黄连总生物碱的提取及其对实验性糖尿病肾病大鼠的保护作用及机制的初步研究
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摘要
糖尿病是慢性进行性内分泌及代谢性疾病,其病情重,发病率高,并伴有多种器官如眼、肾、心、脑等并发症。糖尿病肾病(Diabetic nephropathy,DN)是糖尿病常见的慢性并发症之一,由糖尿病微血管病变所引起。DN确切的发病机制至今未阐明,目前归纳为以下几方面:肾小球血流动力学改变,高血糖相关的生化代谢异常、细胞因子及生长因子的作用等。
肾小球细胞外基质(Extracellular matrix,ECM)沉积是DN主要的病理改变,ECM主要由Ⅳ型胶原、纤维连接蛋白等组成,其生理状态下处于一种快速更新代谢的动态平衡过程中,该过程的失衡是导致肾小球组织结构破坏乃至肾小球硬化的关键性病理过程,最终导致DN的发生。国内外研究发现,DN早期阶段存在肾小球内皮细胞功能异常,肾小球足突细胞及活化的系膜细胞可表达血管内皮生长因子(Vascular endothelialgrowth factor,VEGF),高糖环境中VEGF基因表达升高,而VEGF水平与尿微量白蛋白呈高度相关,并与其他因素一起参与肾小球硬化。结缔组织生长因子(Connecturetissue growth factor,CTGF)具有促进成纤维细胞活化以及ECM产生、积聚等功能,参与糖尿病肾病发生发展的诸多环节,而转化生长因子-β(Transforming growthfactor,TGF-β)是诱导CTGF生成的最重要的因子,TGF-β通过上调蛋白的抑制因子如纤溶酶原激活物抑制因子(PAZ)和金属蛋白酶组织抑制因子(Tissue inhibitor of matrixmetalloproteinases,TIMP)的合成,下调基质降解蛋白酶如胶原酶的合成,阻止新合成的细胞外基质降解,促进基质分子细胞表面受体整合素表达,增强细胞基质相互作用。同时DN与体内山梨醇的聚积有关,多元醇通路的关键限速酶-醛糖还原酶(Aldosereductase,AR)的活性升高是DN发生、发展的重要原因。丝氨酸/苏氨酸激酶蛋白激酶B(PKB/Akt)是原癌基因c-Akt的表达产物,在控制细胞存活,葡萄糖代谢及蛋白质合成等重要细胞功能中起关键作用。Akt作为胰岛素受体的下游分子对胰岛素代谢,激素信号转导和葡萄糖转运起重要作用.
为阐明DN的发病机制,筛选出有效的药物,建立较为理想的糖尿病肾病动物模型显得尤为重要。目前国内外所进行的糖尿病及其并发症实验动物研究多采用有糖尿病遗传倾向的近交系纯种动物,但是这些动物的来源少,饲养条件苛刻,并且其特点是遗传因素在发病过程中占主导地位,与临床上的发病特征不完全吻合。有研究采用高糖高脂饲料、链脲佐菌素(Streptozotocin,STZ)、单肾切除、盐酸多柔比星(Doxorubicinhydrochloride)等因素建立糖尿病肾病动物模型,但对各因素组合对模型建立无系统性比较。我们对这几种因素的糖尿病肾病动物模型建立进行了比较,为建立较理想的糖尿病肾病动物模型提供实验支持和理论依据。
中药复方及提取物治疗DN的实验研究已有较多报道,尝试从分子生物学及基因水平对DN的保护作用探索,也取得一些初步的成果。黄连来源于植物黄连(Coptis chinensisFranch)的根茎,黄连含有多种生物碱,已经分离出来的生物碱主要有盐酸小檗碱、巴马汀、黄连碱、甲基黄连碱、药根碱、木兰碱等。目前针对盐酸小檗碱和巴马汀的研究较多,主要停留在观察其对各种糖尿病症状的改善程度上,具体的分子机制,虽然已经有不少人做了一些工作,但还处在刚刚开始的阶段,且推测居多。对于其他生物碱的研究少见报道。黄连生物碱作用靶点和具体作用机制有待深入探讨。
基于以上分析,结合国内外研究进展,本课题通过体内实验观察对黄连总生物碱保护糖尿病肾病模型大鼠的作用及其机制进行了初步研究,同时通过体外实验观察黄连总生物碱对高糖刺激的肾小球系膜细胞的作用及其机制进行了初步研究。采用乙醇回流提取并减压浓缩得到黄连总生物碱浸膏。同时采用高糖高脂饲料、链脲佐菌素、单肾切除、盐酸多柔比星等因素交叉组合建立糖尿病肾病动物模型,优选出最佳方案。按照优选出的方案建立糖尿病肾病模型大鼠,设立黄连总生物碱低、中、高剂量组,以盐酸二甲双胍为阳性对照,初步研究了黄连总生物碱对糖尿病肾病大鼠的保护作用及机制。采用原代培养的大鼠。肾小球系膜细胞,以盐酸二甲双胍为对照,在体外观察黄连总生物碱对高糖刺激的肾小球系膜细胞增殖的作用及对GMC中TGF-β1、TIMP-1及MMP-9表达的影响。
本课题主要实验结果如下:
1、用乙醇回流提取黄连总生物碱,80%乙醇回流提取3次,合并滤液,减压浓缩蒸干溶剂,3.8g生药得到1克浸膏。用离子对萃取,紫外分光光度法测定浸膏中黄连总生物碱的百分含量为31.49±1.41%;HPLC法测得黄连浸膏中含盐酸小檗碱16.42±1.6%,生药中含盐酸小檗碱4.37±0.4%。
2、成年雄性Sprague-Dawley大鼠分为五组,A为正常组:普通饲料喂养。B为高糖高脂饲料组:高糖高脂饲料喂养。C为高糖高脂饲料+STZ组:高糖高脂饲料喂养六周后,腹腔注射STZ。D为高糖高脂饲料+盐酸多柔比星+STZ组:高糖高脂饲料喂养四周后,腹腔注射盐酸多柔比星,2周后注射STZ。E为高糖高脂饲料+单肾切除+STZ组:高糖高脂饲料喂养四周后,单肾切除,2周后注射STZ。结果发现E组高糖高脂饲料+单肾切除+注射STZ建模的结果是比较理想的建模方法,大鼠明显多食、多饮、消瘦、血糖、血脂升高,尿微量白蛋白、肾小球滤过率及活性氧指标不仅与正常组有显著差异,并且与前几个模型组比较也都有显著性差异:肾/体重比明显增大;醛糖还原酶升高显著。肾脏病理学显示肾小球病变较严重,肾小球系膜增宽,肾小管内皮细胞排列紊乱,空泡变性。肾脏超微病理显示肾小球体积明显增大,基底膜局灶性增厚,系膜区增宽,系膜细胞增生,基质增多,肾小管上皮细胞足突融合,线粒体增多,线粒体肿胀。
3、初步研究发现黄连总生物碱可以降低糖尿病肾病模型SD大鼠的血糖、血脂。本研究表明,大鼠给予高热量的饲料一个月诱发胰岛素抵抗,单肾切除两周后腹腔注射小剂量STZ造糖尿病肾病模型,大鼠造模后血糖升高,体重较正常组增长减慢,肾/体重比显著增大,8周后出现尿微量白蛋白,12周后模型组大鼠血胰岛素、GFR升高,UREA、CREA、TG、糖化血红蛋白、尿肌酐与正常组比较有显著性差异。在氧自由基代谢方面,模型组B组MDA显著增加,SOD显著减小。大鼠造模后分别给予黄连总生物碱高、中、低剂量,盐酸二甲双胍,对大鼠上述指标均有不同程度的改善,其中,以黄连总生物碱中剂量的改善最为显著,推测黄连总生物碱具有胰岛素肽类作用,促进组织细胞再生的作用,从而促进胰岛β—细胞的再生及具有清除自由基作用的SOD等相关,但黄连总生物碱高剂量并不能降低血胰岛素,提示黄连总生物碱可能具有促进胰岛素分泌作用。黄连总生物碱各剂量组均能显著降低大鼠红细胞中AR活性,提示黄连总生物碱可能通过降低大鼠红细胞中AR活性从而保护肾小球的结构和功能。
