Extracellular vesicles from human urine-derived stem cells delay aging through the transfer of PLAU and TIMP1.

Aging increases the risks of various diseases and the vulnerability to death. Cellular senescence is a hallmark of aging that contributes greatly to aging and aging-related diseases. This study demonstrates that extracellular vesicles from human urine-derived stem cells (USC-EVs) efficiently inhibit cellular senescence in vitro and in vivo. The intravenous injection of USC-EVs improves cognitive function, increases physical fitness and bone quality, and alleviates aging-related structural changes in different organs of senescence-accelerated mice and natural aging mice. The anti-aging effects of USC-EVs are not obviously affected by the USC donors' ages, genders, or health status. Proteomic analysis reveals that USC-EVs are enriched with plasminogen activator urokinase (PLAU) and tissue inhibitor of metalloproteinases 1 (TIMP1). These two proteins contribute importantly to the anti-senescent effects of USC-EVs associated with the inhibition of matrix metalloproteinases, cyclin-dependent kinase inhibitor 2A (P16INK4a), and cyclin-dependent kinase inhibitor 1A (P21cip1). These findings suggest a great potential of autologous USC-EVs as a promising anti-aging agent by transferring PLAU and TIMP1 proteins.

Macrophage depletion using clodronate liposomes reveals latent dysfunction of the hematopoietic microenvironment associated with persistently imbalanced M1/M2 macrophage polarization in a mouse model of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH), a hyperinflammatory syndrome, is caused by the incessant activation of lymphocytes and macrophages, resulting in damage to organs, including hematopoietic organs. Recently, we demonstrated that repeated lipopolysaccharide (LPS) treatment induces HLH-like features in senescence-accelerated (SAMP1/TA-1) mice but not in senescence-resistant control (SAMR1) mice. Hematopoietic failure in LPS-treated SAMP1/TA-1 mice was attributed to hematopoietic microenvironment dysfunction, concomitant with severely imbalanced M1 and M2 macrophage polarization. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment. Clodronate liposomes are useful tools for in vivo macrophage depletion. In this study, we depleted macrophages using clodronate liposomes to determine their role in the hematopoietic microenvironment in SAMP1/TA-1 and SAMR1 mice. Under clodronate liposome treatment, the response between SAMR1 and SAMP1/TA-1 mice differed as follows: (1) increase in the number of activated M1 and M2 macrophages derived from newly generated macrophages and M2-dominant and imbalanced M1 and M2 macrophage polarization in the BM and spleen; (2) severe anemia and thrombocytopenia; (3) high mortality rate; (4) decrease in erythroid progenitors and B cell progenitors in the BM; and (5) decrease in the mRNA expression of erythroid-positive regulators such as erythropoietin and increase in that of erythroid- and B lymphoid-negative regulators such as interferon-γ in the BM. Depletion of residual macrophages in SAMP1/TA-1 mice impaired hematopoietic homeostasis, particularly erythropoiesis and B lymphopoiesis, owing to functional impairment of the hematopoietic microenvironment accompanied by persistently imbalanced M1/M2 polarization. Thus, macrophages play a vital role in regulating the hematopoietic microenvironment to maintain homeostasis.

Carotid dysfunction in senescent female mice is mediated by increased α1A-adrenoceptor activity and COX-derived vasoconstrictor prostanoids.

