芩丹胶囊对自发性高血压大鼠血管外膜Ⅰ、Ⅲ胶原表达的影响
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摘要
目的
1.观察芩丹胶囊对自发性高血压大鼠血压的影响。
2.观察芩丹胶囊对血管外膜Ⅰ、Ⅲ胶原表达的影响,并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：32只40周龄雄性SHR大鼠随机分为4组,模型组(SHR组)、芩丹胶囊大剂量组(SHR+QDH组)、芩丹胶囊小剂量组(SHR+QDL组)、氯沙坦组(SHR+Los组),另设40周龄雄性WKY大鼠16只分为空白对照组(WKY空白组)及正常药物对照组(WKY+QD组)。SHR+QDH组给予芩丹胶囊750mg/(kg·d)；SHR+QDL组给予芩丹胶囊150mg/(kg·d)；SHR+Los组给予氯沙坦30mg/(kg·d)；WKY+QD组给予芩丹胶囊750mg/(kg·d)；WKY空白组和SHR组给予相等剂量生理盐水,1次/d,连续给药12周。各组大鼠在末次给药后禁食24 h,不禁水。10%水合氯醛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)免疫组化法观察胸主动脉外膜ⅠⅢ胶原表达。(3)实时定量PCR检测胸主动脉外膜Ⅰ、Ⅲ胶原表达。(4)用western blotting检测Ⅰ胶原蛋白表达。
结果
1.各组大鼠治疗前后收缩压比较
见图1,从给药第4周起,SHR+QDH组、SHR+QDL组、SHR+Los组分别与SHR组比较,血压下降明显,其差异均具有统计学意义(P<0.05)。给药6周到12周间,SHR+QDH组比SHR+QDL组血压下降更为显著,差异具有统计学意义(P<0.05)。
2.胸主动脉组织免疫组织化学测定
结果显示,见表2,图2、3,SHR组与WKY空白组比较,Ⅰ、Ⅲ胶原表达明显增多,差异具有统计学意义(P<0.05)；SHR+QDH组、SHR+QDL组及SHR+Los组表达较SHR组均减少(P<0.05)。WKY空白组与WKY+QD组Ⅰ、Ⅲ胶原表达量比较,其差异无统计学意义(P>0.05),SHR+QDH组与SHR+QDL组比较差异有统计学意义(P<0.05),但与SHR+Los组比较,差异无统计学意义(P>0.05)。
3.主动脉壁Ⅰ、Ⅲ胶原mRNA表达水平比较
采用2-ΔΔCt方法,将Ⅰ、Ⅲ型胶原基因表达Ct值转换为相对于WKY空白组的变化倍数,可以直接得到的目的基因相对于参照基因的定量。如图6和7所示分别为Ⅰ、Ⅲ胶原的扩增、溶解曲线和溶解峰,说明Ⅰ、Ⅲ胶原是特异性基因表达,无引物二聚体及非特异性扩增。研究结果显示：见图4,WKY空白组和WKY+QD组间差异无统计学意义(P>0.05),SHR+QDH组和SHR+QDL组间差异有统计学意义(P<0.05)。SHR组大鼠胸主动脉壁Ⅰ、Ⅲ型胶原mRNA表达量与WKY组之间的差异有统计学意义(P<0.05)。SHR+QDH组、SHR+QDL组及SHR+Los组大鼠胸主动脉壁Ⅰ、Ⅲ型胶原表达与WKY组大鼠相比明显增多,与SHR组相比有所减少,其差异有统计学意义(P<0.05)。
4.Ⅰ胶原在免疫印迹中的蛋白表达
WKY空白组,WKY+QD组,SHR+Los组,SHR+QDH组,SHR+QDL组,SHR组蛋白表达依次增强,Ⅰ型胶原分子量大小在120KD左右,参照actin,分子量为43KD,可看出蛋白表达依次增强的趋势。见图5,用Ⅰ型胶原与actin的比值做统计分析结果显示：WKY空白组和WKY+QD组间差异无统计学意义(P>0.05),SHR+QDH组和SHR+QDL组间差异有统计学意义(P<0.05)。SHR模型组和正常WKY相比,差异有统计学意义(P<0.05)；SHR+QDH组.SHR+QDL组.SHR+Los组分别与SHR组比较,差异均有统计学意义(P<0.05)；SHR+QDH组与SHR+Los组比较差异无统计学意义(P>0.05)。
结论
芩丹胶囊对SHR大鼠有显著的降压疗效,其降压机制与芩丹胶囊降低SHR大鼠Ⅰ、Ⅲ胶原表达水平有关,同时与逆转血管外膜重构,抑制外膜纤维化,减少Ⅰ、Ⅲ胶原在外膜的合成与沉积有关。