4、经过HE及PAS染色光镜下观察,大鼠建模后肾小球体积增大,丝球体系膜区增宽,系膜细胞增生,肾小管内皮细胞空泡变性。系膜区/丝球体增大,电镜下观察,大鼠建模后基底膜增生,系膜区增宽,上皮细胞足突融合,线粒体肿胀,嵴稀疏。经药物治疗后,上述病理学改变有所改善但没有完全逆转,提示大鼠肾脏结构一旦被破坏,用药物并不能完全改善,恢复到正常状态。
5、大鼠建模及用药后肾组织切片免疫组化结果显示VEGF、TGF-β_1、CTGF平均光密度比正常对照组增加,有显著性差异,用药后均值较模型组有所下降,但与正常组相比仍有差异。我们应用RT-PCR方法对黄连总生物碱高、中、低剂量及盐酸二甲双胍对TGF-β_1、TIMP-1、MMP-2mRNA在肾组织中表达的影响分别进行了观察。结果显示,模型组TGF-β_1及TIMP-1 mRNA表达上调,用药后均值较模型组有所下降;模型组MMP-2 mRNA表达下调,用药后均值较模型组有所上升。同时应用免疫印迹方法对黄连总生物碱高、中、低剂量对MMP-9在肾组织中表达的影响进行了观察。结果显示,模型组MMP-9表达下调,用药后均值升高,提示黄连总生物碱有可能是通过影响肾组织中多种细胞因子及基质酶的表达从而起到对糖尿病肾病大鼠的保护作用,但仍然不能恢复到正常水平。
同时我们应用免疫印迹方法对黄连总生物碱高、中、低剂量对MMP-9、Akt、p-Akt在肾组织中表达的影响进行了观察。结果显示,模型组MMP-9表达下调,用药后均值较模型组有所上升。Akt无显著性差异,模型组p-Akt表达上调,用药后均值较模型组下降,提示黄连总生物碱可能影响到肾组织中PI3-K通路从而调节体内的葡萄糖代谢而起到对糖尿病肾病的保护作用。
6、肾小球系膜细胞作为糖尿病肾病致病因子作用的主要靶细胞,研究它在高糖作用下生物学活性的改变,对探讨糖尿病肾病的发病机制具有重要意义。在高糖环境中培养的肾小球系膜细胞已经成为很好的研究糖尿病肾病的离体模型。我们观察并鉴定了原代培养的SD大鼠肾小球系膜细胞的形态,并且用MTT法检测了黄连总生物碱对高糖刺激的GMC增殖的影响,发现GMC的增殖能被黄连总生物碱显著抑制。
初步观察到黄连总生物碱对高糖刺激的系膜细胞表达的TGF-β_1、TIMP-1有一定的下调作用,而对MMP-9有上调作用,体外研究提示黄连总生物碱可能通过影响GMC的TGF-β_1、TIMP-1和MMP-9的表达而起到对系膜细胞的保护作用。
Diabetes mellitus (DM) is a progressive chronic endocrinic and metabolic disease with high morbidity and severity, which usually affects eyes, kidneys, heart and brain and other organs. Diabetic nephropathy (DN), caused by diabetic microvascular lesions, is one of the chronic complications of DM. The exact mechanism of DN is not clear yet. Currently, the blood dynamic changes, hyperglydemic biochemic and metabolic abnormity, effect of cell factor and growth factors and other reasons may play a role in this procedure.
The main pathological changes of DN are the deposit of glomerular extrcellular matrix (ECM). ECM is composed mainly of type IV collagen and fibronectin,which keep metabolic balance by rapid refresh .When the balance is broken,it will lead to glomerular lesion and even glomerular cirrhosis, and causes DN by the end. Researchers indicated that functional disorders of endothelial cells, secretion of vascular endothelial growth factor (VEGF) by the glomerular podocytic cells and the activativation of mesangial cells, occur during the early period of DN. Expression of VEGF gene elevates under hyperglycemic condition. VEGF's level highly correlates with uremia microprotein and participates in the procedure of nephrosclerosis. Connecture tissue growth factor (CTGF) plays a role in activativation of the fibroblasts and generation and deposit of ECM, and participates in many courses of DN, and mainly induced by transforming growth factorβ(TGF-β) .TGF-βcan upregulates the protein inhibitors such as PAZ and TEMP and also downregulates the matrix degradation enzymes, which suppresses the degradation of newly formed extra cellular matrix, promotes the expression of the matrix surface integrin and strengthen the interaction of matrix. The aggregation of sorbitol as well correlates with DN. Activation of Aldose Reductase (AR),the key rate-limiting enzyme of polyalcohol pathway,plays an important role in the initiation and development of DN. The oncogen c-akt's product PKB/Akt has a crucial effect on cell surviving, glucose metabolism, protein synthesis and so on. AKT, as the downstream molecule of insulin receptor, has an important effect on the insulin metabolism, hormone signal conduction and glucose transshipment.
