Tomoko Akase

Skin fragility in obese diabetic mice: possible involvement of elevated oxidative stress and upregulation of matrix metalloproteinases

Abstract:  The purpose of this study was to test the hypothesis that obese diabetic mice exhibit marked skin fragility, which is caused by increased oxidative stress and increased matrix metalloproteinase (MMP) gene expression in the subcutaneous adipose tissue. Scanning electron microscopy of skin samples from Tsumura‐Suzuki obese diabetic (TSOD) mice revealed thinner collagen bundles, and decreased density and convolution of the collagen fibres. Furthermore, skin tensile strength measurements confirmed that the dorsal skin of TSOD mice was more fragile to tensile force than that of non‐obese mice. The mRNA expressions of heme oxygenase 1 (Hmox1), a marker of oxidative stress, Mmp2 and Mmp14 were increased in the adipose tissue of TSOD mice. Antioxidant experiments were subsequently performed to determine whether the changes in collagen fibres and skin fragility were caused by oxidative stress. Strikingly, oral administration of the antioxidant dl‐α‐tocopherol acetate (vitamin E) decreased Hmox1, Mmp2 and Mmp14 mRNA expressions, and improved the skin tensile strength and structure of collagen fibres in TSOD mice. These findings suggest that the skin fragility in TSOD mice is associated with dermal collagen damage and weakened tensile strength, and that oxidative stress and MMP overexpression in the subcutaneous adipose tissue may, at least in part, affect dermal fragility via a paracrine pathway. These observations may contribute to novel clinical interventions, such as dietary supplementation with antioxidants or application of skin cream containing antioxidants, which may overcome skin fragility in obese patients with diabetes.

Preventive Effects of Salacia reticulata on Obesity and Metabolic Disorders in TSOD Mice.

The extracts of Salacia reticulata (Salacia extract), a plant that has been used for the treatment of early diabetes, rheumatism and gonorrhea in Ayurveda, have been shown to have an anti-obesity effect and suppress hyperglycemia. In this study, the effects of Salacia extract on various symptoms of metabolic disorder were investigated and compared using these TSOD mice and non-obese TSNO mice. Body weight, food intake, plasma biochemistry, visceral and subcutaneous fat (X-ray and CT), glucose tolerance, blood pressure and pain tolerance were measured, and histopathological examination of the liver was carried out. A significant dose-dependent decline in the gain in body weight, accumulation of visceral and subcutaneous fat and an improvement of abnormal glucose tolerance, hypertension and peripheral neuropathy were noticed in TSOD mice. In addition, hepatocellular swelling, fatty degeneration of hepatocytes, inflammatory cell infiltration and single-cell necrosis were observed on histopathological examination of the liver in TSOD mice. Salacia extract markedly improved these symptoms upon treatment. Based on the above results, it is concluded that Salacia extract has remarkable potential to prevent obesity and associated metabolic disorders including the development of metabolic syndrome.

Salacia reticulata inhibits differentiation of 3T3-L1 adipocytes.

UNLABELLED ETHOPARMACOLOGICAL RELEVANCE: Salacia reticulata, a herbal medicine which has been used for the treatment of early diabetes in Ayurvedic medicine, is reported to have an anti-obesity effect and to be useful in the treatment of diabetes mellitus, insulin resistance and other metabolic diseases. AIM OF THE STUDY The present study was performed to elucidate the mechanism of action of Salacia reticulata with special attention to the adipocytes as the tissue primarily involved in the pathology of metabolic diseases. MATERIALS AND METHODS Mouse-derived adipocyte precursor 3T3-L1 cells were treated with differentiation inducers in the presence or absence of Salacia reticulata (SRCD). We determined triacylglycerol accumulations, differentiation makers, released glycerol and adiponectin. Mangiferin, the primary component of SRCD, was also treated to 3T3-L1 cells. RESULT Concurrent administration of the extract of SRCD and differentiation inducers resulted in a significant inhibition of differentiation into mature adipocytes. SRCD also exhibited significant inhibitory action on the expression of genes and proteins of peroxisome proliferator-activated receptor (PPAR)γ and CCAAT-enhancer binding protein (C/EBP)α, as well as on the activity of glycerol-3-phosphate dehydrogenase (GPDH), a differentiation marker, and caused a reduction in the concentration of released adiponectin. However, SRCD had no influence on lipolysis as indicated by the release of glycerol into the culture medium. The primary component of SRCD, mangiferin, was investigated for its effect on adipocytes; mangiferin caused no suppression of fat accumulation, suggesting that a component of SRCD other than mangiferin may be involved in the inhibition of adipocyte differentiation. CONCLUSIONS The above results suggest that the inhibitory action of SRCD on adipocyte differentiation, and not the promotion of lipolysis, is involved in the suppression of fat accumulation.

