Nacre extract from pearl oyster shell prevents D-galactose-induced brain and skin aging

doi:10.21203/rs.3.rs-1809986/v1

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Nacre extract from pearl oyster shell prevents D-galactose-induced brain and skin aging

https://doi.org/10.21203/rs.3.rs-1809986/v1

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published 11 Jan, 2023

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Pearl oyster shells comprise two layers, a prismatic and nacreous layer, of calcium carbonate. The nacreous layer has been used in Chinese medicine since ancient times. In this study, we investigated the effects of the extract from the nacreous layer of pearl oysters (nacre extract) on D-galactose-induced brain and skin aging. Treatment with nacre extract led to the recovery of D-galactose-induced memory impairment, as examined using the Barnes maze, novel object recognition, and Y-maze tests. A histological study showed that nacre extract suppressed D-galactose-induced neuronal cell death and the expression of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), which causes apoptosis in the hippocampus. In addition, the expression levels of brain-derived neurotrophic factor, which counteracts age-related brain dysfunction, and nicotinamide adenine dinucleotide-dependent deacetylase (sirtuin 1), which delays aging and extends lifespan, increased after nacre extract treatment. Moreover, the nacre extract showed antiaging effects against D-galactose-induced skin aging; it suppressed D-galactose-induced wrinkle formation, decreased skin moisture, decreased epidermal thickness, and destroyed collagen arrangement associated with aging. Furthermore, the nacre extract suppressed oxidative stress associated with aging in the brain and skin by upregulating the expression of catalase and superoxide dismutase. The expression level of the cellular senescence marker p16, which is induced by oxidative stress, was elevated in the hippocampus and skin epidermal layer of D-galactose-treated mice, and it was suppressed by the administration of nacre extract. These results show that the nacre extract can suppress D-galactose-induced aging by enhancing antioxidant activity and suppressing p16 expression. Thus, the nacre extract may be an effective antiaging agent.

antioxidant

brain aging

nacre extract from pearl oyster shell

skin aging

Aging is defined as the progressive decline in physiological function with increasing age. Aging causes various changes in all tissues. Skin tissue shows the most visible signs of aging, including wrinkle formation and dryness. The skin comprises dermal and epidermal layers. The dermal layer contains fibroblast cells, elastin fibers, and collagen fibers, which support the skin structure, and the epidermal layer contains keratinocytes, proteins, and lipids, which are important for the barrier function. Aging causes a decrease in the thickness of the dermis and epidermis, destruction of collagen and elastin fibers, and a decline in barrier function. Aging also causes cognitive decline, oxidative damage, and inflammation in the brain. Numerous studies have reported that brain aging increases the risk of various diseases, including Parkinson’s and Alzheimer’s diseases (Pan and Nicolazzo 2018; Reeve et al. 2014). The incidence of skin disorders, including dermatitis and skin tears, also increases with age-related changes in skin structure (Blume-Peytavi et al. 2016; Gua et al. 2020).

Several models have been used, such as D-galactose-induced, senescence-accelerated, and naturally aging mouse models. Chronic treatment with D-galactose leads to accelerated aging and induces changes resembling natural aging in the brain, skin, heart, lungs, kidneys, and liver (Azman and Zakaria 2019). D-galactose-induced animal models are effective in identifying antiaging substances.

According to the oxidative stress theory of aging (Beckman and Ames 1998; Liguori et al. 2018), an increase in the production of reactive oxygen species (ROS) can trigger oxidative stress, inflammation, mitochondrial dysfunction, and apoptosis, resulting in aging (Patergnani et al. 2021; Zhu et al. 2018). D-galactose injection induces the production of ROS associated with a decrease in antioxidant enzyme activities, leading to brain aging, including neuronal degeneration and memory impairment, and skin aging, including the appearance of wrinkles and a decrease in the water content and thickness of the dermis and epidermis (Azman and Zakaria 2019; Rusu et al. 2020; Shwea et al. 2018; Umbayev et al. 2020; Zhang et al. 2020). The involvement of ROS in aging has also been supported by the results that antioxidants, such as vitamin C, carotenoids, and vitamin E, suppress brain aging (Dhanjal et al. 2020; Pawlowska et al. 2019).

In the brain, brain-derived neurotrophic factor (BDNF) plays a critical role in the maintenance and survival of neuronal cells and in learning and memory formation. A decrease in BDNF levels has been observed in the brains of mouse models of Alzheimer’s disease and in naturally aging mice (Amidfar et al. 2020; Souza et al. 2015). Several studies have suggested that BDNF is a key factor that suppresses neuronal loss and improves the learning and memory loss associated with brain aging (Xiyang et al. 2020; Zhong et al. 2019)

Sirtuin 1 (nicotinamide adenine dinucleotide-dependent deacetylase) is involved in various cellular processes, such as cell survival, apoptosis, growth, aging, and metabolism. Many studies have shown that sirtuin 1 is associated with an increase in lifespan, and its expression declines with age in animal tissues including the brain and skin. Overexpression of sirtuin 1 in the brain prolongs the lifespan of mice (Li et al. 2018; Lou et al. 2021; Heyward et al. 2012). In addition, the activation of sirtuin 1 suppresses dermal fibroblast aging caused by UV radiation (Chong et al. 2012). The antiaging effect of sirtuin 1 is partially mediated by an increase in its antioxidant activity (Alcendor et al. 2007). Sirtuin 1 promotes mitochondrial biogenesis by activating peroxisome proliferator-activated receptor coactivator 1-α (PGC-1α) (Rodgers et al. 2005). PGC-1α suppresses oxidative stress by upregulating the expression of mitochondrial antioxidant genes, including glutathione peroxidase, catalase, and manganese superoxide dismutase (Mn-SOD) (St-Pierre et al. 2006).

Several studies have reported that p16 is a well-defined senescence-related gene (Rayess et al. 2012). Cell cycle progression is interfered by p16 through inhibiting cyclin-dependent kinases. Elevated p16 expression is observed in aged tissues, and p16 is one of the key factors that regulate aging. Downregulation of p16 expression attenuates the age-induced decline in cell proliferation and survival (Che et al. 2020). Several studies have shown that p16 is involved in oxidative stress response. Melanocytes treated with H₂O₂ upregulated p16 protein levels (Jenkins et al. 2011). Kim and Wong showed that oxidative stress induces senescence in astrocytes via the activation of the p16 pathway (Kim and Wong 2009).