It is the most important thing to establish the ideal DN animal model to illuminate the pathologic mechanisms and choose effective drugs for DN. The heredity DM pure inbred strain animals are the most common choose domesticly or abroadly. But those animals are quite rare and hard to feed. What's more, the pathogenesis of these models is mainly caused by heredity, which does not meet with the clinic characteristic. Feeding with high-calorie food and Streptozotocin, mononephoroctomy, injecting with Doxorubicin hydrochloride and other such kind of methods are used to establish DN model, but the systemic comparison between these methods is not available yet. We compared the models by these methods and find an optimal way to establish DN model.
Compound recipes or extracts of traditional Chinese medicines have being used to treat DN extensively and some of them seem to show effects on DN' s prevention and treatment in molecular biochemic and genetic level. Rhizoma Coptidis is exacted from the rhizome of Coptis chinensis Franch , which contains many types of alkaloids , such as Berberine,Palmatine, Coptisine,Worenine,Jatrorrhizine,Magnoflorine etc. Presently, researches on Berberine and Berberastine are more extensive, mainly on the improvement of clinic symptoms, but the molecular mechanisms are still not clear. Few researches are published on other alkaloids. The alkaloid functional targets and the functional mechanisms of Rhizoma Coptidis need to be explored.
This subject is the preliminary research on the prevention and treatment and the mechanisms of Total Alkaloids of Rhizoma Coptidis administration on DN rat model in vivo and adopt the method of ethanol circumfluence to extract and decompress to obtain the coptis alkaloid. Establishing DN animal model by cross-combination of feeding with high-calorie food and Streptozotocin, mononephoroctomy, and injecting with Adriamycin and choose the best combination,which would be used to establish DN animal model in the following studies. Grouping with low, medium, high dose of Rhizoma Coptidis and set up the positive control by Metformin Hydrochloride. We preliminary studied on protection and treatment and the mechanisms of Total Alkaloids of Rhizoma Coptidis administration on DN rat model in vivo. And we observe the function of Total Alkaloids of Rhizoma Coptidis on glomerular mesangial cells in vitro. The primary cultured glomerular mesangial cell are used to observe the fuction of Total Alkaloids of Rhizoma Coptidis on cell hyperplasia and the expression of TGF-βand TIMP-1 by hyperglycemic condition. The Metformin Hydrochloride act as control.
