药学学报  2018, Vol. 53 Issue (8): 1324-1330   PDF    
和厚朴酚对脂变性状态下HepG2细胞脂合成的影响
翟婷, 刘亚云, 续威, 陈勇     
湖北大学中药生物技术省重点实验室, 药物高通量筛选技术国家地方联合工程研究中心, 湖北 武汉 430062
摘要: 本文采用油红O染色法检测细胞脂滴含量,荧光光度法检测细胞的葡萄糖摄取量,实时荧光定量PCR及蛋白印迹法检测目标基因的表达量,研究了和厚朴酚(honokiol,HN)对正常、TO901317(TO)和油酸(oleic acid,OA)处理的HepG2细胞脂合成、葡萄糖摄取及细胞色素P450酶(cytochrome P450 proteins,CYP)家族的CYP2E1、CYP4A蛋白表达的影响。结果显示:① TO和OA处理能明显提高细胞中脂滴数量,以及固醇调节元件结合蛋白1c(sterol regulatory element binding protein-1,SREBP-1c)和脂肪营养蛋白3(patatin-like phospholipase domain-containing 3,PNPLA3)的mRNA和蛋白表达水平;经HN(10、20、40 μmol·L-1)处理细胞24 h后,肝细胞中脂滴明显减少,SREBP-1c和PNPLA3 mRNA及蛋白表达量降低。② OA处理能明显抑制细胞的糖摄取;经HN处理24 h后,肝细胞对荧光葡萄糖[2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose,2-NBDG]的吸收呈剂量依赖性增加。③正常及TO、OA处理的HepG2细胞经HN处理24 h后,胞内CYP2E1的蛋白表达量较对照组明显降低,但HN给药仅抑制了经OA处理的HepG2细胞中CYP4A的蛋白表达量。上述结果表明,HN可通过抑制脂变性状态下肝细胞内SREBP-1c和PNPLA3的表达而降低肝细胞的脂堆积,通过下调肝细胞内CYP2E1、CYP4A的蛋白表达及促进肝细胞的糖摄取,改善肝细胞的脂质过氧化和胰岛素抵抗。
关键词: 和厚朴酚     HepG2细胞     脂堆积     糖摄取     CYP2E1     CYP4A    
The effects of honokiol on lipid synthesis in HepG2 cells with steatosis
ZHAI Ting, LIU Ya-yun, XU Wei, CHEN Yong     
Hubei Province Key Laboratory of Biotechnology of Chinese Traditional Medicine, National and Local Joint Engineering Research Center of High-throughput Drug Screening Technology, Hubei University, Wuhan 430062, China
Abstract: In this study, the effects of honokiol (HN) treatment for 24 h on lipid synthesis was examined in HepG2 cells. The parameters include intracellular lipid droplet and the expression of SREBP-1c and PNPLA3, glucose uptake, and oxidative stress including the expression of CYP2E1 and CYP4A in normal, TO901317 (TO)- and oleic acid (OA)-treated HepG2 cells. The lipid droplets were detected by oil red O staining. The glucose uptake was measured by fluorescence spectrophotometry using[2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose, 2-NBDG] as probe. The expression levels of target genes were detected by quantitative PCR and Western blot. The results showed that:① TO (5 μmol·L-1) and OA (0.5 mmol·L-1) treatment increased the levels of intracellular lipid accumulation and the mRNA and protein expression of SREBP-1c and PNPLA3. After HN (10, 20, 40 μmol·L-1) treatment for 24 h, the lipid accumulation and the expression of SREBP-1c and PNPLA3 were all decreased in the tested cells. ② OA treatment significantly suppressed glucose uptake, while HN treatment dose-dependently increased the glucose uptake in OA-treated cells. ③ Compared with control group, CYP2E1 protein level significantly decreased in the three tested cells, and CYP4A protein level significantly decreased only in OA-treated cells following HN treatment. The above results suggest that HN may attenuate lipid accumulation by suppressing the expression of SREBP-1c and PNPLA3, and reduce lipid peroxidation and insulin resistance by down-regulation of the protein levels of CYP2E1 and CYP4A in HepG2 cells with steatosis.
Key words: honokiol     HepG2 cell     lipid accumulation     glucose uptake     CYP2E1     CYP4A    

非酒精性脂肪肝(nonalcoholic fatty liver disease, NAFLD)是指在无过量饮酒史的情况下, 肝组织学的改变类似于酒精性肝病, 表现特征主要为肝细胞内脂肪过度沉积和脂肪变性[1]。随着人们生活水平的提高, NAFLD已经成为最常见的肝脏疾病之一, 寻找治疗NAFLD的药物具有十分重要的意义。

