芪蓟肾康颗粒总黄酮对IgA肾病大鼠的治疗及抑制ECM机制的研究

【作者】 杨冠琦；

【导师】 张君；

【作者基本信息】 辽宁中医药大学 ， 中西医结合临床， 2013， 博士

【摘要】 目的：1.本实验采用大孔吸附树脂法纯化了芪蓟肾康颗粒总黄酮，并观察芪蓟肾康颗粒总黄酮对IgA肾病大鼠尿红细胞计数、24小时尿蛋白定量、肾脏病理改变的影响，证明此方治疗由口服牛血清白蛋白所致实验性IgA肾病的有效性；2.采用血管紧张素Ⅱ作为刺激因子，诱导体外培养的大鼠肾小球系膜细胞过度分泌纤维连接蛋白和IV型胶原，使细胞外基质发生积聚。应用血清药理学方法，选择不同的时间点，通过观察芪蓟肾康颗粒总黄酮药理血清对体外培养的肾小球系膜细胞纤维连接蛋白和IV型胶原的影响，探讨芪蓟肾康颗粒总黄酮治疗免疫损伤性肾小球疾病的作用靶点。3.通过检测体外培养的肾小球系膜细胞信号传导通路丝裂原活化蛋白激酶以及核因子-kB的表达，探讨其保护肾脏，治疗免疫损伤性肾小球疾病的作用机制。并为探寻芪蓟肾康颗粒治疗肾小球疾病提供可靠的实验数据，为今后指导临床提供新的思路和方法。材料与方法：1采用大孔吸附树脂法纯化芪蓟肾康颗粒总黄酮：芪蓟肾康颗粒上样，吸附率为1BV·h-1，用2BV水量、3BV的30%乙醇、4BV的50%乙醇洗脱，洗脱流速4BV·h-1，合并30%乙醇和50%乙醇洗脱液，浓缩，即为纯化的总黄酮。纯化后总黄酮含量提高到76%。2采用口服牛血清白蛋白复制实验性IgA肾病大鼠模型及分组治疗：口服免疫原牛血清白蛋白（BSA）剂量较常用剂量增加1倍，为400mg/kg，隔日1次灌胃，四氯化碳（CCl4）注射方式由既往的腹腔注射改为皮下注射，用诱导肝纤维化1/3的剂量（皮下注射蓖麻油0.5mL+CCl40.10mL，每周1次，持续9周），并联合运用脂多糖（LPS），第6，8周予以0.05mg尾静脉注射复制模型。SPF级SD大鼠40只，体重180g-220g，雌雄各半。将大鼠按体重随机分为5组，分别为：正常组、模型组、芪蓟肾康颗粒总黄酮高、芪蓟肾康颗粒总黄酮低剂量组、替米沙坦组，每组8只。正常组、模型组给予生理盐水1ml/100g（体重）每日一次灌胃，芪蓟肾康颗粒总黄酮高、低剂量组分别按生药量60g·kg-1·d、30g·kg-1·d灌胃，替米沙坦组按8mg·kg-1·d灌胃，每日一次。于实验第12周末次给药后，收集标本，测定各项指标。3应用血清药理学方法，制取正常大鼠、芪蓟肾康颗粒总黄酮高、低剂量组的药理血清芪蓟肾康颗粒总黄酮高、低剂量组按生药量30g·kg-1·d、15g·kg-1·d灌胃，每天一次；正常组灌服等量的生理盐水。连续灌胃5天，末次给药1小时后，腹主动脉取血。血液使用离心机，1500rpm离心5分钟，吸取上清液。经56℃水浴，30min灭活处理。用0.22μm的滤器过滤除菌，置-20℃保存备用。4采用血管紧张素Ⅱ作为刺激因子，诱导体外培养的大鼠肾小球系膜细胞，使其发生细胞外基质的积聚。将肾小球系膜细胞接种于25cm2培养瓶中，加入含10%FBS的DMEM培养液，放置于37℃、5%CO2孵箱中进行培养。约2-3天换一次液，待细胞达到80%-90%融合后，按2×104个·孔-1的浓度接种于24孔培养板，每组设置5个复孔。用含0.5%FBS的DMEM培养24小时后，更换含10%药理血清DMEM培养液继续培养24h、48h、72h，分别在不同时间点收集细胞待测。5采用终点法测定IgA肾病大鼠24小时尿蛋白定量末次给药后将大鼠置于代谢笼中单笼饲养(禁食，不禁水)，用代谢笼收集24小时大鼠尿液。24小时候后准确记录每只大鼠24小时的总尿量，并留取尿样5ml，以备测定尿红细胞计数和24小时尿蛋白定量。实验条件：温度：37℃、波长：600nm、光径：1.0cm、吸光度范围：0-2A、反应时间：5min、样品：试剂=1:604。6肾脏组织病理学评价指标将病变的肾组织使用20％甲醛固定，常规石蜡包埋切片，经HE染色，观察IgA肾病病理改变。7免疫荧光染色用直接法进行染色，取大鼠肾皮质常规冰冻切片3μm，用电吹风干燥，切片经丙酮固定5min，PBS液冲洗3次，每次5min，滴加羊抗鼠免疫荧光IgA、IgM和C31：10稀释荧光标记(异硫氰的荧光素标记)的抗体于组织切片上，置温度37℃中孵育45min，取出切片置于PBS液中洗3次，每次5min，取出切片用缓冲液甘油封片。8应用免疫酶联吸附法测定纤维粘连蛋白、IV型胶原的分泌量，丝裂原活化蛋白激酶以及核因子-kB的表达。（1）分别设置空白孔、标准品孔、待测样品孔。在酶标包被板上标准空中加入标准品50μl；在酶标包被板上待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl轻轻晃动均匀，37℃温育30分钟。（2）弃去液体，甩干，每孔加满稀释后洗涤液，震荡30秒，甩去洗涤液，用吸水纸拍干。重复5次，拍干。（3）每孔加入酶标试剂50μl，空白孔除外。轻轻晃动均匀，37℃温育30分钟。（4）弃去液体，甩干，每孔加满稀释后洗涤液，震荡30秒，甩去洗涤液，用吸水纸拍干。重复5次，拍干。（5）每孔先加入显色剂A50μl，再加入显色剂B50μl，轻轻晃动均匀，37℃避光显色10分钟。（6）取出酶标板，每孔加入终止液50μl，终止反应（此时蓝色立转黄色）。（7）测定：以空白孔调零，在450nm波长下测量各孔的吸光度值（OD值）。测定在加终止液后15分钟内进行。（8）根据标准品的浓度及对应的OD值计算样品浓度。结果：1.与正常组（26.30±10.64个/μl，168.38±55.71mg/24小时）比较，模型组大鼠的尿红细胞计数（113.57±32.5个/μl）和24小时尿蛋白排出量（593.51±114.01mg/24小时）显著高于正常组，p＜0.05；经芪蓟肾康颗粒总黄酮高、低剂量和替米沙坦治疗5周后，各治疗组大鼠的尿红细胞计数（高剂量组48.42±22.27个/μl，低剂量组51.38±25.94个/μl，替米沙坦组59.15±11.01个/μl）和24小时尿蛋白排出量（高剂量组286.46±134.33mg/24小时，低剂量组358.43±167.79mg/24小时，替米沙坦组263.91±126.42mg/24小时）显著降低，与模型组比较均有统计学差异（p＜0.05），各治疗组间比较无统计学差异。结果提示，芪蓟肾康颗粒总黄酮能有效的减轻IgA肾病大鼠血尿和蛋白尿的症状。2．肾脏组织病理形态学观察：正常组肾小球结构正常，模型组可见肾小球肿胀、体积增大及重度弥漫性系膜增生，染色显示有部分肾小球呈分叶状，系膜细胞增多，系膜基质增宽。而芪蓟肾康颗粒总黄酮高剂量组系膜增生较轻。芪蓟肾康颗粒总黄酮低剂量组肾小球肿胀，中度系膜增生。替米沙坦组肾小球肿胀较轻，伴中、轻度系膜增生。3.免疫荧光检查示：正常组肾小球系膜区IgA、IgG、IgM和C3均呈阴性。