Antiobesity Effect of Lacticaseibacillus paracasei LM-141 on High-Fat Diet-Induced Rats through Alleviation of Inflammation and Insulin Resistance.

In this study, we set out to evaluate the antiobesity activities of our newly isolated Lacticaseibacillus paracasei LM-141 (LPLM141) using a high-fat diet (HFD)-fed rat model. Male Sprague-Dawley rats were fed with a HFD with or without low-dosage (2 × 107 CFU/day per rat) or high-dosage (2 × 109 CFU/day per rat) LPLM141 for 14 weeks. The results showed that administration of LPLM141 significantly decreased body weight gain, liver weight, adipose tissue weight, and epididymal white adipocyte size increased by HFD feeding. The abnormal serum lipid profile induced by HFD feeding was normalized by administration of LPLM141. The enhanced chronic low-grade inflammation in HFD-fed rats was reduced by LPLM141 supplementation, as reflected by decreased serum lipopolysaccharide (LPS) and monocyte chemoattractant protein-1 (MCP-1) levels, reduced macrophage infiltration in adipose tissue, and increased serum adiponectin concentration. In addition, the elevations of proinflammatory cytokine genes and suppression of PPAR-γ mRNA in adipose tissues of rats fed with a HFD were markedly reversed by LPLM141 administration. Oral administration of LPLM141 induced browning of epididymal white adipose tissue (eWAT) and activation of interscapular brown adipose tissue (iBAT) in rats fed with HFD. Consumption of LPLM141 exhibited a significant amelioration in insulin resistance, which were mechanistically caused by downregulation of the serum leptin level and upregulation of hepatic IRS-1 and p-Akt protein expressions, in HFD treated rats. LPLM141 consumption significantly decreased hepatic lipogenic gene expressions and preserved liver function stimulated by HFD treatment. Administration of LPLM141 obviously mitigated hepatic steatosis observed in HFD feeding rats. Our current findings shed light on LPLM141 supplementation that exhibited an antiobesity effect in HFD-fed rats by alleviating inflammation and insulin resistance, which further highlighted the potential of utilizing LPLM141 as a preventive/therapeutic probiotic agent for obesity.

The effects of UVB irradiance on aberrant epidermal proliferation: Novel insights on how to improve currently available sunscreens.

AIMS: Sunscreen use, which prolonged the time required to develop sunburn by reducing the irradiance (mW/cm2) of the UVB radiation, is thought to protect the skin from developing cancers. Recently, in addition to fluence (mJ/cm2), irradiance of the UVB radiation was demonstrated to play an important role leading to photocarcinogenesis of the skin. After equivalent fluence of UVB exposure, enhanced aberrant keratinocyte proliferation contributes significantly to the photocarcinogenic capacity of low irradiance (LI) UVB as compared to its high irradiance (HI) UVB counterpart. However, the mechanism involved remains unclear. MAIN METHODS: Relevant cell and animal models were employed to investigate the effects of equivalent UVB fluence administered at HI or LI on keratinocyte proliferation. Additionally, the mechanisms involved were also explored. KEY FINDINGS: We found that at equivalent fluence, LIUVB induces significantly higher reactive oxidative species (ROS) production, cell proliferation, as well as phosphorylated AKT (pAKT) expression in both cell and animal models as compare to its HIUVB counterpart. Pretreating cultured keratinocytes with antioxidant or AKT inhibitor significantly reduced the UVB-induced ROS, cell proliferation, and pAKT expression. Additionally, these pretreatments abrogate the difference between the LI and HIUVB treated keratinocytes. Similar findings were noted using animal model treated with AKT inhibitor. SIGNIFICANCE: In summary, at equivalent fluence, LIUVB induces significantly more aberrant epidermal proliferation via enhanced ROS and pAKT signaling. Reducing UVB-induced AKT phosphorylation presents a novel strategy to improve the protective capacity of the currently available sunscreens.

High glucose environment induces M1 macrophage polarization that impairs keratinocyte migration via TNF-α: An important mechanism to delay the diabetic wound healing.

