Serial Passaging of RAW 264.7 Cells Modulates Intracellular AGE Formation and Downregulates RANKL-Induced In Vitro Osteoclastogenesis.

The passage number of cells refers to the number of subculturing processes that the cells have undergone. The effect of passage number on morphological and phenotypical characteristics of cells is of great importance. Advanced glycation end products have also been associated with cell functionality and characteristics. Murine monocyte RAW 264.7 cells differentiate into osteoclasts upon receptor activation caused by nuclear factor-kappa-Β ligand (RANKL) treatment. This study aims to identify the role of passage number on intracellular advanced glycation end products (AGEs) formation and osteoclastogenic differentiation of RAW 264.7 cells. Western blotting was performed to check intracellular AGE formation along with fluorometric analysis using a microplate reader. Tartrate-resistant acid phosphatase (TRAP) staining was performed to check osteoclastogenic differentiation, and qPCR was realized to check the responsible mRNA expression. Immunofluorescence was used to check the morphological changes. Intracellular AGE formation was increased with passaging, and the higher passage number inhibited multinucleated osteoclastogenic differentiation. Osteoclastogenic gene expression also showed a reducing trend in higher passages, along with a significant reduction in F-actin ring size and number. Lower passages should be used to avoid the effects of cell subculturing in in vitro osteoclastogenesis study using RAW 264.7 cells.

Potential Therapeutic Approach of Melatonin against Omicron and Some Other Variants of SARS-CoV-2.

The Omicron variant (B.529) of COVID-19 caused disease outbreaks worldwide because of its contagious and diverse mutations. To reduce these outbreaks, therapeutic drugs and adjuvant vaccines have been applied for the treatment of the disease. However, these drugs have not shown high efficacy in reducing COVID-19 severity, and even antiviral drugs have not shown to be effective. Researchers thus continue to search for an effective adjuvant therapy with a combination of drugs or vaccines to treat COVID-19 disease. We were motivated to consider melatonin as a defensive agent against SARS-CoV-2 because of its various unique properties. Over 200 scientific publications have shown the significant effects of melatonin in treating diseases, with strong antioxidant, anti-inflammatory, and immunomodulatory effects. Melatonin has a high safety profile, but it needs further clinical trials and experiments for use as a therapeutic agent against the Omicron variant of COVID-19. It might immediately be able to prevent the development of severe symptoms caused by the coronavirus and can reduce the severity of the infection by improving immunity.

Identification of Antiglycative Compounds in Japanese Red Water Pepper (Red Leaf Variant of the Persicaria hydropiper Sprout).

Glycation, the nonenzymatic reaction between proteins and excess blood sugar, is implicated in multiple disorders and occurs via the formation and accumulation of advanced glycation end products (AGEs). In our previous studies, we demonstrated that the red-leaf variant of the Persicaria hydropiper sprout (Japanese red water pepper, Benitade) is one of the potent plants that inhibit formation of AGEs. In this study, we aimed to identify antiglycative compounds in Benitade. Benitade extracts were prepared with hot water, then fractionated by using high-performance liquid chromatography (HPLC). The antiglycative efficacy of each fraction was evaluated by measuring the formation of fluorescent AGEs (Ex 370 nm/Em 440 nm). Two fractions, which contained peaks at 26.4 min and 31.8 min, showed potent antiglycative efficacy. When we hydrolyzed these peaks, they shifted to 32.5 and 41.4 min, which are the same retention times as cyanidin and quercetin, respectively. Based on thin-layer chromatography, both compounds contained galactose. Finally, ultrahigh-performance liquid chromatography/quadrupole-time of flight mass spectrometry (UHPLC-QqTOF-MS) analyses were performed to determine the structure of those compounds. Overall, we identified two glycosides, cyanidin 3-O-galactoside (idaein) and quercetin 3-O-galactoside (hyperin), as representative antiglycative compounds in Benitade.

Age-related diseases of the skin and anti-aging medicine.

