畜牧兽医学报  2020, Vol. 51 Issue (12): 3076-3086. DOI: 10.11843/j.issn.0366-6964.2020.12.017    PDF    
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
张才1,2, 邵琦1,2, 徐文浩1,2, 贾哲3, 马璐1,2, 田超1,4, 杨自军1     
1. 河南科技大学动物科技学院, 洛阳 471023;
2. 河南省动物福利与健康养殖国际联合实验室, 洛阳 471023;
3. 皖西学院, 六安 237000;
4. 河南农业职业学院, 郑州 451450
摘要: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PPARαSREBP-1c、ChREBPCPT1、CPT2、MTP和炎症相关基因TNF-αNF-κBIL-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ChREBPPPARαMTP和炎症相关基因TNF-αIL-1β、NF-κBIL-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。
关键词甘草酸单铵盐    肝细胞    油酸钠    炎症    脂肪代谢    
Preventive Effect of Monoammonium Glycyrrhizinate on the Injury Induced by Sodium Oleate in Hepatocytes of Cows in vitro
ZHANG Cai1,2, SHAO Qi1,2, XU Wenhao1,2, JIA Zhe3, MA Lu1,2, TIAN Chao1,4, YANG Zijun1     
1. College of Animal Science&Technology, Henan University of Science and Technology, Luoyang 471023, China;
2. Henan International Joint Laboratory of Animal Welfare and Health Breeding, Luoyang 471023, China;
3. West Anhui University, Liuan 237000, China;
4. Henan Vocational College of Agriculture, Zhengzhou 451450, China
Abstract: 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.
Key words: monoammonium glycyrrhizinate    hepatocytes    sodium oleate    inflammation    lipid metabolism    

脂肪肝病是围产期奶牛发病率较高的营养代谢性疾病,过量的游离脂肪酸在肝细胞中沉积导致的肝细胞损伤是其主要病理特征。据报道,美国奶牛中度或重度脂肪肝发病率可达35%~66%,我国奶牛脂肪肝发病率则超过30%[1],如果根据细胞学检测标准判断[2],脂肪肝发病率还会更高。奶牛脂肪肝病的危害在于降低体况评分、产奶量、繁殖力和缩短使用年限。因此,预防奶牛脂肪肝病的发生对降低围产期疾病的发病率,延长奶牛使用年限具有重要意义[3]。为保证奶牛高产稳产,在开始泌乳的第1个月提高奶牛的肝功能最为关键[4]。有研究表明,奶牛日粮中添加抗氧化剂能够提高亚临床酮病奶牛的恢复率,改善奶牛脂代谢和健康状况[5]。甘草及其提取物因其具有非常强大的抗氧化和清除自由基活性的功能[6-9],而被广泛应用于临床治疗多种肝性疾病。其中,甘草酸制剂可保护肝细胞,降低血清中丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)和血清胆红素(TBIL)水平[10-12]。另外,甘草酸还可通过对肝代谢酶基因的调控,有效改善肝脂肪变性和通过其抗炎症功效减轻细胞脂肪变性引起的炎症损伤[13]。而这些药理作用恰恰为应用甘草酸制剂改善围产期奶牛机体高氧化应激状态、降低肝炎症损伤、提高围产期奶牛适应性和保证围产期奶牛健康提供了可能。研究表明,体外培养的原代肝细胞是研究药物代谢、肝疾病发病机制的良好工具[12, 14]。因此,本研究用油酸钠诱导原代奶牛肝细胞,模拟脂质在奶牛肝细胞中的沉积[14],并用甘草酸单铵盐(MAG)对细胞进行预处理,观察MAG对奶牛原代肝细胞损伤的影响,以期为探索MAG在预防奶牛脂肪肝病中的应用打下试验基础。

1 材料与方法 1.1 试验动物

试验所需1日龄健康荷斯坦犊牛(45~50 kg), 购自洛阳某牛场,试验已通过河南科技大学学术伦理委员会许可。

1.2 主要试剂和材料

油酸钠、油红O、牛胰岛素、双抗购自Sigma公司;RPMI 1640、胎牛血清、IV型胶原酶购自Gibco公司;ALT、AST试剂盒购自南京建成公司;Cell Counting Kit-8(CCK-8)购自DojinDO公司;反转录试剂盒、实时荧光定量PCR试剂盒均购自TaKaRa公司;DAPI(4, 6-二脒基-2-苯基吲哚二盐酸盐)、苏木精、甘油明胶购自北京索莱宝公司;甘草酸单铵盐注射液购自多多药业有限公司。

