甘草酸单铵盐预防油酸钠致奶牛原代肝细胞损伤作用的研究

肝细胞损伤是围产期奶牛脂肪肝病的主要病理特征。本研究旨在探讨甘草酸单铵盐（monoammonium-glycyrrhizinate，MAG）对油酸钠诱导奶牛原代肝细胞损伤的影响。试验以体外培养的肝细胞为研究对象，根据处理不同分为对照组（未做处理的原代肝细胞）和试验组（模型组、MAG组：分别添加0、0.25 mg·L^-1的MAG对原代肝细胞预处理12 h，再使用浓度为0.25 mmol·L^-1的油酸钠诱导肝细胞脂肪变性），随后用CCK-8法检测肝细胞活性、DAPI染色统计凋亡率，紫外比色法检测上清中ALT、AST的含量，油红O染色结合imageJ面积统计法分析细胞内脂肪沉积水平，实时荧光定量PCR（RT-qPCR）检测样品肝细胞脂代谢相关基因PPARα、SREBP-1c、ChREBP、CPT1、CPT2、MTP和炎症相关基因TNF-α、NF-κB、IL-1β、IL-6、IL-8的mRNA表达水平。试验结果显示，经油酸钠诱导后，0.25 mg·L^-1的MAG预处理的肝细胞活性显著高于未经处理的模型组（P<0.05），凋亡率明显降低（P<0.05），培养上清中ALT、AST的释放水平有所降低（P<0.05）。MAG组细胞内脂滴数量和平均脂滴面积较模型组明显减少（P<0.05），脂代谢基因ChREBP、PPARα、MTP和炎症相关基因TNF-α、IL-1β、NF-κB、IL-6、IL-8的mRNA表达水平均显著下调（P<0.05）。结果表明，MAG能通过抑制脂代谢和炎症因子相关基因的表达，有效减少油酸钠诱导的肝细胞内脂肪的沉积，缓解肝细胞损伤。

Preventive Effect of Monoammonium Glycyrrhizinate on the Injury Induced by Sodium Oleate in Hepatocytes of Cows in vitro

Hepatocytes injury is the main pathological feature of fatty liver disease in cows during the perinatal period. The aim of this study was to investigate the preventive effect of monoammonium glycyrrhizinate (MAG) on cell damage induced by sodium oleate in hepatocytes of cows. Primary hepatocytes of cows cultured in vitro were divided into 3 groups according to different treatments:control group (untreated primary hepatocytes) and 2 experimental groups (model group, MAG group:add 0 mg·L^-1, 0.25 mg·L^-1 MAG in culture media respectively, for 12 h and then use sodium oleate at a concentration of 0.25 mmol·L^-1 to induce hepatocytes steatosis), and then the activity of hepatocytes were detected by CCK-8, the cell apoptosis rate was calculated by DAPI fluorescence staining, the content of ALT and AST in the culture solution were detected by ultraviolet colorimetry, the levels of intracellular fat deposition were analyzed by oil red O staining and imageJ. Real-time fluorescence quantification (RT-qPCR) was used to detect the mRNA expression of lipid metabolism-related genes PPARα,SREBP-1c,ChREBP,CPT1,CPT2,MTP and inflammation-related genes TNF-α,NF-κB,IL-1β,IL-6,IL-8. The experimental results showed that hepatocytes preconditioning with 0.25 mg·L^-1 MAG could improve the activity of model cells (P<0.05), and inhibit the apoptosis rate of model cells effectively (P<0.05). The release levels of ALT and AST in the cell culture supernatant of the MAG group were lower than those of the model group (P<0.05). At the same time, compared with the model group, the number of intracellular lipid droplets and the average lipid droplet area of hepatocytes treated with MAG were significantly reduced (P<0.05). And then the levels of lipid metabolism genes ChREBP, PPARα, MTP and inflammation related genes TNF-α, IL-1β, NF-κB, IL-6, IL-8 mRNA were significantly down-regulated in MAG group than the model group (P<0.05). The results showed that MAG could alleviate the impact and relieve lipid deposition induced by sodium oleate in hepatocytes by inhibiting the expression of genes related to lipid metabolism and inflammatory cytokines, which indicated that it could provide protection for the liver.