Wageningen Academic P u b l i s h e r s
Beneficial Microbes, 2015; 6(4): 497-504
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0134 - Thursday, October 05, 2017 9:50:26 PM - Göteborgs Universitet IP Address:130.241.16.16
Effect of Bifidobacterium breve B-3 on skin photoaging induced by chronic UV irradiation in mice T. Satoh*#, M. Murata#, N. Iwabuchi, T. Odamaki, H. Wakabayashi, K. Yamauchi, F. Abe and J.Z. Xiao Food Science and Technology Institute, Morinaga Milk Industry Co., Ltd., 1-83, 5-Chome, Higashihara, 228-8583 ZamaCity, Kanagawa-Pref, Japan; [email protected]; #Both authors contributed equally to this work Received: 22 September 2014 / Accepted: 3 December 2014 © 2015 Wageningen Academic Publishers
RESEARCH ARTICLE Abstract Probiotics have been shown to have a preventative effect on skin photoaging induced by short term UV irradiation, however, the underlying mechanisms and the effect of probiotics on skin photoaging induced by chronic UV irradiation remain unclear. In this study, we investigated the effect of Bifidobacterium breve B-3 on skin photoaging induced by chronic UV irradiation in hairless mice. Mice were irradiated with UVB three times weekly and orally administered B. breve B-3 (2×109 cfu/mouse /day) for 7 weeks. Nonirradiated mice and UVB-irradiated mice without probiotic treatment were used as controls. B. breve B-3 significantly suppressed the changes of transepidermal water loss, skin hydration, epidermal thickening and attenuated the damage to the tight junction structure and basement membrane induced by chronic UVB irradiation. Administration of B. breve B-3 tended to suppress the UV-induced interleukin-1β production in skin (P=0.09). These results suggest that B. breve B-3 could potentially be used to prevent photoaging induced by chronic UV irradiation. Keywords: Bifidobacterium breve, skin barrier, photoaging, chronic UV irradiation
1. Introduction Skin is the most extensive and heaviest organ of the human body. Skin acts as a barrier between the internal and external environment and is constantly exposed to physical, chemical and bacterial challenges. The epidermis plays an important role as a physiological barrier that protects the organism against pathogens, chemical and physiological damage, such as UV irradiation. UV irradiation, the most influential environmental factor associated with skin aging, induces inflammatory responses, impairs skin barrier function (Haratake et al., 1997a,b) and increases epidermal thickness (Yamamoto et al., 2008). UV irradiation induces inflammatory responses, such as the secretion of interleukin (IL)-1β, which negatively regulates homeostasis and enhances degradation of collagen in the abnormal matrix. This includes the disruption of the basement membrane in skin, which exists at the dermal-epidermal junction in skin and plays crucial roles in maintaining a healthy epidermis and dermis (Amano,
2009; Baugé et al., 2007; Fisher et al., 2002; Honda et al., 2008; Jourdan et al., 2011). Additionally, disruption of the basement membrane causes detachment of keratinocytes and keratinocyte abnormality, which leads to epidermal hyperplasia (Amano, 2009; Chen et al., 2013; Kastelan et al., 2006). Previous studies have shown that some probiotic strains improved skin photodamage induced by short-term UV irradiation in animals and humans via their antiinflammatory responses and improved the impaired skin barrier (Guéniche et al., 2006; Ishii et al., 2014; PeguetNavarro et al., 2008; Sugimoto et al., 2012), however, the underlying mechanism has not been well evaluated. In addition, there are no reports on the effect of probiotics on the impaired skin induced by chronic UV irradiation. Acute UV irradiation (such as short-term UV irradiation) induces hyperplasia associated with the increased synthesis of DNA, RNA, and proteins (Epstein et al., 1970; Matsumura and Ananthaswamy, 2004). However, skin cells returned
ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2014.0134497
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0134 - Thursday, October 05, 2017 9:50:26 PM - Göteborgs Universitet IP Address:130.241.16.16
T. Satoh et al.
to normal within 1-2 weeks via the nuclear p53 tumour suppressor protein, which plays a crucial role in DNA repair (Hall et al., 1993; Lane, 1992; Matsumura and Ananthaswamy, 2004). In contrast, chronic UV irradiation, such as chronic sun exposure and long-term UV exposure, induces skin photoaging that causes gradual deterioration of cutaneous structures and function and induces the accumulation of DNA damage resulting from recurrent, acute DNA injury (Matsumura and Ananthaswamy, 2004). Thus, chronic UV irradiation is believed to be more adequate for the simulation of skin photoaging caused by sun exposure. In this study, we investigated the effect of Bifidobacterium breve B-3 on skin photoaging induced by chronic UV irradiation in hairless mice. B. breve B-3 is a probiotic strain that has been shown to exert anti-obesity and anti-inflammatory effects in a diet-induced obese mouse model (Kondo et al., 2010, 2013). Histological and immunohistochemical studies were performed to determine the mechanism of probiotic effects.
