Please wait a minute...
浙江大学学报（医学版）
浙江大学学报（医学版）  2017, Vol. 46 Issue (2): 192-197    DOI: 10.3785/j.issn.1008-9292.2017.04.12
原著     
甘草酸二铵肠溶胶囊对棕榈酸诱导的肝细胞炎症相关基因表达影响
柳银兰(),罗燕,杨文君,施军平,庄振杰*()
杭州师范大学附属医院, 浙江 杭州 310015
Effect of diammonium glycyrrhizinate and phospholipids complex oninflammatory gene expression induced by palmitic acid
LIU Yinlan(),LUO Yan,YANG Wenjun,SHI Junping,ZHUANG ZhenJie*()
The Affiliated Hospital of Hangzhou Normal University, Hangzhou 310015, China
 全文: PDF(1674 KB)   HTML
摘要：

目的：探讨甘草酸二铵肠溶胶囊（商品名天晴甘平）对棕榈酸引起肝细胞模型炎症的改善作用。方法：肝癌细胞株huh7细胞分为对照组、棕榈酸组、天晴甘平+棕榈酸组。对照组给予棕榈酸溶剂，棕榈酸组给予0.2 mmol饱和脂肪酸棕榈酸处理建立脂肪性炎症模型，天晴甘平+棕榈酸组在给予0.2 mmol/L棕榈酸的同时给予20 μmol/L或100 μmol/L天晴甘平。通过RT-PCR检测炎症相关指标环氧合酶2（COX-2）、诱导型一氧化氮合酶（iNOS）及内质网应激相关基因葡萄糖调节蛋白78（GRP78）的表达。另外，以1 mmol/L不饱和脂肪酸油酸和饱和脂肪酸棕榈酸2∶1的混合物处理huh7细胞，建立脂肪变性模型，油红染色观察天晴甘平对脂肪变性细胞的影响。结果：与对照组比较，棕榈酸组COX-2、iNOS、GRP78的表达增加分别为[（6.07±0.73）与（1.00±0.22），P<0.05]、[（3.18±0.91）与（1.00±0.26），P<0.01]、[（3.21±1.00）与（1.00±0.12），均P<0.05]。与棕榈酸组比较，100 μmol/L天晴甘平+棕榈酸组COX-2、iNOS和GRP78的表达下降（分别为2.40±0.76、1.60±0.49和1.17±0.42，均P<0.05）；20 μmol/L天晴甘平+棕榈酸组COX-2和iNOS的表达降低（均P<0.05）。油红染色显示，天晴甘平组细胞浆中油酸棕榈酸混合脂肪酸诱导的脂滴增加，细胞的脂肪变程度加重。结论：天晴甘平能抑制棕榈酸诱导的炎症相关基因COX2及iNOS的增加，同时100 μmol/L天晴甘平抑制棕榈酸诱导的GRP78的表达并增加细胞脂肪变性。推测其抑制炎症的作用可能部分源于天晴甘平中不饱和脂肪酸对内质网应激的改善及增加细胞脂肪变性的作用。
关键词： 甘草次酸/治疗应用;  脂肪酸类;  脂肪酸类，不饱和;  棕榈酸/药理学;  胶囊;  炎症/药物疗法;  环氧化酶2;  一氧化氮合酶;  肝/细胞学    
Abstract:

