仙草水提物对糖尿病大鼠肾脏损伤的保护作用及其作用机制的研究
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摘要
糖尿病肾病(diabetic nephropathy, DN)是糖尿病最常见和严重的微血管并发症之一,也是导致糖尿病患者死亡的主要原因。有资料显示,约有20-30%的1型和2型糖尿病患者发展为DN；在美国和欧洲,DN已成为终末期肾病(end stage renal disease, ESRD)最常见的单一病因。持续的高血糖是糖尿病肾脏损害的始动因素,然而单纯控制血糖并不能使糖尿病患者受到足够的保护。一些我们已经明确或尚未明确的致病因素,即使在血糖受到严密控制的情况下,仍会持续不断地加速糖尿病患者主要脏器的损伤。
近年来,氧化应激机制与抗氧化治疗成为糖尿病及其并发症防治研究的重点。氧化应激是指机体内活性氧簇(reactive oxygen species, ROS)产生增多和／或清除减少,从而导致ROS在体内蓄积而产生的分子、细胞和机体的损伤。正常情况下,ROS的产生和消除处于一种动态平衡,但是在糖尿病持续的高血糖状态下,线粒体活性氧自由基大量增加,同时机体抗氧化防御能力降低,导致自由基在体内过度聚集。糖尿病的统一发病机制学说认为,氧自由基生成增多引起的机体氧化应激损伤是糖尿病各种并发症发生、发展的中心环节。肾脏是对氧化应激高度敏感的器官之一,ROS可通过直接和间接的方式造成肾组织的损伤。目前已应用多种抗氧化剂来防治自由基对机体的损害,如谷胱甘肽,维生素A、C、E、B1、B2、B6、B12、a-硫辛酸,辅酶Q10等。此外,应用于临床的糖尿病药物噻唑烷二酮类、他汀类、血管紧张素转换酶抑制剂及血管紧张素受体拮抗剂均被发现具有不同程度的抗氧化能力。从天然食物中提取高效、低毒的抗氧化剂应用于糖尿病等慢性疾病的防治是当前国内外研究的热点。与人工合成的抗氧化剂相比,天然抗氧化剂更具有资源优势、安全优势和功能优势,而且也更符合人们回归自然的消费时尚。
仙草(Mesona procumbens Hemsl.)是我国传统的药食同源性植物,现代研究发现仙草具有丰富的天然抗氧化剂资源。《中药大辞典》称,仙草能“治中暑,消渴(糖尿病)、高血压和肌肉、关节疼痛”；国内黄钦等报道,仙草制剂具有降血糖和改善糖尿病患者临床症状的作用,但具体机制尚不明确。
仙草已有悠久的食用历史,未曾发现明显的毒副作用。但由于它不在卫生部颁布的84种“药食两用”的中药名单之列,其安全性受到一些质疑。为此,本论文首先参照卫生部《食品安全性毒理学评价程序和方法》要求对仙草水提物进行急性毒性和遗传毒性的研究。在此基础上,应用链脲佐菌素(Streptozotocin, STZ)诱导的糖尿病大鼠模型,通过病理观察、免疫组化、ELISA.实时荧光定量PCR等多种研究手段,从整体、细胞、分子等不同水平系统观察了仙草水提物对实验大鼠血糖、血脂及肾功能的影响,并利用大鼠肾系膜细胞初步探讨了仙草的抗脂质过氧化特性在DN防治中的可能作用机制。我们希望,本研究结果可为天然抗氧化剂早期预防和干预糖尿病及其并发症的研究提供理论依据,并有助于仙草的进一步开发利用。
第一部分仙草水提物的急性毒性和遗传毒性研究
目的：对仙草水提物进行急性毒性和遗传毒性的研究,为其开发利用提供部分毒理学实验依据。
方法：仙草经热水浸提、减压浓缩和干燥粉碎后获得棕色粉末备用。根据卫生部《食品安全性毒理学评价程序和方法》,对仙草水提物进行大、小鼠急性毒性试验,Ames试验,小鼠骨髓微核试验和小鼠精子畸形试验的研究。
结果：染毒后,大、小鼠在2周的观察期内均未出现任何中毒症状和死亡情况；雌、雄性大小鼠对该受试物的经口急性毒性最大耐受剂量(MTD)均大于24.0g／kg体重；Ames试验中,不同剂量受试物在加大鼠肝匀浆代谢活化系统(S9)和不加S9条件下的回变菌落数均未超过空白对照组和溶剂对照组回变菌落数的两倍,且各剂量组间无明显的剂量反应关系；各剂量组小鼠骨髓嗜多染红细胞微核率和精子畸形率明显低于阳性对照组(P<0.01),且与阴性对照组无显著性差异(P>0.05)。以上结果说明在本试验剂量范围内,仙草水提物对实验动物未显示出明显的急性毒性和致突变作用。
第二部分仙草水提物对糖尿病大鼠肾脏损伤的保护作用
目的：应用STZ诱导的糖尿病大鼠模型,观察仙草水提物对实验动物体重、血糖、血脂以及肾脏结构和功能的影响。同时,检测实验动物肾组织中抗氧化指标MDA和SOD、GSH-Px的水平,以及致纤维化因子TGF-β1、TSP-1和FN基因表达的变化,从而探讨仙草水提物在DN防治中的作用及其可能的分子机制。
方法：
糖尿病大鼠模型制备及分组
清洁级雄性SD大鼠,体重180-200g,造模前禁食10小时,以60mg/kg-体重的剂量一次性腹腔注射STZ。正常对照组大鼠腹腔注射等剂量柠檬酸盐缓冲液。72小时后大鼠尾静脉采血,使用血糖仪测定血糖水平,尿糖试纸测定尿糖水平。将血糖≥16.7mmol/L、尿糖阳性(+++～++++)视为糖尿病大鼠模型制备成功。糖尿病成模大鼠随机分为5组,每组7只：
(1)糖尿病对照组(DM)；
(2)低剂量仙草干预组(XC-L,0.75g/kg.d);
(3)中剂量仙草干预组(XC-M,1.5g/kg.d);
(4)高剂量仙草干预组(XC-H,3.0g/kg.d);
(5)吡格列酮干预组(PGLT,10mg/kg.d);另设(6)正常对照组大鼠(Sham).