2. Materials and methods Experimental design Seven-week-old male Hos:HR-1 hairless mice (Japan SLC, Shizuoka, Japan) were housed in single cages (four mice per cage) (22±2 °C and 50±5% humidity with a 12-h light and dark cycle) under specific pathogen-free conditions with free access to water and Lab MR stock diet (Nosan, Tokyo, Japan). After 2 weeks of acclimation, mice were divided into the following three groups (n=8 for each group): UV(-) saline, non-UV irradiated control group; UV(+) saline, UV irradiated control group; and UV(+) B-3, UV irradiated with administration of B. breve B-3. B. breve B-3 (MCC-1274) was cultured in a medium containing glucose, yeast extract and salts. The cells were harvested by centrifugation and washed. After being mixed with a cryoprotectant solution mainly composed of sucrose, the cells were lyophilised. A lyophilised powder of B. breve B-3 suspended in saline was continuously administered per os with a feeding tube at 2×109 cfu/mouse /day (containing approximately 0.5 mg of cryoprotectant) for 7 weeks (6 times per week) to the UV(+) B-3 group. The same volume of saline was administered to the UV(-) and UV(+) saline groups. Mice of the UV irradiated group were treated with UV irradiation three times a week for 7 weeks, and transepidermal water loss (TEWL) and hydration of the stratum corneum in the dorsal skin were measured once per week during the UV irradiation period. Body weight, food intake, and water intake were monitored every week throughout the experimental period. After the 7-week UV irradiation period, mice were fasted for 18 h and then euthanised using inhalation anaesthesia of sevoflurane (Sevoflo, DS Pharma Animal Health, Osaka, Japan). Serum and central dorsal skin samples were harvested for further analysis. All animal experiments were approved by the 498
Animal Research Committee of Morinaga Milk Industry (Kanagawa, Japan). This animal experiment was performed in parallel with another experiment (Murata et al., 2014), and identical control mice were used in both experiments.
UV irradiation UVB was administered using the method described in a previous report (Tanaka et al., 2009). Mice were housed in a stainless steel cage and exposed to UVB irradiation (0.9 mW/cm2) emitted from UVB lamps with peak emission at 306 nm (GL20E; Sankyo Denki, Tokyo, Japan). To avoid the formation of acute erythema, doses of UVB irradiation was started at half of the minimum erythema dose and increased gradually each week. The intensity of UVB was monitored using a Dermaray UV meter DMR-UV-M-2 (Terumo, Tokyo, Japan). The total energy of UVB each mouse received was 3.0 J/cm2 over 7 weeks.
Measurement of TEWL and hydration of the stratum corneum Skin barrier function in the mice was evaluated by measuring TEWL and hydration of the stratum corneum. TEWL of the stratum corneum in the dorsal skin was measured using a Tewameter TM 300 (Courage + Khazaka Electronic, Köln, Germany). The average stabilised TEWL value for 5 s under the mice-holding conditions was adopted as the average value for that mouse. Hydration of the stratum corneum in the dorsal skin was measured with a Corneometer CM 825 (Courage + Khazaka Electronic) under the same condition. Hydration units of the stratum corneum were calculated as the average of more than 10 measured values.
Histological analysis of skin Skin samples (10×10 mm square) were fixed in Bouin’s solution (Wako, Osaka, Japan) and embedded in paraffin after substitution with ethanol. The paraffin block was sliced into 3 μm sections, and the sections were stained with haematoxylin and eosin (H&E). The stained skin samples were observed using a microscope (BX53; Olympus, Tokyo, Japan). Epidermal thickness was analysed using CellSens Dimension software (Olympus), and the average thickness of 30 points per mouse was adopted as the average epidermal thickness.
