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Effect of LTA isolated from bifidobacteria on d -galactose-induced aging
Experimental Gerontology 44 (2009) 760–765
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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Effect of LTA isolated from bifidobacteria on D-galactose-induced aging Zheng-Jun Yi a,1, Yu-Rong Fu b,*,1, Meng Li a, Kun-Shan Gao a, Xu-Guang Zhang a a b
Department of Laboratory Medicine of Clinical Faculty, Weifang Medical University, Weifang, China Department of Medical Microbiology, Weifang Medical University, Weifang, China
a r t i c l e
i n f o
Article history: Received 10 October 2008 Received in revised form 21 August 2009 Accepted 31 August 2009 Available online 6 September 2009 Keywords: Aging Lipoteichoic acid Bifidobacterium Immunity Gene
a b s t r a c t Background: Bifidobacteria are a natural part of the bacterial flora in the human body and have a symbiotic bacteria–host relationship with human beings. Aging is associated with reduced number of beneficial colonic bifidobacteria and impaired immunity. Lipoteichoic acid is a major constituent of the cell wall of bifidobacteria which is important for bacterial survival, growth, and function. The possible anti-aging effects of lipoteichoic acid isolated from bifidobacteria is presently unknown. Objective: The aim of the present study was to investigate possible anti-aging effects of lipoteichoic acid isolated from bifidobacteria on senescent mice artificially induced by chronic injection of D-galactose and explore potential anti-aging’s mechanisms. Methods: Mice were artificially induced senescence by consecutive injection of D-galactose (100 mg/kg) once daily for 7 weeks and lipoteichoic acid from bifidobacterium bifidum, was simultaneously administered to them once a week by intraperitoneal infusion. Mice were sacrificed, blood and other samples were collected at the indicated time. Anti-oxidation activity in brain, histology of tissue, gene expression, lymphocyte’s DNA damage and cytokine production of lymphocytes in vitro and in vivo were measured. Results: Lipoteichoic acid could significantly improve general appearance of the aging model mice, improve anti-oxidation activity in brain, increase IL-2 level and decrease TNF-a level in vitro and in vivo, respectively. Besides, LTA remarkably inhibited DNA damage in the both splenic lymphocytes and circulating lymphocytes. Moreover, LTA could decrease p16 expression while increase c-fos expression in the D-galactose treated mice. Conclusion: Taken together, the results indicated, for the first time, that LTA could suppress the aging process via the following several mechanisms, including enhancement of anti-oxidation activity in brain, improvement of immune function and alteration of gene expression. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Bifidobacteria are a natural part of the bacterial flora in the human body and have a symbiotic bacteria–host relationship with human beings. Bifidobacteria are involved in some physiological functions, including anti-infection action (Ewaschuk et al., 2008; Lomax and Calder, 2009; Graul et al., 2009), anti-tumor action (Capurso et al., 2006; Hidaka et al., 2007; Wei et al., 2007) and anti-aging action (Hopkins and Macfarlane, 2002; Hébuterne, 2003). Aging is associated with reduced number of beneficial colonic bifidobacteria and impaired immunity (He et al., 2001; Woodmansey, 2007). Nevertheless, little is currently known about the molecular basis for the effects of bifidobacteria on aging. Lipoteichoic acid (LTA) is a major constituent of the cell wall of gram-positive bacteria which is important for cell wall remodeling, * Corresponding author. Tel.: +86 0536 8068955. E-mail address: [email protected] (Y.-R. Fu). 1 These authors contributed equally to this work. 0531-5565/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2009.08.011
bacterial survival, growth and function. Many gram-positive bacteria possess common-type LTA consisting of a 1,3-1inked poly (g1ycerophosphate) chain which is bound through a phosphodiester linkage to a membrane glycolipid. However, LTA from bifidobacterium bifidum consists of a 1,2-linked polyglycerophosphate chain (instead of the usual 1,3-), a polysaccharide chain (1,6-linked b-D-glucan or 1,5-linked b-D-galactofuranan) and a glycolipid moiety. To date, little is known about the role of LTA from bifidobacteria. Although several reports suggested that LTA from bifidobacteria was involved in anti-tumor action and anti-aging action (Yue et al., 2007a,b; Yanhua et al., 2009), these studies have been too under-powered to draw definitive conclusion. Rodent chronically injected with D-galactose has been used as an artificially induced animal aging model for anti-aging research (Song et al., 1999). The aim of the present study was to make use of the artificially induced aging model mice to further investigate potential anti-aging effects of LTA from bifidobacteria and explore underlying antiaging molecular mechanisms.
