凉血散瘀法通过抑制巨噬细胞凋亡抗动脉粥样硬化的作用
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摘要
目的:观察凉血散瘀法清心通脉饮含药血清对乙酰化低密度脂蛋白(acetylated low density lipoprotein,ac-LDL)诱导的小鼠单核巨噬细胞系RAW264. 7凋亡率及A型清道夫受体(type A scavenger receptor,SR-A),B细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2),Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax),肌醇需要酶1α(inositol-requiring enzyme lα,IRE1α)表达的影响,探讨清心通脉饮治疗动脉粥样硬化可能的作用机制。方法:8只新西兰兔采用随机数字法分为阿托伐他汀组(2. 6 g·kg~(-1)),清心通脉饮低、中、高剂量组(3. 33,6. 66,13. 32 mg·kg~(-1)),灌胃7 d后颈动脉采血收集含药血清。各组用2. 5,5,10,20%体积分数的含药血清培养液分别刺激RAW264. 7细胞系6,12,24 h,采用细胞增殖与活性检测-8(CCK-8)法观察细胞增殖率。体外培养RAW264. 7细胞系,分为空白组、模型组、阿托伐他汀组、清心通脉饮低、中、高剂量组。空白组用牛血清白蛋白(BSA)培养细胞,模型组用BSA+50 mg·L~(-1)ac-LDL刺激细胞24 h,其他组用BSA+50 mg·L~(-1)ac-LDL+10%含药血清刺激细胞24 h。流式细胞技术检测每组RAW264. 7细胞系凋亡率和SR-A的表达,蛋白免疫印迹法(Western blot)检测Bcl-2,Bax,IRE1α蛋白的表达。结果:与空白组比较,模型组可显著增加RAW264. 7细胞凋亡率(P <0. 01),并增加Bax,SR-A蛋白表达(P <0. 01),减少Bcl-2蛋白表达(P <0. 05)。与模型组比较,清心通脉饮低、中、高剂量组均可降低RAW264. 7细胞系凋亡率(P <0. 05),减少SR-A和IRE1α表达(P <0. 05,P <0. 01)。清心通脉饮低、高剂量组可降低Bax表达(P <0. 05,P <0. 01);清心通脉饮中、高剂量组可降低Bcl-2表达(P <0. 05)。结论:清心通脉饮可降低巨噬细胞凋亡率,其治疗动脉粥样硬化机制可能与调控相关凋促亡蛋白Bax,IREα,SR-A和抗凋亡蛋白Bcl-2的表达有关。
Objective: To observe the effect of serum-containing Qingxin Tongmai decoction( QXTMD)on the apoptosis rate of mouse mononuclear macrophage cell line RAW264. 7 induced by Acetylated low density lipoprotein( ac-LDL) and the expressions of type A scavenger receptor( SR-A),B-cell lymphoma-2( Bcl-2),Bcl-2-associated X protein( Bax),inositol-requiring enzyme 1α( IRE1α),exploring the possible mechanism of QXTMD in the treatment of atherosclerosis. Method: Eight New Zealand rabbits were randomly divided into the atorvastatin group( 2. 6 g·kg~(-1)) low,medium and high-dose QXTMD groups( 3. 33,6. 66,13. 32 mg·kg~(-1)).After 7 days of gavage,the carotid blood was collected to prepare drug-containing serum. The RAW264. 7 cell line was stimulated with 2. 5%,5%,10%,and 20% drug-containing serum culture for 6,12,and 24 h,respectively.The cell proliferation rate was observed by cell counting kit-8( CCK-8) method. The RAW264. 7 cell line was cultured in vitro and divided into blank group,model group,atorvastatin group,and low,medium and high-dose QXTMY groups. The cells in blank group were cultured with bovine serum albumin( BSA). The model group was stimulated with BSA + 50 mg·L~(-1) ac-LDL for 24 h. The other groups were stimulated with BSA + 50 mg·L~(-1) acLDL + 10% drug-containing serum for 24 h. The apoptosis rate and SR-A expression of RAW264. 7 cells were detected by flow cytometry. The expressions of Bcl-2,Bax and IRE1α protein were detected by Western blot.Result: Compared with the blank group,the model group could increase the apoptosis rate of RAW264. 7 cells( P < 0. 01) and the expressions of Bax and SR-A protein( P < 0. 01),but decrease the expression of Bcl-2 protein( P < 0. 05). Compared with the model group,low,medium and high-dose QXTMD groups could decrease the apoptosis rate of RAW264. 7 cell line( P < 0. 05) and the expressions of SR-A and IRE1α( P < 0. 05,P < 0. 01). The low-dose QXTMD group and the high-dose QXTMD group could decrease the expression of Bax( P < 0. 05,P < 0. 01). The middle-dose group and the high-dose group could decrease the expression of Bcl-2( P < 0. 01). Conclusion: QXTMD can reduce the apoptosis rate of macrophages. The mechanism of atherosclerosis may be related to the expressions of Bax,IREα,SR-A and anti-apoptotic protein Bcl-2.