各干预组大鼠于每日上午灌胃给予受试物一次,正常和糖尿病对照组大鼠用等量蒸馏水灌胃。实验期间,所有大鼠自由进食及饮水,每天观察大鼠的一般状况,监测体重和血糖、尿糖水平的变化,持续给予受试物6周后处死。标本收集和指标检测
大鼠麻醉前先收集尿液,并置于4℃保存；然后以40mg/kg·体重的剂量腹腔注射1%戊巴比妥钠,将大鼠充分麻醉后固定于鼠板上,打开腹腔,腹主动脉取血,3000rpm离心15分钟,留取血清,分装后-70℃冻存。迅速采集肾脏标本,取部分肾组织标本,以4%中性甲醛固定,用于HE染色和免疫组化检测。另取肾脏髓质交界处皮质1mm3大小之肾组织数小块,以2.5%戊二醛固定,用于透射电镜检查。余下的肾脏组织分装后液氮冻存,用于肾匀浆中的抗氧化酶活性检测和实时荧光定量PCR (Real time-PCR)检测。
血、尿标本用全自动生化分析仪检测血糖(GLU)、血总胆固醇(TC)、甘油三酯(TG)、低密度胆固醇酯蛋白(LDL)、高密度胆固醇酯蛋白(HDL)、血肌酐(Scr)、血尿素氮(BUN)。
尿微量白蛋白(UMA)采用化学发光法于全自动蛋白分析系统测定。
光镜(HE染色)和电镜观察肾组织病理变化。
采用试剂盒的方法检测大鼠肾匀浆中MDA和SOD、GSH-Px的水平。
采用ELISA试剂盒检测大鼠血清中OxLDL的水平。
Trizol法提取肾组织中总RNA,荧光实时PCR定量检测TGF-β1、TSP-1和FN的mRNA表达。
免疫组化法标记肾组织内TGF-β1、TSP-1和FN的阳性细胞。
结果：
一般状况和体重、血糖的变化
腹腔注射STZ后,实验大鼠均出现了不同程度的血糖升高、尿糖阳性、体重减轻、饮水量和尿量明显增加等糖尿病典型症状。造模6周后,糖尿病组大鼠体重(193.44±53.91g)明显低于正常对照组(430.49±22.41g),血糖(42.06±6.02mmol/L)明显高于正常对照组(14.09±1.89mmol/L),差异具有统计学意义(P<0.05)；各干预组大鼠的一般状况与糖尿病组比较有明显改善,体重明显高于糖尿病组(PGLT 246.00±24.67, XC-L 239.17±50.31, XC-M 248.60±36.95, XC-H257.70±40.40g),高剂量仙草干预组体重差异可见统计学意义(P<0.05)；但各干预组大鼠的血糖水平与糖尿病组比较,差异均未见统计学意义(PGLT41.29±5.03,XCL39.89±5.63, XCM39.63±2.99, XCH38.09±3.29mmol/L, P>0.05).
肾功能的变化
糖尿病组大鼠的血尿素氮、肌酐和尿微量白蛋白水平(12.36±2.14mmol/L, 70.14±4.67μmol/L,54.14±9.29mg/L)均明显高于正常对照组(5.84±0.36mmol/L, 59.57±1.99μmol/L,14.73±11.07mg/L, P<0.05);吡格列酮和仙草低、中、高剂量干预组的尿微量白蛋白水平(17.59±10.82和22.41±11.53、12.40±3.88、13.68±7.64mg/L)明显低于糖尿病组大鼠(P均<0.05)；吡格列酮和仙草各剂量组大鼠的血尿素氮和肌酐水平均未见明显改变,显著高于正常对照组水平(P<0.05)。血脂的变化
糖尿病组大鼠的血清LDL、HDL、TG和TC值(分别为0.66±0.20、1.29±0.15、2.92±2.30、2.30±0.52mmol/L)明显高于正常对照组(分别为0.28±0.07、0.77±0.11、0.42±0.12、1.22±0.13mmol/L),差异有统计学意义(P<0.05)；各剂量仙草干预组的LDL和TC水平,随着仙草受试物浓度增大而存在降低的趋势；吡格列酮和高剂量仙草干预组大鼠的各项血脂指标(PGLT分别为0.42±0.08、1.01±0.27、1.75±0.87、1.74±0.42mmol/L; XC-H为:0.37±0.18,0.97±0.18,1.56±0.84, 1.64±0.41mmol/L)除TG外均明显下降,与糖尿病组比较有统计学意义(P<0.05)。肾组织的病理变化
肉眼观察可见糖尿病大鼠肾脏体积增大,外观苍白、肿胀；在光镜下可见部分肾小球萎缩,囊腔增大；肾小管管腔变窄,上皮细胞肥大、胞浆内出现糖原空泡；电镜下发现,糖尿病大鼠肾小球基底膜厚薄不均匀,部分区域呈局灶性增厚,足突排列紊乱,融合增加,肾小管上皮细胞线粒体肿胀,微绒毛排列紊乱或部分脱落,胞浆内出现较多空泡。吡格列酮和仙草水提物的干预能明显促进糖尿病大鼠肾脏病变的好转,光镜下肾小球结构相对清晰,萎缩少见,囊腔增大和肾小管上皮细胞空泡现象明显改善；而电镜下可见肾小球基底膜变清晰,足突融合、肾小管上皮细胞的线粒体肿胀、绒毛脱落和胞内空泡现象明显改善。
MDA含量和抗氧化酶活性的变化
糖尿病大鼠肾组织的MDA含量(1.16±0.20 nmol/mg.prot)明显高于正常对照组(0.96±0.15 nmol/mg.prot, P<0.05),仙草低、中、高剂量干预组的MDA含量(分别为0.90±0.15、0.86±0.34、0.86±1.01 nmol/mg.prot)均明显低于糖尿病组,差异具有统计学意义(P<0.05)；但SOD和GSH-Px的活性未见明显变化。吡格列酮干预组的上述指标与正常对照组和糖尿病组比较,差异均无统计学意义(P>0.05)。
血清OxLDL的变化
糖尿病大鼠血清中OxLDL的含量(17.97±1.02 ng/ml)明显高于正常对照组(14.16±2.43 ng/ml, P＜0.05);吡格列酮干预组和仙草中剂量干预组的OxLDL水平(分别为14.91±1.41和14.87±3.43 ng/ml)明显低于糖尿病大鼠组,差异具有统计学意义(P<0.05)。
TGF-β1等细胞因子转录水平的变化
实时荧光定量PCR的结果表明：糖尿病大鼠肾组织中TSP-1、TGF-β1和FN的mRNA表达水平均高于正常对照组(分别为2.65±1.28、2.51±1.48、1.42±0.75倍),各干预组三种细胞因子的mRNA表达与糖尿病组比较均有不同程度的下降；且随着仙草受试物浓度的增大,TGF-β1和TSP-1的mRNA表达存在下降趋势,但以上变化均未见统计学意义(P>0.05)。
TGF-β1等细胞因子蛋白水平的变化
免疫组化方法检测到糖尿病大鼠肾组织切片中TGF-β1、TSP-1和FN阳性细胞数量(分别为32.7±6.9、25.44±7.1、18.0±10.5个／mm2)和光密度值(分别为160.6±6.9、186.7±2.5、187.0±16.5)均明显高于正常对照组(分别为1.9±0.4、1.0±1.0、3.1±0.8个／mm2；和149.3±8.7、171.1±5.8、163.6±8.3),差异具有统计学意义(P<0.05)；各剂量仙草干预组受测细胞因子的表达显著下降,其中仙草中剂量组的效果较为明显(分别为9.5±1.0、4.44±1.7、7.1±2.5个／mm2；和148.1±5.2、176.3±5.9、170.6±7.3),与糖尿病组比较差异具有统计学意义(P<0.05)。
第三部分仙草水提物对LDL体外氧化修饰作用和肾系膜细胞TGF-β1mRNA表达的影响
目的：观察仙草水提物对血浆LDL体外氧化修饰作用的影响,以及对OxLDL诱导的大鼠肾系膜细胞TGF-β1等细胞因子转录的影响,探讨仙草水提物对糖尿病大鼠肾脏保护作用的可能机制。
方法：采用序列超速离心和Sepharose 6B凝胶过滤分离纯化的LDL,用10μmol／LCu2+进行氧化诱导,获得OxLDL.在Cu2+诱导过程中加入不同浓度的仙草水提物,观察其对体外Cu2+诱导的LDL氧化修饰作用的影响,结果用琼脂糖凝胶电泳迁移率和硫代巴比妥酸反应物质(TBARS)表示。体外培养大鼠肾小球系膜细胞,随机分为空白对照组、氧化诱导组(OxLDL 50μg／ml)和仙草25、50和125μg/ml干预组,以维生素E(VitE 100μM)作为阳性对照组。细胞共孵育24h后Trizol提取总RNA,荧光定量PCR检测各组细胞TGF-β1(?)、TSP-1、FN的1mRNA表达。
结果：仙草水提物能明显对抗Cu2+诱导的LDL氧化,其中高浓度组仙草水提物的迁移率与LDL未氧化组和VitE干预组几乎相同,在实验浓度范围内存在明显的剂量-效应关系。25-125μg/ml仙草水提物均可显著抑制过氧化脂质(LPO)的生成,与氧化修饰组比较,差异具有统计学意义(P＜0.05)；50μg/ml以上浓度仙草干预组的LPO抑制率趋于稳定,接近氧化修饰组的50%。肾系膜细胞在OxLDL诱导下TGF-β1、FN的1nRNA生成明显增加(分别为空白对照组的2.09±0.19和1.55±0.11倍),仙草低、中、高各剂量组能非常显著地抑制肾系膜细胞在OxLDL诱导下TGF-β1 mRNA的产生(分别为空白对照组的0.74±0.04、0.82±0.03和0.73±0.09倍,P<0.05),抑制效果高于VitE对照组(1.68±0.11倍)；对FN的抑制效果分别为对照组的1.40±0.09,1.33±0.16和1.17±0.21倍,其中高剂量组与诱导组比较,差异有统计学意义(P<0.05)。
结论：
1、仙草水提物在本实验剂量范围内,对实验动物未见急性毒性和致突变作用。
2、仙草水提物可显著增加STZ诱导的糖尿病大鼠的体重,改善一般状况,但对血糖无明显作用。
3、仙草水提物对糖尿病大鼠肾脏损伤具有一定的保护作用。
4、仙草水提物具有显著的抗脂质过氧化、调节血脂和抑制糖尿病大鼠肾组织TGF-β1生成的作用。
5、仙草水提物能有效抑制Cu2+体外诱导的人血清LDL氧化修饰。
6、仙草水提物能明显抑制OxLDL诱导的大鼠肾系膜细胞TGF-β1的转录。
综上,我们认为仙草水提物可能通过调节血脂和抗脂质过氧化作用,从而抑制糖尿病大鼠体内OxLDL的生成；同时,仙草水提物能以不依赖于血糖水平的方式降低TGF-β1的转录,从而发挥对糖尿病大鼠的肾脏保护功能。
Diabetic nephropathy (DN) is one of the most common and serious microvascular complications of diabetes, as well as the leading cause of death in diabetics. According to statistics, diabetes have become the most common single cause of end-stage renal disease (ESRD) in the United States and Europe, about 20-30% of type 1 and type 2 diabetics develop to DN. Metabolic disorder caused by hyperglycaemia is the initiating factor of diabetic renal damage, but the effective control of blood glucose can not completely prevent the occurrence and development of diabetic complications.
