Standardized Kaempferia parviflora Extract Inhibits Intrinsic Aging Process in Human Dermal Fibroblasts and Hairless Mice by Inhibiting Cellular Senescence and Mitochondrial Dysfunction

Intrinsic skin aging is a complex biological phenomenon mainly caused by cellular senescence and mitochondrial dysfunction. This study evaluated the inhibitory effect of Kaempferia parviflora Wall ex. Baker ethanol extract (KPE) on H2O2-stimulated cellular senescence and mitochondrial dysfunction both in vitro and in vivo. KPE significantly increased cell growth and suppressed senescence-associated β-galactosidase activation. KPE inhibited the expression of cell-cycle inhibitors (p53, p21, p16, and pRb) and stimulated the expression of cell-cycle activators (E2F1 and E2F2). H2O2-induced hyperactivation of the phosphatidylinositol 3-kinase/protein kinase B (AKT) signaling pathway was suppressed by KPE through regulated expression of forkhead box O3a (FoxO3a) and mammalian target of rapamycin (mTOR). KPE attenuated inflammatory mediators (interleukin-6 (IL-6), IL-8, nuclear factor kappa B (NF-κB), and cyclooxygenase-2 (COX-2)) and increased the mRNA expression of PGC-1α, ERRα, NRF1, and Tfam, which modulate mitochondrial biogenesis and function. Consequently, reduced ATP levels and increased ROS level were also reversed by KPE treatment. In hairless mice, KPE inhibited wrinkle formation, skin atrophy, and loss of elasticity by increasing the collagen and elastic fibers. The results indicate that KPE prevents intrinsic aging process in hairless mice by inhibiting cellular senescence and mitochondrial dysfunction, suggesting its potential as a natural antiaging agent.


Introduction
Intrinsic skin aging is a complex biological phenomenon mainly caused by intracellular stressors [1]. Among various factors that accelerate intrinsic aging, the major causes are cellular senescence and mitochondrial dysfunction [2]. When proliferating cells are exposed to various types of stressors, they may undergo growth arrest, termed as cellular senescence [3]. Senescent cells exhibit various phenomena, such as cell-cycle arrest, gene expression changes, and secretion of inflammatory cytokines [4]. Reactive oxygen species (ROS) mainly accelerate cellular senescence and also play a role in determining the lifespan of mammalian cells [1]. Although senescent cells are relatively rare in young organisms, their number increases according to the aging. The most obvious sign of cellular senescence is cell-cycle arrest in the G1 phase with an altered gene expression pattern [3].
The phosphatidyl inositol-3 kinase (PI3K)/AKT signaling pathway is known to modulate cellular senescence and longevity in many organisms [6]. Oxidative stress-induced cellular senescence stimulates the PI3K/AKT pathway, subsequently suppressing forkhead box O (FoxO) transcriptional factors and elevated mammalian target of rapamycin (mTOR) [7]. In addition, the accumulation of senescent cells with aging affects neighboring cells resulting in tissue damage and inflammation. The presence of many inflammatory cytokines and mediators secreted by senescent cells can destroy tissue structures and functions [8]. Mitochondria also play a critical role in aging. During the aging process, mitochondria lose their function, leading to a decline in mitochondrial biogenesis and ATP production [9]. Mitochondrial dysfunction or impaired mitochondrial activity can contribute to imbalance in energetic metabolism and oxidative stress resulting in ROS accumulation. ROS are generated as by-products of ATP synthesis in mitochondria [10]. Excessively accumulated ROS negatively influence various cellular components such as proteins, lipids, and nucleic acids, leading to age-associated phenotypes and diseases [11]. Accordingly, antiaging is associated with mitochondrial homeostasis [12]. Peroxisome proliferator-activated receptor coactivator 1 (PGC-1 ) is a key activator of mitochondrial biogenesis and function by stimulating stimulates downstream factors such as estrogen-related receptor alpha (ERR ), nuclear respiratory factor 1 (NRF1), and mitochondrial transcription factor A (Tfam) responsible for replication, transcription, and translation of mitochondrial DNA (mtDNA) [12]. Thus, a low level of PGC-1 gives rise to mitochondrial dysfunction and aging.
Kaempferia parviflora Wall. ex Baker, commonly called black ginger, has been used as a dietary supplement and traditional medicine in tropical countries [13]. K. parviflora is reported to have antioxidative, anti-inflammatory, antiviral, and anticancer activities [13][14][15]. However, its effect on intrinsic skin aging has not been verified. We investigated the inhibitory effect of K. parviflora on intrinsic skin aging process by evaluating its effect on cellular senescence and mitochondrial dysfunction using H 2 O 2 -exposed human dermal fibroblasts. In addition, its effect on skin aging phenotypes was evaluated using hairless mice.