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2. Material and methods 2.1. Isolation and purification of LTA Bifidobacteria bifidum (ATCC 86321) were purchased from microbiology institute of Chinese academy of sciences. LTA was isolated from disintegrated bacteria with aqueous phenol and purified by nuclease digestion and consecutively different chromatography. The purity of LTA was checked by nuclear magnetic resonance and mass spectrometry analysis according to the procedure (Morath et al., 2001; Lynch et al., 2004). The purity of the LTA was greater than 99%. 2.2. Experiment animals Animal housing and all experimental procedures followed the requirements of the Provisions and General Recommendations of Chinese Experimental Animal Administration Legislation. Sevenweek-old female Kun-ming mice (20 ± 2 g) were purchased from the Experimental Animal Center of Weifang Medical University and housed under standard pathogen free condition. During the entire experiment process, they had free access to food and water. After 1-week acclimatization to the home cage, the mice were randomly divided into three groups of 20 each; mice of group one were artificially induced senescence by subcutaneous (s.c.) injection of D-galactose (100 mg/kg) (Sigma) once daily (Wei et al.,2005), and normal saline was simultaneously administered to them by intraperitoneal (i.p.) injection once a week (the aging model mice); mice of group two were treated by i.p. injection of LTA (4 mg/kg) once a week after s.c. injection of D-galactose once daily (the LTA treatment mice); mice of group three were treated by i.p. injection of normal saline once a week and by s.c. injection of normal saline once daily (the young control mice). The mice were sacrificed after treatment for 7 weeks. 2.3. Observation of general appearance, measurement of body weight, organ index and pathological examination During the entire experiment process, general appearance was observed. After treatment for 7 weeks, the mice were sacrificed. The brains, spleens, thymus glands, kidneys and livers were weighted and their weights relative to the final body weight (organ index) were calculated. The thymus glands and kidneys were fixed in neutral-buffered 10% formalin, sectioned and stained with hematoxylin and eosin (HE). For the thymus glands, histopathological examination was observed under a light microscope; for the kidneys, glomeruli were counted and glomerular volume was assessed according to the procedure (Boubred et al., 2007). 2.4. Anti-oxidant measurements in brain Superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO) and nitric oxide synthase (NOS) kits were purchased from Nanjing Jiancheng Biological Technology Company, China. The activities of SOD and NOS, as well as levels of NO and MDA, were determined according to the instructions. Briefly, the brains were separated into two parts for determination of enzymatic activity, NO and MDA levels, respectively. The samples for enzymatic activity analysis were washed and homogenized in phosphate-buffered saline (PBS) (pH 7.4) using Homogenizer. The homogenized samples were then sonicated for 1.5 min (30-s sonications interrupted with a 30-s pause on ice). The samples were then centrifuged at 17,000g for 15 min, and the supernatants, if not used for enzyme assays immediately, were kept in the
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deep freeze at 80 °C. The samples for MDA and NO analysis were washed with ice-cold 0.9% NaCl solution and homogenized in 1.15% KCl. The samples were then centrifuged and supernatants were assayed for NO and MDA levels. The protein content in the brain homogenates was measured by the Lowry method using bovine serum albumin (Sigma, USA) as a standard. SOD and NOS activities were expressed as nU/mg protein and U/mg protein, respectively. MDA and NO levels were expressed as nmol /mg protein. 2.5. Measurement of DNA damage in lymphocytes and cytokines production in vitro and in vivo The spleens were sterilely homogenized and lymphocytes were prepared by ficoll separating medium (Yi et al., 2007). The splenocytes (5 105 cells/well) were stimulated with ConA (5 lg/ml) in a 96-well round bottom plate for 72 h (Wang et al., 2007), and level of IL-2 in the culture supernatant was determined by ELISA (Camarillo, CA). The peripheral blood lymphocytes were prepared by ficoll separating medium and diluted to 1 105 cells/ml. Comet assay was used to determine DNA damage in both circulating lymphocytes and above prepared splenocytes as described (Singh et al., 1988). Under fluorescence microscope, cell morphology was observed and percentage of cells presenting DNA damage was assessed. Studies showed that aging is characterized by a pro-inflammatory state that contributes to the onset of disability and age-related diseases. TNF-a is an important pro-inflammatory mediator that influences host defense against infection and cancer. In the study, TNF-a level in serum was measured using enzyme-linked immunosorbent assay (ELISA) kits (Camarillo, CA) according to the instructions. 2.6. Western blot analysis of gene expression The tissue proteins were separated by 12% SDS–PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and then blotted onto nitrocellulose membrane using a semidry transfer system (Huang 2008; Kurien and Scofield, 2009). Following blocked with 4% nonfat milk, the membranes were probed sequentially with primary mouse-anti human p16, c-fos antibodies and HRP (Peroxidase, Horseradish)-labeled goat-anti mouse secondary antibodies (Santa cruz), and immunoreactive bands were detected with 3,30 diaminobenzidine (DAB) staining. b-actin was served as protein loading control. 2.7. Statistical analysis Data were presented as mean ± standard deviation (SD). ANOVA tests or student’s t tests were used for statistical analysis. The data were regarded as significantly different at P < 0.05. 3. Results 3.1. Changes of general appearance, organ index and morphological structures During the entire experiment process, the aging model mice were habituated to s.c. injection with D-galactose at dose of 100 mg/kg each day and general appearance was observed. The mice were sacrificed at the indicated time and tissues were collected for organ index and histology examination. Results showed that, compared with that of young control mice, hair of the aging model ones gradually lost elasticity and got brittle; skin became thin and sag little by little; body weight, organ indexes, including
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Table 1 Effect of LTA on body weight and organ indexes (x s). Group Aging model Young control LTA treatment * ** #
Body weight (g) 21.67 ± 3.26 29.33 ± 5.19 26.91 ± 5.34
*
Brain index 8.67 ± 0.79 10.59 ± 1.23** 9.89 ± 1.15#
Thymus index 2.37 ± 0.49 3.36 ± 0.65** 3.05 ± 0.62#
Spleen index 4.09 ± 1.21 5.87 ± 1.74 4.78 ± 1.83
*
Kidney index *
13.21 ± 1.69 16.82 ± 2.07 15.53 ± 1.87#
Liver index 50.63 ± 7.68* 59.44 ± 5.06 56.22 ± 6.48
P < 0.05 vs. the young control mice and the LTA treatment mice. P < 0.05 vs. the aging model mice and the LTA treatment mice. P < 0.05 vs. the aging model mice.
