Analgesic Mechanisms of Steroid Ointment against Oral Ulcerative Mucositis in a Rat Model.

Despite the long history of use of steroid ointments for oral mucositis, the analgesic mechanism has not been fully elucidated. In this study, we examined the effects of triamcinolone acetonide (Tmc) on oral ulcerative mucositis-induced pain in conscious rats by our proprietary assay system. Based on evaluations of the physical properties and retention periods in the oral mucosa of human volunteers and rats, we selected TRAFUL® ointment as a long-lasting base. In oral ulcerative mucositis model rats, TRAFUL® with Tmc suppressed cyclooxygenase-dependent inflammatory responses with upregulations of glucocorticoid receptor-induced anti-inflammatory genes and inhibited spontaneous nociceptive behavior. When an ointment with a shorter residual period was used, the effects of Tmc were not elicited or were induced to a lesser extent. Importantly, TRAFUL® with Tmc also improved oral ulcerative mucositis-induced mechanical allodynia, which has been reported to be independent of cyclooxygenase. Ca2+ imaging in dissociated trigeminal ganglion neurons showed that long-term preincubation with Tmc inhibited the hypertonic stimulation-induced Ca2+ response. These results suggest that the representative steroid Tmc suppresses oral ulcerative mucositis-induced pain by general anti-inflammatory actions and inhibits mechanical sensitivity in peripheral nerves. For drug delivery, long-lasting ointments such as TRAFUL® are needed to sufficiently induce the therapeutic effects.

Reliability and Validity of the MD Anderson Dysphagia Inventory Among Japanese Patients.

This study aimed to validate the Japanese version of a dysphagia-specific quality of life questionnaire, the MD Anderson Dysphagia Inventory (MDADI-J), and to verify trends between MDADI-J and Functional Oral Intake Scale (FOIS) scores. The original 20 MDADI items were translated using a forward-backward method following accepted cultural adaptation guidelines. Seventy-two patients with a history of head and neck cancer treatment completed the MDADI-J between October 2015 and August 2016. Concurrent validity was determined by correlations with the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30) and the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Head and Neck 35 (EORTC QLQ-H&N35) instruments. Discriminant validity was examined using cancer stage grouping between stages I-II and III-IV. Additionally, trends between the FOIS and the MDADI-J total scores were analyzed using a trend test. The Cronbach's α coefficient of the MDADI-J total score was 0.92, indicating high internal consistency. The average inter-item correlation coefficients ranged from 0.39 to 0.49. ICC, an indicator of test-retest reliability, was 0.84 for the total score, and 0.58 to 0.81 for individual subscales. The total score and all subscales were significantly associated with the scores for each factor of the EORTC QLQ-C30 and EORTC QLQ-H&N35. The total score and all subscales were significantly different between clinical tumor stages I-II and III-IV. The total scores all increased with the progress of the FOIS. In conclusion, this study validated the Japanese version of the MDADI and showed that as FOIS scores deteriorate, MDADI-J scores tend to constantly decrease.

Mechanisms involved in enhancement of matrix metalloproteinase-9 expression in macrophages by interleukin-33.

Endothelial transmigration of macrophages is accomplished by matrix metalloproteinase (MMP)-induced degradation of the basement membrane and extracellular matrix components. Macrophages upregulate MMP-9 expression and secretion upon immunological challenges and require its activity for migration during inflammatory responses. Interleukin (IL)-33 is a recently discovered pro-inflammatory cytokine that belongs to the IL-1 family. The aim of this study was to elucidate the mechanisms underlying IL-33-induced MMP-9 expression in the mouse monocyte/macrophage line RAW264.7. IL-33 increased MMP-9 mRNA and protein expression in RAW264.7 cells. Blockage of IL-33-IL-33 receptor (ST2L) binding suppressed IL-33-mediated induction of MMP-9. IL-33 induced phosphorylation and nuclear translocation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-kappa B (NF-κB). Chromatin immunoprecipitation indicated that IL-33 increased c-fos recruitment to the MMP-9 promoter. Reporter assay findings also revealed that IL-33 stimulated the transcriptional activity of activator protein 1 (AP-1). Pre-treatment of the cells with a specific inhibitor of ERK1/2 and NF-κB attenuated the IL-33-induced activation of AP-1 subunits, transcriptional activity of AP-1, and expression of MMP-9. We also demonstrated that ERK-dependent activation of cAMP response element binding protein (CREB) is a key step for AP-1 activation by IL-33. These results indicate an essential role of ERK/CREB and NF-κB cascades in the induction of MMP-9 in monocytes/macrophages through AP-1 activation.

