Lactobacillus paracasei subsp. paracasei NTU 101 prevents obesity by regulating AMPK pathways and gut microbiota in obese rat.

This study assessed the anti-obesity effects of Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) both in vitro and in vivo. Initially, the cytotoxicity and lipid accumulation inhibitory effects of NTU 101 on 3T3-L1 cells were evaluated using the MTT assay and oil red O assay, respectively. Subsequently, the anti-obesity effects of NTU 101 were investigated in high-fat diet-induced obese rat. Moreover, western blotting was performed to measure the obesity-related protein expression of PPARα, PPARß, PPARγ, C/EBPα, C/EBPß, ATGL, p-p38 MAPK, p-ERK1/2, p-AMPK and CPT-1 in both 3T3-L1 adipocytes and adipose and liver tissues. Treatment with 16 × 108 CFU/mL NTU 101 reduced lipid accumulation in 3T3-L1 adipocytes by more than 50 %. Oral administration of NTU 101 significantly attenuated body weight gain, as well as adipose tissue weight. NTU 101 administration enhanced fatty acid oxidation increasing expression levels of PPARα, CPT-1, and p-AMPK proteins in liver tissue, while simultaneously inhibited adipogenesis by reducing PPARγ and C/EBPα proteins in adipose tissue. Furthermore, NTU 101 supplementation positively modulated the composition of gut microbiota, notably increasing the abundance of Akkermansiaceae. This present study suggests that NTU 101 exerts anti-obesity effects by regulating gut microbiota, fatty acid oxidation, lipolysis and adipogenesis.

Effects of Perilla frutescens Var. Acuta in Busulfan-Induced Spermatogenesis Dysfunction Mouse Model.

PURPOSE: The leaves of Perilla frutescens var. acuta (PFA) are generally reported to have antioxidant, anti-allergic, anti-inflammatory, and antitumor effects and commonly used as a traditional medicine in East Asia. This study aimed to investigate the protective effect and antioxidant activity of PFA on busulfan-induced testicular dysfunction, histological damage, oxidative stress (OS), sperm quality, and hormone levels using a mouse model. MATERIALS AND METHODS: C57BL/6 male mice were divided into four groups: control, busulfan-only treated, and varying concentrations of PFA (100 and 200 mg/kg) with busulfan. In the busulfan group, 40 mg/kg of busulfan was intraperitoneally injected to induce azoospermia. Mice were orally administered PFA for 35 consecutive days after busulfan administration. Samples were collected and assessed for testis/body weight, testicular histopathology, sperm quality, serum hormone levels, and OS to evaluate the effects of PFA treatment on spermatogenesis dysfunction induced by busulfan. RESULTS: The busulfan-induced testicular dysfunction model showed reduced testis weight, adverse histological changes, significantly decreased sex hormones and sperm quality, and attenuated OS. These results indicate that PFA treatment significantly increased testis weight, testis/body weight, epididymal sperm count, motility, and testosterone level compared with busulfan alone. PFA treatment also attenuated the busulfan-induced histological changes. Furthermore, compared with mice treated with busulfan alone, PFA supplementation upregulated the testicular mRNA expression of the antioxidant enzymes superoxide dismutase 1 (Sod1) and glutathione peroxidase 1 (Gpx1), with a decrease in malondialdehyde (MDA) production and an increase in SOD and GPx activities. CONCLUSIONS: This study shows that PFA exerts a protective effect against testicular damage by attenuating OS induced by busulfan. Our results suggest that PFA is a potentially relevant drug used to decrease the side effects induced by busulfan on testicular function and sperm during cancer chemotherapy.

Enzyme-treated caviar extract ameliorates melanogenesis in UVB-induced SKH-1 hairless mice.