脂质堆积是NAFLD形成的主要特征[2]。许多基因参与了肝脏的脂质代谢, 其中SREBP-1c和PNPLA3对肝脏中脂肪酸的合成具有重要作用。SREBP-1c是脂肪酸合成相关基因的主要转录因子, 它可通过激活脂肪生成相关基因如乙酰辅酶A羧化酶(acetyl CoA carboxylase, ACC)、脂肪酸合酶(fatty acid synthase, FAS)、硬脂酰辅酶A去饱和酶1 (stearoyl-CoA desaturase 1, SCD-1)[3, 4]及调控PNPLA3的表达来调节肝脂代谢[5]。PNPLA3也与肝脂堆积密切相关[6], 过表达PNPLA3可促进小鼠原代肝细胞及人胚肾HEK293细胞中的甘油三酯含量[7, 8]。研究表明在NAFLD状态下肝脏中SREBP-1c和PNPLA3的表达显著升高[6, 9], 提示它们可能是治疗NAFLD的关键靶点。近来研究发现胰岛素抵抗和氧化应激也与NAFLD密切相关, NAFLD患者肌肉、肝脏及脂肪组织的胰岛素敏感性降低[10-12], 葡萄糖氧化和糖原合成减少[11], 细胞脂质过氧化物水平显著提高[13]。细胞色素P450家族中的CYP2E1和CYP4A与脂肪酸代谢密切相关, CYP2E1可通过促进氧化应激、炎症反应、胰岛素抵抗及蛋白修饰作用影响NAFLD的发生发展[14]

和厚朴酚(honokiol, HN)是从传统中药厚朴中提取出来的一种带有烯丙基的连苯二酚类化合物, 具有抗菌、消炎、抗病毒、抗肿瘤等作用[15, 16]。研究表明HN可通过抑制SREBP-1 c的表达而抑制其下游的脂代谢相关基因ACC、FAS及SCD-1的表达, 逆转大鼠酒精性脂肪肝[17]及游离脂肪酸诱导的HepG2细胞脂堆积[18]。联合使用HN与厚朴酚可通过抑制SREBP-1c的表达及诱导某些脂肪酸氧化相关基因的表达, 进而缓解细胞脂堆积[19]。HN还可通过胰岛素信号通路促进前脂肪细胞的分化及脂肪细胞对葡萄糖的摄取[20, 21]。此外, HN还能明显改善高脂饲料诱导的小鼠肝脏脂肪变性[19]及高脂饲料-链脲佐菌素联合诱导的2型糖尿病大鼠血糖血脂及肝脏氧化应激损伤[22]。然而, HN是否可以缓解NAFLD状态下肝细胞的脂堆积目前尚不清楚。本文利用TO901317 (肝X受体激动剂, TO)和油酸(肝脂变性诱导剂, oleic acid, OA)处理HepG2细胞模拟人NAFLD病理状态, 体外研究了HN对NAFLD状态下HepG2细胞脂堆积及胰岛素抵抗的影响及其可能的作用机制。

材料与方法

试剂  HN (纯度大于97%, 武汉泰凯塞公司); DMEM培养基(美国GIBCO公司); 胎牛血清(德国Biochrom公司); Trizol、2-NBDG (美国Invitrogen公司); 逆转录试剂盒、实时荧光定量PCR试剂盒(上海东洋纺生物科技有限公司); DCFH-DA (中国Beyotime Biotechnology公司); β-actin、SREBP-1c、PNPLA3引物(上海桑尼生物科技有限公司); TO、OA、牛血清白蛋白、小鼠抗人β-actin多克隆抗体(美国Sigma-Aldrich公司); 兔抗人PNPLA3、CYP 2E1多克隆抗体、兔抗人CYP 4A单克隆抗体(英国Abcam公司)兔抗人SREBP-1c多克隆抗体(沈阳万类生物公司)。

细胞培养与处置  在37 ℃、5% CO2培养箱中, HepG2细胞(武汉大学典型培养物保藏中心)培养于含10%胎牛血清的DMEM培养基中。取对数生长期细胞接种于6孔板中(2×105细胞/孔), 贴壁后按以下3种方式经HN (10、20和40 μmol·L-1)[18]处理24 h: ①正常HepG2组:设溶剂对照组(仅加入药物助溶剂二甲基亚砜DMSO)及低、中、高浓度HN给药组; ② TO处理的HepG2组:设溶剂对照组(仅加入药物助溶剂DMSO)、TO (5 μmol·L-1)对照组及同时加TO (5 μmol·L-1)和低、中、高浓度HN给药组; ③ OA处理的HepG2组:设溶剂对照组(仅加入药物助溶剂DMSO和甲醇)、OA (0.5 mmol·L-1)对照组(经OA预处理24 h后再加DMSO处理24 h)及OA (0.5 mmol·L-1)预处理后再经低、中、高浓度HN处理组。各溶剂对照组所用溶剂浓度与对应组其他给药组的溶剂浓度保持一致。

