Berberine attenuates experimental autoimmune encephalomyelitis in C57 BL/6 mice

Background

Berberine, an isoquinoline derivative alkaloid, has a wide range of pharmacological properties and is considered to have anti-inflammatory and neuroprotective effects. However, there are no reports about the effects and mechanisms of berberine in experimental autoimmune encephalomyelitis (EAE), an established model of multiple sclerosis (MS).

Methodology/Principal Findings

Female C57 BL/6 mice immunized with myelin oligodendrocyte glycoprotein 35–55 amino acid peptide were treated with berberine at the day of disease onset and medication was administered daily until mice were sacrificed. Blood–brain barrier (BBB) permeability and the alteration of matrix metalloproteinase-2 (MMP-2, 72 kDa) and matrix metalloproteinase-9 (MMP-9, 92 kDa) in the brain and cerebrospinal fluid (CSF) of EAE mice were detected by quantitative measurement for Evan''s blue (EB) content, Western blot and gelatin zymography respectively. The results showed that berberine attenuated clinical and pathological parameters of EAE, reduced the permeability of BBB, inhibited the activity and expression of MMP-9 but not MMP-2 in the CSF and brain of EAE mice.

Conclusions/Significance

These findings suggest that berberine is effective to attenuate the clinical severity of EAE in C57 BL/6 mice by reducing the permeability of BBB, decreasing the expression and activity of MMP-9, and decreasing the inflammatory infiltration. We think that berberine might be a potential therapeutic agent for MS.     

Exendin-4 improves steatohepatitis by increasing Sirt1 expression in high-fat diet-induced obese C57BL/6J mice

The effects of exendin-4 on Sirt1 expression as a mechanism of reducing fatty liver have not been previously reported. Therefore, we investigated whether the beneficial effects of exendin-4 treatment on fatty liver are mediated via Sirt1 in high-fat (HF) diet-induced obese C57BL/6J mice and related cell culture models. Exendin-4 treatment decreased body weight, serum free fatty acid (FA), and triglyceride levels in HF-induced obese C57BL/6J mice. Histological analysis showed that exendin-4 reversed HF-induced hepatic accumulation of lipids and inflammation. Exendin-4 treatment increased mRNA and protein expression of Sirt1 and its downstream factor, AMPK, in vivo and also induced genes associated with FA oxidation and glucose metabolism. In addition, a significant increase in the hepatic expression of Lkb1 and Nampt mRNA was observed in exendin-4-treated groups. We also observed increased expression of phospho-Foxo1 and GLUT2, which are involved in hepatic glucose metabolism. In HepG2 and Huh7 cells, mRNA and protein expressions of GLP-1R were increased by exendin-4 treatment in a dose-dependent manner. Exendin-4 enhanced protein expression of Sirt1 and phospho-AMPKα in HepG2 cells treated with 0.4 mM palmitic acid. We also found that Sirt1 was an upstream regulator of AMPK in hepatocytes. A novel finding of this study was the observation that expression of GLP-1R is proportional to exendin-4 concentration and exendin-4 could attenuate fatty liver through activation of Sirt1.     

IL-10 gene knockout attenuates allergen-induced airway hyperresponsiveness in C57BL/6 mice

Intratracheal administration of interleukin-10 (IL-10) has been reported to inhibit allergic inflammation but augment airway hyperresponsiveness (AHR). In the present study, airway and smooth muscle responsiveness to methacholine (MCh) were compared in wild-type (WT) and IL-10-deficient (IL-10-KO) mice to investigate the role of endogenous IL-10 in AHR development. Naive WT and IL-10-KO mice exhibited similar dose-dependent increases in airway resistance (Raw) to intravenous MCh. Sensitization and challenge with ragweed (RW) induced a twofold increase in responsiveness to intravenous MCh in WT mice, but hyperresponsiveness was not observed in similarly treated IL-10-KO mice. Likewise, tracheal rings from RW-sensitized and -challenged WT mice exhibited a fourfold greater responsiveness to MCh than IL-10-KO tracheal preparations. Measurements of airway constriction by whole body plethysmography further supported the Raw and tracheal ring data (i.e., AHR was not observed in the absence of IL-10). Interestingly, factors previously implicated in the development of AHR, including IL-4, IL-5, IL-13, IgA, IgG1, IgE, eosinophilia, and lymphocyte recruitment to the airways, were upregulated in the IL-10-KO mice. Treatment with recombinant murine IL-10 at the time of allergen challenge reduced the magnitude of inflammation but reinstated AHR development in IL-10-KO mice. Adoptive transfer of mononuclear splenocytes to IL-10-sufficient severe combined immunodeficient mice indicated that lymphocytes were an important source of the IL-10 impacting AHR development. These results provide evidence that IL-10 expression promotes the development of allergen-induced smooth muscle hyperresponsiveness.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Lactobacillus plantarum LRCC 5273 isolated from Kimchi ameliorates diet-induced hypercholesterolemia in C57BL/6 mice