Pearl oysters are widely used to produce pearls for jewelry. However, hundreds of thousands of tons of pearl oyster shells are discarded as industrial waste and recycling pearl oyster shells is strongly desired. Pearl oyster shells comprise a prismatic and nacreous layer, with a composition similar to that of pearls. The nacreous layer mainly contains 91% calcium carbonate, magnesium carbonate, and organic components, such as proteins and saccharides (Pei et al. 2021; Pu et al. 2016).

Pearl powder has been used in traditional Chinese medicine for convulsions, epilepsy, and myopia (Xu et al. 2001; Zhang et al. 2016), as well as in functional foods and cosmetics. Some studies have shown the biological activities of pearl powder in several tissues, such as the skin, bone, and brain (Atlan et al. 1999; Lopez et al. 2000; Mangrulkar et al. 2002). Lee et al. reported that the water-soluble component of the mother of the pearl (nacre) improved second-degree burns in porcine skin (Lee et al. 2012). Nacre has been shown to promote the osteogenic differentiation of human bone cells and induce bone growth in vitro and in vivo (Brion et al. 2015; Chen et al. 2019; Pattapon et al. 2011; Pattapon and Panjit 2012; Rousseau et al. 2003; Westbroek and Marin 1998). These authors suggested that nacre contains signaling molecules that stimulate osteogenesis. Moreover, the administration of water-soluble nacre protein has been shown to be effective in treating brain disorders, including pentylenetetrazol-induced convulsions (Zhang et al. 2016). We also showed that intraperitoneal or oral administration of the extract from the nacreous layer (nacre extract) led to an improvement in scopolamine- and amyloid beta-induced memory impairment by suppressing oxidative stress and increasing the expression of phosphorylated cAMP response element binding protein (CREB) (Fuji et al. 2018; Yamagami et al. 2021; Yotsuya and Hasegawa 2022). In this study, we focused on the antiaging effects of nacre extract and investigated the effect of nacre extract on D-galactose-induced brain and skin aging.

Materials

Shells of pearl oysters (Pinctada maxima) were collected from Iki Bay (Nagasaki, Japan). Antibodies against sirtuin 1, BDNF, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and cyclin-dependent kinase inhibitor 2A (p16) were purchased from Biorbyt (San Francisco, CA, USA).

Preparation of nacre extract

The nacre extract was prepared as previously described (Fuji et al. 2018). Briefly, the nacreous layer was crushed after removal of the prismatic layer from the shells. The nacreous layer was solubilized in 10% acetic acid for decalcification, and the decalcifying solution was dialyzed against deionized water using a dialysis membrane. The solution was lyophilized and extracted again with deionized water. The water-soluble fraction was used as the nacre extract in subsequent experiments.

Animals

Four-week-old male ICR mice were purchased from CLEA (Tokyo, Japan) and kept in a room under the following conditions: temperature, 24 °C; humidity, 50%; and 12 h/12 h light/dark cycle. Five or six mice were housed per cage and acclimatized for at least 7 d. The mice had free access to food (AIN-76A) and water throughout the study. D-galactose was injected intraperitoneally at a dose of 500 mg/kg every day for 8 weeks in the D-galactose and nacre groups according to the schedule presented in Fig. 1. Sterilized water was injected instead of D-galactose into the control group. Nacre extract was administered intraperitoneally every day for 8 weeks at a dose of 40 mg/kg or 80 mg/kg to the nacre 40 and nacre 80 groups, respectively. Dosages were selected based on the results of a previous study. After 8 weeks, behavioral experiments were performed, and the mice were euthanized. The brain and skin tissues were collected immediately and stored at −80 °C until further use. Animal experiments were performed according to the guidelines of the Muroran Institute of Technology (approval number H29KS01) for the care and welfare of mice. All experiments were approved by the Committee on Ethics, Care, and Use of Experimental Animals at the Muroran Institute of Technology.

Senescence grading score

The aging process in mice was evaluated according to the modified method of Hosokawa et al. (2013). Briefly, the six categories of reactivity, passivity, glossiness, coarseness of coat, hair loss, ulcers, and cataracts were assigned to five grades for five or six mice in each group, and the average was determined.

Novel object recognition test

The novel object recognition test was performed as described previously (Fuji et al. 2018; Hasegawa et al. 2016; Yamagami et al. 2021; Yotsuya and Hasegawa 2022). The mouse was placed in a test box (diameter, 60 cm) containing two objects of different shapes and colors and allowed to explore freely for 5 min. After 24 h, one of the familiar objects used in the training session was replaced with a novel object of different shape and color and the mouse was again placed in the box to explore the objects. The time spent exploring the novel and familiar objects was measured for 5 min, and the recognition index was calculated using the following equation:

Recognition index = [(time spent exploring the novel object)/(total time spent exploring the objects)] × 100

Y-maze test

The Y-maze test was performed as described previously (Fuji et al. 2018; Hasegawa et al. 2016; Yamagami et al. 2021; Yotsuya and Hasegawa 2022). Briefly, the mouse was placed in the central area of the Y-maze with three identical arms, each 35 cm long and 25 cm high, and was allowed to move freely. Spontaneous alternation behavior was defined as the number of sequential entries into the three arms. The number and sequence of entries into the arms were recorded for each animal for 10 min. The percentage of spontaneous alternations was calculated using the following equation.

% Spontaneous alternation = (number of correct alternations)/(total number of arm entries − 2)

Barnes maze test

The Barnes maze test was performed as described previously (Yotsuya and Hasegawa 2022). Briefly, on the edge of a white circular maze platform with a diameter of 1.15 m, 12 holes (diameter, 10 cm) were placed, of which only one was a dark escape hole. The mouse was placed at the center of the platform and allowed to search for a dark escape hole for 120 s. If the mouse did not reach the dark escape hole within 120 s, it was guided and placed in the escape hole to memorize its location. The mice were allowed to perform the same exercise once daily for 4 d. In the probe test, the dark escape hole was removed and the mouse was asked to search for a dark escape hole for 60 s. The time required to search the area surrounding the dark escape hole (within 20 cm of the escape hole) was measured.