1. Using ethanol circumfluence 3 times, mixing the filtrate through, decompressing, condensing and vaporing the solvent, obtaining 3.8g crude drugs in per gram of extract ion. Using ion pair to extract and UV-spectrophotometry method to measure the average content of total Alkaloid of Rhizoma Coptidis were 31.4±1.41%, and HPLC method to determine the average content of berberin of its were 16.42±1.6%.
2. Adult male SD rats were divided into five groups: A is normal group: common feed. B is high-lipids and high-sucrose feeding group: high-lipids and high-sucrose feeding. C is high-lipids and high-sucrose feeding+STZ group: high-lipids and high-sucrose feeding for 6w, abdomen administration of STZ. D is high-lipids and high-sucrose feedin g+ Doxorubicin hydrochloride + STZ group: high-lipids and high-sucrose feeding for 4w, abdomen administration of Doxorubicin hydrochloride, abdomen administration of STZ 2 weeks later. E is high-lipids and high-sucrose feeding+mononephroctomy+STZ group: high-lipids and high-sucrose feeding feed for 4w, mononephroctomy, abdomen administration of STZ 2 weeks later. We concluded that E group is the ideal method that the rats appears evidence polydipsia, polyphagia, polyrexia, hypergycemia, hyperglyceremia, microprotein urine, abnormal glomerular filtration ratio and active oxygen, increase of kidney/body weight ratio, marked increase of aldose reductase . The pathology shows the glomerular destroyed, membrane widen, tubular intracellular cell disorders and vacuolization. The super mocropathology showed the glomerular volume increased, base membrane thickened and widened locally, mesangial cell proliferated, extracellular matrix increased, podocytes fused, mitochondria swoll and increased.
3.Our initial studies found that Total Alkaloids of Rhizoma Coptidis can decrease the level of blood sugar and lipid of DN rats. This research shows that feeding rats with high-lipids and high-sucrose food for 1 month induced insulin resistance, and administrating 2 weeks after mononephroctomy STZ can induce DN. The model shows hyperglycemia, elevation of body weight slowing down,high kidney/body weight ratio. After 8 weeks microprotein appeared in urine;while after 12 weeks blood insulin and GFR increase,blood urea,crea,TG,glycohemoglobin and urine creatine disordered. MDA of B group decreases significantly, SOD and all types of NOS increase significantly. low, medium, high doses of Total Alkaloids of Rhizoma Coptidis and Metformin Hydrochloride are given respectively to the models, the targets showed different improvements, and the medium dose group improved most significantly. All doses of Total Alkaloids of Rhizoma Coptidis can decrease the AR activity in rat red blood cells.
4.The HE and PAS staining of glomerular showed that the volume of the glomerules increased,membrane widen,tubular epithelial cell disarranged and vacuolized. The super micropathology shows that in the model of rats,the volume of the glomerule increased,basal membrane widened and thickened locally, mesangial cells proliferated, extracellular matrix increased, podocytes fused,mitochondria swoll and increased. After treatment with Total Alkaloids of Rhizoma Coptidis, the pathology of DN improved, but did not converse completely.
5.In this DN model,the immunohistochemistry result of the sections of organ shows the OD of VEGF、TGF-β1、CTGF increased and have significant difference. The average OD of those factors got lower than models, but didn't return to a normal level. We adopt RT-PCR to observe the effect of high,medium,and low dose of Total Alkaloid and Metformin feeding on expression of TGF-β1、TIMP-1、MMP-2 mRNA. It showed that expression of TGF-β1、TIMP-1 mRNA elevated, while those of the group treated by Total Alkaloids of Rhizoma Coptidis declined. MMP-2 mRNA of the model declined, the average of the group treatd enhanced. We adopt Western blot to observe the effect of high,medium,and low dose of Total Alkaloid and Metformin feeding on the expression of MMP-9.It showed that level of MMP-9 declined, while after being treated,the level of MMP-9 elevated. It prompted that Total Alkaloid may take effects on TGF-β1、VEGF、CTGF、MMP-9、TIMP-1 and MMP-2 , in order to protect rats from getting Diabetic nephropathy.
As the main target cell of DN, glomerular mesangial cells are of high sense in DN pathegenesis by observing its changes under the condition of hyperglycemia. The glomerular mesangial cell cultured in hyperglycemic condition is a good model in vivo. We observed and verified the forms of the primary cultured SD rat renal glomerular mesangial cells. We detected the effect of Total Alkaloids of Rhizoma Coptidis on hyperglycemic GMC and concluded that Total Alkaloids of Rhizoma Coptidis can inhibit the hyperplasia of GMC evidently.
