Introduction: Human amniotic fluid stem (hAFS) cells have been shown to differentiate into multiple lineages, including myoblasts. However, molecular mechanisms underlying the myogenic differentiation of hAFS cells and their regenerative potential for muscle injury remain to be elucidated. Methods: In order to induce myogenic differentiation of hAFS cells, lentiviruses for MYOD were constructed and transduced into hAFS cells. Formation of myotube-like cells was analyzed by immunocytochemistry, and expression of molecular markers for myoblasts was analyzed by reverse transcription polymerase chain reaction and Western blotting. For in vivo muscle regeneration, MYOD transduced hAFS cells were injected into left tibialis anterior (TA) muscles injured with cardiotoxin, and muscle regeneration was analyzed using hematoxylin and eosin, immunocytochemistry and formation of neuro-muscular junction. Results: MYOD expression in hAFS cells successfully induced differentiation into multinucleated myotube-like cells. Consistently, significant expression of myogenic marker genes, such as MYOG, DES, DMD and MYH, was induced by MYOD. Analysis of pre-myogenic factors showed that expression of PAX3, MEOX1 and EYA2 was significantly increased by MYOD. MYOD was phosphorylated and localized in the nucleus. These results suggest that in hAFS cells, MYOD is phosphorylated and localized in the nucleus, thus inducing expression of myogenic factors, resulting in myogenic differentiation of hAFS cells. To test regenerative potential of MYOD-transduced hAFS cells, we transplanted them into injured muscles of immunodeficient BALB/cSlc-nu mice. The results showed a substantial increase in the volume of TA muscle injected with MYOD-hAFS cells. In addition, TA muscle tissue injected with MYOD-hAFS cells has more numbers of neuro-muscular junctions compared to controls, indicating functional restoration of muscle injury by MYOD-hAFS cells. Conclusions: Collectively, our data suggest that transduction of hAFS cells with MYOD lentiviruses induces skeletal myogenic differentiation in vitro and morphological and functional regeneration of injured muscle in vivo.

In this study, we investigated the effects of balanced deep-sea water (BDSW) on hyperglycemia and glucose intolerance in high-fat diet (HFD)-induced diabetic C57BL/6J mice. BDSW was prepared by mixing deep-sea water (DSW) mineral extracts and desalinated water to give a final hardness of 500–2000. Mice given an HFD with BDSW showed lowered fasting plasma glucose levels compared to HFD-fed mice. Oral and intraperitoneal glucose tolerance tests showed that BDSW improves impaired glucose tolerance in HFD-fed mice. Histopathological evaluation of the pancreas showed that BDSW recovers the size of the pancreatic islets of Langerhans, and increases the secretion of insulin and glucagon in HFD-fed mice. Quantitative reverse transcription polymerase chain reaction results revealed that the expression of hepatic genes involved in glucogenesis, glycogenolysis and glucose oxidation were suppressed, while those in glucose uptake, β-oxidation, and glucose oxidation in muscle were increased in mice fed HFD with BDSW. BDSW increased AMP-dependent kinase (AMPK) phosphorylation in 3T3-L1 pre- and mature adipocytes and improved impaired AMPK phosphorylation in the muscles and livers of HFD-induced diabetic mice. BDSW stimulated phosphoinositol-3-kinase and AMPK pathway-mediated glucose uptake in 3T3-L1 adipocytes. Taken together, these results suggest that BDSW has potential as an anti-diabetic agent, given its ability to suppress hyperglycemia and improve glucose intolerance by increasing glucose uptake.

Objective: To determine the effects of balanced deep‐sea water (BDSW) on adipocyte hypertrophy and liver steatosis in high‐fat diet (HFD)‐induced obese C57BL/6J mice.Methods: BDSW was prepared by mixing deep‐sea water (DSW) mineral extracts and desalinated water. C57BL/6J mice were fed a normal diet or HFD with or without BDSW with different hardness (500, 1000, or 2000) for 20 weeks.Results: BDSW suppressed body weight gain in HFD‐fed mice. Histopathologic assays of the fat and liver revealed that BDSW inhibited the increase in adipocyte size and improved severe liver steatosis in HFD‐fed mice. BDSW suppressed the expression of adipogenic, lipogenic, lipolytic, and pro‐inflammatory cytokine genes and increased the expression of adipokines and β‐oxidation genes in fat. In the liver, BDSW suppressed the expression of genes involved in lipogenesis and cholesterol synthesis, and increased the expression of genes related to β‐oxidation. Furthermore, BDSW improved the impaired phosphorylation of IRS‐1, LKB1, AMPK, and mTOR in fat and liver tissues of HFD‐fed mice.Conclusions: These results suggest that BDSW has potential as an anti‐lipidemic agent, given its ability to suppress body weight gain and liver steatosis through the regulation of lipid metabolism by signal molecule activation.

