田黄方抑制ApoE~(-/-)小鼠动脉粥样硬化作用及机制研究
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摘要
目的基于1-磷酸鞘氨醇(S1P)/1型1-磷酸鞘氨醇受体(S1PR1)通路探讨田黄方抑制ApoE~(-/-)小鼠动脉粥样硬化的作用及相关机制。方法将40只ApoE~(-/-)小鼠随机分为模型组、田黄方低剂量组(1.5 g/kg)、田黄方高剂量组(4.5 g/kg)和阿托伐他汀组(10 mg/kg),每组10只,另选具有相同遗传背景的同龄野生型C57BL/6J小鼠10只为对照组。模型组与药物干预组均给予高脂饲料喂养8周,对照组给予普通饲料喂养。干预组每日灌胃相应药物,对照组与模型组给予等量生理盐水灌胃。12周后,取小鼠血清,分离主动脉,保存于-80℃冰箱。全自动生化分析仪检测血清中TG、TC、HDL-C、LDL-C水平,高效液相色谱-质谱联用(HPLC-MS/MS)检测血清中S1P含量;油红O染色法观察主动脉组织形态学变化;RT-PCR法检测肝脏与胆固醇逆转运相关蛋白CD36、ABCA1、SR-BI、LXRa和S1PR1 mRNA表达情况。结果模型组主动脉管腔内及主动脉根部可见较大的AS斑块形成,与模型组比较,干预组主动脉管腔内及主动脉根部的AS斑块面积显著减小(P<0.05)。与对照组比较,模型组小鼠血清中TG、TC、LDL-C水平及CD36 mRNA表达升高(P<0.01),HDL-C、S1P水平及ABCA1、SR-BI、LXRa和S1PR1 mRNA表达降低(P<0.01)。与模型组比较,各给药组TG、TC、LDL-C水平及CD36 mRNA表达降低(P<0.05,P<0.01),HDL-C水平及ABCA1、SR-BI、LXRa mRNA表达升高(P<0.05,P<0.01)。结论田黄方具有调节血脂和抗AS的作用,其机制可能与其调控S1P-S1PR1通路介导胆固醇逆转运有关。
Objective To explore the effect of Tianhuang Formula(THF) on inhibiting atherosclerosis in ApoE~(-/-) mice and its related mechanisms based on sphingosine 1-phosphate(S1P) and sphingosine 1-phosphate receptor 1(S1PR1) pathway. Methods Totally 40 ApoE~(-/-) mice were randomly divided into model group, THF low-dose group(1.5 g/kg), THF high-dose group(4.5 g/kg) and atorvastatin group(AVT,10 mg/kg), 10 in each group. In addition, 10 wild-type C57BL/6J mice with the same age and genetic background were selected as the control group. The model group and intervention group were fed with high-fat diet, the control group was given normal feeding, all for 8 weeks. The intervention group was given daily gavage with THF and AVT, the control group and the model group were given equal volume of normal saline. After 12 weeks, the serum was taken, the aorta was separated, and stored in a refrigerator at-80 ℃. The levels of TG, TC, HDL-C and LDL-C in serum were detected by automatic biochemical analyzer. The S1P content in serum was detected by HPLC-MS/MS. The morphological changes of aorta were observed by oil red O staining. The expression of CD36, ABCA1, SR-BI, LXRa and S1PR1 genes in the liver were detected by RT-PCR. Results The aortic lumen large atherosclerosis plaque formation was observed in the inner and aortic roots in model group. Compared with the model group, the area of atherosclerosis plaque in arterial lumen and aortic root were reduced in intervention group(P<0.05). Compared with the control group, the levels of serum TG, TC and LDL-C and CD36 mRNA expression in model group increased(P<0.01), the serum levels of HDL-C and S1P, ABCA1, SR-BI, LXRa, S1PR1 mRNA expression decreased(P<0.01). Compared with the model group, the levels of serum TG, TC, LDL-C and CD36 mRNA expression in intervention groups decreased(P<0.05, P<0.01), the HDL-C and ABCA1, SR-BI, LXRa mRNA expression increased(P<0.05, P<0.01). Conclusion THF has the function of regulating blood lipids and anti-atherosclerosis, its mechanism may be related to its regulation of mediating cholesterol reverse transport based on S1P-S1PR1 pathway.