In recent years the studies of diabetes and its complications focused on the mechanism of oxidative stress and antioxidant therapy. Oxidative stress is a result of reactive oxygen species (ROS) generation increase and/or removal decrease, leading to ROS accumulation in vivo, and then causes damage on biomolecules, cells, tissues, organs, and/or bodies. Normally, the generation and elimination of ROS is in a dynamic equilibrium, but the sustained hyperglycaemia in diabetes will significantly result in increased generation of mitochondrial reactive oxygen species while the capacity of antioxidant decrease, leading to excessive accumulation of free radicals in the body. The unified theory of the pathogenesis of diabetic complications hypothesized that excessive generation of oxygen free radicals, caused by oxidative stress, is the key of the occurrence and development of various complications in diabetes. Considering that the kidney is one of the organs which are highly sensitive to oxidative stress, it is widely accepted that ROS can cause renal damage through a direct or indirect means. As a matter of fact, a variety of anti-oxidants have currently been applied to prevent free radicals from damaging the body, such as glutathione, vitamin A, C, E, B1, B2, B6, B12, a-lipoic acid, coenzyme Q10, bioflavonoids, and so on. In addition, some diabetic drugs applied in clinic, such as thiazolidinediones, TZDs, statins, angiotensin-converting enzyme inhibitors, and angiotensin receptor antagonists were found to have different degrees of antioxidant activities.
Hsian-tsao(Mesona procumbens Hemsl.) is a traditional Chinese herb used in medicine and daily food. Modern research found Hsian-tsao extracts has a significant anti-oxidation function. "Chinese Medicine Dictionary" said that Hsian-tsao can "rule heat stroke, diabetes, hypertension, muscle, and joint pain". Huang Qin reported that Hsian-tsao agent is effective to reduce hyperglycaemia and to improve the clinical symptoms in diabetic patients, but the mechanism remains elusive.
In the present study, effects of Hsian-tsao water extracts on blood glucose, blood lipids, and renal function were systematically observed in Streptozotocin(STZ)-induced diabetic rats at the whole-body, cellular, and molecular level by various approaches, such as pathological observation, immunohistochemistry, ELISA, and real-time fluorescence quantitative PCR after passing acute toxicity and mutagenicity the evaluation. Then we further explored the antioxidant properties of Hsian-tsao water extracts in prevention and treatment of DN and its possible mechanism. The present results provide scientific data of food-based natural antioxidants on diabetic prevention and to guide the exploitation of Hsian-tsao.
Part I Acute toxicity and mutagenicity evaluation of Hsian-tsao Water Extracts
Objective To evaluate acute toxicity and mutagenicity of Hsian-tsao water extracts.
Method Brown powders of Hsian-tsao water extracts were obtained after extraction with boiling water, vacuum concentration, drying, and pulverizing. According to the "technical specifications of food safety and toxicity evaluation" issued by the Ministry of Health of China, the toxicities of Hsian-tsao water extracts were evaluated by oral toxicity test, Ames test, mouse bone marrow micronucleus test, and mouse teratospermia test.
Results Both rats and mice were not developped any symptom of poisoning or death within 2 weeks observation period after treatment with Hsian-tsao water extracts. The oral maximum tolerance dose of the sample was higher than 24.0g/kg-body weight. Results of Ames test, polychromatic erythrocytes micronucleus test, and sperm deformity test were all negative.
PartⅡRenal Protective Activity of Hsian-tsao Water Extracts in STZ Induced Diabetic Rats
Objective To observe the influence of Hsian-tsao water extracts on the body weight, blood glucose, blood lipids, and renal damages to STZ-induced diabetic rat models, to demonstrate a antioxidant property of Hsian-tsao as a role of prevention of DN development, and to explore its possible mechanism.
Method
Preparation of Diabetic Rats and Grouping
Male SD rats with body weight between 180-200 g were fasted for 10 hours before STZ was intraperitoneally injected (60mg/kg) to induce diabetes; the control group received an injection of same dose of citrate buffer. Seventy-two hours after the injection of STZ, the blood glucose level reached≥16.7mmol/L and urine glucose was positive (+++～++++), indicating the successful induction of diabetes. They were then randomly divided into 5 groups (n=7):
(1) diabetic control group (DM);
(2) low-dose Hsian-tsao treatment group (XC-L,0.75g/kg.d);
(3) medium-dose Hsian-tsao treatment group (XC-M,1.5g/kg.d);
(4) high-dose Hsian-tsao treatment group (XC-H,3.0g/kg.d);
(5) Pioglitazone treatment group (PGLT,10mg/kg.d);
(6) normal control group (Sham).