α-Adrenergic receptors are crucial regulators of vascular hemodynamics and essential pharmacological targets for cardiovascular diseases. With aging, there is an increase in sympathetic activation, which could contribute to the progression of aging-associated cardiovascular dysfunction, including stroke. Nevertheless, there is little information directly associating adrenergic receptor dysfunction in the blood vessels of aged females. This study determined the role of a-adrenergic receptors in carotid dysfunction of senescent female mice (accelerated-senescence prone, SAMP8), compared with a nonsenescent (accelerated-senescence prone, SAMR1). Vasoconstriction to phenylephrine (Phe) was markedly increased in common carotid artery of SAMP8 [area under the curve (AUC), 527 ± 53] compared with SAMR1 (AUC, 334 ± 30, P = 0.006). There were no changes in vascular responses to the vasoconstrictor agent U46619 or the vasodilators acetylcholine (ACh) and sodium nitroprusside (NPS). Hyperactivity to Phe in female SAMP8 was reduced by cyclooxygenase-1 and cyclooxygenase-2 inhibition and associated with augmented ratio of TXA2/PGI2 release (SAMR1, 1.1 ± 0.1 vs. SAMP8, 2.1 ± 0.3, P = 0.007). However, no changes in cyclooxygenase expression were seen in SAMP8 carotids. Selective α1A-receptor antagonism markedly reduced maximal contraction, whereas α1D antagonism induced a minor shift in Phe contraction in SAMP8 carotids. Ligand binding analysis revealed a threefold increase of α-adrenergic receptor density in smooth muscle cells (VSMCs) of SAMP8 vs. SAMR1. Phe rapidly increased intracellular calcium (Cai2+) in VSMCs via the α1A-receptor, with a higher peak in VSMCs from SAMP8. In conclusion, senescence intensifies vasoconstriction mediated by α1A-adrenergic signaling in the carotid of female mice by mechanisms involving increased Cai2+ and release of cyclooxygenase-derived prostanoids.NEW & NOTEWORTHY The present study provides evidence that senescence induces hyperreactivity of α1-adrenoceptor-mediated contraction of the common carotid. Impairment of α1-adrenoceptor responses is linked to increased Ca2+ influx and release of COX-derived vasoconstrictor prostanoids, contributing to carotid dysfunction in the murine model of female senescence (SAMP8). Increased reactivity of the common carotid artery during senescence may lead to morphological and functional changes in arteries of the cerebral microcirculation and contribute to cognitive decline in females. Because the elderly population is growing, elucidating the mechanisms of aging- and sex-associated vascular dysfunction is critical to better direct pharmacological and lifestyle interventions to prevent cardiovascular risk in both sexes.

Anthocyanin-rich blackcurrant extract improves long-term memory impairment and emotional abnormality in senescence-accelerated mice.

Alzheimer's disease (AD) is associated with a progressive worsening in cognitive function, which is often accompanied by emotional disturbance. Recent studies revealed that anthocyanin-rich blackcurrant extract (BCE) can impart health benefits, but it is not known whether BCE is useful in the prevention and/or treatment of AD. Here, we examined the effects of BCE using a senescence-accelerated mouse prone 8 (SAMP8) model. Dietary BCE supplementation for 9 weeks was found to both improve the diminished long-term recognition memory and normalize the anxiety levels of SAMP8 mice. RNA sequencing demonstrated that dietary supplementation with anthocyanin-rich BCE significantly altered the gene expression profile in the hippocampus. According to enrichment analysis, genes regulated by BCE were related to cellular component terms such as "smooth endoplasmic reticulum," "axon," and "glutamatergic synapse." Real-time PCR verified alterations in the expression of AD-related genes. These findings indicate that anthocyanin-rich BCE may be valuable for the prevention and/or treatment of AD. PRACTICAL APPLICATIONS: Blackcurrant contains an abundance of polyphenols, especially anthocyanins. This study demonstrated that anthocyanin-rich BCE improves the long-term recognition memory impairment and emotional abnormality of SAMP8 mice, a mouse model characterized by several pathological features of AD. These findings indicate that anthocyanin-rich BCE may be a useful food supplement or ingredient for the prevention of AD.

Targeting whole body metabolism and mitochondrial bioenergetics in the drug development for Alzheimer's disease.

Aging is by far the most prominent risk factor for Alzheimer's disease (AD), and both aging and AD are associated with apparent metabolic alterations. As developing effective therapeutic interventions to treat AD is clearly in urgent need, the impact of modulating whole-body and intracellular metabolism in preclinical models and in human patients, on disease pathogenesis, have been explored. There is also an increasing awareness of differential risk and potential targeting strategies related to biological sex, microbiome, and circadian regulation. As a major part of intracellular metabolism, mitochondrial bioenergetics, mitochondrial quality-control mechanisms, and mitochondria-linked inflammatory responses have been considered for AD therapeutic interventions. This review summarizes and highlights these efforts.

Gosha-jinki-Gan (GJG) shows anti-aging effects through suppression of TNF-α production by Chikusetsusaponin V.