Histochemistry

The mice were anesthetized with sevoflurane and transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline. Brain and skin tissues were embedded in paraffin, and 5-µm-thick sections were sliced. The brain tissues were sliced at regions including the hippocampus (–1.8 to –2.5 mm from the bregma). Paraffin-embedded sections were deparaffinized and stained with hematoxylin and eosin (HE), Masson Goldner solution (Merck, Tokyo, Japan) for collagen staining, and toluidine blue solution for mast cell staining.

Live neurons were counted in four randomly selected brain slices. The thickness of the dermis and epidermis was measured in 100 randomly selected areas on skin slices from five or six mice after HE staining. Collagen density was calculated in 20 randomly selected areas on four randomly selected skin slices from five or six mice after Masson Goldner staining using the ImageJ software. Density was calculated from the intensity and area of blue color per unit area (Suvik and Effendy 2012). The number of mast cells in the dermis was counted in 10 randomly selected areas of skin slices from five or six mice after toluidine blue staining.

Immunohistochemistry was performed using specific antibodies in the brain hippocampal and skin sections using a Polymer Based 1-Step IHC system (VitroVivo Biotech, Rockville, MD, USA) (Yotsuya and Hasegawa 2022). The 3,3′-diaminobenzidine (DAB)-stained area in the image was determined using ImageJ software (Christensen et al. 2008). The stained images were binarized to 8-bit images, and a fixed intensity threshold was applied to define DAB staining. The relative staining area was expressed as DAB staining area per mm².

Measurement of water content and depth and number of wrinkles in skin

The dorsal skin of the mice was shaved using a hair clipper, and the water content was measured using a moisture checker (MY808S, Scalar, Tokyo, Japan). The water content of 20 areas on the dorsal skin was measured and the average (± standard deviation [SD]) was calculated.

After silicon replicas (Amic Group, Tokyo, Japan) were made from the shaved dorsal skin, fixed replicas were photographed, and pixel measurements of the height were performed along each wrinkle using ImageJ software, according to the method described by Mazzulla et al. (2018). The average (±SD) depth and number of wrinkles (30 fields) in five or six mice were calculated.

Preparation of skin and brain extract

Brain cortical tissue (200 mg) was homogenized in 1 mL of deionized water and centrifuged at 14,000 × g for 10 min at 4 °C. The supernatant was used as the brain extract. Dorsal skin tissue (200 mg) was frozen in liquid nitrogen and crushed. The pieces were homogenized with 1 mL of deionized water and centrifuged at 14,000 × g for 10 min at 4 °C; the supernatant was used as the skin extract.

Real-time polymerase chain reaction (PCR)

Total RNA from the skin and brain cortical tissues was prepared using a Total RNA Purification Kit (Biorbyte, San Francisco, CA, USA) and RNAiso Plus Kit (Takara, Shiga, Japan), respectively. Reverse transcription reactions were performed, and quantitative PCR was conducted using iTaq Universal SYBR Green Supermix (BioRad, Tokyo, Japan). The PCR primer sequences are listed in Table 1. The expression of target genes was normalized to the mean expression of β-actin using the comparative ΔCt method.

Antioxidant activity

The antioxidant activities of the brain and skin extracts were determined, and malondialdehyde (MDA) levels were evaluated using thiobarbituric acid, as described previously (Kariya and Hasegawa 2020). Briefly, a mixture containing brain cortical extract and 50% trichloroacetic acid was prepared and centrifuged at 14,000 × g for 10 min. Thiobarbituric acid solution (0.67%) was incubated with the supernatant at 100 °C for 10 min, and absorbance at 540 nm was measured.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was performed as follows. DPPH solution (0.8 mg/mL) in 50% ethanol was mixed with the brain or skin extract. The absorbance of the solution was measured at 517 nm after 30 min (Kariya and Hasegawa 2020).

Glutathione content was determined using a Bioxytech Gsh 400 Kit (OXIS International, Beverly Hills, CA, USA) according to the manufacturer’s instructions.

The Fe³⁺-reducing activity was measured as described previously. A solution containing 250 mM acetate buffer (pH 3.6), 0.8 mM 2,4,6-tripyridyl-s-triazine, 1.6 mM FeCl₃·6H₂O, and the brain or skin extract was prepared, and absorbance was measured at 593 nm (Kariya and Hasegawa 2020).

Statistical analysis

Each experiment was performed at least twice. Data from five or six mice per group are expressed as mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by the Tukey–Kramer multiple comparison test using Excel Statistics software (SSRI, Tokyo, Japan).

Effect of nacre extract on D-galactose-induced aging

There were differences in the appearance of mice in the different treatment groups (Fig. 2). Control mice had smooth, healthy, and shiny hair with uniform color. In contrast, D-galactose-treated mice had coarse and non-shiny hair, which is a characteristic of aging. Nacre extract treatment improved the appearance of D-galactose-treated mice. Grading scores, which represent senescent status, were lower in nacre extract-treated mice than in D-galactose-treated mice, suggesting that nacre extract attenuated D-galactose-induced aging.

To investigate the effect of nacre extract on D-galactose-induced brain aging, we examined the effect of nacre extract on D-galactose-induced memory impairment. Learning and memory functions were evaluated using the Y-maze, novel object recognition, and Barnes maze tests. In the Y-maze test, compared to control mice, D-galactose-induced mice showed an obvious decrease in spontaneous alternation, and this decrease was reversed by treatment with nacre extract at doses of 40 mg/kg and 80 mg/kg (Fig. 3a). In the novel object recognition test, the relative exploration time of the novel object in D-galactose-treated mice was lower than that in the control mice. However, nacre extract (80 mg/kg) significantly recovered the relative exploration time of the novel object (Fig. 3b). The effect of nacre extract on D-galactose-induced memory impairment was also examined using the Barnes maze test (Fig. 3c). The mice were trained to locate the dark escape hole for 6 d, and a probe test was performed. The time spent searching around the space (approximately 20 cm) where the dark escape hole was initially situated was lower in D-galactose-treated mice than in control mice, but treatment with nacre extract significantly increased the time spent searching around the space. These results suggest that the nacre extract can improve D-galactose-induced memory impairment.