This study investigated anti-melanogenesis effects of enzyme-treated caviar extract (CV) in murine melanoma B16F10 cells and SKH-1 hairless mice. To induce melanin production in vitro and in vivo studies, B16F10 cells were treated with 3-Isobutyl-1-methylxanthine (IBMX), and SKH-1 hairless mice were irradiated with UVB, respectively. The expression of melnogenesis-related factors and signaling molecules were analyzed by ELISA and western blotting. 50, 100 and 200 µg/mL of CV significantly decreased the melanin contents and the activities of tyrosinase, nitric oxide, glutathione, and cAMP, melanogenesis factor, in B16F10 cells treated IBMX. In addition, CV significantly suppressed the expression of melanogenesis proteins such as pPKA, pCREB, MITF, TRP-1and TRP-2. Similarly, results of oral administration of CV (20, 50 and 100 mg/kg) for 8 weeks in UVB-Induced SKH-1 hairless mice, the expression of melanogenesis-related factor tyrosinase, nitric oxide, and cAMP and protein expression of pPKA, pCREBa, MITF, TRP-1and TRP-2 was significantly reduced. In particular, 100 mg/kg of CV exhibited an excellent effect similar to control group. Therefore, we suggest the possibility of developing CV as a food supplement having skin whitening effects by ameliorating melanogenesis.

Immune-Enhancing Effects of Limosilactobacillus fermentum in BALB/c Mice Immunosuppressed by Cyclophosphamide.

This study evaluates the immune-enhancing effects of Limosilactobacillus fermentum on cyclophosphamide (CP)-induced immunosuppression in BALB/c mice. In vitro, the expressions of pro-inflammatory cytokines and MAPK signaling molecules in Raw264.7 cells were analyzed by ELISA and Western blot analysis. Moreover, cell proliferation, surface receptor expression, and cytotoxicity of NK-92 cells were examined by Cell Counting Kit-8, CytoTox96 assay, and flow cytometry, respectively. To investigate the immune-enhancing effects of selected L. fermentum strains in vivo, these strains were orally administered to BALB/c mice for 2 weeks, and CP was intraperitoneally injected. Then, liver, spleen, and whole blood were isolated from each animal. Administration of single L. fermentum strains or their mixture sustained the spleen weight, the counts of white blood cells compared to non-fed group. Splenocyte proliferation and NK cytotoxicity were significantly increased in all L. fermentum-fed groups. The frequency of B220+ cells was also significantly enhanced in splenocytes isolated from L. fermentum groups. In addition, the production of cytokines (TNF-α, IFN-γ) and antibodies was recovered in splenocyte supernatants isolated from L. fermentum groups. In conclusion, L. fermentum could be a suitable functional food additive for immune-enhancing effect.

Skin Anti-Aging Efficacy of Enzyme-Treated Supercritical Caviar Extract: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

Oxidative stress in the skin, induced by an unhealthy lifestyle and exposure to UVB radiation, leads to skin aging, including reduced elasticity, formation of wrinkles, moisture loss, and inflammation. In a previous study, we revealed the photoaging effects of enzyme-treated caviar extract (CV) by regulating collagen and hyaluronic acid synthase, melanogenesis, anti-oxidant mechanisms, and inflammation in a UVB irradiation-induced mice model. HPLC and MALDI-TOF were performed to determine the effect of enzyme treatment on the free amino acid contents and peptide molecular weight in supercritical caviar extract. As results of the analysis, CV is mainly composed of low-molecular-weight peptides consisting of leucine, tyrosine, and phenylalanine. Based on our in vitro and in vivo study, we conducted a clinical trial to assess the skin anti-aging efficacy of CV. In this randomized, double-blind, placebo-controlled trial, we measured indicators related to elasticity, wrinkles, and skin hydration at 4 and 8 weeks after consumption of CV. The subjects were categorized into caviar, combination, and placebo groups. After 4 weeks, skin hydration, dermal hydration, and transepidermal water loss all showed significant improvement. Furthermore, after 8 weeks, skin elasticity indexes-R2 (total elasticity), R5 (net elasticity), and R7 (ratio of elastic recovery to total deformation)-exhibited significant increases. Improvement in wrinkle indicators (Rmax, Ra, and Rz) and the whitening indicator melanin pigment was also observed. This is the first report showing that CV has significant skin anti-aging efficacy on human skin. In conclusion, our study suggests that CV can be used as skin anti-aging nutraceuticals through positive effects on skin condition in clinical trials.

Anti-Tumor Effect of Heat-Killed Bifidobacterium bifidum on Human Gastric Cancer through Akt-p53-Dependent Mitochondrial Apoptosis in Xenograft Models.