Objective: To investigate the effect of glycyrrhizinate and phospholipids (DGPL) complex on inflammatory gene expression in cell inflammation model induced by palmitic acid (PA). Methods: Huh7 cells were divided into control, PA and PA+DGPL groups. For control group, cells were treated with BSA; for PA group, cells were incubated with 0.2 mmol/L saturated fatty acid PA, PA+DGPL group was given 20 μmol/L or 100 μmol/L DGPL in addition to 0.2 μmol/L PA. After 24 h, the expression of inflammation-related genes COX-2 and iNOS and endoplasmic reticulum (ER) stress-related gene GRP78 was determined by RT PCR. Oil red staining was conducted to observe the effect of DGLP on steatosis. Results: Compared with control group, the expression of COX-2, iNOS and GRP78 in PA group was enhanced to 6.07±0.73(P<0.05), 3.18±0.91 (P<0.01) and 3.21±1.00(P<0.05), respectively. Compared to control group, the expression of COX-2,iNOS and GRP78 in 100 μmol/L DGPL group was reduced to 2.40±0.76, 1.60±0.49 and 1.17 ±0.42 (P<0.05); and 20 μmol DGPL had similar inhibition effect on COX-2 and iNOS elevation induced by PA (P<0.01, P<0.05 respectively). In addition, DGLP enhances the steatosis of Huh7 cells as demonstrated by oil red staining. Conclusion: PA can induce the up-regulated expression of inflammation associated genes COX-2, iNOS and ER stress-associated gene GRP78 in Huh7 cells. DGPL is able to protect Huh7 cells from PA induced inflammatory gene expression and the beneficial effect may be partially due to its unsaturated phospholipid component, which may improve ER stress and enhance steatosis.
Key words: Glycyrrhetinic acid/therapeutic use    Fatty acids    Fatty acids, unsaturated    Palmitic acid/pharmacology    Capsules    Inflammation/drug therapy    Cyclooxygenase 2    Nitric oxide synthase    Liver/cytology
收稿日期: 2016-12-13 出版日期: 2017-10-31
基金资助: 浙江省自然科学基金（LQ15H070006，LQ17H070002）；杭州市科学技术委员会基金项目（20150633B03）
通讯作者: 庄振杰     E-mail: 84094294@qq.com;13291410733@163.com
作者简介: 柳银兰(1986—)，女，学士，检验技师，主要从事脂肪性肝病的基础研究和临床试验研究；E-mail: 84094294@qq.com; http://orcid.org/0000-0002-3552-9508
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
柳银兰
罗燕
杨文君
施军平
庄振杰

引用本文:

柳银兰,罗燕,杨文君,施军平,庄振杰. 甘草酸二铵肠溶胶囊对棕榈酸诱导的肝细胞炎症相关基因表达影响[J]. 浙江大学学报（医学版）, 2017, 46(2): 192-197.

LIU Yinlan,LUO Yan,YANG Wenjun,SHI Junping,ZHUANG ZhenJie. Effect of diammonium glycyrrhizinate and phospholipids complex oninflammatory gene expression induced by palmitic acid. J Zhejiang Univ (Med Sci), 2017, 46(2): 192-197.

链接本文:

http://www.zjujournals.com/xueshu/med/CN/10.3785/j.issn.1008-9292.2017.04.12    或    http://www.zjujournals.com/xueshu/med/CN/Y2017/V46/I2/192