In the anti-aging medicine, we recommend to assess the skin aging by five categories: wrinkle age, spot age, yellow tint age, elasticity age, and moisture age. Photo-aging (oxidative stress) and glycative stress are major causes of age-related deterioration in the skin. Gly- cative stress finally causes skin accumulation of advanced glycation end products(AGEs), inducing yellow tint, and cross linkage between collagen fibers inducing less elastic skin. Oxidative stress causes skin dark spots through the various processes; excess pigment forma- tion and DNA damages. It also causes wrinkle formation associated with matrix metallopro- teinase(MMP) activation and degeneration of collagen and elastin fibers. Study of oxidative and glycative stress may help identify new anti-aging treatments so that we can achieve the skin rejuvenation.

Lower levels of insulin-like growth factor-1 standard deviation score are associated with histological severity of non-alcoholic fatty liver disease.

AIM: Growth hormone (GH) deficiency may be associated with histological progression of non-alcoholic fatty liver disease (NAFLD) which includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). Insulin-like growth factor 1 (IGF-1) is mainly produced by hepatocytes and its secretion is stimulated by GH. Our aim was to determine whether more histologically advanced NAFLD is associated with low circulating levels of IGF-1 in Japanese patients. METHODS: Serum samples were obtained in 199 Japanese patients with biopsy-proven NAFLD and in 2911 sex- and age-matched healthy people undergoing health checkups. The serum levels of IGF-1 were measured using a commercially available immunoradiometric assay. The standard deviation scores (SDS) of IGF-1 according to age and sex were also calculated in NAFLD patients. RESULTS: The serum IGF-1 levels in NAFLD patients were significantly lower (median, 112 ng/mL) compared with the control population (median, 121 ng/mL, P < 0.0001). IGF-1 SDS less than -2.0 SD from median were found in 11.6% of 199 patients. NASH patients exhibited significantly lower levels of IGF-1 SDS (n = 130; median, -0.7) compared with NAFL patients (n = 69; median, -0.3; P = 0.026). The IGF-1 SDS values decreased significantly with increasing lobular inflammation (P < 0.001) and fibrosis (P < 0.001). In multiple regressions, the association between the IGF-1 SDS values and the severity of NAFLD persisted after adjusting for age, sex and insulin resistance. CONCLUSION: Low levels of circulating IGF-1 may have a role in the development of advanced NAFLD, independent of insulin resistance. Supplementation with GH/IGF-1 may be a candidate for the treatment of NASH.

[Significance of anti-aging medical checkups for the elderly].

AIM: The purpose of anti-aging medical checkups is to evaluate the functional age of the patient, aiming to prevent functional aging and rejuvenate the functional age. In this study, a comparison of the medical checkup results was conducted in elderly groups with different levels of activities of daily living (ADL). METHODS: There were three groups of subjects; and an independent group that included 43 independently-living subjects (68.9±6.3 years) in the Kyoto Yurin area, a support-requiring group that included 31 subjects (77.8±7.2 years) routinely visiting a day care center and a nursing-requiring group that included 19 subjects (83.7±6.8 years) who were living in a medical care facility for the elderly. We conducted anti-aging medical checkups, and the following five factors were evaluated: muscle mass by the bioelectric impedance method (Physion MD), bone strength by an ultrasonic wave method (A-1000: Yokogawa, AOS-100NW: Aloka), fingertip pulse wave analysis (SDP-100: Fukuda), cognitive function (Wisconsin card sorting test) and the serum insulin-like growth factor-I and dehydroepiandrosterone-sulfate (DHEA-s) level. To calculate the functional age of the subjects, we used the Age Management Check (Ginga Kobo, Nagoya), and the Δage (functional age-chronological age) was analyzed and compared between the groups. RESULTS: The functional ages in the independent group were muscle age, 55.5±7.0; blood vascular age, 65.4±8.5; neural age, 63.6±14.8; hormone age, 70.4±7.6 and bone age, 60.2±14.6 years. As a result of an increase in 1,506 daily steps based on walking instruction with pedometer monitoring, the subjects significantly improved their body weight, BMI, waist size, fasting plasma glucose level and serum DHEA-s in six months. By showing them their functional age, their motivation to participate in the health promotion program was increased, and the compliance was high, with an omission rate of less than 20% in 2.5 years. The support- and nursing-requiring groups showed muscle ages of 58.8±3.3 and 61.9±4.0, blood vascular ages of 75.5±12.6 and 71.8±11.1, neural ages of 86.5±8.8 and 88.5±5.8 and bone ages of 81.0±12.0 and 75.7±12.3, respectively. The Δage analysis in the three groups revealed that the Δneural age increased as the ADL decreased. CONCLUSIONS: The Δage analysis indicated that the decreased ADL in the elderly may be mainly associated with their neural function in the factors examined. Anti-aging medical checkups are high in terms of their cost-performance ratio and are easily accepted by the elderly, and are thus recommended for elderly with all grades of ADL.