Levels of pro inflammatory cytokine in skin and serum Dorsal skin samples were obtained using disposable biopsy punches (Kai Industries, Gifu, Japan) and homogenised using a Polytron homogeniser (Kinematica, Lucerne, Switzerland) in TE buffer (10 mM Tris-HCl, 1 mM EDTA, 0.25 M sucrose, pH 7.4) on ice. Levels of IL-1β in serum in the supernatant of skin homogenate samples were determined using the Quantikine ELISA kit for pro-inflammatory cytokines (R&D systems, Minneapolis, MN, USA). Beneficial Microbes 6(4)
Bifidobacterium breve B-3 prevents skin photoaging
Immunohistochemical study
3. Results
Paraffin embedded sections were stained for claudin-1, laminin and collagen IV. Paraffin embedded sections were deparaffinised and rehydrated, and antigen activation was performed with Target retrieval solution pH 9.0 (Dako, Tokyo, Japan) at 100 °C for 30 min for claudin-1 or with proteinase K (Sigma-Aldrich, St. Louis, MO, USA) for laminin and type IV collagen. Subsequently, the sections were incubated with H2O2-methanol for 5 min to inactivate the internal peroxidase, and then, sections were blocked with 1% goat serum (Nichirei Biosciences, Tokyo, Japan). The sections were incubated with the following primary antibodies: rabbit anti-claudin-1 polyclonal Ab (Abcam, Cambridge, UK) at 4 °C overnight; rabbit anti-laminin polyclonal Ab (Abcam) at room temperature (RT) for 1 h; and rabbit anti-collagen IV polyclonal Ab (Abcam) at RT for 1 h. After washing, the sections were incubated with HRP-conjugated anti-rabbit secondary Ab (Simple stain, MAX PO; Nichirei Biosciences) at RT for 30 min. After washing, sections were stained with Immpact DAB (Vector Laboratories, Inc., Burlingame, CA, USA) substrate and were counterstained with haematoxylin. After dehydration and penetration, the sections were mounted using malinol (Muto Pure Chemicals, Tokyo, Japan).
TEWL and hydration of the stratum corneum after UV irradiation The TEWL of the UV(+) saline group significantly increased and the hydration significantly decreased 2 weeks after UV irradiation in comparison with the UV(-) saline group (Figure 1A, B). Administration of B. breve B-3 significantly suppressed the increase of TEWL and the decrease of hydration of the stratum corneum from 6 weeks after the UV irradiation compared with the UV(+) saline group (Figure 1A, B). Seven weeks after UV irradiation, more reddening and more wrinkle formation were observed in the UV(+) saline group than in the UV(+) B-3 group (data not shown). There were no significant differences in body weight, food intake or water intake among the tested groups (data not shown).
Epidermal thickness after UV irradiation Figure 2A presents histological images of dorsal skin with H&E staining. The epidermal thickness was significantly increased in the UV(+) saline group compared with the UV(-) saline group (P
Beneficial Microbes, 2015; 6(4): 497-504
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0134 - Thursday, October 05, 2017 9:50:26 PM - Göteborgs Universitet IP Address:130.241.16.16
Effect of Bifidobacterium breve B-3 on skin photoaging induced by chronic UV irradiation in mice T. Satoh*#, M. Murata#, N. Iwabuchi, T. Odamaki, H. Wakabayashi, K. Yamauchi, F. Abe and J.Z. Xiao Food Science and Technology Institute, Morinaga Milk Industry Co., Ltd., 1-83, 5-Chome, Higashihara, 228-8583 ZamaCity, Kanagawa-Pref, Japan; [email protected]; #Both authors contributed equally to this work Received: 22 September 2014 / Accepted: 3 December 2014 © 2015 Wageningen Academic Publishers
RESEARCH ARTICLE Abstract Probiotics have been shown to have a preventative effect on skin photoaging induced by short term UV irradiation, however, the underlying mechanisms and the effect of probiotics on skin photoaging induced by chronic UV irradiation remain unclear. In this study, we investigated the effect of Bifidobacterium breve B-3 on skin photoaging induced by chronic UV irradiation in hairless mice. Mice were irradiated with UVB three times weekly and orally administered B. breve B-3 (2×109 cfu/mouse /day) for 7 weeks. Nonirradiated mice and UVB-irradiated mice without probiotic treatment were used as controls. B. breve B-3 significantly suppressed the changes of transepidermal water loss, skin hydration, epidermal thickening and attenuated the damage to the tight junction structure and basement membrane induced by chronic UVB irradiation. Administration of B. breve B-3 tended to suppress the UV-induced interleukin-1β production in skin (P=0.09). These results suggest that B. breve B-3 could potentially be used to prevent photoaging induced by chronic UV irradiation. Keywords: Bifidobacterium breve, skin barrier, photoaging, chronic UV irradiation
