Therapeutic effects of granulocyte-colony stimulating factor on non-alcoholic hepatic steatosis in the rat

Background and rationale. Non-alcoholic hepatic steatosis refers to the accumulation of triglycerides in the liver in the absence of alcohol consumption. Granulocyte colony-stimulating factor (G-CSF) has been reported to be an effective treatment for a variety of liver diseases. We examined the possible therapeutic effects of G-CSF on non-alcoholic hepatic steatosis in rats.Material and methods. Thirty-week-old Otsuka Long Evans Tokushima Fatty (OLETF) rats received water containing 30% sucrose for 8 weeks to promote the development of non-alcoholic hepatic steatosis. After development of the model, the rats were injected with G-CSF (100 Mg/kg/day) or saline for 5 days. Four weeks after this treatment, serum levels of glucose, total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and free fatty acids (FFA) were measured. Histology was examined by hematoxylin and eosin (H-E) and periodic acid Schiff (PAS) staining, and levels of expression of hepatic lipogenic enzymes were determined by RT-PCR.Results. The G-CSF-treated rats displayed significantly fewer lipid droplets than the saline-treated rats (P < 0.01), and their levels of sterol regulatory element-binding protein (SREBP)-1c, fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) mRNAs were also lower (P < 0.01), as were their liver weight and serum levels of TG and FFA (P < 0.05).Conclusion. Our results indicate that G-CSF ameliorated non-alcoholic hepatic steatosis in the OLETF rat, and this therapeutic effect involved a reduction of SREBP-1c expression. Therefore, G-CSF deserves further study as a potential treatment for non-alcoholic hepatic steatosis

Time Course of the Development of Nonalcoholic Fatty Liver Disease in the Otsuka Long-Evans Tokushima Fatty Rat

Nonalcoholic fatty liver disease (NAFLD) is considered a hepatic manifestation of metabolic syndrome. In this study, we investigated histological and biochemical changes in NAFLD and the gene expression involving de novo lipogenesis in Otsuka Long-Evans Tokushima fatty (OLETF) rats. We used OLETF rats and Long-Evans Tokushima Otsuka (LETO) rats as animal models of NAFLD and as controls, respectively. Consistent observations were made at 4-week intervals up to 50 weeks of age, and all rats were fed ad libitum with standard food. Biochemical and histological changes were observed, and gene expression involved in de novo lipogenesis was measured using real-time polymerase chain reactions. As a results hepatic micro- and macrovesicular steatosis and hepatocyte ballooning were evident in the OLETF rats at 22–38 weeks of age but disappeared after 42 weeks; no fibrosis or collagen deposition was observed. Gene expression involved in de novo lipogenesis followed a pattern similar to that of the histological changes. In conclusion, in the absence of dietary manipulation, hepatic steatosis in OLETF rats is evident at 22–38 weeks and declines after 42 weeks. Therefore, OLETF rats at 22–38 weeks are recommended as animal models of hepatic steatosis

The Anti-Stress Effect of Mentha arvensis in Immobilized Rats

Stress can lead to inflammation, accelerated aging, and some chronic diseases condition. Mentha arvensis (MA) is a traditional medicine having antioxidant and anti-inflammatory activities. The present study investigated the anti-stress role of MA and fermented MA (FMA) extract in immobilized rats. We studied the lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells and rats were immobilized for 2 h per day for 14 days using a restraining cage. MA (100 mg/kg) and FMA (100 mg/kg) were orally administered to rats 1 h prior to immobilization. Using high-performance liquid chromatography (HPLC) analysis, we determined the rosmarinic acid content of MA and FMA. The generation of malondialdehyde (MDA) and nitric oxide (NO) in RAW 246.7 cells were suppressed by both MA and FMA. In rats, MA and FMA notably improved the body weight, daily food intake, and duodenum histology. MDA and NO level were gradually decreased by MA and FMA treatment. MA and FMA significantly controlled the stress-related hormones by decreasing corticosterone and β-endorphin and increasing serotonin level. Moreover, protein expression levels of mitogen activated protein kinases (MAPK) and cyclooxygenase-2 (COX-2) were markedly downregulated by MA and FMA. Taken together, MA and FMA could ameliorate immobilized-stress by reducing oxidative stress, regulating stress-related hormones, and MAPK/COX-2 signaling pathways in rats. Particularly, FMA has shown greater anti-stress activities than MA

Expression of the G-CSF receptor (G-CSFR) in kidneys.

<p>(<b>A</b>) RT-PCR analysis of G-CSFR mRNA expression in kidney tissue. Hypothalamus tissue was used as a positive control, together with a no-template negative control. (<b>B</b>) G-CSFR immunostained via antibody (green, a) and DAPI (blue, b) in glomeruli of a kidney section (magnification x400). G-CSF receptor, G-CSFR; positive control, P; negative control, N.</p

Levels of metabolic parameters before treatment with G-CSF or saline.

<p>Long-Evans Tokushima Otsuka rats, LETO; Otsuka Long-Evans Tokushima Fatty rats, OLETF; total cholesterol, TC; triglyceride, TG; urine albumin creatinine ratio, UACR. All data are expressed as mean±SD. *<i>P</i><0.05 vs. LETO rat (LETO, <i>n</i> = 4; OLETF, <i>n</i> = 8).</p

Histological changes in the kidney after treatment.

<p>(<b>A</b>) Stained with periodic acid-Shiff (PAS) (magnification x400). (<b>B</b>) Electron micrograph of a glomerulus (magnification x20.000). Kidney of the LETO rat (a), the saline-treated OLETF rat (b), and the G-CSF-treated OLETF rat (c). (<b>C</b>) Quantitative analysis of images of PAS-stained kidney sections. (<b>D</b>) Quantitative analysis of images of GBM thickness via electron micrographs. (<b>E</b>) Quantitative analysis of foot process width via electron micrographs. All data are expressed as mean±SE. *<i>P</i><0.05 vs. LETO rats. ^†<i>P</i><0.05 vs. untreated OLETF rats (n = 4).</p

FISH imaging and immunostained ED-1 in glomeruli of BMT female rats after treatment.

<p>(<b>A</b>) Stained with hematoxylin and eosin (HE) (magnification x400). (<b>B</b>) Higher magnification views of the boxed regions in (A), stained with FISH using a Cy3-labeled Y-chromosome (red, white arrow) and DAPI-labeled nucleus (blue) (magnification x400). (<b>C</b>) Macrophages immunostained with ED-1 antibody (black arrow). Kidney of the LETO rat (a), the saline-treated OLETF rat (b), and the G-CSF-treated OLETF rat (c). (<b>D</b>) Quantitative analysis of Y-chromosome-positive cells in glomeruli. (<b>E</b>) Quantitative analysis of ED-1-positive cells in glomeruli. Fluorescence <i>in situ</i> hybridization, FISH; 4′–6-Diamidino-2-phenylindole, DAPI; Bone marrow transplantation, BMT. All data are expressed as mean±SD. *<i>P</i><0.05 vs. LETO rats. ^†<i>P</i><0.05 vs. untreated OLETF rats (n = 3).</p

Experimental design.

<p>Experiment 1: a rat model of diabetic nephropathy (male OLETF rats), Experiment 2: a rat model of diabetic nephropathy with bone marrow transplantation (BMT) (donors: male OLETF rats, recipients: female OLETF rats).</p