Preparation of Standardized K. parviflora Extract (KPE).
Rhizomes of K. parviflora were collected in Bangkok, Thailand. The dried rhizomes were ground and extracted with 95% ethanol in a shaking water bath at 60 ∘ C for 3 h. Then, the extract was concentrated using a rotary evaporator (Heidolph Instruments GmbH & Co. KG., Schwabach, Germany) to obtain the KPE with a yield of 8.9% (w/w), including 14.1% (w/w) 5,7-dimethoxyflavone as a bioactive compound [16]. weeks. For the young group, 7-week-old female hairless mice were purchased from Orient Bio Inc. (Seongnam, Korea) at 1 week before they were killed and were allowed to acclimatize for 1 week. After 24 weeks, the mice were killed under anesthesia by using an intraperitoneal injection of a mixture of zoletil (Virbac, Carros, France) and rompun (Bayer Korea Ltd., Seoul, Korea). Sample from the dorsal skin were rapidly frozen in liquid nitrogen and stored at −70 ∘ C. Skin biopsy samples were fixed in 10% buffered formalin for optical microscopy in order to analyze histological changes. All efforts were made to minimize the suffering of mice.

Cell Growth
Assay. Cell growth was evaluated using an MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich]. The cells cultured in 96well plates for 24 h were pretreated with the samples (KPE or resveratrol) for 24 h in a serum-free medium and were subsequently exposed to 600 M H 2 O 2 in order to induce growth arrest. After a further 72 h of treatment, the culture medium was replaced with MTT solution (0.1 mg/ml) and incubated in darkness for an additional 3 h. The insoluble formazan products were dissolved in dimethyl sulfoxide (DMSO) and were measured using a VersaMax tunable microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 540 nm for absorbance.

Senescence-Associated -Galactosidase
Assay. The cellular SA--gal activity was evaluated using a 96-well cellular senescence assay kit (Cell Biolabs, San Diego, CA, USA), following the described assay protocol. The whole lysate of cells treated with cold 1x cell lysis buffer was centrifuged for 10 min at 4 ∘ C. Next, 50 l of the supernatant was mixed with an equal amount of 2x assay buffer and incubated at 37 ∘ C for 1 h. The activity of SA--gal was then measured using a fluorescence plate reader (GloMax5 Multi Microplate Reader; Promega, Madison, WI, USA) at 360 nm (excitation)/465 nm (emission).

Evaluation of Skin Wrinkle Formation.
The dorsal skin surface of anesthetized hairless mice was analyzed with replica (Epigem, Seoul, Korea) and Visioline VL650 (CK Electronics GmbH, Cologne, Germany).

Hydroxyproline Assay.
Skin tissues (100 mg) were homogenized using glass beads in 1 mL of 6 N HCl and were hydrolyzed at 105 ∘ C for 20 h [13]. The hydroxyproline content was then measured using a hydroxyproline assay kit (QuickZyme Biosciences, Leiden, Netherlands).

Evaluation of Skin Elasticity.
Skin elasticity was evaluated using Cutometer5 MPA580 (CK Electronics GmbH). Among the parameters, 2 was used as the main parameter to assess skin elasticity and skin aging.

Statistical Analysis.
Results are expressed as the mean ± standard deviation (SD) of triplicate experiments. All groups were compared via one-way analysis of variance (ANOVA), followed by Scheffe's test using SPSS 21 software (Chicago, IL, USA). values less than 0.05 were marked and considered statistically significant: # p < 0.05 and ## p < 0.01 (normal versus H 2 O 2 control and young versus MA group); * < 0.05 and * * < 0.01 (H 2 O 2 control versus sample-treated cells and young versus MA group).

Effect of KPE on Cell Growth
In Vitro. Cellular senescence inhibits cell proliferation and decreases the number of cells [17]. H 2 O 2 exposure reduced cell proliferation compared to the normal cells; however, KPE treatment significantly reinstated the proliferative activity of Hs68 cells to almost the normal level (Figure 1(a)). KPE at 1, 5, and 10 g/ml showed 1.35-, 1.41-, and 1.40-fold increase in relative cell growth, respectively, compared to that in the H 2 O 2 control.