Objective To explore the effect of Tianhuang Formula(THF) on inhibiting atherosclerosis in ApoE~(-/-) mice and its related mechanisms based on sphingosine 1-phosphate(S1P) and sphingosine 1-phosphate receptor 1(S1PR1) pathway. Methods Totally 40 ApoE~(-/-) mice were randomly divided into model group, THF low-dose group(1.5 g/kg), THF high-dose group(4.5 g/kg) and atorvastatin group(AVT,10 mg/kg), 10 in each group. In addition, 10 wild-type C57BL/6J mice with the same age and genetic background were selected as the control group. The model group and intervention group were fed with high-fat diet, the control group was given normal feeding, all for 8 weeks. The intervention group was given daily gavage with THF and AVT, the control group and the model group were given equal volume of normal saline. After 12 weeks, the serum was taken, the aorta was separated, and stored in a refrigerator at-80 ℃. The levels of TG, TC, HDL-C and LDL-C in serum were detected by automatic biochemical analyzer. The S1P content in serum was detected by HPLC-MS/MS. The morphological changes of aorta were observed by oil red O staining. The expression of CD36, ABCA1, SR-BI, LXRa and S1PR1 genes in the liver were detected by RT-PCR. Results The aortic lumen large atherosclerosis plaque formation was observed in the inner and aortic roots in model group. Compared with the model group, the area of atherosclerosis plaque in arterial lumen and aortic root were reduced in intervention group(P<0.05). Compared with the control group, the levels of serum TG, TC and LDL-C and CD36 mRNA expression in model group increased(P<0.01), the serum levels of HDL-C and S1P, ABCA1, SR-BI, LXRa, S1PR1 mRNA expression decreased(P<0.01). Compared with the model group, the levels of serum TG, TC, LDL-C and CD36 mRNA expression in intervention groups decreased(P<0.05, P<0.01), the HDL-C and ABCA1, SR-BI, LXRa mRNA expression increased(P<0.05, P<0.01). Conclusion THF has the function of regulating blood lipids and anti-atherosclerosis, its mechanism may be related to its regulation of mediating cholesterol reverse transport based on S1P-S1PR1 pathway.
引文
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[2] Ganjali S,Blesso CN,Banach M,et al.Effects of curcumin on HDL functionality [J].Pharmacol Res,2017,119:208-218.
[3] Calkin AC,Tontonoz P.Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR [J].Nat Rev Mol Cell Bio,2012,13(4):213-224.
[4] Rader DJ.Molecular regulation of HDL metabolism and function:implications for novel therapies [J].J Clin Invest,2006,116(12):3090-3010.
[5] Egom EE,Mamas MA,Soran H.HDL quality or cholesterol cargo:what really matters-spotlight on sphingosine-1-phosphate-rich HDL [J].Curr Opin Lipidol,2013,24(4):351-356.
[6] Sattler K,Gr?ler M,Keul P,et al.Defects of high-density lipoproteins in coronary artery disease caused by low sphingosine-1-phosphate content:correction by sphingosine-1-phosphate-loading [J].J Am Coll Cardiol,2015,66(13):1470-1485.
[7] Sattler K,Levkau B.Sphingosine-1-phosphate as a mediator of high-density lipoprotein effects in cardiovascular protection [J].Cardiovasc Res,2009,82(2):201-211.
[8] 张霞,王予娇,曾智桓,等.田黄片对血管内皮损伤后内膜增生的影响[J].中药新药与临床药理,2017,28(5):606-610.
[9] 罗朵生,李坤平,荣向路,等.基于代谢组学技术的田黄方配伍机制分析[J].广州中医药大学学报,2016,33(4):525-530.
[10] 兰天,吴腾,勾红菊,等.检测细胞中1-磷酸鞘氨醇含量LC-MS/MS分析方法的建立[J].中国医药科学,2015,3(9):9-11.
[11] 彭君慧.基于miR-33靶点的天然小分子抗动脉粥样硬化作用研究[D].广东药学院,2013.
[12] Kosmas CE,Dejesus E,Rosario D,et al.CETP inhibition:past failures and future hopes[J].Clin Med Insights Cardiol,2016,10:37-42.
[13] Gom EE,Mamas MA,Soran H.HDL quality or cholesterol cargo:what really matters-spotlight on sphingosine-1-phosphaterich HDL[J].Curr Opin Lipidol,2013,24(4):351-356.
[14] Demina EP,Miroshnikova VV,Schwarzman AL.Role of the ABC transporters A1 and G1,key reverse cholesterol transport proteins,in atherosclerosis[J].Mol Biol,2016,50(2):193-199.
[15] Makoto K,Yutaka.Sphingosine 1-phosphate and atherosclerosis[J].J Atheroscler Thromb,2018,25(1):16-26.
[16] Sattler K,Lehmann I,Gr?ler M,et al.HDL-bound sphingosine 1-phosphate (S1P) predicts the severity of coronary artery atherosclerosis[J].Cell Physiol Biochem,2014,34(1):172-184.
[17] Kappus MS,Murphy AJ,Abramowicz S,et al.Activation of liver X receptor decreases atherosclerosis in Ldlr-/- mice in the absence of ATP-binding cassette transporters A1 and G1 in myeloid cells[J].Arterioscler Thromb Vasc Biol,2014,34(2):279-284.
[18] 王莹,禇扬,李伟,等.三七中皂苷成分及其药理作用的研究进展 [J].中草药,2015,46(9):1381-1392.
[19] 张志辉,邓安珺,于金倩,等.黄连碱药理活性研究进展 [J].中国中药杂志,2013,38(17):2750-2754.