Aim
1. Observe Qindan capsule on blood pressure in spontaneously hypertensive rats.
2. Observe the Qindan capsule on vascular adventitiaⅠ,Ⅲcollagen expression, and to explore the reversal of hypertensive vascular adventitia remodeling mechanism.
Methods
1. Study:32 SHR rats of 40 weeks old were randomly divided into 4 groups, the model group (SHR group), Qindan high dosage group (SHR+QDH group), Qindan low dosage group (SHR+QDL group), the Losartan group (SHR+Los group), Besides, 16 Wistar—Kyoto (WKY) rats of the same weeks were taken as the normal control (WKY control group) and Qindan group (WKY+QD group). SHR+QDH group was treated with high dosage 750mg/(kg·d); SHR+QDL group was treated with low dosage 150mg/(kg·d); SHR+Los group was given losartan 30mg/(kg·d); WKY+QD group were treated with high dosage 750mg/(kg·d); WKY and SHR control group were given an equal dosage of saline, once a day, continuous administration for 12 weeks. Rats in each group after the last administration in the fasted 24h, can not help but water. All the rats were anesthetized with intraperitoneal injection of 10% chloral hydrate follow-up experiments.
2. Research:(1) Determination of rat tail artery systolic pressures.(2)Observed aortic adventitiaⅠ,Ⅲcollagen by immunohistochemistry.(3)Real-time quantitative PCR detection of thoracic aortic adventitiaⅠ,Ⅲcollagen.(4)Western blotting detection of expression of I collagen.
Results
1. Comparison of systolic blood pressure of rats
Fig 1,after 4 weeks from the administration, Qindan high dosage, low dosage group, and losartan group compared with the model group blood pressure decreased, the difference was statistically significant (P<0.05). Between 6 weeks to 12 weeks of administration, the rat blood pressure decreased even more significant in the high dosage group than in the low dosage group, the difference was significant (P<0.05).
2. Thoracic aorta by immunohistochemistry determination
(Table 2, Fig 2, Fig 3)The results showed that the model group than in the control groupⅠ,Ⅲcollagen expression was significantly increased, the difference was statistically significant (P<0.05); Qindan low dosage group and losartan group were lower expression compared with the model group (P<0.05). Qindan control group and normal control groupⅠ,Ⅲcollagen expression was no significant difference (P>0.05), Qindan high dosage group compared with the low dosage group was statistically significant (P<0.05), but compared with the losartan group, the difference was not statistically significant (P>0.05).
3. Aortic wallⅠ,Ⅲcollagen mRNA expression compared
Using 2-ΔΔCt method, typeⅠ,Ⅲcollagen gene expression relative to the Ct is converted to changes in multiples of WKY control group, the target gene can be obtained directly relative to the reference gene quantification. As shown in Figure 6 and 7, respectivelyⅠ,Ⅲcollagen in the amplification curve and dissolution peaks, indicatingⅠ,Ⅲcollagen is a specific gene expression, no primer-dimers and nonspecific amplification. As shown (Fig 4):WKY control group and WKY+QD group was no significant difference (P>0.05), Differences between the SHR+QDH group and SHR+QDL group were statistically significant (P<0.05). There were significant differences between SHR groups and WKY groups in thoracic aorta adventitiaⅠ,Ⅲcollagen mRNA expression (P<0.05). SHR+QDH group, SHR+QDL group and SHR+Los group of rat thoracic aortic adventitiaⅠ,Ⅲcollagen expression compared with the WKY rats increased significantly, reduced compared with the SHR group, the differences were statistical significance (P<0.05).