Rats in treatment groups were given drugs by intragastric administration and the other two groups of rats were given the same amount of distilled water every day for 6 weeks. During the experiment, all the rats were fed a commercial rat feed and water ad libitum. Body weight, blood and urine glucose concentrations were then measured weekly.
Sample Collection and Examination
At the end of the study (6 weeks), all the rats were anesthetized with 1% sodium pentobarbital (40mg/kg-BW) by intraperitoneal injection. Urine, blood sample and kidney tissue were collected and subjected to biochemical measurements or histopathological studies. Blood glucose(GLU), total cholesterol(TC), triglyceride(TG), LDL cholesterol, HDL cholesterol, blood urea nitrogen(BUN), and serum creatinine(Scr) were determined by automatic biochemical analyzer. Microalbuminuria (UMA) was determined with chemoluminescence in the automatic protein analysis system. Pathological changes in kidney were observed in light microscopy (HE staining) and transmission electron microscopy. GSH-Px, SOD, and MDA levels in rat kidney homogenate were measured by commercial kits. Serum OxLDL was determined by an ELISA kit. Total RNA was extracted by TRIzol, and the expressions of TGF-β1, TSP-1 and FN mRNA were detected by real-time fluorescence quantitative PCR. The distributions of TGF-β1, TSP-1 and FN positive cells in renal tissue were detected by immunohistochemical staining.
Results
Changes of Blood Glucose and Body Weight
Diabetic rats had a significant lower body weight and higher blood glucose level versus controls (P＜0.05). The body weight of Pioglitazone and Hsian-tsao intervention rats were higher than diabetic rats, and the high dose of Hsian-tsao group showed significant differences (P<0.05), but blood glucose showed no obvious changes (P＞0.05).
Changes in Renal Function
Serum BUN, Scr and urine microalbuminuria(UMA) were significantly higher in diabetic rats compared to controls (P<0.05). UMA levels were significantly lower in Pioglitazone and Hsian-tsao groups while compared to DM group (P＜0.05), but BUN and Scr showed no obvious changes (P>0.05).
Changes in Blood lipids
Serum LDL、HDL、TG and TC in diabetic rats higher than those in control group (P＜0.05). All the blood lipids except TG were significantly lower in Pioglitazone and High dose of Hsian-tsao groups than in DM group (P＜0.05), but low dose Hsian-tsao group had no obvious changes (P>0.05).
Pathological Changes of Kidney Tissue
Kidneys of diabetic rats were enlarged, with pale and oncotic appearance. Glomerular atrophy, tubular lumen narrowing, and augmentation in capsular space could be seen by a light microscope (with HE staining). It was manifested with a thicken glomerular basement membrane, swelling, shorten and confluence of foot process of podocyte, expansion of mitochondriae of parietal epithelium, and increase of collagen of glomerulus in the ultrastructure in diabetic kidney. In addition, swelling of microvilli and expansion of mitochondriae in the epithelium of glomerular tubule were observed. However, all the phenomena were relieved in Pioglitazone and Hsian-tsao groups.
Changes of MDA concentration and the activity of SOD and GSH-px
Compared with the control group, the concentration of MDA significantly increased in renal tissue of the STZ induced diabetic rats (P<0.05), and MDA level in high, middle and low dose Hsian-tsao groups were significantly lower than DM group (P＜0.05). Pioglitazone treatment could also reduce the MDA level, but had no statistical significance (P>0.05) ersus diabetic rats. There were no significant differences in SOD and GSH-Px among all the five groups.
Changes of Serum OxLDL
The contents of serum OxLDL in diabetic rats were significant higher than the control group. OxLDL level in Pioglitazone and middle dose Hsian-tsao were groups were significantly lower than DM group (P<0.05).
Changes in transcriptional level of cytokines
Fluorescent quantitative PCR demonstrated that the expressions of TGF-β1, TSP-1 and FN mRNA in diabetic rat kidney were significantly higher while comparing to the control group. Hsian-tsao water extracts can significantly reduce the expression of TGF-β1 and TSP-1 mRNA(P＜0.05), and showed a dose-response relationship.
Changes in protein level of cytokines
The TGF-β1, TSP-1 and FN expression in kidneys was examined by the quantitative analysis of the number of positive cells and also the optical density of the immunostaining. The number of positive cells and optical density of TGF-β1, TSP-1 and FN were detected to be much higher in the diabetic kidney than those in control group (P<0.05). The expression of all the cytokines decreased markedly under the treatment of Hsian-tsao water extracts and Pioglitazone. The medium dose of Hsian-tsao group showed the most obvious change.
Part III Effects of Hsian-tsao Water Extracts on the oxidative modification of LDL in vitro and the transcription of TGF-β1 induced by OxLDL in mesangial cells
Objective To evaluate the effect of Hsian-tsao water extracts on Cu2+-mediated LDL oxidative modification in vitro and the transcription of TGF-β1 induced by OxLDL in mesangial cells, with a hope to clarify the possible mechanisms of renal protective effect of Hsian-tsao in DM.
Methods LDL was isolated by ultracenfugation from normal human plasma. The extent of LDL peroxidation was measured in terms of thiobarbituric acid reactive substances (TBARS) expressed as MDA equivalents. The electrophoresis mobility of LDL was determined as relative electrophoresis mobility (REM) on an agarose gel electrophoresis. Rat mesangial cells were randomly divided into blank control group, oxidation-induced group (OxLDL 50μg/ml), and Hsian-tsao groups (25,50, and 125μg/ml), with vitamin E (100μmol/ml) as a positive control. The cells were incubated for 24 hours, and then total RNA was extracted with Trizol, the mRNA levels of TGF-β1, TSP-1, FN mRNA were detected by RT-PCR.
Results Hsian-tsao could protect LDL against Cu2+-mediated LDL oxidative modification in vitro. The TBARS generation and the REM in high concentration Hsian-tsao extract group showed no significant difference with blank and positive control group. Hsian-tsao water extracts (25-125μg/ml)can significantly inhibit the formation of LPO while compared to positive control group (P<0.05). The production of TGF-β1 and FN mRNA induced by OxLDL in mesangial cells were significantly lower than in Hsian-tsao treatment groups, and the inhibition was more effective than the VitE group.
Conclusion
1. Hsian-tsao water extracts has no toxicity and mutagenic effect in the study dose on the experimental animals.
2. Hsian-tsao water extracts can significantly increase the body weight in STZ-induced diabetic rats, but have no effect on blood sugar.
3. Hsian-tsao water extracts has a renal protective effect in diabetic rats.
4. Hsian-tsao water extracts has a significant effect on anti-lipid peroxidation, regulating blood lipids and inhibition of TGF-β1 expression in diabetic rats.
