稳斑汤含药血清对巨噬细胞泡沫化自噬相关蛋白LC3B和Beclin 1表达的影响
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摘要
目的探讨稳斑汤防治动脉粥样硬化的可能作用机制。方法 20只SD大鼠随机分为空白组及稳斑汤低、中、高浓度组各5只。稳斑汤低、中、高浓度组分别给予稳斑汤4.275、8.55、17.1 g/(kg·d)灌胃,空白组给予4 ml蒸馏水灌胃,每天1次,连续灌胃7天。灌胃第8天腹主动脉取血,离心后分离血清。体外培养小鼠巨噬细胞,分为空白对照组、模型组、雷帕霉素组及稳斑汤低、中、高浓度组,每组6孔。除空白对照组外其余各组均进行巨噬细胞泡沫化。空白对照组及模型组予10%大鼠空白血清培养;雷帕霉素组用雷帕霉素10μl/孔(终浓度10 nmol/L)培养;稳斑汤低、中、高浓度组分别用10%稳斑汤低、中、高浓度含药血清200μl/孔培养。48h后收集细胞,在透射电镜下观察各组细胞自噬体的变化,Western Blot法检测各组细胞LC3B和Beclin 1蛋白表达水平。结果透射电镜下观察到,空白对照组无明显自噬体出现,模型组可见少量自噬体出现;与模型组比较,稳斑汤各浓度组与雷帕霉素组的自噬体数量均明显增加,雷帕霉素组数量更多。与空白对照组比较,其余各组LC3B和Beclin 1蛋白表达水平均升高(P<0.05);与模型组比较,稳斑汤各浓度组及雷帕霉素组LC3B和Beclin 1蛋白表达水平均升高(P<0.05);与雷帕霉素组比较,稳斑汤高浓度组LC3B和Beclin 1蛋白表达水平差异无统计学意义(P>0.05)。结论稳斑汤含药血清能够上调巨噬细胞泡沫化自噬相关蛋白LC3B和Beclin 1表达,促进泡沫化的巨噬细胞的自噬,从而抑制巨噬细胞泡沫化的进展,这可能是稳斑汤防治动脉粥样硬化的分子机制之一。
Objective To explore the mechanism of prevention and treatment of atherosclerosis by Wenban Decoction(稳斑汤). Methods Twenty SD rats were randomly divided into blank group and Wenban Decoction group of low,medium and high concentrations,with 5 rats in each group. Wenban Decoction low,medium,high dose groups were given Wenban Decoction 4. 275,8. 55,17. 1 g/( kg·d) gavage,and blank group was given 4 ml distilled water,once a day,continuous gavage for 7 days. Eight days after intragastric gavage,blood was taken from the abdominal aorta and the serum was separated after centrifugation. Mouse macrophages were cultured in vitro and divided into blank control group,model group,rapamycin group,and low-,medium-,and high-concentration group of Wenban Decoction,with 6 holes in each group. Except for the blank control group,macrophage foaming was performed in all other groups. The blank control group and model group were cultured with 10% rat blank serum; the rapamycin group was cultured with 10 μl/hole of rapamycin( final concentration of 10 nmol/L); the low,medium,and high concentrations of Wenban Decoction were treated with 10 % Wenban Decoction low,medium and high concentration of serum containing 200 μl/hole culture. After 48 hours,the cells were collected and the autophagosomes were observed under transmission electron microscope. The expression of LC3 B and Beclin 1 protein in each group was detected by Western Blot. Results Under transmission electron microscope,there were no obvious autophagosomes in the blank control group,and a small amount of autophagosomes were seen in the model group. Compared with the model group,the number of autophagosomes in each concentration of Wenban Decoction group and the rapamycin group was significantly increased,and the number was larger in rapamycin group. Compared with the blank control group,the expression of LC3B and Beclin 1 protein in the other groups increased( P < 0. 05); compared with the model group,the expression levels of LC3B and Beclin 1 protein in each group of Wenban Decoction and rapamycin group elevated( P < 0. 05); Compared with rapamycin group,there was no significant difference in the expression level of LC3B and Beclin 1 protein in the high concentration group of Wenban Decoction( P > 0. 05). Conclusion Serum containing Wenban Decoction can up-regulate the expression of macrophage foaming autophagy-associated proteins LC3B and Beclin 1 and promote autophagy of foamed macrophages,thereby inhibiting the progress of macrophage foaming,which may be one of the molecular mechanisms of prevention and treatment of atherosclerosis by Wenban Decoction.