模型组全部大鼠肾小球内均有IgA沉积,其强度为++～+++，主要在肾小球系膜区呈颗粒状沉积，个别还波及毛细血管壁并伴有IgG、IgM和C3沉积。治疗后各组IgA沉积荧光强度均有不同程度的减弱，尤其是芪蓟肾康颗粒总黄酮高剂量组效果更显著，部分IgA荧光强度在±～-。4.正常组大鼠肾小球系膜细胞分泌了少量的Ⅳ胶原和纤维粘连蛋白，表明在正常情况下，体外培养的肾小球系膜细胞具有分泌Ⅳ胶原和纤维粘连蛋白的功能。经血管紧张素Ⅱ刺激后，Ⅳ胶原和纤维粘连蛋白的分泌量显著增加，约为正常组的2倍，与正常组比较有显著性差异（p＜0.01），提示血管紧张素Ⅱ能明显促进系膜细胞分泌Ⅳ胶原。芪蓟肾康颗粒总黄酮高、低剂量组在24小时、48小时、72小时不同时间点，Ⅳ胶原和纤维粘连蛋白的含量均显著降低，与血管紧张素Ⅱ组比较有显著差异（p＜0.01），并且呈现出一定的量效关系。但各治疗组间比较无统计学差异。芪蓟肾康颗粒总黄酮抑制细胞外基质积聚的作用从24小时开始已经显现，并延续至72小时。5.血管紧张素Ⅱ激活了丝裂原活化蛋白激酶以及核因子-kB的信号转导通路，丝裂原活化蛋白激酶（3.365±0.265）以及核因子-kB（3.355±0.265）的含量与正常组（MAPK,1.805±0.065, NF-kB1.660±0.100）比较有统计学差异，（p＜0.01）。给予芪蓟肾康颗粒总黄酮提取物和氯沙坦干预48小时后，两条通路的信号转导受到了抑制，二者（MAPK高剂量组：2.590±0.150，低剂量组：2.465±0.285，氯沙坦组：2.215±0.015；NF-kB高剂量组：2.435±0.165，低剂量组：2.450±0.160，氯沙坦组：1.995±0.095）的含量显著降低，同时纤维粘连蛋白和Ⅳ型胶原的含量也显著降低，与血管紧张素Ⅱ组比较均有统计学意义，p＜0.01，虽然各治疗组间比较无统计学差异，但是氯沙坦组的作用要稍优于芪蓟肾康颗粒总黄酮高、低剂量组。结论：1.芪蓟肾康颗粒总黄酮能效地降低IgA肾病大鼠尿红细胞计数，减少24小时尿蛋白的排出量，减轻肾脏病理改变，减少免疫复合物的沉积。证实了芪蓟肾康颗粒总黄酮对实验性IgA肾病具有一定的治疗作用。2.芪蓟肾康颗粒总黄酮可以抑制AngⅡ诱导的肾小球系膜细胞细胞外基质—IV型胶原、纤维粘连蛋白的过度表达，通过抑制细胞外基质的过度表达来延缓免疫损伤性肾小球疾病的发展，明确了芪蓟肾康颗粒总黄酮的作用靶点。也为芪蓟肾康颗粒应用于临床治疗肾小球疾病提供了实验数据。3.芪蓟肾康颗粒总黄酮有效的抑制了丝裂原活化蛋白激酶和核因子-kB两条信号转导通路的转导，减少细胞外基质的过度积聚，减轻肾小球的损伤，从而防止免疫损伤性肾小球疾病的进一步发展，对肾脏起到保护作用。更多还原

【Abstract】 Purpose：This experiment use macroporous purification Qiji shenkang granule extract flavonesadsorption resin method, and to observe the effects of Qiji shenkang granule flavonoidsextracts on urine red blood cell count、24hour urinary protein quantitative、pathologicalchanges in kidney and renal immunofluorescence examination in rats with IgA nephropathy,proving the Qiji shenkang granule can treat the rats with IgA nephropathy.The angiotensin IIas a stimulating factor, rat glomerular mesangial cells cultured in vitro, induced excessivesecretion of fibronectin and collagen type IV, leading to the extracellular matrix accumulationoccurs. Choose a different time point, by observing Qiji shenkang granule effect on thesecretion of fibronectin and collagen type IV, to investigate the role of the target by Qijishenkang granule of total flavonoids extract on glomerular disease. The signal transductionpathway through glomerulus mesangial cell in vitro, to detect the expression of activatedmitogen activated protein kinase and nuclear factor kappa b, and to explore the role ofprotecting kidney and the mechanism of prevention and treatment of glomerular diseases.And to provide reliable experimental data for the treatment of diseases of glomerular by Qijishenkang granule, in order to guidance clinical provide new ideas and methods in future.Material and method：1Using purification technology of total flavonoids from Qiji shenkang granule bymacroporous resinThe sample of Qi ji shen kang granule was passed through the column with flow rate of1BV·h-1，eluted by2BV water，3BV30%ethanol，4BV50%ethanol respectively，elutionflow rate was2BV·h-1.Combined30%and50%ethanol elutes were concentrated to yield thepurification of total flavonoids. The purity of total flavonoids was up to76%.2. The use