Frailty develops due to multiple factors, such as sarcopenia, chronic pain, and dementia. Go-sha-jinki-Gan (GJG) is a traditional Japanese herbal medicine used for age-related symptoms. We have reported that GJG improved sarcopenia, chronic pain, and central nervous system function through suppression of tumor necrosis factor-alpha (TNF-α) production. In the present study, GJG was found to reduce the production of TNF-α in the soleus muscle of senescence-accelerated mice at 12 weeks and 36 weeks. GJG did not change the differentiation of C2C12 cells with 2% horse serum. GJG significantly decreased the expression of Muscle atrophy F-box protein (MAFbx) induced by TNF-α in C2C12 cells on real-time PCR. TNF-α significantly decreased the expression of PGC-1α and negated the enhancing effect of GJG for the expression of PGC-1α on digital PCR. Examining 20 chemical compounds derived from GJG, cinnamaldehyde from cinnamon bark and Chikusetsusaponin V (CsV) from Achyrantes Root dose-dependently decreased the production of TNF-⍺ in RAW264.7 cells stimulated by LPS. CsV inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB) p65 in RAW264.7 cells. CsV showed low permeability using Caco-2 cells. However, the plasma concentration of CsV was detected from 30 min to 6 h and peaked at 1 h in the CD1 (ICR) mice after a single dose of GJG. In 8-week-old SAMP8 mice fed 4% (w/w) GJG from one week to four weeks, the plasma CsV concentration ranged from 0.0500 to 10.0 ng/mL. The evidence that CsV plays an important role in various anti-aging effects of GJG via suppression of TNF-⍺ expression is presented.

Aging-Related Phenotypic Conversion of Medullary Microglia Enhances Intraoral Incisional Pain Sensitivity.

Activated microglia involved in the development of orofacial pain hypersensitivity have two major polarization states. The aim of this study was to assess the involvement of the aging-related phenotypic conversion of medullary microglia in the enhancement of intraoral pain sensitivity using senescence-accelerated mice (SAM)-prone/8 (SAMP8) and SAM-resistant/1 (SAMR1) mice. Mechanical head-withdrawal threshold (MHWT) was measured for 21 days post palatal mucosal incision. The number of CD11c-immunoreactive (IR) cells [affective microglia (M1)] and CD163-IR cells [protective microglia (M2)], and tumor-necrosis-factor-α (TNF-α)-IR M1 and interleukin (IL)-10-IR M2 were analyzed via immunohistochemistry on days 3 and 11 following incision. The decrease in MHWT observed following incision was enhanced in SAMP8 mice. M1 levels and the number of TNF-α-IR M1 were increased on day 3 in SAMP8 mice compared with those in SAMR1 mice. On day 11, M1 and M2 activation was observed in both groups, whereas IL-10-IR M2 levels were attenuated in SAMP8 mice, and the number of TNF-α-IR M1 cells increased, compared to those in SAMR1 mice. These results suggest that the mechanical allodynia observed following intraoral injury is potentiated and sustained in SAMP8 mice due to enhancement of TNF-α signaling, M1 activation, and an attenuation of M2 activation accompanying IL-10 release.

Improvement of Learning and Memory in Senescence-Accelerated Mice by S-Allylcysteine in Mature Garlic Extract.

S-allylcysteine (SAC), a major thioallyl compound contained in mature garlic extract (MGE), is known to be a neuroactive compound. This study was designed to investigate the effects of SAC on primary cultured hippocampal neurons and cognitively impaired senescence-accelerated mice prone 10 (SAMP10). Treatment of these neurons with MGE or SAC significantly increased the total neurite length and number of dendrites. SAMP10 mice fed MGE or SAC showed a significant improvement in memory dysfunction in pharmacological behavioral analyses. The decrease of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, N-methyl-d-aspartate (NMDA) receptor, and phosphorylated α-calcium/calmodulin-dependent protein kinase II (CaMKII) in the hippocampal tissue of SAMP10 mice fed MGE or SAC was significantly suppressed, especially in the MGE-fed group. These findings suggest that SAC positively contributes to learning and memory formation, having a beneficial effect on brain function. In addition, multiple components (aside from SAC) contained in MGE could be useful for improving cognitive function by acting as neurotrophic factors.

Carbohydrate-restricted diet alters the gut microbiota, promotes senescence and shortens the life span in senescence-accelerated prone mice.

