凝集素样氧化型低密度脂蛋白受体-1下调对糖尿病小鼠肝损伤及Ⅲ型纤维连接蛋白域蛋白5的影响研究

Effects of lectin-like oxidized low density lipoprotein receptor-1 down-regulation on liver injury and fibronectin type Ⅲ domain-containing protein 5 in diabetic mice

目的探讨凝集素样氧化型低密度脂蛋白受体-1(LOX-1)下调对糖尿病小鼠肝损伤及Ⅲ型纤维连接蛋白域蛋白5(FNDC5)的影响。方法30只7~8周龄的雄性db/db小鼠进行糖尿病造模,按照随机数字表法将其分为3组:db/db LOX-1敲减[AAV(腺相关病毒)-shLOX-1]组、db/db空载体(AAV-shCON)组和空白对照(db/db)组,每组10只;10只同龄的雄性db/m小鼠为正常对照(NC)组。采用HE染色观察小鼠肝脏的病理形态,实时荧光定量PCR(qRT-PCR)、Western blotting法检测小鼠肝脏中LOX-1、白细胞介素-6(IL-6)及FNDC5的mRNA和蛋白表达水平,酶联免疫吸附测定(ELISA)法检测血清丙氨酸转氨酶(ALT)、甘油三酯(TG)及FNDC5的水平。采用单因素方差分析进行组间比较。结果HE染色显示,与NC组相比,AAV-shLOX-1组、AAV-shCON组和db/db组小鼠肝小叶结构紊乱,肝细胞内可见大量的空泡样脂滴,呈显著的脂肪变性;与AAV-shCON组和db/db组相比,AAV-shLOX-1组小鼠肝细胞中脂滴数量明显减少,脂肪变性明显减轻。qRT-PCR和Western blotting结果显示,与NC组相比,AAV-shLOX-1组、AAV-shCON组和db/db组小鼠肝脏中LOX-1、IL-6的mRNA和蛋白表达均升高,FNDC5的mRNA和蛋白表达均降低;与AAV-shLOX-1组相比,AAV-shCON组和db/db组小鼠肝脏中LOX-1、IL-6的mRNA和蛋白表达均升高,FNDC5的mRNA和蛋白表达均降低,差异均有统计学意义(P<0.05)。与NC组相比,AAV-shLOX-1组、AAV-shCON组和db/db组小鼠血清中ALT、TG均升高,FNDC5降低;与AAV-shLOX-1组相比,AAV-shCON组和db/db组小鼠血清中ALT、TG均升高,FNDC5均降低,差异均有统计学意义(P<0.05)。结论LOX-1参与调控了db/db小鼠肝损伤和FNDC5的变化,LOX-1下调可减轻db/db小鼠肝脏的脂肪变性及炎症反应,提高db/db小鼠FNDC5的组织和血清水平,糖尿病小鼠的肝损伤可能与LOX-1/FNDC5的调控有关。

Objective To investigate the effects of lectin-like oxidized low density lipoprotein receptor-1(LOX-1)on liver injury and fibronectin typeⅢdomain-containing protein 5(FNDC5)in diabetic mice.Methods Thirty 7-8 week-old male db/db mice were divided into 3 groups according to the random number table,db/db LOX-1 knockdown[AAV(adeno-associated virus)-shLOX-1]group,db/db negative control virus(AAV-shCON)and blank control group(db/db group),10 in every group.Ten male db/m mice of the same age were used as normal control group(NC).The HE staining was used to observe the pathological morphology of mouse liver.The mRNA and protein expression levels of LOX-1,interleukin-6(IL-6)and FNDC5 in liver of mice were detected by the quantitative real-time PCR(qRT-PCR)and Western blotting.Serum levels of glutamic pyruvic transaminase(ALT),triglyceride(TG)and FNDC5 were detected by the enzyme-linked immunosorbent assay(ELISA).One-way analysis of variance was used for the comparison between groups.Results HE staining showed that compared with NC group,the hepatic lobule structure of mice in the AAV-shLOX-1,AAV-shCON and db/db groups were disordered,a large number of vacuolate-like lipid droplets were seen in the hepatocytes,showing significant steatosis.Compared with AAV-shCON and db/db groups,the number of lipid droplets in hepatocytes of mice in AAV-shLOX-1 group was reduced,and steatosis was reduced.Compared with the NC group,the mRNA and protein expressions of LOX-1 and IL-6 in the liver of mice in the AAV-shLOX-1,AAV-shCON and db/db groups were increased,while the mRNA and protein expressions of FNDC5 were decreased.Compared with AAV-shLOX-1 group,the mRNA and protein expressions of LOX-1 and IL-6 in liver of mice in AAV-shCON and db/db groups were increased,while the mRNA and protein expressions of FNDC5 were decreased,and the differences were statistically significant(P<0.05).Compared with NC group,ALT and TG were increased and FNDC5 was decreased in AAV-shLOX-1,AAV-shCON and db/db groups.Compared with AAV-shLOX-1 group,ALT and TG were increased and FNDC5 was decreased in AAV-shCON and db/db groups,and the differences were statistically significant(P<0.05).Conclusions LOX-1 was involved in the regulation of liver injury and the change of FNDC5 in db/db mice.LOX-1 down-regulation reduced liver steatosis and inflammation in db/db mice and improve the tissue and serum levels of FNDC5 in db/db mice.The liver injury in diabetic mice might be related to the regulation of LOX-1/FNDC5.