4. I collagen protein expression by Western blotting
The order in expression of collagenⅠfrom low to high:WKY control group, WKY+QD group, SHR+Los group, SHR+QDH group, SHR+QDL group, SHR group.Ⅰcollagen in 120KD molecular weight around, with reference to actin, a molecular weight of 43KD, can be seen followed the trend of increased protein expression. TypeⅠcollagen and actin by the ratio of statistical analysis were shown in Figure 5:WKY control group and WKY+QD have no significant difference (P>0.05), SHR+QDH group and SHR+QDL differences between the groups have statistical significance (P<0.05). SHR group compared with WKY group, the difference was statistically significant (P<0.05); SHR+QDH group, SHR+QDL group, SHR+Los group were compared with SHR group, the difference was statistically significant (P<0.05); SHR+QDH group and the SHR+Los group difference was not statistically significant (P>0.05).
Conclusion
Qindan capsule on SHR rats had significant antihypertensive effects, and its step-down mechanism was possibly associated with reducing SHR ratsⅠ,Ⅲcollagen expression level, and also with the reversal of vascular adventitial remodeling, inhibition of membrane fibrosis, and reducingⅠ,Ⅲcollagen synthesis and deposition of the vascular adventitia.
1.观察芩丹胶囊对自发性高血压大鼠血压的影响。
2.观察芩丹胶囊对血管外膜Ⅰ、Ⅲ胶原表达的影响,并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：32只40周龄雄性SHR大鼠随机分为4组,模型组(SHR组)、芩丹胶囊大剂量组(SHR+QDH组)、芩丹胶囊小剂量组(SHR+QDL组)、氯沙坦组(SHR+Los组),另设40周龄雄性WKY大鼠16只分为空白对照组(WKY空白组)及正常药物对照组(WKY+QD组)。SHR+QDH组给予芩丹胶囊750mg/(kg·d)；SHR+QDL组给予芩丹胶囊150mg/(kg·d)；SHR+Los组给予氯沙坦30mg/(kg·d)；WKY+QD组给予芩丹胶囊750mg/(kg·d)；WKY空白组和SHR组给予相等剂量生理盐水,1次/d,连续给药12周。各组大鼠在末次给药后禁食24 h,不禁水。10%水合氯醛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)免疫组化法观察胸主动脉外膜ⅠⅢ胶原表达。(3)实时定量PCR检测胸主动脉外膜Ⅰ、Ⅲ胶原表达。(4)用western blotting检测Ⅰ胶原蛋白表达。
结果
1.各组大鼠治疗前后收缩压比较
见图1,从给药第4周起,SHR+QDH组、SHR+QDL组、SHR+Los组分别与SHR组比较,血压下降明显,其差异均具有统计学意义(P<0.05)。给药6周到12周间,SHR+QDH组比SHR+QDL组血压下降更为显著,差异具有统计学意义(P<0.05)。
2.胸主动脉组织免疫组织化学测定
结果显示,见表2,图2、3,SHR组与WKY空白组比较,Ⅰ、Ⅲ胶原表达明显增多,差异具有统计学意义(P<0.05)；SHR+QDH组、SHR+QDL组及SHR+Los组表达较SHR组均减少(P<0.05)。WKY空白组与WKY+QD组Ⅰ、Ⅲ胶原表达量比较,其差异无统计学意义(P>0.05),SHR+QDH组与SHR+QDL组比较差异有统计学意义(P<0.05),但与SHR+Los组比较,差异无统计学意义(P>0.05)。
3.主动脉壁Ⅰ、Ⅲ胶原mRNA表达水平比较
采用2-ΔΔCt方法,将Ⅰ、Ⅲ型胶原基因表达Ct值转换为相对于WKY空白组的变化倍数,可以直接得到的目的基因相对于参照基因的定量。如图6和7所示分别为Ⅰ、Ⅲ胶原的扩增、溶解曲线和溶解峰,说明Ⅰ、Ⅲ胶原是特异性基因表达,无引物二聚体及非特异性扩增。研究结果显示：见图4,WKY空白组和WKY+QD组间差异无统计学意义(P>0.05),SHR+QDH组和SHR+QDL组间差异有统计学意义(P<0.05)。SHR组大鼠胸主动脉壁Ⅰ、Ⅲ型胶原mRNA表达量与WKY组之间的差异有统计学意义(P<0.05)。SHR+QDH组、SHR+QDL组及SHR+Los组大鼠胸主动脉壁Ⅰ、Ⅲ型胶原表达与WKY组大鼠相比明显增多,与SHR组相比有所减少,其差异有统计学意义(P<0.05)。
4.Ⅰ胶原在免疫印迹中的蛋白表达
WKY空白组,WKY+QD组,SHR+Los组,SHR+QDH组,SHR+QDL组,SHR组蛋白表达依次增强,Ⅰ型胶原分子量大小在120KD左右,参照actin,分子量为43KD,可看出蛋白表达依次增强的趋势。见图5,用Ⅰ型胶原与actin的比值做统计分析结果显示：WKY空白组和WKY+QD组间差异无统计学意义(P>0.05),SHR+QDH组和SHR+QDL组间差异有统计学意义(P<0.05)。SHR模型组和正常WKY相比,差异有统计学意义(P<0.05)；SHR+QDH组.SHR+QDL组.SHR+Los组分别与SHR组比较,差异均有统计学意义(P<0.05)；SHR+QDH组与SHR+Los组比较差异无统计学意义(P>0.05)。