BACKGROUND: Macrophages play important roles during wound healing, and delayed healing in diabetics is associated with sustained inflammation. M1 type macrophage is recognized to secrete excessive amount of tumor necrosis factor-alpha (TNF-α) as compared to its M2 counterpart. OBJECTIVES: We hypothesized that macrophage polarization is different between diabetic and normal rats during skin wounding and has direct impact on keratinocyte function in the context of re-epithelialization. METHODS: Skin wounds were created in diabetic and control rats. The phenotypes of infiltrating macrophages, the levels of TNF-α, and the rate of wound closure were determined. Using cell model, the effects of M1 type macrophage on keratinocyte migration were evaluated, and the potential regulatory pathways were determined. RESULTS: The percentage of M1 macrophages and the levels of TNF-α expression were significantly higher in the perilesional area of diabetic rats as compared to control. The condition media (CM) from M1 type macrophage upregulated tissue inhibitor metalloproteinases (TIMP)-1 expression in keratinocytes and significantly reduced keratinocyte migratory capacity. Addition of neutralizing TNF-α antibody to the CM or gene-silencing of TIMP1 in keratinocytes restored the keratinocyte migratory capacity. Treating wounds of diabetic rats with TNF-α antagonist improved the wound healing process. CONCLUSIONS: In summary, high glucose wound environment harbored more M1 macrophages infiltration, an event that created excess TNF-α micro-environment. TNF-α upregulated TIMP1 expression in keratinocytes and resulted in impaired keratinocyte migration. Taken together, these events contributed to impaired wound healing during diabetic condition, and targeting TNF-α is a potential therapeutic option to improve diabetic wound healing.

Dose-Dependent Effect of Hyperbaric Oxygen Treatment on Burn-Induced Neuropathic Pain in Rats.

Hyperbaric oxygen treatment (HBOT) has been used to reduce neuropathic pain. Melatonin and opioid receptors are involved in neuropathic pain, but it is not known if HBOT works through these pathways to achieve its antinociceptive effect. We divided anesthetized rats into two treatment and three sham groups. The two treatment groups received third-degree burns on their right hind paws, one treated in a hyperbaric chamber for a week and the other for two weeks. We evaluated the mechanical paw-withdrawal threshold (MWT) and expression of melatonin receptor 1 (MT1), melatonin receptor 2 (MT2), µ (MOR) and κ (KOR) opioid receptor, brain-derived neurotrophic factor (BDNF), Substance P, and calcitonin gene-related peptide (CGRP) in cuneate nucleus, dorsal horn, and hind paw skin by immunohistochemical, immunofluorescence assays and real-time quantitative polymerase chain reaction (RT-PCR). The group receiving one-week HBOT had increased expressions of MT1, MT2, MOR and KOR and decreased expressions of BDNF, Substance P, and CGRP. Their mechanically measured pain levels returned to normal within a week and lasted three weeks. This anti-allodynia effect lasted twice as long in those treated for two weeks. Our findings suggest that increasing the duration of HBOT can reduce burn-induced mechanical allodynia for an extended period of time in rats. The upregulation of melatonin and opioid receptors observed after one week of HBOT suggests they may be partly involved in attenuation of the mechanical allodynia. Downregulation of BDNF, substance P and CGRP may have also contributed to the overall beneficial effect of HBOT.

The impact of irradiance on UVB-induced cutaneous immunosuppression: Implications on administering most efficient phototherapy.

BACKGROUND: Ultraviolet B (UVB) is commonly used for treating dermatologic conditions. Recently, high irradiance UVB (HIUVB) has been suggested to be more effective for treating skin conditions as compared to its low irradiance (LI) counterpart. The biological impact of UVB radiation emitted at different irradiance on cutaneous immunity remains obscure. OBJECTIVE: This study aimed to explore the impacts of UVB radiation administered at equivalent fluence (mJ/cm2) but different irradiance (mW/cm2) on cutaneous immune response. METHODS: Cultured bone marrow derived dendritic cell (BMDC) were treated with equivalent fluence of UVB radiation with HIUVB or LIUVB. The phenotypic and functional alterations of BMDCs were documented. Animal models were used to validate the in vitro results in vivo and explore the mechanisms involved. RESULTS: After equivalent fluence of UVB radiation, the HIUVB treated BMDC showed significantly lower MHCII and CD86 expressions, reduced capacity to stimulate T cell proliferation, and enhanced activation of aryl hydrocarbon receptor (AhR)-activated genes as compared to control while their LIUVB treated counterpart showed no significant change. Using animal model, the HIUVB induced significantly higher immune suppressive effect in mice as compared to their LIUVB counterpart after equivalent fluence of UVB treatment. The superior immune suppressive effect of HIUVB over LIUVB radiation was not observed when similar experiments were performed using AhR-deficient mice. CONCLUSION: We propose irradiance played an important role modulating UVB-induced cutaneous immune suppression. Future works on UVB phototherapy, both clinical and research, should incorporate this important parameter into consideration.