ABSTRACT

This study was designed to select potent cholesterol-lowering probiotic strains on HepG2 cell and investigate the effect of selected strain, Lactobacillus plantarum LRCC 5273 and LRCC 5279 in hypercholesterolemic mice. In the results, LP5273 group showed significantly reduced total and LDL cholesterol compared to HCD group. In addition to significantly up-regulated hepatic mRNA expression of LXR-α and CYP7A1, intestinal LXR-α and ABCG5 were significantly up-regulated in LP5273 group. With activation of hepatic and intestinal LXR-α and its target genes, fecal cholesterol and bile acid excretion were increased in LP5273 fed mice. These results suggest that LP5273 ameliorates hypercholesterolemia in mice through the activation of hepatic and intestinal LXR-α, resulting in enhancement of fecal cholesterol and bile acids excretion in the small intestine. The results of present study suggest mechanistic evidences for hypocholesterolemic effects of L. plantarum spp., and may contribute to future researches for prevention of hypercholesterolemia and cardiovascular disease.     

Elimination of macrophage-specific apolipoprotein E reduces diet-induced atherosclerosis in C57BL/6J male mice

Apolipoprotein (apo)E is synthesized in atherosclerotic lesions by macrophages, however, its role in lesions is not known. Whereas apoE could exacerbate atherosclerosis by promoting macrophage uptake of cholesterol-rich lipoproteins or modulating protective inflammatory responses, it could also restrict lesion formation by facilitating cholesterol efflux out of lesions. The role of apoE was examined in lethally irradiated male C57BL/6J wild-type (WT) mice that were repopulated with bone marrow cells (BMT) from either identical C57BL/6J mice (WT+WT BMT) or C57BL/6J apoE-deficient mice (WT+E-/- BMT). This enabled us to compare normal mice with mice possessing macrophages that did not express apoE. The participation of macrophage-derived apoE in atherosclerosis was assessed by placing the mice on an atherogenic diet. Male WT+E-/- BMT mice had significantly reduced lesion area in the aortic valves (P < 0.01) compared with male WT+WT BMT mice ( approximately 22,000 vs. approximately 49,000 microm2/section, respectively). Further evaluation revealed that plasma cholesterol, lipoprotein cholesterol distribution, and plasma apoE were similar between the two groups, indicating that these known risk factors did not account for the differences in lesion area. However, the two groups were distinguished by the amount of apoE found in the lesions. ApoE antigen was expressed abundantly in WT+WT BMT lesions, whereas WT+E-/- BMT lesions contained little apoE. These findings indicate that the majority of apoE in lesions is synthesized locally by resident macrophages, and suggest that locally produced apoE can promote diet-induced atherosclerosis in male wild-type mice.     

Differences in anxiety-related behavior between apolipoprotein E-deficient C57BL/6 and wild type C57BL/6 mice

The influence of ApoE gene deletion on the anxiety state has not been previously investigated. The elevated plus maze was used in this study to determine differences in anxiety-related behavior between apoE-deficient and wild type C57BL/6 mice. The apoE-deficient mice demonstrated less anxiety on the elevated plus maze by spending more time in the open arms of the elevated plus maze compared to wild type mice (p<0.001). Additionally, female apoE-deficient mice visited the open arm of the maze more often than their apoE-deficient male counterpart (p<0.05). The anxiety state and/or sex are possible variables to be considered when designing physiological and/or behavioral studies involving mice that are apoE-deficient.     

