黄芪卫矛合剂对糖尿病肾病大鼠肾脏内皮细胞损伤的干预研究

【作者】 杨敏；

【导师】 赵进喜；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2007， 博士

【摘要】 糖尿病肾病(diabeticnephropathy,DN)是糖尿病(diabetesmellitus,DM)重要的微血管并发症之一,也是导致慢性肾功能不全的主要原因。目前DN的发病机制尚不清楚,亦缺乏有效的治疗手段。因此,积极地寻找有效方法治疗DN对于延长患者寿命,提高生活质量具有重大意义。本论文从文献综述、理论和实验研究三方面加以探讨,实验研究方面选用了体现益气行血、消癥化积治法的黄芪卫矛合剂,重点观察其对单侧肾切除合并注射链脲佐菌素DN大鼠模型肾脏内皮细胞损伤的干预作用。1文献研究:就近年来现代医学对于DN的发病机理、治疗用药、研究方法;中医药防治DN作用机制进展;内皮细胞和DN关系;实验性DN动物模型的建立及研究方法等方面研究动态进行了较为系统的总结和综述。2理论研究:在中医理论指导下,结合现代研究成果,对消渴病肾病的病名、病机理论进行了阐述,认为消渴病肾病是以肾元亏虚、气阴两伤为本,气血瘀滞、痰湿内停为标,久病入络、肾生癥瘕为发病关键;初步阐明了微型癥瘕假说与肾脏内皮细胞损伤机制的相关性,确立了“益气行血、消癥散结”为DN基本治法,为消渴病肾病的研究思路及治法提供了理论依据。3实验研究:目的:观察黄芪卫矛合剂对实验性糖尿病肾病大鼠肾脏内皮细胞损伤的干预作用及机制。方法:采用单侧肾切除加链脲佐菌素(STZ)尾静脉注射建立糖尿病肾病动物模型,随机分为模型组(M)、黄芪卫矛合剂治疗组(H)、科素亚治疗组(K),另设假手术组为空白对照组(F)。0、4、8、12周动态观察大鼠一般状态、体重、尿量、空腹血糖(FBG)、糖化血红蛋白(HbA1c)、血脂、24小时尿蛋白定量、肾功能的变化,光镜观察肾小球及肾小管间质形态学和肾小管周围血管(PCT)网改变;采用双抗体夹心酶联免疫吸附(ELISA)法检测血清层粘连蛋白(LN)和Ⅳ型胶原(typeⅣcollagen)含量,硝酸还原酶法检测肾组织一氧化氮(NO)含量;放射免疫法检测内皮素-1(ET-1)的含量;采用Western-blot的方法测定肾组织中肝细胞生长因子(HGF)和转化生长因子β1(TGF-β1)的蛋白表达。结果:①黄芪卫矛合剂能显著降低DN大鼠空腹血糖和糖化血红蛋白,并具有降低血清胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL-C)而调节脂代谢的作用,与模型组比较有显著差异(P<0.05,P<0.01);能够降低DN大鼠升高的血清BUN和Scr,与模型组比较有显著差异(P<0.05);降低实验性DN大鼠24小时尿蛋白排泄量,与模型组比较有极显著差异(P<0.01)。提示黄芪卫矛合剂对实验性DN大鼠具有良好的调节糖、脂代谢作用,并具有减少尿蛋白排出、保护肾功能的作用。尤其在降糖、调脂、改善肾功能方面疗效优于科素亚组。其降低24小时尿蛋白的作用不如科素亚,但差异无统计学意义(P>0.05)。病理形态观察显示:模型组光镜下可见肾小球基底膜增厚,系膜区弥漫性增宽,细胞外基质(ECM)增多,肾小管纤维化。经黄芪卫矛合剂治疗后,上述病理改变有显著减轻,提示其对DN大鼠肾脏具有保护作用。科素亚对肾小球也有一定保护作用。②ELISA结果表明:糖尿病肾病大鼠血清LN和Ⅳ型胶原的合成和分泌明显增多,揭示了DN大鼠肾脏受到一定程度损害。在抑制肾小球合成和分泌LN和Ⅳ型胶原方面,黄芪卫矛合剂和科素亚皆能明显降低造模后LN和Ⅳ型胶原(与模型组相比P<0.05),在降低Ⅳ型胶原方面黄芪卫矛合剂优于科素亚组,但在降低LN方面两组之间差异不显著(P>0.05)。表明黄芪卫矛合剂能减少肾小球合成和分泌LN和Ⅳ型胶原,是其防治DN的作用机制之一。③硝酸还原酶法检测表明糖尿病肾病模型组NO含量显著降低,放射免疫法检测显示其ET-1水平比假手术组显著升高,提示DN大鼠NO与ET-1的平衡失调,内皮功能紊乱,揭示了DN大鼠肾脏损害进一步发展。中药黄芪卫矛合剂和科素亚能明显提高NO含量,降低ET-1水平,与模型组比较差异均有显著性统计学意义(P<0.05,P<0.01),两组之间比较差异不显著。提示黄芪卫矛合剂能显著改善糖尿病肾病模型所造成的NO表达减少和ET-1释放增多,从而发挥对肾脏内皮细胞的保护作用。表明黄芪卫矛合剂能显著改善糖尿病肾病大鼠模型所致NO的降低和ET-1增高,一定程度恢复二者的平衡紊乱、改善肾脏内皮功能,在此方面与科素亚比较作用相接近。④采用Westernblot蛋白印迹方法检测各组大鼠肾组织HGF和TGF-β1蛋白表达,研究结果显示:模型组大鼠肾组织中TGF-β1蛋白表达较假手术组明显升高(P<0.05),而HGF蛋白表达较假手术组明显降低(P<0.05),黄芪卫矛合剂和科素亚治疗后可明显下调TGF-β1蛋白表达,上调HGF蛋白表达,以调节HGF和TGF-β1的动态平衡,进一步抑制肾脏内皮细胞损伤,促进肾组织修复。结论:本课题中黄芪卫矛合剂采用“益气行血、消癥散结”之法,针对DN“微型癥瘕”病机设立,可有效防治糖尿病肾病,其作用机理可能是通过降糖、调脂、降低DN模型大鼠尿蛋白,减轻肾组织病理损伤,抑制细胞外基质主要成分LN和Ⅳ型胶原的合成,调节内皮细胞相关细胞因子NO和ET-1,HGF和TGF-β1的动态平衡发挥作用。其中,部分检测结果显示黄芪卫矛合剂优于科素亚,可能与中药多环节、多靶点治疗作用有一定关系。更多还原

【Abstract】 Diabetic nephropathy, (DN) is an important microvascular complication of diabeticmellitus (DM) as well as the common reason resulting in chronic renal failure. DN whosepathogenesis has not been fully understood, doesn’t been well treated in the days. It is verysignificance to find a effective method of treatment to DN. Systematic studies on theprevention and treatment for DN by HuangQiWeiMao Mixture (HQWM Mixture) had beenperformed from theaspects of referencereviews, theoryand experimental studyinthis essay.westudytheHuangQiWeiMaoMixturepreventDNkidneyendothelialcellsfromlesion.1ReferencereviewThis