Effect of nacre extract on neuronal degeneration in the brain hippocampus region

To determine whether hippocampal neuronal degeneration is involved in D-galactose-induced memory impairment and whether the nacre extract can suppress degeneration, a histological study was performed (Fig. 4). Treatment with D-galactose increased the number of degenerated neurons with cytomorphological shrinkage and pyknotic nuclei in the CA1, CA2, CA3, and dentate gyrus regions compared to those in control mice. Treatment with nacre extract significantly suppressed neuronal degeneration. This result was also supported by the expression of Bcl-2 and Bax, which regulate apoptosis. Strong immunostaining for Bax was observed in the hippocampal CA1 region of the D-galactose-treated mice (Fig. 4c). In contrast, less immunostaining was observed in the hippocampus of the control and nacre-treated mice. The expression of Bcl-2 was also observed in degenerated neurons in the hippocampus of D-galactose-treated mice, as well as in live neurons of control and nacre-treated mice. These results show that nacre extract protects against neuronal cell death by suppressing D-galactose-induced apoptosis.

Effect of nacre extract on the expression of sirtuin 1 and BDNF

The levels of BDNF have been reported to be reduced in the hippocampus of naturally aging mice (Xiyang et al. 2020), which may contribute to synaptic and cellular loss and memory deficits. We investigated changes in BDNF expression using real-time PCR. Although the difference was not significant, the expression level of BDNF in the brain showed a tendency to increase with nacre extract treatment compared with that in D-galactose-induced mice (Fig. 5). The expression of BDNF in the hippocampus was confirmed by immunohistochemistry. BDNF expression levels were clearly decreased in the hippocampus of D-galactose-induced mice compared to control mice. In contrast, BDNF expression levels in nacre-treated mice were similar to those in control mice, suggesting that nacre extract treatment restored BDNF expression in the hippocampus of D-galactose-treated mice.

Next, we investigated sirtuin 1 expression levels using real-time PCR and immunohistochemistry. Sirtuin 1 is highly expressed in brain neurons and its deficiency causes cognitive decline and neurodegeneration during aging (Li et al. 2018). D-galactose treatment did not reduce the expression level of sirtuin 1 compared with that in control mice, whereas administration of nacre extract showed a tendency to increase sirtuin 1 expression level compared to that in D-galactose-treated mice (Fig. 5). This result was also supported by the immunohistochemistry results. These results suggest that nacre extract can increase the expression levels of BDNF and sirtuin 1, which are involved in memory function in the brain, and that nacre extract may suppress D-galactose-induced aging by increasing the expression of BDNF and sirtuin 1.

Effect of nacre extract on D-galactose-induced skin aging

Aging skin is characterized by a decline in water content and epidermal barrier function. First, we examined the effects of nacre extract on epidermal barrier function by measuring water content in the epidermal layer. D-galactose treatment decreased the epidermal water content to approximately 70% of that in the control mice, but nacre extract reversed this decrease (Fig. 6a). We examined the effects of nacre extract on the expression of cornified cell envelope proteins, including involucrin, filaggrin, and loricrin, which form the epidermal barrier (Pyun et al. 2012; Rosenthal et al. 1992). D-galactose treatment tended to decrease the mRNA expression levels of these proteins; however, treatment with nacre extract upregulated the mRNA expression of these proteins (Fig. 6b). The expression of ceramide and fatty acid elongase 1, which are responsible for skin hydration (Eckl et al. 2013; Jennemann et al. 2012; Mueller et al. 2019) also increased after treatment with the nacre extract. To confirm these results, immunostaining for ceramide synthase, elongase 1, and involucrin was performed (Fig. 6c). Expression of these proteins in the epidermis decreased in D-galactose-treated mice and recovered in nacre extract-treated mice, similar to the results of mRNA expression analysis. These results show that nacre extract can improve D-galactose-induced epidermal barrier dysfunction and prevent loss of skin water content.

To investigate the effect of the nacre extract on skin thickness, dorsal skin slices were histochemically examined. Staining with HE revealed that epidermal and dermal thickness in D-galactose-treated mice was lower than that in control mice (Fig. 7a). A decrease in the thickness of the dermis and epidermis layers is characteristic of naturally aging skin (Branchet et al. 1990). Nacre extract suppressed the decrease in epidermal layer thickness, but not in the dermal layer. A decrease in the thickness of the epidermis is also associated with a decline in skin barrier function. This result also supports the idea that nacre extract is effective in maintaining the skin barrier function.

Next, we investigated the effect of the nacre extract on D-galactose-induced wrinkle formation, a consequence of skin aging (Fig. 7b). Wrinkle formation was examined based on wrinkle depth and the number per unit area using silicon replica images of the skin. D-galactose treatment increased wrinkle formation compared to that in the control mice. Compared to control mice, treatment with D-galactose increased wrinkle depth and number by approximately 1.7-fold and 1.9-fold, respectively. However, treatment with nacre extract suppressed the depth and number of wrinkles. Wrinkle formation is caused by destruction of skin collagen (Oba and Edwards 2006). To estimate the effect of nacre extract on D-galactose-induced collagen degradation, dorsal skin slices of mice were stained with Masson’s trichrome (Fig. 7c). Treatment with D-galactose destroyed the collagen fiber arrangement, leading to a reduction in the spatial density of collagen in the dermis. However, nacre extract treatment restored this destruction. These results suggest that nacre extract can suppress wrinkle formation by suppressing D-galactose-induced collagen destruction.

Finally, we determined the number of mast cells in the dermis. Several studies have revealed an increasing number of mast cells in aging skin (Gunin et al. 2011; Luo et al. 2002). D-galactose treatment resulted in an increase in the number of mast cells, and nacre extract suppressed this increase (Fig. 7d). These results suggest that nacre extract suppresses D-galactose-induced skin aging.