Bacterial diversity in saliva and oral health-related conditions: the Hisayama Study.

This population-based study determined the salivary microbiota composition of 2,343 adult residents of Hisayama town, Japan, using 16S rRNA gene next-generation high-throughput sequencing. Of 550 identified species-level operational taxonomic units (OTUs), 72 were common, in ≥75% of all individuals, as well as in ≥75% of the individuals in the lowest quintile of phylogenetic diversity (PD). These "core" OTUs constituted 90.9 ± 6.1% of each microbiome. The relative abundance profiles of 22 of the core OTUs with mean relative abundances ≥1% were stratified into community type I and community type II by partitioning around medoids clustering. Multiple regression analysis revealed that a lower PD was associated with better conditions for oral health, including a lower plaque index, absence of decayed teeth, less gingival bleeding, shallower periodontal pockets and not smoking, and was also associated with tooth loss. By contrast, multiple Poisson regression analysis demonstrated that community type II, as characterized by a higher ratio of the nine dominant core OTUs, including Neisseria flavescens, was implicated in younger age, lower body mass index, fewer teeth with caries experience, and not smoking. Our large-scale data analyses reveal variation in the salivary microbiome among Japanese adults and oral health-related conditions associated with the salivary microbiome.

Cognitive function and 10 year mortality in an 85 year-old community-dwelling population.

The relationship between mortality and impaired cognitive function has not been thoroughly investigated in a very elderly community-dwelling population, and little is known about the association of disease-specific mortality with Mini-Mental State Examination (MMSE) subscale scores. Here we evaluated these data in Japanese community-dwelling elderly. In 2003, 85 year-olds (n=207) were enrolled; 205 completed the MMSE for cognitive function and were followed-up for 10 years, during which time 120 participants died, 70 survived, and 17 were lost to follow-up. Thirty-eight deaths were due to cardiovascular disease, 22 to senility, 21 to respiratory disease, and 16 to cancer. All-cause mortality decreased by 4.3% with a 1-point increase in the global MMSE score without adjustment, and it decreased by 6.3% with adjustment for both sex and length of education. Cardiovascular mortality decreased by 7.6% and senility mortality decreased by 9.2% with a 1-point increase in the global MMSE score with adjustment for sex and education. No association was found between respiratory diseases or cancer mortality and global MMSE score. All-cause mortality also decreased with increases in MMSE subscale scores for time orientation, place orientation, delayed recall, naming objects, and listening and obeying. Cardiovascular mortality was also associated with the MMSE subscale of naming objects, and senility mortality was associated with the subscales of time orientation and place orientation. Thus, we found that impaired cognitive function determined by global MMSE score and some MMSE subscale scores were independent predictors of all-cause mortality or mortality due to cardiovascular disease or senility in 85 year-olds.

Serum total cholesterol concentration and 10-year mortality in an 85-year-old population.