1. Introduction Skin is the most extensive and heaviest organ of the human body. Skin acts as a barrier between the internal and external environment and is constantly exposed to physical, chemical and bacterial challenges. The epidermis plays an important role as a physiological barrier that protects the organism against pathogens, chemical and physiological damage, such as UV irradiation. UV irradiation, the most influential environmental factor associated with skin aging, induces inflammatory responses, impairs skin barrier function (Haratake et al., 1997a,b) and increases epidermal thickness (Yamamoto et al., 2008). UV irradiation induces inflammatory responses, such as the secretion of interleukin (IL)-1β, which negatively regulates homeostasis and enhances degradation of collagen in the abnormal matrix. This includes the disruption of the basement membrane in skin, which exists at the dermal-epidermal junction in skin and plays crucial roles in maintaining a healthy epidermis and dermis (Amano,
2009; Baugé et al., 2007; Fisher et al., 2002; Honda et al., 2008; Jourdan et al., 2011). Additionally, disruption of the basement membrane causes detachment of keratinocytes and keratinocyte abnormality, which leads to epidermal hyperplasia (Amano, 2009; Chen et al., 2013; Kastelan et al., 2006). Previous studies have shown that some probiotic strains improved skin photodamage induced by short-term UV irradiation in animals and humans via their antiinflammatory responses and improved the impaired skin barrier (Guéniche et al., 2006; Ishii et al., 2014; PeguetNavarro et al., 2008; Sugimoto et al., 2012), however, the underlying mechanism has not been well evaluated. In addition, there are no reports on the effect of probiotics on the impaired skin induced by chronic UV irradiation. Acute UV irradiation (such as short-term UV irradiation) induces hyperplasia associated with the increased synthesis of DNA, RNA, and proteins (Epstein et al., 1970; Matsumura and Ananthaswamy, 2004). However, skin cells returned
ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2014.0134497
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0134 - Thursday, October 05, 2017 9:50:26 PM - Göteborgs Universitet IP Address:130.241.16.16
T. Satoh et al.
to normal within 1-2 weeks via the nuclear p53 tumour suppressor protein, which plays a crucial role in DNA repair (Hall et al., 1993; Lane, 1992; Matsumura and Ananthaswamy, 2004). In contrast, chronic UV irradiation, such as chronic sun exposure and long-term UV exposure, induces skin photoaging that causes gradual deterioration of cutaneous structures and function and induces the accumulation of DNA damage resulting from recurrent, acute DNA injury (Matsumura and Ananthaswamy, 2004). Thus, chronic UV irradiation is believed to be more adequate for the simulation of skin photoaging caused by sun exposure. In this study, we investigated the effect of Bifidobacterium breve B-3 on skin photoaging induced by chronic UV irradiation in hairless mice. B. breve B-3 is a probiotic strain that has been shown to exert anti-obesity and anti-inflammatory effects in a diet-induced obese mouse model (Kondo et al., 2010, 2013). Histological and immunohistochemical studies were performed to determine the mechanism of probiotic effects.
2. Materials and methods Experimental design Seven-week-old male Hos:HR-1 hairless mice (Japan SLC, Shizuoka, Japan) were housed in single cages (four mice per cage) (22±2 °C and 50±5% humidity with a 12-h light and dark cycle) under specific pathogen-free conditions with free access to water and Lab MR stock diet (Nosan, Tokyo, Japan). After 2 weeks of acclimation, mice were divided into the following three groups (n=8 for each group): UV(-) saline, non-UV irradiated control group; UV(+) saline, UV irradiated control group; and UV(+) B-3, UV irradiated with administration of B. breve B-3. B. breve B-3 (MCC-1274) was cultured in a medium containing glucose, yeast extract and salts. The cells were harvested by centrifugation and washed. After being mixed with a cryoprotectant solution mainly composed of sucrose, the cells were lyophilised. A lyophilised powder of B. breve B-3 suspended in saline was continuously administered per os with a feeding tube at 2×109 cfu/mouse /day (containing approximately 0.5 mg of cryoprotectant) for 7 weeks (6 times per week) to the UV(+) B-3 group. The same volume of saline was administered to the UV(-) and UV(+) saline groups. Mice of the UV irradiated group were treated with UV irradiation three times a week for 7 weeks, and transepidermal water loss (TEWL) and hydration of the stratum corneum in the dorsal skin were measured once per week during the UV irradiation period. Body weight, food intake, and water intake were monitored every week throughout the experimental period. After the 7-week UV irradiation period, mice were fasted for 18 h and then euthanised using inhalation anaesthesia of sevoflurane (Sevoflo, DS Pharma Animal Health, Osaka, Japan). Serum and central dorsal skin samples were harvested for further analysis. All animal experiments were approved by the 498
Animal Research Committee of Morinaga Milk Industry (Kanagawa, Japan). This animal experiment was performed in parallel with another experiment (Murata et al., 2014), and identical control mice were used in both experiments.