Effect of nacre extract on antioxidant activity in the brain and skin

To investigate the antiaging mechanism of nacre extract, we estimated its antioxidant activity in brain and skin tissues. Real-time PCR analysis showed that the expression levels of antioxidant enzymes, including Mn- and Cu/Zn-SOD and catalase, were increased after treatment with nacre extract in the brain and skin tissues (Fig. 8a). To further confirm these results, we measured MDA content, radical scavenging activity, and iron-reducing activity in the brain and skin tissues (Fig. 8b). MDA content increased in D-galactose-treated mice, and this increase was suppressed by treatment with nacre extract. Radical-scavenging and iron-reducing activities of the brain and skin tissues were decreased in D-galactose-treated mice; however, treatment with nacre extract significantly increased these activities. These results suggest that nacre extract can suppress oxidative stress in brain and skin tissues. Glutathione content and glutathione peroxidase activity decreased in the brain tissues of D-galactose-treated mice, and this decrease was reversed after treatment with nacre extract. In contrast, no change in glutathione content was observed in skin tissues after D-galactose treatment.

Oxidative stress induces the expression of senescence mediator p16. Thus, we investigated p16 expression in skin and brain tissues using immunohistochemistry (Fig. 9). D-galactose treatment significantly upregulated p16 expression in the skin epidermis and brain hippocampus compared to that in control mice. Treatment with nacre extract decreased the expression levels in the skin and brain tissues. These results also show that nacre extract can suppress oxidative stress in skin and brain tissues.

D-galactose treatment caused memory and learning impairments in the brain, decreased skin water content, and caused wrinkle formation. In addition, the appearance changed to coarse and non-shiny hair, which is characteristic of aging mice (Hosokawa et al. 2013). Treatment with the nacre extract suppressed these changes, suggesting that it can suppress D-galactose-induced brain and skin aging.

D-galactose accelerates the brain and skin aging processes by elevating free radical generation in vivo (Umbayev et al. 2020). According to the free-radical theory of aging, ROS generation can trigger mitochondrial dysfunction and cellular impairment, resulting in cellular senescence and aging (Beckman and Ames 1998; Liguori et al. 2018). In the present study, D-galactose treatment increased MDA content in the brain and skin tissues, which is an index of lipid peroxidation induced by oxidative stress. However, nacre extract decreased MDA content by increasing the production of antioxidant enzymes such as SOD and catalase. Oxidative stress induces p16 expression, which promotes cellular senescence (Che et al. 2020; Kim and Wong 2009). D-galactose treatment increased p16 expression in brain and skin tissues, and nacre extract suppressed D-galactose-induced p16 expression. These results also suggest that the nacre extract can suppress brain and skin aging by decreasing D-galactose-induced oxidative stress.

BDNF plays an important role in the survival, growth, and synaptic plasticity of neurons in the brain and can improve learning and memory capacity in aging mice (Amidfar et al. 2020; Souza et al. 2015; Xiyang et al. 2020; Zhong et al. 2019). In the present study, the expression level of BDNF decreased in the brains of D-galactose-induced aging mice, and nacre extract restored the expression level in the brain. The protective effect of the nacre extract against memory impairment associated with brain aging might have been mediated by increased BDNF levels.

Moreover, treatment with the nacre extract increased sirtuin 1 expression in the brain. Recent studies have shown that sirtuin 1 promotes the expression of CREB and BDNF, which regulate memory function via an miR-134-mediated post-transcriptional mechanism, leading to improvements in learning and memory impairment (Gao et al. 2010). Increased sirtuin 1 activity alters neuronal transcription profiles to induce antioxidant genes, such as Mn-SOD, a potent antioxidant enzyme in mitochondria, resulting in a decrease in ROS production (Meng et al. 2018). Additionally, sirtuin 1 expression inhibits apoptosis (Luo et al. 2019). Nacre extract treatment increased BDNF expression, inhibited apoptosis, and increased antioxidant activity by increasing the expression level of sirtuin 1 in the brain. In contrast, we did not observe an increase in sirtuin 1 expression in the skin following nacre extract treatment (data not shown). Differences in responses between skin and brain tissues following nacre extract treatment were also observed with respect to glutathione content. The mechanism of action of nacre extract appears to be different in brain and skin tissues. Further identification of the bioactive substances acting on the brain and skin may be necessary to clarify this.

Skin aging can be classified into two types: natural and photoaging. The clinical features of natural and photoaging differ. Natural aging decreases the thickness of the epidermis and dermis, whereas photoaging increases it (Farage et al. 2013; Fischer et al. 2018). In the present study, D-galactose treatment decreased the thickness of the dermis and epidermis, suggesting that D-galactose-induced aging resembles natural aging. There have been many studies on skin photoaging and its prevention; however, few studies have investigated the natural aging of the skin and its prevention. Nacre extract may be an effective substance to suppress natural aging of the skin. In this study, nacre extract reversed the D-galactose-induced decrease in the thickness of the skin epidermis, but not that of the dermis. Although the reason for this remains unclear, D-galactose treatment caused destruction of the arrangement of collagen fibers, resulting in the generation of some spaces in the dermis. The thickness of the dermis measured in D-galactose-induced mice may be higher than the actual values. Therefore, the reversal effect of the nacre extract may not have been observed.

Although pearl powder has been used in Chinese medicine, there have been few studies on its effects in humans. Recently, in a randomized placebo-controlled trial involving 20 participants, Chiu et al. showed that protein-rich pearl powder had antioxidant properties (Chiu et al. 2018). In the present study, we showed that nacre extract is effective in suppressing the aging effect by enhancing antioxidant activity, suggesting that nacre extract may also be effective against the human aging process. In the future, it will be interesting to examine the antiaging effects of pearl powder in interventional studies.

We previously showed that treatment with sulfated polysaccharides from nacre extract activates CREB signaling, increases BDNF expression in the brain, and improves scopolamine- and amyloid beta-induced memory impairment (Yamagami et al. 2021). In the present study, nacre extract increased BDNF expression levels in D-galactose-induced aging mice, suggesting that sulfated polysaccharides may also contribute to the preventive effect of nacre extract against D-galactose-induced brain aging. The identification of antiaging substances present in the nacre extract against brain and skin aging is ongoing.

The results of this study suggest that pearl powder may be useful as an antiaging agent in the brain and skin.

Conflict of Interest The authors declare no competing interests.

Findings This study received no specific grants from any funding agency.

Ethical Approval This study was approved by the Committee on Ethics, Care, and Use of Experimental Animals at the Muroran Institute of Technology (number H29KS01).

Acknowledgements The authors would like to thank Kaneko Pearl Farming Company Limited for pearl oyster shell collection. The authors would like to thank the Department of Molecular Medicine, Akita University, for their assistance with immunohistochemistry.