of oral bovine serum albumin replication of experimental IgA nephropathy modelrats and the treatment groupThe dose of oral immunogen bovine serum albumin (BSA) increased one times (400mg·kg-1every other day), the method of injecting carbon tetrachloride CCl4was subcutaneousinjection instead of intraperitoneal injection, which dose was one third of content to induce hepatic fibrosis (benne oil0.5ml once a week and CCl40.10ml once a week, continue9weeks). Combined with0.05mg lipopolysaccharide once a everyday, at the sixth and ninthweek once.forty SD rats (specific pathogen free), in which the half was male and the other half wasfemale and200±20g body weight. The rats were randomly divided into five groups, namely:the control group, model group, the Qiji shenkang granule total flavonoids high does group,the Qiji shenkang granule total flavonoids low does group.the telmisartan group. N=8.Thenormal group, model group were given normal saline1ml/100g (weight) once daily. the Qijishenkang granule total flavonoids high does group was treated by60g·kg-1of Qiji shenkanggranule total flavonoids everyday and Qiji shenkang granule total flavonoids low does groupby30g·kg-1of Qiji shenkang granule total flavonoids everyday. The telmisartan group wastreated by8mg·kg-1of telmisartan everyday. In the twelfth week, after the last administration，collection of samples, determination all kinds of indexs.3. Through the pharmacology of serum method,we got the control group，the Qiji shenkanggranule total flavonoids high does group, the Qiji shenkang granule total flavonoids low doesgroups’ pharmacological serum．The Qiji shenkang granule total flavonoids high does group was treated by30g·kg-1·d ofQiji shenkang granule total flavonoids and Qiji shenkang granule total flavonoids low doesgroup by15g·kg-1·d of Qiji shenkang granule total flavonoids. The normal group was givennormal saline1ml/100g (weight) once daily for five days.1hour after the last administration,blood was taken from the abdominal aorta. Blood using a centrifuge, condition is1500rpmcentrifugation for5minutes, taking supernatant. After56℃water bath,30min inactivation.By filter sterilization of0.22μ m,-20℃preservation reserve.4. The angiotensin II as a stimulating factor, rat glomerular mesangial cells in vitro, make itsproduce extracellular matrix accumulation.The glomerular mesangial cells were cultured in25cm2culture flask, with mediumcontained10%FBS DMEM. The cells were incubated at37℃in humidified5%CO2-95%air. The cell medium was left untouched for2-3days and changed every other day untilconfluence. Glomerulus mesangial cell were grown to80%-90%confluence, washed oncewith serum-free DMEM.According to the concentration of2x104cells/hole, MCs cells was seeded in24well plates, each set of5hole. Cells were made arrested in0.5%FBS DMEM for24h to synchronize the cell growth. After this time period, the media was changed to DMEMcontaining10%pharmacological serum for24h、48h、72h, Cells were collected at differenttime points to be measured。