Fig. 1. Observation of morphology. Tissues were collected at the indicated time, fixed in l0% neutral-buffered formalin, embedded in paraffin, and then stained with hematoxylin and eosin (H&E) for histology examination. (a) The young control group; (b) the aging model group; (c) the LTA-treated group. (A) kidney morphology: global glomerular number in the LTA-treated mice was higher than that in the aging model mice, while lower than that in the young control mice. Mean glomerular volume in the LTA-treated mice was lower than that in the aging model mice, while higher than that in the young control mice. (B) Thymus morphology: border between cortex and medulla of thymus gland showed obscure in the aging model mice and fewer lymphocytes were remained in the cortex section than that in the LTA-treated mice and the young control mice. Data shown was a representative from twenty separate experiments. (magnification, 200).
brain, thymus gland, spleen, kidney and liver, showed significant decrease in the aging model mice than that in the LTA-treated mice and in the young control mice (Table 1). Global glomerular number in the LTA-treated mice (n = 3500) was higher than that in the aging model ones (n = 2600), while lower than that in the young control ones (n = 4200). Mean glomerular volume in the LTA-treated mice (v = 0.43 103) was lower than that in the aging model ones (v = 0.65 103), while higher than that in the young control ones (v = 0.28 103) (Fig. 1 as arrow showed). Border between cortex and medulla of the thymus gland showed obscure in the aging model mice and fewer lymphocytes were remained in the cortex section than that in the LTA-treated mice and the young control mice (Fig. 1 as arrow showed).The results above mentioned indicated that LTA had obvious anti-aging effect and its underlying molecular mechanisms were also explored and described as follows.
that compared with that of young control mice, SOD activity in the aging model mice were remarkably reduced, whereas NO, MDA levels and NOS activity were noticeably increased; LTA could increase SOD activity and decrease NO, MDA levels and NOS activity (Table 2). The results suggested that LTA could improve antioxidation ability in brain. 3.3. Improvement of immune function Immune senescence means impaired immune response (Murciano et al., 2006) and altered immune organ structure (Larbi et al., 2004). Many studies showed that DNA damage in lymphocytes increased with age (Ribeiro et al., 2007). In the study, DNA damage in both circulating lymphocytes and splenic lymphocytes was anaTable 2 Effect of LTA on anti-oxidant systems in brain x s.
3.2. Enhancement of activity of anti-oxidant systems in brain According to free radical theory of aging, senescence is mainly due to accumulation of free radicals which do harm to all kinds of cells and make organs senescent. It was found that oxidative stress of organs increases gradually with age (Boveris and Fraga, 2004). In order to explore possible mechanisms of LTA anti-aging, anti-oxidant activity in brain was evaluated. The result showed
Group
SOD (nU/mg)
MDA (nmol/mg)
NO (nmol/mg)
NOS (U/mg)
Aging model 104.51 ± 5.77* 12.06 ± 1.59* 1.742 ± 0.321* 1.137 ± 0.278* Young control 122.31 ± 6.86 7.21 ± 1.08 1.135 ± 0.212 0.609 ± 0.211 LTA treatment 117.82 ± 7.63 8.86 ± 1.78 1.493 ± 0.208 0.974 ± 0.244 *
P < 0.05 vs. the young control mice and the LTA treatment mice. There was no difference between the young control mice and the LTA treatment mice for above parameters.
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Table 3 Effect of LTA on DNA lesion and on cytokines level in vitro and in vivo x s. Group
Percentage of splenic lymphocytes with DNA lesion (100%)
Percentage of circulating lymphocytes with DNA lesion (100%)
IL-2 (pg/mL)
TNF-a (pg/mL)
Young control Aging model LTA treatment
12.8 ± 5.4**
15.4 ± 6.1**
166.6 ± 24.8
11.51 ± 4.11
32.3 ± 5.5 22.1 ± 5.4#
38.3 ± 7.3 25.1 ± 6.4#
87.8 ± 15.1* 114.2 ± 17.9
17.37 ± 5.92* 9.13 ± 2.03
#
P < 0.05 vs. the aging model mice. P < 0.05 vs. the control mice and the LTA-treated mice. There was no difference between the control mice and the LTA-treated mice for TNF-a and IL-2 levels. ** P < 0.05 vs. the aging model mice and the LTA-treated mice. *
lyzed by alkaline comet assay. Under fluorescence microscope, for normal lymphocyte, DNA was characterized by locating inside the cell, whereas for damaged cell, DNA was characterized by obvious migration to outside cell. The percentage of cells exhibiting DNA migration in the aging model mice was higher than that in the young control ones. DNA damage percentage was significantly reduced after the aging model mice were treated by LTA (Table 3). Many studies have been shown that T cell function decreases with age. In order to evaluate the effect of LTA on T cell function, splenocytes were stimulated and IL-2 level in the culture supernatant was determined by ELISA. Compared with that in the young control mice, IL-2 level in the aging model ones was significantly reduced, while significantly increased after the aging model mice were treated by LTA. However, no difference was found between the young control mice and the LTA-treated mice (Table 3). Aging is characterized by a pro-inflammatory state that contributes to the onset of disability and age-related diseases (Huang et al., 2005; Opal et al., 2005). TNF-a is a potent pro-inflammatory cytokine and can produce tissue inflammatory actions. Influence of LTA on TNF-a in serum was studied. The data showed that TNF-a level significantly increased in the aging model mice than that in the control mice and in the LTA-treated mice, whereas no difference was found between the young control mice and the LTA-treated mice (Table 3). The results both in vitro and in vivo indicated that LTA could significantly improve immune function. 3.4. Regulation of aging-related gene expression A lot of studies have found that, many genes are closely related to mammalian aging, and among them, both p16 and c-fos are well defined senescence-related genes (Krishnamurthy et al., 2004). In the study, there were more p16 expression while less c-fos expression in many organs in the aging model mice than that in the young control ones. After treatment by LTA, p16 expression was remarkably down-regulated whereas c-fos expressiosn was upregulated in the organs (Fig. 2). The results suggested that LTA could modulate age-related genes expression.