5. Hsian-tsao water extracts can effectively inhibit the Cu2+-mediated LDL oxidative modification in vitro.
6. Hsian-tsao water extracts can obviously inhibit the TGF-β1 transcription induced by OxLDL in mesangial cells.
Based on above results, we infer that Hsian-tsao water extracts can reduce the generation of OxLDL in diabetic rats by regulating lipids and anti-lipid peroxidation. Hsian-tsao water extracts can also inhibit the TGF-β1 transcription in the diabetic kidney independent with the blood glucose. Hsian-tsao water extracts may be able to use as a functional food or nutraceuticle to protect renal function for DM.
近年来,氧化应激机制与抗氧化治疗成为糖尿病及其并发症防治研究的重点。氧化应激是指机体内活性氧簇(reactive oxygen species, ROS)产生增多和／或清除减少,从而导致ROS在体内蓄积而产生的分子、细胞和机体的损伤。正常情况下,ROS的产生和消除处于一种动态平衡,但是在糖尿病持续的高血糖状态下,线粒体活性氧自由基大量增加,同时机体抗氧化防御能力降低,导致自由基在体内过度聚集。糖尿病的统一发病机制学说认为,氧自由基生成增多引起的机体氧化应激损伤是糖尿病各种并发症发生、发展的中心环节。肾脏是对氧化应激高度敏感的器官之一,ROS可通过直接和间接的方式造成肾组织的损伤。目前已应用多种抗氧化剂来防治自由基对机体的损害,如谷胱甘肽,维生素A、C、E、B1、B2、B6、B12、a-硫辛酸,辅酶Q10等。此外,应用于临床的糖尿病药物噻唑烷二酮类、他汀类、血管紧张素转换酶抑制剂及血管紧张素受体拮抗剂均被发现具有不同程度的抗氧化能力。从天然食物中提取高效、低毒的抗氧化剂应用于糖尿病等慢性疾病的防治是当前国内外研究的热点。与人工合成的抗氧化剂相比,天然抗氧化剂更具有资源优势、安全优势和功能优势,而且也更符合人们回归自然的消费时尚。
仙草(Mesona procumbens Hemsl.)是我国传统的药食同源性植物,现代研究发现仙草具有丰富的天然抗氧化剂资源。《中药大辞典》称,仙草能“治中暑,消渴(糖尿病)、高血压和肌肉、关节疼痛”；国内黄钦等报道,仙草制剂具有降血糖和改善糖尿病患者临床症状的作用,但具体机制尚不明确。
仙草已有悠久的食用历史,未曾发现明显的毒副作用。但由于它不在卫生部颁布的84种“药食两用”的中药名单之列,其安全性受到一些质疑。为此,本论文首先参照卫生部《食品安全性毒理学评价程序和方法》要求对仙草水提物进行急性毒性和遗传毒性的研究。在此基础上,应用链脲佐菌素(Streptozotocin, STZ)诱导的糖尿病大鼠模型,通过病理观察、免疫组化、ELISA.实时荧光定量PCR等多种研究手段,从整体、细胞、分子等不同水平系统观察了仙草水提物对实验大鼠血糖、血脂及肾功能的影响,并利用大鼠肾系膜细胞初步探讨了仙草的抗脂质过氧化特性在DN防治中的可能作用机制。我们希望,本研究结果可为天然抗氧化剂早期预防和干预糖尿病及其并发症的研究提供理论依据,并有助于仙草的进一步开发利用。
第一部分仙草水提物的急性毒性和遗传毒性研究
目的：对仙草水提物进行急性毒性和遗传毒性的研究,为其开发利用提供部分毒理学实验依据。
方法：仙草经热水浸提、减压浓缩和干燥粉碎后获得棕色粉末备用。根据卫生部《食品安全性毒理学评价程序和方法》,对仙草水提物进行大、小鼠急性毒性试验,Ames试验,小鼠骨髓微核试验和小鼠精子畸形试验的研究。
结果：染毒后,大、小鼠在2周的观察期内均未出现任何中毒症状和死亡情况；雌、雄性大小鼠对该受试物的经口急性毒性最大耐受剂量(MTD)均大于24.0g／kg体重；Ames试验中,不同剂量受试物在加大鼠肝匀浆代谢活化系统(S9)和不加S9条件下的回变菌落数均未超过空白对照组和溶剂对照组回变菌落数的两倍,且各剂量组间无明显的剂量反应关系；各剂量组小鼠骨髓嗜多染红细胞微核率和精子畸形率明显低于阳性对照组(P<0.01),且与阴性对照组无显著性差异(P>0.05)。以上结果说明在本试验剂量范围内,仙草水提物对实验动物未显示出明显的急性毒性和致突变作用。
第二部分仙草水提物对糖尿病大鼠肾脏损伤的保护作用
目的：应用STZ诱导的糖尿病大鼠模型,观察仙草水提物对实验动物体重、血糖、血脂以及肾脏结构和功能的影响。同时,检测实验动物肾组织中抗氧化指标MDA和SOD、GSH-Px的水平,以及致纤维化因子TGF-β1、TSP-1和FN基因表达的变化,从而探讨仙草水提物在DN防治中的作用及其可能的分子机制。
方法：
糖尿病大鼠模型制备及分组
清洁级雄性SD大鼠,体重180-200g,造模前禁食10小时,以60mg/kg-体重的剂量一次性腹腔注射STZ。正常对照组大鼠腹腔注射等剂量柠檬酸盐缓冲液。72小时后大鼠尾静脉采血,使用血糖仪测定血糖水平,尿糖试纸测定尿糖水平。将血糖≥16.7mmol/L、尿糖阳性(+++～++++)视为糖尿病大鼠模型制备成功。糖尿病成模大鼠随机分为5组,每组7只：
(1)糖尿病对照组(DM)；
(2)低剂量仙草干预组(XC-L,0.75g/kg.d);
(3)中剂量仙草干预组(XC-M,1.5g/kg.d);
(4)高剂量仙草干预组(XC-H,3.0g/kg.d);
(5)吡格列酮干预组(PGLT,10mg/kg.d);另设(6)正常对照组大鼠(Sham).
各干预组大鼠于每日上午灌胃给予受试物一次,正常和糖尿病对照组大鼠用等量蒸馏水灌胃。实验期间,所有大鼠自由进食及饮水,每天观察大鼠的一般状况,监测体重和血糖、尿糖水平的变化,持续给予受试物6周后处死。标本收集和指标检测
大鼠麻醉前先收集尿液,并置于4℃保存；然后以40mg/kg·体重的剂量腹腔注射1%戊巴比妥钠,将大鼠充分麻醉后固定于鼠板上,打开腹腔,腹主动脉取血,3000rpm离心15分钟,留取血清,分装后-70℃冻存。迅速采集肾脏标本,取部分肾组织标本,以4%中性甲醛固定,用于HE染色和免疫组化检测。另取肾脏髓质交界处皮质1mm3大小之肾组织数小块,以2.5%戊二醛固定,用于透射电镜检查。余下的肾脏组织分装后液氮冻存,用于肾匀浆中的抗氧化酶活性检测和实时荧光定量PCR (Real time-PCR)检测。
血、尿标本用全自动生化分析仪检测血糖(GLU)、血总胆固醇(TC)、甘油三酯(TG)、低密度胆固醇酯蛋白(LDL)、高密度胆固醇酯蛋白(HDL)、血肌酐(Scr)、血尿素氮(BUN)。
尿微量白蛋白(UMA)采用化学发光法于全自动蛋白分析系统测定。
光镜(HE染色)和电镜观察肾组织病理变化。
采用试剂盒的方法检测大鼠肾匀浆中MDA和SOD、GSH-Px的水平。
采用ELISA试剂盒检测大鼠血清中OxLDL的水平。
Trizol法提取肾组织中总RNA,荧光实时PCR定量检测TGF-β1、TSP-1和FN的mRNA表达。
免疫组化法标记肾组织内TGF-β1、TSP-1和FN的阳性细胞。
结果：
一般状况和体重、血糖的变化
腹腔注射STZ后,实验大鼠均出现了不同程度的血糖升高、尿糖阳性、体重减轻、饮水量和尿量明显增加等糖尿病典型症状。造模6周后,糖尿病组大鼠体重(193.44±53.91g)明显低于正常对照组(430.49±22.41g),血糖(42.06±6.02mmol/L)明显高于正常对照组(14.09±1.89mmol/L),差异具有统计学意义(P<0.05)；各干预组大鼠的一般状况与糖尿病组比较有明显改善,体重明显高于糖尿病组(PGLT 246.00±24.67, XC-L 239.17±50.31, XC-M 248.60±36.95, XC-H257.70±40.40g),高剂量仙草干预组体重差异可见统计学意义(P<0.05)；但各干预组大鼠的血糖水平与糖尿病组比较,差异均未见统计学意义(PGLT41.29±5.03,XCL39.89±5.63, XCM39.63±2.99, XCH38.09±3.29mmol/L, P>0.05).