UV irradiation UVB was administered using the method described in a previous report (Tanaka et al., 2009). Mice were housed in a stainless steel cage and exposed to UVB irradiation (0.9 mW/cm2) emitted from UVB lamps with peak emission at 306 nm (GL20E; Sankyo Denki, Tokyo, Japan). To avoid the formation of acute erythema, doses of UVB irradiation was started at half of the minimum erythema dose and increased gradually each week. The intensity of UVB was monitored using a Dermaray UV meter DMR-UV-M-2 (Terumo, Tokyo, Japan). The total energy of UVB each mouse received was 3.0 J/cm2 over 7 weeks.
Measurement of TEWL and hydration of the stratum corneum Skin barrier function in the mice was evaluated by measuring TEWL and hydration of the stratum corneum. TEWL of the stratum corneum in the dorsal skin was measured using a Tewameter TM 300 (Courage + Khazaka Electronic, Köln, Germany). The average stabilised TEWL value for 5 s under the mice-holding conditions was adopted as the average value for that mouse. Hydration of the stratum corneum in the dorsal skin was measured with a Corneometer CM 825 (Courage + Khazaka Electronic) under the same condition. Hydration units of the stratum corneum were calculated as the average of more than 10 measured values.
Histological analysis of skin Skin samples (10×10 mm square) were fixed in Bouin’s solution (Wako, Osaka, Japan) and embedded in paraffin after substitution with ethanol. The paraffin block was sliced into 3 μm sections, and the sections were stained with haematoxylin and eosin (H&E). The stained skin samples were observed using a microscope (BX53; Olympus, Tokyo, Japan). Epidermal thickness was analysed using CellSens Dimension software (Olympus), and the average thickness of 30 points per mouse was adopted as the average epidermal thickness.
Levels of pro inflammatory cytokine in skin and serum Dorsal skin samples were obtained using disposable biopsy punches (Kai Industries, Gifu, Japan) and homogenised using a Polytron homogeniser (Kinematica, Lucerne, Switzerland) in TE buffer (10 mM Tris-HCl, 1 mM EDTA, 0.25 M sucrose, pH 7.4) on ice. Levels of IL-1β in serum in the supernatant of skin homogenate samples were determined using the Quantikine ELISA kit for pro-inflammatory cytokines (R&D systems, Minneapolis, MN, USA). Beneficial Microbes 6(4)
Bifidobacterium breve B-3 prevents skin photoaging
Immunohistochemical study
3. Results
Paraffin embedded sections were stained for claudin-1, laminin and collagen IV. Paraffin embedded sections were deparaffinised and rehydrated, and antigen activation was performed with Target retrieval solution pH 9.0 (Dako, Tokyo, Japan) at 100 °C for 30 min for claudin-1 or with proteinase K (Sigma-Aldrich, St. Louis, MO, USA) for laminin and type IV collagen. Subsequently, the sections were incubated with H2O2-methanol for 5 min to inactivate the internal peroxidase, and then, sections were blocked with 1% goat serum (Nichirei Biosciences, Tokyo, Japan). The sections were incubated with the following primary antibodies: rabbit anti-claudin-1 polyclonal Ab (Abcam, Cambridge, UK) at 4 °C overnight; rabbit anti-laminin polyclonal Ab (Abcam) at room temperature (RT) for 1 h; and rabbit anti-collagen IV polyclonal Ab (Abcam) at RT for 1 h. After washing, the sections were incubated with HRP-conjugated anti-rabbit secondary Ab (Simple stain, MAX PO; Nichirei Biosciences) at RT for 30 min. After washing, sections were stained with Immpact DAB (Vector Laboratories, Inc., Burlingame, CA, USA) substrate and were counterstained with haematoxylin. After dehydration and penetration, the sections were mounted using malinol (Muto Pure Chemicals, Tokyo, Japan).
TEWL and hydration of the stratum corneum after UV irradiation The TEWL of the UV(+) saline group significantly increased and the hydration significantly decreased 2 weeks after UV irradiation in comparison with the UV(-) saline group (Figure 1A, B). Administration of B. breve B-3 significantly suppressed the increase of TEWL and the decrease of hydration of the stratum corneum from 6 weeks after the UV irradiation compared with the UV(+) saline group (Figure 1A, B). Seven weeks after UV irradiation, more reddening and more wrinkle formation were observed in the UV(+) saline group than in the UV(+) B-3 group (data not shown). There were no significant differences in body weight, food intake or water intake among the tested groups (data not shown).
Epidermal thickness after UV irradiation Figure 2A presents histological images of dorsal skin with H&E staining. The epidermal thickness was significantly increased in the UV(+) saline group compared with the UV(-) saline group (P
