【作者】 冯利；

【导师】 张继东；

【作者基本信息】 山东大学 ， 中西医结合临床， 2010， 硕士

【摘要】 目的1.观察芩丹胶囊对SHR大鼠主动脉壁形态学及超微结构的改变,及对大鼠血压的影响。2.观察芩丹胶囊对血管外膜TGF-p1.SMα-actin表达的影响及二者之间的关系,探讨芩丹胶囊逆转高血压血管外膜重构的机制。方法1.研究对象：40周龄雄性SHR大鼠32只和40周龄Wistar-Kyoto大鼠(WKY)16只。随机分为6组,每组8只：(1)WKY对照组；(2)WKY+芩丹胶囊大剂量组(WKY+QCH)；(3)SHR对照组；(4)SHR+芩丹胶囊大剂量组(SHR+QCH)；(5)SHR+芩丹胶囊小剂量组(SHR+QCL)；(6)SHR+氯沙坦组(SHR+Losartan).WKY+QCH组给予芩丹胶囊750mg／(kg·d),SHR+QCH组给予给予芩丹胶囊750mg/(kg·d),SHR+QCL组给予芩丹胶囊150mg/(kg·d),SHR+Losartan组给予氯沙坦30mg／(k·d)。WKY对照组和SHR对照组分别给予等量生理盐水。各组大鼠均灌胃给药,每日1次,连续给药12周。各组大鼠于末次给药后禁食24 h,不禁水。3%戊巴比妥钠(30mg／kg,腹腔注射)麻醉后进行后续实验。2.研究内容：(1)大鼠尾动脉收缩压测定；(2)胸主动脉壁病理学观察以及外膜细胞核密度变化；(3)胸主动脉壁形态学指标检测：激光共聚焦显微镜观测胸主动脉内、径(luminal diameter,LD).中膜(medial thickness,MT).外膜的比例变化；(4)免疫组化法观察以及实时定量PCR检测胸主动脉外膜TGF-β1.SMα-actin的表达。结果1.各组大鼠治疗前后收缩压动态变化与40周龄正常血压对照组WKY大鼠比较,40周龄SHR各组大鼠血压已经明显升高(P<0.01)。SHR组大鼠收缩压在实验过程中呈持续升高状态。从给药第4周起,芩丹胶囊大、小剂量组、氯沙坦组大鼠的血压与模型组相比均有下降,其差异具有统计学意义(P<0.05)。给药7周后,芩丹胶囊大剂量组较之小剂量组血压下降更为显著(P<0.05),给药12周后,各给药组大鼠收缩压与SHR组相比均有明显下降(P<0.05),但各组下降程度不同；其中芩丹胶囊小剂量组下降程度最小,氯沙坦组下降程度最大,两组相比差异具有显著性(P<0.05)(见表1)。2.胸主动脉壁切片光镜下HE染色观察对照组WKY大鼠胸主动脉血管壁内膜、中膜、外膜三层结构明显,均无增厚；外膜厚度均一,细胞排列致密有序,无细胞增生,血管壁无增厚。SHR组大鼠外膜细胞增生活跃,血管管壁增厚,有些出现断裂、剥脱,内弹力层破坏,内径、中膜、外膜比值降低,细胞核密度明显增加。QC (qindan capsule)组、氯沙坦组病理改变较少,但较WKY仍有明显差异。(见图1)3.胸主动脉形态学指标比较(1)与40周龄正常血压对照组WKY大鼠相比,40周龄SHR组大鼠主动脉MT明显增加(P<0.01)。与SHR组相比,SHR+QCL组大鼠主动脉MT略有下降(P<0.05)；SHR+QCH组、SHR+Losartan组则下降明显(P均<0.01),但三组之间差异无显著性(P均>0.05)。(2)与40周龄正常血压对照组WKY大鼠相比,SHR组及各给药组大鼠主动脉LD虽有不同程度下降,但差异无显著性(P均>0.05)。(3)与对照组WKY大鼠相比,SHR组大鼠主动脉MT／LD显著增加(P<0.01)。与SHR组相比,SHR+QCH、SHR+QCL、SHR+Losartan组三组MT／LD下降明显(P均<0.05),但三组之间差异无显著性(P均>0.05)(4)与40周龄正常血压对照组WKY大鼠相比,40周龄SHR组大鼠主动脉外膜明显增厚(P<0.01)。与SHR组相比, SHR+QCH组、SHR+QCL组和SHR+Losartan组下降明显(P均<0.01),但三组之间差异无显著性(P均>0.05)。(5)与对照组WKY大鼠相比,SHR组大鼠主动脉LD／MT／AT比值显著降低(P<0.01)。与SHR组相比,SHR+QCH、SHR+QCL、SHR+Losartan组三组LD／MT／AT下降则不明显(P均<0.05),且三组之间差异无显著性(P均>0.05) (见表2)。4.胸主动脉壁切片光镜下免疫组化观察免疫组织化学检测显示,TGF-β1为胞浆表达,胞浆内棕黄色颗粒为TGF-β1抗原抗体复合物；切片于光镜下×400放大,经计算机图像分析仪随机选择8个视野,计算TGF-β1光密度值,结果显示,SHR组较WKY组TGF-β1表达明显增多；SHR+QCH组,SHR+QCL组,氯沙坦组表达较SHR组均减少,但较WKY组明显(P<0.05,见图2)。SM a-actin亦为胞浆表达,观测结果显示, WKY组外膜SM a-actin几乎不表达,而SHR组则明显增多,SHR+QCH组,SHR+QCL组,氯沙坦组表达较SHR组均减少,但较WKY组明显(P<0.05,见图3)。5.主动脉壁TGF-p1、SM a-actin mRNA表达水平比较(1)TGF-p1mRNA表达比较：与40周龄的WKY组大鼠相比,同周龄的SHR组大鼠血管壁TGF-β1mRNA表达显著升高(P<0.05)。与SHR组相比,SHR+QCH组、SHR+QCL、SHR+Losartan组TGF-P1mRNA表达显著下降(P均<0.05)(见表3)。(2) SM a-actin mRNA表达比较：与40周龄的WKY组大鼠相比,同周龄的SHR组大鼠血管壁SM a-actin mRNA表达显著升高(P<0.01)。与SHR组大鼠相比,SHR+QCH组、SHR+QCL、SHR+Losartan组SM a-actin mRNA与SHR无显著性差异(P均>0.05)(见表4)。结论芩丹胶囊对SHR大鼠具有显著的降压疗效,其降压机制与芩丹胶囊降低SHR大鼠TGF-β1、SM a-actin水平有关,同时与逆转血管外膜重构,抑制TGF-β1转移和活化及SM a-actin表型转化有关。更多还原

【Abstract】 Aim1. To investigate the anti-hypertensive effects of Qindan Capsule (QC) on spontaneous hypertensive rats (SHR), as well as to observe the changes of the morphology and the ultrastructure of the arteries.2. To investigate the possible mechanism of QC in reversing vascular adventitia remodeling in hypertension through detecting the variation of TGF-β1 and SM a-actin mRNA and protein expression.Methods32 male (40 weeks old) SHRs and 16 male (40 weeks old) Wistar Kyoto(WKY) rats were randomly divided into 6 groups (n=8 per group):the WKY control group, the WKY+QCH group, the SHR control group, the high dosage QC group (SHR+QCH), and the low dosage QC group (SHR+QCL), and the Losartan group (SHR+Losartan). The SHR+QCH group was treated with high dosage (750 mg/kg per day) of QC; the SHR+QCL group was treated with low dosage (150 mg/kg per day) of QC; the SHR+Losartan group was