肾功能的变化
糖尿病组大鼠的血尿素氮、肌酐和尿微量白蛋白水平(12.36±2.14mmol/L, 70.14±4.67μmol/L,54.14±9.29mg/L)均明显高于正常对照组(5.84±0.36mmol/L, 59.57±1.99μmol/L,14.73±11.07mg/L, P<0.05);吡格列酮和仙草低、中、高剂量干预组的尿微量白蛋白水平(17.59±10.82和22.41±11.53、12.40±3.88、13.68±7.64mg/L)明显低于糖尿病组大鼠(P均<0.05)；吡格列酮和仙草各剂量组大鼠的血尿素氮和肌酐水平均未见明显改变,显著高于正常对照组水平(P<0.05)。血脂的变化
糖尿病组大鼠的血清LDL、HDL、TG和TC值(分别为0.66±0.20、1.29±0.15、2.92±2.30、2.30±0.52mmol/L)明显高于正常对照组(分别为0.28±0.07、0.77±0.11、0.42±0.12、1.22±0.13mmol/L),差异有统计学意义(P<0.05)；各剂量仙草干预组的LDL和TC水平,随着仙草受试物浓度增大而存在降低的趋势；吡格列酮和高剂量仙草干预组大鼠的各项血脂指标(PGLT分别为0.42±0.08、1.01±0.27、1.75±0.87、1.74±0.42mmol/L; XC-H为:0.37±0.18,0.97±0.18,1.56±0.84, 1.64±0.41mmol/L)除TG外均明显下降,与糖尿病组比较有统计学意义(P<0.05)。肾组织的病理变化
肉眼观察可见糖尿病大鼠肾脏体积增大,外观苍白、肿胀；在光镜下可见部分肾小球萎缩,囊腔增大；肾小管管腔变窄,上皮细胞肥大、胞浆内出现糖原空泡；电镜下发现,糖尿病大鼠肾小球基底膜厚薄不均匀,部分区域呈局灶性增厚,足突排列紊乱,融合增加,肾小管上皮细胞线粒体肿胀,微绒毛排列紊乱或部分脱落,胞浆内出现较多空泡。吡格列酮和仙草水提物的干预能明显促进糖尿病大鼠肾脏病变的好转,光镜下肾小球结构相对清晰,萎缩少见,囊腔增大和肾小管上皮细胞空泡现象明显改善；而电镜下可见肾小球基底膜变清晰,足突融合、肾小管上皮细胞的线粒体肿胀、绒毛脱落和胞内空泡现象明显改善。
MDA含量和抗氧化酶活性的变化
糖尿病大鼠肾组织的MDA含量(1.16±0.20 nmol/mg.prot)明显高于正常对照组(0.96±0.15 nmol/mg.prot, P<0.05),仙草低、中、高剂量干预组的MDA含量(分别为0.90±0.15、0.86±0.34、0.86±1.01 nmol/mg.prot)均明显低于糖尿病组,差异具有统计学意义(P<0.05)；但SOD和GSH-Px的活性未见明显变化。吡格列酮干预组的上述指标与正常对照组和糖尿病组比较,差异均无统计学意义(P>0.05)。
血清OxLDL的变化
糖尿病大鼠血清中OxLDL的含量(17.97±1.02 ng/ml)明显高于正常对照组(14.16±2.43 ng/ml, P＜0.05);吡格列酮干预组和仙草中剂量干预组的OxLDL水平(分别为14.91±1.41和14.87±3.43 ng/ml)明显低于糖尿病大鼠组,差异具有统计学意义(P<0.05)。
TGF-β1等细胞因子转录水平的变化
实时荧光定量PCR的结果表明：糖尿病大鼠肾组织中TSP-1、TGF-β1和FN的mRNA表达水平均高于正常对照组(分别为2.65±1.28、2.51±1.48、1.42±0.75倍),各干预组三种细胞因子的mRNA表达与糖尿病组比较均有不同程度的下降；且随着仙草受试物浓度的增大,TGF-β1和TSP-1的mRNA表达存在下降趋势,但以上变化均未见统计学意义(P>0.05)。
TGF-β1等细胞因子蛋白水平的变化
免疫组化方法检测到糖尿病大鼠肾组织切片中TGF-β1、TSP-1和FN阳性细胞数量(分别为32.7±6.9、25.44±7.1、18.0±10.5个／mm2)和光密度值(分别为160.6±6.9、186.7±2.5、187.0±16.5)均明显高于正常对照组(分别为1.9±0.4、1.0±1.0、3.1±0.8个／mm2；和149.3±8.7、171.1±5.8、163.6±8.3),差异具有统计学意义(P<0.05)；各剂量仙草干预组受测细胞因子的表达显著下降,其中仙草中剂量组的效果较为明显(分别为9.5±1.0、4.44±1.7、7.1±2.5个／mm2；和148.1±5.2、176.3±5.9、170.6±7.3),与糖尿病组比较差异具有统计学意义(P<0.05)。
第三部分仙草水提物对LDL体外氧化修饰作用和肾系膜细胞TGF-β1mRNA表达的影响
目的：观察仙草水提物对血浆LDL体外氧化修饰作用的影响,以及对OxLDL诱导的大鼠肾系膜细胞TGF-β1等细胞因子转录的影响,探讨仙草水提物对糖尿病大鼠肾脏保护作用的可能机制。
方法：采用序列超速离心和Sepharose 6B凝胶过滤分离纯化的LDL,用10μmol／LCu2+进行氧化诱导,获得OxLDL.在Cu2+诱导过程中加入不同浓度的仙草水提物,观察其对体外Cu2+诱导的LDL氧化修饰作用的影响,结果用琼脂糖凝胶电泳迁移率和硫代巴比妥酸反应物质(TBARS)表示。体外培养大鼠肾小球系膜细胞,随机分为空白对照组、氧化诱导组(OxLDL 50μg／ml)和仙草25、50和125μg/ml干预组,以维生素E(VitE 100μM)作为阳性对照组。细胞共孵育24h后Trizol提取总RNA,荧光定量PCR检测各组细胞TGF-β1(?)、TSP-1、FN的1mRNA表达。
结果：仙草水提物能明显对抗Cu2+诱导的LDL氧化,其中高浓度组仙草水提物的迁移率与LDL未氧化组和VitE干预组几乎相同,在实验浓度范围内存在明显的剂量-效应关系。25-125μg/ml仙草水提物均可显著抑制过氧化脂质(LPO)的生成,与氧化修饰组比较,差异具有统计学意义(P＜0.05)；50μg/ml以上浓度仙草干预组的LPO抑制率趋于稳定,接近氧化修饰组的50%。肾系膜细胞在OxLDL诱导下TGF-β1、FN的1nRNA生成明显增加(分别为空白对照组的2.09±0.19和1.55±0.11倍),仙草低、中、高各剂量组能非常显著地抑制肾系膜细胞在OxLDL诱导下TGF-β1 mRNA的产生(分别为空白对照组的0.74±0.04、0.82±0.03和0.73±0.09倍,P<0.05),抑制效果高于VitE对照组(1.68±0.11倍)；对FN的抑制效果分别为对照组的1.40±0.09,1.33±0.16和1.17±0.21倍,其中高剂量组与诱导组比较,差异有统计学意义(P<0.05)。
结论：
1、仙草水提物在本实验剂量范围内,对实验动物未见急性毒性和致突变作用。
2、仙草水提物可显著增加STZ诱导的糖尿病大鼠的体重,改善一般状况,但对血糖无明显作用。
3、仙草水提物对糖尿病大鼠肾脏损伤具有一定的保护作用。
4、仙草水提物具有显著的抗脂质过氧化、调节血脂和抑制糖尿病大鼠肾组织TGF-β1生成的作用。
5、仙草水提物能有效抑制Cu2+体外诱导的人血清LDL氧化修饰。
6、仙草水提物能明显抑制OxLDL诱导的大鼠肾系膜细胞TGF-β1的转录。
综上,我们认为仙草水提物可能通过调节血脂和抗脂质过氧化作用,从而抑制糖尿病大鼠体内OxLDL的生成；同时,仙草水提物能以不依赖于血糖水平的方式降低TGF-β1的转录,从而发挥对糖尿病大鼠的肾脏保护功能。
Diabetic nephropathy (DN) is one of the most common and serious microvascular complications of diabetes, as well as the leading cause of death in diabetics. According to statistics, diabetes have become the most common single cause of end-stage renal disease (ESRD) in the United States and Europe, about 20-30% of type 1 and type 2 diabetics develop to DN. Metabolic disorder caused by hyperglycaemia is the initiating factor of diabetic renal damage, but the effective control of blood glucose can not completely prevent the occurrence and development of diabetic complications.
