Inbred mice are an essential animal strain for research as they can improve the reproducibility and reliability of study results. The establishment of new inbred lines is continuing, and new inbred lines are being used in many research fields. C57BL/6 is a mouse laboratory animal that has been developed and used as an inbred strain since early stage of mouse strain development, and, in the 1950s, C57BL/6 was separated into substrains by the Jackson Laboratory (C57BL/6J) and the National Institutes of Health (C57BL/6N). C57BL/6 mice have been used in immunology and antitumor activity studies since the early strain development stage. After the mouse genome was fully described, C57BL/6 mice use in many areas of research has expanded. In particular, immunological characteristics such as those related to cell-mediated immunity and NK cell activity are relatively higher in C57BL/6 mice than in other mice. The C57BL/6NKorl is a stock of C57BL/6N established as part of a localization of experimental animal strategy of the Korean Food and Drug Administration. Based on analysis of single nucleotide polymorphisms (SNPs), C57BL/6NKorl is considered a genetically distinct inbred stock from other C57BL/6N. Various research efforts have been made to describe the characteristics and increase knowledge of the characteristics of C57BL/6Nkorl. The results obtained through these efforts are expected to increase the utilization of C57BL/6Nkorl as a domestic laboratory animal resource and to enhance the reliability of mouse based studies.

The therapeutic effects of mulberry (Morus alba) leaves on lipid metabolism, including lipogenesis, lipolysis and hyperlipidemia are widely known, although their fermented products are yet to be applied. To investigate the therapeutic effects of a novel extract of mulberry leaves fermented with Cordyceps militaris (EMfC) on lipid metabolism, the lipid profile of serum, lipid accumulation, lipolytic activity and lipogenesis regulation were measured in high fat diet (HFD)‑induced obese C57BL/6 mice treated with EMfC for 12 weeks. Briefly, the concentrations of low‑density lipoprotein, triglyceride, total cholesterol and glucose significantly decreased in the serum of the HFD+EMfC treated group when compared with the HFD+Vehicle treated group, while the levels of high‑density lipoprotein increased in the HFD+EMfC group. The amount of abdominal fat and the size of adipocytes were significantly lower in the HFD+EMfC treated group when compared with the HFD+Vehicle treated group. The weight and number of lipid droplets of liver tissue exhibited a similar decrease. Furthermore, the mRNA levels of peroxisome proliferator‑activated receptor‑γ for adipogenesis as well as adipocyte protein 2 and Fas cell surface death receptor for lipogenesis reduced following EMfC treatment for 12 weeks. Phosphorylation of perilipin and hormone‑sensitive lipase, and in the adipose triglyceride lipase expression showed a significant increase in the HFD+EMfC treated group. These results indicated that EMfC may prevent fat accumulation in the HFD‑induced obese C57BL/6 mice through the inhibition of lipogenesis and by stimulating lipolysis. Thus, the results provide evidence for the potential use of EMfC as an anti‑obesity complex in the treatment of obesity.

Gallarhois (GR) is a traditional oriental herbal medicine with various pharmacological effects; however, its effect on gastric ulcer has not been previously explored. We firstly investigated the component and antioxidant activity of GR extract (EtGR) by HPLC analysis and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The results showed that EtGR consisted of gallotannin (68.7%), gallic acid (27.2%) and methyl gallate (4.1%) and that it had a high antioxidant value (IC50 value; 1.93 μg/mL). To evaluate the possible anti-gastric ulcer potential of EtGR, we investigated the effects of EtGR in the model of ethanol/hydrochloric acid (EtOH/HCl)-induced gastric ulcer. Gross and histological gastric lesions, biochemical and gene expression parameters were taken into consideration. The results showed that EtOH/HCl treatment produced mucosal injuries with morphological and histological damage, whereas EtGR co-treatment reduced the gastric injuries. EtGR treatment also decreased the contents of malonaldehyde (MDA) activity relative to the vehicle group. Moreover, EtGR decreased the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and cyclo-oxygenase-2 (COX-2) expression. Finally, EtGR did not induce any specific toxicity in the livers or kidneys of the EtOH/HCl-induced gastric ulcer model. These results suggest that EtGR had stronger antioxidant activity and could be a new useful natural drug for gastroprotection against gastric ulcer. Moreover, these findings provide a scientific basis for the development of drugs from traditional oriental herbal medicines.