Paraprobiotics, inactivated microbial cells, regulate immune system and exhibit antioxidant and anti-inflammatory activities in patients with weakened immunity or the elderly. This study evaluated the anti-tumor effects of heat-killed Bifidobacterium and Lactobacillus on human gastric cancer MKN1 cells in vitro and in vivo in xenograft animal models. First, cytotoxicity and apoptosis in MKN1 cells of 11 different heat-killed Bifidobacterium or Lactobacillus strains were examined using the MTT assay or flow cytometry, respectively. Then, BALB/c nude mice xenograft animal models were implanted with human gastric cancer MKN1 cells and orally administered a selected single or a mixture of heat-killed bacterial strains to investigate their inhibitory effect on tumor growth. In addition, the expression of p-Akt, p53, Bax, Bak, cleaved caspase-9, -3, and PARP in the tumor tissues was analyzed using Western blotting assay or immunohistochemistry staining. The results show that heat-killed B. bifidum MG731 (MG731), L. reuteri MG5346 (MG5346), and L. rhamnosus MG5200 (MG5200) induced relatively greater apoptosis than other strains in MKN1 cells. Oral administration of a single dose or a mixture of MG731, MG5346, or MG5200 significantly delayed tumor growth, and MG731 had the most effective anti-tumor effect in the xenograft model. Protein expression of p-Akt, p53, Bax, cleaved caspase-3 and -9, and PARP in tumors derived from the xenograft model correlated with the results of the immunohistochemistry staining.

Anti-Tumor Effects of Heat-Killed L. reuteri MG5346 and L. casei MG4584 against Human Colorectal Carcinoma through Caspase-9-Dependent Apoptosis in Xenograft Model.

In this study, we examined the anti-tumor effects of heat-killed Bifidobacterium and Lactobacillus strains on human colorectal carcinoma RKO cells in in vitro and in vivo xenograft models. First, the cytotoxic and apoptotic effects of 11 different strains were examined using an MTT assay and flow cytometry, respectively. Then, xenograft BALB/c nude mice were implanted with RKO cells and orally administered with single or mixed heat-killed bacterial strains to examine their inhibitory effects on tumor growth. Additionally, the levels of cleaved caspase-9, -3, and -7 and PARP in tumor tissues were analyzed using Western blotting or immunohistochemistry staining. The results showed that RKO cells were highly susceptible to heat-killed B. bifidum MG731 and L. reuteri MG5346 and that L. casei MG4584 induced apoptosis to a greater extent than other strains. The oral administration of individual MG731, MG5346, or MG4584 significantly delayed tumor growth, and mixtures of MG5346 and MG4584 or MG731, MG5346, and MG4584 synergistically inhibited the tumor growth in the xenograft model. The expression of cleaved caspase-3, -7, and -9 and PARP in the tumor tissues was increased in Western blotting, and the expression of cleaved caspase-3 and PARP in immunohistochemistry staining was also increased. Therefore, we suggest that the use of the combination of MG5346 and MG4584 as parabiotics could effectively inhibit the growth of colorectal cancer.

Weissella cibaria MG5285 and Lactobacillus reuteri MG5149 attenuated fat accumulation in adipose and hepatic steatosis in high-fat diet-induced C57BL/6J obese mice.

BACKGROUND: Excessive consumption of dietary fat is closely related to obesity, diabetes, insulin resistance, cardiovascular disease, hypertension, and non-alcoholic fatty liver disease. Recently, probiotics have been highly proposed as biotherapeutic to treat and prevent diseases. Previously, there are studies that demonstrated the beneficial effects of probiotics against metabolic disorders, including obesity and diabetes. OBJECTIVE: We investigated the anti-obesity effect and mechanism of action of four human-derived lactic acid bacterial (LAB) strains (Lacticaseibacillus rhamnosus MG4502, Lactobacillus gasseri MG4524, Limosilactobacillus reuteri MG5149, and Weissella cibaria MG5285) in high-fat diet (HFD)-induced obese mice. DESIGN: Obesity was induced in mice over 8 weeks, with a 60% HFD. The four human-derived LAB strains (2 × 108 CFU/mouse) were orally administered to male C57BL/6J mice once daily for 8 weeks. Body weight, liver and adipose tissue (AT) weights, glucose tolerance, and serum biochemistry profiles were determined. After collecting the tissues, histopathological and Western blot analyses were conducted. RESULTS: Administration of these LAB strains resulted in decreased body weight, liver and AT weights, and glucose tolerance. Serum biochemistry profiles, including triglyceride (TG), total cholesterol, low-density lipoprotein cholesterol, and leptin, pro-inflammatory cytokines, improved. Hepatic steatosis and TG levels in liver tissue were significantly reduced. In addition, the size of adipocytes in epididymal tissue was significantly reduced. In epididymal tissues, Limosilactobacillus reuteri MG5149 and Weissella cibaria MG5285 groups showed a significantly reduced expression of lipogenic proteins, including peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α, fatty acid synthase (FAS), and adipocyte-protein 2. In addition, sterol regulatory element-binding protein 1-c and its downstream protein FAS in the liver tissue were significantly decreased. These strains attenuated fat accumulation in the liver and AT by upregulating the phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase in HFD-fed mice. CONCLUSION: We suggest that L. reuteri MG5149 and W. cibaria MG5285 could be used as potential probiotic candidates to prevent obesity.

