丹红注射液对动脉粥样硬化小鼠主动脉自噬基因Atg13启动子区甲基化及PI3K/Akt/mTORC1信号通路的影响
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摘要
目的观察丹红注射液对动脉粥样硬化(AS)小鼠自噬基因Atg13启动子区甲基化及自噬信号通路PI3K/Akt/mTORC1的影响,探讨其可能的作用机制。方法 40只8周龄雄性ApoE~(-/-)小鼠高脂饲料喂养6周,随机分为模型组、阳性对照组和丹红注射液低、高剂量组,每组10只,各给药组给予相应药物干预。6周后RT-PCR检测AS小鼠主动脉自噬基因Atg13、Atg3和Atg4a的mRNA表达;Westernblot检测AS小鼠主动脉Beclin1、LC3Ⅱ/Ⅰ、PI3K、Akt、p-Akt、mTORC1和p-mTORC1的蛋白表达;采用亚硫酸氢盐测序法检测AS小鼠主动脉自噬基因Atg13启动子区甲基化水平。结果与模型组比较,丹红注射液高、低剂量组小鼠主动脉Beclin1蛋白表达明显升高,丹红注射液低剂量组小鼠主动脉LC3Ⅱ/Ⅰ表达明显升高(P<0.01),丹红注射液高、低剂量组小鼠主动脉Atg13、Atg3和Atg4a mRNA表达显著升高(P<0.01),丹红注射液高、低剂量组小鼠主动脉自噬基因Atg13启动子区甲基化水平明显降低(P<0.01),丹红注射液高、低剂量组小鼠主动脉p-Akt蛋白表达明显降低(P<0.01),丹红注射液高剂量组小鼠主动脉p-mTORC1蛋白表达明显降低(P<0.01)。结论丹红注射液防治AS的机制可能与降低模型小鼠主动脉Atg13启动子区甲基化水平、抑制PI3K/Akt/mTORC1信号通路和促进细胞自噬有关。
Objective To study the effects of Danhong Injection on methylation of promoter region of autophagy gene Atg13 and autophagy signaling pathway PI3K/Akt/mTORC1 in atherosclerotic(AS) mice, to explore possible action mechanism. Methods Totally forty eight-week-old male ApoE-/-mice were fed with high-fat diet for 6 weeks.They were randomly divided into model group, positive control group, Danhong Injection low-and high-dosage groups(n=10). The administration groups were given the corresponding drug for intervention. After 6 weeks, the mRNA expressions of the autophagy genes Atg13, Atg3 and Atg4 a in AS mice were detected by RT-PCR; Western blot method was used to detect the protein expressions of Beclin1, LC3Ⅱ/Ⅰ, PI3K, p-Akt, mTORC1 and p-mTORC1 in the aorta of AS mice; Methylation level of the promoter region of autophagy gene Atg13 in the aorta of AS mice was detected by bisulfile sequencing PCR. Results Compared with model group, the protein expressions of Beclin1 in aorta of Danhong Injection groups significantly increased, LC3Ⅱ/Ⅰ expressions in aorta of Danhong Injection low-dosage group significantly increased(P<0.01), and the mRNA expressions of Atg13, Atg3 and Atg4 a in Danhong Injection groups also increased significantly(P<0.01); The level of methylation of promoter region of autophagy gene Atg13 in Danhong Injection groups significantly decreased(P<0.01), and the protein expressions of p-Akt in aorta of Danhong Injection groups significantly decreased(P<0.01), the protein expressions of p-mTORC1 Danhong Injection in aorta of Danhong Injection groups high-dosage groups significantly decreased(P<0.01). Conclusion The mechanism of Danhong Injection in preventing and treating AS may be related to decrease of methylation level of Atg13 in the promoter region in the aorta of model mice, reducing signaling pathway PI3K/Akt/mTORC1 and promoting autophagy.