基因 引物序列
      COX-2：环氧合酶2；iNOS：一氧化氮合酶；GRP-78:葡萄糖调节蛋白78.
    β-actin 正向：5′-CATGTACGTTGCTATCCAGGC-3′
反向：5′-CTCCTTAATGTCACGCACGAT-3′
    COX-2 正向：5′-CAAATTGCTGGCAGGGTTGC-3′
反向：5′-AGGGCTTCAGCATAAAGCGT-3′
    iNOS 正向：5′-TCACTCAGCTGTGCATCGAC-3′
反向：5′-CCTCAAGCACAAGGTCAGGT-3′
    GRP-78 正向：5′-GAAAGAAGGTTACCCATGCAGT-3′
反向：5′-CAGGCCATAAGCAATAGCAGC-3′
表 1  基因引物序列
图 1  不同浓度天晴甘平对细胞乳酸脱氢酶活性的影响
图 2  不同浓度天晴甘平对细胞炎症相关基因表达的影响
($\bar x \pm s$)
组别 n 100 μmol/L 20 μmol/L
  GRP-78:葡萄糖调节蛋白78；与对照组比较，*P < 0.05；与棕榈酸组比较，#P < 0.05.
对照组 3 1.00±0.12 1.00±0.07
棕榈酸组 3 3.21±1.00* 2.87±0.46
棕榈酸+天晴甘平组 3 1.17±0.42# 3.41±0.34
表 2  不同浓度天晴甘平对棕榈酸诱导的内质网相关基因GRP-78表达的影响
图 3  各组油红染色结果
1 潘 浩 , 窦 爱霞 , 张 燕华 et al. 甘草酸二铵脂质配位体治疗高脂饮食大鼠非酒精性脂肪性肝病疗效观察[J]. 肝脏, 2009, 14 (5): 401- 403
PAN Hao , DOU Aixia , ZHANG Yanhua et al. Effect of diammonium glycurrhizinate lipid ligand on rat non-alcoholic fatty liver disease induced by high fat diet[J]. Chinese Hepatology, 2009, 14 (5): 401- 403
2 马 巧玉 . α-甘草酸二铵磷脂酰胆碱脂质复合物胶囊治疗脂肪肝的临床观察[J]. 中华传染病杂志, 2009, 27 (1): 55- 57
MA Qiaoyu . Observation of Alpha diammonium glycyrrhizinate phosphatidylcholine compound capsule in the treatment of fatty liver[J]. Chinese Journal of Infectious Diseases, 2009, 27 (1): 55- 57
3 于 滨 . 甘草酸二铵肠溶胶囊的保肝降酶作用[J]. 实用药物与临床, 2006, 9 (3): 166-
YU Bin . The protective effect of diammonium glycyrrhizinate phosphatidylcholine compound capsule on liver enzyme[J]. Practical Pharmacy and Clinical Remedies, 2006, 9 (3): 166-
4 NOUREDDIN M , RINELLA M E . Nonalcoholic Fatty liver disease, diabetes, obesity, and hepatocellular carcinoma[J]. Clin Liver Dis, 2015, 19 (2): 361- 379
doi: 10.1016/j.cld.2015.01.012
5 SINGH S , ALLEN A M , WANG Z et al. Fibrosis progression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: a systematic review and meta-analysis of paired-biopsy studies[J]. Clin Gastroenterol Hepatol, 2015, 13 (4): 643- 654
doi: 10.1016/j.cgh.2014.04.014
6 RINELLA M E . Nonalcoholic fatty liver disease: a systematic review[J]. JAMA, 2015, 313 (22): 2263- 2273
doi: 10.1001/jama.2015.5370
7 茹 仁萍 , 吴 锡铭 . 18α甘草酸及其脂质配位体的生物利用度与抗肝损害作用的比较[J]. 浙江医学, 2001, 23 (8): 18- 20
RU Renping , WU Ximing . The bioavailability and anti-hapati damage effect of 18α-glycyrrhizic acids with phospholopids[J]. Zhejiang Medical Journal, 2001, 23 (8): 18- 20
8 刘 春 , 解 炜 , 于 锋 . 天然活性成分磷脂复合物的研究新思路-α-甘草酸二铵脂质复合物[J]. 抗感染药学, 2004, 1 (3): 97- 100, 109
LIU Chun , XIE Wei , YU Feng et al. Phytosomes-α-Diammonium glycyrrhizate phosphatidylcholine complex[J]. Anti-infection Pharmacy, 2004, 1 (3): 97- 100, 109
