炎症对C57BL／6J小鼠肝脏胆固醇积聚及纤维化的影响

Effect of inflammatory stress on hepatic cholesterol accumulation and hepatic fibrosis in C57BL/6Jmice

目的观察炎症对小鼠肝脏内胆固醇蓄积、内质网应激及纤维化的影响。方法将8周龄雄性C57BL／6J小鼠随机分为对照组∞=6）和炎症组∞=6），均以高脂饮食，炎症组小鼠隔天皮下注射0．5ml10％酪蛋白以建立慢性炎症模型，对照组注射等量等渗盐水，14周处死。收集血清和肝组织样本，测定血清及肝组织中炎症因子及脂质水平；油红O、天狼猩红和Masson三色染色观察肝脂质沉积、形态改变以及纤维化程度；荧光实时定量PeR检测肝组织单核细胞趋化蛋白1、肿瘤坏死因子（TNF）仅、胆固醇调节元件结合蛋白（SREBP）2、低密度脂蛋白受体、3-羟-3-甲基戊二酰辅酶A还原酶、活化转录因子6、葡萄糖调节蛋白（GRP）78、骨形成蛋白（BMP）7、转化生长因子（TGF）D、层黏连蛋白、I／Ⅳ型胶原的mRNA水平。对数据进行两样本均数t检验或近似t检验。结果血清中，炎症组白细胞介素（IL）6的水平[（32．41土7．42）pg／m1]高于对照组[（12．55±4．75）pg／nat]，f=5．522，P〈0．01；总胆固醇水平为（10．62±0．48）mmoL／L，低于对照组的（14．82土1．56）mmol／L，t=6．303，尸〈0．01。肝组织中，炎症组TNFa的mRNA相对表达水平为2．12±0．72，高于对照组的1．05±0．35，t=3．132，P〈0．01；总胆固醇水平为（23．21±8．75）μg／mg，高于对照组的（12．10±2．57）ug／mg，t=2．984，P〈0．05。油红O染色表明，炎症组肝脏脂滴沉积异常增多；天狼猩红和masson三色染色可见炎症组有更为明显的肝纤维化改变。荧光定量PCR检测表明炎症组SREBP-2、GRP78的mRNA相对表达水平分别为2．63±0．13、2．21±0．99，高于对照组的1．01±0．19、1．07土0．47，t值分别为15．641和2．031，P值均〈0．05。TGFβ、I型胶原的mRNA相对表达水平分别为1．38±0．28、1．71士0．51，高于对照组的1．01±0．14、1．02±0．27，f值分别为3．032和3．152，P值均〈0．05。BMP-7的mRNA相对表达水平为0．55±0．25，低于对照组的1．01±0．15，f=3．765，P〈0．01。结论慢性炎症可以明显促进小鼠肝脏胆固醇沉积而加重肝细胞损害，刺激内质网应激，从而增加促纤维化因子表达、抑制抗纤维化因子表达，促进肝纤维化发生。

Objective To investigate whether inflammatory stress exacerbates hepatic cholesterol accumulation and liver fibrosis using a C57BL/6J mouse model of chronic inflammation. Methods Twelve male C57BL/6J mice were given a high-fat diet （15.0% fat, 1.25% cholesterol, 0.5% cholic acid） and randomly assigned to the normal control group （n = 6; subcutaneously injected with 0.5 mL of isotonic saline, every other day for 14 weeks） or the chronic inflammation model group （n = 6; subcutaneously injected with of 0.5 mL of 10% casein, every other day for 14 weeks）. At the end of week 14, the animals were sacrificed and blood was collected from theleft ventricle for serological analysis of inflammatory markers and lipid profile, including serum amyloid A （SAA）, interleukin-6 （IL-6）, total cholesterol （TC） and free cholesterol （FC）, low-density lipoprotein （LDL）, and high- density lipoprotein （HDL））. Extracted liver tissues were divided for use in histological analysis （lipid accumulation and fibrosis evaluated by Oil Red O, Sirius red and Masson＇s trichrome staining） and quantitative fluorescence real-time PCR （to measure b-actin normalized expression of TNF-q MCP1, SREBP-2, LDLr, HMGCoA-r, ATF-6, GRP78, BMP-7, TGF-13, and collagens type I and type IV）. Comparisons between groups were made by the two- samples t-test or Satterthwaite t-approximation test, collagen type I and type IV. Results Compared to the normal control group, the inflammation model group showed elevated serum IL-6 （12.55± 4.75 vs. 32.41±7.42 pg/ mL, P 〈 0.01）, reduced serum TC （14.82 ＋ 1.56 vs. 10.62 ＋ 0.48 mmol/L, P 〈 0.01）, up-regulated hepatic TNF-ct mRNA expression （1.05±0.35 vs. 2.12±0.72, P 〈 0.01）, and elevated hepatic TC （12.10 ±2.57 vs. 23.21 ± 8.75 mmol/L, P 〈 0.05）. In addition, the inflammation group showed abnormal lipid deposition, and increased and thickened reticular fibers. The livers of the infammafion group also showed up-regulated mRNA expression of SREBP-2 （normal control： 1.01± 0.19 vs. 2.63±0.13, P〈 0.05）, GRP78 （1.07± 0.47 vs. 2.21±0.99, P 〈 0.05）, TGF-13 （1.01±0.14 vs. 1.38 ± 0.28,P〈 0.05）, and collagen type I （1.02±0.27 vs. 1.71±0.51, P〈 0.05） and down-regulation of BMP-7 （ 1.01±0.15 vs. 0.55± 0.25, P 〈 0.01）. Conclusion Activation of the inflammatory system exacerbates hepatic cholesterol accumulation and hepatic fibrosis in C57BL/6J mice.