Suppression of bone resorption by Mori Radicis Cortex through NFATc1 and c-Fos signaling-mediated inhibition of osteoclast differentiation.

BACKGROUND: Mori Radicis Cortex (MRC) is the root bark of the mulberry family as Morus alba L. In Korea, it is known as "Sangbaegpi". Although MRC has demonstrated anti-inflammatory and antioxidant effects, its specific mechanisms of action and impact on osteoporosis remain poorly understood. METHODS: To investigate the antiosteoporosis effect of MRC, we examined the level of osteoclast differentiation inhibition in receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced-RAW 264.7 cells and animal models of ovariectomy (OVX) with MRC. Serum analysis in OVX animals was investigated by enzyme-linked immunosorbent assay (ELISA), and bone density analysis was confirmed by micro-computed tomography (micro-CT). The expression analysis of nuclear factor of activated T cells 1 (NFATc1) was confirmed by immunohistochemistry (IHC) in femur tissue. In addition, osteoclast differentiation inhibition was measured using tartrate-resistant acid phosphatase (TRAP). mRNA analysis was performed using reverse transcription-polymerase chain reaction (RT-PCR), and the protein expression analysis was investigated by western blot. RESULTS: Micro-CT analysis showed that MRC effectively inhibited bone loss in the OVX-induced rat model. MRC also inhibited the expression of alkaline phosphatase (ALP) and TRAP in serum. Histological analysis showed that MRC treatment increased bone density and IHC analysis showed that MRC significantly inhibited the expression of NFATc1. In RANKL-induced-RAW 264.7 cells, MRC significantly reduced TRAP activity and actin ring formation. In addition, MRC significantly inhibited the expression of NFATc1 and c-Fos, and suppressed the mRNA expression. CONCLUSION: Based on micro-CT, serum and histological analysis, MRC effectively inhibited bone loss in an OVX-induced rat model. In addition, MRC treatment suppressed the expression of osteoclast differentiation, fusion, and bone resorption markers through inhibition of NFATc1/c-Fos expression in RANKL-induced RAW 264.7 cells, ultimately resulting in a decrease in osteoclast activity. These results demonstrate that MRC is effective in preventing bone loss through inhibiting osteoclast differentiation and activity.

Antiobesity Effects of Lactobacillus paracasei Subsp. paracasei, L. casei 431 on High-Fat Diet-Induced Obese Rats.

Obesity is currently regarded as a global concern, and the key objectives of the global health strategy include its prevention and control. Probiotic supplementation can help achieve these objectives. This study aimed to assess whether a probiotic strain Lactobacillus paracasei ssp. paracasei, Lactobacillus casei 431 (henceforth, L. casei 431) possesses antiobesogenic properties. High-fat diet-induced obese Sprague-Dawley rats were treated with L. casei 431 for 10 weeks, and the outcomes were compared with those of rats treated with the antiobesity medication orlistat. Body weights, epididymal fat, and tissues from mice were assessed. Furthermore, serological and histological analyses were performed. Epididymal fat accumulation was significantly reduced in groups administered L. casei 431 and orlistat. Furthermore, L. casei 431 and orlistat treatments lowered serum alanine transaminase, aspartate aminotransferase, and triglyceride (TG) levels. Hematoxylin and eosin staining of the liver and epididymal adipose tissues showed that the L. casei 431-treated groups exhibited reduced lipid buildup and adipocyte size. Furthermore, sterol regulatory element-binding protein 1c, adipose TG lipase, and lipoprotein lipase messenger RNA (mRNA) levels were upregulated, leading to lipid oxidation and degradation, in L. casei 431-supplemented groups. Furthermore, carnitine palmitoyltransferase 1, a major factor in lipolysis, was consistently upregulated at the protein level after L. casei 431 administration. Collectively, these results demonstrate the potential of L. casei 431 in alleviating obesity in rats through optimizing lipid metabolism and some related biomarkers.

Effects of Pear Extracts on Microbiome and Immunocytokines to Alleviate Air Pollution-Related Respiratory Hypersensitivity.