We initially observed in a hyperglycemic circumstance, the total Alkaloids of Rhizoma Coptidis system stimulated mesangial cells to express TGF-β1 , activity of TIMP-1 declined, while that of MMP-9 elevated. Those prompt Total Alkaloids of Rhizoma Coptidis possibly upregulates the expression of TGF-β1 , MMP-9 and the TIMP-1 through affecting GMC in vitro.
肾小球细胞外基质(Extracellular matrix,ECM)沉积是DN主要的病理改变,ECM主要由Ⅳ型胶原、纤维连接蛋白等组成,其生理状态下处于一种快速更新代谢的动态平衡过程中,该过程的失衡是导致肾小球组织结构破坏乃至肾小球硬化的关键性病理过程,最终导致DN的发生。国内外研究发现,DN早期阶段存在肾小球内皮细胞功能异常,肾小球足突细胞及活化的系膜细胞可表达血管内皮生长因子(Vascular endothelialgrowth factor,VEGF),高糖环境中VEGF基因表达升高,而VEGF水平与尿微量白蛋白呈高度相关,并与其他因素一起参与肾小球硬化。结缔组织生长因子(Connecturetissue growth factor,CTGF)具有促进成纤维细胞活化以及ECM产生、积聚等功能,参与糖尿病肾病发生发展的诸多环节,而转化生长因子-β(Transforming growthfactor,TGF-β)是诱导CTGF生成的最重要的因子,TGF-β通过上调蛋白的抑制因子如纤溶酶原激活物抑制因子(PAZ)和金属蛋白酶组织抑制因子(Tissue inhibitor of matrixmetalloproteinases,TIMP)的合成,下调基质降解蛋白酶如胶原酶的合成,阻止新合成的细胞外基质降解,促进基质分子细胞表面受体整合素表达,增强细胞基质相互作用。同时DN与体内山梨醇的聚积有关,多元醇通路的关键限速酶-醛糖还原酶(Aldosereductase,AR)的活性升高是DN发生、发展的重要原因。丝氨酸/苏氨酸激酶蛋白激酶B(PKB/Akt)是原癌基因c-Akt的表达产物,在控制细胞存活,葡萄糖代谢及蛋白质合成等重要细胞功能中起关键作用。Akt作为胰岛素受体的下游分子对胰岛素代谢,激素信号转导和葡萄糖转运起重要作用.
为阐明DN的发病机制,筛选出有效的药物,建立较为理想的糖尿病肾病动物模型显得尤为重要。目前国内外所进行的糖尿病及其并发症实验动物研究多采用有糖尿病遗传倾向的近交系纯种动物,但是这些动物的来源少,饲养条件苛刻,并且其特点是遗传因素在发病过程中占主导地位,与临床上的发病特征不完全吻合。有研究采用高糖高脂饲料、链脲佐菌素(Streptozotocin,STZ)、单肾切除、盐酸多柔比星(Doxorubicinhydrochloride)等因素建立糖尿病肾病动物模型,但对各因素组合对模型建立无系统性比较。我们对这几种因素的糖尿病肾病动物模型建立进行了比较,为建立较理想的糖尿病肾病动物模型提供实验支持和理论依据。
中药复方及提取物治疗DN的实验研究已有较多报道,尝试从分子生物学及基因水平对DN的保护作用探索,也取得一些初步的成果。黄连来源于植物黄连(Coptis chinensisFranch)的根茎,黄连含有多种生物碱,已经分离出来的生物碱主要有盐酸小檗碱、巴马汀、黄连碱、甲基黄连碱、药根碱、木兰碱等。目前针对盐酸小檗碱和巴马汀的研究较多,主要停留在观察其对各种糖尿病症状的改善程度上,具体的分子机制,虽然已经有不少人做了一些工作,但还处在刚刚开始的阶段,且推测居多。对于其他生物碱的研究少见报道。黄连生物碱作用靶点和具体作用机制有待深入探讨。
基于以上分析,结合国内外研究进展,本课题通过体内实验观察对黄连总生物碱保护糖尿病肾病模型大鼠的作用及其机制进行了初步研究,同时通过体外实验观察黄连总生物碱对高糖刺激的肾小球系膜细胞的作用及其机制进行了初步研究。采用乙醇回流提取并减压浓缩得到黄连总生物碱浸膏。同时采用高糖高脂饲料、链脲佐菌素、单肾切除、盐酸多柔比星等因素交叉组合建立糖尿病肾病动物模型,优选出最佳方案。按照优选出的方案建立糖尿病肾病模型大鼠,设立黄连总生物碱低、中、高剂量组,以盐酸二甲双胍为阳性对照,初步研究了黄连总生物碱对糖尿病肾病大鼠的保护作用及机制。采用原代培养的大鼠。肾小球系膜细胞,以盐酸二甲双胍为对照,在体外观察黄连总生物碱对高糖刺激的肾小球系膜细胞增殖的作用及对GMC中TGF-β1、TIMP-1及MMP-9表达的影响。
本课题主要实验结果如下:
1、用乙醇回流提取黄连总生物碱,80%乙醇回流提取3次,合并滤液,减压浓缩蒸干溶剂,3.8g生药得到1克浸膏。用离子对萃取,紫外分光光度法测定浸膏中黄连总生物碱的百分含量为31.49±1.41%;HPLC法测得黄连浸膏中含盐酸小檗碱16.42±1.6%,生药中含盐酸小檗碱4.37±0.4%。