In recent years the studies of diabetes and its complications focused on the mechanism of oxidative stress and antioxidant therapy. Oxidative stress is a result of reactive oxygen species (ROS) generation increase and/or removal decrease, leading to ROS accumulation in vivo, and then causes damage on biomolecules, cells, tissues, organs, and/or bodies. Normally, the generation and elimination of ROS is in a dynamic equilibrium, but the sustained hyperglycaemia in diabetes will significantly result in increased generation of mitochondrial reactive oxygen species while the capacity of antioxidant decrease, leading to excessive accumulation of free radicals in the body. The unified theory of the pathogenesis of diabetic complications hypothesized that excessive generation of oxygen free radicals, caused by oxidative stress, is the key of the occurrence and development of various complications in diabetes. Considering that the kidney is one of the organs which are highly sensitive to oxidative stress, it is widely accepted that ROS can cause renal damage through a direct or indirect means. As a matter of fact, a variety of anti-oxidants have currently been applied to prevent free radicals from damaging the body, such as glutathione, vitamin A, C, E, B1, B2, B6, B12, a-lipoic acid, coenzyme Q10, bioflavonoids, and so on. In addition, some diabetic drugs applied in clinic, such as thiazolidinediones, TZDs, statins, angiotensin-converting enzyme inhibitors, and angiotensin receptor antagonists were found to have different degrees of antioxidant activities.
Hsian-tsao(Mesona procumbens Hemsl.) is a traditional Chinese herb used in medicine and daily food. Modern research found Hsian-tsao extracts has a significant anti-oxidation function. "Chinese Medicine Dictionary" said that Hsian-tsao can "rule heat stroke, diabetes, hypertension, muscle, and joint pain". Huang Qin reported that Hsian-tsao agent is effective to reduce hyperglycaemia and to improve the clinical symptoms in diabetic patients, but the mechanism remains elusive.
In the present study, effects of Hsian-tsao water extracts on blood glucose, blood lipids, and renal function were systematically observed in Streptozotocin(STZ)-induced diabetic rats at the whole-body, cellular, and molecular level by various approaches, such as pathological observation, immunohistochemistry, ELISA, and real-time fluorescence quantitative PCR after passing acute toxicity and mutagenicity the evaluation. Then we further explored the antioxidant properties of Hsian-tsao water extracts in prevention and treatment of DN and its possible mechanism. The present results provide scientific data of food-based natural antioxidants on diabetic prevention and to guide the exploitation of Hsian-tsao.
Part I Acute toxicity and mutagenicity evaluation of Hsian-tsao Water Extracts
Objective To evaluate acute toxicity and mutagenicity of Hsian-tsao water extracts.
Method Brown powders of Hsian-tsao water extracts were obtained after extraction with boiling water, vacuum concentration, drying, and pulverizing. According to the "technical specifications of food safety and toxicity evaluation" issued by the Ministry of Health of China, the toxicities of Hsian-tsao water extracts were evaluated by oral toxicity test, Ames test, mouse bone marrow micronucleus test, and mouse teratospermia test.
Results Both rats and mice were not developped any symptom of poisoning or death within 2 weeks observation period after treatment with Hsian-tsao water extracts. The oral maximum tolerance dose of the sample was higher than 24.0g/kg-body weight. Results of Ames test, polychromatic erythrocytes micronucleus test, and sperm deformity test were all negative.
PartⅡRenal Protective Activity of Hsian-tsao Water Extracts in STZ Induced Diabetic Rats
Objective To observe the influence of Hsian-tsao water extracts on the body weight, blood glucose, blood lipids, and renal damages to STZ-induced diabetic rat models, to demonstrate a antioxidant property of Hsian-tsao as a role of prevention of DN development, and to explore its possible mechanism.
Method
Preparation of Diabetic Rats and Grouping
Male SD rats with body weight between 180-200 g were fasted for 10 hours before STZ was intraperitoneally injected (60mg/kg) to induce diabetes; the control group received an injection of same dose of citrate buffer. Seventy-two hours after the injection of STZ, the blood glucose level reached≥16.7mmol/L and urine glucose was positive (+++～++++), indicating the successful induction of diabetes. They were then randomly divided into 5 groups (n=7):
(1) diabetic control group (DM);
(2) low-dose Hsian-tsao treatment group (XC-L,0.75g/kg.d);
(3) medium-dose Hsian-tsao treatment group (XC-M,1.5g/kg.d);
(4) high-dose Hsian-tsao treatment group (XC-H,3.0g/kg.d);
(5) Pioglitazone treatment group (PGLT,10mg/kg.d);
(6) normal control group (Sham).