This study examined the effects of a carbohydrate-restricted diet on aging, brain function, intestinal bacteria and the life span to determine long-term carbohydrate-restriction effects on the aging process in senescence-accelerated prone mice (SAMP8). Three-week-old male SAMP8 were divided into three groups after a week of preliminary feeding. One group was given a controlled diet, while the others fed on high-fat and carbohydrate-restricted diets, respectively. The mice in each group were further divided into two subgroups, of which one was the longevity measurement group. The other groups fed ad libitum until the mice were 50 weeks old. Before the test period termination, passive avoidance test evaluated the learning and memory abilities. Following the test period, serum and various mice organs were obtained and submitted for analysis. The carbohydrate-restricted diet group exhibited significant decrease in the survival rate as compared to the other two diet groups. The passive avoidance test revealed a remarkable decrease in the learning and memory ability of carbohydrate-restricted diet group as compared to the control-diet group. Measurement of lipid peroxide level in tissues displayed a marked increase in the brain and spleen of carbohydrate-restricted diet group than the control-diet and high-fat diet groups. Furthermore, notable serum IL-6 and IL-1ß level (inflammation indicators) elevations, decrease in Enterobacteria (with anti-inflammatory action), increase in inflammation-inducing Enterobacteria and lowering of short-chain fatty acids levels in cecum were observed in the carbohydrate-restricted diet group. Hence, carbohydrate-restricted diet was revealed to promote aging and shortening of life in SAMP8.

Low-Dose Ethanol Has Impacts on Plasma Levels of Metabolites Relating to Chronic Disease Risk in SAMP8 mice.

The effects of low-dose alcohol on experimental animals are unclear. This study examined plasma metabolites in senescence-accelerated mice 8 (SAMP8) given low-dose ethanol, and compared them with aging progress and skeletal muscle strength. Male SAMP8 mice (10-wk-old) were given drinking water containing 0% (control), 1%, 2%, or 5% (v/v) ethanol for 14 wk. Compared with the control group, only mice who consumed 1% ethanol experienced a lower senescence score at 18 and 23 wk, as well as an increased limb grip strength at 21 wk. Plasma metabolites of control, 1% and 2% ethanol groups were analyzed by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS). Among the 7 metabolites affected by ethanol, notewhorthy is the positive association of the ethanol levels in drinking water with the levels of α-ketoglutarate (antioxidant and anti-inflammatory metabolite) and hippurate (antioxidant and microbial co-metabolite) (p<0.05). Intriguingly, the levels of 2-hydroxyisobutyrate (the biomarker of energy metabolism and microbial co-metabolite) were higher in the 1% ethanol group (p<0.05), but not in the 2% ethanol group as compared to the control. Furthermore, the levels of some of the metabolites affected were correlated with some variables in the grading score of senescence and muscle strength. This study provides a novel insight into how low-dose ethanol in SAMP8 mice modulates the levels of circulating metabolites relating to chronic disease risk.

[Using bioinformatics tools to explore cellular biological mechanisms of "Triple Energizer Acupuncture Method" in treating senile dementia].

OBJECTIVE: To observe the effect of needling "Danzhong" (CV17), "Zhongwan"(CV12), "Qihai" (CV6), "Xuehai"(SP10)and "Zusanli"(ST36) (Triple Energizer Acupuncture Method) on the learning-memory ability and profile of hippocampal differentially-expressed genes and proteins of SAMP8 mice (rapid aging mice), so as to explore its underlying mechanisms in improving Alzheimer's disease (AD)．. METHODS: A total of 60 SAMP8 were used as the dementia model and randomly divided into blank control, acupoint and non-acupoint groups (n＝20 per group). The 5 acupuncture points and non-acupoints (subcostal region) on the bilateral sides were needled with filiform needles and manipulated manually for 30 s at each one，once daily, 6 times a week for 4 weeks. The Morris water maze tests (location navigation tests and space probing trials) were used to evaluate the mouse's learning-memory ability. The hippocampal tissue was extracted to detect differentially expressed genes and proteins related to acupuncture intervention by gene chip and isobaric tags for relative and absolute quantitation (iTRAQ) techniques as well as bioinformatic analysis, separately. The information is analyzed through bioinformatics database tools. Finally, immunofluorescence staining was used to verify theresults of microarray analysis. RESULTS: Compared with mice of the control and non-acupoint groups, the escape latency of location navigation task of Morris water maze tests on 4th and 5th day of training was significantly shortened in mice of the acupoint group, and the duration of stay in the original safe-platform quadrant was significantly increased in the acupoint group (P<0.05). Gene microarray displayed that in comparison with the control group, 898 differentially expressed genes were up-regulated, 418 genes were down-regulated in the hippocampus of acupoint group. The iTRAQ analysis indicated that in the acupoint and non-acupoint groups, 286 and 299 differentially expressed proteins were up-regulated, 319 and 179 proteins down-regulated, respectively. Of the 34 terms containing 47 proteins up-regulated by acupoint needling, including intermediate filament, keratin filament, myelin sheath, postsynaptic density, neuron projection were related with neurite and cytoskele-ton. While in the non-acupoint group, of the 24 terms were listed by the system, only the myelin sheath involving 11 differentially expressed proteins functions in activities of neurite and cytoskeleton. Immunofluorescence staining of the hippocampal tissue showed that the high-density distribution areas of neurons and neurite fibers were characterized by decentralization and disordering, with the highlighted areas being mainly near the cell body parts in control mice, but in mice of the acupoint group, the highlighted areas at the neurite were relatively dense, the morphology of hippocampal cells was complete, the fiber structure was clear, dense and orderly, and the neurites were closely arranged and in order, indicating an improvement of the distribution and arrangement of nerve fibers after acupuncture. The height of neurite highlight area of the acupoint group was significantly higher than that of the non-acupoint group (P<0.05)．. CONCLUSION: The "Triple Energizer Acupuncture" of acupoints is able to improve the learning-memory ability in SAMP8, which may be related to its effects in regulating the expression and function of hippocampal genes and proteins related to neurite and cytoskeleton.

