双蓣调脂汤对高脂血症模型大鼠小肠组织NPC1L1及ABCG8表达的影响
|
摘要
目的:观察高脂血症模型大鼠小肠组织中尼曼-匹克C1型类蛋白1(NPC1L1)及三磷酸腺苷结合盒转运体G8(ABCG8)的表达水平,探讨双蓣调脂汤对高脂血症的治疗机制。方法:选取40只雄性SD大鼠,随机选取8只大鼠为正常组,其余32只建立高脂血症大鼠模型,造模成功后随机分为模型组(等量生理盐水),双蓣调脂汤高、低剂量组(15. 6,7. 8 g·kg~(-1)),辛伐他汀组(4 mg·kg~(-1)),共4组,每组8只,灌胃给药8周。生化法和酶法测定各组大鼠血清总胆固醇(TC),甘油三酯(TG)及肝脏总胆固醇(TTC),游离胆固醇(FTC)的含量;采用苏木素-伊红(HE)染色光镜下观察肝组织形态学的变化;运用逆转录-聚合酶链式反应(RT-PCR)和蛋白免疫印迹法(Western blot)检测大鼠小肠组织NPC1L1,ABCG8及肝X受体α(LXR-α)表达水平;免疫组化法测定ABCG8蛋白表达的水平。结果:高脂血症大鼠模型复制成功。与正常组比较,模型组血脂水平明显增高,肝脏脂肪变性明显,NPC1L1,LXR-α及ABCG8表达水平明显升高(P <0. 05,P <0. 01);与模型组比较,双蓣调脂汤组血脂水平明显下降,NPC1L1和LXR-α的表达明显下调,ABCG8的表达明显上调,呈现一定的剂量依赖性(P <0. 05,P <0. 01)。免疫组化法检测结果显示,与正常组比较,模型组小肠中ABCG8蛋白表达水平升高(P <0. 01);与模型组比较,各给药组ABCG8蛋白表达水平显著上调(P <0. 05,P <0. 01),其中双蓣调脂汤高剂量组效果最为显著(P <0. 05)。结论:双蓣调脂汤通过减少胆固醇的吸收来降低高脂血症模型大鼠的血脂水平,其机制可能与下调NPC1L1的表达,上调ABCG8的表达有关。
Objective: To observe the expression levels of niemann-pick C1-like 1( NPC1 L1) and adenosine triphosphate-binding cassette transporters G8( ABCG8) in intestine of hyperlipidemic model rats,in order to investigate the therapeutic mechanism of Shuangyu Tiaozhi decoction on hyperlipidemia. Method: A total of 40 SD rats were selected,including 8 for normal control group. The remaining 32 rats were used to establish hyperlipemic model. After modeling,the rats were randomly divided into the model group( equivalent normal saline),the high and low-dose Shuangyu Tiaozhi groups( 15. 6,7. 8 g·kg~(-1)),and the Simvastatin group( 4 mg·kg~(-1)),with 8 in each group. They were given drugs by gavage for 8 weeks. The levels of total cholesterol( TC),triglyceride( TG) in serum and total cholesterol( TTC),free cholesterol( FTC) in liver of rats in each group were determined by biochemical and enzymatic methods. The morphological changes of liver were observed by hematoxylin-eosin( HE) staining,and the levels of expressions of NPC1 L1,ABCG8 and liver X receptor-α( LXR-α) in intestine were detected by Real-time fluorescence quantitative polymerase chain reaction( Real-time PCR) and Western blot. The expression of ABCG8 protein was determined by immunohistochemistry. Result:After successful replication of the hyperlipidemia model,the blood lipid level was abnormally increased,and the liver steatosis became obvious in the model group compared with the normal control group. The expression levels of NPC1 L1,LXR-α and ABCG8 increased significantly( P < 0. 05,P < 0. 01). Compared with the model group,the blood lipid levels were significantly reduced in the Shuangyu Tiaozhi decoction group. The expressions of NPC1 L1 and LXR-α were significantly down-regulated,but ABCG8 was obviously up-regulated in a dose-dependent manner( P < 0. 05,P < 0. 01). The results of immunohistochemistry indicated that the protein expression of ABCG8 in intestine of the model group increased significantly( P < 0. 01). Compared with the model group,the expression of ABCG8 in each drug-administered groups increased obviously( P < 0. 05,P < 0. 01),especially in high-dose Shuangyu Tiaozhi group( P < 0. 05). Conclusion: Shuangyu Tiaozhi decoction can reduce the blood lipid level of hyperlipemic rat model by reducing the absorption of cholesterol. Its mechanism may be correlated with the downregulation of NPC1 L1 expression and the up-regulation of ABCG8 expression.