Fig. 2. Examination of p16 and c-fos expressions. Western blot was used to detect expressions of p16 and c-fos. Compared with that of young control group, there was more p16 expression whereas less c-fos expression in aging model mice; After being treated by LTA, p16 expression was remarkably decreased whereas c-fos expression increased. (a) The young control group; (b) the LTA-treated group; (c) the aging model group. Data shown was a representative of twenty independent experiments.
It was reported that LTA from bifidobacteria could delay cell aging induced by H2O2, markedly decreased p21 expression, and increased cyclin E and CDK2 expressions (Yanhua et al., 2009). LTA could postpone aging by improving anti-oxidation activity and klotho gene expression in aging mice kidney (Yue et al., 2007a,b). These studies have been too under-powered to draw definitive conclusion on LTA function. The aim of the present study was to make use of the artificially induced aging model mice to further investigate potential anti-aging effects of LTA from bifidobacteria. The aging model mice showed aging-related appearance changes, significant decrease in body weight and organ indexes. However, after the aging model mice treated with LTA, both body weight and organ indexes increased. The data indicated that LTA could suppress the aging process. Kidney is an important organ and function was declined gradually due to its structure changes with age. The global glomerular number decreased while mean glomerular volume increased with age. Otherwise, immune system also presents physiological diminution. Thymus is an important immune organ which shows senescent signs firstly. Histology of thymus is characterized as rare lymphocytes remained in cortex, some connective tissue and fatty tissue instead with age. Our results showed that LTA could increase the global glomerular number and decrease the mean glomerular volume. Otherwise, LTA could increase the lymphocytes in cortex. The data above mentioned suggested that LTA of bifidobacterium could inhibit the aging process and its underlying molecular mechanisms were also explored and described as follows. 4.2. Enhancement of activity of anti-oxidant systems
4. Discussion 4.1. LTA inhibiting the aging process Accelerated senescence in mice can be induced by D-galactose (D-gal) and D-gal treated mice were found close to those of 21-month mice showing neurological impairment, decreased activity of anti-oxidant enzymes, and poor immune responses (Wei et al., 2005). D-gal injection has been usually used to establish an aging model for anti-aging research.
According to free radical theory of aging, senescence is mainly due to accumulation of free radicals which do harm to all kinds of cells and make organs senescent. A large of studies revealed that the balance between reactive oxygen species system and anti-oxidation system determines the degree of oxidative stress (Villamor et al., 2004). It was found that oxidative stress of organs increases gradually with age (Boveris and Fraga, 2004). MDA level usually reflects degree of lipid peroxidation and means indirect impairment level of cell. SOD is one of critical enzyme that can protect cells
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from damage and its activity reflects indirectly ability of anti-oxidation. NO, generated by NOS, plays an ubiquitous role in the body in controlling the function of almost every, if not every, organ system. NO can cause cell death by oxidant damage and inactivating enzymes which may play a role in the progression of Alzheimer’s disease and parkinsonism (McCann et al., 1998, 2005). LTA could increase SOD activity and decrease NOS activity, MDA and NO levels in the brain. The results indicated that LTA could enhance antioxidant activity in brain.
and alter gene expression. Although the report supports the potential anti-aging action of LTA from bifidobacteria, further longer-term investigations should be conducted to substantiate its anti-aging action.
Acknowledgements The authors thank Mu Liang and Tan tei for helpful discussions and suggestions and Ding Zhou, Qian Yong for technical support.
4.3. Improvement of immune function It has been well-established that immunity declines with aging (Linton and Dorshkind, 2004). The effects of aging on the immune system are widespread and result in a diminution of immune responsiveness in the elderly. Many studies showed that DNA damage in lymphocytes increased with age (Jeyapalan and Sedivy, 2008). In the study, DNA damage in circulating lymphocytes and splenic lymphocytes was analyzed by alkaline comet assay and the percentage of DNA damage was significantly reduced after the aging model mice treated by LTA. It is well known that advancing age is associated with significant alternations in T cells function. The aging process has been associated with a reduction in T cell responsiveness to a variety of antigens, which may contribute to the significant increase in the incidence of infectious diseases observed in elder individuals (Sharma et al., 2006). IL-2 is an important, powerful T cell growth factor and plays important role in inmmune response, immunoloregulation and anti-tumor. Our results showed that IL-2 level in the aging model mice was significantly reduced than that in the young control ones, while IL-2 level was obviously increased after the aging model mice were treated by LTA. Aging is characterized by a pro-inflammatory state that contributes to the onset of disability and age-related diseases (Huang et al., 2005; Opal et al., 2005). TNF-a is a potent pro-inflammatory cytokine and can produce tissue inflammatory actions. Influence of LTA on TNF-a in serum was studied in the mice. The data showed that TNF-a level was significantly increased in the aging model mice than that in the control ones and in the LTA-treated ones. There was no difference between the control mice and the LTAtreated mice for TNF-a and IL-2 levels. The data both in vitro and in vivo indicated that LTA could significantly improve immune function. 