1.3 仪器设备

Scientific Multiskan FC型酶标仪(美国Thermo公司),超微量核酸蛋白测定仪(Thermo公司),CFX96荧光定量PCR仪(美国伯乐公司),倒置显微镜CKX41(日本奥林巴斯公司),BSC-1000 II B2型生物安全柜(浙江苏净净化有限公司),CO2培养箱(德国Thermo公司),台式高速冷冻离心机(德国Thermo Fisher),超低温冰箱(日本SANYO公司)。

1.4 原代肝细胞的分离培养

注射戊巴比妥钠将犊牛麻醉后,无菌采取犊牛肝尾状突,使用改良的两步灌流法获取奶牛肝细胞[15],并使用Percoll工作液进行纯化,将分离得到的肝细胞于细胞培养箱中培养。

1.5 试验分组和处理

原代肝细胞在5%CO2、37 ℃饱和湿度条件下培养72 h后,将肝细胞分为对照组(使用无血清、无双抗的RPMI 1640培养基,持续饥饿培养);脂肪肝模型组(饥饿处理12 h后使用0.25 mmol·L-1的油酸钠诱导9 h);MAG组(饥饿处理,处理时培养基含0.25 mg·L-1的MAG,12 h后使用0.25 mmol·L-1的油酸钠诱导9 h),每组设6个重复。倒置相差显微镜记录诱导前后细胞状态,诱导完成后收集培养上清和细胞样品。

1.6 MAG对肝细胞活力和凋亡的影响

将肝细胞接种在96孔板上,按照“1.5”所述将细胞进行分组诱导培养,按CCK-8细胞增殖-毒性检测试剂盒说明书操作检测细胞活力。用10 μg·mL-1的DAPI染色液对肝细胞进行活染30 min,倒置荧光显微镜(同一曝光强度下,每孔随机选取5个视野)拍照,image J计数统计凋亡细胞比率(凋亡率=凋亡细胞个数/视野总细胞数*100%)。

1.7 肝细胞AST和ALT释放水平的测定

试验处理结束后,收集各组细胞培养液,按试剂盒说明测定培养液中AST、ALT的含量。

1.8 油红O染色检测肝细胞内脂肪沉积情况

试验处理结束后,使用4 ℃预冷PBS对去除培养基的细胞清洗3遍,4%的多聚甲醛固定30 min,PBS再清洗3遍,用中性滤纸过滤后的油红工作液进行染色处理(加入体积以覆盖板底为宜),避光染色45 min后用60%异丙醇进行脱色处理。双蒸水清洗2遍,苏木精复染1 min,PBS漂洗,甘油明胶封片观察。

单个细胞内脂滴面积的计数:每组样品在相同条件下制备3个细胞爬片,每个细胞爬片随机选取3个视野,拍照之后使用image J软件对每个视野细胞个数和脂滴总面积进行统计(脂滴面积参数见表 1),然后使用Excel得出平均每个细胞内脂滴的面积。

表 1 脂滴面积参数表 Table 1 Lipid droplet area parameter
1.9 实时荧光定量PCR检测肝细胞脂代谢相关基因mRNA的表达

试验处理结束后,使用4 ℃预冷PBS对去除培养基的细胞清洗1~2遍,加入Trizol并轻微吹打收集细胞,参照TaKaRa总RNA提取试剂盒说明书的方法和步骤进行操作,提取总RNA并反转录。以β-actin为内参对照,采用荧光定量PCR方法对过氧化物酶增殖激活的受体α(PPARα)、固醇调节元件结合蛋白-1c(SREBP-1c)、碳水化合物反应元件结合蛋白(ChREBP)、肉毒碱棕榈酰基转移酶1(CPT1)、肉毒碱棕榈酰基转移酶2(CPT2)和微粒体甘油三酯转移蛋白(MTP)的转录水平进行测定。引物序列见表 2

表 2 脂代谢相关引物序列及靶基因大小 Table 2 Lipid metabolism-related primer sequences and target gene sizes
1.10 实时荧光定量PCR检测肝细胞炎症因子相关基因mRNA的表达

样品收集同上“1.9”。参照TaKaRa总RNA提取试剂盒说明书的方法和步骤进行操作,提取总RNA并反转录。以GAPDH为内参对照,采用荧光定量PCR方法对肿瘤坏死因子(TNF-α)、白细胞介素1β(IL-1β)、核因子κB(NF-κB)、白细胞介素6(IL-6)、白细胞介素8(IL-8)的转录水平进行测定。序列见表 3

