活血化瘀“生新层面”之“化旧生新”法干预细胞凋亡机制研究

Study on the Intervention Mechanism of Apoptosis by Removing Damaged Tissue and Generating New Tissue on the Basis of Generating New Level Tissue Through Activating Blood Circulation and Removing Blood Stasis

目的:探讨活血化瘀"生新层面"之"化旧生新"干预细胞凋亡的机制。方法:清洁级SD成年雄性健康大鼠采用Oli-vette[5]方法建立AMI模型,造模24 h后存活大鼠随机分成5组,活血化瘀高剂量组:血塞通600 mg.kg-1灌胃;活血化瘀中剂量组:血塞通400 mg.kg-1灌胃;活血化瘀低剂量组:血塞通200 mg.kg-1灌胃,模型组和假手术组:生理盐水10 mL.kg-1,连续灌胃15 d,每天1次,直到每组处死动物前保证8只存活大鼠,采用免疫组化法检测梗死边沿区Bax和Bcl-2蛋白表达、蛋白caspase-3的表达和采用TUNEL染色检测梗死边沿区心肌细胞凋亡的变化。结果:活血化瘀高、中剂量组与模型组明显下调Bax蛋白表达(P<0.05),上调Bcl-2蛋白表达(P<0.05)。模型组与假手术组相比Bax蛋白阳性表达有所升高,Bcl-2蛋白表达有所下降(P<0.05);活血化瘀高、中剂量组与假手术组和模型组比较,能明显抑制大鼠缺血后心肌细胞凋亡(P<0.05)。模型组大鼠急性心肌梗死边沿区蛋白caspase-3呈现过度表达,活血化瘀高、中剂量组与模型组比较能明显抑制caspase-3的表达,有显著意义(P<0.05)。结论:活血化瘀生新层面之"化旧生新"作用机制可能与其通过多靶点抑制心肌细胞凋亡、多途径降低细胞凋亡指数有关,从而发挥保护心肌作用。

Objective：To discuss the mechanism of apoptosis intervened by removing damaged tissue and generating new tissue on the basis of generating new level tissue through activating blood circulation and removing blood stasis.Methods：Healthy adult male SD rats（clean class） were built into AMI model by adopting the method of Olivette[5],and were randomly divided into five groups after modeling for 24 hours,namely,activating blood circulation and removing blood stasis in high-dose group（gastric irrigation with Xueshuangtong 6 000 mg·kg-1）,middle-dose group（gastric irrigation with Xueshuangtong 4 000 mg·kg-1）,low-dose group（gastric irrigationwith Xueshuangtong 2 000 mg·kg-1）,model group（gastric irrigation with physiological saline 100 mL·kg-1）,sham operation group（gastric irrigation with physiological saline 100 mL·kg-1）.Rats in each group were made gastric irrigation for 15 days,once a day.8 rats should be certainly ensured survival before other rats in every sub-group were all put to death and then immunohistochemistry method was used to detect protein expression of Bax,Bc1-2,caspase-3 in the edge of infarction area.TUNEL staining was used to detect changes of cardiac muscle apoptosis in the edge of infarction area.Results：Protein expression of Bax obviously declined（P0.05）;protein expression of Bc1-2 obviously ascended（P0.05） in the high-dose group and middle-dose group compared with the model group;protein positive expression of Bax slightly ascended and protein expression of Bc1-2 slightly declined in the model group compared with the sham operation group（P0.05）;the high-dose group and middle-dose group of activating blood circulation and removing blood stasis could obviously restrain cardiac muscle apoptosis of rats after blood insufficiency compared with the model group and the sham operation group（P0.05）;protein caspase-3 in the model group showed excessive expression in the edge of acute cardiac muscle.The high-dose group and middle-dose group of activating blood circulation and removing blood stasis could obviously restrain protein caspase-3 expression and had significant difference（P0.05）.Conclusion：The mechanism of removing damaged tissue and generating new tissue on the basis of generating new level tissue through activating blood circulation and removing blood stasis can protect cardiac muscle,through restraining cardiac muscle apoptosis in multiple target spots and reducing index of apoptosis with multiple ways.