【作者】 张旗；

【导师】 谢桂权；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2012， 博士

【摘要】 目的本课题以中医“痰瘀互结”是肾纤维化发生发展的主要病理特点为立论基础,采取临床与动物实验相结合的方法,探讨观察祛瘀化痰法对慢性肾衰大鼠模型的影响及防治肾纤维化患者的临床效果。并主要从TGF-β/Smads信号通路,深入研究祛瘀化痰法对慢性肾衰大鼠模型的影响和可能机制。总体评判在祛瘀化痰法基础上防治肾纤维化的临床效果,为临床防治肾纤维化提供新的临床证据与指导理论。方法1.祛瘀化痰法防治肾纤维化的临床研究通过观察祛瘀化痰中药对慢性肾衰患者的临床症状及实验室指标如血肌酐、尿素氮、肾纤维化相关因子水平(TGF-β1.TIMP-1).肝纤四项、血脂、凝血四项等的影响,实事求是评判祛瘀化痰中药对肾纤维化的临床防治效果。2.祛瘀化痰法防治肾纤维化的作用机理研究通过建立慢性肾衰大鼠模型,观察经过使用祛瘀化痰中药后,对大鼠模型肾单位结构的病理组织学及肾功能实验室指标影响,判断祛瘀化痰中药对大鼠模型肾纤维化的有效性。并应用间接免疫荧光法测定各组大鼠肾组织TGF-β1.p-Smad2/3和α—SMA.E-cadherin及p-Smad7蛋白的表达,揭示祛瘀化痰中药对调节TGF-β/Smads信号通路与拮抗肾纤维化的关系,初步阐明祛瘀化痰法防治肾纤维化的作用机制。结果(一)祛瘀化痰法防治肾纤维化的临床研究1.治疗组与对照组中医症候积分比较研究结果显示,用药前两组中医症候积分比较差异无显著性(P>0.05)；治疗后,治疗组的面色晦暗,困怠身重,脘闷腹胀,肌肤甲错,肢体麻木或刺痛,口干不欲饮或口中粘腻,恶心呕吐,食少纳呆,大便粘滞不爽等症状,较治疗前有明显改善(P<0.05或P<0.01)；对照组经治疗后,面色晦暗,肌肤甲错,肢体麻木或刺痛,口中粘腻,反胃泛酸、恶心呕吐,大便粘滞不爽,较治疗前有明显改善(P<0.05或P<0.01)；治疗后,两组中医症候积分组间比较,治疗组面色晦暗,肌肤甲错,肢体麻木或刺痛,困怠身重,脘闷腹胀,口干不欲饮,口中粘腻,纳呆食少,大便粘滞不爽的改善作用明显优于对照组(P<0.05或P<0.01),表明祛瘀化痰中药在提高中医痰瘀互阻症候疗效方面,治疗组优于对照组。2.两组血清中TGF-β1.TIMP-1因子水平比较结果显示,治疗组与对照组血清中TGF-β1.TIMP-1因子水平治疗前比较,差异无显著性(P>0.05)；TGF-β1治疗组治疗前后之间比较差异有非常显著性(P<0.01),对照组治疗前后之间比较差异有显著性(P＜0.05)；TIMP-1两组治疗前后差异均有显著性(P<0.05)；治疗后两组组间比较,治疗组血清中TGF-β1与TIMP-1因子水平降低程度均优于对照组(P＜0.05)。3.肝纤四项、肾功能、血脂、凝血功能指标的治疗前后比较结果显示,反映纤维化程度的指标肝纤四项,两组治疗前比较,差异无显著性(P>0.05)；两组治疗后与疗前比较,均有所降低,但治疗组4项指标差异均有统计学意义(P<0.05),对照组仅HA一项有统计学意义(P<0.05)；治疗后,两组组间比较,差异有显著性(P<0.05或<0.01)；表明慢性肾衰患者服用祛瘀化痰中药对肝纤四项指标有一定的降低,且优于对照组。结果显示,两组尿素氮(BUN)、血肌酐(Scr)治疗前比较,差异无显著性(P>0.05)；两组治疗后比较, BUN及Scr均有所降低,但仅治疗组的差异有统计学意义(P<0.05)：治疗后两组比较,治疗组的治疗结果优于对照组(P<0.05或<0.01)；表明慢性肾衰患者服用祛瘀化痰中药,对其肾功能有更好的保护作用。两组治疗前低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)、甘油三酯(TG)、总胆固醇(TC)比较,差异无显著性(P>0.05)；两组治疗前后比较则均有明显改善(P<0.05)；治疗后两组比较,治疗组的改善作用优于对照组(P<0.05),表明祛瘀化痰中药在改善血脂方面,有更好的效果。治疗后两组患者纤维蛋白原(FIB)含量较治疗前降低,活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)均有所延长。两组治疗后与治疗前比较差异均有显著性(P<0.05),且治疗组的改善明显优于对照组(P<0.05,或<0.01)。4.两组药物治疗后安全性观察指标的比较结果显示,治疗组患者未发现各种不良症状和体征,治疗组反映心脏功能、肝功能的指标肌酸激酶-MB同工酶(CK-MB)、谷丙转氨酶(ALT)、谷草转氨酶(AST),治疗前后,差异无统计学意义(P>0.05),表明慢性肾衰患者服用祛瘀化痰中药,对心脏功能及肝功能无明显影响。(二)祛瘀化痰法防治肾纤维化的作用机理研究1.各组大鼠肾组织学观察HE染色镜下显示：正常对照组大鼠’肾组织无异常病理改变,肾小球、肾小管无异常,小管管腔无结晶物沉积,肾间质无异常改变。模型组肾皮质区肾小球数量减少,萎缩呈分叶状,大多数肾小球毛细血管基底膜增厚,MC增多,肾小管上皮细胞坏死、变性、脱落,大量肾小管萎缩、破坏、消失,可见较多肉芽肿形成。肾小管管腔内可见蛋白管型、细胞管型,部分小管代偿性扩张。肾皮质可见炎症细胞浸润,及异物巨细胞反应,间质纤维化明显。而中药组肾小球MC轻度增生,。肾小管轻度扩张,上皮细胞颗粒变性,腔内可见管型形成,脱落较少,肾间质少量炎症细胞浸润与纤维化。尤以中药高剂量组差异明显。2.各组实验结果比较四组大鼠血清中尿素氮、血肌酐检测结果比较结果显示,实验前各组尿素氮、血肌酐平均浓度之间分别比较,差异均无显著性(P>0.05)。手术后8周,正常对照组大鼠尿素氮与血肌酐均值分别为6.42±1.33mmol/L.46.65士5.90umol／L,与文献正常参考值基本接近。而模型组分别为24.11士6.37mmol/L.130.71士14.52umol/L,经统计差异具有非常显著性(P<0.01),说明慢性肾功能衰竭大鼠建模成功。而用药组中,中药高剂量组尿素氮与血肌酐均值分别为16.50±5.38mmol/L.115.93±11.45umol/L；中药低剂量组尿素氮与血肌酐均值分别为18.16士5.41mmol/L.118.06±12.89umol/L.中药高剂量组的尿素氮和血肌醉均值与模型组相比均下降,差异具有显著性(P<0.05)；中药低剂量组的尿素氮均值与模型组相比下降,差异具有显著性(P<0.05)。说明经过8周的祛瘀化痰中药复方灌胃治疗后尿素氮和血肌酐均值明显降低,肾功能有所改善。但中高组和中低组的尿素氮、肌酐均值之间分别比较,中高组均值虽有下降,但差异均无显著性(P>0.05)。免疫荧光结果显示Smad2/3主要表达在胞核,Smad7、α-SMA和TGF-β1主要表达在胞浆。正常对照组Smad2/3.TGF-β1. α-SMA.Smad7的表达很少,而E-cadherin的表达十分明显；模型组Smad2/3.Smad7.TGF-β1和α-SMA表达较高,E-cadherin表达极低,与正常对照组比较差异有非常显著性(P<0.01)。中高组、中低组Smad2／3.TGF-β1和α-SMA表达均较模型组减少,而Smad7. E-cadherin表达均较模型组增加,两组与模型组相比,差异均有显著性(P<0.05)。中高组Smad2／3.TGF-β1和α-SMA表达均较中低组明显减少,两者比较差异有显著性(P<0.05)；中高组Smad7.E-cadherin表达均较中低组明显增加,两组比较差异有显著性(P<0.05)。结论1临床研究(1)祛瘀化痰中药复方可以改善慢性肾衰患者临床症状,提高慢性肾衰治疗效果。(2)祛瘀化痰中药复方能明显降低反映肾纤维化的相关指标(肝纤四项、血清中TGF-β1.TIMP-1因子)水平,初步显示祛瘀化痰中药在防治肾纤维化中有较好的作用。(3)祛瘀化痰中药复方应用于肾纤维化患者,临床未见明显不良反应,心肝功能未见异常。2实验研究(1)祛瘀化痰中药可以改善肾纤维化大鼠模型的临床症候,及肾功能指标,以中药高剂量组效果更明显。(2)祛瘀化痰中药可以明显减轻慢性肾衰大鼠模型肾组织的炎症反应及病理改变程度,保护肾组织形态结构的完整,从而起到拮抗肾纤维化的作用,这一作用呈量效关系,以中药高剂量组效果更明显。(3)祛瘀化痰中药是通过对TGF-β/Smads信号通路的影响来拮肾纤维化的,其作用机制可是通过下调TGF-β1水平、上调TGF-β抑制性信号蛋白Smad7的表达,进而抑制TGF-β受体调控信号蛋白(Smad2/3)的活化,阻止肾纤维化动物模型肾组织间皮细胞转分化的发生,从而阻止或减轻肾纤维化的发生和进展。更多还原