Little is known about the association between total cholesterol (TC) and all-cause mortality in the elderly (especially the very elderly). Here we examined the association between TC and all-cause mortality in 207 very elderly (85-year-old) participants. In 2003, we performed a baseline laboratory blood examination, and blood pressure (BP) and body mass index (BMI) measurements, and lifestyle questionnaires were completed by the participants. The participants were followed for the subsequent 10 years. As of 2013, of the 207 participants in 2003, 70 participants had survived, 120 individuals had died, and 17 were lost to follow up. The TC values were divided into high-TC (≥209 mg/dL), intermediate-TC (176-208 mg/dL), and low-TC (≤175 mg/dL) categories. With the Kaplan-Meier method, we found that both the high-TC and intermediate-TC participants survived longer than the low-TC participants. The men with high TC survived longer than those with low TC, but no corresponding difference was found for the women. A multivariate Cox proportional hazards regression model, with adjustment for gender, smoking, alcohol intake, history of stroke or heart disease, serum albumin concentration, BMI, and systolic BP, revealed that the total mortality in the low-TC group was 1.7-fold higher than that in the high-TC group. Mortality, adjusted for the same factors, decreased 0.9% with each 1 mg/dL increase in the serum TC concentration and decreased 0.8% with each 1 mg/dL increase in the serum (low-density lipoprotein) LDL-cholesterol (LDL-C) concentration. Our results indicate an association between lower serum TC concentrations and increased all-cause mortality in a community-dwelling, very elderly population. Mortality decreased with the increases in both TC and LDL-C concentrations, after adjustment for various confounding factors. These findings suggest that low TC and low LDL-C may be independent predictors of high mortality in the very elderly.

Gender differences in the association between metabolic syndrome and periodontal disease: the Hisayama Study.

AIMS: Periodontal disease and metabolic syndrome (MS) are more prevalent in males than in females. However, whether there is a gender difference in the association between these health conditions has not yet been investigated. This study examined the gender difference in this association, considering the definition of periodontal disease. MATERIALS AND METHODS: We recruited 1040 males and 1330 females, aged ≥40 years, with at least ten teeth from subjects of the 2007 Hisayama health examination. We performed a logistic regression analysis with various definitions of periodontal disease the dependent variable and MS as the independent variable. Following the analysis, the data were reanalysed with the structural equations model. RESULTS: The logistic regression analysis suggested a stronger association between periodontal disease and MS in females than that in males when periodontal disease was more severely defined. When we constructed the structural equations model in each gender, the model showed a good fit to the data of females, suggesting the association between periodontal disease and MS in females, but not in males. CONCLUSIONS: Gender differences seem to exist in the association between periodontal disease and MS; MS might show a stronger association with periodontal disease in females than in males.

High-level activities of daily living and disease-specific mortality during a 12-year follow-up of an octogenarian population.

BACKGROUND: Little is known about the relationship between disease-specific mortality and high-level activities of daily living in the elderly. We examined whether mortality is associated with high-level activities of daily living in an octogenarian population. METHODS: We conducted a population-based cross-sectional and prospective cohort study in 693 older persons aged 80 years and living in Japan's Fukuoka Prefecture. We then evaluated the association between 12-year disease-specific mortality and high-level functional capacity as measured by the Tokyo Metropolitan Institute of Gerontology Index of Competence, which is a standardized multidimensional 13-item instrument; items 1 through 5 are classified as instrumental self-maintenance activity, items 6 through 9 as intellectual activity, items 10 through 13 as social roles activity, and all 13 items together yield total functional capacity. RESULTS: By the 12-year follow-up of the 693 participants, 413 had died, 242 survived, and 38 were unable to be located. Of the 413 who died, 105 died of cardiovascular disease, 73 of respiratory tract disease, 71 of cancer, and 39 of senility. Of the other 125 deaths, 59 were due to other diseases, and the cause of death for 66 participants is not known. The hazard ratio (HR) for all-cause mortality, adjusted for confounding factors with multivariate Cox analyses, fell by 6% (HR 0.937, 95% confidence interval [CI] 0.899-0.978, P = 0.003) with each one-point increase in participants' scores on the Tokyo Metropolitan Institute of Gerontology Index of total functional capacity. With one-point increases in instrumental self-maintenance activity and in intellectual activity, the HRs for all-cause mortality decreased by 14% (HR 0.856, 95% CI 0.787-0.930, P = 0.000) and 12% (HR 0.884, 95% CI 0.794-0.983, P = 0.023), respectively. Respiratory mortality with HR adjustment fell by 11% (HR 0.887, 95% CI 0.804-0.978, P = 0.016) and 24% (HR 0.760, 95% CI 0.627-0.922, P = 0.005) with one-point increases in the scores of total functional capacity and instrumental self-maintenance activity, respectively. Similarly, mortality due to senility fell by 16% (HR 0.838, 95% CI 0.743-0.946, P = 0.004), 29% (HR 0.707, 95% CI 0.564-0.886, P = 0.003), and 29% (HR 0.710, 95% CI 0.522-0.966, P = 0.029) with one-point increases in the scores of total functional capacity, instrumental self-maintenance activity, and intellectual activity, respectively. CONCLUSION: These findings suggest that high-level activities of daily living may be an independent predictor of mortality due to all causes, respiratory disease and senility in older persons.