A Standardized Extract of Asparagus officinalis Stem (ETAS®) Ameliorates Cognitive Impairment, Inhibits Amyloid ß Deposition via BACE-1 and Normalizes Circadian Rhythm Signaling via MT1 and MT2.

The prevalence of cognitive impairments and circadian disturbances increases in the elderly and Alzheimer's disease (AD) patients. This study investigated the effects of a standardized extract of Asparagus officinalis stem, ETAS® on cognitive impairments and circadian rhythm status in senescence-accelerated mice prone 8 (SAMP8). ETAS® consists of two major bioactive constituents: 5-hydroxymethyl-2-furfural (HMF), an abundant constituent, and (S)-asfural, a novel constituent, which is a derivative of HMF. Three-month-old SAMP8 male mice were divided into a control, 200 and 1000 mg/kg BW ETAS® groups, while senescence-accelerated resistant mice (SAMR1) were used as the normal control. After 12-week feeding, ETAS® significantly enhanced cognitive performance by an active avoidance test, inhibited the expressions of amyloid-beta precursor protein (APP) and BACE-1 and lowered the accumulation of amyloid ß (Aß) in the brain. ETAS® also significantly increased neuron number in the suprachiasmatic nucleus (SCN) and normalized the expressions of the melatonin receptor 1 (MT1) and melatonin receptor 2 (MT2). In conclusion, ETAS® enhances the cognitive ability, inhibits Aß deposition and normalizes circadian rhythm signaling, suggesting it is beneficial for preventing cognitive impairments and circadian rhythm disturbances in aging.

Abnormal platelet amyloid-ß precursor protein metabolism in SAMP8 mice: Evidence for peripheral marker in Alzheimer's disease.

Senescence-accelerated mouse strains have proved to be an accelerated-aging model, which mimics numerous features with Alzheimer's disease (AD). Three, six, and nine-month senescence-accelerated resistant 1 and senescence-accelerated prone 8 (SAMP8) mice were used in the current study, to unravel potential mechanisms for dementia and explore new diagnostic approaches for AD. The amyloid-ß (Aß40) and Aß42 levels were elevated in hippocampi and platelets from SAMP8, along with a reduced α-secretase expression and an enhanced ß-secretase expression extent with age, compared to control mice. Furthermore, hippocampal Aß40 and Aß42 of SAMP8 were positively correlated with platelet of these mice with aging progression. In addition, ß-γ-secretase-modulated proteolytic proceeding of amyloid precursor protein in platelet might work through the PI3K/Akt/GSK3ß pathway. These results indicate that platelet could be a potential early marker in the periphery to study the age-correlative aggregation of the amyloid-ß peptide in patients with AD, while still requiring the considerable study.

Sake cake (sake-kasu) ingestion increases branched-chain amino acids in the plasma, muscles, and brains of senescence-accelerated mice prone 8.

