【作者】 陈艳；

【导师】 邓虹珠；

【作者基本信息】 南方医科大学 ， 中药学， 2008， 博士

【摘要】 慢性肾小球肾炎是临床上的常见病,多发病,疑难病,慢性肾小球肾炎患者病情迁延,可以临床缓解,但很难完全康复,最后发展为终末期肾衰导致死亡。因此,探讨慢性肾小球肾炎的发病机制,掌握病情进展的环节,寻求最佳的治疗方案和有效药物,治疗早期肾脏疾病并发症,从而延缓疾病的进展,为最终改善肾脏病患者治疗质量和预后奠定基础。Harris等在动物肾脏缺血再灌注实验中发现,健存肾单位代偿性功能亢进,耗氧量明显增加,由此提出了高代谢学说,这使得研究人员对肾炎发病机制有了突破性的进展:许多与肾小球肾炎发生发展密切相关的因素和环节,如多种细胞因子、血管活性物质和化学趋化因子表达过多、细胞外基质积聚、肾小球系膜细胞异常增殖或肾小管上皮细胞肥大等都涉及到一种重要的机制——氧化应激。豆科植物细梗胡枝子（Lespedeza virgata（Thurb）DC）是临床上用于治疗慢性肾小球肾炎的中成药肾炎四味片中的君药,国内外学者对细梗胡枝子进行化学成分分析发现,其中富含大量的黄酮及酚酸类化合物,而黄酮类与酚酸类化合物因其强大的抗氧化能力而普遍受到关注。我们前期的实验研究表明,细梗胡枝子的总黄酮部位对阿霉素诱导的大鼠微小病变性肾病综合征有一定的治疗作用,但是,细梗胡枝子中究竟是哪些成分在发挥有效作用,且其药效机制是否与抑制氧化应激有关,迄今为止国内外尚无研究报告。阐明这些,不仅能从该植物中寻找到治疗肾小球肾炎的有效成分,还能从抗氧化的角度进一步明确细梗胡枝子对肾脏的保护作用及其作用机制。肾小球系膜细胞,是肾小球固有的三大细胞之一,具有活跃的生理功能,在肾小球炎症及硬化的发生发展中具有重要的作用。实验表明,氧自由基作为信号分子,通过激活蛋白激酶C（PKC）,调控细胞增殖水平而导致肾小球硬化。肾小球疾病中,肾小管间质纤维化的严重性不容忽视。大量无法及时清除的氧自由基可刺激肾小管上皮细胞分泌大量的细胞外基质（ECM）,其主要组成成分如纤维连接蛋白（FN）,层粘连蛋白（LN）,胶质等可直接导致肾间质纤维化,也可活化核转录因子KappB（NF-κB）,刺激纤维化生长因子,如转化生长因子β（TGF-β）,单核细胞趋化蛋白1（MCP-1）的过表达,放大肾脏局部免疫反应,进一步损伤肾小管间质。本课题采用药理学追踪指导化学活性分离的方法,应用现代层析技术及波谱方法从细梗胡枝子中分离活性成分并鉴定化学结构;同时,运用快速测定抗氧化能力指数、细胞培养、MTT、免疫组织化学、Western blotting、流式细胞术等实验手段,从体外的角度观察细梗胡枝子总黄酮对炎症介质脂多糖（LPS）刺激大鼠肾小球系膜细胞异常增殖的抑制作用;分析了细梗胡枝子活性成分对TGF-β1诱导的人类肾小管上皮细胞内氧化应激的影响,并初步探讨了这种作用所涉及的可能机制,以期明确其对病理状态下肾小管上皮细胞的抗氧化保护作用,阐明了其作为一种抗氧化剂防治肾小球肾炎的重要意义,为临床治疗肾小球肾病以及其它肾脏疾病的防治提供了新的理论线索和实验依据。第一部分胡枝子属植物资源调查目的概述胡枝子属植物的种属分类,形态特征及地理分布,了解胡枝子化学成分的研究进展及其药理活性。方法采用本草考证和文献调研的方法对胡枝子属植物的研究进展做一综述。结果我国产胡枝子属植物26种,分布广泛;迄今为止共从该属植物中分离得到黄酮、生物碱、有机酸等化学成分,具有极高的药用价值。结论胡枝子属植物资源丰富,价廉易得,药理活性明确,具有很好的开发利用价值。第二部分胡枝子总黄酮抑制大鼠肾小球系膜细胞增殖的作用研究目的观察胡枝子总黄酮诱导异常增殖的大鼠肾小球系膜细胞凋亡并探讨可能介导凋亡的途径。方法四甲基偶氮唑（MTT）比色法测定胡枝子总黄酮对系膜细胞增殖的抑制作用:Heochst 33342/PI双染色后荧光显微镜下观察细胞形态;AnnexinV/PI双染色流式细胞仪（FCM）定量分析系膜细胞的凋亡;试剂盒测定细胞上清MDA含量、SOD及GSH-PX活性:casepase活性检测试剂盒定量分析细胞内半胱天冬氨酸酶3（casepase-3）的表达。结果MTT实验检测证明胡枝子总黄酮可显著抑制大鼠系膜细胞的增殖（P＜0.05）。Hochest33342/PI双染条件下,荧光显微镜可明显区分胡枝子总黄酮作用细胞24小时后的早期凋亡细胞和中晚期凋亡细胞。FCM定量分析发现系膜细胞凋亡数量与胡枝子总黄酮浓度呈剂量依赖性（P＜0.05）。与对照组比较,1000、200及40μg/ml胡枝子总黄酮组的SOD和GSH-PX活性显著增高（P＜0.05）,而MDA含量则显著降低（P＜0.05）。Casepase活性检测发现胡枝子总黄酮可增加细胞内casepase-3的表达,并且呈浓度依赖性增加。结论胡枝子总黄酮诱导肾小球系膜细胞凋亡,抑制其增殖可能与抗氧化效应相关。第三部分细梗胡枝子化学活性成分的研究目的研究豆科植物细梗胡枝子（Lespedeza virgata）的化学成分。方法将乙醇提取物的正丁醇部分进行反复柱色谱,利用光谱方法对分离得到的单体进行结构鉴定。结果分离并鉴定了19个化合物,分别为β-谷甾醇（A1）,硬脂酸（A2）,7-O-α-吡喃鼠李糖基山萘酚苷（A3）,7-O-α-L吡喃鼠李糖基-山萘酚-3-O-β-D-吡喃葡萄糖苷（A4）,槲皮素（A5）,对羟基反式肉桂酸（A6）,原儿茶酸（A7）,对羟基苯甲酸（A8）,苜蓿素（A9）,山奈酚（A10）,胡枝子酚（A11）,芹菜素（A12）,胡枝子醛（A13）,山奈酚苷（A14）,桑色素（A15）,木犀草素（A16）,槲皮素-3-O-α-L鼠李糖苷（A17）,胡枝子内酯（A18）,柯伊利素H-7-O-β-D-芦丁糖苷（A19）。结论化合物A11、A13、A18为三个结构新颖的新化合物,A19为首次从胡枝子属植物中分离获得,化合物A1、A2、A3、A4、A5、A6、A7、A8、A9、A10、A12、A15、A16为首次从该植物中分离获得。第四部分细梗胡枝子中三个新化合物的抗氧化活性研究目的研究细梗胡枝子正丁醇部位三个新化合物体外抗氧化能力方法以维生素C（Vitamin C）为阳性对照,采用抗氧化能力指数（ORAC）方法测定胡枝子酚、胡枝子醛及胡枝子内酯体外抗氧化活性;复制溴酸钾（0.7mmol/kg）诱导大鼠肾损伤模型,TBARS法测定三个新化合物体外清除大鼠肾匀浆及血浆中过氧化脂质的能力。结果三个新化合物抗氧化能力均较Vitamin C强;TBARS方法检测发现,三个新化合物抑制大鼠肾匀浆及血浆中MDA的半数抑制率（IC50）由强至弱顺序为:胡枝子酚＞胡枝子内酯＞胡枝子醛。结论三个新化合物均有很强清除氧自由基的能力。