Therapeutic effects of exon skipping and losartan on skeletal muscle of mdx mice.

Various attempts have been made to find treatments for Duchenne muscular dystrophy (DMD) patients. Exon skipping is one of the promising technologies for DMD treatment by restoring dystropin protein, which is one of the muscle components. It is well known that losartan, an angiotensin II type1 receptor blocker, promotes muscle regeneration and differentiation by lowering the level of transforming growth factor-beta1 signaling. In this study, we illustrated the combined effects of exon skipping and losartan on skeletal muscle of mdx mice. We supplied mdx mice with losartan for 2 weeks before exon skipping treatment. The losartan with the exon skipping group showed less expression of myf5 than the losartan treated group. Also the losartan with exon skipping group recovered normal muscle architecture, in contrast to the losartan group which still showed many central nuclei. However, the exon skipping efficiency and the restoration of dystrophin protein were lower in the losartan with exon skipping group compared to the exon skipping group. We reveal that losartan promotes muscle regeneration and shortens the time taken to restore normal muscle structure when combined with exon skipping. However, combined treatment of exon skipping and losartan decreases the restoration of dystrophin protein meaning decrease of exon skipping efficiency.

ENA-A actimineral resource A extends lifespan associated with antioxidant mechanism in SMP30 knockout mice.

ENA-actimineral resource A (ENA-A) is an alkaline mineral water and has a few biological activities such as antioxidant activity. The aim of this study was to examine the effects of ENA-A on lifespan in mice using senescence marker protein-30 knockout mice. The present study had groups of 18-week-old mice (n = 24), 26-week-old mice (n = 12), and 46-week-old mice (n = 20). Each differently aged mice group was divided into three subgroups: a control group, a 5 % ENA-A-treated group, and a 10 % ENA-A-treated group. Mice in the 18-week-old group were treated with vitamin C drinking water 1.5 g/L. However, the mice in the 26-week-old and 46-week-old groups were not treated with vitamin C. The experiments were done for 18 weeks. All vitamin C-treated mice were alive at week 18 (100% survival rate). In the non-vitamin C group, the 10% ENA-A-treated mice were alive at week 18. The control and 5% ENA-A-treated mice died by week 15. As expected, vitamin C was not detected in the non-vitamin C-treated group. However, vitamin C levels were increased in an ENA-A dose-dependent manner in the vitamin C-treated group. In the TUNEL assay, a number of positive hepatocytes significantly decreased in an ENA-A dose-dependent manner. Periodic acid Schiff positive hepatocytes were significantly increased in an ENA-A dose-dependent manner. In addition, the expression level of CuZnSOD was increased by the ENA-A treatment. These data suggest that the intake of ENA-A has a critical role in the anti-aging mechanism and could be applied toward the lifespans of humans.

Alcohol induced hepatic degeneration in a hepatitis C virus core protein transgenic mouse model.

Hepatitis C virus (HCV) has become a major public health issue. It is prevalent in most countries. HCV infection frequently begins without clinical symptoms, before progressing to persistent viremia, chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) in the majority of patients (70% to 80%). Alcohol is an independent cofactor that accelerates the development of HCC in chronic hepatitis C patients. The purpose of the current study was to evaluate ethanol-induced hepatic changes in HCV core-Tg mice and mutant core Tg mice. Wild type (NTG), core wild-Tg mice (TG-K), mutant core 116-Tg mice (TG-116) and mutant core 99-Tg mice (TG-99) were used in this investigation. All groups were given drinking water with 10% ethanol and 5% sucrose for 13 weeks. To observe liver morphological changes, we performed histopathological and immunohistochemical examinations. Histopathologically, NTG, TG-K and TG-116 mice showed moderate centrilobular necrosis, while severe centrilobular necrosis and hepatocyte dissociation were observed in TG-99 mice with increasing lymphocyte infiltration and piecemeal necrosis. In all groups, a small amount of collagen fiber was found, principally in portal areas. None of the mice were found to have myofibroblasts based on immunohistochemical staining specific for α-SMA. CYP2E1-positive cells were clearly detected in the centrilobular area in all groups. In the TG-99 mice, we also observed cells positive for CK8/18, TGF-ß1 and phosphorylated (p)-Smad2/3 and p21 around the necrotic hepatocytes in the centrilobular area (p < 0.01). Based on our data, alcohol intake induced piecemeal necrosis and hepatocyte dissociation in the TG-99 mice. These phenomena involved activation of the TGF-ß1/p-Smad2/3/p21 signaling pathway in hepatocytes. Data from this study will be useful for elucidating the association between alcohol intake and HCV infection.

Effects of vitamin C on cytotherapy-mediated muscle regeneration.