Glutathione deficient C57BL/6J mice are not sensitized to ozone-induced lung injury

In this study we examined the role of the antioxidant glutathione (GSH) in pulmonary susceptibility to ozone toxicity, utilizing GSH deficient C57BL/6J mice that lack the expression of glutamate-cysteine ligase modifier subunit (GCLM). Gclm(−/−) knockout mice had 70% GSH depletion in the lung. Gclm(+/+) wild-type and Gclm(−/−) mice were exposed to either 0.3 ppm ozone or filtered air for 48 h. Ozone-induced lung hyperpermeability, as measured by total protein concentration in bronchoalveolar lavage fluid, was surprisingly lower in Gclm(−/−) mice than in wild-type mice. Lung hyperpermeability did not correlate with the degree of neutrophilia or with inflammatory gene expression. Pulmonary antioxidant response to ozone, assessed by increased mRNA levels of metallothionein 1 and 2, α-tocopherol transporter protein, and solute carrier family 23 member 2 (sodium-dependent vitamin C transporter) was greater in Gclm(−/−) mice than in Gclm(+/+) mice. These results suggest that compensatory augmentation of antioxidant defenses in Gclm(−/−) mice may confer increased resistance to ozone-induced lung injury.     

Effects of pre-germinated brown rice treatment high-fat diet-induced metabolic syndrome in C57BL/6J mice

To investigate using pre-germinated brown rice (PGBR) to treat metabolic syndrome, we fed one group of mice standard-regular-diet (SRD) for 20 weeks and another group of mice high-fat-diet (HFD) for 16 weeks. We subdivided them into HFD group and HFD + PGBR group whose dietary carbohydrate was replaced with PGBR for 4 weeks. The HFD group gained more weight, had higher blood pressure, heart rate, blood glucose and lipids, liver levels of TG, feces TG and bile acid, lower adipose levels of adipocytokine, lower skeletal muscle IR, IRS-1, IRS-2, PI3 K, Akt/PKB, GLUT-1, GLUT-4, GCK and PPAR-γ; higher liver SREBP-1, SCD-1, FAS, HMGCR, LDLR, CYP7α1 and PPAR-α, and higher adipose SREBP-1, SCD-1, FAS, and lower adipose PPAR-α and adiponectin. The HFD + PGBR group had clearly improved blood pressure, biochemical parameters and above proteins expressions. PGBR successful treatment of metabolic syndrome was achieved through improvements in glucose and lipid synthesis and metabolism.     

Substrains matter in phenotyping of C57BL/6 mice

The inbred mouse strain C57BL/6 has been widely used as a background strain for spontaneous and induced mutations. Developed in the 1930s, the C57BL/6 strain diverged into two major groups in the 1950s, namely, C57BL/6J and C57BL/6N, and more than 20 substrains have been established from them worldwide. We previously reported genetic differences among C57BL/6 substrains in 2009 and 2015. Since then, dozens of reports have been published on phenotypic differences in behavioral, neurological, cardiovascular, and metabolic traits. Substrains need to be chosen according to the purpose of the study because phenotypic differences might affect the experimental results. In this paper, we review recent reports of phenotypic and genetic differences among C57BL/6 substrains, focus our attention on the proper use of C57BL/6 and other inbred strains in the era of genome editing, and provide the life science research community wider knowledge about this subject.     

Delayed matching-to-position performance in C57BL/6N mice

Delayed matching-to-sample is one of the most frequently employed behavioral tasks for assessing spatial working memory in animals. Although the advantages of the task have been widely acknowledged and it is used in the study of a variety of species, its application to mice has been rare. In the present study, we reported the efficacy of a delayed matching-to-position task in C57BL mice lever-pressing in an operant-conditioning chamber. Each trial started with the press of a back lever, followed by the presentation of either a left or right front lever. When the ratio requirement for presses to the front lever (sample) was met, a delay interval started. Delay interval continued until the mice made the first response after the elapse of the programmed delay interval. This was followed by the presentation of a choice of left or right front levers. The choice of the same front lever as the sample was reinforced, whereas the other was not. The proportion of correct choices showed a delay-dependent decrement. A higher ratio of response requirement to the sample resulted in increased accuracy, but the duration of the intertrial interval had no effect. Preceding trials also influenced response accuracy, indicating proactive interference. Overall, the results replicated the effects of parametric manipulations reported in other species, and thus, our findings validate the efficacy of the task for assessing spatial working memory in laboratory mice.     

