Effects of dietary intervention versus metformin treatment on retinol binding protein 4 expressions in rats with non-alcoholic fatty liver disease

doi:10.3969/j.issn.2095-4344.2014.49.024

Abstract

Abstract:

BACKGROUND: Conflicting data have been reported regarding the expression of retinol-binding protein 4 in non-alcoholic fatty liver disease.

OBJECTIVE: To evaluate the impact of dietary intervention versus metformin treatment on expression of retinol-binding protein 4 in rats with non-alcoholic fatty liver disease.

METHODS: Fifty male Sprague-Dawley rats were randomized to six groups, including two normal control groups (rats were killed after 8 and 16 weeks of normal diet), two HFD groups (rats were killed after 8 and 16 weeks of high-fat diet), one dietary intervention group (rats were killed after 8 weeks of high-fat diet and 8 weeks of normal diet) and one metformin treatment group (rats were killed after 8 weeks of high-fat diet and 8 weeks of high-fat diet and metformin treatment). The levels of retinol-binding protein 4 in serum and biochemical indexes were detected through enzyme-linked immunosorbent assay. The expression of retinol-binding protein 4 mRNA in liver tissues was measured via western blot analysis, immunohistochemistry and RT-PCR.

RESULTS AND CONCLUSION: Non-alcoholic fatty liver disease models were successfully established by high-fat diet. Liver tissues of high-fat diet fed rats showed progressing non-alcoholic fatty liver disease histology, from non-alcoholic fatty liver to non-alcoholic steatohepatitis. Dietary intervention increased retinol-binding protein 4 expression in liver tissue as well as improving liver enzyme, dyslipidemia and insulin resistance, and alleviated impaired liver histology. Metformin treatment only alleviated hepatic steatosis caused by high-fat diet. The results indicated that retinol-binding protein 4 expression might play a role in the pathogenesis of non-alcoholic fatty liver disease. Metformin treatment can alleviate non-alcoholic fatty liver disease histology,dietary intervention should be the fundamental treatment.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

全文链接：

Key words: rat, fatty liver, retinol-binding proteins

CLC Number:

R318

Ma Hong, Guo Chun-hua, Yang Xiang-jiu, Lu Hua-dong, Chen Li-gang, Huang Yan-ling, Zhang Wen-qiang. Effects of dietary intervention versus metformin treatment on retinol binding protein 4 expressions in rats with non-alcoholic fatty liver disease [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(49): 8007-8014.