Rats in treatment groups were given drugs by intragastric administration and the other two groups of rats were given the same amount of distilled water every day for 6 weeks. During the experiment, all the rats were fed a commercial rat feed and water ad libitum. Body weight, blood and urine glucose concentrations were then measured weekly.
Sample Collection and Examination
At the end of the study (6 weeks), all the rats were anesthetized with 1% sodium pentobarbital (40mg/kg-BW) by intraperitoneal injection. Urine, blood sample and kidney tissue were collected and subjected to biochemical measurements or histopathological studies. Blood glucose(GLU), total cholesterol(TC), triglyceride(TG), LDL cholesterol, HDL cholesterol, blood urea nitrogen(BUN), and serum creatinine(Scr) were determined by automatic biochemical analyzer. Microalbuminuria (UMA) was determined with chemoluminescence in the automatic protein analysis system. Pathological changes in kidney were observed in light microscopy (HE staining) and transmission electron microscopy. GSH-Px, SOD, and MDA levels in rat kidney homogenate were measured by commercial kits. Serum OxLDL was determined by an ELISA kit. Total RNA was extracted by TRIzol, and the expressions of TGF-β1, TSP-1 and FN mRNA were detected by real-time fluorescence quantitative PCR. The distributions of TGF-β1, TSP-1 and FN positive cells in renal tissue were detected by immunohistochemical staining.
Results
Changes of Blood Glucose and Body Weight
Diabetic rats had a significant lower body weight and higher blood glucose level versus controls (P＜0.05). The body weight of Pioglitazone and Hsian-tsao intervention rats were higher than diabetic rats, and the high dose of Hsian-tsao group showed significant differences (P<0.05), but blood glucose showed no obvious changes (P＞0.05).
Changes in Renal Function
Serum BUN, Scr and urine microalbuminuria(UMA) were significantly higher in diabetic rats compared to controls (P<0.05). UMA levels were significantly lower in Pioglitazone and Hsian-tsao groups while compared to DM group (P＜0.05), but BUN and Scr showed no obvious changes (P>0.05).
Changes in Blood lipids
Serum LDL、HDL、TG and TC in diabetic rats higher than those in control group (P＜0.05). All the blood lipids except TG were significantly lower in Pioglitazone and High dose of Hsian-tsao groups than in DM group (P＜0.05), but low dose Hsian-tsao group had no obvious changes (P>0.05).
Pathological Changes of Kidney Tissue
Kidneys of diabetic rats were enlarged, with pale and oncotic appearance. Glomerular atrophy, tubular lumen narrowing, and augmentation in capsular space could be seen by a light microscope (with HE staining). It was manifested with a thicken glomerular basement membrane, swelling, shorten and confluence of foot process of podocyte, expansion of mitochondriae of parietal epithelium, and increase of collagen of glomerulus in the ultrastructure in diabetic kidney. In addition, swelling of microvilli and expansion of mitochondriae in the epithelium of glomerular tubule were observed. However, all the phenomena were relieved in Pioglitazone and Hsian-tsao groups.
Changes of MDA concentration and the activity of SOD and GSH-px
Compared with the control group, the concentration of MDA significantly increased in renal tissue of the STZ induced diabetic rats (P<0.05), and MDA level in high, middle and low dose Hsian-tsao groups were significantly lower than DM group (P＜0.05). Pioglitazone treatment could also reduce the MDA level, but had no statistical significance (P>0.05) ersus diabetic rats. There were no significant differences in SOD and GSH-Px among all the five groups.
Changes of Serum OxLDL
The contents of serum OxLDL in diabetic rats were significant higher than the control group. OxLDL level in Pioglitazone and middle dose Hsian-tsao were groups were significantly lower than DM group (P<0.05).
Changes in transcriptional level of cytokines
Fluorescent quantitative PCR demonstrated that the expressions of TGF-β1, TSP-1 and FN mRNA in diabetic rat kidney were significantly higher while comparing to the control group. Hsian-tsao water extracts can significantly reduce the expression of TGF-β1 and TSP-1 mRNA(P＜0.05), and showed a dose-response relationship.
Changes in protein level of cytokines
The TGF-β1, TSP-1 and FN expression in kidneys was examined by the quantitative analysis of the number of positive cells and also the optical density of the immunostaining. The number of positive cells and optical density of TGF-β1, TSP-1 and FN were detected to be much higher in the diabetic kidney than those in control group (P<0.05). The expression of all the cytokines decreased markedly under the treatment of Hsian-tsao water extracts and Pioglitazone. The medium dose of Hsian-tsao group showed the most obvious change.
Part III Effects of Hsian-tsao Water Extracts on the oxidative modification of LDL in vitro and the transcription of TGF-β1 induced by OxLDL in mesangial cells
Objective To evaluate the effect of Hsian-tsao water extracts on Cu2+-mediated LDL oxidative modification in vitro and the transcription of TGF-β1 induced by OxLDL in mesangial cells, with a hope to clarify the possible mechanisms of renal protective effect of Hsian-tsao in DM.
Methods LDL was isolated by ultracenfugation from normal human plasma. The extent of LDL peroxidation was measured in terms of thiobarbituric acid reactive substances (TBARS) expressed as MDA equivalents. The electrophoresis mobility of LDL was determined as relative electrophoresis mobility (REM) on an agarose gel electrophoresis. Rat mesangial cells were randomly divided into blank control group, oxidation-induced group (OxLDL 50μg/ml), and Hsian-tsao groups (25,50, and 125μg/ml), with vitamin E (100μmol/ml) as a positive control. The cells were incubated for 24 hours, and then total RNA was extracted with Trizol, the mRNA levels of TGF-β1, TSP-1, FN mRNA were detected by RT-PCR.
Results Hsian-tsao could protect LDL against Cu2+-mediated LDL oxidative modification in vitro. The TBARS generation and the REM in high concentration Hsian-tsao extract group showed no significant difference with blank and positive control group. Hsian-tsao water extracts (25-125μg/ml)can significantly inhibit the formation of LPO while compared to positive control group (P<0.05). The production of TGF-β1 and FN mRNA induced by OxLDL in mesangial cells were significantly lower than in Hsian-tsao treatment groups, and the inhibition was more effective than the VitE group.
Conclusion
1. Hsian-tsao water extracts has no toxicity and mutagenic effect in the study dose on the experimental animals.
2. Hsian-tsao water extracts can significantly increase the body weight in STZ-induced diabetic rats, but have no effect on blood sugar.
3. Hsian-tsao water extracts has a renal protective effect in diabetic rats.
4. Hsian-tsao water extracts has a significant effect on anti-lipid peroxidation, regulating blood lipids and inhibition of TGF-β1 expression in diabetic rats.
5. Hsian-tsao water extracts can effectively inhibit the Cu2+-mediated LDL oxidative modification in vitro.
6. Hsian-tsao water extracts can obviously inhibit the TGF-β1 transcription induced by OxLDL in mesangial cells.
Based on above results, we infer that Hsian-tsao water extracts can reduce the generation of OxLDL in diabetic rats by regulating lipids and anti-lipid peroxidation. Hsian-tsao water extracts can also inhibit the TGF-β1 transcription in the diabetic kidney independent with the blood glucose. Hsian-tsao water extracts may be able to use as a functional food or nutraceuticle to protect renal function for DM.
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