Author Contribution Y.H. designed the research; H.Y, N.S, and K.O carried out the experiments and data analysis; Y.H. was responsible for manuscript preparation; all authors reviewed and approved the manuscript.

Data Availability Statement: All data generated or analyzed during this study are included in this published article.

Alcendor RR, Gao S, Zhai P, Zablocki D, Holle E, Yu X, Tian B, Wagner T, Vatner SF, Sadoshima J (2007). Sirt1 regulates aging and resistance to oxidative stress in the heart. Cir Res 100: 1512–1521.
Amidfar M, Oliveira J, Kucharska E, Budni J, Kim YK (2020). The role of CREB and BDNF in neurobiology and treatment of Alzheimer’s disease. Life Sci 257: 118020.
Atlan G, Delattre O, Berland S, LeFaou A, Nabias G, Cot D, Lopez E (1999). Interface between bone and nacre implants in sheep. Biomaterials 20: 1017–1022.
Azman KF, Zakaria R (2019). D-Galactose-induced accelerated aging model: an overview. Biogerontology 20: 763–782.
Beckman KB, Ames BN (1998). The free radical theory of aging matures. Physiol Rev 78: 547–581.
Blume-Peytavi U, Kottner J, Sterry W, Hodin M, Giffiths TW, Watson REB, Hay RJ, Griffiths CEM (2016). Age-associated skin conditions and diseases: current perspectives and future options. Gerontologist 56 (Suppl 2): S230–S242.
Branchet MC, Boisnic S, Frances C, Robert AM (1990). Skin thickness changes in normal aging skin. Gerontology 36: 28–35.
Brion A, Zhang G, Dossot M, Moby V, Dumas D, Hupont S, Piet MH, Bianchi A, Mainard D, Galois L, Gillet P, Rousseau M (2015). Nacre extract restores the mineralization capacity of subchondral osteoarthritis osteoblasts. J Struct Biol 192: 500–509.
Che H, Li J, Li Y, Ma C, Liu H, Qin J, Dong J, Zhang Z, Xian CJ, Miao D, Wang L, Ren Y (2020). p16 deficiency attenuates intervertebral disc degeneration by adjusting oxidative stress and nucleus pulposus cell cycle. Elife 9: e52570.
Chen X, Peng LH, Chee SS, Shan YH, Liang WQ, Gao JQ (2019). Nanoscaled pearl powder accelerates wound repair and regeneration in vitro and in vivo. Drug Dev Ind Pharm 45: 1009–1016.
Chiu HF, Hsiao SC, Lu YY, Han YC, Shen YC, Venkatakrishnan K, Wang CK (2018). Efficacy of protein rich pearl powder on antioxidant status in a randomized placebo-controlled trial. J Food Drug Anal 26: 309–317.
Chong ZZ, Shang YC, Wang S, Maiese K (2012). SIRT1: new avenues of discovery for disorders of oxidative stress. Expert Opin Ther Targets 16: 167–178.
Christensen DZ, Kraus SL, Flohr A, Cotel MC, Wirths O, Bayer TA (2008). Transient intraneuronal Ab rather than extracellular plaque pathology correlates with neuron loss in the frontal cortex of APP/PS1KI mice. Acta Neuropathol 116: 647–655.
Dhanjal DS, Bhardwaj S, Sharma R, Bhardwaj K, Kumar D, Chopra C, Nepovimova E, Singh R, Kuca K (2020). Plant fortification of the diet for anti-ageing effects: A review. Nutrients 12: 3008.
Eckl KM, Tidhar R, Thiele H, Oji V, Hausser I, Brodesser S, Preil ML, Onal-Akan A, Stock F, Müller D, Becker K, Casper R, Nürnberg G, Altmüller J, Nürnberg P, Traupe H, Futerman AH, Hennies HC (2013). Impaired epidermal ceramide synthesis causes autosomal recessive congenital ichthyosis and reveals the importance of ceramide acyl chain length. J Invest Dermatol 133: 2202–2211.
Farage MA, Miller KW, Elsne P, Maibach HI (2013). Characteristics of the aging skin. Adv Wound Care 2: 5–10.
Fisher GJ, Wang ZQ, Datta SC, Varani J, Kang S, Voorhees JJ (1997). Pathophysiology of premature skin aging induced by ultraviolet light. N Engl J Med 337: 1419–1428.
Fuji T, Inoue T, Hasegawa Y (2018). Nacre extract prevents scopolamine-induced memory deficits in rodents. Asian Pac J Trop Med 11: 202–208.
Gao J, Wang WY, Mao YM, Gräff J, Guan JS, Pan L, Mak G, Kim D, Su SC, Tsai LH (2010). A novel pathway regulates memory and plasticity via SIRT1 and miR-134. Nature 466: 1105–1109.
Gua Y, Hana J, Jiang C, Zhang Y (2020). Biomarkers, oxidative stress and autophagy in skin aging. Ageing Res Rev 59: 101036.
Gunin AG, Kornilova NK, Vasilieva OV, Petrov VV (2011). Age-related changes in proliferation, the numbers of mast cells, eosinophils, and cd45-positive cells in human dermis. J Gerontol: Series A 66A: 385–392.
Hasegawa Y, Inoue T, Kawaminami S, Fujita M (2016). Effects of scallop shell extract on scopolamine-induced memory impairment and MK801-induced locomotor activity. Asian Pac J Trop Med 9: 662–667.
Heyward FD, Walton RG, Carle MS, Coleman MA, Garvey WT, Sweatt JD (2012). Adult mice maintained on a high-fat diet exhibit object location memory deficits and reduced hippocampal SIRT1 gene expression. Neurobiol Learn Mem 98: 25–32.
Hosokawa M, Sakura M, Chiba Y (2013). The grading score system: a method for evaluating the degree of senescence in SAM strains of mice. In: Takeda T (ed) The senescence-accelerated mouse (SAM): achievements and future directions. Elsevier, Amsterdam 561–567.
Jennemann R, Rabionet M, Gorgas K, Epstein S, Dalpke A, Rothermel U, Bayerle A, van der Hoeven F, Imgrund S, Kirsch, Nickel W, Willecke K, Riezman H, Grone HJ, Sandhoff R (2012). Loss of ceramide synthase 3 causes lethal skin barrier disruption. Hum Mol Genet 21: 586–608.
Jenkins NC, Liu T, Cassidy P, Leachman SA, Boucher KM, Goodson AG, Samadashwily G, Grossman D (2011) The p16^INK4A tumor suppressor regulates cellular oxidative stress. Oncogene 30: 265–274.
Kariya T, Hasegawa Y (2020). Scallop mantle toxin induces apoptosis in liver tissues of mice. Food Sci Nutr 8: 3308–3316.
Kim J, Wong PKY (2009). Oxidative stress is linked to ERK1/2-p16 signaling-mediated growth defect in ATM-deficient astrocytes. J Biol Chem 284: 14396–14040.
Lee K, Kim H, Kim JM, Chung YH, Lee TY, Lim H, Lim JH, Kim T, Bae JS, Woo CH, Kim KJ, Jeong D (2012). Nacre-driven water-soluble factors promote wound healing of deep burn porcine skin by recovering angiogenesis and ﬁbroblast function. Mol Biol Rep 39: 3211–3218.
Li Q, Zeng J, Su M, He Y, Zhu B (2018). Acetylshikonin from Zicao attenuates cognitive impairment and hippocampus senescence in d-galactose-induced aging mouse model via upregulating the expression of SIRT1. Brain Res Bull 137: 311–318.
Liguori I, Russo G, Curcio F, Bulli G, Aran L, Della-Morte D, Gargiulo G, Testa G, Cacciatore FD, Abete P (2018). Oxidative stress, aging, and diseases. Clin Interv Aging 13: 757–772.
Lopez E, Le Faou A, Borzeix S, Berland S (2000). Stimulation of rat cutaneous fibroblasts and their synthetic activity by implants of powdered nacre (mother of pearl). Tissue Cell 32: 95–101.
Lou T, Huang Q, Su H, Zhao D, Li X (2021). Targeting Sirtuin 1 signaling pathway by ginsenosides. J Ethnopharmacol 268: 113657.
Luo G, Jian Z, Zhu Y, Zhu Y, Chen B, Ma R, Tang F, Xiao Y (2019). Sirt1 promotes autophagy and inhibits apoptosis to protect cardiomyocytes from hypoxic stress. Int J Mol Med 43: 2033–2043.