treated with losartan for 30 mg/kg per day; and the WKY+QCH group were treated with high dosage (750 mg/kg per day) of QC. The WKY control group and the SHR control group were treated with equal volume of normal saline instead. All the treatments were administered by gastrogavage once a day for 12 successive weeks.After the last administration, all rats were fasted with free access to water for 24 h, and anesthetized with intraperitoneal injection of 3% pentobarbital (30 mg/kg) which was followed by other experiments. The detection contents listed below:(1) Measurement of the systolic blood pressure (SBP); (2) Observation of the pathological structure in aortic wall, including the density of the nuclei in the adventitia (3) Measurement of the morphological index of aortic wall by laser scanning confocal microscope:the luminal diameter (LD), the medial thickness (MT), the adventitia thickness (AT), and the ratio of MT to AT; (4) Analysis of TGF-β1 and SM a-actin mRNA expression by real-time PCR, as well as protein expression by immuno-histochemical staining.Results1. Comparison of SBPThe SBP of rats in the SHR group with 40 weeks old was much higher than those in the WKY group (P＜0.01). The rats in the SHR group continuously developed further hypertension to a high pressure level. From the fourth week of treatment, the SBP was lower in the SHR+QCH group, the SHR+QCL group and the SHR+ Losartan group than that in the SHR control group (all P＜0.05). After seven weeks of treatment, the SBP in the SHR+QCH group was decreased significantly compared with that in the SHR+QCL group (P<0.05). After twelve weeks of treatment, the SBP in each group was decreased in different degree compared with the SHR control group (all P<0.05). Among all groups, the strongest decrease of SBP was shown in the SHR+ Losartan group, while the weakest decrease was shown in the SHR+QCL group. A statistically significant difference was shown between the SHR+ Losartan group and the SHR+QCL group. (P<0.05) (table 1).2. Observation of sections of thoracic aortic wall stained with HEThe structure of the intima, the media and the adventitia of thoracic aortic wall was very clear and was no thickening in the WKY rats. The thickness of adventitia was uniform, and the cells were arranged densely and regularly without proliferation. The thoracic aortic walls were shown obviously thickened in the SHR group, with active cell proliferation in the adventitia. Some sections showed that the adventitia was ruptured and even exfoliated. The medial elastic fibers were destroyed. The ratio of MT/LD/AT was decreased, while the density of the nuclei was obviously increased. After treatment, the pathologic alterations of the adventitia in the SHR+QCH, the SHR+QCL and the SHR+Losartan groups were ameliorated. But a significant difference was shown compared with the WKY group.(fig.l)3. Comparison of morphological index of thoracic aortic wall(1) MT was increased significantly in the SHR group compared with that in the WKY group (P＜0.01). The SHR+QCL group showed a lower MT than the SHR group (P＜0.05). MT was decreased significantly in the SHR+QCH and the SHR+Losartan groups compared with that in the SHR group (all P<0.01). However, no significant difference was shown in comparison among these three above groups (all P>0.05).(2) LD was decreased in different degree in the SHR group and other treatment groups compared with the WKY group, but no significant difference was shown in comparison among these six experimental groups (all P>0.05).