To examine metabolic effects of sake cake ingestion, plasma and tissues were analyzed in senescence-accelerated mice prone 8 (SAMP8) fed a sake cake diet. As a result, branched-chain amino acids (BCAA) were found to be significantly higher in the plasma, gastrocnemius muscles and brains of the sake cake group than in the control group. Mice in the sake cake group showed stronger grip strength than the control group. High levels of circulating BCAA have been reported to be associated with pathological states, such as metabolic diseases, but the parameters of glucose and lipid metabolism were not affected between the two groups. Otherwise, pyridoxal was significantly higher and nicotinamide as well as 1-methylnicotinamide showed a tendency to be higher in the plasma of the sake cake group than in the control group. These findings indicate that intake of sake cake increases the levels of BCAA, vitamin B6, and vitamin B3. Abbreviation: CE-TOFMS: capillary electrophoresis time-of-flight mass spectrometry.

Carbohydrate-restricted diet promotes skin senescence in senescence-accelerated prone mice.

This study used senescence-accelerated prone mice (SAMP8) to examine the effects of a carbohydrate-restricted diet on aging and skin senescence, to determine how long-term carbohydrate restriction affects the aging process. Three-week-old male SAMP8 mice were divided into three groups after 1 week of preliminary feeding: one was given a controlled diet, the other was given a high-fat diet, and the third was given a carbohydrate-restricted diet. Ad libitum feeding was administered until the mice reached 50 weeks of age. Before the end of the test period, a grading test was used to evaluate visible aging in the mice. After the test period, serum and skin samples in mice were obtained and submitted for analysis. As a result, the grading test demonstrated that there was significant progression of visible aging in the carbohydrate-restricted group, as well as a decreased survival rate. Histological examination of the skin revealed that the epidermis and dermis in the carbohydrate-restricted group had become thinner. Analysis of the mechanisms involved demonstrated an increase in serum interleukin-6, aggravated skin senescence, inhibition of skin autophagy and activation of skin mTOR. Therefore, this study proved that a carbohydrate-restricted diet promoted skin senescence in senescence-accelerated mice.

Using bioinformatics tools to explore cellular biological mechanisms of "Triple Energizer Acupuncture Method" in treating senile dementia / 针刺研究

OBJECTIVE: To observe the effect of needling "Danzhong" (CV17), "Zhongwan"(CV12), "Qihai" (CV6), "Xuehai"(SP10)and "Zusanli"(ST36) (Triple Energizer Acupuncture Method) on the learning-memory ability and profile of hippocampal differentially-expressed genes and proteins of SAMP8 mice (rapid aging mice), so as to explore its underlying mechanisms in improving Alzheimer's disease (AD)．. METHODS: A total of 60 SAMP8 were used as the dementia model and randomly divided into blank control, acupoint and non-acupoint groups (n＝20 per group). The 5 acupuncture points and non-acupoints (subcostal region) on the bilateral sides were needled with filiform needles and manipulated manually for 30 s at each one，once daily, 6 times a week for 4 weeks. The Morris water maze tests (location navigation tests and space probing trials) were used to evaluate the mouse's learning-memory ability. The hippocampal tissue was extracted to detect differentially expressed genes and proteins related to acupuncture intervention by gene chip and isobaric tags for relative and absolute quantitation (iTRAQ) techniques as well as bioinformatic analysis, separately. The information is analyzed through bioinformatics database tools. Finally, immunofluorescence staining was used to verify theresults of microarray analysis. RESULTS: Compared with mice of the control and non-acupoint groups, the escape latency of location navigation task of Morris water maze tests on 4th and 5th day of training was significantly shortened in mice of the acupoint group, and the duration of stay in the original safe-platform quadrant was significantly increased in the acupoint group (P<0.05). Gene microarray displayed that in comparison with the control group, 898 differentially expressed genes were up-regulated, 418 genes were down-regulated in the hippocampus of acupoint group. The iTRAQ analysis indicated that in the acupoint and non-acupoint groups, 286 and 299 differentially expressed proteins were up-regulated, 319 and 179 proteins down-regulated, respectively. Of the 34 terms containing 47 proteins up-regulated by acupoint needling, including intermediate filament, keratin filament, myelin sheath, postsynaptic density, neuron projection were related with neurite and cytoskele-ton. While in the non-acupoint group, of the 24 terms were listed by the system, only the myelin sheath involving 11 differentially expressed proteins functions in activities of neurite and cytoskeleton. Immunofluorescence staining of the hippocampal tissue showed that the high-density distribution areas of neurons and neurite fibers were characterized by decentralization and disordering, with the highlighted areas being mainly near the cell body parts in control mice, but in mice of the acupoint group, the highlighted areas at the neurite were relatively dense, the morphology of hippocampal cells was complete, the fiber structure was clear, dense and orderly, and the neurites were closely arranged and in order, indicating an improvement of the distribution and arrangement of nerve fibers after acupuncture. The height of neurite highlight area of the acupoint group was significantly higher than that of the non-acupoint group (P<0.05)．. CONCLUSION: The "Triple Energizer Acupuncture" of acupoints is able to improve the learning-memory ability in SAMP8, which may be related to its effects in regulating the expression and function of hippocampal genes and proteins related to neurite and cytoskeleton.

