【作者】 秦书芝；

【导师】 肖洪彬；

【作者基本信息】 黑龙江中医药大学 ， 方剂学， 2014， 博士

【摘要】 目的：以补阳还五汤、少腹逐瘀汤、丹参饮三首活血化瘀方药为探针,以气滞血瘀模型为研究对象,以红细胞膜生物学指标为靶点,探讨三方对气滞血瘀模型红细胞膜生物学指标的影响,明确气滞血瘀模型的证候本质,并揭示三方在红细胞膜分子生物学水平的作用机制。方法：以大鼠为研究对象,采用不可预知的刺激配合肾上腺素皮下注射的方法复制大鼠气滞血瘀模型,同时灌胃给予补阳还五汤、丹参饮、少腹逐瘀汤高低剂量。持续刺激、给药两周至大鼠出现气滞血瘀表征,颈动脉取血,检测全血粘度、血浆粘度、血沉K值、红细胞变形性、凝血四项、TC及TG含量；取血制备红细胞膜,检测红细胞膜唾液酸、巯基及膜MDA、胆固醇含量,检测膜Na+-K+-ATP酶及SOD活力；采用SDS-PAGE及凝胶成像分析系统检测膜蛋白的组成及含量。结果：同空白组比较,气滞血瘀模型组全血黏度、血浆黏度血沉K值、血浆胆固醇(CHO)及FIB含量显著升高,红细胞变形指数、血浆甘油三酯(TG)显著降低,APTT、PT显著缩短,红细胞膜MDA、胆固醇含量显著升高(P<0.05,P<0.01)。与模型组比较,补阳还五汤、少腹逐瘀汤及丹参饮三方均能显著升高气滞血瘀模型大鼠高、低切变率下的红细胞变形指数,均可显著延长APTT(P<0.05,P<0.01),显著提高红细胞膜Na+-K+-ATP酶活力(P<0.05)；三方均可显著降低血沉K值及FIB含量(P<0.05,P<0.01)。少腹逐瘀汤和丹参饮可显著降低血浆粘度及膜胆固醇含量(P<0.05,P<0.01)。补阳还五汤和丹参饮可显著升高中切变率下的红细胞变形指数,显著降低血浆CHO及膜MDA含量(P<0.05,P<0.01)。丹参饮可显著降低中、低切变率下的全血粘度(P<0.05)。补阳还五汤可显著延长PT,显著缩短TT(P<0.05)。给药组同模型组比较,补阳还五汤可显著降低带4.2蛋白含量,显著提高带6蛋白含量(P<0.05)；少腹逐瘀汤可显著提高原肌球调节蛋白含量及带5蛋白含量(P<0.05,P<0.01),可显著降低带4.2蛋白含量(P<0.05)；丹参饮可显著提高带4.1蛋白含量(P<0.05)。结论：1.模型组全血粘度、血浆粘度、红细胞变形指数、血沉K值、FIB等宏观血液流变学指标均出现明显改变,符合血瘀证的客观诊断标准,说明本模型符合中医血瘀证模型。2.补阳还五汤、少腹逐瘀汤及丹参饮三方均能提高气滞血瘀模型红细胞变形能力、提高红细胞膜Na+-K+-ATP酶活力、延长APTT、降低FIB含量、降低血沉K值,从而改善血液高凝状态,提高红细胞变形能力,阻止红细胞聚集。在提高红细胞变形能力、降低FIB含量、延长APTT方面,丹参饮作用优于其他两方。3.少腹逐瘀汤和丹参饮可降低气滞血瘀模型血浆粘度及膜胆固醇含量；补阳还五汤和丹参饮可降低血浆CHO及膜MDA含量；丹参饮对血浆CHO及膜胆固醇的作用优于其他两方。丹参饮可降低气滞血瘀模型的全血粘度；补阳还五汤可显著延长PT,显著缩短TT。4.补阳还五汤可作用于红细胞膜的带4.2蛋白及带6蛋白；少腹逐瘀汤作用于原肌球调节蛋白、带5蛋白及带4.2蛋白；丹参饮作用于带4.1蛋白。更多还原