Effects of chronic oral rimonabant administration on energy budgets of diet-induced obese C57BL/6 mice

The endocannabinoids have been recognized as an important system involved in the regulation of energy balance. Rimonabant (SR141716), a selective inverse agonist of cannabinoid receptor 1 (CB1), has been shown to cause weight loss. However, its suppressive impact on food intake is transient, indicating a likely additional effect on energy expenditure. To examine the effects of rimonabant on components of energy balance, we administered rimonabant or its vehicle to diet-induced obese (DIO) C57BL/6 mice once daily for 30 days, by oral gavage. Rimonabant induced a persistent weight reduction and a significant decrease in body fatness across all depots. In addition to transiently reduced food intake, rimonabant-treated mice exhibited decreased apparent energy absorption efficiency (AEAE), reduced metabolizable energy intake (MEI), and increased daily energy expenditure (DEE) on days 4-6 of treatment. However, these effects on the energy budget had disappeared by days 22-24 of treatment. No chronic group differences in resting metabolic rate (RMR) or respiratory quotient (RQ) (P > 0.05) were detected. Rimonabant treatment significantly increased daily physical activity (PA) levels both acutely and chronically. The increase in PA was attributed to elevated activity during the light phase but not during the dark phase. Taken together, these data suggested that rimonabant caused a negative energy balance by acting on both energy intake and expenditure. In the short term, the effect included both reduced intake and elevated PA but the chronic effect was only on increased PA expenditure.     

Protective effects of azelaic acid against high-fat diet-induced oxidative stress in liver, kidney and heart of C57BL/6J mice

Excess fat intake induces hyperinsulinaemia, increases nutrient uptake and lipid accumulation, amplifies ROS generation, establishes oxidative stress and morphological changes leading to tissue injury in the liver, kidney and heart of high-fat diet (HFD)-fed mice. The effect of azelaic acid (AzA), a C9 α,ω-dicarboxylic acid, against HFD-induced oxidative stress was investigated by assaying the activities and levels of antioxidants and oxidative stress markers in the liver, kidney and heart of C57BL/6J mice. Mice were segregated into two groups, one fed standard diet (NC) and the other fed high-fat diet (HFD) for 15 weeks. HFD-fed mice were subjected to intragastric administration of AzA (80 mg/kg BW)/RSG (10 mg/kg BW) during 11-15 weeks. Glucose, insulin, triglycerides, hepatic and nephritic markers were analysed in the plasma and the activity of enzymatic, non-enzymatic antioxidants and lipid peroxidation markers were examined in the plasma/erythrocytes, liver, kidney and heart of normal and experimental mice. We inferred significant decrease in enzymatic and non-enzymatic antioxidants along with significant increase in glucose, insulin, hepatic and nephritic markers, triglycerides and lipid peroxidation markers in HFD-fed mice. Administration of AzA could positively restore the levels of plasma glucose, insulin, triglycerides, hepatic and nephritic markers to near normal. AzA increased the levels of enzymatic and nonenzymatic antioxidants with significant reduction in the levels of lipid peroxidation markers. Histopathological examination of liver, kidney and heart substantiated these results. Hence, we put forward that AzA could counteract the potential injurious effects of HFD-induced oxidative stress in C57BL/6J mice.     

Conjugated linoleic acid protects against age-associated bone loss in C57BL/6 female mice

Osteoporosis is one of the major causes of morbidity in the elderly. Inflammation exerts a significant influence on bone turnover, inducing the chronic form of osteoporosis. Dietary nutrition has the capacity to modulate inflammatory response. Therefore, nutritional strategies and lifestyle changes may prevent age-related osteoporosis, thereby improving the quality of life of the elderly population. Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism. Hence, this study was undertaken to examine the effect of CLA on bone mineral density (BMD) in middle-aged C57BL/6 female mice. After 10 weeks on diet, CLA-fed mice (14 months) maintained a higher BMD in different bone regions than corn oil (CO)-fed mice. The increased BMD was accompanied by a decreased activity of proinflammatory cytokines (such as tumor necrosis factor alpha, interleukin-6 and the receptor activator of NF-kappaB ligand) and decreased osteoclast function. Furthermore, a significant decrease in fat mass and an increase in muscle mass were also observed in CLA-fed mice compared to CO-fed mice. In conclusion, these findings suggest that CLA may prevent the loss of bone and muscle mass by modulating markers of inflammation and osteoclastogenic factors.     

