蒙药阿给炭促进胃溃疡出血创面愈合改善粘膜下组织修复状态的机制研究

【作者】 刘伟志；

【导师】 崔箭；

【作者基本信息】 中央民族大学 ， 中国少数民族传统医学， 2013， 博士

【摘要】 消化性溃疡是一种全球性的常见疾病,而胃溃疡是消化性溃疡中最为常见的类型。一般来说胃溃疡本身不会引起患者死亡,但重症患者多有伴有出血、穿孔等并发症,若不及时处理病死率很高。在胃溃疡的并发症中出血最为常见、也是上消化道大出血最常见的病因。胃溃疡可发生于任何年龄,尤以青壮年居多,一般经药物治疗后,症状缓解或消失,但溃疡愈合质量差,停药后极易复发,给患者的身心健康带来极大的危害。止血与促进溃疡愈合是胃溃疡合并出血治疗的关键手段,然而止血留瘀严重影响了溃疡的愈合质量,如何解决溃疡出血后的止血以及止血后的溃疡愈合质量问题是困扰本病治疗的瓶颈。蒙医学在治疗胃溃疡出血治疗方面积累了大量宝贵的用药经验和特色药物,阿给就是其代表之一。蒙药阿给系菊科蒿属植物冷蒿(Arteisia frigida Willd)的全草,又名小白蒿、菟毛蒿,是蒙古族常用的止血消肿药,在内蒙古自治区、西藏自治区、蒙古国等处广泛应用,疗效确切。在前期研究中我们发现蒙药阿给炭既能止血又能活血,有“止血不留瘀”的治疗特点,该特性与胃溃疡合并出血的病机具有较强的吻合度。藉此本研究提出：“止血活血并举是治疗胃溃疡出血,提升溃疡愈合质量的关键”的假说,拟通过对模型大鼠凝血关键指标的检测,进一步研究蒙药阿给炭促凝止血的作用机制；同时观察溃疡组织VEGF、 VEGFRmRNA、 bFGF等指标的变化,探讨其促血管新生,提高溃疡愈合质量的机制,结合免疫组化、病理学等方法,观测溃疡部位新生血管数量、密度及其构相,最终阐明溃疡与出血病理机制的内在关系,建立止血活血双向调节治法与胃溃疡出血治疗的统一性认识,为活血止血类民族药在胃溃疡出血疾病中的应用提供实验学依据。实验方法：1.SPF级SD大鼠随机分为8组,即空白对照组、模型对照组、云南白药组(阳性药)、雷尼替丁组(阳性药)、阿给生药组、阿给炭药低剂量组、阿给炭药中剂量组和阿给炭药高剂量组。采用水浸应激束缚法复制大鼠急性胃溃疡合并出血模型。连续灌胃给药7d,空白对照组、模型对照组给予溶媒灌胃。2.利用普通显微镜结合电镜观察和分析溃疡面积、溃疡指数、炎性细胞浸润情况,从宏观和微观形态学角度明确阿给炭促进胃溃疡出血创口愈合的疗效。3.通过大鼠断尾出血实验及玻璃毛细管凝血实验,观察阿给炭对模型大鼠出血时间及凝血时间作用的影响,全血生化检测仪测定血小板计数,揭示阿给炭止血作用效果。4.采用免疫组织化学法,Ⅷ因子标记血管,光镜观察,计算新生血管数量,SP法双标记检测微血管密度(MVD),观察阿给炭对溃疡基底粘膜组织重建的作用。5. ELISA法检测大鼠血清纤溶系统t-PA和PAI-1活性、TXA2、6-ke to-PGF1a、 GMP-140等指标,全自动血流变仪检测红细胞聚集指数、红细胞压积、全血粘度,探讨阿给炭对模型大鼠止血促凝的机制。6. RT-PCR结合·Western blot检测VEGF及其受体VEGFR、 bFGF及其受体FGFR的mRNA及蛋白表达水平,从分子水平探讨阿给炭促进血管新生和肉芽组织生长的作用机制。实验结果：1.蒙药阿给炭能显著降低胃溃疡模型大鼠的溃疡面积和溃疡指数(p<0.05),减少黏膜下组织炎性细胞润外,对其它病理改变均较模型组有不同程度的减轻。2.蒙药阿给炭能降低胃溃疡模型大鼠溃疡出血点数(P<0.05)；缩短模型大鼠出血时间和凝血时间(P<0.01),效果优于生药组；能提升模型鼠血小板计数(P<0.05)。3.蒙药阿给炭高剂量组能显著增加黏膜下组织新生血管数(P<0.05),低剂量组对模型大鼠微血管密度的影响有显著差异(P<0.05)。4.蒙药阿给炭高剂量组能增加胃溃疡出血模型大鼠血清TXB2水平(P<0.05),对大鼠血清6-keto-PGF1α没有影响(P>0.05)；能显著提高大鼠血清GMP-140水平(P<0.01)。5.蒙药阿给炭中、高剂量组能显著提高模型大鼠血清t-PA的水平(P<0.05),低剂量组、高剂量组均能降低模型大鼠血清PAI-1的含量水平(P<0.05)。6.蒙药阿给炭对胃溃疡出血模型大鼠红细胞聚集指数、红细胞压积、全血粘度的影响无统计学意义(P>0.05),而雷尼替丁能显著降低大鼠红细胞压积和不同切变率下全血粘度(P<0.05)。7.蒙药阿给炭低剂量组能显著增加VEGF及其受体蛋白表达水平(P<0.05),上调VEGFmRNA表达；阿给炭能提高bFGF及其受体bFGFR蛋白和mRNA的表达。结论：1.蒙药阿给炭对模型大鼠溃疡出血创面愈合疗效作用显著：蒙药阿给炭能降低胃溃疡出血模型大鼠的溃疡指数、减小溃疡部位炎性细胞浸润,对溃疡面的愈合有促进作用；蒙药阿给炭能降低胃溃疡出血模型大鼠的出血点数、缩短大鼠出、凝血时间,并通过增加血小板计数等方式,加快出血部位止血促凝,对溃疡出血有良好的治疗作用；蒙药阿给炭能促进胃溃疡出血模型大鼠溃疡黏膜下的新生血管数和微血管密度的增加,对溃疡部位肉芽组织生长有促进作用,同时提高溃疡愈合质量,在一定程度上能避免溃疡复发。2.蒙药阿给炭具有止血、活血的双向调节作用：一方面能通过影响凝血系统发挥止血促凝作用；另一方面能通过调整纤溶系统来防止血栓形成,同时阿给炭对模型大鼠血液流变学指标没有影响,不会因为止血太过而形成血瘀。3.蒙药阿给炭能上调模型大鼠胃溃疡组织VEGF及其受体、bFGF及其受体蛋白及mRNA的表达,揭示此可能为其通过促进血管新生和粘膜下组织重建来修复胃溃疡的分子机制之一。更多还原

