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

Effect of Cooling Blood Method in Inhibiting Macrophage Apoptosis Against Atherosclerosis

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^-1ac-LDL刺激细胞24 h,其他组用BSA+50 mg·L^-1ac-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^-1ac-LDL for 24 h. The other groups were stimulated with BSA + 50 mg·L^-1acLDL + 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.