表 3 炎症相关引物序列及靶基因大小 Table 3 Inflammation-related primer sequences and target gene sizes
1.11 数据处理

试验数据以“平均数±标准差”表示,采用SPSS 20.0统计学软件对试验原始数据进行统计分析,用单因素方差分析进行多重比较(Duncan),P<0.05为差异有统计学意义。

2 结果 2.1 MAG对油酸钠诱导的奶牛原代肝细胞活性的影响

试验分离的奶牛原代肝细胞在接种4 h后贴壁,继续培养12 h可见细胞形态完全伸展,呈铺路石样紧密排列,与以往试验结果一致[15]。如图 1-Ⅰ所示,MAG预处理12 h的肝细胞贴壁良好,几乎完全汇合,呈单层整齐排列。经油酸钠诱导9 h后,不同组别间细胞形态出现差异:对照组图细胞(图 1-Ⅱ-d)有少量脱落,细胞形态正常,折光率高;模型组(图 1-Ⅱ-e)细胞间隙变大,边界模糊,大面积脱落死亡;MAG组细胞(图 1-Ⅱ-f)脱落较少,细胞间隙仅少量增加,细胞边界清晰,结构相对完整。CCK-8法检测各组细胞活性比率可见(图 1-j)脂肪肝模型组细胞活性显著低于对照组,而使用0.25 mg·L-1 MAG培养基进行预处理的肝细胞细胞活性较模型组显著升高(P < 0.05),但与对照组无显著差异。DAPI染色(图 1-Ⅲ)可见模型组凋亡细胞(视野中小亮点)个数明显高于对照组,凋亡率也显著高于对照组(P < 0.05)(图 1-k),MAG组凋亡细胞个数相对模型组显著降低(P < 0.05),与对照组无明显差异(P>0.05)。

Ⅰ.MAG预处理肝细胞12 h(油酸钠诱导前);Ⅱ.油酸钠诱导9 h后;Ⅲ.油酸钠诱导9 h后DAPI荧光染色,a、d、g为对照组,b、e、h为模型组,c、f、i为MAG组;j.CCK-8检测细胞活性;k.DAPI染色检测细胞凋亡率(image J计数)。数据标注相同小写字母表示差异不显著(P>0.05),标注不同小写字母者表示差异显著(P<0.05),(n=6),下同 Ⅰ. Hepatocytes were pretreated with MAG for 12 h (before sodium oleate induction); Ⅱ. Hepatocytes after being induced by sodium oleate for 9 h; Ⅲ. With DAPI fluorescence staining after being induced by sodium oleate for 9 h, a, d, g are control group, b, e, h are model groups, c, f, i are MAG group; j. Cell activity detected by CCK-8; k. Apoptosis rate detected by DAPI staining (count with image J). The data marked with the same lowercase letter means no significant difference(P > 0.05), marked with different lowercase letters indicate significant differences (P < 0.05), (n=6), the same as below 图 1 MAG对肝细胞活性和凋亡率的影响 Fig. 1 The effect of MAG on hepatocytes activity and apoptosis rate
2.2 MAG对肝细胞释放ALT、AST的影响

表 4所示,脂肪肝模型组细胞释放ALT、AST的水平显著高于对照组,而添加0.25 mg·L-1 MAG培养基进行预处理,肝细胞ALT、AST释放量与模型组相比显著降低(P < 0.05)。

表 4 MAG对体外脂肪肝模型的影响 Table 4 Effect of monoammonium glycyrrhizinate on fatty liver model in vitro
2.3 MAG对肝细胞模型中脂肪蓄积的影响

经油红O染色可见对照组(图 2a)肝细胞细胞核清晰完整,核大,胞浆丰富,细胞内有微量细小的红色脂滴;模型组(图 2b)细胞发生脂肪变性,脂滴数量较多,脂滴大小不均匀;MAG组(图 2c)细胞脂肪变性程度明显降低,与模型组相比体积较大的脂滴基本消失,脂滴体积分布均匀。用imageJ测定平均单个细胞内脂滴面积,发现模型组显著高于对照组(P<0.05),MAG组虽然高于对照组,但显著低于模型组(P<0.05)。

a.对照组; b.模型组; c.MAG组; d~f.对应imageJ处理的脂滴区域; g.计算的平均单个细胞内脂滴面积。(n=3) a. Control group; b. Model group; c. MAG group; d-f. correspond to the area of lipid droplets processed by imageJ; g. The calculated average intracellular lipid droplet area. (n=3) 图 2 MAG对肝细胞脂肪蓄积的影响 Fig. 2 The effect of MAG on fat accumulation in hepatocytes
2.4 MAG对脂肪肝细胞模型中脂代谢基因mRNA水平的影响