Anti-adipogenic effect of Lactobacillus fermentum MG4231 and MG4244 through AMPK pathway in 3T3-L1 preadipocytes.

This study evaluated the anti-adipogenic effects and mechanisms underlying the action of Lactobacillus fermentum MG4231 and MG4244 strains on adipogenesis and lipid accumulation in 3T3-L1 preadipocytes. Treatment with cell-free extracts (CFEs) from the two strains reduced lipid accumulation and intracellular triglyceride production in 3T3-L1 adipocytes by more than 50%. The inhibitory effects of L. fermentum on lipid accumulation were mediated by the downregulation of FAS and aP2 resulting from the inhibition of PPARγ and C/EBPα gene expression. Moreover, AMPK and HSL phosphorylation was upregulated by CFE treatment. These results indicated that the anti-adipogenic and lipolysis activities of L. fermentum strains were caused by increased AMPK and HSL phosphorylation. Both strains displayed high leucine arylamidase and ß-galactosidase enzymatic activity, with excellent adhesion to epithelial cells. Therefore, we identified L. fermentum as potential new probiotics for the prevention of obesity.

Morphologic Changes in Patient with Drusen and Drusenoid Pigment Epithelial Detachment after Intravitreal Ranibizumab for Choroidal Neovascular Membrane : A Case Report.

The authors present a case of morphologic changes of drusen and drusenoid pigment epithelial detachment (DPED) after treating choroidal neovascularization (CNV) using ranibizumab in age-related macular degeneration (AMD). A 71-year-old woman has noticed mild visual acuity deterioration in the right eye for several months. She was presented with some drusen and DPED associated with CNV. This patient was given intravitreal injection of 0.5 mg of ranibizumab five times at monthly intervals for treating CNV. DPED in the temporal and drusen in the superior to macula were diminished, which continued up to 2 months. Intravitreal ranibizumab injection may have influenced with diminishment of drusen and DPED. After 2 months, CNV was recurred.

Nasal-type NK/T-cell lymphomas are more frequently T rather than NK lineage based on T-cell receptor gene, RNA, and protein studies: lineage does not predict clinical behavior.

Extranodal natural killer (NK)/T-cell lymphoma (ENKTL), nasal type, comprises NK or cytotoxic T cells. We evaluated the clinical impact of cell type and the usefulness of T-cell receptor (TCR) gene transcripts in distinguishing cell lineage. One hundred and eight cases of ENKTL were analyzed for TCR gene rearrangements using the BIOMED-2 protocol and for TCR gene expression using immunohistochemistry for TCR-ßF1 and TCR-cγM1, and RNA in situ hybridization for TCR gene transcripts. Prognostic factors were analyzed. Among the 108 cases, 44 were monoclonal for a TCR rearrangement (40%) while 64 (60%) were undefinable. The monoclonal cases expressed TCR-ßF1 in 14 out of 40 cases (35%) and TCR-cγM1 in 1 out of 44 cases (2%). The 64 undetermined cases expressed TCR-ßF1 in 15 cases (23%) and TCR-cγM1 in 1 (2%). Thirteen of 40 TCR-ß constant gene transcript-positive cases (33%) expressed TCR-ßF1 and one of nine TCR-γ constant gene transcript-positive cases (11%) expressed TCR-cγM1. TCR gene transcripts were not useful in the distinction of cell lineages. TCR gene transcripts were positive in ENKTLs as well as in normal B cells and aggressive NK-cell leukemia. Based on gene rearrangements and immunohistochemistry for TCR, there were 60 T-cell type cases (56%), 32 NK-cell type cases (30%), and 16 cases with an undetermined cell type (14%). TCR protein was expressed in 30/60 T-ENKTLs (50%) in a variable fraction of tumor cells. There were no significant differences in clinical findings or overall patient survival between T- or NK-cell types of ENKTL, although those with a T-cell type tended to show a better prognosis for those with localized nasal lymphomas. Univariate and multivariate analysis showed that a non-nasal ENKTL, age >60 years, high level of lactate dehydrogenase, bone marrow involvement, and the absence of radiotherapy were independent prognostic factors.