Objective: To observe the effect of serum-containing Qingxin Tongmai decoction( QXTMD)on the apoptosis rate of mouse mononuclear macrophage cell line RAW264. 7 induced by Acetylated low density lipoprotein( ac-LDL) and the expressions of type A scavenger receptor( SR-A),B-cell lymphoma-2( Bcl-2),Bcl-2-associated X protein( Bax),inositol-requiring enzyme 1α( IRE1α),exploring the possible mechanism of QXTMD in the treatment of atherosclerosis. Method: Eight New Zealand rabbits were randomly divided into the atorvastatin group( 2. 6 g·kg~(-1)) low,medium and high-dose QXTMD groups( 3. 33,6. 66,13. 32 mg·kg~(-1)).After 7 days of gavage,the carotid blood was collected to prepare drug-containing serum. The RAW264. 7 cell line was stimulated with 2. 5%,5%,10%,and 20% drug-containing serum culture for 6,12,and 24 h,respectively.The cell proliferation rate was observed by cell counting kit-8( CCK-8) method. The RAW264. 7 cell line was cultured in vitro and divided into blank group,model group,atorvastatin group,and low,medium and high-dose QXTMY groups. The cells in blank group were cultured with bovine serum albumin( BSA). The model group was stimulated with BSA + 50 mg·L~(-1) ac-LDL for 24 h. The other groups were stimulated with BSA + 50 mg·L~(-1) acLDL + 10% drug-containing serum for 24 h. The apoptosis rate and SR-A expression of RAW264. 7 cells were detected by flow cytometry. The expressions of Bcl-2,Bax and IRE1α protein were detected by Western blot.Result: Compared with the blank group,the model group could increase the apoptosis rate of RAW264. 7 cells( P < 0. 01) and the expressions of Bax and SR-A protein( P < 0. 01),but decrease the expression of Bcl-2 protein( P < 0. 05). Compared with the model group,low,medium and high-dose QXTMD groups could decrease the apoptosis rate of RAW264. 7 cell line( P < 0. 05) and the expressions of SR-A and IRE1α( P < 0. 05,P < 0. 01). The low-dose QXTMD group and the high-dose QXTMD group could decrease the expression of Bax( P < 0. 05,P < 0. 01). The middle-dose group and the high-dose group could decrease the expression of Bcl-2( P < 0. 01). Conclusion: QXTMD can reduce the apoptosis rate of macrophages. The mechanism of atherosclerosis may be related to the expressions of Bax,IREα,SR-A and anti-apoptotic protein Bcl-2.
引文
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[12]伦琦星,刘炳琳,李军,等.猫耳刺皂苷ilexpernoside C抑制低密度脂蛋白多聚体诱导泡沫细胞形成的活性及机制研究[J].中国中药杂志,2016,41(3):498-503.
[13] Kzhyshkowska J, Neyen C, Gordon S. Role of macrophage scavenger receptors in atherosclerosis[J].Immunobiology,2012,217(5):492-502.
[14] Tsuzuki S,Kimoto Y,Lee S,et al. A novel role for scavenger receptor B1 as a contributor to the capture of specific volatile odorants in the nasal cavity[J]. Biomed Res,2018,39(3):117-129.
[15] ZHU G Y,ZHU X L,LI R T,et al. Atorvastatin inhibits scavenger receptor A and monocyte chemoattractant protein-1 expressions in foam cell[J].Chin J Cardiol Med,2007,35(7):666-669.
[16] DAI X Y,CAI Y,MAO D D,et al. Increased stability of phosphatase and tensin homolog by intermedin leading to scavenger receptor A inhibition of macrophages reduces atherosclerosis in apolipoprotein E-deficient mice[J]. J Mol Cell Cardiol,2012,53(4):509-520.