第五部分胡枝子酚对TGF-β₁诱导肾小管上皮细胞氧化应激的抑制作用目的观察胡枝子酚（Lespedezavirgat01）对TGF-β₁诱导的大鼠肾小管上皮细胞内氧化应激的影响并探讨其可能的作用机制。方法采用双氢一乙酞乙酸二氯荧光黄（DCFH-DA）捕获细胞内ROS,应用流式细胞仪和共聚焦显微镜检测细胞内生成ROS的情况。应用免疫组织化学法检测细胞内纤维连接蛋白（FN）的表达;Western blotting方法观察TGF-β₁诱导人肾小管上皮细胞MMP-9和NF-κB的蛋白表达。结果Lespedezavirgatol可抑制TGF-β₁作用下ROS水平的升高,模型组FN强阳性表达,苯那普利组FN中等量表达,Lespedezavirgatol低、中、高剂量组FN轻度表达,明显少于模型组,并呈剂量依赖关系。药物干预24小时后,MMP-9的表达明显上调,NF-κB的表达较正常水平低（P＜0.05）。结论Lespedezavirgatol能显著抑制TGF-β₁作用下肾小管上皮细胞内ROS水平的升高。其抑制细胞内FN的表达并下调NF-κB,上调MMP-9的蛋白表达,这可能是Lespedezavirgatol减缓肾小球硬化和肾纤维化发展的机制之一。总结（1）细梗胡枝子中富含大量抗氧化活性成分;（2） Lespedezavirgatol具有强大的抗氧化能力,且能够抑制ECM的主要成分FN的过表达;（3） Lespedezavirgatol通过下调病理状态下NF-κB的过表达、上调MMP-9来修复损伤的肾组织;（4） Lespedezavirgatol可能是胡枝子治疗肾小球肾炎的有效成分。更多还原

【Abstract】 Chronic glomerulonephritis（CGN） is a frequently occurring and stubborn disease in the clinic.With the development of CGN,it can be relieved,but can not recovery completely,and died for terminal stage renal failure at last.Therefore,it is necessary to approach the pathogenesis of CGN,to seek the optimal therapeutic regimen and active drug.It can cure earlier period complication of kidney disease, delay the development of it,improve cure quality of patients,and decrease the danger of death.In the experimentation of ischemia-reperfusion for animal kidney,Harris found that the compensated function of alive nephrons was accentuation and its oxygen-consumption increased greatly,so he put forward hypermetabolism theory, which make this researches get a breakthrough in the pathogenesis of nephritis that many etiological factors correlating with glomerulonephritis（GN）.For example,the overexpression of cytokine,vasoactive substance and chemotatic factor,the accumulation of extracellar matrix,the proliferation of Mesangial cell and the hypertrophy of renal tubular epithelial cell,are all concerned in an important mechanism-oxidative stress. Lespedeza virgata（Thurb） DC is the principal drug of Shen Yan Si Wei Pian, which is used to cure CGN in the clinic.After analyzing the chemical composition of lespedeza virgata（Thurb） De,we find that it has abundant flavonoids and phenolic acid.And the powerful anti-oxygenation capability of flavonoids and phenolic acid are engaged popular attention.We have described that the flavonoids of lespedeza virgata（Thurb） De has definite therapeutic action for minimal change nephrotic syndrome of rat induced by Adriamycin in our previous experimentations.However, as for ours knowledge,there have been no reports concerning the chemical constituents of this plant are available and whether their phannacodynamic mechanism has relationship with inhibiting oxidative stress.After elucidate the above questions,we can not only find the available compositions for curving CGN, but also further discern the mechanism of protecting kidney from the aspect of anti-oxidation.Mesangial cell,as one of the three inherent