Resveratrol counteracts bone loss via mitofilin-mediated osteogenic improvement of mesenchymal stem cells in senescence-accelerated mice.

Rational: Senescence of mesenchymal stem cells (MSCs) and the related functional decline of osteogenesis have emerged as the critical pathogenesis of osteoporosis in aging. Resveratrol (RESV), a small molecular compound that safely mimics the effects of dietary restriction, has been well documented to extend lifespan in lower organisms and improve health in aging rodents. However, whether RESV promotes function of senescent stem cells in alleviating age-related phenotypes remains largely unknown. Here, we intend to investigate whether RESV counteracts senescence-associated bone loss via osteogenic improvement of MSCs and the underlying mechanism. Methods: MSCs derived from bone marrow (BMMSCs) and the bone-specific, senescence-accelerated, osteoblastogenesis/osteogenesis-defective mice (the SAMP6 strain) were used as experimental models. In vivo application of RESV was performed at 100 mg/kg intraperitoneally once every other day for 2 months, and in vitro application of RESV was performed at 10 µM. Bone mass, bone formation rates and osteogenic differentiation of BMMSCs were primarily evaluated. Metabolic statuses of BMMSCs and the mitochondrial activity, transcription and morphology were also examined. Mitofilin expression was assessed at both mRNA and protein levels, and short hairpin RNA (shRNA)-based gene knockdown was applied for mechanistic experiments. Results: Chronic intermittent application of RESV enhances bone formation and counteracts accelerated bone loss, with RESV improving osteogenic differentiation of senescent BMMSCs. Furthermore, in rescuing osteogenic decline under BMMSC senescence, RESV restores cellular metabolism through mitochondrial functional recovery via facilitating mitochondrial autonomous gene transcription. Molecularly, in alleviating senescence-associated mitochondrial disorders of BMMSCs, particularly the mitochondrial morphological alterations, RESV upregulates Mitofilin, also known as inner membrane protein of mitochondria (Immt) or Mic60, which is the core component of the mitochondrial contact site and cristae organizing system (MICOS). Moreover, Mitofilin is revealed to be indispensable for mitochondrial homeostasis and osteogenesis of BMMSCs, and that insufficiency of Mitofilin leads to BMMSC senescence and bone loss. More importantly, Mitofilin mediates resveratrol-induced mitochondrial and osteogenic improvements of BMMSCs in senescence. Conclusion: Our findings uncover osteogenic functional improvements of senescent MSCs as critical impacts in anti-osteoporotic practice of RESV, and unravel Mitofilin as a novel mechanism mediating RESV promotion on mitochondrial function in stem cell senescence.

Elucidation of the mechanism of changes in the antioxidant function with the aging in the liver of the senescence-accelerated mouse P10 (SAMP10).

