南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (7): 1037-1043.doi: 10.12122/j.issn.1673-4254.2021.07.10

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甲状旁腺激素相关蛋白加重蛋氨酸胆碱缺乏饲料诱导的小鼠非酒精性脂肪性肝病进展

覃碧艳，刁 娜，白 岚   

  • 出版日期:2021-07-20 发布日期:2021-07-19

Parathyroid hormone-related protein aggravates nonalcoholic fatty liver disease induced by methionine choline-deficient diet in mice

  • Online:2021-07-20 Published:2021-07-19

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摘要： 目的 研究甲状旁腺激素相关蛋白（PTHrP）对蛋氨酸胆碱缺乏饲料（MCD）诱导的小鼠非酒精性脂肪性肝病（NAFLD）的影响。方法 构建小鼠 NAFLD 模型并分为空白对照组（普通饲料喂养 4 周）、AAV-Vehicle 组（注射空载腺相关病毒 AAV-Vehicle）和AAV-PTHrP组（注射PTHrP 过表达腺相关病毒）。实验期间监测小鼠体质量。收集小鼠肝脏组织进行HE染色、油红染色和天狼星红染色评估肝脏组织病理学改变；检测肝脏及血清中谷草转氨酶、谷丙转氨酶、甘油三酯和游离脂肪酸浓度评估小鼠肝脏损伤及脂肪代谢水平。250 μmol/LFFAs诱导小鼠正常肝细胞和人正常肝细胞24 h构建NAFLD细胞。根据有无PTHrP处理分为PTHrP组、对照组。油红及尼罗红染色检测细胞内脂质沉积程度；CCK8试剂盒检测PTHrP对脂肪变性肝细胞的毒性作用。结果 动物实验：对比AAV-Vehicle组，AAV-PTHrP组小鼠体重下降更为快速；AAV-PTHrP组小鼠肝脏谷草转氨酶（P<0.05）、谷丙转氨酶（P<0.05）、甘油三酯（P<0.01）、游离脂肪酸（P<0.05）水平，NAS评分以及SAF评分均显著升高（P<0.01， P<0.05），肝脏脂滴堆积更为明显。细胞实验：同时加入PTHrP，小鼠及人NAFLD细胞模型的脂质沉积更为明显（P<0.01），且细胞活性下降（P<0.05）。结论 PTHrP可能通过促进肝细胞脂滴沉积，加重MCD诱导的小鼠NAFLD。

关键词: 非酒精性脂肪性肝病；甲状旁腺激素相关蛋白；蛋氨酸胆碱缺乏饲料

Abstract: Objective To study the effect of parathyroid hormone-related protein (PTHrP) on nonalcoholic fatty liver disease (NAFLD) induced by methionine choline-deficient diet (MCD) in mice. Methods Twelve male C57BL/6J mice were randomized into blank control group, vehicle group and PTHrP group (n=4). The mice in vehicle group and PTHrP group received injections of a control adeno-associated virus (AAV) vector and an AVV vector carrying PTHrP (AAV-PTHrP) gene, respectively, followed one week later by MCD feeding for 3 weeks; the mice in the blank control were fed a normal diet for 4 weeks. Body weight changes of the mice were monitored during the experiment. At the end of the experiment, liver tissues were harvested from the mice for histological analysis using HE staining, oil red O staining, and Sirius red staining. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride, and free fatty acids (FFAs) in the liver and serum were detected to assess hepatic impairment and lipid metabolism of the mice. Cell models of NAFLD were established in mouse and human normal liver cells by treatment with 250 μmol/L FFAs for 24 h, and the effect of AAV-PTHrP on lipid deposition and viability of the cells were tested using Oil Red O and Nile red staining and CCK8 assay. Results Treatment with AAV-PTHrP, as compared with the control AVV vector, caused more rapid reduction of body weight in mice with MCD feeding and significantly increased the levels of AST (P<0.05), ALT (P<0.05), triglyceride (P<0.01) and FFA (P<0.05) in the liver and the scores of NAS (P<0.01) and SAF (P<0.05). HE and Oil red O staining of the liver tissue revealed obvious lipid deposition after MCD feeding, which was more serious in PTHrP group. In the cell experiment, FFAs induced steatosis in both mouse and human hepatocytes, and treatment with PTHrP increased the accumulation of lipid droplets and lowered the viability of the cell model of NAFLD (P<0.01 or 0.05). Conclusion PTHrP may aggravate MCD-induced NAFLD in mice by promoting the deposition of lipid droplets in the hepatocytes.

Key words: nonalcoholic fatty liver disease; parathyroid hormone-related protein; methionine choline-deficient diet