cells of renal glomerulus,has active physiologic function,and also play an important role in the occurrence and development process of renal glomerulus inflammation and sclerosis.Previous experimentations indicated that oxygen radicals,as a signaling molecule,gives rise to renal glomerulus sclerosis from activating protein kinase C（PKC） to adjust and control cell proliferation level.In the phase of renal glomerulus diseases,the severity of renal tubule interstitial fibrosis can not be ignored.A great deal of oxygen radicals,which can not be eliminated in time,it can stimulus renal tubular epithelial cell to excrete a mass of extracellular matrix（ECM）,for example,fibronectin（FN）,laminin（LN） and colloidal matter,which induce renal interstitium fibrosis.In addition,they can also activate nuclear factor KappB（NF-κB）,excite the overexpression of fibrosis growth factor,suah as transforming growth factorβ（TGF-β）,monocyte chemoattractant protein 1（MCP-1）,enlarge the immunological response of kidney, and further damage the interstitial tissue of renal tubule.The topic adopts a chemical activity separation method guided by pharmacology,and uses advanced chromatography technology to isolate active components from lespedeza virgata（Thurb） DC.The structures of these compounds were elucidated on the basis of spectroscopic analysis.At the same time,it applies many experimental medias,such as determine oxygen radical absorbance capacity, cell culture,MTT,immunohistochemistry,Western blotting,flow cytometry and so on,in order to observe the inhibitory action of lespedeza virgata（Thurb） DC to the rat mesangial cell proliferation that caused by lipopolysaccharide（LPS）.Analyzed the effect of the active components of lespedeza virgata（Thurb） DC to renal tubular epithelial cell oxidative stress induced by TGF-β₁,discusses the possible mechanism of anti-oxygenation function for renal tubular epithelial cell under pathologic status, and proves its important significance as an antioxidant to prevention and cure GN.PartⅠ:Research of lespedeza virgata（Thurb） DC resource.Objective:To know the research progression,pharmacody activity,genera, morphological characteristic and geographical distribution of lespedeza virgata （Thurb） DC.Methods:Summarize the research progression of lespedeza virgata（Thurb） DC by adopting herbal textual and literature investigation method.Results:There are about 26 kinds of lespedeza plant in China,and they are distributed different region.There are flavone,alkaloid,organic acid and other elemental composition in this plant,which have skyscraping medicine value.Conclusions:The resource of lespedeza plant is very exuberant,the price is low, and the pharmacody activity is exact.Therefore,it has good exploitation and utilization value.PartⅡEffects of flavonoids from Lespedeza virgata（Thunb） on the