图 3所示,脂氧化关键基因PPARα在模型组中的mRNA表达水平显著高于对照组,而在MAG组中则显著低于模型组;其下游靶基因 CPT1、CPT2在模型组中mRNA的表达水平显著高于对照组(P<0.05),MAG组与模型组相比表现出下降趋势,但差异不显著(P>0.05)。肝细胞脂合成基因 SREBP-1c的mRNA表达水平在模型组明显降低(P<0.05),而MAG组与模型组相比差异不显著(P>0.05)。肝细胞另一参与调控的脂合成基因ChREBP与转运蛋白重要原件MTP在模型组中的mRNA表达水平与对照组相比无显著差异(P>0.05);但与模型组相比,MAG组显著下调了ChREBPMTP的mRNA表达水平(P<0.05)。

图 3 MAG对肝细胞脂代谢基因表达水平的影响 Fig. 3 The effect of MAG on the expression level of hepatic lipid metabolism genes
2.5 MAG对脂肪肝细胞模型中炎症相关基因mRNA水平的影响

图 4所示,模型组肝细胞中炎症相关因子的mRNA表达水平与对照组相比显著增加(P < 0.05),MAG组与模型组相比显著降低(P < 0.05)。模型组肝细胞中炎症介导先质TNF-αIL-1β的mRNA表达水平与对照组相比显著升高(P<0.05),MAG组与模型组相比显著降低(P<0.05)。模型组NF-κBIL-6、IL-8的mRNA表达水平显著高于对照组(P < 0.05),MAG组显著低于模型组(P < 0.05),其中MAG组IL-6的表达低于对照组(P<0.05)。

图 4 MAG对肝细胞炎症相关基因表达水平的影响 Fig. 4 The effect of MAG on the expression level of hepatocyte inflammation-related genes
3 讨论

人类医学研究认为,非酒精性脂肪肝的发生及发展与胰岛素抵抗、脂质代谢异常、炎性细胞因子和氧化应激等多种因素相关[16]。而奶牛脂肪肝病的发生主要由于围产期奶牛能量负平衡,为满足日常消耗需要,大量动员自身储存的脂肪,引起肝对脂质的代谢失衡使大量脂质在肝中蓄积。根据“二次打击学说”,脂肪在肝中的蓄积会使肝细胞线粒体产生大量自由基,而使细胞发生氧化应激[17-18],脂肪酸还能通过减少细胞内的谷胱甘肽,进而损伤内源性的抗氧化防御系统[19],最终导致围产期奶牛不能及时代谢多余的自由基。奶牛机体内抗氧化系统的失衡,进一步引起细胞的炎症反应和肝细胞线粒体DNA的损伤,促进肝细胞的凋亡,加剧脂肪在肝中蓄积。

肝细胞是体外研究物质代谢、肝毒性评价和药物筛选的首选工具。肝细胞的来源主要由原代肝细胞、永生化肝细胞系和其他干细胞诱导分化而来[20]。国内奶牛原代肝细胞体外培养最早报道于2007年[15],随后,此技术在国内被广泛应用,是主要的奶牛肝细胞来源[21-24]。近年来,也有奶牛干细胞分离的报道[25-28],这可为奶牛肝细胞提供新来源。本试验选择原代分离的犊牛肝细胞,纯度和活力很高,在体外培养72 h后进行诱导试验,满足试验对细胞和细胞功能的要求。

现代研究表明,甘草具有非常强大的抗氧化和清除自由基活性的功能[29-31]。其中,甘草酸作为甘草重要的活性成分可通过作用于激素受体,影响离子通道(抑制钙离子内流)[32],激活或抑制酶的活性,调节物质代谢和胆碱能神经的兴奋性,具有肾上腺素皮质激素样作用,发挥抗炎、抗纤维化、抗氧化和抗细胞凋亡等功效[33]。Wang等[34]通过向蛋氨酸和胆碱缺乏引起的非酒精性脂肪肝模型小鼠饲喂甘草酸发现,甘草酸能够显著降低小鼠血清及肝脂质含量及血清中AST和ALT的水平,改善脂肪肝模型脂肪的沉积情况,从而降低脂肪肝的发生。18α-甘草酸[29]和甘草酸二铵[35]均能显著降低肝中TG和总胆固醇含量,减少肝脂肪沉积。本试验结果发现,经油酸钠诱导后肝细胞的活性与对照组相比显著降低,MAG预处理组显著高于模型组。同时,MAG组肝细胞脂肪变性的程度与模型组相比明显减轻,细胞内脂滴的面积显著减少,表明MAG可以提高肝细胞的活性,预防油酸钠引起的肝细胞脂肪变性。