[17] To M,Peterson C W,Roberts M A,et al. Lipid disequilibrium disrupts ER proteostasis by impairing ERAD substrate glycan trimming and dislocation[J].Mol Biol Cell,2017,28(2):270-284.
[18] Abdullah A,Ravanan P. The unknown face of IRE1alpha-Beyond ER stress[J]. Eur J Cell Biol,2018,97(5):359-368.
[19]刘斯文,郭晓辰,张军平.内质网应激中IRE1级联反应对动脉粥样硬化的作用[J].中国病理生理杂志,2016,32(6):1147-1152.
[20] ZHOUA X,Tabas I. The UPR in atherosclerosis[J].Semin Immunopathol,2013,35(3):321-332.
[2] Perrotta P,Veseli B E,Van der Veken B,et al.Pharmacological strategies to inhibit intra-plaque angiogenesis in atherosclerosis[J]. Vascul Pharmacol,2018,doi:10. 1016/j. vph. 2018. 06. 014.
[3] Andres V,Pello O M,Silvestre-Roig C. Macrophage proliferation and apoptosis in atherosclerosis[J]. Curr Opin Lipidol,2012,23(5):429-438.
[4] Orekhov A N. LDL and foam cell formation as the basis of atherogenesis[J]. Curr Opin Lipidol,2018,29(4):279-284.
[5] Gonzalez L,Trigatti B L. Macrophage apoptosis and necrotic core development in atherosclerosis:a rapidly advancing field with clinical relevance to imaging and therapy[J]. Can J Cardiol,2017,33(3):303-312.
[6]殷小杰,马晓静,王岚,等.三黄泻心汤活血化瘀优势方抗动脉粥样硬化的作用机制[J].中国实验方剂学杂志,2018,24(22):83-88.
[7]陈宁,宋囡,贾连群,等.化瘀祛痰方对AS家兔肝脏线粒体呼吸链酶复合物的影响[J].中国实验方剂学杂志,2018,24(7):171-176.
[8]周仲瑛.瘀热相搏证的系列研究(一)[J].天津中医药大学学报,2008,27(3):151-155.
[9]刘美之,郎艳松,张鑫月,等.从痰、瘀、毒论治动脉粥样硬化研究进展[J].中医杂志,2014,55(9):800-803.
[10]孙少卫,童文娟,谢雪娇,等.动脉粥样硬化的中西医观[J].中国动脉硬化杂志,2016,24(5):524-528.
[11]钟伟,王永刚,于远望,等.中医防治颈动脉粥样硬化的研究进展[J].中华中医药学刊,2017,35(8):2043-2045.
[12]伦琦星,刘炳琳,李军,等.猫耳刺皂苷ilexpernoside C抑制低密度脂蛋白多聚体诱导泡沫细胞形成的活性及机制研究[J].中国中药杂志,2016,41(3):498-503.
[13] Kzhyshkowska J, Neyen C, Gordon S. Role of macrophage scavenger receptors in atherosclerosis[J].Immunobiology,2012,217(5):492-502.
[14] Tsuzuki S,Kimoto Y,Lee S,et al. A novel role for scavenger receptor B1 as a contributor to the capture of specific volatile odorants in the nasal cavity[J]. Biomed Res,2018,39(3):117-129.
[15] ZHU G Y,ZHU X L,LI R T,et al. Atorvastatin inhibits scavenger receptor A and monocyte chemoattractant protein-1 expressions in foam cell[J].Chin J Cardiol Med,2007,35(7):666-669.
[16] DAI X Y,CAI Y,MAO D D,et al. Increased stability of phosphatase and tensin homolog by intermedin leading to scavenger receptor A inhibition of macrophages reduces atherosclerosis in apolipoprotein E-deficient mice[J]. J Mol Cell Cardiol,2012,53(4):509-520.
[17] To M,Peterson C W,Roberts M A,et al. Lipid disequilibrium disrupts ER proteostasis by impairing ERAD substrate glycan trimming and dislocation[J].Mol Biol Cell,2017,28(2):270-284.
[18] Abdullah A,Ravanan P. The unknown face of IRE1alpha-Beyond ER stress[J]. Eur J Cell Biol,2018,97(5):359-368.
[19]刘斯文,郭晓辰,张军平.内质网应激中IRE1级联反应对动脉粥样硬化的作用[J].中国病理生理杂志,2016,32(6):1147-1152.
[20] ZHOUA X,Tabas I. The UPR in atherosclerosis[J].Semin Immunopathol,2013,35(3):321-332.