The effect of interaction of heat and UVB on human keratinocyte: Novel insights on UVB-induced carcinogenesis of the skin.

BACKGROUND: Skin cancer is an important environmentally-related health issue. Although sun exposure is closely associated with increasing environmental heat, the effects of environmental heat on the skin, especially in the context of photocarcinogenesis, has not been carefully examined. OBJECTIVES: This study aimed to explore the effects and interactions of UVB radiation and environmental heat on photocarcinogenesis of the skin using cell and animal models. METHODS: Cultured keratinocytes and hairless mice were exposed to different treatment conditions including UVB radiation and environmental heat. The effects of treatment on keratinocyte and mice skin were evaluated at indicated time points. RESULTS: UVB induced DNA damage was significantly lower in keratinocytes that were pretreated in an environment with slightly elevated temperature followed by UVB treatment (Heat-UVB) as compared to UVB and UVB radiation followed by exposure to equivalent increase in environmental heat (UVB-Heat) groups. Similar phenomenon was observed in terms of keratinocyte viability. In the animal model, it was found that Heat-UVB treated mice showed delayed and reduced tumor formation as compared to the UVB and UVB-Heat treated groups. Quantum simulation analyses demonstrated that the energy required for CPD formation at environment with higher temperature required considerable higher energy as compared to CPD formation at lower temperature. CONCLUSION: Taken together, our results demonstrated that with equivalent UVB exposure, higher temperature environment may protect cells against subsequent UVB-induced DNA damages.

High-glucose environment induced intracellular O-GlcNAc glycosylation and reduced galectin-7 expression in keratinocytes: Implications on impaired diabetic wound healing.

BACKGROUND: Diabetes is an important global health issue due to its increasing prevalence and association with various complications. Impaired wound healing is a serious complication associated with diabetes that frequently results in infection and amputation. Galectin-7 (Gal-7) has been reported to play an important role during skin wound healing. Previously, we had demonstrated that high glucose environment alters physiologic functions of keratinocytes and contributes to impaired wound healing in diabetic condition. OBJECTIVE: In this study, we hypothesized that Gal-7 expression of keratinocytes may be involved in delayed wound healing of diabetics. METHODS: Using cultured human keratinocytes and diabetic mice model, the Gal-7 expression was evaluated under high glucose environment. RESULTS: Our results demonstrated that high-glucose environment reduced Gal-7 expression, a molecule that plays an important role in keratinocyte migration. Additionally, we found that increased O-linked N-Acetyl-glucosamine (O-GlcNAc) is responsible for reduced Gal-7 expression in keratinocytes exposed to high glucose environment. CONCLUSION: Taken together, restoring the levels of Gal-7 and O-GlcNAc glycosylation may present novel therapeutic approach to promote wound healing in diabetic patients.

Irradiance-dependent UVB Photocarcinogenesis.

Ultraviolet B (UVB) radiation from the sun may lead to photocarcinogenesis of the skin. Sunscreens were used to protect the skin by reducing UVB irradiance, but sunscreen use did not reduce sunburn episodes. It was shown that UVB-induced erythema depends on surface exposure but not irradiance of UVB. We previously showed that irradiance plays a critical role in UVB-induced cell differentiation. This study investigated the impact of irradiance on UVB-induced photocarcinogenesis. For hairless mice receiving equivalent exposure of UVB radiation, the low irradiance (LI) UVB treated mice showed more rapid tumor development, larger tumor burden, and more keratinocytes harboring mutant p53 in the epidermis as compared to their high irradiance (HI) UVB treated counterpart. Mechanistically, using cell models, we demonstrated that LI UVB radiation allowed more keratinocytes harboring DNA damages to enter cell cycle via ERK-related signaling as compared to its HI UVB counterpart. These results indicated that at equivalent exposure, UVB radiation at LI has higher photocarcinogenic potential as compared to its HI counterpart. Since erythema is the observed sunburn at moderate doses and use of sunscreen was not found to associate with reduced sunburn episodes, the biological significance of sunburn with or without sunscreen use warrants further investigation.

Heat-killed and live Lactobacillus reuteri GMNL-263 exhibit similar effects on improving metabolic functions in high-fat diet-induced obese rats.