【Abstract】 ObjectiveThis study based on the theory of "the phlegm ties stasis mutually" is the pathogenesis key which the renal fibrosis occurs.Uses the clinical method which unifies with the animal experimentation, observes and discusses the clinical effect by the method of eliminating phlegm and removing stasis to prevent the renal fibrosis in renal treatment patients. And further discusses the function and the mechanism by the method of eliminating phlegm and removing stasis to prevent the big mouse model of renal fibrosis from TGF-β/Smads signal passage. Comprehensive evaluation of the clinical efficacy by use of eliminating phlegm and removing stasis to prevent and treat the renal fibrosis, and provide a new theoretical basis and methods of Chinese medicine for the prevention of renal fibrosis in renal treatment patients.MethodsThrough the collection of our hospital cases met the inclusion criteria of the renal treatment patients from January2011to January2012, We observe the change in patients on of clinical symptoms and laboratory parameters such as serum creatinine, urea nitrogen, albumin, blood lipids,blood analysis, liver filament four items and the level of TGF-β1,TIMP-1expression in renal fluid of the impact of herbs which can eliminate phlegm and remove stasis. We also can objective evaluate herbs clinical efficacy of prevention and treatment of renal fibrosis. Through establishing the big mouse model of renal flbrosis, observation the mouse model renal structure’s histology change after the intervention of herbs which can eliminate phlegm and remove stasis. To expound the validity of herbs to oppress the big mouse model renal fibrosis. And applies the indirect immunity fluorescence method to determine the protein expression of each group of big mouse somatopleura renal such as TGF-β1, p-Smad2/3and α-SMA, E-cadherin and the p-Smad7, to discuss relations of phlegm reduction removes extravasted blood the traditional Chinese medicine to oppress the renal fibrosis with the regulative TGF-β/Smad signal passage’s, promulgates initially the function mechanism.Result(一)The clinical research of prevention renal fibrosis by the law phlegm reduction and removal extravasted blood1、Phlegm and blood stasis influnce syndrome score of Chinese medicineAfter the treatment the improvement function of the treatment group clinical symptoms such as the facial color is gloomy, the flesh armor is wrong, the body numb or the stabbing pain, the feeling of heaviness in the limbs is sleepy, wan abdomen puffiness, in the mouth sticks greasily, dry mouth do not want to drink, losses of appetite, stool viscosity surpasses the control group obviously (P<0.05), which show phlegm reduction removes extravasted blood the traditional Chinese medicine to improve the symptoms, the treatment group better than the control group.2、Phlegm and blood stasis