4.4. Alteration of aging-related gene expression Many genes are closely related to mammalian aging. P16 is an inhibitor of cell cycle progression, increases with age and contributes to the impaired cellular regeneration of an aging organism (Sharpless, 2004). P16 deficiency partially prevented the age-induced decline in cell proliferation and tissue function. c-fos plays an important role in cell growth, differentiation, regeneration and remodeling. Moreover, c-fos is essential for cell entry into S phase. c-fos was remarkably down-regulated in many tissues with age (Cayetanot et al., 2005). In the study, we found that LTA could remarkably decrease p16 expression while increase c-fos expression. The results suggested that LTA could modulate age-related gene expression. 5. Conclusions Taken together, the results mentioned above suggested that LTA isolated from bifidobacteria could be involved in the anti-aging action, and anti-aging mechanisms might be as follows: LTA could enhance anti-oxidant activity in brain, improve immune function
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Effect of LTA isolated from bifidobacteria on D-galactose-induced aging Zheng-Jun Yi a,1, Yu-Rong Fu b,*,1, Meng Li a, Kun-Shan Gao a, Xu-Guang Zhang a a b
Department of Laboratory Medicine of Clinical Faculty, Weifang Medical University, Weifang, China Department of Medical Microbiology, Weifang Medical University, Weifang, China
a r t i c l e
i n f o
Article history: Received 10 October 2008 Received in revised form 21 August 2009 Accepted 31 August 2009 Available online 6 September 2009 Keywords: Aging Lipoteichoic acid Bifidobacterium Immunity Gene
a b s t r a c t Background: Bifidobacteria are a natural part of the bacterial flora in the human body and have a symbiotic bacteria–host relationship with human beings. Aging is associated with reduced number of beneficial colonic bifidobacteria and impaired immunity. Lipoteichoic acid is a major constituent of the cell wall of bifidobacteria which is important for bacterial survival, growth, and function. The possible anti-aging effects of lipoteichoic acid isolated from bifidobacteria is presently unknown. Objective: The aim of the present study was to investigate possible anti-aging effects of lipoteichoic acid isolated from bifidobacteria on senescent mice artificially induced by chronic injection of D-galactose and explore potential anti-aging’s mechanisms. Methods: Mice were artificially induced senescence by consecutive injection of D-galactose (100 mg/kg) once daily for 7 weeks and lipoteichoic acid from bifidobacterium bifidum, was simultaneously administered to them once a week by intraperitoneal infusion. Mice were sacrificed, blood and other samples were collected at the indicated time. Anti-oxidation activity in brain, histology of tissue, gene expression, lymphocyte’s DNA damage and cytokine production of lymphocytes in vitro and in vivo were measured. Results: Lipoteichoic acid could significantly improve general appearance of the aging model mice, improve anti-oxidation activity in brain, increase IL-2 level and decrease TNF-a level in vitro and in vivo, respectively. Besides, LTA remarkably inhibited DNA damage in the both splenic lymphocytes and circulating lymphocytes. Moreover, LTA could decrease p16 expression while increase c-fos expression in the D-galactose treated mice. Conclusion: Taken together, the results indicated, for the first time, that LTA could suppress the aging process via the following several mechanisms, including enhancement of anti-oxidation activity in brain, improvement of immune function and alteration of gene expression. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Bifidobacteria are a natural part of the bacterial flora in the human body and have a symbiotic bacteria–host relationship with human beings. Bifidobacteria are involved in some physiological functions, including anti-infection action (Ewaschuk et al., 2008; Lomax and Calder, 2009; Graul et al., 2009), anti-tumor action (Capurso et al., 2006; Hidaka et al., 2007; Wei et al., 2007) and anti-aging action (Hopkins and Macfarlane, 2002; Hébuterne, 2003). Aging is associated with reduced number of beneficial colonic bifidobacteria and impaired immunity (He et al., 2001; Woodmansey, 2007). Nevertheless, little is currently known about the molecular basis for the effects of bifidobacteria on aging. Lipoteichoic acid (LTA) is a major constituent of the cell wall of gram-positive bacteria which is important for cell wall remodeling, * Corresponding author. Tel.: +86 0536 8068955. E-mail address: [email protected] (Y.-R. Fu). 1 These authors contributed equally to this work. 0531-5565/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2009.08.011
bacterial survival, growth and function. Many gram-positive bacteria possess common-type LTA consisting of a 1,3-1inked poly (g1ycerophosphate) chain which is bound through a phosphodiester linkage to a membrane glycolipid. However, LTA from bifidobacterium bifidum consists of a 1,2-linked polyglycerophosphate chain (instead of the usual 1,3-), a polysaccharide chain (1,6-linked b-D-glucan or 1,5-linked b-D-galactofuranan) and a glycolipid moiety. To date, little is known about the role of LTA from bifidobacteria. Although several reports suggested that LTA from bifidobacteria was involved in anti-tumor action and anti-aging action (Yue et al., 2007a,b; Yanhua et al., 2009), these studies have been too under-powered to draw definitive conclusion. Rodent chronically injected with D-galactose has been used as an artificially induced animal aging model for anti-aging research (Song et al., 1999). The aim of the present study was to make use of the artificially induced aging model mice to further investigate potential anti-aging effects of LTA from bifidobacteria and explore underlying antiaging molecular mechanisms.