Activities of daily living dependency and disease-specific mortality during 12-year follow-up in an 80-year-old population.

BACKGROUND AND AIMS: Although total mortality is likely to be higher in elderly individuals with frailty or impaired activities of daily living (ADL), little is known about the relationships between disease-specific mortality and ADL dependency in the elderly. Therefore, we examined whether 12-year disease-specific mortality may be associated with ADL dependency in an 80-year-old population. METHODS: In 1998, of 1,282 community-dwelling residents of Japan's Fukuoka Prefecture, 824 (64.3 %) (309 males and 515 females) participated, the remaining 458 subjects did not participate, and their deaths and causes of death were followed up for 12 years after the baseline examination. ADL dependency was determined according to the guidelines for disabled elderly from the Health, Labor, and Welfare Ministry of Japan, and ADL dependency was measured only at baseline. RESULTS: During the 12-year follow-up, 506 died, 276 did not die, and 42 were lost. Of the 506 who died, 128 died due to cardiovascular disease, 96 to respiratory tract disease, 87 to cancer, and 51 to senility. The subjects were classified into three groups as follows: ADL-1 (independent group, n = 600), ADL-2 (almost-independent group, n = 113), and ADL-3 (dependent group, n = 93).Total-cause mortality was 2.8 times higher in ADL-3 subjects, respiratory disease mortality was 4.1 times higher in ADL-3 subjects, and senility mortality was 5.7 times higher in ADL-3 subjects than in ADL-1 subjects, after adjusting for various confounding factors. There was no association between mortality due to cancer or cardiovascular disease and ADL dependency. CONCLUSIONS: We found an independent association between ADL dependency and mortality due to all causes, respiratory disease or senility, but no association with mortality due to cancer or cardiovascular disease. These findings suggest that improving ADL dependency may reduce all mortality and mortality due to respiratory disease or senility.

Body mass index and disease-specific mortality in an 80-year-old population at the 12-year follow-up.

Although many investigations examined the relationship between body mass index (BMI) and mortality, little is known about the possible associations between BMI and disease-specific mortality in very elderly people. Here we evaluated this association in an 80-year-old population. In 1998, 675 residents in Japan's Fukuoka Prefecture participated. They were followed up for 12 years after the baseline examination; 37 subjects (5.5%) were lost to follow-up. The subjects were divided into six groups by their BMI values: <19.5 (most-thin), 19.5 to <21.1 (relatively thin), 21.1 to <22.5 (thin/normal), 22.5 to <23.8 (normal/overweight), 23.8 to <26.0 (relatively obese), ≥26.0 (most-obese). The most-thin group had the highest mortality from all-causes, and from respiratory disease. The normal/overweight group had the lowest overall mortality among the six BMI groups. These associations were found in the men, but not in the women. The most-obese group did not have higher mortality from all-causes or cardiovascular disease compared to the normal/overweight group. Respiratory disease-related mortality was lowest in the most-obese group. No association was found between BMI group and mortality from cancer. In conclusion, in an 80-year-old Japanese population, mortality from all-causes or respiratory disease was highest in the most-lean group (BMI <19.5), and mortality from all-causes or cardiovascular disease was lowest in the group with BMI 22.5 to <23.8.

Essential role of JAK/STAT pathway in the induction of cell cycle arrest in macrophages infected with periodontopathic bacterium Aggregatibacter actinomycetemcomitans.