油红O染色  细胞经给药处理后, 用PBS缓冲液清洗3次, 4%多聚甲醛溶液固定30 min, 60%异丙醇润洗10 s, 油红O染色液染色30 min, 吸弃染色液后经60%异丙醇漂洗及PBS清洗后, 用倒置显微镜观察细胞内红色脂滴。

实时荧光定量PCR  细胞经给药处理后, 提取总RNA, 按照逆转录试剂盒说明逆转录成cDNA。用SYBR Green Ⅰ荧光染料法进行实时荧光定量PCR, 反应条件为95 ℃预变性3 min、95 ℃变性30 s、58.3 ℃退火30 s、72 ℃延伸30 s, 共40个循环; 定量PCR的引物序列如下: SREBP-1c: Forward 5'-CGACATCGAAGACATGCTTCAG-3', Reverse 5'-GGAAGGCTTCAAGAGAGGAGC-3'; PNPLA3: Forward 5'-CTGTACCCTGCCTGTGGAAT-3', Reverse 5'-TCGAGTGAACACCTGTGAGG-3'; β-actin: Forward 5'-TCACCCACACTGTGCCCATCTACGA-3', Reverse 5'-CAGCGGAACCGCTCATTGCCAATGG-3';各基因表达水平的定量采用2-ΔΔCt法计算。

蛋白印迹  细胞经给药处理后, 提取总蛋白, 经SDS-PAGE电泳分离目的蛋白并转至PVDF膜上, 封闭后4 ℃温孵一抗过夜, 用TBST洗膜后置于辣根过氧化酶标记的二抗中室温孵育1 h, 使用增强化学发光法(ECL)进行化学发光反应。

葡萄糖摄取量检测  细胞经给药处理后, 换无糖DMEM培养基饥饿3 h, 然后加入50 μmol·L-1 2-NBDG处理30 min, 最后用预冷的PBS清洗细胞3次, 收集细胞后用多功能荧光酶标仪检测荧光强度, 激发光波长为485 nm, 发射光波长为535 nm。

数据处理  数据以x±s表示。实验数据采用SPSS 17.0软件进行单因素方差分析, 多组之间两两进行t-检验, P < 0.05为有统计学意义。

结果 1 HN对细胞内脂堆积的影响

各组细胞经油红O染色法检测细胞内脂滴含量结果见图 1图 1A显示, 当HN给药浓度为40 μmol·L-1时, 细胞内脂滴明显减少; 图 1BC显示, TO组和OA组细胞内脂滴含量较图 1A组显著增加, 但经低、中、高浓度HN处理后, 细胞内脂滴逐渐减少。结果表明, HN能缓解经TO及OA诱导的HepG2细胞内脂堆积状况。

Figure 1 Effect of HN treatment for 24 h on the lipid accumulation in normal (A), TO-treated (B) and OA-pretreated (C) HepG2 cells detected by oil-red O staining (400×). HN: Honokiol; TO: TO901317; OA: Oleic acid
2 HN对SREBP-1c和PNPLA3表达的影响

各组细胞经实时荧光定量PCR及Western blot检测的SREBP-1c和PNPLA3 mRNA及蛋白表达的结果见图 2图 2A显示, 与溶剂对照组相比, HN能抑制HepG2细胞PNPLA3 mRNA和蛋白的表达, 以及SREBP-1c的蛋白表达, 且中高剂量组有显著性差异。图 2BC显示, TO及OA对照组与对应的溶剂对照组相比, SREBP-1c及PNPLA3的mRNA和蛋白表达明显增加, 当使用HN处理后, SREBP-1c、PNPLA3 mRNA和蛋白表达减少, 且与模型组相比, HN给药组均有显著性差异。结果表明, HN能抑制经TO及OA诱导的HepG2细胞SREBP-1c、PNPLA3 mRNA和蛋白表达。

Figure 2 Effect of HN treatment for 24 h on the expression levels of mRNA and protein of SREBP-1c and PNPLA3 in normal (A), TO-treated (B) and OA-pretreated (C) HepG2 cells. n = 3, x±s. *P < 0.05, **P < 0.01 vs vehicle; #P < 0.05, ##P < 0.01 vs TO or OA
3 HN对细胞葡萄糖摄取的影响

各组细胞对荧光葡萄糖2-NBDG的摄取结果见图 3图 3AB显示, HN对正常及TO处理的HepG2细胞荧光葡萄糖摄取无明显影响。图 3C显示OA对照组细胞对2-NBDG的摄取显著少于溶剂对照组, 但经低、中、高浓度HN处理24 h后, 细胞对2-NBDG的摄取呈剂量依赖性升高, 且低、中剂量组与OA组相比有显著性差异。结果表明, TO处理不影响HepG2细胞的糖摄取能力, OA处理能抑制HepG2细胞的糖摄取能力, 而HN处理能拮抗OA诱导的对肝细胞糖摄取的抑制作用。

