益气解毒活络法防治早期糖尿病肾病的实验研究

【作者】 田国伟；

【导师】 李敬林；

【作者基本信息】 辽宁中医药大学 ， 中西医结合临床， 2011， 博士

【摘要】 目的:通过观察益气解毒活络中药复方对链脲佐菌素(STZ)所致早期糖尿病肾病(DN)大鼠一般状况的影响;对药效学指标(体重、空腹血糖、糖化血红蛋白、尿微量白蛋白、血β2微球蛋白及肾脏病理改变等)变化的影响;对药物作用机制指标【血清脂联素(ADPN)、血清超敏C-反应蛋白(hs-CRP)、肾组织单核细胞趋化蛋白(MCP)-1及肾组织肿瘤坏死因子-α(TNF-α)】变化的影响,以探讨益气解毒活络中药复方防治早期糖尿病肾病的效果及作用机制,并深入探讨早期糖尿病肾病发生发展的病理机制。材料与方法:1.实验材料:选择SPF级的健康SD雄性大鼠;益气解毒活络中药复方由黄精、黄芪、黄连、虎杖、泽兰、水蛭粉组成,用免煎中药配方颗粒;西药选用盐酸贝那普利(洛汀新)片。2.动物分组及造模方法:将大鼠按体重随机分为六组,即A组-正常对照组、B组-模型空白组、C组-中药预防组、D1组-中药低剂量治疗组、D2组-中药高剂量治疗组及E组-阳性西药对照组。所有造模大鼠采用高脂高糖饮料喂养4周,在注射STZ后72小时,将空腹血糖≥16.7mmol/L确定为DM模型,纳入到正式实验中,根据血糖值高低进行随机分组。3.给药方法:正常对照组、模型空白组给生理盐水2ml;中药颗粒剂溶于蒸馏水中,中药预防组、中药低剂量治疗组及阳性西药对照组的大鼠每日用药量按人体(g/kg)的等效剂量,即6.3倍计算;中药高剂量治疗组的用药量按中药低剂量组的3倍计算。中药预防组在血糖达标后即给予中药。DM模型复制成功两周后,中药低剂量治疗组、中药高剂量治疗组及阳性西药对照组开始给药。灌胃,每日1次,给药疗程共4周。4.观测指标:药效学指标,其中包括:大鼠的一般情况、体重、血糖、糖化血红蛋白、血β2微球蛋白、24h尿微量白蛋白及肾脏组织病理形态学。观测益气解毒活络中药复方的药物作用机制指标,其中包括:血清脂联素和血清超敏C-反应蛋白,肾组织单核细胞趋化蛋白-1(MCP-1)和肿瘤坏死因子-α(TNF-α)的mRNA含量及其蛋白表达的变化。5.统计学分析:统计分析采用SPSS11.5统计软件进行分析,组间比较用单因素方差分析(One-Way ANOVE),P<0.05为有显著性差异。结果:1.对大鼠一般状况的影响:正常对照组大鼠体重明显增加,精神状态良好,反应灵敏,动作敏捷,毛色光泽。模型空白组大鼠表现为多饮、多食、多尿,精神萎靡,反应迟钝,动作迟缓,毛枯黄稀少,拱背蜷卧,2只出现尾部溃烂,2只大鼠的眼睛出现病理变化。实验组也有类似表现,经治疗后,程度均有所缓解。2.对大鼠体重的影响:模型空白组大鼠的体重在成模后4周末明显下降,与正常对照组比较有显著性差异(P<0.01);经药物干预后的各组大鼠,体重明显增加,与模型空白组比较都有显著性的差异(P<0.01)。3.对血糖的影响:模型空白组大鼠的血糖在成模后4周末比正常组明显升高,两组之间存在显著性差异(P<0.01);用药物干预后的各组大鼠,血糖值与模型空白组相比均呈显著性下降(P<0.01)。4.对糖化血红蛋白的影响:与正常对照组组比较,模型空白组大鼠的糖化血红蛋白含量明显升高,组间存在显著性差异(P<0.01);经药物干预后的各组大鼠,其糖化血红蛋白含量与模型组相比均呈显著性下降(P<0.01)。5.