related indicators of the impact of renal fibrosisAfter treatment the treatment group TGF-β1levels effluent decreased significantly, compared with before treatment, there was significant difference (P<0.05);the control group compared with pre-treatment showed no significant difference (P>0.05);After treatment, comparison between the two groups, the treatment group TGF-β1levels renal treatment effluent decreased was significantly higher (P <0.01). After treatment the treatment group TIMP-1levels renal treatment effluent were significantly decreased, compared with before treatment, there was significant difference (P<0.05); the control group compared with pre-treatment showed no significant difference (P>0.05); After treatment, comparison between the two groups, the treatment group factor of decline in effluent TIMP-1levels were significantly higher (P<0.01). Indicators reflect the degree of fibrosis:hepatic-fibrosis four items, treatment and control groups before treatment compared after treatment were decreased,but only the treatment group has statistically significant difference (P<0.05), no statistical difference between the control group significance (P>0.05);After treatment between the two groups was statistically significant difference (P<0.01); showed that patients treated with phlegm reduction and stasis removal herbs renal treatment on hepatic-fibrosis four items have some improvement. In conclusion, the results suggest that phlegm and blood stasis can significantly reduce the level of renal fibrosis-related indicators, play antagonistic renal fibrosis, and protect the renal function.3、Phlegm and blood stasis effect on laboratory parametersTreatment group after treatment with phlegm and blood stasis, the improvement of creatinine, urea nitrogen, total cholesterol, triglycerides, low density lipoprotein, fibrinogen better than the control group (P<0.05), indicating that the phlegm and blood stasis in improving renal functi on, blood lipid and cogulation have a good effect.4、Phlegm and blood stasis indicators of drug safetyTreatment group during the medication, the symptoms do not appear all kinds of adverse reactions, and reflecting the heart and liver function indicators of CK-MB, AST, ALT, before and after treatment, the difference was not statistically significant (P>0.05), showed that patients treated with phlegm and blood stasis, on cardiac function and liver no functional effect, taking safety.(二)Experimental study of phlegm and blood stasis to Prevent and Treat the renal fibrosis1、Observation renal tissue of rats and comparison dense layer thickness Control group, HE staining showed thin and dense renal smooth, flat layer of mesothelial cells covering intact, thin basement membrane between the skin and connective tissue attached to the skin, between the collagen fibers had no significant deposition, capillary dilatation and congestion-free, non-inflammatory cell infiltration. Model group was significantly thicker renal, mesothelial cell loss, spindle fiber cells, fibroblasts, generated by multi-layer, between the loose skin matrix, a large number of collagen fiber deposition, capillary dilatation and congestion, accompanied by inflammatory cell infiltration, suggesting that renal fibrosis model success. The western