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2. Material and methods 2.1. Isolation and purification of LTA Bifidobacteria bifidum (ATCC 86321) were purchased from microbiology institute of Chinese academy of sciences. LTA was isolated from disintegrated bacteria with aqueous phenol and purified by nuclease digestion and consecutively different chromatography. The purity of LTA was checked by nuclear magnetic resonance and mass spectrometry analysis according to the procedure (Morath et al., 2001; Lynch et al., 2004). The purity of the LTA was greater than 99%. 2.2. Experiment animals Animal housing and all experimental procedures followed the requirements of the Provisions and General Recommendations of Chinese Experimental Animal Administration Legislation. Sevenweek-old female Kun-ming mice (20 ± 2 g) were purchased from the Experimental Animal Center of Weifang Medical University and housed under standard pathogen free condition. During the entire experiment process, they had free access to food and water. After 1-week acclimatization to the home cage, the mice were randomly divided into three groups of 20 each; mice of group one were artificially induced senescence by subcutaneous (s.c.) injection of D-galactose (100 mg/kg) (Sigma) once daily (Wei et al.,2005), and normal saline was simultaneously administered to them by intraperitoneal (i.p.) injection once a week (the aging model mice); mice of group two were treated by i.p. injection of LTA (4 mg/kg) once a week after s.c. injection of D-galactose once daily (the LTA treatment mice); mice of group three were treated by i.p. injection of normal saline once a week and by s.c. injection of normal saline once daily (the young control mice). The mice were sacrificed after treatment for 7 weeks. 2.3. Observation of general appearance, measurement of body weight, organ index and pathological examination During the entire experiment process, general appearance was observed. After treatment for 7 weeks, the mice were sacrificed. The brains, spleens, thymus glands, kidneys and livers were weighted and their weights relative to the final body weight (organ index) were calculated. The thymus glands and kidneys were fixed in neutral-buffered 10% formalin, sectioned and stained with hematoxylin and eosin (HE). For the thymus glands, histopathological examination was observed under a light microscope; for the kidneys, glomeruli were counted and glomerular volume was assessed according to the procedure (Boubred et al., 2007). 2.4. Anti-oxidant measurements in brain Superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO) and nitric oxide synthase (NOS) kits were purchased from Nanjing Jiancheng Biological Technology Company, China. The activities of SOD and NOS, as well as levels of NO and MDA, were determined according to the instructions. Briefly, the brains were separated into two parts for determination of enzymatic activity, NO and MDA levels, respectively. The samples for enzymatic activity analysis were washed and homogenized in phosphate-buffered saline (PBS) (pH 7.4) using Homogenizer. The homogenized samples were then sonicated for 1.5 min (30-s sonications interrupted with a 30-s pause on ice). The samples were then centrifuged at 17,000g for 15 min, and the supernatants, if not used for enzyme assays immediately, were kept in the
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deep freeze at 80 °C. The samples for MDA and NO analysis were washed with ice-cold 0.9% NaCl solution and homogenized in 1.15% KCl. The samples were then centrifuged and supernatants were assayed for NO and MDA levels. The protein content in the brain homogenates was measured by the Lowry method using bovine serum albumin (Sigma, USA) as a standard. SOD and NOS activities were expressed as nU/mg protein and U/mg protein, respectively. MDA and NO levels were expressed as nmol /mg protein. 2.5. Measurement of DNA damage in lymphocytes and cytokines production in vitro and in vivo The spleens were sterilely homogenized and lymphocytes were prepared by ficoll separating medium (Yi et al., 2007). The splenocytes (5 105 cells/well) were stimulated with ConA (5 lg/ml) in a 96-well round bottom plate for 72 h (Wang et al., 2007), and level of IL-2 in the culture supernatant was determined by ELISA (Camarillo, CA). The peripheral blood lymphocytes were prepared by ficoll separating medium and diluted to 1 105 cells/ml. Comet assay was used to determine DNA damage in both circulating lymphocytes and above prepared splenocytes as described (Singh et al., 1988). Under fluorescence microscope, cell morphology was observed and percentage of cells presenting DNA damage was assessed. Studies showed that aging is characterized by a pro-inflammatory state that contributes to the onset of disability and age-related diseases. TNF-a is an important pro-inflammatory mediator that influences host defense against infection and cancer. In the study, TNF-a level in serum was measured using enzyme-linked immunosorbent assay (ELISA) kits (Camarillo, CA) according to the instructions. 2.6. Western blot analysis of gene expression The tissue proteins were separated by 12% SDS–PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and then blotted onto nitrocellulose membrane using a semidry transfer system (Huang 2008; Kurien and Scofield, 2009). Following blocked with 4% nonfat milk, the membranes were probed sequentially with primary mouse-anti human p16, c-fos antibodies and HRP (Peroxidase, Horseradish)-labeled goat-anti mouse secondary antibodies (Santa cruz), and immunoreactive bands were detected with 3,30 diaminobenzidine (DAB) staining. b-actin was served as protein loading control. 2.7. Statistical analysis Data were presented as mean ± standard deviation (SD). ANOVA tests or student’s t tests were used for statistical analysis. The data were regarded as significantly different at P < 0.05. 3. Results 3.1. Changes of general appearance, organ index and morphological structures During the entire experiment process, the aging model mice were habituated to s.c. injection with D-galactose at dose of 100 mg/kg each day and general appearance was observed. The mice were sacrificed at the indicated time and tissues were collected for organ index and histology examination. Results showed that, compared with that of young control mice, hair of the aging model ones gradually lost elasticity and got brittle; skin became thin and sag little by little; body weight, organ indexes, including
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Table 1 Effect of LTA on body weight and organ indexes (x s). Group Aging model Young control LTA treatment * ** #
Body weight (g) 21.67 ± 3.26 29.33 ± 5.19 26.91 ± 5.34
*
Brain index 8.67 ± 0.79 10.59 ± 1.23** 9.89 ± 1.15#
Thymus index 2.37 ± 0.49 3.36 ± 0.65** 3.05 ± 0.62#
Spleen index 4.09 ± 1.21 5.87 ± 1.74 4.78 ± 1.83
*
Kidney index *
13.21 ± 1.69 16.82 ± 2.07 15.53 ± 1.87#
Liver index 50.63 ± 7.68* 59.44 ± 5.06 56.22 ± 6.48
P < 0.05 vs. the young control mice and the LTA treatment mice. P < 0.05 vs. the aging model mice and the LTA treatment mice. P < 0.05 vs. the aging model mice.
