Positive metabolic effects of selected probiotic bacteria on diet-induced obesity in mice are associated with improvement of dysbiotic gut microbiota.

Given the rising evidence that gut malfunction including changes in the gut microbiota composition, plays a major role in the development of obesity and associated metabolic diseases, the exploring of novel probiotic bacteria with potential health benefits has attracted great attention. Recently Lactobacillus spp., exert potent anti-obesity effects by regulating key transcriptional and translational factors in adipose tissues. However, the molecular mechanism behind the anti-obesity effect of probiotics is not yet fully understood. Therefore, we investigated the effect of Lactobacillus plantarum A29 on the expression of adipogenic and lipogenic genes in 3T3-L1 adipocytes and high-fat diet (HFD)-fed mice. We observed that the treatment of 3T3-L1 adipocytes with the cell-free metabolites of L plantarum inhibited their differentiation and fat depositions via downregulating the key adipogenic transcriptional factors (PPAR-γ, C/EBP-α, and C/EBP-ß) and their downstream targets (FAS, aP2, ACC, and SREBP-1). Interestingly, supplementation with L plantarum reduced the fat mass and serum lipid profile concurrently with downregulation of lipogenic gene expression in the adipocytes, resulting in reductions in the bodyweight of HFD-fed obese mice. L plantarum treatment attenuated the development of obesity in HFD-fed mice via the activation of p38MAPK, p44/42, and AMPK-α by increasing their phosphorylation. Further analysis revealed that A29 modulated gut-associated microbiota composition. Thus, A 29 potential probiotic strain may alleviate the obesity development and its associated metabolic disorders via inhibiting PPARγ through activating the p38MAPK and p44/42 signaling pathways.

Evaluation of probiotic Lactobacillus plantarum against foodborne pathogens and its fermentation potential in improving Lolium multiflorum silage quality.

The objective of this study was to isolate the lactic acid bacteria from fermented silage sample and analyze their antibacterial activities, probiotic properties, and fermentation potential in silage. Eleven lactic acid bacteria (LAB) were selected based on distinct morphologies and preliminary studies. Cell-free supernatant (CFS) was then prepared from the selected strains for antibacterial analysis. L-30 strain and its CFS showed highest inhibition (> 10 mm) against tested foodborne pathogens as compared to other strains. Hereafter, the strain L-30 was named as KCC-30 and used for further studies. KCC-30 can survive in the harsh conditions of GIT such as low pH ( 2) and bile salt environment (oxgal) than standard L. plantarum KACC-91016 (pH 2: 27.2% vs 20.5%; oxgal: 72.3% vs 57.7%, both p < 0.05). In addition, KCC-30 exhibited strong auto-aggregation (68.3% vs 51.5%) and co-aggregation (33% vs 23.9%) properties. For silage experiment, KCC-30 treatment did not alter the nutrient profiles of silage. At the same time, KCC-30 treatment increased the lactic acid content of silage as compared to untreated silage (5.55 DM% vs 3.11 DM%). An increase of lactic acid content in the silage is due to higher lactic acid bacteria population in KCC-30 treated silage (15.33 × 107 CFU/g vs 7.66 × 107 CFU/g) than untreated silage (p < 0.05). Overall data suggested that KCC-30 exhibited strong probiotic potential and improved the quality of Lolium multiflorum silage by increasing the lactic acid level. Therefore, KCC-30 could be considered as potential strain to improve the fermentation quality of L. multiflorum silage.

R-Limonene Enhances Differentiation and 2-Deoxy-D-Glucose Uptake in 3T3-L1 Preadipocytes by Activating the Akt Signaling Pathway.

Adipocyte is an important place for lipid storage. Defects in lipid storage in adipocytes can lead to lipodystrophy and lipid accumulation in muscle, liver, and other organs. It is the condition of mixed dyslipidemia which may favor the development of insulin resistance via lipotoxic mechanisms. Our objective of the study was to investigate the potential role of R-limonene (LM) on differentiation, lipid storage, and 2-deoxy-D-glucose (2DG) uptake in 3T3-L1 preadipocytes. Genes and proteins associated with differentiation, lipid accumulation, 2DG uptake and its signaling pathways in the adipocytes were analyzed using qPCR and western blot methods. LM treatment increased differentiation, lipid accumulation, and the expression of adipogenic and lipogenic markers such as C/EBP-α, C/EBP-ß, PPARγ, SREBP-1, RXR, FAS, and adiponectin. However, the LM concentration at 10µM decreased (p < 0.05) adipogenesis and lipogenesis via regulating key transcriptional factors. LM treatment increased activation of Akt by increasing its phosphorylation, but p44/42 activation was not altered. MK-2206, an Akt specific inhibitor, reduced the activation of Akt phosphorylation whereas LM treatment aborted the MK-2206 mediated inhibition of Akt activation. LM enhanced glucose uptake in differentiated adipocytes. Overall data suggested that LM treatment favored lipid storage and glucose uptake in adipocytes via activation of key transcriptional factors through activation of Akt phosphorylation in 3T3-L1 adipocytes.