Pears are ancient functional foods for modern times. Particularly, Korean pears (Pyrus pyrifolia cv.) have been used as folk medicine for respiratory diseases and have strong potential for the treatment of hazardous aerosol-related diseases. Thus, the effects of pear ethanol extracts on air pollution-related respiratory hypersensitivity were studied by toxicokinetics, pro-inflammatory cytokines, and microbiomics in preclinical and randomized double-blind clinical studies. The mild-asthma subjects, who lived in the same city, Seoul, Korea, were separated into the placebo and the treatment (pear extracts, as brix 55; arbutin 5.01 mg and chlorogenic acid 0.18 mg/3 mL per day) groups for 4 weeks (n = 20). As results, there were positive associations between urinary 2-naphthol (NT) or 1-hydroxypyrene (OHP), exposure biomarkers for polyaromatic hydrocarbons in PM2.5, and pro-inflammatory cytokines, interleukin (IL)-4 or IgE, respectively, in the human subjects. The pear extracts somewhat reduced 2-NT and 1-OHP levels. The proportions of fiber-degrading bacteria that stimulate growth of beneficial microflora for immune defense, that is, Bifidobacterium and Eubacterium, were significantly higher in the pear consuming group than in the placebo group. Moreover, pro-inflammatory cytokines, including IgE, IL-4, IL-5, and IL-13, were significantly suppressed by the pear extracts in the preclinical tests of the ovalbumin-induced asthma mice. Thus, we suggest that air pollution-related respiratory hypersensitivity can be alleviated by Korean pear extracts by modulation of microbiome and immunocytokines.

Postbiotics Enhance NK Cell Activation in Stress-Induced Mice through Gut Microbiome Regulation.

Recent studies have revealed that probiotics and their metabolites are present under various conditions; however, the role of probiotic metabolites (i.e., postbiotics in pathological states) is controversial. Natural killer (NK) cells play a key role in innate and adaptive immunity. In this study, we examined NK cell activation influenced by a postbiotics mixture in response to gut microbiome modulation in stress-induced mice. In vivo activation of NK cells increased in the postbiotics mixture treatment group in accordance with Th1/Th2 expression level. Meanwhile, the Red Ginseng treatment group, a reference group, showed very little expression of NK cell activation. Moreover, the postbiotics mixture treatment group in particular changed the gut microbiome composition. Although the exact role of the postbiotics mixture in regulating the immune system of stress-induced mice remains unclear, the postbiotics mixture-induced NK cell activation might have affected gut microbiome modulation.

Caesalpinia sappan induces apoptotic cell death in ectopic endometrial 12Z cells through suppressing pyruvate dehydrogenase kinase 1 expression.

Endometriosis is a common gynecological disease defined as the growth of endometrial tissues outside the uterus. Although the mechanism underlying the progression of endometriosis has not been fully elucidated, cancer-like aerobic glycolysis is considered to mediate the elevated growth and resistance to apoptosis of endometriotic cells. The heartwood of Caesalpinia sappan L. (family Leguminosae) is a herbal medicinal product used to treat gynecological symptoms, including algomenorrhea and amenorrhea. The results of the present study revealed that endometriotic 12Z cells exhibited more rapid growth than normal endometrial cells (THES). The expression levels of pyruvate dehydrogenase kinase (PDK)1 and 3 and lactate production were higher in 12Z cells than in THES cells. In addition, the 12Z cells were more sensitive to the cytotoxicity of the aqueous extract of C. sappan heartwood (CS) than the THES cells. CS inhibited lactate production and phosphorylation of pyruvate dehydrogenase A by reducing the expression of PDK1. CS also increased mitochondrial reactive oxygen species (ROS) levels, decreased mitochondrial membrane potential and consequently stimulated the apoptosis of 12Z cells. CS-induced cell death was substantially inhibited by exogenous PDK1 expression. In conclusion, CS may be a novel drug candidate for treating endometriosis by inhibiting aerobic glycolysis and inducing ROS-mitochondria-mediated apoptotic cell death.

Solanum nigrum Linne improves DNCB­induced atopic dermatitis­like skin disease in BALB/c mice.