对24h尿微量白蛋白的影响:模型空白组大鼠24h尿微量白蛋白含量比正常对照组明显上升,组间存在显著性差异(P<0.01);经药物干预后的各组大鼠,其24h尿微量白蛋白含量明显下降,与模型空白组比较均有显著性差异(P<0.01)。6.对血β2微球蛋白的影响:与正常对照组比较,模型空白组大鼠的血β2微球蛋白含量明显上升,存在显著性的差异(P<0.01);经各药物干预后的大鼠,其血β2微球蛋白含量明显下降,与模型空白组比较均有显著性差异(P<0.01)。7.对大鼠肾脏病理变化的影响:光镜下,正常组可见正常肾小球,细胞无明显增生,系膜区较窄,毛细血管腔开放,充满红细胞。模型组系膜区增宽,肾小球囊与毛细血管襻发生粘连;肾脏皮质变窄;髓质肾小管上皮空泡变;管腔内可见蛋白样物;肾小管破坏,部分上皮细胞核消失。各中药组和西药组大鼠经治疗后,上述病变有不同程度的改善,光镜下可见部分肾小球增大,皮质变窄,髓质肾小管上皮空泡变及肾小球系膜细胞增生等改变均减轻。8.对血清脂联素的影响:模型空白组的血清脂联素含量与正常对照组相比明显下降,存在着明显的差异(P<0.01);经药物干预后,各组血清脂联素水平比模型空白组均呈现上升趋势,且与模型空白组都有显著性差异(P<0.01)。9.对血清hs-CRP的影响:模型空白组的血清hs-CRP含量明显高于正常对照组,具有显著性差异(P<0.01)。经药物干预治疗后,各组血清hs-CRP含量与模型空白组相比均下降,且与模型空白组间均存在显著性差异(P<0.01)。10.对肾组织单核细胞趋化蛋白-1的影响10.1对肾组织MCP-1mRNA含量的影响:模型空白组大鼠肾组织的MCP-1mRNA含量比正常对照组明显上升,并具有显著性差异(P<0.01);经药物干预后,各组大鼠肾组织的MCP-1mRNA含量比模型空白组明显下降(P<0.01)。10.2对肾组织MCP-1蛋白表达的影响:模型空白组大鼠肾组织的MCP-1蛋白表达比正常对照组明显上升,并具有显著性差异(P<0.01);经药物干预后,各组大鼠肾组织的MCP-1蛋白表达比模型空白组均有显著性下降(P<0.01)。11.对肾组织肿瘤坏死因子-α的影响11.1对肾组织TNF-αmRNA含量的影响:模型空白组大鼠肾组织的TNF-αmRNA含量明显上升,与正常对照组相比具有显著性差异(P<0.01);经药物干预后的各组大鼠,其肾组织的TNF-αmRNA含量表达水平比模型空白组均显著性下降(P<0.01)。11.2对肾组织TNF-α蛋白表达的影响:模型空白组大鼠肾组织的TNF-α蛋白表达比正常对照组明显上升,具有显著性差异(P<0.01);经药物干预后的各组大鼠,其肾组织的TNF-α蛋白表达水平比模型组均显著性下降(P<0.01)。结论:1.益气解毒活络中药复方对早期DN大鼠的药效学指标(大鼠的一般情况、体重、血糖、糖化血红蛋白、24h尿微量白蛋白、血β2微球蛋白及肾脏的病理改变)有显著的改善作用,能够有效控制早期DN大鼠的血糖变化、保护受损的肾组织,对早期DN有明显的预防和治疗作用。2.益气解毒活络中药复方对早期DN大鼠药物作用机制指标(血清脂联素、血清超敏C-反应蛋白、肾组织MCP-1含量及肾组织TNF-α含量)的异常变化有显著的调节作用,能够有效控制早期DN发生发展过程中的炎症反应,该中药复方防治早期DN发生发展的药理机制是通过改善上述指标的异常表达来实现。3.早期DN的发生发展与脂联素表达水平降低、hs-CRP含量升高、MCP-1表达升高及TNF-α表达升高密切相关,提示早期DN的发病机制可能与上述指标的异常变化相关。更多还原