Staphylococcus aureus Biofilm Inhibiting Activity of Advanced Glycation Endproduct Crosslink Breaking and Glycation Inhibiting Compounds.

Staphylococcus aureus is a Gram-positive bacterium that plays a role in the pathogenesis of skin lesions in diabetes mellitus, atopic dermatitis, and psoriasis, all of which are associated with elevated non-enzymatic glycation biomarkers. The production of biofilm protects resident bacteria from host immune defenses and antibiotic interventions, prolonging pathogen survival, and risking recurrence after treatment. Glycated proteins formed from keratin and glucose induce biofilm formation in S. aureus, promoting dysbiosis and increasing pathogenicity. In this study, several glycation-inhibiting and advanced glycation endproduct (AGE) crosslink-breaking compounds were assayed for their ability to inhibit glycated keratin-induced biofilm formation as preliminary screening for clinical testing candidates. Ascorbic acid, astaxanthin, clove extract, n-phenacylthiazolium bromide, and rosemary extract were examined in an in vitro static biofilm model with S. aureus strain ATCC 12600. Near complete biofilm inhibition was achieved with astaxanthin (ED50 = 0.060 mg/mL), clove extract (ED50 = 0.0087 mg/mL), n-phenacylthiazolium bromide (ED50 = 5.3 mg/mL), and rosemary extract (ED50 = 1.5 mg/mL). The dosage necessary for biofilm inhibition was not significantly correlated with growth inhibition (R2 = 0.055. p = 0.49). Anti-glycation and AGE breaking compounds with biofilm inhibitory activity are ideal candidates for treatment of S. aureus dysbiosis and skin infection that is associated with elevated skin glycation.

Inhibitory Effects of Astaxanthin on CML-HSA-Induced Inflammatory and RANKL-Induced Osteoclastogenic Gene Expression in RAW 264.7 Cells.

OBJECTIVE: Elevated levels of serum Nε-carboxymethyllysine (CML), a well-known advanced glycation end-product (AGE), were observed in patients with inflammation or osteoporosis. Astaxanthin was reported to possess anti-inflammatory and antioxidant effects. In the present study, we investigated the effects of commercially available dietary supplement AstaReal ACTR (ASR) capsule content as astaxanthin on CML-HSA-induced inflammatory and receptor activator of nuclear factor-kappa-Β ligand (RANKL)-induced osteoclastogenic gene expression. METHODS: RAW 264.7 murine macrophage cells were stimulated with CML-HSA to trigger inflammatory gene expression and treated with either a vehicle control or varied concentrations of astaxanthin. Inflammatory gene expression was measured using an enzyme-linked immunosorbent assay (ELISA) or qPCR. We triggered osteoclastogenesis using RANKL, and osteoclastogenic gene expression was measured through tartrate-resistant acid phosphatase (TRAP) activity, staining, immunofluorescence, and qPCR analyses. RESULTS: CML-HSA showed a stimulatory effect on inflammatory gene expression, and astaxanthin reduced the expression by at least two-fold. The levels of autoinflammatory gene expression were reduced by astaxanthin. The RANKL-induced osteoclastogenesis was significantly inhibited by astaxanthin, with reductions in the activation of nuclear factor-κB (NF-κB), the expression of NFATc1 (nuclear factor of activated T cells 1), multinucleated cell formation, and the expression of mature osteoclast marker genes. CONCLUSION: Astaxanthin has potential as a remedy for CML-HSA-induced inflammation and RANKL-induced excessive bone loss.

Comprehensive NMR analysis of two kinds of post-fermented tea and their anti-glycation activities in vitro.