In the present study, phosphorylation of signal transducers and activators of transcription 3 (STAT3) was found to be important in the induction of G1 cell cycle arrest in murine macrophages infected with Aggregatibacter actinomycetemcomitans. First, we focused on suppressor of cytokine signaling 3 (SOCS3) as a negative regulator of the JAK/STAT pathway. Flow cytometric analysis showed that A. actinomycetemcomitans infection eliminated G1 cell cycle arrest in SOCS3-overexpressing RAW 264.7 macrophage cells. Western blotting analysis demonstrated expression of cell cycle-associated protein p21 and hypophosphorylation of retinoblastoma protein (Rb) was decreased in SOCS3-overexpressing RAW 264.7 cells. AG490, a specific inhibitor of JAK2, inhibited the expression of p21 and degradation of cyclin D1 in A. actinomycetemcomitans-infected RAW 264.7 cells, resulting in suppression of STAT3 phosphorylation. These results indicated that constitutive SOCS3 expression and AG490 inhibited the expression of JAK2 and phosphorylation of STAT3, and prevented cell cycle arrest in A. actinomycetemcomitans-infected RAW 264.7 cells. These findings suggest that the JAK/STAT pathway plays crucial roles in the cell cycle regulation of macrophages infected with periodontopathic bacteria through the suppression of p21 expression and degradation of cyclin D1.

Relationship between receiving a workplace oral health examination including oral health instruction and oral health status in the Japanese adult population.

OBJECTIVES: Dental caries and periodontal disease are highly prevalent in the Japanese adult population. Oral examination is an effective method to find various oral health problems in their early stages. However, workplace oral examination is not common in Japan. The purpose of this study was to examine the relationship between receiving workplace oral health examination, including oral health instruction, and oral health status in the Japanese adult population. METHODS: This study was performed using data from 4,484 Japanese employees aged 35-74 yr. The proportion of teeth with a probing depth (PD) ≥4 mm and the number of decayed teeth were used for periodontal disease and dental caries parameters. The subjects were asked by questionnaire about past experiences with workplace oral health examination. RESULTS: The subjects who received a workplace oral health examination every year had better periodontal health status than those receiving an examination for the first time. The odds ratio for having ≥10% of teeth with PD ≥4 mm in the subjects who received workplace oral health examination every year was 0.63 (p<0.05) after adjustment for age, sex, smoking habits, tooth-brushing habits, routine visits to dental clinics, number of missing teeth, and oral hygiene status, in a multivariate, multinomial logistic regression analysis. On the other hand, no significant relationship was found between workplace oral health examination and number of decayed teeth. CONCLUSIONS: These results suggest that workplace oral health examination accompanied by oral health instruction may be effective for maintenance of periodontal health.

Diacylglycerol kinase alpha regulates globular adiponectin-induced reactive oxygen species.

It has previously been reported that the globular form of adiponectin (gAd), mature adipocyte-derived cytokine, induced generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. This study investigated whether diacylglycerol kinases (DGKs), enzymes functioning in sub-cellular signalling pathways, had a role on gAd-induced ROS generation in RAW 264 cells. Administration of R59022, a specific inhibitor for DGK, reduced gAd-induced ROS generation and NO release. RAW 264 cell expressed DGKα mRNA. Depression of DGKα mRNA by RNA interference significantly reduced the ROS generation in response to gAd treatment. Interestingly, transfection with the DGKα-specific small interfering RNA attenuated the expression level of Nox1 mRNA in gAd-treated RAW 264 cells. In addition, the DGKα knockdown with siRNA suppressed gAd-induced NO release.

Effectiveness of the salivary occult blood test as a screening method for periodontal status.

BACKGROUND: Community-based periodontal examinations are not popular despite the high prevalence of periodontal disease among adults. This study examines the effectiveness of a novel salivary occult blood test (SOBT) as a screening method for periodontal status. METHODS: Comprehensive health examinations were conducted in adult residents aged ≥40 years in Hisayama, Fukuoka, Japan; 1,998 subjects, each with ≥20 teeth, were analyzed. A paper test strip was used to perform the SOBT and was followed by a periodontal examination. Results were ranked as negative or positive. Subjects with ≥15% of teeth with bleeding on probing (BOP) or ≥1 tooth with a probing depth (PD) ≥4 mm were defined as having a poor periodontal status. The relationship between the results of the SOBT and periodontal parameters and among other variables was examined. RESULTS: The sensitivity and specificity of the SOBT in screening for poor periodontal status were 0.72 and 0.52, respectively. In a multivariate logistic regression analysis, the results of the SOBT were significantly associated with the proportion of teeth with BOP and the proportion of teeth with PD ≥4 mm, independent of age, sex, use of antihypertensive medication, use of antidiabetic medication or insulin therapy, and the number of decayed or filled teeth. CONCLUSION: The SOBT may offer a simple screening method for periodontal status when a thorough periodontal examination is not possible, although it is not sufficiently specific to be a reasonable substitute for a periodontal examination.

