大鼠非酒精性脂肪性肝病与内质网应激关系的研究

Study on relationship between fat-rich diet-induced nonalcoholic fatty liver disease and endoplasmic reticulum stress in rats

目的通过建立大鼠非酒精性脂肪性肝病（NAFLD）模型，探讨内质网应激（ERS）在NAFLD中的作用。方法36只雄性SD大鼠分为对照1组（n=8）、对照2组（n=8）、模型1组（n=10）和模型2组（n=10）。对照组大鼠给予普通饲料，模型组大鼠则喂饲高脂饲料。分别于第12周末处死对照1组和模型1组，第20周末处死对照2组和模型2组大鼠。测定血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、γ-谷氨酰转移酶（GGT）、碱性磷酸酶（ALP）、总蛋白（TP）、三酰甘油（TG）、总胆固醇（TC）、低密度脂蛋白（LDL）及高密度脂蛋白（HDL）水平。H—E和Masson染色观察肝组织脂肪变、炎症和纤维化的变化。实时定量PCR测定肝组织内GRP78、CHOP及procaspase-12 mRNA表达情况。Western印迹法检测肝组织内procaspase-12和caspase-12蛋白的变化。结果第12周末，模型1组大鼠ALT、AST、ALP、TC及LDL水平均较对照1组显著增高（P〈0．01），而HDL水平显著降低（P〈0．01）；第20周末模型2组大鼠TC和LDL水平较模型1组显著增加（P〈0．05）。与对照1组相比，模型1组肝组织的脂肪变和炎症程度均明显增加（P值均〈0．01）；模型2组肝组织的脂肪变、炎症程度和纤维化分期较对照2组均明显增加（P值均〈0．01），炎症程度和纤维化分期较模型1组明显加重（P值分别〈0．05和0．01）。模型组大鼠肝组织GRP78、CHOP及procaspase-12 mRNA水平在第12周末及第20周末时与对照组比较差异均无统计学意义（P值均〉0．05）。模型组caspase-12及其前体蛋白水平与对照组相比也无显著变化（P值均〉0．05）。结论成功建立高脂饮食大鼠NAFLD模型。研究中未发现ERS，提示此模型中ERS诱导的肝脏损伤可能未参与NAFLD的发病机制。

Objective To establish the nonalcoholic fatty liver disease（NAFLD） model in SD rats and to investigate if endoplasmic reticulum stress （ERS） plays a role in the pathogenesis of NAFLD. Methods A total of 36 male SD rats were randomly divided into 4 groups： normal control group 1（N1,n=8）, normal control group 2 （N2,n=8）, model group 1（M1,n=10） and model group 2（M2,n=10）. The two control groups were fed with normal diets, whereas the two model groups were provided diets enriched in fat （10% lard oil and 2% cholesterol）. The rats in N1 and M1 groups were sacrificed at the 12th week, and those in N2 and M2 groups were sacrificed at the 20th week. The serum alanine aminotransferase （ALT）, aspartate aminotransferase（AST）, alkaline phosphatase （ALP）, total protein （TP）, triglycerides （TG）, total cholesterol （TC）, low-density lipoprotein （LDL）, high-density lipoprotein （HDL） were measured. The steatosis, immflamation and fibrosis of the liver were observed with HE and Masson staining. The expressions of GRP78, CHOP and procaspase-12 mRNA were tested using real-time PCR and the activation of procaspase-12 protein was detected by Western blot. Results At the 12th week, the liver index and the serum levels of ALT, AST,ALP,TP,TC,LDL in M1 group were significantly higher than those in N1 group （P〈0.01）, but HDL level was significantly lower in M1 group than that in N1 group （P〈0.01）. At the 20th week, the concentrations of TC and LDL in M2 group were significantly increased in comparison with N2 group. The histochemical study revealed that the hepatic steatosis and inflammation in M1 and M2 groups were more serious than those in N1 and N2 goups（P〈0.01）. The slight fibrosis was seen in M2 group. At the 12th and 20th weeks, the expressions of GRP78,CHOP and procaspase-12 mRNA and protein in M1 and M2 groups did not differ in N1 and N2 groups. Conclusions The fat-rich diet might successfully induce NAFLD in rats and there is no ERS observed in the study, which suggests that ERS may not be involved in the pathogenesis of NAFLD.