The present study aimed to investigate the effects of Solanum nigrum Linne (SNL) in a model of 1­chloro­2,4­dinitrobenzene (DNCB)­induced atopic dermatitis (AD) and in TNF­α/IFN­Î³­stimulated HaCaT cells. AD is a chronic inflammatory skin disease and is characterized by erythema, edema, increased pruritus and eczema. Steroids are most commonly used for anti­inflammatory therapy; however, their long­term use is limited due to side­effects, such as osteoporosis, brittle skin, muscle weaknesses and diabetes. Therefore, patients with AD require alternative treatment strategies. In previous studies, SNL has been reported to be effective against oxidants and cancer. However, to the best of our knowledge, the effects of SNL on AD have not yet been investigated. The present study examined the effects of SNL ethanol extract on a model of DNCB induced AD and on TNF­α/IFN­Î³­stimulated HaCaT cells. The skin tissue was sectioned to measure the thicknesses of the epidermis and dermis, as well as the numbers of eosinophils, mast cells and CD8 infiltration by H&E, toluidine blue, Masson's trichrome and IHC staining. ELISA was performed using serum to measure IgE levels. The present study also examined the expression of various inflammatory cytokines, MAPK and NF­κB in TNF­α/IFN­Î³­stimulated HaCaT cells. SNL significantly reduced the levels of cytokines released from HaCaT cells stimulated with TNF­α/IFN­Î³. SNL also significantly reduced the levels of p­p38 at 30 min and significantly reduced the activation of NF­κB in a time course experiment. In addition, SNL significantly reduced the level of serum IgE and dermal thickness and the infiltration of mast cells and CD8 in the BALB/c mouse model of DNCB­induced AD. The results of the current study suggest that SNL exerts a suppressive effect on pro­inflammatory cytokines in vitro and in vivo through the regulation of the immune system.

Novel hepatoprotective peptides derived from protein hydrolysates of mealworm (Tenebrio molitor).

In the present study, we hypothesized that protein hydrolysates of mealworm (Tenebrio molitor) which is known to exert significant scavenging activity toward reactive oxygen species (ROS) might protect liver cells against ROS-induced cytotoxicity. Therefore, hepatoprotective effects of protein hydrolysates of mealworm and their underlying mechanisms were investigated in AML12 mouse liver cells and the responsible peptides were further identified. Pretreatment with the mealworm alcalase hydrolysate (MAH; <1 kDa) showed the highest protective effect against H2O2-induced cytotoxicity in AML12 cells among three mealworm hydrolysates produced by different proteases (alcalase, flavourzyme, and neutrase). Further mechanistic studies demonstrated that MAH reduces ROS levels through increasing NF-E2-related factor 2-mediated expression of catalase, heme oxygenase-1, and genes involved in glutathione synthesis. Moreover, two novel hepatoprotective peptides, Ala-Lys-Lys-His-Lys-Glu and Leu-Glu, which shared similar mechanisms of action with MAH were identified. These results suggest that MAH and the two peptides represent potential sources of natural hepatoprotective agents.

Dual targeting of Nur77 and AMPKα by isoalantolactone inhibits adipogenesis in vitro and decreases body fat mass in vivo.

BACKGROUND: Suppression of adipogenesis has been considered as a potential target for the prevention and treatment of obesity and associated metabolic disorders, and the nuclear receptor 4A1 (NR4A1/Nur77) and AMPKα are known to play important roles during early and intermediate stages of adipogenesis. Therefore, we hypothesized that dual targeting Nur77 and AMPKα would show strong inhibitory effect on adipogenesis. METHODS: We screened a herbal medicine-based small molecule library to identify novel natural compounds dual targeting Nur77 and AMPKα, and the antiadipogenic effects and mechanisms of action of a "hit" compound were studied in 3T3-L1 cells. In vivo antiobesity effects of the compound were also investigated in high-fat diet (HFD)-induced obese mice. RESULTS: We identified isoalantolactone (ISO) as a new NR4A1 inactivator that also activates AMPKα in 3T3-L1 cells. ISO, as expected, inhibited adipogenic differentiation of 3T3-L1 preadipocytes, accompanied by reduced mitotic clonal expansion (MCE) which occurs in the early stage of adipogenesis and decreased expression of genes required for MCE and cell cycle markers including cyclin A, cyclin D1. Furthermore, ISO reduced body weight gain and fat mass (epididymal, subcutaneous, perirenal, and inguinal white adipose tissues) in the high-fat diet-fed C57BL/6 N mice. Serum levels of triglycerides, aspartate transaminase, and alanine transaminase and hepatic steatosis were also significantly improved in the ISO-treated group compared to the high-fat diet control group. CONCLUSIONS: These results suggest that ISO dual targeting Nur77 and AMPKα during adipogenesis represents a novel class of mechanism-based antiadipogenic agents for treatment of obesity and associated metabolic disorders, including hyperlipidemia and fatty liver.