essay reviewed from the advances of such fields as follwed:The results andprogresses of studyon the pathogenesis and Medication for DN, the method of investigate ofmodern medicine, mechanism of action of prevention and cure DN with traditional Chinesemedicine, the relationship of DN and EC, foundation of the experimental DN animal model.2TheorystudyBy discussing the pathogenesis theory, principle and method of treatment of Xiao keBing nephrosis with Chinese medicine theories quoting from classics works and combiningmodern research result, we put forward and perfect the theory that deficiency of kidney andimpairment of both QI and YIN are the origin, however qi-stagnancy and blood stasis andphlegmatic hygrosis stay in the body are indications, pathogen usually intruding intocollateral in protracted disease and ZhengJia generate in kidney are the key point.We initialIlluminate the relationship of WeiXingZhengJia hypothesis and endothelial cell lesionmechanism, quibus establish the effective way of“Method of invigorate Qi and activateblood circulation, removing cumulation and stasis”is the fundamental therapeutics to DN ,gisttoprovidthetheoryofstudymethodandresearchtocureDN.3ExperimentalstudyPURPOSE: Study the HuangQiWeiMao Mixture prevent DN kidney endothelial cellsfromlesionandmechanism.METHODS: We used DM glomerulosclerosis rats model induced by unilateralnephrectom and injecting streptozotocin(STZ) , then divide them randomly into modelgroup(M),experimental group (H) in HQWM Mixture, positive control group(K) , shamoperated group(F) was blank group . Observed the rats common condition , and examinedrats weight, urine volume , fasting blood glucose, glycosylated hemoglobin(HbA1c),blood-fat , 24h Urine protein quantitation, renal function per month dynamicly . Observeglomerulus and renal tubule mesenchymal morph, peripheral vessels(PCT) use lightmicroscope; detected serum laminin(LN)and typeⅣcollagen content used enzyme linkedimmunosorbent assay(ELISA) method; examined nitrogen monoxidum (NO) content ofnephridial tissue through nitrate reductase; deted endothelin-1(ET-1) by radio-immunity method; observed the express of hepatocyte growth factor(HGF) and transforming growthfactorbeta1(TGF-β1)innephridialtissuebywesternblot.RESULTS:①The results showed HQWM Mixture obviouslyimprove the rats’generalcondition and had a certain effect on reducing blood sugar, serum cholesterol (TC) andtriglyceride (TG), low density lipoprotein (LDL), which had a significant statistic sense incomparing with diabetic model group (P<0.05, P<0.01). as well as it can decrease theexperimental rats’higher serum urea nitrogen, creatinine,it also reduced experimental rats’24h Urine protein evacuation, Which had a significant statistic sense in comparing withdiabetic model group (P<0.05). The results indicated that HQWM Mixture was beneficial forregulating glucose and lipid metabolism, decreasing Urine proteinand excretion improvingrenal