5.24hour urinary protein quantitative was determined by end-point method。After the last administration, rats were placed in metabolic cages with single cagefeeding (fasting, but the water), collected24hours urine of rats in metabolic cage.24hourslater, accurate records of each rat urinary total24hours of content, and collected the urine5ml, for determination of urine red blood cell count and24hour urinary protein quantitative.Experimental conditions: Temperature:37°C, wavelength:600nm, optical diameter:1.0cmthe absorbance range to:0-2A, reaction time:5min Sample: Reagents=1:604.6. Pathological evaluation of renal tissueThe renal lesions using20%formalin-fixed, paraffin embedded sections, and throughHE, to object pathological changes of IgA nephropathy.7. Immunofluorescence stainingA direct method for dyeing, and renal cortex of rats with routine frozen sections of3μ m,with a hair dryer drying, sections by acetone fixed5min, PBS liquid rinse3times, each time5min, drop of sheep anti-mouse immune fluorescence IgA, IgM and C31:10dilutionfluorescence labeling (labeled with fluorescein isothiocyanate.) antibody in tissue section, thetemperature of37℃incubating45min, remove the slices in PBS solution and wash3times,each time5min, remove the slices with buffer glycerol.8. The secretion of fibronectin、collagen type Ⅳand the expression of mitogen activatedprotein kinase、nuclear factor-kappa b were tested by enzyme-linked immunosorbentassay(ELISA)．（1）add the standard: according to the order on board hole concentration in50μlstandard product configured. add samples: a blank hole（blank respectively controlled holewithout samples, affinity,and standard reagents, the rest of the enzyme each step for the sameoperation）, sample hole. The enzymes standard bag was board to be added sample and then40μl add meat10μl Sample billogical. And when the Sample billogical and in a sample ofenzyme panels at the bottom of the hole, try not to touch the hole wall, gently shaking blending. Incubate: After closing plate with Closure plate membrane, incubate for30min at37℃.（2）Configurate liquid:30-fold wash solution diluted30-fold with distilled water andreserve. washing: Uncover Closure plate membrane dry by swing, add washing buffer toevery well, still30s then drain, repeat5times, dry by pat.（3）add enzyme: Add ELISAreagents50μl to each well, except the blank well.（4）Uncover Closure plate membrane dryby swing, add washing buffer to every well, still30s then drain, repeat5times, dry by pat（.5）color: add color reagent A50μl and color B50μl to each well. Gently mix, incubate for15min at37℃.（6）Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction（the blue color change to yellow color Immediately）.（7）assay: take blank well as zero,measure the optical densit（OD）at450nm after Adding Stop Solution and within15min.