proliferation of rat mesangial cells in vitroObjective:To investigate the apoptotic effects of lespedeza flavonoids in rat glomerular mesangial cells（GMCs） and the mechanism of apoptosis.Methods:MTT-based cytotoxicity assay was to test the GMCs proliferation. GMCs were double stained by Heochst33342/PI and observed under fluorescence microscope.AnnexinV/PI staining and flow cytometry were employed for quantitative measurement of apoptosis on GMCs.The content of MDA,activitive of SOD and GSH-PX in GMCs supernatant was measured.Casepase-3 activation was determined by casepase-3 staining Kit.Results:Lespedeza flavonoids significantly inhibited proliferation of GMCs compared with control（P＜0.05）.Heochst33342/PI images could discriminate normal cells,apoptosis cells and necrotic cells under fluorescence microscope.The quantitative assay for apoptosis by flow cytometry method,which showed obvious concentration-dependent relationship.The activities of SOD and GSH-PX in 1000 g/ml;200μg/ml;40μg/ml lespedeza flavonoids groups were significantly higher than that in the control group（P＜0.05）,as same as concentrations of MDA were significantly lower than that in the control group.Casepase-3 expression was activated by lespedeza flavonoids in a dose-dependent manner.Conclusions:Proliferation inhibition and apoptosis induction of mesangial cells are involved in the mechanisms of the antioxidation effects of lespedeza flavonoids. The latter may be induced by activation of caspase-3. PartⅢ:Research on chemical active component of lespedeza virgata （Thurb） DC.Objective:To investigate the chemical constituents of lespedeza virgata（Thurb） DC.Methods:Repeated column chromatography and HPLC of the n-BuOH-soluble fraction in the EtOH extract of lespedeza virgata（Thurb） DC.Their structures were elucidated by chemical properties and spectroscopic evideuce.Results:Nineteen components were isolated and identified asβ-rhamno（A1）, octadecoic-acid（A2）,kaempferol-7-O-L-rhamnopyranoside（A3）,7-O--L-rhamnopyra-nsyl -Kaempeferol-3-O-β-D- glucopyranoside（A4）,quereetin（A5）,E-β-hydroxycinnamic acid（A6）,p-rotoeatechuic acid（A7）,p-hydroxybenzoic（A8）, tricin（A9）,kaempferol（A10）,lespedezavirgatol（A11）,apigenin（A12）, lespedezavirgatal（A13）,kaempfedtrin（A14）,morin（A15）,luteolol（A16）,quercetin-3-O -α-L-rhamnoside（A17）,lespedezacoumestan（A18） and chrysoeriol-7-rutinoside （A19）.Conclusions:A11,A13 and A18 are new compounds,A19 was isolated from Lespedeza Michx for the first time,A1,A2,A3,A4,A5,A6,A7,A9,A10,A12,A15 and A16 are isolated from lespedeza virgata（Thurb） DC for the first time.PartⅣ:Research on the anti-oxygenation activity of the three new compositions isolated from lespedeza virgata（Thurb） DC.Objective:To investigate the anti- oxygenation capability of the three new compounds.Methods:We adopt VitaminC as control drug.Determine the