Limonene promotes osteoblast differentiation and 2-deoxy-d-glucose uptake through p38MAPK and Akt signaling pathways in C2C12 skeletal muscle cells.

BACKGROUND: Limonene is a cyclic monoterpene (CTL) found in citrus fruits and many plant kingdoms. It has attracted attention as potential molecule due to its diverse biological activities. However, molecular mechanism involved in the osteogenic induction of CTL in C2C12 skeletal muscle cells remain unclear. PURPOSE: Skeletal development maintains the bone homeostasis through bone remodeling process. It coordinated between the osteoblast and osteoblast process. Osteoporosis is one of the most common bone diseases caused by a systemic reduction in bone mass. Recent osteoporosis treatment is based on the use of anti-resorptive and bone forming drugs. However, long term use of these drugs is associated with serious side effects and strategies on the discovery of lead compounds from natural products for osteoblast differentiation are urgently needed. Therefore, we planned to find out the role of CTL on osteoblast differentiation and glucose uptake in C2C12 cells and its effect on signaling pathways. METHODS: Cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, genes, and proteins associated with osteoblast activation and glucose utilization were analysed. RESULTS: CTL did not affect the cell viability. CTL significantly increased ALP activity, calcium depositions and the expression of osteogenic specific genes such as Myogenin, Myogenic differentiation 1 (MyoD), ALP, Run-related transcription factor 2(RUNX2), osteocalcin (OCN). In addition, CTL induced the mRNA expression of bone morphogenetic proteins (BMP-2 BMP-4 BMP-6 BMP-7 BMP-9). CTL treatment enhanced 2-Deoxy-d-glucose (2DG) uptake. Moreover, CTL stimulated the activation of p38 mitogen activated protein kinase (p38MAPK), Protein kinase B (Akt), Extracellular signal related kinase (ERKs) by increasing phosphorylation. CTL treatment abolished p38 inhibitor (SB203580) mediated inhibition of osteoblast differentiation, but no effect was noted by ERKs specific inhibitor (PD98059). CONCLUSION: These results suggest that limonene induces osteoblast differentiation and glucose uptake through activating p38MAPK and Akt signaling pathways, confirming the molecular basis of the osteoblast differentiation by limonene in C2C12 skeletal muscle cells.

Application of customised bacterial inoculants for grass haylage production and its effectiveness on nutrient composition and fermentation quality of haylage.

The present study aimed to investigate the efficacy of customised Lactobacillus plantarum KCC-10, KCC-19 and K-46 on nutrient composition and fermentation quality of low moisture Italian ryegrass (IRG) forage. An addition of customised bacterial inoculants (CBI) did not affect the nutrient compositions and digestibility rates of haylage. The lactic acid content was higher in CBI-inoculated haylage, whereas the amount of acetic acid and butyric acid production was significantly reduced than the control. CBI-inoculated haylage exhibited higher numbers of bacterial colonies that reduced the pH of the haylage. Low pH in haylage is an important criterion for preventing undesirable microbial growth and improves fermentation quality of haylage. PCR studies indicated that the DNA of L. plantarum was predominantly amplified. It evidenced that the CBI is the main reason behind the improvement of haylage fermentation as compared to control. Overall results suggested that KCC-10, KCC-19 and K-46 are considered as potent strains for improving fermentation quality of low moisture forage and preserve its stability for a long time.

Antitumor Effect of the Mannich Base(1,3-bis-((3-Hydroxynaphthalen-2-yl)phenylmethyl)urea) on Hepatocellular Carcinoma.