medicine group and the morphological changes of rat renal have different levels of mitigation to reduce, the level of medicine in high dose group significantly. The group compared the thickness of dense layer parietal renal, renal thickening model rats in which the most obvious level, compared with the control group were significantly different (P<0.01); Chinese medicine high dose group and low dose group and western medicine group were significantly different (P<0.05); western medicine group and low dose group, significant differences (P<0.05). Phlegm and blood stasis can be effectively shown to improve renal fibrosis pathological changes, especially in the high dose of Chinese medicine.2、Comparison of immunohistochemistryImmunofluorescence showed that Smad2/3was mainly expressed in the nucleus, Smad7, α-SMA and TGF-β1expressed mainly in the cytoplasm. Control group, Smad2/3, TGF-β1, α-SMA, Smad7expression is low, and the expression of E-cadherin is obvious; Model group Smad2/3, Smad7, TGF-β1and α-SMA expression in a high, E-cadherin expression is very low; the two groups there was a significant difference(P<0.01); In the high group and low group and western medicine group Smad2/3, TGF-β1and α-SMA expression compared with model group decreased, but Smad7, E-cadherin expression compared with model group increased, compared with model group, three groups were significantly different (P<0.05); in the high group Smad2/3, TGF-β1and α-SMA expression than those in the low group and western medicine group significantly reduced. In the high group were compared with the latter two groups were significantly different(P<0.05); In the high group of Smad7, E-cadherin expression than those in the low group and western medicine group was significantly increased. In the high group were compared with the latter two groups were significantly differences (P<0.05); Western medicine group Smad2/3, TGF-β1and α-SMA expression decreased than those in the low group, Smad7, E-cadherin expression was increased by lower than those in the two groups were significantly different(P<0.05).Conclusion1、Through January2011to January2012,40patients met the criteria of the standard treatment of clinical studies suggest that: phlegm and blood stasis can improve clinical symptoms of patients and treatment effection.2、Blood stasis and phlegm can significantly reduce the level of relevant indicator of renal fibrosis (liver fiber four, renal fluid TGF-β1, TIMP-1factor), can protect the structural integrity of renal morphology and reduce the rat model of renal fibrosis, play the role of renal fibrosis antagonist. This effect was dose-effect relationship, the best high-dose group.Phlegm and blood stasis initially showed better prevention and treatment of renal fibrosis. Through5/6nephrectomy rats application of caused by renal fibrosis were found: phlegm and blood stasis on the impact of TGF-β/Smad signaling pathway possibly by increased TGF-β signaling inhibition protein expression of Smad7and inhibit TGF-β receptor signaling protein regulated (Smad2/3) activation, prevents renal fibrosis in animal models of renal mesothelial cell transdifferentiation occurs, thereby preventing or reducing the incidence and progression of renal fibrosis, Protecting the renal function.更多还原