Aging-Like Skin Changes Induced by Ultraviolet Irradiation in an Animal Model of Metabolic Syndrome:

Both physiological skin aging and pathologic photo-aging caused by ultraviolet (UV) irradiation are mediated by latent inflammation and oxidative stress. Although numerous animal skin-aging models have used UV irradiation, most require massive doses or long-term irradiation. To establish a more refined skin-aging model, we focused on an animal model of metabolic syndrome (MS) because MS involves damage to various organs via oxidative stress or inflammation, similar to the changes associated with aging. We hypothesized that MS skin might exhibit more aging-like changes after milder, shorter-term UV irradiation than would normal animal skin under similar conditions, thus providing a useful model for skin aging. The authors therefore examined the skin from Tsumura Suzuki obese diabetic (TSOD) mice (MS model) and control Tsumura Suzuki non-obese (TSNO) mice before and after UV irradiation. Skin from TSOD mice had a thinner epidermis and dermis, a thicker fatty layer, reduced density and convolution of the fragmented collagen fibers, and upregulated expression of tumor necrosis factor (TNF)-α, a dual marker for inflammation and aging, compared to the skin from TSNO mice. UV irradiation affected TSOD skin more severely than TSNO skin, resulting in various changes resembling those in aged human skin, including damage to the dermis and subcutaneous fatty tissue, infiltration of inflammatory cells, and further upregulation of TNF-α expression. These results suggest that UV-irradiated TSOD mice may provide a new model of skin aging and imply that skin from humans with MS is more susceptible to UV- or aging-related damage than normal human skin.

Aging-Like Skin Changes in Metabolic Syndrome Model Mice Are Mediated by Mineralocorticoid Receptor Signaling

Aging is accelerated, at least in part, by pathological condition such as metabolic syndrome (MetS), and various molecular pathways such as oxidative stress are common mediators of aging and MetS. We previously developed the aging‐like skin model by single ultraviolet (UV) irradiation on the MetS model mice. Recent studies revealed that mineralocorticoid receptor (MR) signaling plays a pivotal role for various tissue inflammation and damages in MetS. Although previous studies reported that MR is expressed in the skin and that overexpression of MR in the skin resulted in the skin atrophy, the physiological or pathological functions of MR in the skin are not fully elucidated. Here, we show the involvement of MR signaling in the aging‐like skin changes in our own model. Elevations of oxidative stress and inflammation markers were observed in the MetS mice, and the UV‐evoked aging‐like skin damages were attenuated by topical antioxidant. MR expression was higher in the MetS mouse skin, and notably, expression of its effecter gene Sgk1 was significantly upregulated in the aging‐like skin in the UV‐irradiated MetS mice. Furthermore, topical application of MR antagonist spironolactone suppressed Sgk1 expression, oxidative stress, inflammation, and the aging‐like changes in the skin. The 2‐week UV onto the non‐MetS mice, the more usual photoaging model, resulted in the skin damages mostly equivalent to the MetS mice with single UV, but they were not associated with upregulation of MR signaling. Our studies suggested an unexpected role of MR signaling in the skin aging in MetS status.

Aging enhances maceration-induced ultrastructural alteration of the epidermis and impairment of skin barrier function.

BACKGROUND Skin maceration is recognized as a risk factor for the development of certain skin lesions. In health care settings, incontinence-associated skin maceration is highly prevalent in the elderly. However, the effect of senescence on maceration has not been fully elucidated. OBJECTIVE To reveal the enhancement of the maceration-induced ultrastructural alteration and barrier function of the epidermis by aging. METHODS Skin maceration was reproduced by exposure to agarose gel in human and rat. The ultrastructural alterations in human and rat tissue were observed by transmission electron microscopy. The skin barrier function was evaluated by noninvasive methods in human, and by the transdermal penetration of small- and large-fluorescent molecules in rat. In order to reveal the effect of aging on the skin maceration, we compared these parameters between young and aged rats. RESULTS In macerated skin, we observed expansion of the interstices of the stratum corneum, spinosum, and basale of the epidermis; disruption of the intercellular lipid structure in the stratum corneum; a decreased number of cell processes in the stratum spinosum and basale. The transdermal penetration test in the rat using two types of fluorescein indicated that maceration disrupted skin barrier function. Furthermore, senescence-enhanced ultrastructural and functional alterations were revealed in the rodent studies. CONCLUSION This study demonstrates that aging enhances skin maceration. Considering that maceration is a risk factor for the skin damage, the development of technology to promote skin barrier recovery after maceration in the elderly is warranted.