anti-oxygenation activity of lespedezavirgatol,Lespedezavirgatal and Lespedezacoumestan by using ORAC method.At the same time,we establish rat kidney injury model inducted by kalitan bromicum（0.7mmol/kg）,and determine the capability of eliminating lipid peroxidation by the three new compositions in both plasma and kidney homogenate of renal failure rats.Results:In an oxygen radical absorbance capacity（ORAC）,the three new compounds exhibited dose-dependent oxygen radical scavenging activity.All were higher than that of vitamin C.The IC₅₀ values of three new compounds and vitamin C toward malondialdehyd（MDA） levels in plasma and kidney homogenates of kidney failurerats shown the three new compounds could potently counteract oxidative damage on kidney.Conclusions:All of the three new compounds have strong capability to eliminate oxygen radical.PartⅤ:Research on the function of lespedezavirgatol inhibits the oxidative stress in renal tubular epithelial cell that inducted by TGF-β₁.Objective:To investigate the role of lespedezavirgatol inhibits the reactive oxygen species（ROS） on TGF-β₁ induced accumulation of extracellular matrix （ECM） in human kidney proximal tubular cell-2（HK-2）,and discuss its possible mechanisms.Methods:The DCFH-sensitive cellular ROS was measured by flow-cytometry technology（FCM） and confocal microscopy.Immunohistochemical method was employed to detect the expression of FN in intracellular.MNP-9 and NF-κB protein expression were examined by Western blotting.Results:TGF-β₁（8ng/ml） significantly induced the production of intracellular ROS compared with control（P＜0.01）.DPI and Lespedezavirgatol can inhibit the TGF-β₁-induced ROS generation（P＜0.01）.TGF-β₁ stimulated fibronectin（FN） protein overexpression.Both benazepril and Lespedezavirgatol inhibited the up-regulation of FN（P＜0.05）.TGF-β₁ stimulated NF-κB protein overexpression in HK-2 and down-regulated MMP-9 protein-expression,which were ameliorated by lespedezavirgatol and benazepril（P＜0.05）.Conclusions:Lespedezavirgatol can inhibit the increasing of ROS level in human kidney proximal tubular cell-2（HK-2） inducted by TGF-β₁.It can inhibit the FN expression and reduce the expression of NF-κB and upregulating the expression of MMP-9,which is likely to be one of mechanisms why lespedezavirgatol can delay glomerular sclerosis and kidney fibrosis.Conclusions（1） There are many anti-oxygenation activity components in the lespedeza virgata（Thurb） DC.（2） Lespedezavirgatol has very strong anti-oxygenation capability,and it can also inhibit the overexpression of FN for the key components in ECM.（3） Lespedezavirgatol can repair the impaired nephridial tissue by decreasing the overexpression of NF-κB and increased MMP-9 protein expression.（4） Lespedezavirgatol is likely to be the active component to curve GN in the lespedeza virgata（Thurb） DC.更多还原