(3) The ratio of MT/LD was significantly higher in the SHR group than the WKY group (P＜0.01). The ratio of MT/LD was significantly lower in the SHR+QCH, the SHR+QCL and the SHR+Losartan groups than the SHR group (all P<0.05), but no significant difference was shown in comparison among these three above groups (all P>0.05).(4) The adventitia thickness was increased significantly in the SHR group compared with that in the WKY group (P＜0.01). The adventitia thickness decreased significantly in the SHR+QCH, SHR+QCL and the SHR+Losartan groups compared with that in the SHR group (all P＜0.01). However, no significant difference was shown in comparison among these three above groups (all P>0.05).(5) The ratio of LD/MT/AT was significantly decereased in the SHR group than the WKY group (P＜0.01). The ratio of LD/MT/AT was not significantly lower in the SHR+QCH, the SHR+QCL and the SHR+Losartan groups than the SHR group (all P<0.05), and no significant difference was shown in comparison among these three above groups (all P>0.05).(table 2)4. Observation of sections of thoracic aortic wall stained with immunohistochemistryTGF-β1 was expressed in the cytoplasm, and stained in brown. Eight non-overlapping fields per tissue section, chosen at random, were scanned, and the optimal density (OD) values of TGF-β1 were calculated. Compared with the WKY group, the expression of TGF-(31 was significantly increased in the SHR group. After treatment, the expressions of TGF-P1 in the SHR+QCH, the SHR+QCL and the SHR+Losartan groups were all decreased compared with the SHR group. However, the levels of TGF-P1 in those above groups were still higher than the WKY group. (P< 0.05, Fig.2)SM a-actin was also expressed in the cytoplasm. However, the positive staining of SM a-actin could hardly be detected in the vascular adventitia in the WKY group. The expression of SM a-actin was obviously increased in the SHR group. After treatment, the expressions of SM a-actin in the SHR+QCH, the SHR+QCL and the SHR+Losartan groups were all decreased compared with the SHR group. However, the levels of SM a-actin in those above groups were still higher than the WKY group. (P< 0.05, Fig.3)5. Comparison of expression of TGF-β1 and SM a-actin mRNA in aortic wall(1) Comparison of expression of TGF-β1 mRNA:The expression of TGF-β1 mRNA was higher in the SHR group than that in the WKY group (P＜0.05). The expression of TGF-β1 mRNA was decreased significantly in the SHR+QCH group, the SHR+QCL group and the SHR+Losartan group compared with that in the SHR group (all P＜0.05, table 3).(2) Comparison of expression of SM a-actin mRNA:The expression of SM a-actin mRNA was higher in the SHR group than that in the WKY group (P<0.01). There was no significant difference in the SM a-actin mRNA expression in the SHR+QCH group, the SHR+QCL group and the SHR+Losartan group compared with that in the SHR group (all P>0.05, table 4).ConclusionA significant anti-hypertensive effect of QC on SHR was observed in our present study. QC could decrease the local TGF-β1 and SM a-actin level in the vascular adventitia, which was possibly associated with the anti-hypertensive effect of QC. Also, QC could reverse vascular adventitia remodeling in hypertension, the mechanisms were possibly associated with the suppressive effect of QC on the migration and activation of TGF-β1 and the phenotypic transition of SM a-actin.更多还原