结论
芩丹胶囊对SHR大鼠有显著的降压疗效,其降压机制与芩丹胶囊降低SHR大鼠Ⅰ、Ⅲ胶原表达水平有关,同时与逆转血管外膜重构,抑制外膜纤维化,减少Ⅰ、Ⅲ胶原在外膜的合成与沉积有关。
Aim
1. Observe Qindan capsule on blood pressure in spontaneously hypertensive rats.
2. Observe the Qindan capsule on vascular adventitiaⅠ,Ⅲcollagen expression, and to explore the reversal of hypertensive vascular adventitia remodeling mechanism.
Methods
1. Study:32 SHR rats of 40 weeks old were randomly divided into 4 groups, the model group (SHR group), Qindan high dosage group (SHR+QDH group), Qindan low dosage group (SHR+QDL group), the Losartan group (SHR+Los group), Besides, 16 Wistar—Kyoto (WKY) rats of the same weeks were taken as the normal control (WKY control group) and Qindan group (WKY+QD group). SHR+QDH group was treated with high dosage 750mg/(kg·d); SHR+QDL group was treated with low dosage 150mg/(kg·d); SHR+Los group was given losartan 30mg/(kg·d); WKY+QD group were treated with high dosage 750mg/(kg·d); WKY and SHR control group were given an equal dosage of saline, once a day, continuous administration for 12 weeks. Rats in each group after the last administration in the fasted 24h, can not help but water. All the rats were anesthetized with intraperitoneal injection of 10% chloral hydrate follow-up experiments.
2. Research:(1) Determination of rat tail artery systolic pressures.(2)Observed aortic adventitiaⅠ,Ⅲcollagen by immunohistochemistry.(3)Real-time quantitative PCR detection of thoracic aortic adventitiaⅠ,Ⅲcollagen.(4)Western blotting detection of expression of I collagen.
Results
1. Comparison of systolic blood pressure of rats
Fig 1,after 4 weeks from the administration, Qindan high dosage, low dosage group, and losartan group compared with the model group blood pressure decreased, the difference was statistically significant (P<0.05). Between 6 weeks to 12 weeks of administration, the rat blood pressure decreased even more significant in the high dosage group than in the low dosage group, the difference was significant (P<0.05).
2. Thoracic aorta by immunohistochemistry determination
(Table 2, Fig 2, Fig 3)The results showed that the model group than in the control groupⅠ,Ⅲcollagen expression was significantly increased, the difference was statistically significant (P<0.05); Qindan low dosage group and losartan group were lower expression compared with the model group (P<0.05). Qindan control group and normal control groupⅠ,Ⅲcollagen expression was no significant difference (P>0.05), Qindan high dosage group compared with the low dosage group was statistically significant (P<0.05), but compared with the losartan group, the difference was not statistically significant (P>0.05).