【Abstract】 Peptic ulcer is a common disease worldwide, and gastric ulcer is the most common type of peptic ulcer. Generally speaking, gastric ulcer itself does not cause the patient to die, but it can associated with complications such as bleeding and perforation in severe cases, and if not treated timely, the mortality rate will be very high. In gastric ulcer, bleeding is the most common complication, and it also the most common cause of upper gastrointestinal bleeding. Gastric ulcer occurs at any age, especially in young adults, and symptoms can relieve or disappeare after medication. However, qualities of gastric ulcer healing are poor and gastric ulcer can re-emerge easily, which bring great harm to the patients both physically and mentally.Hemostasis and ulcer healing promotion are key treatment methods of gastric ulcer with hemorrhage, but stagnant blood have serious impact on the quality of ulcer healing. For this reason, how to resolve problems of hemostasis after bleeding and ulcer healing after hemostasis are choke points in the treatment of gastric ulcer. Mongolian medicine have accumulated a lot of valuable experience and drugs in the treatment of gastric ulcer haemorrhage, and Agei is one of representatives. Agei which also named Xiao-Bai-Hao is the entire plants of Arteisia frigida Willd, and it is a common drug used to reduce swelling and hemostasis in Mongolian. Arteisia frigida Willd are widely used in Inner Mongolia Autonomous Regionand, Tibet Autonomous Region and Mongolia, and it has definite effect in clinical use.In preliminary studies, we found that carbonized Agei have effect on hemostasis and invigorating the circulation of blood, and this feature, which can also smmerize by "hemostasis but no stagnat of blood", is similar to the pathogenisis of gastric ulcer accompanied with haemorrhage. In this study, we put forward the hypothesis that combining hemostasis and invigorating the circulation of blood palys key role in the treatment of gastric ulcer bleeding and promotion of quality of ulcer healing. We observed the hemostatic effects of carbonized Agei and explore the hemostatic mechanism through detecting relative index in model rats, and by observing the change of VEGF, VEGFRmRNA and bFGF in ulser tissue, we also explore the mechanism of promotion of angiogenesis and ulcer healing quality. Besides, combining with immunohistochemistry and pathological method, we observed the number of new vessels, vascular density and vascular conformation, then we want to illuminate the inner relationship between ulcer and pathogenisis of haemorrhage, and we also want to establish a unified understanding between two-ways regulation treatment and treatment of gastric ulcer accompanied with haemorrhage. Eventully, this study will provide experimental basis to clinical application for ethnodrugs in the treatment of gastric ulcer accompanied with haemorrhage. Method:1. SD rats were randomly divided into8groups, named control group, model group, Yunnanbaiyao group (Positive drug), ranitidine group, Agei crude drug group, low dose carbonized Agei group, middle dose carbonized Agei group and high dose carbonized Agei group. Model rats which have acute gastric ulcer accompanied with haemorrhage were duplicated by restraint and watered stress, and intragastric administration were given7d continuously. Solvent were administrated by intragastric for control group and model group.2. Observe and analyse the area of ulcer, ulcer index and inflammatory cell infiltration by combining electron microscopy with ordinary microscope, then clear and definite the effect of carbonized Agei on promoting healing of gastric ulcer accompanied with haemorrhage.3. The mouse bleeding time was measured by severing tails test, the clotting time was measured by capillary test, and the platelet count was measured by blood biochemical detection instrument.4. Immunohistochemical method, Ⅷ factor maker, light microscope observation, new vesscels calculation and MVD detection were