当归四逆颗粒对冠心病寒凝血瘀证模型大鼠心肌细胞凋亡及血管活性因子的影响

Effects of Danggui Sini Granules(当归四逆颗粒)on Cardiomyocyte Apoptosis and Vasoactive Factors in Rat Models of Coronary Heart Disease with Cold Congealing and Blood Stasis Syndrome

目的探讨当归四逆颗粒治疗冠心病寒凝血瘀证的可能作用机制。方法50只SD大鼠随机分为空白组、模型组、丹参滴丸组及当归四逆颗粒低、高剂量组,每组10只。除空白组外,其余各组大鼠通过低温反复冷刺激结合腹腔注射垂体后叶素法建立大鼠冠心病寒凝血瘀证模型。造模第6周丹参滴丸组给予复方丹参滴丸0.073 g·kg^(-1)·d^(-1)灌胃,当归四逆颗粒低、高剂量组分别给予当归四逆颗粒20.2、40.4 g·kg^(-1)·d^(-1)灌胃,空白组及模型组给予无菌水0.2 ml/10 g灌胃,均连续灌胃2周。记录大鼠一般情况,比较每周体质量,干预结束后检测心电图、血液流变学指标(包括全血低切黏度、全血高切黏度、红细胞聚集指数)及心脏指数评价药物作用效果,并采用HE染色观察心肌组织病理学变化,TUNEL染色检测心肌细胞凋亡情况,ELISA法检测血清促甲状腺激素(TSH)、游离三碘甲状腺原氨酸(FT_(3))、游离四碘甲状腺原氨酸(FT_(4))水平及血栓素B_(2)(TXB_(2))、6-酮前列腺素F1α(6-Keto-PGF_(1α))、内皮素1(ET-1)、一氧化氮(NO)水平。结果与空白组同时间点比较,其余各组大鼠各时间点体质量均降低(P<0.05或P<0.01);与模型组同时间点比较,当归四逆颗粒高剂量组及丹参滴丸组大鼠在第6、7周时体质量增加(P<0.05或P<0.01)。空白组大鼠心电图S-T段接近等电位线,心肌结构规则,纤维排列紧密,细胞核完整且排列整齐;模型组大鼠心电图S-T段显著升高,心肌纤维排列明显紊乱,心肌细胞出现肿大、坏死且有炎症细胞浸润,心肌细胞凋亡阳性细胞明显增多;各给药组心电图S-T段降低,心肌纤维排列整齐,心肌细胞结构及形态改善,炎症细胞减少,凋亡阳性细胞数明显降低。与空白组比较,模型组大鼠心脏指数、全血高切黏度、全血低切黏度、红细胞聚集指数升高,血清中TSH、FT_(3)、FT_(4)、NO、6-Keto-PGF_(1α)含量显著降低,ET-1、TXB_(2)含量显著升高(P<0.05或P<0.01)。与模型组比较,各给药组大鼠心脏指数、全血高切黏度、全血低切黏度、红细胞聚集指数、ET-1、TXB_(2)水平均显著降低,NO、6-Keto-PGF_(1α)含量显著升高,当归四逆颗粒高剂量组及丹参滴丸组血清中TSH、FT_(3)、FT_(4)水平均升高(P<0.05或P<0.01)。当归四逆颗粒低剂量组大鼠血清TSH水平低于丹参滴丸组和当归四逆颗粒高剂量组(P<0.01)。结论当归四逆颗粒具有减轻冠心病寒凝血瘀证心肌损伤的作用,其作用机制可能与减少心肌细胞凋亡、提高能量代谢、调节血管活性因子水平有关。

Objective To explore the possible mechanism of Danggui Sini Granules(当归四逆颗粒)in treat⁃ment of coronary heart disease with cold congealing and blood stasis syndrome.Methods Fifty SD rats were randomly divided into a blank group,a model group,a Danshen Pill(丹参滴丸)group,and a low-and high-dosage Danggui Sini Granules group,with 10 rats in each group.Except for the blank group,the rat model of coronary heart disease with cold congealing and blood stasis syndrome was established by repeated cold stimulation at low temperature com⁃bined with intraperitoneal injection of posterior pituitary hormone in all other groups.In the 6th week of modelling,0.073 g·kg^(-1)·d^(-1)of compound Danshen Pill was given to the Danshen Pill group,20.2 and 40.4 g·kg^(-1)·d^(-1)of Danggui Sini Granules were given to the low-and high-dose Danggui Sini Granules groups,respectively,and 0.2 ml/10 g of sterile water was given to the blank group and the model group,all for 2 consecutive weeks.The general conditions of the rats were recorded,and the body mass was compared weekly.At the end of the intervention,electrocardiogram,blood rheological indexes,including whole blood low-cut viscosity,whole blood high-cut viscosity,erythrocyte aggre⁃gation index,and cardiac index were detected to evaluate the effect of the medication,and HE staining was used to observe the myocardial histopathological changes,TUNEL staining to detect the apoptotic situation of cardiomyo⁃cytes,and ELISA to detect the serum thyroid-stimulating hormone(TSH),free triiodothyronine(FT_(3)),free tet⁃raiodothyronine(FT_(4))levels and thromboxane B_(2)(TXB_(2)),6-keto-prostaglandin F1α(6-Keto-PGF_(1α)),endothelin 1(ET-1),and nitric oxide(NO)levels.Results Compared with the blank group at the same time point,the body mass of rats in the remaining groups decreased at all time points(P<0.05 or P<0.01);compared with the model group at the same time point,the body mass of rats in high-dose Danggui Sini Granules group and Danshen Pill group increased at the 6th and 7th weeks(P<0.05 or P<0.01).In the blank group,the S-T segment of the electrocardio⁃gram was close to the isoelectric line,the myocardial structure was regular,the fibres were closely arranged,and the nuclei of the cells were intact and neatly aligned;in the model group,the S-T segment of the electrocardiogram signifi⁃cantly elevated,and the arrangement of the myocardial fibres was obviously disordered,with myocardial cells appear⁃ing to be swollen,necrotic,and infiltrated by inflammatory cells,and the apoptosis-positive cells of the cardiac mus⁃cle cells obviously increased;in each of the medication groups,the electrocardiogram had a lowered S-T segment,and myocardial fibres were aligned,myocyte structure and morphology were improved,inflammatory cells reduced,and the number of apoptosis-positive cells significantly reduced.Compared with the blank group,the cardiac index,whole blood high cut viscosity,whole blood low cut viscosity,erythrocyte aggregation index increased in the model group,and the serum levels of TSH,FT_(3),FT_(4),NO,6-Keto-PGF_(1α)significantly reduced,and the levels of ET-1 and TXB_(2) significantly increased(P<0.05 or P<0.01).Compared with the model group,the levels of cardiac index,whole blood high cut viscosity,whole blood low cut viscosity,erythrocyte aggregation index,ET-1 and TXB_(2) signifi⁃cantly decreased and the levels of NO and 6-Keto-PGF_(1α)significantly increased in each medication group,and the se⁃rum levels of TSH,FT_(3),and FT_(4)elevated in the high-dose Danggui Sini Granules group and Danshen Pill group(P<0.05 or P<0.01).The serum TSH level of rats in the low-dose Danggui Sini Granules group was lower than that in Danshen Pill group and the high-dose Danggui Sini Granules group(P<0.01).Conclusion Danggui Sini Granules have the effect of alleviating myocardial injury in coronary heart disease with cold congealing and blood stasis syn⁃drome,and its mechanism may be related to reducing myocardial cell apoptosis,improving energy metabolism,and regulating the level of vasoactive factors.