Effect of KPE on H 2 O 2 -Induced SA--Gal Expression In
Vitro. Senescent cells are featured by the activation of SA--gal, the most important biomarker to identify cellular senescence [3,18]. H 2 O 2 exposure caused almost a 1.39-fold increase in the SA--gal activity in Hs68 cells, but this activity was dose-dependently decreased by KPE (Figure 1(b)). The relative SA--gal activity after treatment with 1, 5, and 10 g/ml KPE showed a 17.6%, 20.5%, and 33.0% reduction, respectively, compared to that in the H 2 O 2 control.

Effect of KPE on H 2 O 2 -Induced Cell-Cycle Arrest In Vitro.
Cell-cycle arrest in the senescent state results from alteration of markers associated with the cell cycle, including cell-cycle inhibitors (p53, p21, p16, and pRb) and cell-cycle activators (E2F1 and E2F2) [5]. The mRNA levels of cell-cycle inhibitors were significantly reduced by KPE treatment (Figure 2(a)). The protein expression of cell-cycle inhibitors was dosedependently reduced upon KPE treatment (Figure 2(b)). The mRNA expression of cell-cycle activators was downregulated upon H 2 O 2 exposure but was highly elevated by KPE (Figure 2(c)).

Effect of KPE on H 2 O 2 -Induced Activation of the PI3K/ AKT Pathway In Vitro.
Oxidative stress activates the PI3K/ AKT pathway, triggering cellular senescence. PI3K/AKT downstream markers such as FoxO3a and mTOR are also intimately associated with cellular senescence [7]. The H 2 O 2induced expression of PI3K and phospho-AKT was decreased by KPE without any visible changes in the total AKT level compared to that in the H 2 O 2 control (Figure 3(a)). In addition, KPE treatment increased FoxO3a (an active form) and decreased phospho-FoxO3a (an inactive form) levels. Phospho-mTOR protein expression was increased by H 2 O 2induced cellular senescence, but KPE significantly reduced its expression without changing the level of total mTOR (Figure 3(b)).

Effect of KPE on SIRT1 Expression
In Vitro. SIRT1 is known as an effective antiaging factor that plays a central role in cellular senescence [4]. SIRT1 mRNA expression was significantly upregulated by KPE compared to that in the H 2 O 2 control. The protein level of SIRT1 was also increased in KPE treated Hs68 cells (Figure 4).

Effect of KPE on H 2 O 2 -Induced Inflammatory Responses
In Vitro. Senescent cells show various increased senescenceassociated secretory phenotypes (SASP), including cytokines, and other factors [8]. Senescence-associated inflammatory markers including interleukin-6 (IL-6), IL-8, nuclear factor kappa B (NF-B), and cyclooxygenase-2 (COX-2) were investigated to verify the anti-inflammatory effect of KPE in senescent fibroblasts. H 2 O 2 -induced senescent Hs68 cells exhibited higher IL-6 and IL-8 mRNA levels than that in normal cells; however, KPE treatment markedly reduced these mRNA levels ( Figure 5(a)). Additionally, the protein expression of other inflammatory markers, NF-B and COX-2, was also decreased by KPE treatment as compared to that in the H 2 O 2 control ( Figure 5(b)).     cells showed 13% reduction in ROS levels compared to those in the H 2 O 2 control ( Figure 6).

KPE Increases Mitochondrial Biogenesis Transcription
Factor Expression In Vitro. To clarify whether KPE treatment regulates mitochondrial biogenesis transcription factors, the mRNA expression of PGC-1 , ERR , NRF1, and Tfam was measured in human dermal fibroblasts. The H 2 O 2 control showed lower mRNA expression of PGC-1 than that in normal cells. The expression of other transcription factors including ERR , NRF1, and Tfam also decreased in the H 2 O 2 control because these transcription factors are regulated via PGC-1 activation. However, KPE treatment elevated the mRNA expression of PGC-1 and its downstream genes, ERR , NRF1, and Tfam. Based on these data, KPE stimulates the expression of mitochondrial biogenesis transcription factors by upregulating PGC-1 expression (Figure 7).

KPE Attenuates SA--Gal Activity In Vivo.
In the intrinsically MA group, SA--gal activity is significantly elevated; however, this activity showed 25.5% reduction upon oral administration of KPE compared to that in the intrinsically MA group (Figure 8(a)). The results demonstrate that KPE treatment results in pronounced attenuation of age-related SA--gal activation, indicating its inhibitory effect on cellular senescence.

KPE Recovers Cell-Cycle Arrest In
Vivo. Compared to young mice, intrinsically MA mice showed increased mRNA and protein levels of cell-cycle inhibitors, including p53, p21, p16, and pRb. In the KPE administered group, the p53, p21, p16, and pRb levels exhibited 33.1%, 44.4%, 40.8%, and 37.4% reduction, respectively, compared to those in the intrinsically MA group. The protein levels of cell-cycle inhibitors were also attenuated by KPE treatment (Figures 8(a) and 8(c)).