ALT和AST是临床上判断肝功能的指标,ALT反映肝细胞有无受损,AST反映肝细胞有无坏死,因此,可以通过测定细胞培养上清中ALT、AST酶的含量来判断肝细胞损伤的程度。刘露等[36]统计评价了异甘草酸镁注射液和复方甘草酸苷注射液治疗肝炎患者,其中86.95%的试验都表明,使用这两种注射液可以明显降低ALT和AST,对治疗肝病有一定的效果。复方甘草酸单铵和甘草酸制剂可有效促进胆色素代谢,降低肝细胞中AST、ALT活性,使脂肪性肝炎患者的肝功能得到改善,从而起到保护肝细胞的功能。在本研究中,模型组细胞培养上清中的AST、ALT的水平显著高于对照组,而MAG组低于模型组,表明MAG具有保护肝细胞减轻细胞损伤的作用。

对肝细胞损伤机制及对其保护和治疗药物的研究,一直是国内外研究的热点。有学者通过大鼠试验证实,MAG对利福平和异烟肼引起的肝损伤有保护作用,其机制可能与转运蛋白Mrp2、Ntcp、Oatp1a4及氧化应激的调节作用有关[37-38]。此外,甘草酸还可通过对肝代谢酶基因的调控,改善肝脂肪变性,降低脂肪肝的发生[34]。本试验中,MAG组细胞脂合成关键基因SREBP-1c的表达水平与模型组细胞相比差异不显著;ChREBP的表达水平较模型组显著降低,表明MAG可能通过下调脂合成基因来降低脂质的合成,从根源上减少了脂质来源。转运蛋白重要原件MTP的mRNA表达水平在MAG组较对照组降低,可能是由于细胞内TG含量降低导致转运蛋白减少。脂代谢关键基因PPARα的mRNA表达水平在模型组显著高于对照组,而MAG组较模型组显著降低,而其下游靶基因CPT1、CPT2 的mRNA表达水平虽无显著性差异但仍有降低。李钦佩[39]在对PKG-1过表达小鼠的研究中认为,肝内脂肪合成减少可抑制CPT、GC1α等下游基因的表达,这与本试验的结果相似。这表明,MAG可能通过抑制脂代谢关键基因的表达来延缓细胞内脂质过氧化过程。

脂肪在肝细胞中大量积累会加剧机体内的氧化应激和脂质过氧化。而脂肪酸的代谢分解主要依赖线粒体的β氧化,也是活性氧ROS的主要来源,因此,线粒体也是最容易受到打击的部位,线粒体损伤会导致细胞色素C和炎症因子释放到细胞质中,最终引起细胞的凋亡。Chen等[40]用高脂饮食建立小鼠NAFLD模型时发现,与正常组相比,小鼠肝脂肪堆积严重,血清炎性因子(TNF-α、IL-1β、IL-6和NF-κB)表达水平明显增高。抗炎作用是MAG的主要效应之一。Huang等[41]在小鼠气管内注射LPS模拟急性肺损伤模型,在注射LPS前于腹腔内注入MAG,结果发现,与LPS组相比,MAG组可以改善LPS引起的炎性细胞浸润、肺泡壁增厚、肺泡出血等病理变化,并且能够显著降低小鼠肺泡灌洗液中白细胞介素1、肿瘤坏死因子-α(TNF-α)的表达水平,减少NF-κB、p65的表达,表明MAG可能通过抑制NF-κB信号通路来发挥抗炎保护作用。Xie等[42]发现,甘草酸镁能够下调LPS诱导RAW264.7细胞炎症模型中促炎性介质TNF-α、IL-6、IL-1β、IL-8等的表达。本试验发现,经过油酸钠诱导后,细胞内TNF-αIL-1β、NF-κB和炎性因子IL-6、IL-8的mRNA表达水平较对照组显著升高,在油酸钠诱导细胞之前使用MAG预处理细胞12 h,这些基因的mRNA表达水平都显著降低,表明MAG可以通过抑制炎症因子表达,减轻细胞炎症损伤来保护肝细胞。

4 结论

研究结果表明,MAG能通过抑制脂代谢和炎症因子相关基因的mRNA表达水平,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,起到保护肝的作用。

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