used in order to observe the effect of carbonized Agei on restruction of basal mucosal tissue of ulcer.5. To investigate the mechanism of hemostasis and coagulation of carbonized Agei on model rats, t-PA, PAI-1, TXA2,6-ke to-PGFla and GMP-140were measured by ELISA, and red cell assembling index, hematocrit and whole blood viscosity were measured by automatic blood rheometer.6. To explore the molecular mechanism for carbonized Agei to promote angiogenesis and the growth of granulation tissue, the level of protein expression and mRNA expression of VEGF, VEGFR, bFGF and FGFR were measured respectively by using RT-PCR and Western blot.Result:1. Carbonized Agei can significantly reduce the gastric ulcer model rat’s ulcer area and the ulcer index (p<0.05), decrease the submucosal tissue inflammatory cells embellish. Other pathological changes compared with model group have different degree of ease2. Carbonized Agei can reduce ulcer bleeding points (P<0.05) of gastric ulcer model rat; Shorten the bleeding time and blood coagulation time in rat models (P<0.01). The effect is better than that of dried medicinal herbs group; the model rat platelet count (P<0.05) is improved.3. Carbon medicine high dose group could significantly increase the number of new blood vessels of submucosal tissue (P<0.05) and carbon medicine low dose group of model rat have significant different influence of microvascular density (P<0.05).4. Carbonized Agei high dose group could increase rats serum TXB2level (P<0.05) in gastric ulcer hemorrhage model and has no effect on6-keto-PGFla (P> 0.05) in rat serum. It can significantly improve the level of GMP-140(P<0.01) in rat serum.5. Carbonized Agei high and middle dose group could significantly improve the level of t-PA (P<0.05) in model rats serum, low and high dose group can reduce the level of PAI-1(P<0.05) in model rat serum.6. Carbonized Agei has no statistical significance on erythrocyte aggregation index, hematokrit and Whole blood viscosity (P>0.05) in the gastric ulcer rat model. Ranitidine can significantly reduce rat red blood cell volume and whole blood viscosity under different shear rate (P<0.05)7. Carbonized Agei low dose group could significantly increase VEGF and its receptor protein expression levels (P<0.05) and increase VEGFmRNA expression. It can improve the expression level of bFGF, its receptor protein and mRNA.Conclusion:1. Carbonized Agei has significant effect on wound healing on the ulcer bleeding model rats:it can significantly reduce the gastric ulcer model rat’s the ulcer index, decrease the submucosal tissue inflammatory cells embellish and promote the healing of the ulcer surface; Carbonized Agei can reduce ulcer bleeding points of gastric ulcer model rat; Shorten the bleeding time and blood coagulation time in rat models; and through the increased platelet count, the bleeding part’s hemostatic and coagulant is sped up and Carbonized Agei has a good therapeutic effect on ulcer bleeding; Carbonized Agei could significantly increase the number of new blood vessels of submucosal tissue and microvascular density; stimulates the growth of granulation tissue in the ulcer area and improve the quality of ulcer healing. Carbonized Agei could improve the quality of ulcer healing.2. Carbonized Agei has a two-way adjustment to hemostatic and invigorating the circulation of blood:On the one hand it can affect blood coagulation system and play a role in hemostasis and coagulanting; On the other hand by adjusting the fibrinolytic system it could prevent thrombus formation, at the same time it has no effect on model rats hemorheology indexes and no blood stasis formation when there is too much hemostasis.3. Carbonized Agei could significantly increase VEGF and its receptor protein expression levels and increase VEGFmRNA expression. It can improve the expression level of bFGF, its receptor protein and mRNA. It reveals one of the possible molecular mechanisms of submucosal tissue reconstruction and promoting angiogenesis.更多还原