Involvement of the JAK-STAT pathway and SOCS3 in the regulation of adiponectin-generated reactive oxygen species in murine macrophage RAW 264 cells.

Adiponectin is a protein hormone produced by differentiating adipocytes and has been proposed to have anti-diabetic and immunosuppressive properties. We previously reported that the globular form of adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO), followed by caspase-dependent apoptotic cell death in RAW 264 cells. Here, we demonstrate that gAd-induced ROS generation and apoptosis were diminished by suppressor of cytokine signaling 3 (SOCS3). The phosphorylation level of signal transducer and activator of transcription (STAT) 3 detected by Western blotting was highest at 20 min in gAd-treated RAW 264 cells. This phosphorylation was inhibited by AG490, a specific inhibitor of janus-activator kinase (JAK). The gAd-induced ROS and NO were reduced by administration of AG490 and Jak-2-specific siRNA in RAW 264 cells. The gAd stimulation transiently induced SOCS3 mRNA expression and protein production. We examined SOCS3-overexpressing RAW 264 cells to investigate the role of the JAK-STAT pathway in gAd-induced ROS and NO generation. SOCS3 overexpression significantly reduced both ROS and NO generation. Additionally, gAd-induced caspase activation and apoptotic cell death were reduced in SOCS3 transfectants compared with vector control transfectants. These results suggest that the JAK-STAT pathway, which can be suppressed by SOCS3 expression, is involved in gAd-induced ROS and NO generation followed by apoptotic cell death.

Relationship between obesity and physical fitness and periodontitis.

BACKGROUND: Obesity and exercise are important elements associated with lifestyle-related diseases, and studies suggested that these factors may also be related to periodontitis. This study investigates the relationship between obesity and physical fitness and periodontitis. METHODS: One thousand, one hundred and sixty Japanese subjects, aged 20 to 77 years, who participated in health examinations at Fukuoka Health Promotion Center were analyzed. Periodontal conditions were evaluated using the Community Periodontal Index (CPI), and subjects with > or =3 sextants of CPI code 3 or 4 were defined as having severe periodontitis. We used the body mass index (BMI) and percentage of body fat as indicators of obesity and estimated the maximal oxygen consumption (VO(2max)) during exercise as an indicator of physical fitness. We divided these variables into quintiles. We examined the single effect and interactions of the obesity index and VO(2max) on severe periodontitis. RESULTS: The lowest quintile in BMI and the highest quintile in VO(2max) were inversely associated with severe periodontitis, singly, in multivariate logistic regression analyses. Subjects with the combined lowest quintile in BMI and the highest quintile in VO(2max) had a significantly lower risk of severe periodontitis compared to subjects with other combined quintiles in BMI and in VO(2max) (odds ratio: 0.17; 95% confidence interval: 0.05 to 0.55). CONCLUSION: This study suggests that obesity and physical fitness may have some interactive effect on periodontal health status.

Serum albumin levels as an independent predictor of 4-year mortality in a community-dwelling 80-year-old population.

BACKGROUND AND AIMS: Although serum albumin levels are associated with mortality in non-institutionalized elderly people under 80 years old, as well as in the institutionalized very elderly, little is known about the relationship in community-dwelling very elderly people. We, therefore, examined the association in a Japanese population of 80-year-old community residents. METHODS: Serum albumin levels were measured in 672 (267 men, 405 women) out of 1282 80-year-old individuals. Over the following 4 years, the dates and causes of death were recorded from resident registration cards and official death certificates. RESULTS: Of the above individuals, 107 subjects died (58 men, 49 women: 27 due to cancer, 27 cardiovascular disease, and 22 pneumonia). Survival rates were compared among 4 groups (highest >or=45 g/L, higher than 43-44 g/L, lower than 41-42 g/L, lowest

Involvement of mTOR in globular adiponectin-induced generation of reactive oxygen species.