Fig. 1. Observation of morphology. Tissues were collected at the indicated time, fixed in l0% neutral-buffered formalin, embedded in paraffin, and then stained with hematoxylin and eosin (H&E) for histology examination. (a) The young control group; (b) the aging model group; (c) the LTA-treated group. (A) kidney morphology: global glomerular number in the LTA-treated mice was higher than that in the aging model mice, while lower than that in the young control mice. Mean glomerular volume in the LTA-treated mice was lower than that in the aging model mice, while higher than that in the young control mice. (B) Thymus morphology: border between cortex and medulla of thymus gland showed obscure in the aging model mice and fewer lymphocytes were remained in the cortex section than that in the LTA-treated mice and the young control mice. Data shown was a representative from twenty separate experiments. (magnification, 200).
brain, thymus gland, spleen, kidney and liver, showed significant decrease in the aging model mice than that in the LTA-treated mice and in the young control mice (Table 1). Global glomerular number in the LTA-treated mice (n = 3500) was higher than that in the aging model ones (n = 2600), while lower than that in the young control ones (n = 4200). Mean glomerular volume in the LTA-treated mice (v = 0.43 103) was lower than that in the aging model ones (v = 0.65 103), while higher than that in the young control ones (v = 0.28 103) (Fig. 1 as arrow showed). Border between cortex and medulla of the thymus gland showed obscure in the aging model mice and fewer lymphocytes were remained in the cortex section than that in the LTA-treated mice and the young control mice (Fig. 1 as arrow showed).The results above mentioned indicated that LTA had obvious anti-aging effect and its underlying molecular mechanisms were also explored and described as follows.
that compared with that of young control mice, SOD activity in the aging model mice were remarkably reduced, whereas NO, MDA levels and NOS activity were noticeably increased; LTA could increase SOD activity and decrease NO, MDA levels and NOS activity (Table 2). The results suggested that LTA could improve antioxidation ability in brain. 3.3. Improvement of immune function Immune senescence means impaired immune response (Murciano et al., 2006) and altered immune organ structure (Larbi et al., 2004). Many studies showed that DNA damage in lymphocytes increased with age (Ribeiro et al., 2007). In the study, DNA damage in both circulating lymphocytes and splenic lymphocytes was anaTable 2 Effect of LTA on anti-oxidant systems in brain x s.
3.2. Enhancement of activity of anti-oxidant systems in brain According to free radical theory of aging, senescence is mainly due to accumulation of free radicals which do harm to all kinds of cells and make organs senescent. It was found that oxidative stress of organs increases gradually with age (Boveris and Fraga, 2004). In order to explore possible mechanisms of LTA anti-aging, anti-oxidant activity in brain was evaluated. The result showed
Group
SOD (nU/mg)
MDA (nmol/mg)
NO (nmol/mg)
NOS (U/mg)
Aging model 104.51 ± 5.77* 12.06 ± 1.59* 1.742 ± 0.321* 1.137 ± 0.278* Young control 122.31 ± 6.86 7.21 ± 1.08 1.135 ± 0.212 0.609 ± 0.211 LTA treatment 117.82 ± 7.63 8.86 ± 1.78 1.493 ± 0.208 0.974 ± 0.244 *
P < 0.05 vs. the young control mice and the LTA treatment mice. There was no difference between the young control mice and the LTA treatment mice for above parameters.
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Table 3 Effect of LTA on DNA lesion and on cytokines level in vitro and in vivo x s. Group
Percentage of splenic lymphocytes with DNA lesion (100%)
Percentage of circulating lymphocytes with DNA lesion (100%)
IL-2 (pg/mL)
TNF-a (pg/mL)
Young control Aging model LTA treatment
12.8 ± 5.4**
15.4 ± 6.1**
166.6 ± 24.8
11.51 ± 4.11
32.3 ± 5.5 22.1 ± 5.4#
38.3 ± 7.3 25.1 ± 6.4#
87.8 ± 15.1* 114.2 ± 17.9
17.37 ± 5.92* 9.13 ± 2.03
#
P < 0.05 vs. the aging model mice. P < 0.05 vs. the control mice and the LTA-treated mice. There was no difference between the control mice and the LTA-treated mice for TNF-a and IL-2 levels. ** P < 0.05 vs. the aging model mice and the LTA-treated mice. *
lyzed by alkaline comet assay. Under fluorescence microscope, for normal lymphocyte, DNA was characterized by locating inside the cell, whereas for damaged cell, DNA was characterized by obvious migration to outside cell. The percentage of cells exhibiting DNA migration in the aging model mice was higher than that in the young control ones. DNA damage percentage was significantly reduced after the aging model mice were treated by LTA (Table 3). Many studies have been shown that T cell function decreases with age. In order to evaluate the effect of LTA on T cell function, splenocytes were stimulated and IL-2 level in the culture supernatant was determined by ELISA. Compared with that in the young control mice, IL-2 level in the aging model ones was significantly reduced, while significantly increased after the aging model mice were treated by LTA. However, no difference was found between the young control mice and the LTA-treated mice (Table 3). Aging is characterized by a pro-inflammatory state that contributes to the onset of disability and age-related diseases (Huang et al., 2005; Opal et al., 2005). TNF-a is a potent pro-inflammatory cytokine and can produce tissue inflammatory actions. Influence of LTA on TNF-a in serum was studied. The data showed that TNF-a level significantly increased in the aging model mice than that in the control mice and in the LTA-treated mice, whereas no difference was found between the young control mice and the LTA-treated mice (Table 3). The results both in vitro and in vivo indicated that LTA could significantly improve immune function. 3.4. Regulation of aging-related gene expression A lot of studies have found that, many genes are closely related to mammalian aging, and among them, both p16 and c-fos are well defined senescence-related genes (Krishnamurthy et al., 2004). In the study, there were more p16 expression while less c-fos expression in many organs in the aging model mice than that in the young control ones. After treatment by LTA, p16 expression was remarkably down-regulated whereas c-fos expressiosn was upregulated in the organs (Fig. 2). The results suggested that LTA could modulate age-related genes expression.