Post-fermented tea (dark tea) is produced from enzyme-inactivated fresh tea leaves by microbial fermentation. Batabata tea and Awaban tea are two major dark teas fermented under aerobic and anaerobic conditions, respectively. However, how their chemical compositions and functionalities change during different post-fermentation processes remains unclear. Nuclear magnetic resonance (NMR)-based analyses showed that (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC) decreased in Batabata tea during post-fermentation with aerobic molds. In contrast, EGC and EC increased, and pyrogallol was produced in Awaban tea during post-fermentation with lactic acid bacteria (LAB). The anti-glycation activities of two dark teas were investigated using an in vitro assay system with human serum albumin (HSA). The anti-glycation activity decreased in Batabata tea, but it was retained in Awaban tea during post-fermentation. Our results showed that post-fermentation with LAB was an efficient way to enhance phenol content and that pyrogallol contributed to anti-glycation activity of Awaban tea.

Effects on the Human Body of a Dietary Supplement Containing L-Carnitine and Garcinia cambogia Extract: A Study using Double-blind Tests.

The effect of a dietary supplement with L-carnitine (600 mg/day) and Garcinia cambogia extract (500 mg/day as hydroxycitric acid) as main ingredients was studied in 35 healthy volunteers {48.3 +/- 6.9 years, body mass index (BMI): 26.3 +/- 1.7} in a double-blind test (18 subjects in the Test Group and 17 in the Control Group). The yearly examination includes the standard yearly medical tests done in Japan, tests for assessing hormonal age, and a survey for assessing physical and mental fitness of the subjects, called the Anti-Aging QOL Common Questionnaire (AAQol). Use of this supplement significantly improved the level of lipid peroxides (-12.8%) in the blood as well as physical symptoms such as "tired eyes," "blurry eyes," "muscle pain/stiffness," "early satiety," "epigastralgia," "dizziness," "arthralgia" and "easily breaking into a sweat." The Control Group showed a significantly favorable improvement rate, especially for "dizziness." On the other hand, groups of subjects using the test compounds saw a significant rise in total cholesterol (4.5%), fasting blood sugar (4.1%) and HbA1c (3.4%). Our findings suggest that the consumption of the supplement can reduce the oxidative damage; however, the effect on QOL was equivocal. Garcinia cambogia extract did not show dietary efficacy.

[Medical treatment for calcium metabolism and balance in terms of the anti-aging medicine].

The aging balance and the risk factor are considered from a whole body viewpoint whenever it thinks about the anti-aging treatment for calcium metabolism. The aging balance attaches to the muscles, the blood vessel, the nerve, the hormone, and the bone, and the aging risk factor evaluates immunity, the oxidation stress, the mental stress, the lifestyle, and the metabolic states. The basis of treatment is improvement of life styles in daily meal, physical activities, and the spirit. The drug therapy is used to the end assisting. It thought about the aging risk factor by describing the calcium abnormal metabolizing according to aging influence on the whole body, the prevention, and treatment in this text.

Effect of Passion Fruit Seed Extract Rich in Piceatannol on the Skin of Women: A Randomized, Placebo-Controlled, Double-Blind Trial.

Piceatannol has been reported to have a wide variety of effects on the skin, including promoting collagen production, inhibiting melanin synthesis, inducing the antioxidant glutathione, and eliminating reactive oxygen species. In this study, a randomized, placebo-controlled, double-blind trial was conducted to clinically evaluate the effects of piceatannol-rich passion fruit seed extract on the skin of healthy Japanese women (age, 35-54 y). Thirty-two women with dry skin received either passion fruit seed extract (5 mg piceatannol) or a placebo (dextrin) for 8 wk. Skin hydration and other parameters on the face were assessed at 0, 4, and 8 wk by using specialized equipment. Furthermore, questionnaire interviews were conducted regarding the physical condition of subjects at 0, 4, and 8 wk. The results showed that consumption of passion fruit seed extract led to significant increases in the moisture content of human skin after 4 and 8 wk compared with that before the trial. The amount of transepidermal water loss decreased over time, although the differences were not significant. Moreover, a stratified analysis of subjects with moisture values of ≤200 µS revealed increased moisture content in the passion fruit seed extract group as compared with the placebo group. Furthermore, the results of questionnaires showed significant reductions in "perspiration" and "fatigue" in the passion fruit seed extract group as compared with the placebo group. These results indicate that oral intake of passion fruit seed extract that is rich in piceatannol could improve the moisture of dry skin and reduce fatigue.