Luo Y, Toyoda M, Nakamura M, Morohashi M (2002). Morphological analysis of skin in senescence-accelerated mouse P10. Med Electron Microsc 35: 31–45.
Mangrulkar RS, Saint S, Chu S, Tierney LM (2002). What is the role of the clinical “pearl”? Am J Med 113: 617–624.
Mazzulla S, Anile D, De Sio S, Scaglione A, De Seta M, Anile A (2018). In vivo evaluations of emulsion O/W for a new topical anti-aging formulation: Short-term and long-term efficacy. J Cosmet Dermatol Sci Appl 8: 110–125.
Meng Q, Guo T, Li G, Sun S, He S, Cheng B, Shi B, Shan A (2018). Dietary resveratrol improves antioxidant status of sows and piglets and regulates antioxidant gene expression in placenta by Keap1-Nrf2 pathway and Sirt1. J Anim Sci Biotechnol 9: 34.
Mueller N, Sassa T, Morales-Gonzalez S, Schneider J, Salchow DJ, Seelow D, Knierim E, Stenzel W, Kihara A, Schuelke M (2019). De novo mutation in ELOVL1 causes ichthyosis, acanthosis nigricans, hypomyelination, spastic paraplegia, high frequency deafness and optic atrophy. J Med Genet 56: 164–175.
Oba A, Edwards C (2006). Relationships between changes in mechanical properties of the skin, wrinkling, and destruction of dermal collagen fiber bundles caused by photoaging. Skin Res Technol 12: 283–288.
Pan Y, Nicolazzo JA (2018). Impact of aging, Alzheimer’s disease and Parkinson’s disease on the blood-brain barrier transport of therapeutics. Adv Drug Deliv Rev 135: 62–74.
Patergnani S, Bouhamida E, Leo S, Pinton P, Rimessi A (2021). Mitochondrial oxidative stress and “Mito-Inflammation”: Actors in the diseases. Biomedicines 9: 216.
Pattapon A, Panjit C (2012). Alveolar bone regeneration by implantation of nacre and B-tricalcium phosphate in guinea pig. Implant Dent 21: 248–253.
Pattapon A, Panjit C, Theeralaksna S (2011). Potential induction of bone regeneration by nacre: An in vitro study. Implant Dent 20: 32–39.
Pawlowska E, Szczepanska J, Koskela A, Kaarniranta K, Blasiak J (2019). Dietary polyphenols in age-related macular degeneration: Protection against oxidative stress and beyond. Oxid Med Cell Longev 2019: 9682318.
Pei J, Wang Y, Zou X, Ruan H, Tang C, Liao J, Si G, Sun P (2021). Extraction, purification, bioactivities and application of matrix proteins from pearl powder and nacre powder: A review. Front Bioeng Biotechnol 9: 649665.
Pu YH, He JF, Gao ZS, Zeng M, Liao YB, Tong YH (2016). Study on the characteristics of trace elements in pearl powder and pearl layer powder. Food R. D. 37: 125–128.
Pyun HB, Kim M, Park J, Sakai Y, Numata N, Shin JY, Shin HJ, Kim DU, Hwang JK (2012). Effects of collagen tripeptide supplement on photoaging and epidermal skin barrier in UVB-exposed hairless mice. Prev Nutr Food Sci 17: 245–253.
Rayess H, Wang MB, Srivatsan ES (2012). Cellular senescence and tumor suppressor gene p16. Int J Cancer 130: 1715–1725.
Reeve A, Simcox E, Turnbull D (2014). Ageing and Parkinson’s disease: Why is advancing age the biggest risk factor? Ageing Res Rev 14: 19–30.
Rodgers JT, Lerin C, Haas W, Gygi SP, Spiegelman BM, Puigserver P (2005). Nutrient control of glucose homeostasis through a complex of PGC-1a and sirt1. Nature 434: 113–118.
Rosenthal DS, Griffiths CEM, Yuspa SH, Roop DR, Voorhees JJ (1992). Acute or chronic topical retinoic acid treatment of human skin in vivo alters the expression of epidermal transglutaminase, loricrin, involucrin, filaggrin, and keratins 6 and 13 but not keratins 1, 10, and 14. J Invest Dermatol 98: 343–350.
Rousseau M, Pereira-Mouriès L, Almeida MJ, Milet C, Lopez E (2003). The water-soluble matrix fraction from the nacre of Pinctada maxima produced earlier mineralization of MC3T3-E1 mouse pre-osteoblasts. Comp Biochem Physiol B Biochem Mol Biol 135: 1–7.
Rusu ME, Georgiu C, Pop A, Mocan A, Kiss B, Vostinaru O, Fizesan I, Stefan MG, Gheldiu AM, Mates L, Moldovan R, Muntean DM, Loghin F, Vlase L, Popa DS (2020). Effects of walnut (Juglans regia L.) kernel and walnut septum extract in a D-galactose-induced aging model and in naturally aged rats. Antioxidant 9: 424.
Shwea T, Pratchayasakul W, Chattipakorn N, Chattipakorna SC (2018). Role of D-galactose-induced brain aging and its potential used for therapeutic interventions. Exp Gerontol 101: 13–36.
Souza LS, Antunes M, Filho CB, Fabbro LD, Gomes MG, Goes ATR, Donato F, Prigol M, Boeira SP, Jesse CR (2015). Flavonoid Chrysin prevents age-related cognitive decline via attenuation of oxidative stress and modulation of BDNF levels in aged mouse brain. Pharmacol Biochem Behav 134: 22–30.
St-Pierr J, Drori S, Uldry M, Silvaggi JM, Rhee J, Jager S, Handshin C, Zheng K, Lin J, Yang W, Simon D, Bachoo R, Spiegelman BM (2006). Suppression of Reactive Oxygen Species and Neurodegeneration by the PGC-1 Transcriptional Coactivators. Cell 127: 397–408.
Suvik A, Effendy AWM (2012). The use of modified Masson’s trichrome staining in collagen evaluation in wound healing study. Malays J Vet Res 3: 39–47.
Umbayev B, Askarova S, Almabayeva A, Saliev T, Masoud AR, Bulanin D (2020). Galactose-induced skin aging: The role of oxidative stress. Oxid Med Cell Longev 2020: 7145656.
Westbroek P, Marin F (1998). A marriage of bone and nacre. Nature 392: 861–862.
Xiyang YB, Liu R, Wang XY, Li S, Zhao Y, Lu BT, Xiao ZC, Zhang LF, Wang TH, Zhang J (2020). COX5A plays a vital role in memory impairment associated with brain aging via the BDNF/ERK1/2 signaling pathway. Front Aging Neurosci 12: 215.
Xu H, Huang K, Gao Q, Gao Z, Han XA (2001). A study on the prevention and treatment of myopia with nacre in chicks. Pharmacol Res 44: 1–6.
Yamagami H, Fuji T, Wako M, Hasegawa Y (2021). Sulfated polysaccharide isolated from the nacre of pearl oyster improves scopolamine-induced memory impairment. Antioxidants (Basel) 10: 505.
Yotsuya Y, Hasegawa Y (2022). Nacre extract from pearl oyster attenuates amyloid beta-induced memory impairment. J Nat Med 76: 419–434.
Zhang JX, Li SR, Yao S, Bi QR, Hou JJ, Cai LY, Han SM, Wu WY, Guo DA (2016). Anticonvulsant and sedative–hypnotic activity screening of pearl and nacre (mother of pearl). J Ethnopharmacol 181: 229–235.
Zhang Z, Zhu H, Zheng Y, Zhang L, Wang X, Luo Z, Tang J, Lin L, Du Z, Dong C (2020). The effects and mechanism of collagen peptide and elastin peptide on skin aging induced by D-galactose combined with ultraviolet radiation. J Photochem Photobiol B: Biol 210: 111964.
Zhong SJ, Wang L, Wu HT, Lan R, Qin XY (2019). Coeloglossum viride var. bracteatum extract improves learning and memory of chemically-induced aging mice through upregulating neurotrophins BDNF and FGF2 and sequestering neuroinflammation. J Funct Foods 57: 40–47.
Zhu MJ, Wang X, Shi L, Liang LY, Wang Y (2018). Senescence, oxidative stress and mitochondria dysfunction. Med Res Innov 2: 1–5.