KPE Increases Mitochondrial Biogenesis In Vivo.
The level of PGC-1 , a crucial regulator of mitochondrial function as well as mitochondrial biogenesis, is decreased with aging [2]. The mRNA levels of PGC-1 and its downstream genes were reduced in intrinsically MA mice; however, KPE treatment upregulated the expression of these genes ( Figures  9(b) and 9(c)), suggesting an enhancing effect of KPE on mitochondrial function in MA mice. The protein level of PGC-1 was consistent with its mRNA level. Consequently, KPE increased the mtDNA involved in mitochondrial function and biogenesis, supporting the observation that KPE improved mitochondrial function and biogenesis through PGC-1 stimulation (Figure 9(a)).

KPE Reduces Wrinkle Formation.
Wrinkle formation is a major characteristic of intrinsic skin aging [19]. Compared to young mice, intrinsically MA mice showed elevated wrinkle values, such as total area, number, length, and depth of wrinkles; however, the KPE administered group showed lower wrinkle values than those of the intrinsically MA group, suggesting that oral KPE administration alleviated wrinkle formation ( Figure 10).

KPE Increases Skin Thickness.
Photoaging is characterized by skin hypertrophy; in contrast, intrinsically aged skin shows skin thinning, also called skin atrophy [20]. Skin thickness and H&E staining were used to evaluate the extent of skin atrophy. The results showed the presence of skin atrophy in intrinsically MA mice; however, oral administration of KPE was found to attenuate skin atrophy ( Figure 11). of collagen, thus accelerating wrinkle formation [21]. The hydroxyproline content and M&T staining were used to measure the collagen content in skin. According to the results of the hydroxyproline assay and M&T staining, intrinsically MA mice showed low collagen content, whereas oral administration of KPE recovered the loss of collagen (Figure 12).

KPE Increases Elasticity.
In intrinsically aged skin, elastic fiber atrophy occurs, thereby reducing skin elasticity [22]. Loss of collagen and elastic fibers results in wrinkling [20]. EVG staining showed that the elastic fiber content in intrinsically MA mice decreased; however, elastic fiber content of KPE administered mice was much higher as compared to that in the intrinsically MA mice. In addition, oral KPE administration increased the skin elasticity, indicating that skin elasticity improved after KPE treatment through reduction of age-related elastic fiber atrophy (Figure 13).