The superior olivary complex in C57BL/6 mice.

The cellular and cytoarchitectural features of the lateral superior olive, the medial superior olive, the superior paraolivary nucleus and the medial, lateral and ventral nuclei of the trapezoid body are described in C57BL/6 mice using Nissl, Bodian and Golgi techniques. Principal, spindle and marginal cells are present in a well-defined lateral superior olive. The dendrites of these cells run primarily within rostrocaudal sheets as in the cat. The principal cells of the medial nucleus of the trapezoid body are similar to the principal cells in the cat. Large multipolar cells characterize the lateral nucleus of the trapezoid body and bipolar cells with a medial-lateral orientation are found in the medial superior olive. The largest neurons are found in the superior paraolivary nucleus and the lateral superior olive, and the medial and ventral nuclei of the trapezoid body. While brain weight and neuronal packing density change with development, the characteristic location of cell groups and the shape and Nissl-staining pattern of neurons in the youngest brains examined were essentially unchanged in the adult mice, although dendritic maturation had occurred. The homologies of the C57BL/6 superior olivary complex nuclei with the same areas described in other mouse strains, rat and cat are discussed. This study expands our understanding of the organization of the superior olivary complex in an inbred strain of Mus musculus and relates it to other species. The data about changes occurring during postnatal maturation may aid in the interpretation of behavioral and physiological studies of neonatal plasticity of the auditory system.     

Self-mutilation of the penis in C57BL/6N mice

A major cause of male reproductive failure in a C57BL/6N mouse production colony is self-inflicted mutilation of the penis. The extent of the damage ranged from loss of the distal end to loss of the entire penis. From January 1974 to August 1976, 645 adult male mice with mutilated penis were removed from this colony--where the monthly census was 9500 mice, mated 1 male to 4 females using a continuous mating system. On necropsy, it was observed that the substance blocking the urethra in the penile stump resulted in urine retention, grossly distending the urinary bladder. Proteus mirabilis and other bacteria isolated from the urethral plugs were considered secondary invaders.     

Mast cells and macrophages in normal C57/BL/6 mice

Mast cells and macrophages have an important role in immunity and inflammation. Because mice are used extensively for experimental studies investigating immunological and inflammatory responses, we examined mast cell and macrophage distribution in normal murine tissues. Mast cells were abundant in the murine dermis, tongue, and skeletal muscle but were rarely found in the heart, lung, spleen, kidney, liver, and the bowel mucosa. In contrast, dogs exhibited large numbers of mast cells in the lung parenchyma, liver, and bowel. Some murine dermal mast cells had long cytoplasmic projections filled with granular content. Mouse mast cells demonstrated intense histamine immunoreactivity and were identified with histochemical enzymatic techniques for tryptase and chymase. Macrophages, identified using the monoclonal antibody F4/80, were abundant in the spleen, lung, liver, kidney, and bowel but relatively rare in the heart, tongue, and dermis. Using a nuclease protection assay we investigated mRNA expression of stem cell factor (SCF), a crucial survival factor for mast cells, and the macrophage growth factors macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Stem cell factor mRNA was highly expressed in the murine lung. Relatively low levels of SCF mRNA expression were found in the tongue and earlobe, which are tissues containing a high number of mast cells. Macrophage CSF and GM-CSF mRNA was highly expressed in the lung and spleen. The murine heart, an organ with a low macrophage content, expressed high levels of M-CSF but negligible levels of GM-CSF mRNA. Constitutive growth factor mRNA expression in murine tissues without significant populations of mast cells and macrophages may suggest an alternative role for these factors in tissue homeostasis.