蓝莓对大鼠肝纤维化的作用及其对核转录因子-κB表达的影响

Effects of blueberry on hepatic fibrosis and expression of nuclear transcription factor-κB in rats

目的观察蓝莓对大鼠肝纤维化的作用及核转录因子-κB（NF—κB）p65在实验性大鼠肝纤维化肝组织中的表达情况。方法45只SD大鼠随机分为等渗盐水对照组（A）、模型组（B）、蓝莓汁预防组（C，15g／kg）、丹芍化纤胶囊预防组（D，1g／kg）、蓝莓汁加丹芍化纤胶囊预防组（E）。用四氯化碳（CCl4）复合因素制备大鼠肝纤维化模型，各预防组给予相应剂量蓝莓汁或（和）丹芍化纤胶囊灌胃，8周后处死大鼠。HE染色观察大鼠肝组织纤维化程度；免疫组织化学法检测肝组织NF—κBp65、转化生长因子（TGF）D1蛋白的表达情况；Westernblot法检测肝组织NF—κBD65蛋白的表达情况。多组问数据比较采用单因素方差分析，等级资料用秩和检验进行统计学分析。结果免疫组织化学检测到TGFp1蛋白主要表达于肝星状细胞及间质，A组（3．75±1．67）表达很少，B组（9．00±2．07）、C组（7．33±1．00）、D组（6．00±1．51）、E组（3．5±1．41）的相对表达水平增高，但B组肝组织内TGFD1蛋白表达水平明显高于C、D、E组（F=18．350，P〈0．05），且D组也高于E组（P〈0．05）。NF—κBp65蛋白的表达情况很复杂，在不同组中表达模式不一样（其实验数据是以表达在肝细胞中的量来统计），与B组（4．37±2．13）比较，A组（0．46±0．25）、C组（2．76±1．01）、D组（2．13±1．51）、E组（1．88±0．99）NF—κBp65蛋白相对表达水平均明显减低（F=27．490，P值〈0．05），其表达趋势与TGFp1蛋白表达一致。Westernblot检测各组大鼠肝组织中NF—κBp65蛋白：与A组比较，B、C、D、E组水平显著升高（F=96．983，P〈0．05），C、D、E组显著低于B组垆值均〈0．05），而E组低于C组（P〈0．05）。各预防组肝纤维化程度比B组减轻（T=24．19，P〈0．05）。结论蓝莓对CCl4所致大鼠肝纤维化具有预防作用，蓝莓预防肝纤维化的机制可能与抑制NF—κBp65在肝细胞中的表达、活化，进而减少其介导的TGFp1产生或活化有关。

Objective To observe the effects of blueberry and nuclear expression of transcription factor-Kb （NF-Kb） p65 in an experimental rat model of liver fibrosis. Methods Forty-five Sprague-Dawley rats were randomly divided into isotonic saline control group （A）, model group （B）, blueberry juice prevention group （C, 15 g/kg）; dan-shao-hua-xian capsule prevention group （D, 1 g/kg）; and blueberry juice ＋ dan-shao- hua-xian capsule prevention group （E）. Rat liver fibrosis model was established by covalent compound carbon tetrachloride （CCl4）. Each prevention group was given the corresponding dose of blueberry juice or （and） dan- shao-hua-xian capsule, and the rats were sacrificed after 8 weeks. The degree of liver fibrosis was evaluated by hematoxylin and eosin stain. A liver tissue of NF-kBp65 was detected by immunohistochemical method. The NF-kBp65 protein expression of liver tissue and transforming growth factor （TGF） 131 was detected by Western blot. Data of multiple groups were compared by one-way analysis of variance, and rank sum test. Results Immunohistochemistry detected that TGFβ1 protein was mainly expressed in mesenchymal origin of hepatic stellate cells. The expression level of group A （3.75 ± 1.67） was low, while those of group B （9.00 ± 2.07）, C （7.33 ± 1.00）, D （6.00 ± 1.51）, and E （3.5 ± 1.41） were high. However, the expression level of TGF-131 protein in hepatic tissues of group B was significantly higher than that of group C, D and E [group E： 3.5 ± 1.41, F = 18.350, P 〈 0.05]. In addition, group D was higher than group E （P 〈 0.05）. The expression of NF- kappa Bp65 protein was very complex, and the expression patterns in different groups were different （Statistical calculation of experimental data were based on expression of liver cells）. Compared with group B （4.37 ± 2.13）, the relative expression levels of NF-ld3p65 protein in-group A （0.46 ± 0.25）, group C （2.76 ± 1.01）, group D （2.13 ± 1.51）, group E （1.88 ± 0.99） were significantly decreased （F= 27.490,P 〈 0.05）, and the expression trend was consistent with TGFβ1 protein. Westem blot detected NF-k Bp65 protein in liver tissues of rats. Compared with group A, levels in groups B, C, D and E were significantly increased （F = 96.983, P 〈 0.05）, and groups C, D and E were significantly lower. The E group was significantly lower than the C group （F= 96.983,P 〈 0.05）, and the degree of hepatic fibrosis was lower in each prevention group than in the B group （T = 24.1, P 〈 0.05）. Conclusion Blueberries have preventive effect on CCl4-induced hepatic fibrosis in rats, and its preventive mechanism may inhibit the expression and activation of NF-kBp65 in hepatocytes, thereby reducing TGFβ1- mediated production or activation.