（8）To calculate the sample concentration according to the standard concentration and thecorresponding OD.Results：1Compared with the normal group,(the urine red blood cell count:26.30±10.64/μ L,24hour urine protein:168.38±55.71mg/24hours), the urine red blood cell count of modelgroup rats (113.57±32.5/μ L) and24hour urine protein excretion (593.51±114.01mg/24hours) was significantly higher than that in normal group, p＜0.05; The Qiji shenkang granuletotal flavonoids high, low dose and telmisartan treatment after5weeks, urine red blood cellcount of rats in all treatment groups (the high dose group48.42±22.27/μ L, the low dosegroup51.38±25.94/μ L, the telmisartan group59.15±11.01/μ L) and24hour urine proteinexcretion (the high dose group was286.46±134.33mg/24hours, the low dosage group was358.43±167.79mg/24hours, the telmisartan group263.91±126.42mg/24hours) decreasedsignificantly, which has statistical difference compared with model group (p <0.05), thetreatment group showed no statistical difference. The results suggest that, Qiji Shenkanggranule total flavonoids can effectively improve the rat IgA nephropathy hematuria andproteinuria symptoms.2. Pathological observation: normal group kidney glomerular structure normal, model groupshowed glomerular swelling, enlargement and severe diffuse mesangial proliferation,glomerular staining showed some lobulated, mesangial cells, mesangial matrix increased. Total flavonoids of Qiji Shenkang granula high dose group mesangial proliferative lighter.Total flavonoids of Qiji Shenkang granule low dose group glomerular swelling, moderatemesangial proliferation. Telmisartan group glomerular swelling and lighter, with moderate,mild mesangial proliferation.3. Immunofluorescence examination showed: normal group glomerular mesangial areas IgA,IgG, IgM and C3were negative. The model group rats were all glomerular IgA deposition, itsstrength is+++, mainly in the mesangium granular deposition, the individual also affectedthe capillary wall and associated with IgG, IgM and C3deposition. Each IgA depositiondecreased fluorescence intensity of treatment in different degree, especially the totalflavonoids of Qiji Shenkang granule effect of high dose group was more significant, part ofthe IgA fluorescence intensity in±~-.4. The normal rat glomerular mesangial cells can secrete a small amount of collagen type IVand fibronectin, show that under normal circumstances, glomerular mesangial cells in vitrocan secrete collagen type IV and fibronectin. After the angiotensin II stimulation, the ratglomerular mesangial cells secretion of collagen IV and fibronectin increased significantly,about2times the normal group, there was significant difference compared with the normalgroup (p <0.01), suggesting that angiotensin II can obviously promote mesangial cellssecrete collagen type IV and fibronectin. At different time points, the Qiji Shenkang granuletotal flavone high, low dose