Table 1. Primer sequences

Gene name	Sequence (5′ to 3′)
Fatty acid elongase 1	F-TGATGTCTGGTTGGCTGAGT
	R-AGTGGTGGTGGAAGACATGGAGG
BDNF	F-AGAGCTGTTGGATGAGGACCAG
	R-CAAAGGCACTTGACTACTGAGCA
Sirtuin 1	F-TCCTTGGAGACTGCGATGTT
	R-AATTCCTTTTGTGGGCGTGG
Ceramide synthase 2	F-CCTCTGCTTCTCCTGGTTTG
	R-GGCGAACACAATGAAGAGGT
Ceramide synthase 3	F-ATCTCGAGCCCTTCTTCTCC
	R-CTCTGTCTCTTTGCCCTTGG
Filaggrin	F-ACAGAAGACCCAGAGCAGAC
	R-GGAGATGGTTTGGAGTGGGA
Involucrin	F-CAGCAGCAACAGATAGAGCG
	R-GTCCGGTTCTCCAATTCGTG
Loricrin	F-GCTACGGAGGCGGTTCTAG
	R-GTCTGACTGGTCTGCTGAGA
Catalase	F-AGGTGTTGAACGAGGAGGAG
	R-TGCGTGTAGGTGTGAATTGC
CuZn-SOD	F-CGGATGAAGAGAGGCATGTT
	R-CACCTTTGCCCAAGTCATCT
MnSOD	F-GGCCAAGGGAGATGTTACAA
	R-GCTTGATAGCCTCCAGCAAC
HO1	F-CACGCATATACCCGCTACCT
	R-CCAGAGTGTTCATTCGAGCA
β-actin	F-GGCTGTATTCCCCTCCATCG
	R-CCAGTTGGTAACAATGCCATGT

No competing interests reported.

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Nacre extract from pearl oyster shell prevents D-galactose-induced brain and skin aging

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