Discussion
Based on our results, KPE was found to attenuate cellular senescence in H 2 O 2 -treated fibroblasts and intrinsically MA mice by suppressing SA--gal activity and the expression of cell-cycle inhibitors. The restrained expression of cellcycle inhibitors upon KPE treatment activates the E2F group, which is responsible for cell proliferation. In addition, KPE prevents cellular senescence by regulating the PI3K/AKT signaling pathway. Hyperactivation of AKT in the senescent state increases ROS generation. Recently, mTOR, one of the downstream regulators of AKT, has become a leading target for slowing aging and age-related diseases [23]. Wellknown mTOR inhibitors such as rapamycin and resveratrol effectively suppress cellular senescence in vitro and in vivo [24]. Furthermore, AKT activation suppresses FoxO3a which decreases ROS levels and cellular senescence [7]. As shown in Figure 3, KPE regulates the H 2 O 2 -induced PI3K/AKT signaling pathway by affecting the levels of the downstream markers, FoxO3a and mTOR. These results indicate that KPE inhibits oxidative stress-induced cellular senescence by regulating the PI3K/AKT pathway in vitro; our study also demonstrates the inhibitory effect of KPE on cellular senescence using an in vivo model.
In pharmacological/nutritional approaches to longevity, there has been a great deal of interest in calorie restriction (CR) because it can increase the lifespan of model organisms [4]. Physiological changes under CR largely depend on SIRT1 expression, leading to resistance against senescence and aging. Accordingly, the search for natural CR mimetics has become a promising area in antiaging studies. Resveratrol is known as an effective CR mimetic. The senescence-reversing activity of resveratrol is mainly SIRT1-dependent [25,26]. In the same vein, KPE considerably elevated expression of SIRT1 (Figure 4), indicating that KPE can also serve as an effective CR mimetic and is a potential natural antiaging agent. Senescent cells secrete various factors known as the senescence-associated secretory phenotype (SASP), which affects neighboring cells. The SASP is a clear indication of senescence that is observed both in vitro and in vivo, including the presence of inflammatory cytokines, chemokines, proteases, growth factors, and insoluble compounds. In the present study, KPE effectively attenuated the senescenceassociated inflammatory responses ( Figure 5). Senescent fibroblasts stimulate neighboring cells by secreting inflammatory cytokines, including IL-6 and IL-8. In this state, normal neighboring cells can become dysfunctional, and premalignant cells can enter the malignant state, thus promoting cancer formation. Accordingly, senescence-associated chronic inflammation contributes to age-related diseases, a process known as inflamm-aging [2]. Our data suggest that KPE can serve as an effective antiaging agent through its antiinflammatory activities.
In terms of mitochondrial dysfunction, H 2 O 2 -treated human skin fibroblasts and intrinsically MA hairless mice exhibited low levels of PGC-1 and its downstream genes such as ERR , NRF-1, and Tfam. Reduced expression of PGC-1 , which is responsible for mitochondrial function and biogenesis, was recovered by KPE treatment (Figure 7). In addition, KPE reversed the decreased ATP levels and the increased ROS generation, which suggest mitochondrial dysfunction ( Figure 6). A previous study has reported 12 Evidence-Based Complementary and Alternative Medicine that age-related mitochondrial dysfunction alters the energy metabolism pathway. Senescent cells with mitochondrial dysfunction tend to produce energy via anaerobic glycolysis. In this process, advanced glycation end products (AGEs) are generated as by-products and negatively affect the extracellular matrix proteins, collagen, and elastin, leading to loss of contractile capacity [27]. Although cellular senescence and mitochondrial dysfunction are important targets of antiaging agents, natural products that have such efficacy in the skin are not as well known.
Several studies demonstrated that cellular senescence and mitochondrial dysfunction are closely interconnected via p53. Elevated p53 activation suppresses PGC-1 , resulting in mitochondrial dysfunction. On the contrary, mitochondrial dysfunction causes oxidative stress that induces cellular senescence. Both FoxO3, a downstream transcription factor of the PI3K/AKT signaling pathway, and SIRT1 have a relationship with p53 and PGC-1 . SIRT1 and FoxO3 increase the level of PGC-1 , whereas they decrease the p53 level [28,29].
These two distinct causes of aging play an essential role in remodeling the ECM. During aging, the skin shows structural and functional changes [19]. The skin of intrinsically MA mice exhibited wrinkling, loss of elasticity, collagen and elastic fiber atrophy, and skin thinning. These changes were diminished by the oral administration of KPE (Figures 10-13). The aging process drives the degeneration of collagen and elastic fibers, which is generally considered to be involved in wrinkle formation, loss of elasticity, and skin thinning. Accordingly, the regulation of collagen and elastic fiber degeneration is essential to prevent the intrinsic skin aging phenotype. Histological staining indicated increased collagen and elastic fiber content in KPE administered mice.
Our previous study on the antiphotoaging effect of KPE showed decreased wrinkling and loss of collagen fiber in KPE (100 or 200 mg/kg/day)-treated UVB-irradiated hairless mice [30]. Because the high-dose KPE group gave better preventive effect on UVB-induced photoaging than that of lower-dose KPE group, we use the dose of KPE 200 mg/kg/day in this research. KPE prevents both extrinsic and intrinsic aging process in hairless mice. However, when hairless mice were subjected to UVB irradiation, their skin thickness was significantly increased [30] in association with an inflammatory response. UVB-induced skin inflammation elicits epidermal proliferation leading to skin thickening [30]. In contrast to that in extrinsic aging, the skin thickness of intrinsically aged mice decreased. Skin thinning in intrinsic skin aging is reportedly associated with the degeneration of collagen and elastic fibers. In addition, several studies have supported an intimate relationship between cellular senescence and skin atrophy. An investigation using superoxide dismutase 2-(Sod2-) deficient mice has verified that mitochondrial oxidative stress promotes cellular senescence and contributes to the skin thinning aging phenotype [20,31].

Conclusion
In conclusion, KPE delays intrinsic skin aging process by inhibiting cellular senescence and mitochondrial dysfunction. KPE does not only attenuate cellular senescence through inhibition of the p53/p21, p16/pRb, and PI3K/AKT signaling pathways but also improve mitochondrial biogenesis through PGC-1 stimulation in H 2 O 2 -exposed human dermal fibroblasts and MA hairless mice. Consequently, KPE prevents wrinkle formation, skin atrophy, and loss of elasticity by increasing collagen and elastic fibers in MG hairless mice.
Evidence-Based Complementary and Alternative Medicine 13 Therefore, KPE can serve as an antiaging nutraceutical and cosmeceutical agent.