Skeletal muscles are the largest organs in the human body, and several therapeutic trials have been conducted that included stem cell transplantation to regenerate damaged or wasted muscles. It is well known that it is essential to make a favorable microenvironment (stem cell niche) to induce the proper differentiation of the transplanted stem cells. Some drugs, such as losartan (angiotensin II type I blocker), enhance the therapeutic effects of transplanted stem cells by inhibiting fibrosis. In this study, we hypothesized that another substance, vitamin C (ascorbic acid), might improve the niche for stem cell transplantation based on its potent antioxidant effects. In both gross and microscopic observations, vitamin C-depleted mice exhibited more incomplete regeneration of damaged muscles than those treated with vitamin C. Carbonylated protein groups, which are the end products of oxidative stress, were detected in all experimental groups; however, the vitamin C-depleted groups exhibited a more potent positive reaction than that of the vitamin C-supplied groups. The difference is clearer in the presence of transplanted stem cells. Moreover, the serum total vitamin C level and the ascorbic acid (AA) to dehydroascorbic acid (DHA) ratio also were decreased in the presence of transplanted adipose-derived stem cells (ASCs). Taken together, these data can be considered as proof of vitamin C utilization by cells in vivo. The vitamin C-supplied groups displayed more severe fibrosis than that of the vitamin C-depleted groups. Since vitamin C is a major cofactor for the collagen synthesis, its deficiency resulted in reduced fibrosis. In conclusion, we demonstrated that vitamin C not only has a positive effect on adjusting the stem cell niche to boost muscle regeneration but also has an adverse aspect due to its profibrotic effect.

Vitamin C deficiency accelerates bone loss inducing an increase in PPAR-γ expression in SMP30 knockout mice.

Senescence marker protein (SMP) 30 knockout (KO) mice display symptoms of scurvy, including spontaneous bone fractures, and this was considered to be induced by a failure of collagen synthesis owing to vitamin C deficiency. However, low bone mineral density is also known to be associated with spontaneous bone fracture. Therefore, we investigated the effects of vitamin C deficiency on the balance between osteoblasts and osteoclasts in SMP30 KO mice as evidenced by histopathology. All mice were fed a vitamin C-free diet, and only one group (KV) mice were given water containing 1.5 g/l of vitamin C, whereas wild-type (WT) and KO mice were given normal drinking tap water without vitamin C for 16 weeks. After 16 weeks, all femur samples were removed for histopathological examination. The femurs of KO mice showed significantly reduced bone area and decreased number of osteoblasts compared with those of WT mice and KV mice. KO mice also exhibited the lowest level of alkaline phosphatase (ALP) expression in their femurs. However, KO mice showed the most elevated expression of the receptor activator of nuclear factor kappa-B ligand (RANKL). Moreover, KO mice had the strongest peroxisome proliferator-activated receptor (PPAR)-γ expression level in their osteoblasts and the highest number of TUNEL-positive bone marrow cells. Therefore, we concluded that vitamin C deficiency plays an important role in spontaneous bone fracture by inhibiting osteoblast differentiation and promoting transition of osteoblasts to adipocytes, and this could in turn be related to the increased PPAR-γ expression.

Vitamin C deficiency increases the binucleation of hepatocytes in SMP30 knock-out mice.

BACKGROUND AND AIMS: The binucleation of hepatocytes, which was known as an important feature of liver growth and physiology, has been reported to be increased during the chronic oxidative injury stage and has been regarded as an age-related change of hepatic structures. Therefore, we investigated the binuclearity pattern in the livers of senescence marker proteins-30 (SMP30) knock-out (KO) mice compared with wild-type (WT) mice and vitamin C-treated KO (KO + VC) mice. METHODS: The WT, KO and KO + VC mice were fed a vitamin C free diet and VC(+) group mice were given vitamin C water containing 1.5 g/L of vitamin C, whereas VC(-) group was given normal drinking water without vitamin C, for 16 weeks. RESULTS: In microscopic examination, the livers of KO mice showed a significantly increased number of binuclear hepatocytes compared with that of WT mice and KO + VC mice. KO mice also showed the most increased expression level of CYP2E1 and PCNA determined by immunohistochemistry and immunoblot analysis. Moreover, KO mice indicated the highest level of serum alanine aminotransferase and aspartate aminotransferase level in serum biochemical analysis. Accordingly, significantly decreased levels of reactive oxygen species, MDA (malondialdehyde) and HAE (4-hydroxyalkenals) were detected in KO + VC mice compared with KO mice. CONCLUSION: Therefore, it is concluded that vitamin C deficiency induces an increase of CYP2E1 expression and elevated ROS production, which causes oxidative liver injury and the elevation of hepatocyte binucleation in SMP30 KO mice.