Objective: To observe the expression levels of niemann-pick C1-like 1( NPC1 L1) and adenosine triphosphate-binding cassette transporters G8( ABCG8) in intestine of hyperlipidemic model rats,in order to investigate the therapeutic mechanism of Shuangyu Tiaozhi decoction on hyperlipidemia. Method: A total of 40 SD rats were selected,including 8 for normal control group. The remaining 32 rats were used to establish hyperlipemic model. After modeling,the rats were randomly divided into the model group( equivalent normal saline),the high and low-dose Shuangyu Tiaozhi groups( 15. 6,7. 8 g·kg~(-1)),and the Simvastatin group( 4 mg·kg~(-1)),with 8 in each group. They were given drugs by gavage for 8 weeks. The levels of total cholesterol( TC),triglyceride( TG) in serum and total cholesterol( TTC),free cholesterol( FTC) in liver of rats in each group were determined by biochemical and enzymatic methods. The morphological changes of liver were observed by hematoxylin-eosin( HE) staining,and the levels of expressions of NPC1 L1,ABCG8 and liver X receptor-α( LXR-α) in intestine were detected by Real-time fluorescence quantitative polymerase chain reaction( Real-time PCR) and Western blot. The expression of ABCG8 protein was determined by immunohistochemistry. Result:After successful replication of the hyperlipidemia model,the blood lipid level was abnormally increased,and the liver steatosis became obvious in the model group compared with the normal control group. The expression levels of NPC1 L1,LXR-α and ABCG8 increased significantly( P < 0. 05,P < 0. 01). Compared with the model group,the blood lipid levels were significantly reduced in the Shuangyu Tiaozhi decoction group. The expressions of NPC1 L1 and LXR-α were significantly down-regulated,but ABCG8 was obviously up-regulated in a dose-dependent manner( P < 0. 05,P < 0. 01). The results of immunohistochemistry indicated that the protein expression of ABCG8 in intestine of the model group increased significantly( P < 0. 01). Compared with the model group,the expression of ABCG8 in each drug-administered groups increased obviously( P < 0. 05,P < 0. 01),especially in high-dose Shuangyu Tiaozhi group( P < 0. 05). Conclusion: Shuangyu Tiaozhi decoction can reduce the blood lipid level of hyperlipemic rat model by reducing the absorption of cholesterol. Its mechanism may be correlated with the downregulation of NPC1 L1 expression and the up-regulation of ABCG8 expression.
引文
[1] Lopez A D,Mathers C D,Ezzati M,et al. Global and regional burden of disease and risk factors, 2001:systematic analysis of population health data[J].Lancet,2006,367(9524):1747-1757.
[2]冯利民,李立凤,杜武勋,等.高脂血症基本病机与证型规律研究进展[J].时珍国医国药,2012,23(12):3101-3103.
[3] Kamishikiryo J,Haraguchi M,Nakashima S,et al. Nterminal domain of the cholesterol transporter NiemannPick C1-like 1(NPC1L1)is essential forα-tocopherol transport[J]. Biochem Biophys Res Commun,2017,486(2):476-480.
[4] Nihei W,Nagafuku M,Hayamizu H,et al. NPC1L1-dependent intestinal cholesterol absorption requires ganglioside GM3 in membrane microdomains[J]. J Lipid Res,2018,59(11):2181-2187.
[5] YU X H,QIAN K,JIANG N,et al. ABCG5/ABCG8 in cholesterol excretion and atherosclerosis[J]. Clinica Chimica Acta,2014,428(2):82-88.
[6] Bonamassa B,Moschetta A. Atherosclerosis:lessons from LXR and the intestine[J]. Trends Endocrinol Metab,2013,24(3):120-128.
[7]姜春玲,原佺.他汀类药物的副作用与临床观察[J].中国实用医药,2016,11(1):193-194.
[8]孙吉叶.治疗高脂血症的新药研究进展[J].现代药物与临床,2012,27(5):435-441.
[9]徐凤,肖韩艳,周淑芬,等.柴朴汤对哮喘模型大鼠气道炎症及ERK/p38 MAPK信号通路的影响[J].中国实验方剂学杂志,2018,24(2):104-109.
[10]韦乃球,郝二伟,冼寒梅,等.白子菜对Hep G2细胞胰岛素抵抗作用的分子机制[J].中国实验方剂学杂志,2018,24(2):110-115.