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. This study investigated the role of the mammalian target of rapamycin (mTOR) in gAd-induced ROS and NO generation. gAd stimulation induced phosphorylation of mTOR, which peaked at 20 min and dissolved rapidly. Inhibition of phosphatidylinositol 3-kinase activity with wortmannin suppressed gAd-induced phosphorylation of Akt and mTOR. Administration of rapamycin partially reduced gAd-induced generation of intracellular and mitochondrial ROS, but not release of NO. To further confirm the role of mTOR in gAd stimulation, the effect of the activators of AMP-activated protein kinase (AMPK) on gAd-induced mTOR phosphorylation was examined. Pre-treatment with three kinds of AMPK activators, AICAR, 2-deoxy-D-glucose and A-769662, suppressed gAd-induced mTOR phosphorylation. Furthermore, these AMPK activators significantly reduced gAd-evoked intracellular and mitochondrial ROS generation and NO release.

Anti-inflammatory activity of a globular adiponectin function on RAW 264 cells stimulated by lipopolysaccharide from Aggregatibacter actinomycetemcomitans.

Adiponectin is an adipokine with potent anti-inflammatory properties. We previously reported that a globular adiponectin (gAd) suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced nuclear factor-kappaB activity, suggesting an anti-inflammatory effect of gAd. In this study, we investigated whether gAd is able to modulate the effect of A. actinomycetemcomitans lipopolysaccharide on cytokine induction in a murine macrophage cell line (RAW 264). The phosphorylation of p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and IkappaB kinase alpha/beta and the degradation of IkappaB, which were induced by A. actinomycetemcomitans lipopolysaccharide intoxication, were clearly reduced in gAd-pretreated RAW 264 cells compared with the untreated cells. Expression levels of tumor necrosis factor (TNF)-alpha and interleukin-10 (IL-10) mRNA were assessed by real-time PCR. Cell-free supernatants were collected after 12 h of stimulation and analyzed by enzyme-linked immunosorbent assay for TNF-alpha and IL-10. Pretreatment with gAd significantly inhibited the A. actinomycetemcomitans lipopolysaccharide-induced TNF-alpha mRNA expression and protein secretion. In contrast, pretreatment with gAd significantly enhanced the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 mRNA expression and protein secretion. These data suggest a mechanism for the anti-inflammatory activity of gAd in local inflammatory lesions, such as periodontitis.

Globular adiponectin-induced RAW 264 apoptosis is regulated by a reactive oxygen species-dependent pathway involving Bcl-2.

Globular adiponectin (gAd), a truncated form of adipocyte-derived cytokine, stimulates RAW 264 cells to produce reactive oxygen species (ROS), which trigger an apoptotic cascade. In this study, we investigated the generation of intracellular and mitochondrial ROS in gAd-stimulated RAW 264 cells. Treatment with gAd efficiently induced the generation of intracellular and mitochondrial ROS, as detected by dichlorodihydrofluorescein diacetate and MitoSOX fluorescence, respectively. Furthermore, gAd treatment significantly increased 8-oxoguanine, a specific indicator of oxidative DNA damage. The transfection of RAW 264 cells with iNOS- and gp91(phox)-specific small interfering RNA reduced markedly the generation of intracellular, but not mitochondrial, ROS. Quantitative PCR revealed that the expression ratio of Bcl-2 to Bax was reduced in a time-dependent manner in gAd-treated RAW 264 cells. The overexpression of Bcl-2 markedly inhibited gAd-induced apoptosis in RAW 264 cells and also reduced both the intracellular and the mitochondrial ROS generation induced by gAd treatment. Moreover, the overexpression of Bcl-2 significantly suppressed gAd-induced NO secretion and NOS activity. In addition, the inhibition of NOS activity partially reduced the oxidative DNA damage induced by gAd. Taken together, these results demonstrate that the gAd-induced apoptotic pathway acting via ROS/RNS generation involves Bcl-2.