The aim of this study was to determine the effects of balanced deep-sea water (BDSW) on hyperglycemia and glucose intolerance in streptozotocin (STZ)-induced diabetic mice. BDSW was prepared by mixing DSW mineral extracts and desalinated water to yield a final hardness of 1000–4000 ppm. Male ICR mice were assigned to 6 groups; mice in each group were given tap water (normal and STZ diabetic groups) or STZ with BDSW of varying hardness (0, 1000, 2000, and 4000 ppm) for 4 weeks. The STZ with BDSW group exhibited lowered fasting plasma glucose levels than the STZ-induced diabetic group. Oral glucose tolerance tests showed that BDSW improves impaired glucose tolerance in STZ-induced diabetic mice. Histopathological evaluation of the pancreas showed that BDSW restores the morphology of the pancreatic islets of Langerhans and increases the secretion of insulin in STZ-induced diabetic mice. Quantitative real-time PCR assay revealed that the expression of hepatic genes involved in gluconeogenesis, glucose oxidation, and glycogenolysis was suppressed, while the expression of the genes involved in glucose uptake, β-oxidation, and glucose oxidation in muscle were increased in the STZ with BDSW group. BDSW stimulated PI3-K, AMPK, and mTOR pathway-mediated glucose uptake in C₂C₁₂ myotubes. BDSW increased AMPK phosphorylation in C₂C₁₂ myotubes and improved impaired AMPK phosphorylation in the muscles of STZ-induced diabetic mice. Taken together, these results suggest that BDSW is a potential anti-diabetic agent, owing to its ability to suppress hyperglycemia and improve glucose intolerance by modulating glucose metabolism, recovering pancreatic islets of Langerhans and increasing glucose uptake.

Colorectal adenocarcinoma is the most common type of gastrointestinal cancer. Colon adenocarcinoma is a major health problem worldwide due to the high prevalence and mortality rates associated with the disease. The majority of colorectal carcinomas are adenocarcinomas, which originate from the epithelial cells of the colorectal mucosa. HT-29 cells, which originate from human colon adenocarcinoma, are used as an in vitro model to investigate the effect of malignant transformation on the expression of cellular constituents and functions of the intestinal epithelium. Nitric oxide (NO) is a signaling molecule, which is involved in inflammation and carcinogenesis. It has been reported that enhanced inducible NO synthase (iNOS) activity and the resulting NO concentrations in human colon carcinoma contribute to tumor progression and vascular invasion. The present study investigates the effect of pro‑inflammatory cytokine‑induced nitric oxide (NO) production and iNOS expression on the invasion of human colorectal adenocarcinoma HT‑29 cells, and the effect of extract from Cnidii Rhizoma on NO production and the invasiveness of HT‑29 cells. Treatment of HT‑29 cells with cytokines, 100 U/ml interferon γ, 10 ng/ml interleukin‑1 α and 25 ng/ml tumor necrosis factor α was found to increase NO production. Pretreatment of the cells with Cnidii Rhizoma (0.1‑5 mg/ml) resulted in an inhibition of cytokine‑induced NO production and iNOS expression. The invasiveness of HT‑29 cells through Matrigel was significantly increased by treatment with cytokines. Cnidii Rhizoma inhibited the invasiveness of cytokine‑treated HT‑29 cells through the Matrigel‑coated membrane in a concentration‑dependent manner. Matrix metalloproteinase (MMP) activity in HT‑29 cells increased following the treatment with cytokines, and pretreatment of the cells with Cnidii Rhizoma inhibited cytokine‑induced MMP‑2 activity. These results provide sufficient information for the further development of Cnidii Rhizoma as an antitumor metastatic agent for the treatment of colon cancer.