Our objective was to investigate and compare the effects of heat-killed (HK) and live Lactobacillus reuteri GMNL-263 (Lr263) on insulin resistance and its related complications in high-fat diet (HFD)-induced rats. Male Sprague-Dawley rats were fed with a HFD with either HK or live Lr263 for 12 weeks. The increases in the weight gain, serum glucose, insulin, and lipid profiles in the serum and liver observed in the HFD group were significantly reduced after HK or live Lr263 administration. Feeding HK or live Lr263 reversed the decreased number of probiotic bacteria and increased the number of pathogenic bacteria induced by high-fat treatment. The decreased intestinal barrier in the HFD group was markedly reversed by HK or live Lr263 treatments. The elevations of pro-inflammatory associated gene expressions in both adipose and hepatic tissues by high-fat administration were markedly decreased by HK or live Lr263 treatments. The increased macrophage infiltration noticed in adipose tissue after high-fat treatment was effectively suppressed by HK or live Lr263 consumption. The insulin resistance associated gene expressions in both adipose and hepatic tissues, which were downregulated in the HFD group, were markedly enhanced after HK or live Lr263 administration. HK or live Lr263 consumption significantly decreased hepatic lipogenic gene expressions stimulated by high-fat treatment. Administration of HK or live Lr263 significantly reduced hepatic oil red O staining and ameliorated the hepatic steatosis observed in high-fat treated rats. Our data suggested that similar to live Lr263, HK Lr263 exerted significant effects on attenuating obesity-induced metabolic abnormalities by reducing insulin resistance and hepatic steatosis formation.

High-glucose environment increased thrombospondin-1 expression in keratinocytes via DNA hypomethylation.

Diabetes is an important health issue because of its increasing prevalence and association with impaired wound healing. Epidermal keratinocytes with overexpressed antiangiogenic molecule thrombospondin-1 (TSP1) have been shown to impair proper wound healing. This study examined the potential involvement of keratinocyte-derived TSP1 on diabetic wound healing. Cultured human keratinocytes and diabetic rat model were used to evaluate the effect of high-glucose environment on TSP1 expression in epidermal keratinocytes, and the molecular mechanisms involved in the process were also studied. We demonstrated that high-glucose environment increased TSP1 expression in keratinocytes. In addition, increased oxidative stress induced DNA hypomethylation at the TSP1 promoter region in keratinocytes exposed to high-glucose environment. Similar findings were found in our diabetic rat model. Early antioxidant administration normalized TSP1 expression and global DNA methylation status in diabetic rat skin and improved wound healing in vivo. Because oxidative stress contributed to TSP1 DNA hypomethylation, early recognition of diabetic condition and timely administration of antioxidant are logical approaches to reduce complications associated with diabetes as alterations in epigenome may not be reversible by controlling glucose levels during the later stages of disease course.

LED 590 nm photomodulation reduces UVA-induced metalloproteinase-1 expression via upregulation of antioxidant enzyme catalase.

BACKGROUND: Light at visible spectrum has been associated with anti-inflammatory and anti-aging effects. Ultraviolet A (UVA) radiation is the most important environmental factor associated with exogenous aging via induction of reactive oxygen species (ROS). OBJECTIVE: In this study, we focused on elucidating the molecular mechanisms involved in biological effects associated with 590 nm light delivered from light emitting diode (LED). METHODS: UVA-induced metalloproteinase-1 (MMP-1) expression in dermal fibroblast was used as a model system for investigation. RESULTS: Pretreating cultured human fibroblasts with 590 nm light attenuated UVA-induced ROS, phosphorylated Jun N-terminal kinases, and MMP-1 expressions in a sequential manner. Pretreatment with potent antioxidant N-acetylcysteine produced similar effect, suggesting enhanced antioxidant capacity induced by 590 nm photomodulation. Further experiments demonstrated that 590 nm photomodulation attenuated UVA-induced ROS and MMP-1 expressions via mitochondrial retrograde signaling that augments the antioxidant enzyme expression in a peroxisome proliferators-activated receptor γ coactivator-1α-dependent manner. CONCLUSION: Our results provided possible mechanistic insights explaining the effect of visible light on treating clinical conditions associated with ROS-mediated dysfunctions.

Oral administration of Lactobacillus reuteri GMNL-263 improves insulin resistance and ameliorates hepatic steatosis in high fructose-fed rats.