【Abstract】 Objective:Using3formulas for activating blood circulation as probe, Qi-stagnation and blood stasis model as research object, and bioindicator of erythrocyte membrane as target, to discuss the effect of three formulas on bioindicator of erythrocyte membrane of Qi-stagnation and blood stasis model. In order to clear syndrome essence of Qi-stagnation and blood stasis model, and reveal action mechanism of3formulas at the molecular biology level of erythrocyte membrane.Methods:Rats were used as research objects. We used the method of unpredictable stimulation with adrenaline subcutaneous injection to copy Qi-stagnation and blood stasis model of rats. At the same time, rats received gastric gavage of Buyang Huanwu Decoction, Danshen Decoction, and Shaofu Zhuyu Decoction with high and low dose. Constant stimulation and drug administration for two weeks until rats showed characterization of Qi-stagnation and blood stasis. We took blood from carotid artery, and detected whole blood viscosity, plasma viscosity, blood sedimentation K value, erythrocyte deforma-bility, four coagulation tests, TC and TG content. We took blood to prepare erythrocyte membrane, and detected erythrocyte membrane sialic acid, sulfydryl and content of membrane SOD, MDA, cholesterol. We tested Na+-K+-ATP enzyme activity membrane; Using SDS-PAGE and SYNGENE to detect the composition and content of the membrane protein.Results:Compared with blank group, whole blood viscosity, plasma viscosity, blood sedimentation K value, content of serum cholesterol (CHO) and FIB in Qi-stagnation and blood stasis model group increased significantly, and HBX, triglyceride (TG) decreased significantly, APTT and PT shortened significantly, erythrocyte membrane MDA and cholesterol increased signifi-cantly (P<0.05, P<0.01).Compared with model group, three formulas of Buyang Huanwu Decoction, Danshen Decoction, and Shaofu Zhuyu Decoction all could increase HBX of Qi-stagnation and blood stasis model under low and high shear rate significantly, and prolong APTT significantly (P<0.05, P<0.01), increase Na+-K+-ATP enzyme activity on erythrocyte membrane significantly (P<0.05); Three formulas all could decrease blood sedimentation K value (P<0.05) and content of FIB significantly (P<0.05, P<0.01). Shaofu Zhuyu Decoction and Danshen Decoction all could significantly reduce blood viscosity and membrane cholesterol (P<0.05, P<0.01). Buyang Huanwu Decoction, Danshen Decoction could significantly increase HBX under low and high shear rate, and significantly reduce plasma CHO and MDA content on membrane (P<0.05, P<0.01). Danshen Decoction could significantly reduce whole blood viscosity under middle and low shear rate (P<0.05). Buyang Huanwu Decoction could significantly prolong PT, and significantly shorten TT (P<0.05).Compared with model group, Buyang Huanwu Decoction could significantly reduce the content of band4.2protein, while significantly increase the content of band6protein (P<0.05). Shaofu Zhuyu Decoction could significantly reduce the content of band4.2protein, while significantly increase the content of band5protein and the original ball regulatory proteins (P<0.05, P<0.01). Danshen Decoction could significantly increase the content of band4.1protein (P<0.05).Conclusion:1. Macroscopic hemorheology indexes such as whole blood viscosity, plasma viscosity, HBX, blood sedimentation K value, FIB in model group all showed significant changes and measured up to diagnostic criteria of blood stasis syndrome.2. Three formulas of Buyang Huanwu Decoction, Danshen Decoction, and Shaofu Zhuyu Decoction all could improve erythrocyte deformation ability of Qi-stagnation and blood stasis model; They could increase Na+-K+-ATP enzyme activity on erythrocyte membrane, prolong APTT, reduce the content of FIB and reduce high blood coagulation state; They could reduce blood sedimentation K value, and prevent erythrocyte aggregation. Danshen Decoction was better than other two formulas in aspects of raising erythrocyte deformation ability, reducing the content of FIB and prolonging APTT. 3. Shaofu Zhuyu Decoction and Danshen Decoction could correct high blood viscosity and high membrane cholesterol content in Qi-stagnation and blood stasis model group; Buyang Huanwu Decoction and Danshen Decoction could decrease the plasma CHO content and MDA content on membrane; In terms of reducing plasma CHO and membrane cholesterol, Danshen Decoction was better than that of the other two. Danshen Decoction could improve whole blood viscosity increasing of Qi-stagnation and blood stasis model; Buyang Huanwu Decoction could significantly prolong PT and significantly shorten the TT.4. Buyang Huanwu Decoction could act on erythrocyte membrane band4.2protein and band6protein; Shaofu Zhuyu Decoction could act on the original ball regulatory proteins, band4.2protein and band5protein; Danshen Decoction could act on band4.1protein.更多还原