Effects of Bevacizumab on Bcl-2 Expression and Apoptosis in Retinal Pigment Epithelial Cells under Oxidative Stress.

PURPOSE: To evaluate the effects of bevacizumab on expression of B-cell leukemia/lymphoma (Bcl)-2 and apoptosis in retinal pigment epithelial (RPE) cells under oxidative stress conditions. METHODS: RPE cells were treated with H(2)O(2) (0, 100, 200, 300, and 400 µM) and bevacizumab at or above the doses normally used in clinical practice (0, 0.33, 0.67, 1.33, and 2.67 mg/mL). Cell apoptosis was measured using flow cytometry with annexin V-fluorescein isothiocyanate. The expression of Bcl-2 mRNA was determined using reverse transcription polymerase chain reaction. RESULTS: Under low oxidative stress conditions (H(2)O(2) 100 µM), cell apoptosis was not significantly different at any concentration of bevacizumab, but Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL). Under moderate oxidative stress conditions (H(2)O(2) 200 µM), Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL), but cell apoptosis increased only at 2.67 mg/mL of bevacizumab. Under high oxidative stress (300 µM) conditions, cell apoptosis increased at high concentrations of bevacizumab (1.33 and 2.67 mg/mL), but it did not correlate with Bcl-2 expression. CONCLUSIONS: Withdrawal of vascular endothelial growth factor can lead to RPE cell apoptosis and influences the expression of anti-apoptotic genes such as Bcl-2 under oxidative stress conditions. Since oxidative stress levels of each patient are unknown, repeated injections of intravitreal bevacizumab, as in eyes with age-related macular degeneration, might influence RPE cell survival.

Insufficient bilateral femoral subtrochanteric fractures in a patient receiving imatinib mesylate.

We present a case of insufficient bilateral femoral subtrochanteric fractures in a patient who was treated with imatinib mesylate, an anticancer drug, for 1 year after a diagnosis of chronic myelogenous leukemia (CML). A 60-year-old woman presented with bilateral thigh pain for 6 months. A plain radiograph revealed bilateral progressive insufficient fractures on the subtrochanteric areas of the femurs. MRI of the femurs revealed incomplete stress fractures and no evidence of bone metastasis on either femur. Bone densitometry showed normal T-scores around the hip joint and spine. The patient had normal serum levels of calcium, vitamin D derivatives, and thyroid hormones. Serum phosphate levels were decreased, and parathyroid hormone levels were increased. Serum osteocalcin and urinary N-telopeptide of collagen cross-links (NTx) were both decreased. A bone biopsy demonstrated normocellular marrow without leukemic cells. A histomorphometric evaluation of her bones revealed reduced bone turnover despite secondary hyperparathyroidism. The serum markers for bone metabolism and histomorphometric evaluations in this patient suggest that the drug may have an effect on bone metabolism. These effects could be seen for both bone formation and resorption: this could result in impaired bone mineralization, a severely suppressed bone turnover rate, insufficient fractures, and bone necrosis, which are sometimes seen with long-term use of bisphosphonates. To our knowledge, this is the first case of an insufficient bilateral femoral shaft fracture that is potentially related to the use of imatinib mesylate in a patient with CML. Careful examination of bone metabolism should be performed in patients with CML because imatinib mesylate treatment is a lifelong process.