Fig. 2. Examination of p16 and c-fos expressions. Western blot was used to detect expressions of p16 and c-fos. Compared with that of young control group, there was more p16 expression whereas less c-fos expression in aging model mice; After being treated by LTA, p16 expression was remarkably decreased whereas c-fos expression increased. (a) The young control group; (b) the LTA-treated group; (c) the aging model group. Data shown was a representative of twenty independent experiments.
It was reported that LTA from bifidobacteria could delay cell aging induced by H2O2, markedly decreased p21 expression, and increased cyclin E and CDK2 expressions (Yanhua et al., 2009). LTA could postpone aging by improving anti-oxidation activity and klotho gene expression in aging mice kidney (Yue et al., 2007a,b). These studies have been too under-powered to draw definitive conclusion on LTA function. The aim of the present study was to make use of the artificially induced aging model mice to further investigate potential anti-aging effects of LTA from bifidobacteria. The aging model mice showed aging-related appearance changes, significant decrease in body weight and organ indexes. However, after the aging model mice treated with LTA, both body weight and organ indexes increased. The data indicated that LTA could suppress the aging process. Kidney is an important organ and function was declined gradually due to its structure changes with age. The global glomerular number decreased while mean glomerular volume increased with age. Otherwise, immune system also presents physiological diminution. Thymus is an important immune organ which shows senescent signs firstly. Histology of thymus is characterized as rare lymphocytes remained in cortex, some connective tissue and fatty tissue instead with age. Our results showed that LTA could increase the global glomerular number and decrease the mean glomerular volume. Otherwise, LTA could increase the lymphocytes in cortex. The data above mentioned suggested that LTA of bifidobacterium could inhibit the aging process and its underlying molecular mechanisms were also explored and described as follows. 4.2. Enhancement of activity of anti-oxidant systems
4. Discussion 4.1. LTA inhibiting the aging process Accelerated senescence in mice can be induced by D-galactose (D-gal) and D-gal treated mice were found close to those of 21-month mice showing neurological impairment, decreased activity of anti-oxidant enzymes, and poor immune responses (Wei et al., 2005). D-gal injection has been usually used to establish an aging model for anti-aging research.
According to free radical theory of aging, senescence is mainly due to accumulation of free radicals which do harm to all kinds of cells and make organs senescent. A large of studies revealed that the balance between reactive oxygen species system and anti-oxidation system determines the degree of oxidative stress (Villamor et al., 2004). It was found that oxidative stress of organs increases gradually with age (Boveris and Fraga, 2004). MDA level usually reflects degree of lipid peroxidation and means indirect impairment level of cell. SOD is one of critical enzyme that can protect cells
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from damage and its activity reflects indirectly ability of anti-oxidation. NO, generated by NOS, plays an ubiquitous role in the body in controlling the function of almost every, if not every, organ system. NO can cause cell death by oxidant damage and inactivating enzymes which may play a role in the progression of Alzheimer’s disease and parkinsonism (McCann et al., 1998, 2005). LTA could increase SOD activity and decrease NOS activity, MDA and NO levels in the brain. The results indicated that LTA could enhance antioxidant activity in brain.
and alter gene expression. Although the report supports the potential anti-aging action of LTA from bifidobacteria, further longer-term investigations should be conducted to substantiate its anti-aging action.
Acknowledgements The authors thank Mu Liang and Tan tei for helpful discussions and suggestions and Ding Zhou, Qian Yong for technical support.
4.3. Improvement of immune function It has been well-established that immunity declines with aging (Linton and Dorshkind, 2004). The effects of aging on the immune system are widespread and result in a diminution of immune responsiveness in the elderly. Many studies showed that DNA damage in lymphocytes increased with age (Jeyapalan and Sedivy, 2008). In the study, DNA damage in circulating lymphocytes and splenic lymphocytes was analyzed by alkaline comet assay and the percentage of DNA damage was significantly reduced after the aging model mice treated by LTA. It is well known that advancing age is associated with significant alternations in T cells function. The aging process has been associated with a reduction in T cell responsiveness to a variety of antigens, which may contribute to the significant increase in the incidence of infectious diseases observed in elder individuals (Sharma et al., 2006). IL-2 is an important, powerful T cell growth factor and plays important role in inmmune response, immunoloregulation and anti-tumor. Our results showed that IL-2 level in the aging model mice was significantly reduced than that in the young control ones, while IL-2 level was obviously increased after the aging model mice were treated by LTA. Aging is characterized by a pro-inflammatory state that contributes to the onset of disability and age-related diseases (Huang et al., 2005; Opal et al., 2005). TNF-a is a potent pro-inflammatory cytokine and can produce tissue inflammatory actions. Influence of LTA on TNF-a in serum was studied in the mice. The data showed that TNF-a level was significantly increased in the aging model mice than that in the control ones and in the LTA-treated ones. There was no difference between the control mice and the LTAtreated mice for TNF-a and IL-2 levels. The data both in vitro and in vivo indicated that LTA could significantly improve immune function. 4.4. Alteration of aging-related gene expression Many genes are closely related to mammalian aging. P16 is an inhibitor of cell cycle progression, increases with age and contributes to the impaired cellular regeneration of an aging organism (Sharpless, 2004). P16 deficiency partially prevented the age-induced decline in cell proliferation and tissue function. c-fos plays an important role in cell growth, differentiation, regeneration and remodeling. Moreover, c-fos is essential for cell entry into S phase. c-fos was remarkably down-regulated in many tissues with age (Cayetanot et al., 2005). In the study, we found that LTA could remarkably decrease p16 expression while increase c-fos expression. The results suggested that LTA could modulate age-related gene expression. 5. Conclusions Taken together, the results mentioned above suggested that LTA isolated from bifidobacteria could be involved in the anti-aging action, and anti-aging mechanisms might be as follows: LTA could enhance anti-oxidant activity in brain, improve immune function
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