外源性脂联素对大鼠非酒精性脂肪肝病影响的研究

Effect of exogenous adiponectin on nonalcoholic fatty liver in rats

目的探讨外源性脂联素对大鼠非酒精性脂肪肝病(NAFLD)的影响。方法成年SD(Sprague Dawley)远交群大鼠82只,分别予普通饮食(普通饮食组,n=12)、高脂饮食(高脂饮食组,n=70)。所有大鼠喂养4w,并处死2只大鼠做肝脏病理HE染色,以确定造模成功。将造模成功大鼠随机分为高脂饮食对照组(n=10)、脂联素给药组(10、20、30μg·kg^(-1)·d^(-1))腹腔注射,每组10只,持续4w,分组处理期间,大鼠仍继续给予高脂饮食。4w后以3%戊巴比妥腹腔注射麻醉(2mL/kg),腹主动脉采血。每只大鼠取50g肝脏备用,肝组织石蜡切片苏木素-伊红(HE)常规染色。采用酶联免疫吸附试验(ELISA)检测血肿瘤坏死因子α(TNF-α)水平、检测各组大叔血清总胆固醇游离脂肪酸(TC)、甘油三脂(TG)、血丙氨酸转氨酶(ALT)水平。RT-PCR法检测AdipoR2mRNA表达。结果高脂饮食组大鼠肝细胞肿胀,体积较正常明显增大,出现脂肪变性,胞质内可见大小不等的脂滴空泡,以大泡性脂肪滴为主,汇管区有以单核、淋巴细胞为主的炎症细胞浸润,部分出现点状坏死和灶状坏死,已进展为NAFLD阶段,大鼠NAFLD的模型制备成功。高脂饮食对照组、脂联素10μg·kg^(-1)·d^(-1)组、脂联素20μg·kg^(-1)·d^(-1)组、脂联素30μg·kg^(-1)·d^(-1)组体质量、TG、TC、ALT高于普通饮食组(P<0.05)。脂联素20μg·kg^(-1)·d^(-1)组、脂联素30μg·kg^(-1)·d^(-1)组体质量、TG、TC、ALT低于高脂饮食对照组(P<0.05)。随着脂联素浓度的升高,大鼠体质量、TG、TC、ALT逐渐增加,但各给药组间以上指标差异无统计学意义(P>0.05)。与高脂饮食对照组比较,脂联素10μg·kg^(-1)·d^(-1)组、20μg·kg^(-1)·d^(-1)组、30μg·kg^(-1)·d^(-1)组AdipoR2基因表达均增加,差异有统计学意义(P<0.05)。脂联素10μg·kg^(-1)·d^(-1)组AdipoR2mRNA表达量明显高于其他脂联素给药组(P<0.05)。高脂饮食对照组TNF-α明显升高,脂联素各给药组TNF-α低于高脂饮食对照组(P<0.05)。结论高脂饮食能使大鼠发生肝脂肪变性,具有脂肪细胞的特征,同时血脂、TNF-α水平升高。脂联素能降低大鼠血脂、TNF-α水平,改善肝功能,升高adipoR2 mRNA水平,脂联素可能在NAFLD的治疗中发挥作用。

Objective To investigate the effect of exogenous adiponectin on nonalcoholic fatty liver disease（NAFLD）in rats.Methods 82 adult SD（Sprague Dawley）rats were provided with normal diet（normal diet group,n =12）and high fat diet（high fat diet group,n =70）.All rats were fed for 4weeks and two rats were sacrificed for liver pathologic HE staining to make sure the success of the model.Adiponectin（10,20,30μg·kg-1·d-1）were injected intraperitoneally for 4weeks（n =10）,the rats were still given high-fat diet during the treatment.After 4weeks,3% pentobarbital intraperitoneal injection was used for anesthesia（2mL/kg）,abdominal aorta blood were collected.Each rat received 50 g of liver for liver,paraffin sections of liver tissue hematoxylin-eosin（HE）was routinely stained.Levels of total cholesterol（TC）,triglyceride（TG）,serum alanine aminotransferase（ALT）,tumor necrosis factor alpha（TNF-α）were measured.The expression of AdipoR2 mRNA was detected by RT-PCR.Results The hepatocyte swelling in the high fat diet group was significantly higher than that in the normal diet group.There were lipid vacuoles present in the cytoplasm and the size of the lipid droplets,presence of Lymphocyte-like inflammatory cell infiltration,part of the occurrence of point necrosis and focal necrosis,which progressed to NAFLD stage.All the presence indicates that rat NAFLD model was successfully established.The body weight,TG,TC and ALT in the high fat diet group,adiponectin 10μg·kg-1·d-1 group,adiponectin20μg·kg-1·d-1 group,adiponectin 30μg·kg-1·d-1 group were significantly higher than those in the normal diet group（P〈0.05）.TG,TC and ALT were significantly lower than those in the normal diet group（P〈0.05）.With the increase of adiponectin concentration,the body weight,TG,TC and ALT of rats increased gradually,but no significant difference was found between three groups（P〈0.05）.Compared with the high-fat diet control group,AdipoR2 gene expression in adiponectin 10μg·kg-1·d-1group,20μg·kg-1·d-1 group,30μg·kg-1·d-1 group significantly increased（P〈0.05）.The expression of AdipoR2 mRNA in adiponectin 30μg·kg-1·d-1 group was significantly higher than that in other adiponectin group（P〈0.05）.The levels of TNF-αin the high-fat diet group were significantly higher than those in the high-fat diet group（P〈0.05）.Conclusion High fat diet can make liver steatosis in rats,characterized by fat cells,while blood lipids,TNF-αlevels increased.Adiponectin can reduce blood lipid,TNF-αlevels,improving liver function and increasing AdipoR2 mRNA levels,which may play a role in the treatment of NAFLD.