[Hormone replacement Up-to-date. Hormone replacement therapy in anti-aging medicine].

The purpose of anti-aging medicine is to raise quality of life (QOL) and to aim at healthy longevity. Melatonin, DHEA(-s) (dehydroepiandrosterone [sulfate]), somatotropin/IGF-I (insulin-like growth factor-I), male and female hormone decrease with aging, that can be prevented by hormone replacement therapy. This therapy has two sides of supplement of the deficit and a healthy increase. Hormone age is "the function age that expressed whether you are equivalent to a hormone secretion state of how old average" and 30 years old or 70-80% of chronological age regard the targeted value of hormone age. We do not decide quantity of hormone replacement uniformly. The reasonable quantity should be determined based on each figure, active mass, life level, and/or blood levels of hormones. Data accumulation for a long term in multiple institutions is important.

Effects of a year-long pedometer-based walking program on cardiovascular disease risk factors in active older people.

We assessed the relationship between the number of daily steps and changes in cardiovascular disease (CVD) risk factors in a year-long pedometer-based walking program for physically active older people. A total of 36 physically active older people (68.3 ± 5.8 years) completed this 59-week program. The CVD risk parameters were measured at baseline and at weeks 21 and 59. The mean number of steps increased by week 21 and was maintained at week 59 (approximately 10 000 steps, increase in 1500 steps from the baseline; P < .05). Following a significant reduction in body mass index at week 21, systolic blood pressure levels were significantly reduced, and high-density lipoprotein cholesterol levels were significantly increased at week 59 (P < .05). In summary, even active older people can achieve further protection against CVD risks by minor, but sustained, physical activity using pedometers for a period of more than 1 year.

The effects of rabeprazole on parietal cells and enterochromaffin-like cells in rats: a comparison with omeprazole.

BACKGROUND: We investigated the effects of rabeprazole compared with those of omeprazole on enterochromaffin-like cells and parietal cells in rats. METHODS: Rabeprazole or omeprazole was administered for 7 days by intraperitoneal injection (100 mg/kg or 20mg/kg once a day) and the serum gastrin concentration, the antral density of G cells and D cells, fundic histamine content, fundic H+, K+-ATPase mRNA level, and parietal cell morphology were determined. RESULTS: Both rabeprazole and omeprazole inhibited gastric acid secretion and increased the intragastric pH to over 6.5, as well as causing a marked increase in the serum gastrin concentration. The serum gastrin level was lower with rabeprazole treatment than with omeprazole treatment at both doses. Also, the antral G-cell density was higher with omeprazole than with rabeprazole, while the increase in both the histamine content and the H+, K-ATPase mRNA level in the fundic mucosa was higher with omeprazole treatment at both doses, with the difference being significant at 100 mg/kg. Ultrastructural examination indicated that the stimulation of parietal cells by omeprazole was stronger than that by rabeprazole. CONCLUSIONS: Rabeprazole treatment does not drive enterochromaffin-like cells and parietal cells as strongly as omeprazole treatment despite its potent acid suppressive effect, suggesting that it represents a new generation of proton pump inhibitors.

Current status of bariatric surgery in Japan and effectiveness in obesity and diabetes.

The rate of obesity in Japan, defined as having a body mass index (BMI) of 25 kg/m(2) or greater, is reportedly at 24 %, a lower level of severe obesity than in the EU and US. However, the incidence of obesity-related health problems is reportedly higher among Asians. Laparoscopic sleeve gastrectomy (LSG) is the most frequently performed bariatric surgery in Japan and accounted for 54 % of such surgeries in 2011; procedures such as laparoscopic adjustable gastric banding and laparoscopic Roux-en-Y gastric bypass (LRYGB), practiced frequently worldwide, were uncommon. Possible reasons include concern over delayed postoperative discovery of gastric cancer in LRYGB, and rapid adoption of the comparatively simple LSG procedure. In type 2 diabetes mellitus (T2DM) patients, where continued pursuit of medical treatment is difficult and a potential exists for future deterioration of diabetes-complicated diseases, the criterion for surgical indication in the EU and US is a BMI of 30-35 kg/m(2), with priority given to BMI >35 kg/m(2). For Asian patients, the recommendation is to lower this indication criterion by 2.5 kg/m(2). Efficacy of metabolic surgery is anticipated particularly among T2DM patients with obesity complication, a short history of insulin treatment, and intact insulin secreting ability, and in these cases bariatric surgery should be contemplated.