Figures/Tables 3

2.1 实验动物数量分析实验纳入大鼠50只，喂养过程顺利，全部进入结果分析。 2.2 大鼠体质量、肝湿质量及肝指数的变化与正常对照组相比，高脂饮食组大鼠体质量明显增加，并随着高脂喂养时间的延长呈逐渐上升的趋势。虽然8周高脂组较8周对照组动物体质量增长无统计学意义(P=0.067)，但8周高脂组大鼠的肝湿质量和肝指数都较8周对照组明显增加(P < 0.001)。与16周对照组大鼠相比，16周高脂组的大鼠体质量(P < 0.01)，肝湿质量和肝指数均明显增加(P < 0.001)。同时，与8周高脂组的大鼠相比，16周高脂组大鼠体质量(P < 0.001)，肝湿质量(P < 0.01)和肝指数(P < 0.05)也均显著增加。与16周高脂组相比，饮食干预组和二甲双胍治疗组大鼠体质量均呈下降的趋势，分别下降约6%和4.6%，虽然均没有统计学意义(P > 0.05)，但饮食干预和二甲双胍治疗后大鼠的肝湿质量和肝指数均出现显著下降(P < 0.05)，见表1。 2.3 血清RBP4及其他生化指标的变化与正常对照组相比，高脂饮食组大鼠血清RBP4水平有升高的趋势，但未达统计学意义。8周高脂组大鼠空腹血糖水平较8周对照组有所增高，16周高脂组大鼠空腹血糖与16周对照组大鼠相比无明显变化，但胰岛素水平及胰岛素抵抗指数均较相应对照组大鼠明显增高。分别予饮食干预和二甲双胍治疗后，虽然空腹血糖、血浆胰岛素和胰岛素抵抗变化均未达统计学意义，但胰岛素水平和胰岛素抵抗指数均呈现出下降趋势，且饮食干预组大鼠下降趋势更明显。 8周高脂组大鼠血清谷丙转氨酶水平明显高于8周对照组；16周高脂组大鼠谷丙转氨酶和谷草转氨酶水平均明显高于16周对照组大鼠；饮食干预组大鼠谷丙转氨酶和谷草转氨酶水平与16周高脂组大鼠相比均显著降低，二甲双胍治疗组大鼠谷丙转氨酶水平与16周高脂组大鼠相比显著增高(P < 0.05)。与正常对照组相比，高脂饮食组大鼠脂质代谢参数如总胆固醇、三酰甘油、低密度脂蛋白胆固醇均升高，高密度脂蛋白胆固醇降低。与16周高脂组大鼠相比，饮食干预能改善以上所有指标，但二甲双胍治疗对以上脂质代谢参数并无明显影响(表2)。 2.4 肝组织病理变化正常对照组肝组织细胞形态、小叶结构及汇管区均未见异常，肝细胞脂肪变性几不可见。高脂饮食组全肝小叶脂质沉积明显，8周高脂组大部分为小脂滴，伴有气球样变，肝窦间隙变窄；16周高脂组肝细胞均出现中、重度大泡性脂肪变，多数肝细胞排列紊乱，肿胀、气球样变，可见成群炎性细胞浸润，肝窦间隙明显变窄。与16周高脂组相比，饮食干预组肝细胞脂肪变性明显好转，脂肪变细胞减少，肝窦间隙无明显变窄，无炎性细胞浸润，无明显坏死；二甲双胍治疗组肝细胞脂肪变性好转，脂肪变细胞未见明显减少，但各细胞脂滴变小，气球样变减少，无明显坏死，肝窦间隙仍变窄。各组肝组织均未见明显肝纤维化(图1)。8周高脂组NAFLD勾当度积分4.67±0.52比8周对照组评分0.67±0.82明显增高(P < 0.001)；16周高脂组NAFLD勾当度积分6.83±1.33较16周对照组评分1.0±0.89明显升高(P < 0.001)。与16周高脂组相比，饮食干预组NAFLD勾当度积分3.00±1.26及二甲双胍治疗组评分5.33±0.52均明显降低。 2.5 肝脏RBP4免疫组织化学结果图2免疫组织化学结果显示，抗RBP4棕褐色免疫颗粒在肝细胞中特异性广泛表达，其他细胞类型包括胆管细胞、肝星状细胞、内皮细胞和炎症细胞中无表达，阳性染色位于肝细胞胞质。高脂饮食组较正常对照组肝脏RBP4蛋白表达下降，8周高脂组较8周对照组降低(t=3.674，P < 0.01)，16周高脂组较16周对照组降低(z=-3.338，P=0.001)，16周高脂组较8周高脂组略降低，但无统计学差异；与16周高脂组相比，饮食干预组RBP4表达升高(t=2.319，P < 0.05)，二甲双胍治疗组RBP4蛋白表达无太大变化(t=0.767，P > 0.05)，见图3。 2.6 肝组织RBP4的免疫组化评分与肝组织学变化的相关性分析运用Spearman相关分析法分析肝组织RBP4的免疫组化评分与肝组织学病理变化的相关性，得出结果：肝组织RBP4的免疫组织化学评分与肝脏的炎症分级呈显著负相关(r=-0.354，P=0.029)，与脂肪百分比(r=-0.281，P=0.092)、气球样变(r=0.034，P=0.843)无明显相关性。 2.7 肝脏RBP4 Western blotting检测结果该法所测肝脏RBP4蛋白表达结果与免疫组织化学法所测结果一致，正常对照组肝脏RBP4蛋白表达量高，高脂饮食组表达下降，8周高脂组较8周对照组降低(t=3.301，P < 0.01)，16周高脂组较16周对照组降低(t=5.573，P < 0.001)，16周高脂组较8周高脂组略降低，但无统计学差异；与16周高脂组相比，饮食干预组明显恢复肝组织RBP4蛋白的表达(t=4.769，P < 0.01)，二甲双胍治疗无法恢复RBP4蛋白的表达(t=-0.382，P > 0.05)，见图4。 2.8 肝脏RBP4 mRNA检测结果用RT-PCR法检测大鼠肝脏RBP4 mRNA的表达，发现肝脏RBP4 mRNA在正常对照组中表达量高，在高脂组表达量降低，8周高脂组较8周对照组降低(t=4.405，P < 0.01)，16周高脂组较16周对照组降低(z=-2.309，P < 0.05)，16周高脂组较8周高脂组进一步降低(t=6.278，P < 0.01))；与16周高脂组相比，饮食干预组明显恢复肝组织RBP4 mRNA的表达(z=-2.309，P < 0.05)，二甲双胍治疗未恢复RBP4 mRNA的表达(t=0.767，P > 0.05)，见图5。"

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