Sepsis, one of the most fatal diseases in the world, is known to culminate in multiple organ failure due to an uncontrolled inflammatory response. Hence, the use of animal models in sepsis research is very important to study complex immune responses. The current study was undertaken to compare commercial stocks with KFDA stocks of DBA/2 mice as an animal model for sepsis study. To compare responses of DBA/2 mice to lipopolysaccharides (LPS)-induced sepsis, we measured altered characteristics of various factors associated with sepsis, including survival curves, organ failure and inflammatory response, in DBA/2Korl stock and two commercial stocks (DBA/2A and DBA/2B). Survival rates after LPS exposure were similar for DBA/2Korl and DBA/2B; however, for times over 20 h, survival rates were reduced and concentration dependent in DBA/2A. In order to evaluate multiple organ failure caused by sepsis, H&E stains were evaluated for liver and spleen tissues obtained in the early (2 h) and later (20 h) stages after exposure to LPS; no significant differences were observed between the three stocks. mRNA and protein levels of proinflammatory cytokines were assessed for evaluating inflammatory reactions, and were found to increase in a dose-dependent manner in most DBA/2 mice after LPS treatment. However, no changes were observed in the mRNA levels of proinflammatory cytokines at 20 h after LPS exposure in the DBA/2A stock. The induction of inflammation-mediated factors by LPS exposure did not induce alterations in the mRNA levels of COX-2 and iNOS in all three DBA/2 stocks. Our results indicate that response of DBA/2Korl to LPS-induced sepsis is similar to the two commercial DBA/2 stocks, thus representing its potential as a useful biological resource established in Korea.

Mulberry leaves have antioxidant activity and anti‑inflammatory effects in several types of cells. However, the efficacy of mulberry leaves fermented with Cordyceps militaris remains unknown. Therefore, the present study aimed to investigate whether the ethanol extracts of mulberry leaves fermented with C. militaris (EMfC) can prevent lipopolysaccharide (LPS)‑induced inflammation and autophagy in macrophages. To achieve this, RAW264.7 cells pretreated with three different dose of EMfCs were subsequently stimulated with LPS, and examined for alterations in the regulatory factors of inflammatory responses and key parameters of the autophagy signaling pathway. EMfC treatment inhibited the generation of reactive oxidative species; however, significant activity was observed for 2,2‑diphenyl‑1‑picrylhydrazyl (DPPH) radical scavenging (IC50=579.6703 mg/ml). Most regulatory factors in inflammatory responses were significantly inhibited following treatment with EMfC, without any significant cellular toxicity. EMfC‑treated groups exhibited marked suppression of nitrogen oxide (NO) levels, mRNA expression levels of iNOS/COX‑2, levels of all inflammatory cytokines (TNF‑α, IL‑1β and IL‑6) and phosphorylation of MAPK members, as well as recovery of cell cycle progression. Furthermore, similar effects were observed in the LPS‑induced autophagy signaling pathway of RAW264.7 cells. The expression levels of microtubule‑associated protein 1A/1B‑light chain 3 (LC3) and Beclin exhibited a dose‑dependent decrease in the EMfC+LPS‑treated groups compared with in the Vehicle+LPS‑treated group, whereas the phosphorylation of PI3K and mTOR were enhanced in a dose‑dependent manner in the same groups. Overall, the results of the present study provide evidence that exposure to EMfC protects against LPS‑induced inflammation and autophagy in RAW264.7 cells. These results indicated that EMfC is a potential candidate for treatment of inflammatory diseases.

Along with de novo resistance, continued exposure to trastuzumab, an anti-human epidermal growth factor receptor 2 (HER2/neu) antibody, can lead to acquired resistance. In this study, we characterize a new anti-HER2/neu antibody resistant and metastatic mouse breast carcinoma cell line, TUBO-P2J. This cell line was developed during in vivo experiments using the antibody sensitive and non-metastatic tumor line TUBO. In addition, TUBO-P2J was used to establish an intratumoral HER2 heterogenous animal tumor model to evaluate the therapeutic effects of anti-HER2/neu antibody. After establishing the cell line, TUBO-P2J was characterized regarding its susceptibility to anti-neu antibody and chemotherapeutics, as well as its metastatic potential in vitro and in vivo. In addition, expression profiles of metastasis related genes were also evaluated. A clinically relevant intratumoral HER2 heterogenous tumor model was established by inoculating mice with tumor cells consisting of TUBO and TUBO-P2J at a ratio of 1,000:1 or 10,000:1. Tumor growth and mouse survival were used to evaluate the therapeutic effects of anti-neu antibody. The TUBO-P2J cell line is a HER2/neu negative and highly metastatic variant of TUBO. This cell line was resistant to anti-neu antibody therapy, and when inoculated subcutaneously, metastasized to the lungs within 14 days. Compared to the parental TUBO cell line, TUBO-P2J displayed an epithelial-mesenchymal transition (EMT) related gene expression profile including: the loss of E-cadherin, and increased Vimentin, Snail, and Twist1 expression. In addition, TUBO-P2J exhibited increased invasion and migration activity, and was resistant to chemotherapy drugs. Finally, mixed tumor implantations experiments revealed that an increased percentage of TUBO-P2J rendered tumors less responsive to anti-neu antibody therapy. This study describes a novel model of intratumoral heterogenous metastatic breast cancer in immune competent mice that can be used to develop novel or combined immunotherapies to overcome antibody resistance.