2、成年雄性Sprague-Dawley大鼠分为五组,A为正常组:普通饲料喂养。B为高糖高脂饲料组:高糖高脂饲料喂养。C为高糖高脂饲料+STZ组:高糖高脂饲料喂养六周后,腹腔注射STZ。D为高糖高脂饲料+盐酸多柔比星+STZ组:高糖高脂饲料喂养四周后,腹腔注射盐酸多柔比星,2周后注射STZ。E为高糖高脂饲料+单肾切除+STZ组:高糖高脂饲料喂养四周后,单肾切除,2周后注射STZ。结果发现E组高糖高脂饲料+单肾切除+注射STZ建模的结果是比较理想的建模方法,大鼠明显多食、多饮、消瘦、血糖、血脂升高,尿微量白蛋白、肾小球滤过率及活性氧指标不仅与正常组有显著差异,并且与前几个模型组比较也都有显著性差异:肾/体重比明显增大;醛糖还原酶升高显著。肾脏病理学显示肾小球病变较严重,肾小球系膜增宽,肾小管内皮细胞排列紊乱,空泡变性。肾脏超微病理显示肾小球体积明显增大,基底膜局灶性增厚,系膜区增宽,系膜细胞增生,基质增多,肾小管上皮细胞足突融合,线粒体增多,线粒体肿胀。
3、初步研究发现黄连总生物碱可以降低糖尿病肾病模型SD大鼠的血糖、血脂。本研究表明,大鼠给予高热量的饲料一个月诱发胰岛素抵抗,单肾切除两周后腹腔注射小剂量STZ造糖尿病肾病模型,大鼠造模后血糖升高,体重较正常组增长减慢,肾/体重比显著增大,8周后出现尿微量白蛋白,12周后模型组大鼠血胰岛素、GFR升高,UREA、CREA、TG、糖化血红蛋白、尿肌酐与正常组比较有显著性差异。在氧自由基代谢方面,模型组B组MDA显著增加,SOD显著减小。大鼠造模后分别给予黄连总生物碱高、中、低剂量,盐酸二甲双胍,对大鼠上述指标均有不同程度的改善,其中,以黄连总生物碱中剂量的改善最为显著,推测黄连总生物碱具有胰岛素肽类作用,促进组织细胞再生的作用,从而促进胰岛β—细胞的再生及具有清除自由基作用的SOD等相关,但黄连总生物碱高剂量并不能降低血胰岛素,提示黄连总生物碱可能具有促进胰岛素分泌作用。黄连总生物碱各剂量组均能显著降低大鼠红细胞中AR活性,提示黄连总生物碱可能通过降低大鼠红细胞中AR活性从而保护肾小球的结构和功能。
4、经过HE及PAS染色光镜下观察,大鼠建模后肾小球体积增大,丝球体系膜区增宽,系膜细胞增生,肾小管内皮细胞空泡变性。系膜区/丝球体增大,电镜下观察,大鼠建模后基底膜增生,系膜区增宽,上皮细胞足突融合,线粒体肿胀,嵴稀疏。经药物治疗后,上述病理学改变有所改善但没有完全逆转,提示大鼠肾脏结构一旦被破坏,用药物并不能完全改善,恢复到正常状态。
5、大鼠建模及用药后肾组织切片免疫组化结果显示VEGF、TGF-β_1、CTGF平均光密度比正常对照组增加,有显著性差异,用药后均值较模型组有所下降,但与正常组相比仍有差异。我们应用RT-PCR方法对黄连总生物碱高、中、低剂量及盐酸二甲双胍对TGF-β_1、TIMP-1、MMP-2mRNA在肾组织中表达的影响分别进行了观察。结果显示,模型组TGF-β_1及TIMP-1 mRNA表达上调,用药后均值较模型组有所下降;模型组MMP-2 mRNA表达下调,用药后均值较模型组有所上升。同时应用免疫印迹方法对黄连总生物碱高、中、低剂量对MMP-9在肾组织中表达的影响进行了观察。结果显示,模型组MMP-9表达下调,用药后均值升高,提示黄连总生物碱有可能是通过影响肾组织中多种细胞因子及基质酶的表达从而起到对糖尿病肾病大鼠的保护作用,但仍然不能恢复到正常水平。
同时我们应用免疫印迹方法对黄连总生物碱高、中、低剂量对MMP-9、Akt、p-Akt在肾组织中表达的影响进行了观察。结果显示,模型组MMP-9表达下调,用药后均值较模型组有所上升。Akt无显著性差异,模型组p-Akt表达上调,用药后均值较模型组下降,提示黄连总生物碱可能影响到肾组织中PI3-K通路从而调节体内的葡萄糖代谢而起到对糖尿病肾病的保护作用。
6、肾小球系膜细胞作为糖尿病肾病致病因子作用的主要靶细胞,研究它在高糖作用下生物学活性的改变,对探讨糖尿病肾病的发病机制具有重要意义。在高糖环境中培养的肾小球系膜细胞已经成为很好的研究糖尿病肾病的离体模型。我们观察并鉴定了原代培养的SD大鼠肾小球系膜细胞的形态,并且用MTT法检测了黄连总生物碱对高糖刺激的GMC增殖的影响,发现GMC的增殖能被黄连总生物碱显著抑制。
初步观察到黄连总生物碱对高糖刺激的系膜细胞表达的TGF-β_1、TIMP-1有一定的下调作用,而对MMP-9有上调作用,体外研究提示黄连总生物碱可能通过影响GMC的TGF-β_1、TIMP-1和MMP-9的表达而起到对系膜细胞的保护作用。
Diabetes mellitus (DM) is a progressive chronic endocrinic and metabolic disease with high morbidity and severity, which usually affects eyes, kidneys, heart and brain and other organs. Diabetic nephropathy (DN), caused by diabetic microvascular lesions, is one of the chronic complications of DM. The exact mechanism of DN is not clear yet. Currently, the blood dynamic changes, hyperglydemic biochemic and metabolic abnormity, effect of cell factor and growth factors and other reasons may play a role in this procedure.