Minor effects of green tea catechin supplementation on cardiovascular risk markers in active older people: a randomized controlled trial.

AIM: Although previous studies have shown that consumption of green tea catechins (GTC) and walking might prevent development of cardiovascular disease (CVD), the effects of GTC supplementation on CVD risk in active older people are unknown. METHODS: A total of 52 older adults (male/female 20/32, mean age 69.1 ± 5.9 years) participating in a pedometer-based walking program were randomly assigned to a GTC group with an intake of 630.9 mg GTC daily (n=26) or a control group (n=26) for 14 weeks. Cardiovascular risk markers were measured before and after this trial. RESULTS: In the GTC group, values of the following markers were significantly reduced (P<0.05) from the beginning to the end of the trial: waist circumference (from 84.2 ± 8.4 to 82.2 ± 8.5 cm), hip circumference (from 95.1 ± 6.9 to 92.2 ± 6.3 cm), total cholesterol (from 233.0 ± 46.3 to 218.8 ± 42.3 mg/dL), low-density lipoprotein cholesterol (from 130.4 ± 36.2 to 119.1 ± 33.4 mg/dL) and low-density lipoprotein cholesterol to high-density lipoprotein cholesterol ratio (from 2.0 ± 1.7 to 1.7 ± 0.5); only hip circumference (from 95.6 ± 8.1 to 94.1 ± 7.6 cm) was significantly reduced (P<0.05) in the control group. No significant between-group differences were found for any parameter measured. CONCLUSIONS: Although GTC might reduce cholesterol levels, the present randomized control trial suggests that GTC supplementation in active older participants did not significantly affect cardiovascular risk markers. Future studies should identify more effective combinations of GTC supplementation and physical activity.

Lower circulating levels of dehydroepiandrosterone, independent of insulin resistance, is an important determinant of severity of non-alcoholic steatohepatitis in Japanese patients.

AIM: The biological basis of variability in histological progression of non-alcoholic fatty liver disease (NAFLD) remains unknown. Dehydroepiandrosterone (DHEA), the most abundant steroid hormone, has been shown to influence sensitivity to reactive oxygen species, insulin sensitivity and expression of peroxisome proliferator-activated receptor-α. Our aim was to determine whether more histologically advanced NAFLD is associated with low circulating levels of DHEA in Japanese patients. METHODS: Serum samples were obtained in 133 Japanese patients with biopsy-proven NAFLD and in 399 sex- and age-matched healthy people undergoing health checkups. Serum levels of sulfated DHEA (DHEA-S) were measured by chemiluminescent enzyme immunoassay. RESULTS: Serum DHEA-S levels in NAFLD patients were similar to those in the control group. Of 133 patients, 90 patients were diagnosed as non-alcoholic steatohepatitis (NASH): 73 patients had stage 0-2, and 17 had stage 3 or 4. Patients with advanced NAFLD (NASH with fibrosis stage 3 or 4) had lower plasma levels of DHEA-S than patients with mild NAFLD (simple steatosis or NASH with fibrosis stage 0-2). The area under the receiver operating characteristic curve for DHEA in separating patients with and without advanced fibrosis was 0.788. A "dose effect" of lower DHEA-S and incremental fibrosis stage was observed with a mean DHEA-S of 170.4 ± 129.2, 137.6 ± 110.5, 96.2 ± 79.3, 61.2 ± 46.3 and 30.0 ± 32.0 µg/dL for fibrosis stages 0, 1, 2, 3, and 4, respectively. The association between DHEA-S and severity of NAFLD persisted after adjusting for age, sex and insulin resistance. CONCLUSION: Low circulating DHEA-S might have a role in the development of advanced NASH.