Figure 3 Effect of HN treatment for 24 h on the uptake of 2-NBDG in normal (A), TO-treated (B) and OA-pretreated (C) HepG2 cells. n = 3, x±s. *P < 0.05 vs vehicle; #P < 0.05 vs OA. 2-NBDG: 2-(N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-2-deoxyglucose
4 HN对HepG2细胞CYP2E1、CYP4A蛋白表达的作用

各组细胞内CYP2E1与CYP4A的蛋白表达量结果见图 4。结果显示, HN处理对正常及经TO诱导的HepG2细胞CYP4A蛋白表达无明显影响, 但能显著下调各组细胞CYP2E1的蛋白表达, 及经OA诱导的HepG2细胞CYP4A蛋白的表达, 且与空白或模型组相比, HN给药组均有极显著性差异。

Figure 4 Effect of HN treatment for 24 h on the levels of CYP2E1 and CYP4A protein in normal (A), TO-treated (B) and OA-pretreated (C) HepG2 cells. n = 3, x±s. **P < 0.01 vs vehicle; ##P < 0.01 vs TO or OA
讨论

NAFLD的发病机制目前尚无定论, “二次打击”学说得到较多认可。该学说认为第一次打击是由胰岛素抵抗导致的肝细胞脂堆积和脂变性, 第二次打击则是由第一次打击引起的肝细胞二次损伤, 包括氧化应激紊乱和线粒体功能障碍等[23]。OA是一种不饱和脂肪酸, 不仅能诱导HepG2细胞脂变性及TNF-α和PNPLA3的表达, 抑制PPARγ和SOD-1的表达[24, 25], 也能通过激活p38诱导肝细胞胰岛素抵抗[26]。TO是肝X受体激动剂, 体内能显著诱导小鼠肝脏SREBP-1c的表达[27], 体外也能诱导HaCaT细胞和HepG2细胞SREBP-1c及其下游脂肪合成关键基因FAS等的表达, 促进脂质合成增多[28-30]

SREBP-1c和PNPLA3是NAFLD的敏感基因。在NAFLD、饥饿及2型糖尿病状态下, 肝脏中SREBP-1c和PNPLA3的表达显著升高[6, 9]。小鼠、大鼠及人肝脏中的PNPLA3基因受到SREBP-1c的直接调控, SREBP-1c的上调表达会引起PNPLA3转录水平的显著上调[5, 25]。本文研究发现, HN能减少正常及NAFLD状态下HepG2细胞内的脂堆积, 抑制脂代谢关键基因SREBP-1c和PNPLA3的表达。胰岛素抵抗与NAFLD密切相关, 它能导致肝脂变性, 促进肝脏对游离脂肪酸的吸收并通过酯化作用生成甘油三酯[31, 32]。本文发现OA能抑制肝细胞的糖摄取, HN处理能拮抗OA诱导的这种作用, 但在TO处理组则没有发现该现象, 这可能是因为TO只通过调控SREBP-1c而并非通过胰岛素信号通路来调节脂代谢。上述结果表明, HN能降低NAFLD状态下肝细胞的脂堆积, 促进对葡萄糖的摄取, 从而改善肝细胞的胰岛素抵抗。

氧化应激紊乱是NAFLD发生发展的重要原因[33, 23]。肝脏中细胞色素P450家族中的CYP2E1和CYP4A在代谢一些低分子质量化合物如乙醇、脂肪酸、对乙酰氨基酚等过程中产生活性氧自由基(ROS), 引发氧化应激反应、炎性反应与胰岛素抵抗, 进而诱发脂肪性肝病[14, 34, 35]。ROS虽是氧化应激的标志物, 但它的产生机制十分复杂, 体内ROS的主要来源是线粒体呼吸链的氧化还原反应, 其他如NADPH的氧化还原反应, CYP2E1/4A对小分子化合物的代谢, 以及脂代谢等过程也是ROS生成的重要途径, 总ROS的含量并不能反映CYP2E1/4A与NAFLD的关系。此外, 文献报道芹菜素、草质素处理HepG2细胞, 能诱导ROS的生成导致肝癌细胞凋亡, 从而发挥抗癌作用[36, 37], HN也有抗肿瘤作用, 能促进淋巴瘤细胞Raji细胞ROS的生成[38], 因此, 本文没有报道HN对OA诱导的HepG2细胞ROS生成的影响, 而是根据CYP2E1/4A与NAFLD的关系, 探讨HN通过氧化应激途径对NAFLD的影响。本文发现HN可抑制脂变性状态下HepG2细胞CYP2E1和4A的蛋白表达, 这可能是HN缓解NAFLD模型细胞氧化应激损伤与胰岛素抵抗的重要原因。

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