Senescence-accelerated mice are known to display a variety of deficits and signs of accelerated aging, but the specific mechanisms involved in this process are still unclear. In this study, we examined the expression levels of antioxidant enzymes, transcription factors responsible for the regulation of expression of these enzymes, and mitochondrial proteins in the liver of SAMP10 and SAMR1 mice at 3 and 12 months of age using western blotting analysis. To investigate the amount of oxidative damage to DNA, levels of 8-OHdG were measured in the liver of these mice. At 3 months of age, the levels of catalase, Mn-SOD, GPx, UQCRC2 and COXIV were significantly upregulated in SAMP10 mice compared with that in SAMR1 mice. However, NDUFS3 levels were not significantly different at this young age. In contrast, the expression level of catalase was significantly lower, and the levels of phosphorylated FoxO-1a and UQCRC2 were significantly higher in SAMP10 mice compared to those in SAMR1 mice; however, at 12 months of age, there were no significant differences in Mn-SOD, GPx, total -FoxO-1a, COXIV, and NDUFS3 expression between the two groups of mice. The levels of 8-OHdG in the liver were markedly higher in 12-month-old SAMP10 mice than those in 3-month-old SAMP10 and SAMR1 mice. These results suggest that an increase in number of mitochondria or a collapse in the balance between the levels of complexes I and III results in an increase in the amount of ROS and induces the expression of antioxidant enzymes in the liver of SAMP10 mice at 3 months of age. Although young SAMP10 mice produce a large amount of ROS, they also produce suitable levels of antioxidant enzymes that decompose ROS; consequently accelerated aging does not occur in young SAMP10 mice. In addition to excessive ROS production which is an important cause of aging, the level of catalase was significantly lower in SAMP10 than that in SAMR1 mice. These results suggested that overexpression of ROS and a decrease in the levels of catalase resulted in the accelerated aging observed in older SAMP10 mice. Moreover, the level of phosphorylated FoxO-1a was increased in SAMP10 compared to that in SAMR1 mice though the total amount of FoxO-1a was not significantly different between the two groups in old age. These results suggest that some impairment in the regulation mechanism of FoxO-1a phosphorylation is responsible for abnormal catalase expression and that a significant decrease in the level of catalase with aging decisively affects the metabolic balance of ROS; thus, ROS that cannot be metabolized contributes to the accelerated aging of SAMP10 mice.

The peel of Citrus kawachiensis (kawachi bankan) ameliorates microglial activation, tau hyper-phosphorylation, and suppression of neurogenesis in the hippocampus of senescence-accelerated mice.

We previously reported that the dried peel powder of Citrus kawachiensis, one of the citrus products of Ehime, Japan, exerted anti-inflammatory effects in the brain of a lipopolysaccharide-injected systemic inflammation animal model. Inflammation is one of the main mechanisms underlying aging in the brain; therefore, we herein evaluated the anti-inflammatory and other effects of the dried peel powder of C. kawachiensis in the senescence-accelerated mouse-prone 8 (SAMP8) model. The C. kawachiensis treatment inhibited microglial activation in the hippocampus, the hyper-phosphorylation of tau at 231 of threonine in hippocampal neurons, and ameliorated the suppression of neurogenesis in the dentate gyrus of the hippocampus. These results suggest that the dried peel powder of C. kawachiensis exert anti-inflammatory and neuroprotective effects.

Decreased bioavailability of nitric oxide in aorta from ovariectomized senescent mice. Role of cyclooxygenase.

This study investigates the effects of aging and/or ovariectomy on vascular reactivity to thromboxane A2 (TXA2) receptor stimulation with U46619, and the modulation by nitric oxide (NO) and cyclooxygenase (COX) in aorta from female senescence-accelerated mice (SAMP8) and from senescence resistant mice (SAMR1). Five-month-old female SAMR1 and SAMP8 were divided into three groups: sham-operated, ovariectomized and ovariectomized plus estradiol. Twenty-eight days after surgery, thoracic aortic rings were mounted for isometric recording of tension and concentration-response curves for U46619 (10(-10)-3 × 10(-7) M) were performed in the absence and in the presence of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) and/or COX inhibitor indomethacin (10(-5)M). Vascular superoxide production was detected by dihydroethidium staining on sections of thoracic aorta. NO bioavailability in response to U46619 was suppressed by estrogen withdrawn in young and senescent mice and was restored by the administration of estradiol. In the presence of indomethacin, contractions to U46619 decreased in all groups indicating an aging- and estrogen-dependent modulation of contractile prostanoids. The simultaneous incubation of L-NAME and indomethacin did not change the maximal responses and sensitivities to TXA2 in any group in comparison with untreated aortic segments. The superoxide generation induced by TXA2 was greater in aorta from SAMP8 than in SAMR1. Moreover, in ovariectomized groups superoxide production was further increased and treatment with 17ß-estradiol reverted the effects of the ovariectomy. Inhibition of COX with indomethacin prevented the U46619-induced increase in superoxide formation. Our results indicate that NO bioavailability in response to TP receptor activation is both estrogen- and aging-dependent. TXA2 induced contractions are partially mediated by COX activation. Both aging and ovariectomy enhanced COX-dependent component of the TXA2-induced contraction. It is noteworthy that in the absence of estrogen, COX inhibition induces an increase of NO bioavailability. Therefore, in senescent female mice with an experimental menopause, TP-receptor stimulation is responsible for COX activation and enhanced superoxide generation, which may result in reduced NO bioavailability. These effects were reversed by estrogen administration.