3. Aortic wallⅠ,Ⅲcollagen mRNA expression compared
Using 2-ΔΔCt method, typeⅠ,Ⅲcollagen gene expression relative to the Ct is converted to changes in multiples of WKY control group, the target gene can be obtained directly relative to the reference gene quantification. As shown in Figure 6 and 7, respectivelyⅠ,Ⅲcollagen in the amplification curve and dissolution peaks, indicatingⅠ,Ⅲcollagen is a specific gene expression, no primer-dimers and nonspecific amplification. As shown (Fig 4):WKY control group and WKY+QD group was no significant difference (P>0.05), Differences between the SHR+QDH group and SHR+QDL group were statistically significant (P<0.05). There were significant differences between SHR groups and WKY groups in thoracic aorta adventitiaⅠ,Ⅲcollagen mRNA expression (P<0.05). SHR+QDH group, SHR+QDL group and SHR+Los group of rat thoracic aortic adventitiaⅠ,Ⅲcollagen expression compared with the WKY rats increased significantly, reduced compared with the SHR group, the differences were statistical significance (P<0.05).
4. I collagen protein expression by Western blotting
The order in expression of collagenⅠfrom low to high:WKY control group, WKY+QD group, SHR+Los group, SHR+QDH group, SHR+QDL group, SHR group.Ⅰcollagen in 120KD molecular weight around, with reference to actin, a molecular weight of 43KD, can be seen followed the trend of increased protein expression. TypeⅠcollagen and actin by the ratio of statistical analysis were shown in Figure 5:WKY control group and WKY+QD have no significant difference (P>0.05), SHR+QDH group and SHR+QDL differences between the groups have statistical significance (P<0.05). SHR group compared with WKY group, the difference was statistically significant (P<0.05); SHR+QDH group, SHR+QDL group, SHR+Los group were compared with SHR group, the difference was statistically significant (P<0.05); SHR+QDH group and the SHR+Los group difference was not statistically significant (P>0.05).
Conclusion
Qindan capsule on SHR rats had significant antihypertensive effects, and its step-down mechanism was possibly associated with reducing SHR ratsⅠ,Ⅲcollagen expression level, and also with the reversal of vascular adventitial remodeling, inhibition of membrane fibrosis, and reducingⅠ,Ⅲcollagen synthesis and deposition of the vascular adventitia.
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29.邱志兵,陈鑫,万松.血管外膜和平滑肌细胞增殖活性及胶原分布对血管重塑影响[J].南京医科大学学报(自然科学版).2008,28(6)：752-757.
30. Cai WJ, Koltai S, Kocsis E, et al. Remodeling of the adventitia during coronary arteriogenesis. Am J Physiol Heart Circ Physiol.2003,284(1):31-40.
31. Arribas SM, Hinek A, Gonzalez MC. Elastic fibres and vascular structure in hypertension. Pharmacol Ther.2006,111(3):771-791.
32.邱志兵,陈鑫,万松.血管外膜和胶原分布对内膜增生及血管重塑的影响[J].中国胸心血管外科临床杂志.2008,15(4)：272-276.
33.吴宗贵,梁春.关注“由内而外”的转变：重视血管外膜在动脉粥样硬化中的研究[J].疑难病杂志.2009,8(1)：1-2.
34.牟华明,祝之明.外膜对血管重塑和功能的调控.国外医学·心血管疾病分册.2004,31(5)：267-269.
35. Broder WA, Ruoslahti e. Transforming growth factor β in disease:the dark side of tissue repair. J Clin Invest.1992,90(1):1-7.
36. Tuan TL, Nichter LS. The molecular basis of keloid and hypertrophic scar formation. Mol Med Today.1998,4(1):19-24.
37.杨方,赵培真,佘铭鹏,等PDGF与肝素对动脉SMC Ⅰ,Ⅲ型前胶原mRNA表达及胶原蛋白合成的作用.中国医学科学院学报[J].1999,21(1)：13-18.