Objective To study the effects of Danhong Injection on methylation of promoter region of autophagy gene Atg13 and autophagy signaling pathway PI3K/Akt/mTORC1 in atherosclerotic(AS) mice, to explore possible action mechanism. Methods Totally forty eight-week-old male ApoE-/-mice were fed with high-fat diet for 6 weeks.They were randomly divided into model group, positive control group, Danhong Injection low-and high-dosage groups(n=10). The administration groups were given the corresponding drug for intervention. After 6 weeks, the mRNA expressions of the autophagy genes Atg13, Atg3 and Atg4 a in AS mice were detected by RT-PCR; Western blot method was used to detect the protein expressions of Beclin1, LC3Ⅱ/Ⅰ, PI3K, p-Akt, mTORC1 and p-mTORC1 in the aorta of AS mice; Methylation level of the promoter region of autophagy gene Atg13 in the aorta of AS mice was detected by bisulfile sequencing PCR. Results Compared with model group, the protein expressions of Beclin1 in aorta of Danhong Injection groups significantly increased, LC3Ⅱ/Ⅰ expressions in aorta of Danhong Injection low-dosage group significantly increased(P<0.01), and the mRNA expressions of Atg13, Atg3 and Atg4 a in Danhong Injection groups also increased significantly(P<0.01); The level of methylation of promoter region of autophagy gene Atg13 in Danhong Injection groups significantly decreased(P<0.01), and the protein expressions of p-Akt in aorta of Danhong Injection groups significantly decreased(P<0.01), the protein expressions of p-mTORC1 Danhong Injection in aorta of Danhong Injection groups high-dosage groups significantly decreased(P<0.01). Conclusion The mechanism of Danhong Injection in preventing and treating AS may be related to decrease of methylation level of Atg13 in the promoter region in the aorta of model mice, reducing signaling pathway PI3K/Akt/mTORC1 and promoting autophagy.
引文
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[12]王和峰,翟纯刚,庞文会,等.PI3K/Akt/mTOR信号通路在巨噬细胞自噬及动脉粥样硬化斑块不稳定中的作用[J].中国病理生理杂志,2013,29(3):390-397.
[13]任攀,周明学,刘卫红,等.丹红注射液治疗心脑血管疾病的药理作用研究进展[J].现代生物医学进展,2016,16(31):6197-6200.
[14]SANG E P,HYE J Y,NAYOUNG S,et al.Inhibition of EHMT2/G9a epigenetically increases the transcription of Beclin1 via an increase in ROS and activation of NF-κB[J].Oncotarget,2016,7(26):39796-39808.
[2]LIAO X,SLUIMER J C,WANG Y,et al.Macrophage autophagy plays a protective role in advanced atherosclerosis[J].Cell Metab,2012,15(4):545-553.
[3]MIKAELA M B,ROSS T M,ANTHONY W R.Epigenetic modulation in the treatment of atherosclerotic disease[J].Frontiers in Genetics,2014,364:1-7.
[4]孙源超,秦训思,陈宏,等.细胞自噬发生的表观遗传调节[J].遗传,2014,36(5):447-455.
[5]徐叔云,卞如濂,陈修.药理实验方法学[M].3版.北京:人民卫生出版社,2002:202-204.
[6]陈光,高嘉良,刘咏梅,等.冠心病血瘀证表观遗传学研究进展[J].中国中医基础医学杂志,2017,23(9):1336-1339.
[7]KHAN S,REHMAN R.DNA methylation:Atherosclerosis leading to congenital heart diseases[J].J Pak Med Assoc,2019,69(1):137-138.
[8]YU M,MEISSA D T,COHEN R A,et al.Autophagy and oxidative stress in cardiovascular diseases[J].Biochimica et Biophysica Acta,2015,185:243-251.
[9]张雨晴,张丽慧,魏尔清.Beclin1在凋亡和自噬中的调节作用[J].2015,31(4):331-338.
[10]晏浩,徐建军,李文林,等.MicroRNA-30a调控Beclin-1对缺氧复氧乳鼠心肌细胞的保护效应[J].中国病理生理杂志,2012,28(4):583-588.
[11]吕燕妮,付龙生,魏筱华.丹参与红花配伍机制的系统生物学分析[J].中国实验方剂学杂志,2015,21(5):220-224.
[12]王和峰,翟纯刚,庞文会,等.PI3K/Akt/mTOR信号通路在巨噬细胞自噬及动脉粥样硬化斑块不稳定中的作用[J].中国病理生理杂志,2013,29(3):390-397.
[13]任攀,周明学,刘卫红,等.丹红注射液治疗心脑血管疾病的药理作用研究进展[J].现代生物医学进展,2016,16(31):6197-6200.
[14]SANG E P,HYE J Y,NAYOUNG S,et al.Inhibition of EHMT2/G9a epigenetically increases the transcription of Beclin1 via an increase in ROS and activation of NF-κB[J].Oncotarget,2016,7(26):39796-39808.