[11]赵梓邯,张琳,李文斌,等.中药毒性与安全性评价研究进展[J].中国实验方剂学杂志,2018,24(20):208-216.
[12]席加秋,王中琳.化浊行血汤治疗高脂血症30例临床研究[J].山东中医药大学学报,2017,41(4):338-340.
[13]张风霞,张铭,孙西庆,等.化浊行血汤治疗2型糖尿病40例[J].河南中医,2013,33(11):1919-1920.
[14]张珊,尹立祥,王爱迪,等.穿龙薯蓣皂苷对再生障碍性贫血小鼠PPARγ,C/EBPα及脂肪分泌因子表达的影响[J].中国实验方剂学杂志,2019,25(5):126-134.
[15]张风霞,王新陆,高聆,等.双蓣调脂汤治疗高脂血症118例[J].山东中医杂志,2013,32(2):91-92.
[16]刁婷婷,闵清.高脂血症动物模型研究进展[J].湖北科技学院学报:医学版,2018,32(6):541-545.
[17]赵媛媛,覃骊兰,郝二伟.高血脂症动物模型研究进展[J].中国实验方剂学杂志,2018,24(18):215-221.
[18]邵礼梅,许世伟.山药化学成分及现代药理研究进展[J].中医药学报,2017,45(2):125-127.
[19]刘梦萱,张风霞,荀丽英,等.薯蓣皂苷元降血脂作用及其机制研究进展[J].中华中医药学刊,2016,34(4):798-800.
[20]苏瑾,王丹,胡孟洋,等.山药总多糖脂质体的制备及表征[J].中国实验方剂学杂志,2016,22(16):18-21.
[21] Mckoy M L,Thomas P G,Asemota H,et al. Effects of Jamaican Bitter Yam(Dioscorea polygonoides)and diosgenin on blood and fecal cholesterol in rats[J]. J Med Food,2014,17(11):1183-1188.
[22] Losacco M C,de Almeida C F T,Hijo A H T,et al.High-fat diet affects gut nutrients transporters in hypo and hyperthyroid mice by PPAR-a independent mechanism[J]. Life Sci,2018,202:35-43.
[23] WANG H, Garruti G, LIU M. Cholesterol and lipoprotein metabolism and atherosclerosis recent advances in reverse cholesterol transpor[J]. Ann Hepatol,2017,16(1):s27-s42.
[24] Davis H R,ZHU L J,Hoos L M,et al. Niemann-Pick C1 like 1(NPC1L1)is the intestinal phytosterol and cholesterol transporter and a key modulator of wholebody cholesterol homeostasis[J]. J Biol Chem,2004,279(32):33586-33592.
[25] Duval C,Touche V,Tailleux A,et al. Niemann-pick C1 like 1 gene expression is down-regulated by LXR activators in the intestine[J]. Biochem Biophys Res Commun,2006,340:1259-1263.
[26] Bonamassa B,Moschetta A. Atherosclerosis:lessons from LXR and the intestine[J]. Trends Endocrinol Metab,2013,24:120-128.
[27] YU L, Lihawkins J, Hammer R E, et al.Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol[J]. J Clin Invest,2002,110(5):671-680.
[28]杨丽丽,王淼,柳涛,等.降脂颗粒对非酒精性脂肪性肝病大鼠肝X受体α和固醇调节元件结合蛋白1c表达的影响[J].结合医学学报:英文版,2011,9(9):998-1004.
[29]赵敏,徐安莉,陈会敏,等.右归丸对肾阳虚高脂血症大鼠JAK/STATs通路的实验探讨[J].中国实验方剂学杂志,2015,21(21):108-112.
[2]冯利民,李立凤,杜武勋,等.高脂血症基本病机与证型规律研究进展[J].时珍国医国药,2012,23(12):3101-3103.
[3] Kamishikiryo J,Haraguchi M,Nakashima S,et al. Nterminal domain of the cholesterol transporter NiemannPick C1-like 1(NPC1L1)is essential forα-tocopherol transport[J]. Biochem Biophys Res Commun,2017,486(2):476-480.
[4] Nihei W,Nagafuku M,Hayamizu H,et al. NPC1L1-dependent intestinal cholesterol absorption requires ganglioside GM3 in membrane microdomains[J]. J Lipid Res,2018,59(11):2181-2187.
[5] YU X H,QIAN K,JIANG N,et al. ABCG5/ABCG8 in cholesterol excretion and atherosclerosis[J]. Clinica Chimica Acta,2014,428(2):82-88.