Preventive Effect of Boiogito on Metabolic Disorders in the TSOD Mouse, a Model of Spontaneous Obese Type II Diabetes Mellitus

“Boiogito” is a Kampo preparation which has been used since ancient times in patients with obesity of the “asthenic constitution” type, so-called “watery obesity”, and its effect has been recognized clinically. In this study, we investigated the anti-obesity effect of Boiogito in the TSOD (Tsumura Suzuki Obese Diabetes) mouse, a model of spontaneous obese type II diabetes mellitus. Boiogito showed a significant anti-obesity effect in TSOD mice by suppressing body weight gain in a dosage-dependent manner. In addition, Boiogito showed significant ameliorative effects on features of metabolic syndrome such as hyperinsulinemia, fasting hyperglycemia and abnormal lipid metabolism. Regarding lipid accumulation in TSOD mice, Boiogito showed a significant suppressive effect on accumulation of subcutaneous fat, but the effect on the visceral fat accumulation that constitutes the basis of metabolic syndrome was weak, and the suppressive effect on insulin resistance was also weak. Furthermore, Boiogito did not alleviate the abnormal glucose tolerance, the hypertension or the peripheral neuropathy characteristically developed in the TSOD mice. In contrast, in the TSNO (Tsumura Suzuki Non-Obesity) mice used as controls, Boiogito suppressed body weight gain and accumulation of subcutaneous and visceral fat. The above results suggested that Boiogito is effective as an anti-obesity drug against obesity of the “asthenic constitution” type in which subcutaneous fat accumulates, but cannot be expected to exert a preventive effect against various symptoms of metabolic syndrome that are based on visceral fat accumulation.

Novel models for bacterial colonization and infection of full‐thickness wounds in rats

An animal model is needed to study the pathophysiology of wound infections; however, an animal model that is reproducible and clinically relevant has not previously been available. In addition, an animal model of wound colonization generated in a manner similar to the wound infection model would be useful. Here, we describe new animal models of the wound infection continuum for the characterization of essential host–pathogen relationships. We determined the conditions needed to establish rat models of stable wound colonization and infection, without the use of disturbing factors (e.g., foreign bodies or induction of diabetes mellitus). We found that the age of the rats, bacterial inoculum size, and wound location were important elements in generating reproducible, obvious, spreading wound infections. We inoculated approximately 6‐month‐old rats with 2.06 × 109 or 4.12 × 109 colony‐forming units of Pseudomonas aeruginosa to generate the wound colonization and wound infection models, respectively. Wounds were made 2 cm cranial to the greater trochanter. These clinically relevant and highly reproducible animal models can be used to investigate the mechanisms of wound infection and monitor the effect of therapeutic agents in vivo.

Altered Expression of Matrix Metalloproteinases and Their Tissue Inhibitors in Matured Rat Adipocytes in Vitro

Obesity is recognized as a risk factor for delayed cutaneous wound healing. The authors hypothesized that the secretion of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) from subcutaneous adipose tissue correlates with disorder of the healing process in obese subjects. Findings from previous studies on the expression of MMPs and TIMPs in obese adipose tissue are inconsistent. Since these conflicting results could be due to the effect of several intrinsic factors, the authors conducted a simple in vitro experiment to clarify the change in profile of MMPs and TIMPs in excessively matured adipocytes. The authors cultured the induced adipocytes under conditions of high or low glucose and with or without insulin supplementation. Oil red O staining and its dye extraction assay revealed excessive lipid accumulation in high glucose and insulin-supplemented adipocytes. Additionally, there was altered expression of adipokines, similar to the change in adipose tissue in obese subjects. Under these conditions, the expression/activity of MMP8 was promoted and the expression of MMP3 and TIMP3 was inhibited. Further studies to determine the effect of other obesity-related factors, such as insulin resistance, on MMPs and TIMPs are required.

Novel biomarkers for the detection of wound infection by wound fluid RT-PCR in rats

Abstract:  Wound infection is a form of host damage resulting from an imbalance in pathogen virulence and the host immune response. However, at present, diagnosis is based solely on bacterial numbers or inflammatory signs and is therefore not precise. Thus, infection diagnosis requires indicators of both of these factors. We focused on wound fluid because it includes both bacteria and host cells. The purpose of this study was to establish biomarkers that reflect both bacterial and host factors using the reverse transcription‐polymerase chain reaction method on the centrifugal precipitation of wound fluids (wound fluid RT‐PCR). We created full thickness wounds in animal models of the three groups: control, colonization and infection, which were conditioned by administration of different concentrations of Pseudomonas aeruginosa dispersion. Messenger RNA expression in bacteria and host cells was analysed. Expression of bacterial housekeeping genes was detected in the samples in the colonization and infection groups. Expression of host housekeeping genes was detected in all samples from the three groups. Expression of toxA, encoding the virulence factor exotoxin A, was detected in 90% of samples in the infection group only. Expression of Foxp3, encoding the transcription factor forkhead box P3, was detected in 100% of samples only in the colonization group. These results revealed that wound fluid RT‐PCR analysis reflected both bacterial virulence and the host immune status, and we determined the combination of novel biomarkers that can discriminate these three groups. We anticipate that wound fluid RT‐PCR could be applied in the future to diagnose wound infection.