38. Ryan ST, Koteliansky VE, Gotwals PJ, et al. Transforming growth factor-beta-dependent events in vascular remodeling following arterial injury. J Vase Res.2003,40(1):37-46.
39.张亚文,张国元,吴宗贵.动脉损伤后Ⅰ型胶原蛋白基因表达在再狭窄形成中的时相性变化[J].第二军医大学学报.2001,22(2)：133-137.
40. Sakata N, Kawamura K, Takebayashi S. Effects of collagen matrix on proliferation and differentiation of vascular smooth muscle cells in vitro [J]. Exp Mo 1 Pathol.1990,52(2):179-191.
41. Barnes MJ. Collagens in atherosclerosis [J]. Coll Relat Res.1985,5(1):65-97.
42.万新红,陈金龙,陈朝霞,等.高血压大鼠血管外膜血管紧张素转化酶活性与胶原关系的初步研究[J].热带医学杂志.2007,7(9)：863-865.
43.韩启德,文允镒主编.血管生物学[M].第一版,北京：北京医科大学,中国协和医科大学联合出版社.1997,218.
44. Dickhout JG, Lee RMKW. Structural and functional analysis of small arteries from young spontaneously hypertensive rats. Hypertension.1997,29(3):781-789.
45. Falloon BJ, Bund SJ, Tulip JR, et al. In vitro perfusion studies of resistance artery function in genetic hypertension. Hypertension.1993,22(4):486-495.
46. Groot E, Jukema JW, Montauban AD, et al. B-Mode ultrasound assessment of pravastatin treatment effect on carotid and femoral artery walls and its correlations with coronany arterographic findings:a report of the regression growth evaluation statin study(REGRESS). JACC.1998,31(7):1561-1567.
47. Lee RMKW, Owens GK, Scott-Burden T, et al. Pathophysiology of smooth muscle in hypertension. Can J Physiol Pharmacol.1995,73(5):574-584.
48.陶婷,朱鼎良,龚兰生.血管紧张素Ⅱ对血管外膜成纤维细胞Ⅰ型胶原合成及其mRNA表达的影响.中国病理生理杂志.2000,16(5)：405-408.
49. Wilcox J N, Okamoto E I, Nakahara K I, et al. Perivascular responses after angioplasty which may contribute to postangioplasty restenosis:a role for circulating myofibroblast precursors? [J]. Ann N Y Acad Sci.2001,947:68-90.
50. Tao T, Gao PJ, Zhang YZ, et al. Collagen content in aortic adventitia of spont-aneously hypertensive rats. Fundam Clin Pharmacol.1999,13:274.
51.陶婷,朱鼎良,龚兰生.自发性高血压大鼠胸主动脉外膜胶原分布及含量的 变化[J].高血压杂志.2001,9(1)：50-53.
52. Kim S, Ohta K, Hanaguchi A, et al. Angiotensin Ⅱ type Ⅰ receptor antagonist inhibits the gene expression of transforming growth factor-betal and extracellular matrix in cardiac and vascular tissues of hypertensive rats. J Pharmacol Exp Ther. 1995,273(1):509-515.
53.贡元,朱鼎良.外膜在血管重塑中的作用机制[J].国外医学·生理病理科学与临床分册.1999,19(6)：516-518.
54.范金汝,王行宽,熊国强.高血压左室肥厚中医病名、病位、病因病机探讨.中医药通报.2005,4(2)：19-21.
55.鞠大宏,韩学杰,谢雁鸣,等.高血压病从络论治探讨[J].2001,7(9)：43-44.
56.黄力,史载祥.高血压病的病因病机和辨证论治.中医临床医生.1999,27(12)：10-12.
57.张莹莹,郑星宇.高血压病从瘀论治研究概况.陕西中医学院学报.2010,33(4)：108-110.
58.肖梅芳,周端.应用活血潜阳法治疗高血压病经验[J].上海中医药杂志.2004,38(4)：12-13.
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60.陈锋,熊伟,何庆.黄芩对皮肤瘢痕形成过程中转化生长因子-β1及胶原蛋白Ⅲ表达的影响[J].实用医院临床杂志.2007,4(5)：32-33.
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