| Background and aimsNonalcoholic fatty liver disease (NAFLD) is a well recognized liver disorder which is commonly characterized by hepatocyte fat accumulation,lobular inflammation, ballooning degeneration with or without fibrosis.NAFLD refers to a wide spectrum of liver damage, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH),advanced fibrosis, and cirrhosis. The primary NAFLD is an increasingly recognized condition linked to metabolic syndrome (MS),however, the secondary NAFLD is linked to viral hepatitis,drugs,surgical operation, long-term intravenous hyperalimentation. In recent years, NAFLD is now considered to be the commonest liver problem in the western world. The prevalence of obesity and2type diabetes mellitus are dramatically increasing, leading to the rise of liver related mortality and total mortality.The prevalent hypothesis is the "two-hit" hypothesis proposed by Day et al, with the "first-hit" being the accumulation of triglycerides in hepatocytes (steatosis) followed by the "second-hit" that triggers the progression of steatosis to NASH.NAFLD is an acquired diseases by multiple pathogenesis, therefore, wipe off the pathogenesis and predisposition,prevent protopathy and risk factors,abstinence are effective measures. The basic premise is to change life-style and prevent related risk factors, including rational diet, aerobic exercise, correct harmful life-style. An animal experiment have confirmed that supplying GSH could relieve oxidative stress and lipid peroxidation, reducing the express of TNF-α, suppressing hepatocellular apoptosis. It could protect liver function and recover dysfunctional liver.The seeds of Sinapis alba Linn.(commonly called white or yellow mustard), cruciferous brassica mustard seeds, containing isothiocyanate, erucic acid, polysaccharides, phenols and other active ingredients, the processing of mustard oil and mustard powder products is a commonly used condiment in our lives, possessing the physiological function of anticancer, anti-inflammatory, antibiosis, antioxidant etal. ITCs can inhibit oxidation process by inducing the hydrolytic enzymes.Our previous study had proved that Mustard seed could enhance SOD levels and inhibit macrophage respiratory burst,which is more effective than coriolus versicolor polysaccharide.However, there is limited study regarding the use of mustard seed to prevent CRC, and the anticancer mechanism of mustard seed is unclear.Based on the above background, the purpose of this study is to investigate the preventive effects of mustard seed on high fatty diet-induced NAFLD in mices and to explore its mechanisms. The effects of mustard seed on cellular immunity, cytokines, will be tested.The prevention mechanism of mustard seed on NAFLD is to be investigated. This study will lay the foundation for the feasibility study for the mustard seed used in the prevention of NAFLD in future.Materials and Methods1. In vitro experiment:The HepG2cells was used to form lipoid degeneratiaon. Cells were divided into five groups:(1) control group;(2) ox-LDL group;(3) ox-LDL+SB;(4) ox-LDL+PD;(5) ox-LDL+SP。 In vivo experiment:NAFLD was induced consistently in experimental ApoE-/-mice by high fatty diet.60mice were randomly divided into five groups and twelve of each:(1)Normal control group;(2)7.5%MS only group;(3)HF+5%MS group;(4)HF+7.5%MS group;(5)HF only group The first week from the start the experiment, the mice in HF group, HF+5%MS group and HF+7.5%MS group are given high fatty diet. The mice in normal control group and7.5%MS only group given regular feeding, low-dose intervention group and the high dose intervention group were respectively treated with5%and7.5%diet of mustard seeds until the end of the experiment25weeks.2. Observe the general condition of mice, such as eating and drinking, nutritceion, weight, physical activity, coat color, gloss change, stool, etal.The misces were sacrificed and blood sample, spleen, liver were collected after25weeks.3. Liver were conventional fixed and dehydrated, paraffin-embedded sections, H.E staining to determine the histological type and tumor metastasis.4. The CD4+、CD25+、CD8+、CD11c+cells ratio of each mice spleen lymphocytes were tested by flow cytometry (FCM) at the end of the experiment.5. Liquid multi-chip analysis system (1uminex200) assay for levels of IL-5, IL-12, IFN-γ in serum of each mice.6. ELISA assay for