9 ARENDT B M , COMELLI E M , MA D W et al. Altered hepatic gene expression in nonalcoholic fatty liver disease is associated with lower hepatic n-3 and n-6 polyunsaturated fatty acids[J]. Hepatology, 2015, 61 (5): 1565- 1578
doi: 10.1002/hep.27695
10 KAKISAKA K , CAZANAVE S C , FINGAS C D et al. Mechanisms of lysophosphatidylcholine-induced hepatocyte lipoapoptosis[J]. Am J Physiol Gastrointest Liver Physiol, 2012, 302 (1): 77- 84
doi: 10.1152/ajpgi.00301.2011
11 RICCHI M , ODOARDI M R , CARULLI L et al. Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes[J]. J Gastroenterol Hepatol, 2009, 24 (5): 830- 840
doi: 10.1111/jgh.2009.24.issue-5
12 SALVADó L , COLL T , GóMEZ-FOIX A M et al. Oleate prevents saturated-fatty-acid-induced ER stress, Inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism[J]. Diabetologia, 2013, 56 1372- 1382
doi: 10.1007/s00125-013-2867-3
13 BORRADAILE N M , HAN X , HARP J D et al. Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death[J]. J Lipid Res, 2006, 47 (12): 2726- 2737
doi: 10.1194/jlr.M600299-JLR200
14 ARIYAMA H , KONO N , MATSUDA S et al. Decrease in membrane phospholipid unsaturation induces unfolded protein response[J]. J Biol Chem, 2010, 285 (29): 22027- 22035
doi: 10.1074/jbc.M110.126870
15 RAMADORI P , KROY D , STREETZ K L . Immunoregulation by lipids during the development of non-alcoholic steatohepatitis[J]. Hepatobiliary Surg Nutr, 2015, 4 (1): 11- 23
[1] 马婷婷,王毅,陈晓倩,赵筱萍. 液相色谱-质谱联用导向的黄葵胶囊肾保护活性物质研究[J]. 浙江大学学报（医学版）, 2017, 46(1): 66-73.
[2] 白石 等. 雷公藤甲素缓解局灶性脑组织缺血再灌注损伤诱发的炎症及其相关凋亡的研究[J]. 浙江大学学报（医学版）, 2016, 45(5): 493-500.
[3] 陈滟珊 等. 转瓶变速培养对微胶囊肝细胞聚集体形成及活性的影响[J]. 浙江大学学报（医学版）, 2016, 45(4): 403-409.
[4] 宋顺德，汤慧芳 . 环腺苷酸磷酸二酯酶4抑制剂靶向治疗炎症性疾病研究新进展[J]. 浙江大学学报（医学版）, 2014, 43(3): 353-358.
[5] 钱莺, 傅旭春, 扈荣, 沈丽美, 白海波. 百令疏肝胶囊对二甲基亚硝胺诱导的大鼠肝纤维化作用[J]. 浙江大学学报（医学版）, 2013, 42(5): 561-566.
[6] 万磊，刘健，黄传兵，汪元，谌曦，张皖东，王桂珍，范海霞，葛瑶，陈瑞莲，曹云祥，纵瑞凯. 新风胶囊对佐剂关节炎大鼠肺功能及Treg、Foxp3、TGF-β1/Smads的作用[J]. 浙江大学学报（医学版）, 2013, 42(4): 418-.
[7] . 百令疏肝胶囊治疗大鼠酒精损伤性肝纤维化的作用[J]. 浙江大学学报（医学版）, 2012, 41(5): 564-568.
[8] . 七氟醚预处理对大鼠产生延迟相的心肌保护作用[J]. 浙江大学学报（医学版）, 2012, 41(5): 553-558.
[9] . 胃复安对胶囊内镜检查影响的临床研究[J]. 浙江大学学报（医学版）, 2012, 41(2): 206-209.
[10] . 尼克酰胺对帕金森病模型小鼠的保护作用[J]. 浙江大学学报（医学版）, 2012, 41(2): 146-152.
[11] . GAD67-GFP基因敲入小鼠GFP与nNOS在嗅球中的表达和共存研究[J]. 浙江大学学报（医学版）, 2012, 41(2): 159-165.
[12] 陆建锋,厉旭云,沈春红,陆源,李芝,叶治国,王琼,夏强,王会平. 西红花酸部分内皮依赖性血管舒张作用及其机制[J]. 浙江大学学报（医学版）, 2010, 39(6): 559-565.
[13] 傅佳寅,夏满莉,陆建锋,柳琪,蔡新,杨洁,王会平,夏强. 白桦脂酸改善氧化应激损伤大鼠血管的内皮依赖性舒张功能[J]. 浙江大学学报（医学版）, 2010, 39(5): 523-529.
[14] 张蓓,薛长湖,冯婷玉,胡晓倩,任兵兴,徐杰,王玉明. 海参脑苷脂对脂肪肝大鼠脂肪代谢的调节作用[J]. 浙江大学学报（医学版）, 2010, 39(5): 493-498.
[15] 李一乔，陈浩浩，尹一飞，韩飞，叶学松，凌树才. 甲醛吸入对大鼠嗅球和海马神经细胞的影响[J]. 浙江大学学报（医学版）, 2010, 39(3): 272-277.