The main pathological changes of DN are the deposit of glomerular extrcellular matrix (ECM). ECM is composed mainly of type IV collagen and fibronectin,which keep metabolic balance by rapid refresh .When the balance is broken,it will lead to glomerular lesion and even glomerular cirrhosis, and causes DN by the end. Researchers indicated that functional disorders of endothelial cells, secretion of vascular endothelial growth factor (VEGF) by the glomerular podocytic cells and the activativation of mesangial cells, occur during the early period of DN. Expression of VEGF gene elevates under hyperglycemic condition. VEGF's level highly correlates with uremia microprotein and participates in the procedure of nephrosclerosis. Connecture tissue growth factor (CTGF) plays a role in activativation of the fibroblasts and generation and deposit of ECM, and participates in many courses of DN, and mainly induced by transforming growth factorβ(TGF-β) .TGF-βcan upregulates the protein inhibitors such as PAZ and TEMP and also downregulates the matrix degradation enzymes, which suppresses the degradation of newly formed extra cellular matrix, promotes the expression of the matrix surface integrin and strengthen the interaction of matrix. The aggregation of sorbitol as well correlates with DN. Activation of Aldose Reductase (AR),the key rate-limiting enzyme of polyalcohol pathway,plays an important role in the initiation and development of DN. The oncogen c-akt's product PKB/Akt has a crucial effect on cell surviving, glucose metabolism, protein synthesis and so on. AKT, as the downstream molecule of insulin receptor, has an important effect on the insulin metabolism, hormone signal conduction and glucose transshipment.
It is the most important thing to establish the ideal DN animal model to illuminate the pathologic mechanisms and choose effective drugs for DN. The heredity DM pure inbred strain animals are the most common choose domesticly or abroadly. But those animals are quite rare and hard to feed. What's more, the pathogenesis of these models is mainly caused by heredity, which does not meet with the clinic characteristic. Feeding with high-calorie food and Streptozotocin, mononephoroctomy, injecting with Doxorubicin hydrochloride and other such kind of methods are used to establish DN model, but the systemic comparison between these methods is not available yet. We compared the models by these methods and find an optimal way to establish DN model.
Compound recipes or extracts of traditional Chinese medicines have being used to treat DN extensively and some of them seem to show effects on DN' s prevention and treatment in molecular biochemic and genetic level. Rhizoma Coptidis is exacted from the rhizome of Coptis chinensis Franch , which contains many types of alkaloids , such as Berberine,Palmatine, Coptisine,Worenine,Jatrorrhizine,Magnoflorine etc. Presently, researches on Berberine and Berberastine are more extensive, mainly on the improvement of clinic symptoms, but the molecular mechanisms are still not clear. Few researches are published on other alkaloids. The alkaloid functional targets and the functional mechanisms of Rhizoma Coptidis need to be explored.
This subject is the preliminary research on the prevention and treatment and the mechanisms of Total Alkaloids of Rhizoma Coptidis administration on DN rat model in vivo and adopt the method of ethanol circumfluence to extract and decompress to obtain the coptis alkaloid. Establishing DN animal model by cross-combination of feeding with high-calorie food and Streptozotocin, mononephoroctomy, and injecting with Adriamycin and choose the best combination,which would be used to establish DN animal model in the following studies. Grouping with low, medium, high dose of Rhizoma Coptidis and set up the positive control by Metformin Hydrochloride. We preliminary studied on protection and treatment and the mechanisms of Total Alkaloids of Rhizoma Coptidis administration on DN rat model in vivo. And we observe the function of Total Alkaloids of Rhizoma Coptidis on glomerular mesangial cells in vitro. The primary cultured glomerular mesangial cell are used to observe the fuction of Total Alkaloids of Rhizoma Coptidis on cell hyperplasia and the expression of TGF-βand TIMP-1 by hyperglycemic condition. The Metformin Hydrochloride act as control.