CHO、TG、LDL-C、HDL-C、Adiponectin、Leptin、Resistin、 TNF-a expression levels of the plasma each mice.7. The ERK, JNK, p38MAPK pathway of each mice spleen lymphocytes were tested by flow cytometry (FCM) at the end of the experiment.Results1. At the termination of experiment, the mice in high fatty model group showed mental fatigue, anorexia, hair removal. There was no diarrhea and stool abnomal.2. The liver degeneration showed more lighten in5%MS+HF group and7.5%MS+HF group than HF group, and there was no significant change in7.5%MS group and control group.3. The CD11cCD45RB positive spleen cell ratio of mice in each group showed significant difference by statistical (P=0.000). The CD11cCD45RB-positive spleen cell ratios of mice in7.5%MS and7.5%MS intervention group were1.89±0.91%and2.03±0.23%respectively, as compared to HF group(4.22±0.36%) was significantly decreased, the difference was significant (P<0.05).4. The CD4+CD25+spleen cell ratios of mice in7.5%MS and7.5%MS intervention group was46.93±5.99%and49.00±4.71%respectively, and that was24.10±3.23%in HF group. The difference among the experimental groups was significant (P=0.000), of which7.5%MS and7.5%MS intervention group were both increased compared with the HF group, the difference significant (P<0.05).5. The value of CD4+/CD8+of mice spleen among the groups showed significant difference by statistical (P=0.000), in which the normal control group,7.5%MS,5%MS and7.5%MS intervention group mice spleen CD4+/CD8+values were1.98±0.79、1.92±0.93、1.88±0.81and1.96±0.69,as compared to the HF group (1.73±0.13) were all increased, the difference was significant (P<0.01).6. The serum IL-5levels of7.5%MS group was207.17±36.41pg/ml, as compared to that of HF group(16.79±7.25pg/ml) was increased, the difference was significant (P<0.01). The serum cytokines INF-r and IL-12levels of7.5%MS intervention group, as compared to that of HF group was decreased, the difference was significant (P<0.01).7. The level of LDL in7.5%MS intervention group,5%MS intervention group,7.5%MS group were0.29±0.004、0.39±0.04、0.021±0.003respectively,it was decreased significantly compared with the HF group(p<0.01); The plasm HDL-c in groups showed significant difference by statistical (P=0.000),7.5%MS group,5%MS intervention group and7.5%MS intervention group were 0.053±0.01,0.029±0.01,0.055±0.01respectively, increased compared to HF model group (P<0.01); However,there was no difference of the total cholesterol (P>0.05)。8. The level of Leptin in7.5%MS group and control group were0.035±0.006and0.020±0.008respectively,it was decreased significantly compared with the HF group(0.075±0.07)(p<0.01); The plasm Resistin in groups showed significant difference by statistical (P=0.000),7.5%MS group,7.5%MS intervention group and control group were0.045±0.006、0.054±0.007'0.041±0.004respectively, decreased compared to HF model group (P<0.05); The plasm TNF-a in groups showed significant difference by statistical (P=0.000),7.5%MS group,7.5%MS intervention group and control group were0.022±0.008、0.035±0.008'0.024±0.006respectively, decreased compared to HF model group (P<0.05); The level of Adiponectin in groups showed significant difference by statistical (P=0.000),7.5%MS group,7.5%MS intervention group and control group were0.082±0.007、0.059±0.010'0.083±0.009respectively, increased compared to HF model group (P<0.05).9. In vitro, the mechanism of prevent Ox-LDL induced lipid degeneration maybe related to blocking the differentiation by ERK-MAPK pathway.Conclusions1. The mustard seeds possess chemopreventive activity against NAFLD induced by high fatty diet in mice. The concentration of mustard seed is higher, and the effect of prevention of NAFLD is stronger.2. The mustard seeds can prevent NAFLD which is related to ERK-MAPK pathway.3. In the process of high fatty diet induced NAFLD, the mustard seeds can increase the content of HDL, reduce LDL and TG, can improve leptin, resistin, TNF-α,can reduce adiponectin, improve the ratio of CD4+/CD8+to maintenance of T cell subsets balance, enhance immune function, and increase lymphocyte activity. The mustard seeds can improve CD4+CD25+cell level, so that can diminish inflammation and induce immune tolerance. The prevention mechanism of mustard seeds on NAFLD is related with regulation by enhancing immune cell function, regulate blood fat and adipokines. |
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