function. Surpass positive control therapeutic effect especially in regulating glucose,lipid metabolism and improving renal function. But it didn’t as good as positive control indepress 24h Urine protein, have no statistically significant (P>0.05). The changes of renaltissue morphology in experimental diabetic rats were observed by optical microscope , theconsequence showed that the model group Glomerular mesangium diffuse widen ,extracellular matrix (ECM) increased, basement membrane became thicken.The above-mentioned pathology changes relieved significantly after the treatment by HQWM Mixture,which hinting that it can protect DN rats kidney. While Cozaar can also protect renalglomeruli.②TheconsequenceofELISAtestshowedthat DNrats secretedandsynthesised LN andtypeⅣcollagen increasingly, which revealingthe kidneyof DN rats damaged at some level.HQWM Mixture and Cozaar can both degrade LN and typeⅣcollagen contents,HQWMMixture group have an advantage effect in reducing typeⅣcollagen, while it didn’t showbetter in LN (P>0.05). We can conclude that HQWM Mixture can decrease glomerulisecreting LN and typeⅣcollagen contents, which maybe one of its mechanism of action topreventandcureDN.③Studying the content of NO in the experimental animals’renal tissue with nitratereductasetestandET-1withradioimmunoassay,theresultsshowed:thecontentofNOinDNmodelgroup’srenaltissueobviouslydecreased(P<0.01),whilethecontentofET-1increasedsignifycantly. It hinted that kidney endodermis cell lesion and imbalance of NO and ET-1.After the therapy of HQWM Mixture and Cozaar, the content of NO increased andET-1deduced obviously, which have remarkable difference from the model group (P <0.05);WecanconcludethatHQWMMixturecanblockatleastpartlyblockDNratsEClesion.④ItwasdonethatassayingtheproteinexpressionofHGF,TGF-β1 inexperimentalrats’renaltissueofeachgroupbywestern-blotmethod.Theresultsshowed:theproteinexpressionof HGF in DN model group’s renal tissue obviously decreased, while the protein expressionof TGF-β1 increased significantly comparing with sham operated group (P<0.05). After thetherapyofHQWMMixtureandCozaar,theproteinexpressionofHGFincreasedandTGF-β1 increased significantly, which can adjust the dynamic equilibrium of HGF and TGF-β1,furthermore,itcanhold-backtheECdamageofkidneyandcanpromotekidneytissuerepair.Conclusion: HQWM Mixture of“method of invigorate Qi and activate bloodcirculation, removing cumulation and stasis”, which aimed directly at“WeiXingZhengJia”,pathogenesis, canpreventionandcurediabeticnephropathyutilitily. Thepossiblemechanismof action is beneficial for regulating glucose and lipid metabolism, decreasing Urineproteinand excretion improving renal function, lessen nephridial tissuedamage, suppressormajor extracellular matrix such as LN and typeⅣcollagen, modulation EC correlated cellfactors dynamic equilibrium such as NO, ET-1, HGF, TGF-β1. This study explores themechanismofHQWMMixtureintreatingDNonamultitargetandmulti-elementbasis.更多还原