The present study was designed to evaluate the antitumor effects of the synthetic Mannich base 1,3-bis-((3-hydroxynaphthalen-2-yl)phenylmethyl)urea (1,3-BPMU) against HEP-G2 hepatoma cells and diethylnitrosamine (DEN)-induced hepatocarcinoma (HCC) in albino rats. In vitro analysis results revealed that 1,3-BPMU showed significant cytotoxicity and cell growth inhibition in HEP-G2 hepatoma cells in a concentration-dependent manner. Furthermore, flow cytometry results indicated that 1,3-BPMU enhanced early and late apoptosis. The maximum apoptosis was exhibited at a concentration of 100 µg/mL of 1,3-BPMU. In in vivo analysis, DEN treatment increased the content of nucleic acids, LPO and the activities of AST, ALT, ALP, LDH, γGT and 5'NT with decreased antioxidant activity as compared to control rats. However, 1,3-BPMU treatment to DEN-induced rats decreased the content of nucleic acids, LPO and the activities of AST, ALT, ALP, LDH, γGT and 5'NT and increased the activities of SOD, CAT, GPx, GST and GR (p < 0.05). Furthermore, 1,3-BPMU enhanced the apoptosis via upregulation of caspase-3 and caspase-9 and the downregulation of Bcl-2 and Bcl-XL mRNA expression as compared to DEN-induced rats. Histological and ultrastructural investigation showed that 1,3-BPMU treatment renovated the internal architecture of the liver in DEN-induced rats. In this study, the molecular and pre-clinical results obtained by treatment of DEN-induced rats with 1,3-BPMU suggested that 1,3-BPMU might be considered as an antitumor compound in the future.

Trigonelline protects the cardiocyte from hydrogen peroxide induced apoptosis in H9c2 cells

Objective: To elucidate the key parameters associated with hydrogen peroxide induced oxidative stress and investigates the mechanism of trigonelline (TG) for reducing the H2O2 induced toxicity in H9c2 cells. Methods: Cytotoxicity and antioxidant activity of TG was assessed by EZ-CYTOX kit. RNA extraction and cDNA synthesized according to the kit manufacture protocol. Apoptosis was measured by the Flowcytometry, general PCR and qPCR. Results: It was found that the TG significantly rescued the morphology of the H9c2 cells. Treatment of cells with TG attenuated H2O2 induced cell deaths and improved the antioxidant activity. In addition, TG regulated the apoptotic gene caspase-3, caspase-9 and anti-apoptotic gene Bcl-2, Bcl-XL during H2O2 induced oxidative stress in H9c2 cells. These results were comparable with quercetin treatment. For evident, flow cytometer results also confirmed the TG significantly reduced the H2O2 induced necrosis and apoptosis in H9c2 cells. However, further increment of TG concentration against H2O2 could induce the necrosis and apoptosis along with H2O2. Conclusions: It is suggested that less than 125 μ M of TG could protect the cells from H2O2 induced cell damage by down regulating the caspases and up regulating the Bcl-2 and Bcl-XL expression. Therefore, we suggest the trigonelline could be useful for treatment of oxidative stress mediated cardiovascular diseases in future.

Trigonelline protects the cardiocyte from hydrogen peroxide induced apoptosis in H9c2 cells

OBJECTIVE@#To elucidate the key parameters associated with hydrogen peroxide induced oxidative stress and investigates the mechanism of trigonelline (TG) for reducing the H2O2 induced toxicity in H9c2 cells.@*METHODS@#Cytotoxicity and antioxidant activity of TG was assessed by EZ-CYTOX kit. RNA extraction and cDNA synthesized according to the kit manufacture protocol. Apoptosis was measured by the Flowcytometry, general PCR and qPCR.@*RESULTS@#It was found that the TG significantly rescued the morphology of the H9c2 cells. Treatment of cells with TG attenuated H2O2 induced cell deaths and improved the antioxidant activity. In addition, TG regulated the apoptotic gene caspase-3, caspase-9 and anti-apoptotic gene Bcl-2, Bcl-XL during H2O2 induced oxidative stress in H9c2 cells. These results were comparable with quercetin treatment. For evident, flow cytometer results also confirmed the TG significantly reduced the H2O2 induced necrosis and apoptosis in H9c2 cells. However, further increment of TG concentration against H2O2 could induce the necrosis and apoptosis along with H2O2.@*CONCLUSIONS@#It is suggested that less than 125 μ M of TG could protect the cells from H2O2 induced cell damage by down regulating the caspases and up regulating the Bcl-2 and Bcl-XL expression. Therefore, we suggest the trigonelline could be useful for treatment of oxidative stress mediated cardiovascular diseases in future.