1. Using ethanol circumfluence 3 times, mixing the filtrate through, decompressing, condensing and vaporing the solvent, obtaining 3.8g crude drugs in per gram of extract ion. Using ion pair to extract and UV-spectrophotometry method to measure the average content of total Alkaloid of Rhizoma Coptidis were 31.4±1.41%, and HPLC method to determine the average content of berberin of its were 16.42±1.6%.
2. Adult male SD rats were divided into five groups: A is normal group: common feed. B is high-lipids and high-sucrose feeding group: high-lipids and high-sucrose feeding. C is high-lipids and high-sucrose feeding+STZ group: high-lipids and high-sucrose feeding for 6w, abdomen administration of STZ. D is high-lipids and high-sucrose feedin g+ Doxorubicin hydrochloride + STZ group: high-lipids and high-sucrose feeding for 4w, abdomen administration of Doxorubicin hydrochloride, abdomen administration of STZ 2 weeks later. E is high-lipids and high-sucrose feeding+mononephroctomy+STZ group: high-lipids and high-sucrose feeding feed for 4w, mononephroctomy, abdomen administration of STZ 2 weeks later. We concluded that E group is the ideal method that the rats appears evidence polydipsia, polyphagia, polyrexia, hypergycemia, hyperglyceremia, microprotein urine, abnormal glomerular filtration ratio and active oxygen, increase of kidney/body weight ratio, marked increase of aldose reductase . The pathology shows the glomerular destroyed, membrane widen, tubular intracellular cell disorders and vacuolization. The super mocropathology showed the glomerular volume increased, base membrane thickened and widened locally, mesangial cell proliferated, extracellular matrix increased, podocytes fused, mitochondria swoll and increased.
3.Our initial studies found that Total Alkaloids of Rhizoma Coptidis can decrease the level of blood sugar and lipid of DN rats. This research shows that feeding rats with high-lipids and high-sucrose food for 1 month induced insulin resistance, and administrating 2 weeks after mononephroctomy STZ can induce DN. The model shows hyperglycemia, elevation of body weight slowing down,high kidney/body weight ratio. After 8 weeks microprotein appeared in urine;while after 12 weeks blood insulin and GFR increase,blood urea,crea,TG,glycohemoglobin and urine creatine disordered. MDA of B group decreases significantly, SOD and all types of NOS increase significantly. low, medium, high doses of Total Alkaloids of Rhizoma Coptidis and Metformin Hydrochloride are given respectively to the models, the targets showed different improvements, and the medium dose group improved most significantly. All doses of Total Alkaloids of Rhizoma Coptidis can decrease the AR activity in rat red blood cells.
4.The HE and PAS staining of glomerular showed that the volume of the glomerules increased,membrane widen,tubular epithelial cell disarranged and vacuolized. The super micropathology shows that in the model of rats,the volume of the glomerule increased,basal membrane widened and thickened locally, mesangial cells proliferated, extracellular matrix increased, podocytes fused,mitochondria swoll and increased. After treatment with Total Alkaloids of Rhizoma Coptidis, the pathology of DN improved, but did not converse completely.
5.In this DN model,the immunohistochemistry result of the sections of organ shows the OD of VEGF、TGF-β1、CTGF increased and have significant difference. The average OD of those factors got lower than models, but didn't return to a normal level. We adopt RT-PCR to observe the effect of high,medium,and low dose of Total Alkaloid and Metformin feeding on expression of TGF-β1、TIMP-1、MMP-2 mRNA. It showed that expression of TGF-β1、TIMP-1 mRNA elevated, while those of the group treated by Total Alkaloids of Rhizoma Coptidis declined. MMP-2 mRNA of the model declined, the average of the group treatd enhanced. We adopt Western blot to observe the effect of high,medium,and low dose of Total Alkaloid and Metformin feeding on the expression of MMP-9.It showed that level of MMP-9 declined, while after being treated,the level of MMP-9 elevated. It prompted that Total Alkaloid may take effects on TGF-β1、VEGF、CTGF、MMP-9、TIMP-1 and MMP-2 , in order to protect rats from getting Diabetic nephropathy.
As the main target cell of DN, glomerular mesangial cells are of high sense in DN pathegenesis by observing its changes under the condition of hyperglycemia. The glomerular mesangial cell cultured in hyperglycemic condition is a good model in vivo. We observed and verified the forms of the primary cultured SD rat renal glomerular mesangial cells. We detected the effect of Total Alkaloids of Rhizoma Coptidis on hyperglycemic GMC and concluded that Total Alkaloids of Rhizoma Coptidis can inhibit the hyperplasia of GMC evidently.
We initially observed in a hyperglycemic circumstance, the total Alkaloids of Rhizoma Coptidis system stimulated mesangial cells to express TGF-β1 , activity of TIMP-1 declined, while that of MMP-9 elevated. Those prompt Total Alkaloids of Rhizoma Coptidis possibly upregulates the expression of TGF-β1 , MMP-9 and the TIMP-1 through affecting GMC in vitro.
引文
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