BACKGROUND: Type 2 diabetes mellitus (DM), characterized by peripheral insulin resistance, is the most common form of diabetes. Probiotics are live micro-organisms that, when administered in adequate amounts, confer delaying effect on DM development. In this study, the effects Lactobacillus reuteri GMNL-263 (Lr263), a new probiotic strain developed by our laboratory, on insulin resistance and the development of hepatic steatosis in high-fructose fed rats were explored. Furthermore, the relevant regulatory pathways involved were also investigated. METHOD: Male Sprague-Dawley rats were fed a high-fructose diet with or without Lr263 administration for 14 weeks. The composition of fecal microbiota, oral glucose tolerance, glycated haemoglobin, insulin, leptin, C-peptide, and incretins were measured. The markers of liver injury, serum and hepatic lipids profile, activity of hepatic antioxidant enzyme, and proinflammatory cytokines in adipose tissue were investigated. Additionally, the expression of hepatic lipogenic genes and insulin signaling related genes in adipose tissue were also studied. Liver sections were examined for hepatic steatosis using hematoxylin-eosin staining. RESULTS: The levels of serum glucose, insulin, leptin, C-peptide, glycated hemoglobin, GLP-1, liver injury markers, lipid profile in serum and liver were significantly increased in high-fructose-fed rats. However, after Lr263 administration, the elevation of these parameters was significantly suppressed. Feeding of Lr263 reversed the decreased number of bifidobacterium species and lactobacillus species and increased number of clostridium species induced by high fructose treatment. The decreased activities of hepatic antioxidant enzymes in HFD rats were dramatically reversed by Lr263 treatment. Concentrations of IL-6 and TNF-α in adipose tissue which were elevated in high fructose treatment were markedly decreased after Lr263 feeding. Decreased levels of PPAR-γ and GLUT4 mRNA after high fructose treatment were significantly enhanced by Lr263 administration. Lr263 consumption normalized the increased lipogenic gene (Srebp-1c, FAS, and Elvol6) expressions stimulated by high fructose. Administration of Lr263 significantly ameliorated hepatic steatosis observed in high fructose treated rats. CONCLUSION: Our study provided evidences clarifying the effectiveness of Lr263 on reducing insulin resistance as well as hepatic steatosis formation in high-fructose-fed rats and suggested that Lr263 may be a promising therapeutic agent in treating type 2 diabetes.

Irradiance, but not fluence, plays a crucial role in UVB-induced immature pigment cell development: new insights for efficient UVB phototherapy.

Light exposure modulates development of living organisms. In the field of medicine, light has frequently been used for regenerative purposes. Excimer light (308 nm) has demonstrated superior efficacy in treating vitiligo, a condition requiring development of melanoblasts and a model for studying nerve cell regeneration, as compared to narrow-band ultraviolet B (NBUVB; 311 nm). Using mouse-derived melanoblast cells to examine the pro-differentiation effects of these two light sources, we demonstrated that at equivalent fluence, excimer light induces melanoblast differentiation, while NBUVB failed to so. Mechanistically, activation of aryl hydrocarbon receptor pathway and nuclear translocation of epidermal growth factor receptor are involved in pro-differentiation effects of excimer light. Reduction in irradiance by filter abrogated the effects of excimer light in melanoblasts, even when equivalent fluence was delivered by the same light source. As ultraviolet B (UVB) irradiation is closely associated pigment cell development, future therapy employing UVB for pigmentation purposes should incorporate irradiance as a crucial specification.

High-glucose environment enhanced oxidative stress and increased interleukin-8 secretion from keratinocytes: new insights into impaired diabetic wound healing.

Impaired wound healing frequently occurs in patients with diabetes. Interleukin (IL)-8 production by keratinocyte is responsible for recruiting neutrophils during healing. Intense inflammation is associated with diabetic wounds, while reduction of neutrophil infiltration is associated with enhanced healing. We hypothesized that increased neutrophil recruitment by keratinocytes may contribute to the delayed healing of diabetic wounds. Using cultured human keratinocytes and a diabetic rat model, the current study shows that a high-glucose environment enhanced IL-8 production via epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase (ERK) pathway in a reactive oxygen species (ROS)-dependent manner in keratinocytes. In addition, diabetic rat skin showed enhanced EGFR, ERK, and IL-8 expression compared with control rats. The dermal neutrophil infiltration of the wound, as represented by expression of myeloperoxidase level, was also significantly higher in diabetic rats. Treating diabetic rats with dapsone, an agent known to inhibit neutrophil function, was associated with improved healing. In conclusion, IL-8 production and neutrophil infiltration are increased in a high-glucose environment due to elevated ROS level and contributed to impaired wound healing in diabetic skin. Targeting these dysfunctions may present novel therapeutic approaches.