Objective To explore the mechanism of prevention and treatment of atherosclerosis by Wenban Decoction(稳斑汤). Methods Twenty SD rats were randomly divided into blank group and Wenban Decoction group of low,medium and high concentrations,with 5 rats in each group. Wenban Decoction low,medium,high dose groups were given Wenban Decoction 4. 275,8. 55,17. 1 g/( kg·d) gavage,and blank group was given 4 ml distilled water,once a day,continuous gavage for 7 days. Eight days after intragastric gavage,blood was taken from the abdominal aorta and the serum was separated after centrifugation. Mouse macrophages were cultured in vitro and divided into blank control group,model group,rapamycin group,and low-,medium-,and high-concentration group of Wenban Decoction,with 6 holes in each group. Except for the blank control group,macrophage foaming was performed in all other groups. The blank control group and model group were cultured with 10% rat blank serum; the rapamycin group was cultured with 10 μl/hole of rapamycin( final concentration of 10 nmol/L); the low,medium,and high concentrations of Wenban Decoction were treated with 10 % Wenban Decoction low,medium and high concentration of serum containing 200 μl/hole culture. After 48 hours,the cells were collected and the autophagosomes were observed under transmission electron microscope. The expression of LC3 B and Beclin 1 protein in each group was detected by Western Blot. Results Under transmission electron microscope,there were no obvious autophagosomes in the blank control group,and a small amount of autophagosomes were seen in the model group. Compared with the model group,the number of autophagosomes in each concentration of Wenban Decoction group and the rapamycin group was significantly increased,and the number was larger in rapamycin group. Compared with the blank control group,the expression of LC3B and Beclin 1 protein in the other groups increased( P < 0. 05); compared with the model group,the expression levels of LC3B and Beclin 1 protein in each group of Wenban Decoction and rapamycin group elevated( P < 0. 05); Compared with rapamycin group,there was no significant difference in the expression level of LC3B and Beclin 1 protein in the high concentration group of Wenban Decoction( P > 0. 05). Conclusion Serum containing Wenban Decoction can up-regulate the expression of macrophage foaming autophagy-associated proteins LC3B and Beclin 1 and promote autophagy of foamed macrophages,thereby inhibiting the progress of macrophage foaming,which may be one of the molecular mechanisms of prevention and treatment of atherosclerosis by Wenban Decoction.
引文
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[17]CAO Y,KLIONSKY DJ.Physiological functions of Atg6/Beclin-1:a unique autophagy-related protein[J].Cell Res,2007,17(10):839-849.
[18]WIRAWAN E,LIPPENS S,VANDEN BERGHE T,et al.Beclin 1:a role in membrane dynamics and beyond[J].Autophagy,2012,8(1):6-17.
[19]FINGAR DC,BLENIS J.Target of rapamycin(TOR):an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression[J].Oncogene,2004,23(18):3151-3171.
[2]NOUROOZ-ZADEH J,SMITH CC,BETTERIDGE DJ.Measures of oxidative stress in heterozygous familial hypercholesterolaemia[J].Atherosclerosis,2001,156(2):435-441.
[3]HAN J,PAN XY,XU Y,et al.Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage[J].Autophagy,2012,8(5):812-825.
[4]LIU H,CAO Y,TONG T,et al.Autophagy in atherosclerosis:a phenomenon found in human carotid atherosclerotic plaques[J].Chin Med J,2015,128(1):69-74.
[5]OUIMET M,MARCEL YL.Regulation of lipid droplet cholesterol efflux from macrophage foam cells[J].Arterioscler Thromb Vasc Biol,2012,32(3):575-581.
[6]TANIDA I,UENO T,KOMINAMI E.LC3 conjugation system in mammalian autophagy[J].Int J Biochem Cell Biol,2004,36(12):2503-2518.
[7]DJAVAHERI-MERGNY M,MAIURI MC,KROEMER G.Cross talk between apoptosis and autophagy by casepasemediated cleavage of Beclin 1[J].Oncogene,2010,29(12):1717-1719.
[8]倪小鸥,宫丽鸿,张湜,等.搜风祛痰中药对Apo E基因敲除小鼠动脉粥样硬化不稳定斑块自噬相关蛋白Beclin 1和LC3的影响[J].中西医结合心脑血管病杂志,2016,14(5):488-491.
[9]张君涛,王平,刘爱峰,等.中药含药血清制备方法的研究概述[J].中华中医药杂志,2015,30(11):4006-4009.
[10]薛偕华,魏伟,陈彤,等.泽泻汤对巨噬细胞泡沫化脂质沉积及其LXRα和ABCA1表达的影响[J].中国动脉硬化杂志,2013,21(11):971-976.
[11]LEY K,MILLER YI,HEDRICK CC.Monocyte and macrophage dynamics during atherogenesis[J].Arterioscler Thromb Vasc Biol,2011,31(7):1506-1516.
[12]JOHANSEN T,LAMARK T.Selective autophagy mediated by autophagic adapter proteins[J].Autophagy,2011,7(3):279-296.
[13]LIAO X,SLUIMER JC,WANG Y,et al.Macrophage autophagy plays a protective role in advanced atherosclerosis[J].Cell Metab,2012,15(4):545-553.
[14]SINGH R,KAUSHIK S,WANG Y,et al.Autophagy regulates lipid metabolism[J].Nature,2009,458(7242):1131-1135.
[15]OUIMET M,FRANKLIN V,MAK E,et al.Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase[J].Cell Metab,2011,13(6):655-667.
[16]WU J,DANG Y,SU W,et al.Molecular cloning and characterization of rat LC3A,and LC3B—Two novel markers of autophagosome[J].Biochem Biophys Res Commun,2006,339(1):437-442.
[17]CAO Y,KLIONSKY DJ.Physiological functions of Atg6/Beclin-1:a unique autophagy-related protein[J].Cell Res,2007,17(10):839-849.
[18]WIRAWAN E,LIPPENS S,VANDEN BERGHE T,et al.Beclin 1:a role in membrane dynamics and beyond[J].Autophagy,2012,8(1):6-17.
[19]FINGAR DC,BLENIS J.Target of rapamycin(TOR):an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression[J].Oncogene,2004,23(18):3151-3171.