[6] Bonamassa B,Moschetta A. Atherosclerosis:lessons from LXR and the intestine[J]. Trends Endocrinol Metab,2013,24(3):120-128.
[7]姜春玲,原佺.他汀类药物的副作用与临床观察[J].中国实用医药,2016,11(1):193-194.
[8]孙吉叶.治疗高脂血症的新药研究进展[J].现代药物与临床,2012,27(5):435-441.
[9]徐凤,肖韩艳,周淑芬,等.柴朴汤对哮喘模型大鼠气道炎症及ERK/p38 MAPK信号通路的影响[J].中国实验方剂学杂志,2018,24(2):104-109.
[10]韦乃球,郝二伟,冼寒梅,等.白子菜对Hep G2细胞胰岛素抵抗作用的分子机制[J].中国实验方剂学杂志,2018,24(2):110-115.
[11]赵梓邯,张琳,李文斌,等.中药毒性与安全性评价研究进展[J].中国实验方剂学杂志,2018,24(20):208-216.
[12]席加秋,王中琳.化浊行血汤治疗高脂血症30例临床研究[J].山东中医药大学学报,2017,41(4):338-340.
[13]张风霞,张铭,孙西庆,等.化浊行血汤治疗2型糖尿病40例[J].河南中医,2013,33(11):1919-1920.
[14]张珊,尹立祥,王爱迪,等.穿龙薯蓣皂苷对再生障碍性贫血小鼠PPARγ,C/EBPα及脂肪分泌因子表达的影响[J].中国实验方剂学杂志,2019,25(5):126-134.
[15]张风霞,王新陆,高聆,等.双蓣调脂汤治疗高脂血症118例[J].山东中医杂志,2013,32(2):91-92.
[16]刁婷婷,闵清.高脂血症动物模型研究进展[J].湖北科技学院学报:医学版,2018,32(6):541-545.
[17]赵媛媛,覃骊兰,郝二伟.高血脂症动物模型研究进展[J].中国实验方剂学杂志,2018,24(18):215-221.
[18]邵礼梅,许世伟.山药化学成分及现代药理研究进展[J].中医药学报,2017,45(2):125-127.
[19]刘梦萱,张风霞,荀丽英,等.薯蓣皂苷元降血脂作用及其机制研究进展[J].中华中医药学刊,2016,34(4):798-800.
[20]苏瑾,王丹,胡孟洋,等.山药总多糖脂质体的制备及表征[J].中国实验方剂学杂志,2016,22(16):18-21.
[21] Mckoy M L,Thomas P G,Asemota H,et al. Effects of Jamaican Bitter Yam(Dioscorea polygonoides)and diosgenin on blood and fecal cholesterol in rats[J]. J Med Food,2014,17(11):1183-1188.
[22] Losacco M C,de Almeida C F T,Hijo A H T,et al.High-fat diet affects gut nutrients transporters in hypo and hyperthyroid mice by PPAR-a independent mechanism[J]. Life Sci,2018,202:35-43.
[23] WANG H, Garruti G, LIU M. Cholesterol and lipoprotein metabolism and atherosclerosis recent advances in reverse cholesterol transpor[J]. Ann Hepatol,2017,16(1):s27-s42.
[24] Davis H R,ZHU L J,Hoos L M,et al. Niemann-Pick C1 like 1(NPC1L1)is the intestinal phytosterol and cholesterol transporter and a key modulator of wholebody cholesterol homeostasis[J]. J Biol Chem,2004,279(32):33586-33592.
[25] Duval C,Touche V,Tailleux A,et al. Niemann-pick C1 like 1 gene expression is down-regulated by LXR activators in the intestine[J]. Biochem Biophys Res Commun,2006,340:1259-1263.
[26] Bonamassa B,Moschetta A. Atherosclerosis:lessons from LXR and the intestine[J]. Trends Endocrinol Metab,2013,24:120-128.
[27] YU L, Lihawkins J, Hammer R E, et al.Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol[J]. J Clin Invest,2002,110(5):671-680.
[28]杨丽丽,王淼,柳涛,等.降脂颗粒对非酒精性脂肪性肝病大鼠肝X受体α和固醇调节元件结合蛋白1c表达的影响[J].结合医学学报:英文版,2011,9(9):998-1004.
[29]赵敏,徐安莉,陈会敏,等.右归丸对肾阳虚高脂血症大鼠JAK/STATs通路的实验探讨[J].中国实验方剂学杂志,2015,21(21):108-112.