significantly, the MB-treated fibroblasts in both cell lines repopulated significantly faster than their vehicle-treated counterparts (Fig. 7A–C), suggesting that MB treatment promotes wound healing.

Figure 7 MB promotes wound healing in dermal fibroblasts. (A,B) Representative images showing skin fibroblasts migration in the scratch-wounded area at 0 and 24 hours post wound. The cells were pre-treated with Vehicle (PBS) or 100 nM MB for one week. The wound region was then manually created by scrapping a straight line across the cultured cells. Fibroblasts from a middle-aged donor (A, HGFDFN168, 40 yrs) and an old-aged donor (B, AG11725, 84 yrs) were tested. Scale bar = 200 μm. (C) Quantification of the cell number in each wounded region at 24 hours post wound. (*p < 0.05, **p < 0.01). Full size image

In summary, our analyses using the 2D dermal fibroblasts and 3D reconstructed skin models support the idea that MB is a safe and potent anti-oxidant, and has great potential to be used in skin care.