group the content of collagen type IV and fibronectin weresignificantly reduced in24hours,48hours,72hours, compared with angiotensin II group，there were significant differences between them (p <0.01), and showing a certaindose-effect relationship. But the treatment group showed no statistical difference. Inhibitionof extracellular matrix accumulation effect by Qiji shenkang granule total flavonoids hasemerged from the24hours, and continued to72hours.5. Ang II activates mitogen-activated protein kinase and nuclear nuclear factor kappa b signaltransduction pathway. The contents of mitogen-activated protein kinase (3.365±0.265)andnuclear factor kappa b(3.355±0.265)compared with the normal group(mitogen-activatedprotein kinase1.805±0.065, nuclear factor kappa b1.660±0.100), there were significantdifferences (p <0.01). Given the Qiji shenkang granule total flavonoids and losartan intervention for48hours, two signal transduction pathways were inhibited in glomerulusmesangial cell in vitro. The content of mitogen-activated protein kinase(the high group2.590±0.150, the low group2.465±0.285, the losartan group2.215±0.015) and nuclearnuclear factor kappa b(the high group2.435±0.165, the low group2.450±0.160, the losartangroup1.995±0.095) were significantly decreased, at the same time content of fibronectin andcollagen type IV was significantly lower. Compare with the angiotensin II group, all of themwere statistically significant, p <0.01. Although every treatment group showed no statisticaldifference between them, but the effect of losartan group is better than the Qiji Shenkanggranule total flavonoids high and low dose group.Conclusion：1. Qiji shenkang granule total flavonoids can effectively reduce urinary red blood cell countin and the excretion of urine protein in24hours in rats with IgA nephropathy. Reduce therenal pathological changes, reduce the deposition of immune factors. Confirm the QijiShenkang granule total flavonoids have a certain therapeutic effect on experimental IgAnephropathy.2. Qiji Shenkang granule total flavonoids can inhibit Ang II induced glomerular mesangialcell extracellular matrix–fibronectin and collagen type IV expression. Through inhibitexcessive expression of extracellular matrix to delay the occurrence and development of renaldisease. The target was clear about Qiji Shenkang granule total flavonoids. Also providingexperimental data, the aim is useing Qiji Shenkang granule in clinical treatment of glomerulardisease.3. Both of the mitogen-activated protein kinase and nuclear factor kappa b two signaltransduction pathways were effectively inhibited by Qiji Shenkang granule total flavonoids,and it prevent the excessive accumulation of extracellular matrix, meanwhile reduce theglomerular injury. Play a role in renal protection. Qiji Shenkang granule total flavonoidsprevent the occurrence of glomerular disease and further development of glomerular disease.更多还原