【Abstract】 Purpose:We took the DN rat model, built with HG & HF feeding and once injection through vena caudalis with STZ as the research object, to investigate the pathological mechanism of DN in early period of occurrence and development and to approach the effect and acting mechanism of pescription of invigorate vital energy, detoxicating and activating collaterals (IDAP) in preventing and curing DN by evaluating the general condition of rats, detecting the related figure such as weight, BFS, glycosylated hemoglobin, uria albumin, serum beta 2-microglobulin and MCP-1 and TNF-αin nephridial tissue and observing the pathological change of kidney.Material and method:1.MaterialsWe took healthy and male, SD rats in SPF Level as research object. Pescription of invigorate vital energy, detoxicating and activating collaterals (IDAP) was composed of the detocting free powder of herbs including solomonseal rhizome, astragali,radix, coptis chinensis, giant knotweed rhizome, eupatorium and sanguisuge. benazepril hydrochloride pellet was taken as the positive controlling factor.2.Grouping dividing and DN rat model building:72 SD rats were randomly divided into 6 groups as following, control group (A), blank group (B), LD group (D1), HD group (D2) and positive control group (E). We built up DN rat model by 4-week HG & HF feeding and once injection through vena caudalis with STZ and recognized the model by the standard of FBG≥16.7mmol/L.3.MedicationWe daily intragastricly administrated the rats of Group A and B with NS 2ml, the rats of Group C, D1, and D2 with IDAP, drug granules dissolved in distilled water, at respectively dose as 14.7g/kg, 14.7g/kg and 44.1g/kg and the rats of group E with Benazepril Hydrochloride Tablets at dose of 1.05mg/kg. The procedure administrating with drug lasted 4weeks in total.4.Figure and Assay methodThe pharmacodynamics figure we took to observe the effect and mechanism of IDAP includes general condition, weight, blood glucose, serum beta2-microglobulin, uria-albumin (24h), pathomorphism change of nephridial tissue, hs-CRP and ADPN in serum (ELISA), MCP-1 and TNF-α.5.Statistics analysisMeasurement data was taken by using SPSS11.5 software and P<0.05 was recognized as significant difference existing.Results:1.Influences to general conditions of rats:Group A- obviously weight gained, in good mood and sensitive, agile moving, tramosericeous color pattern and no death.Group B- polydipsia, polyphagia, hyperdiuresis, depressed, dull, sluggishly moving, dry and rare fur, bending, tow rats got ulcerate in tail.Group C, D1, D2, E– similar manifestation to Group B but less in degree.2.Influences to weight: Compared to group A, result of group B is significantly lower when 4w after pattern achieved (P<0.01). Compared to group B, result of group C, D1, D2, E is significantly higher (P<0.01).3.Influences to blood glucose: 4w after pattern achieved, result of group B is significantly higher than group A (P<0.01). Compared to group B, result of group C, D1, D2, E is significantly lower (P<0.01).4.Influences to glycosylated hemoglobin: Result of group B is significantly higher than group A, interclass significant difference shown (P<0.01). Compared to group B, results of group C, D1, D2, E are all significantly lower (P<0.01).5.influences to results of uria-albumin (24h): Result of group B is significantly higher than group A, interclass significant difference shown(P<0.01). Compared to group B, results of group C, D1, D2, E are all significantly lower (P<0.01).6.Influences to Beta 2-Microglobulin in serum: Result of group B is significantly higher than group A, significant difference shown(P<0.01). Compared to group B, results of group C, D1, D2, E are all significantly lower (P<0.01).7.Influence to nephric pathological change: Observed with light microscope, nephridial tissue of group A appears normal acinus renis, no cellular proliferation, tight mesangial region, open capillary lumen, RBC suffusion. In contrast, nephridial tissue of group B appears accretion between capsula glomeruli and loop of micrangium, cortical substance narrowed, vacuolus-like change in endepidermis of nephric tubule, protein-like mas in lumina, nephric tubule ruined and cellula epithelialis nucleus partly disappeared. Nephridial tissue of group C, D1, D2 and E are all observed better than group B and all the pathological changes got improved. We can see abatement in acinus renis partly accreted, cort narrowed, vacuolus-like change in endepidermis of nephric tubule and mesangial cellular proliferation.8.Influences to serum ADPN: Result of group B is significantly lower than group A, significant difference shown(P<0.01). Compared to group B, results of group C, D1, D2, E show significantly upgrading tendency (P<0.01).9.Influences to hs-CRP in serum: Result of group B is significantly higher than group A, significant difference shown(P<0.01). Compared to group B, results of group C, D1, D2, E show significantly descending tendency (P<0.01).10.Influence to MCP-1 in nephridial tissue:A. m-RNA content: Result of group B is significantly higher than group A, significant difference shown (P<0.01). Compared to group B, results of group C, D1, D2, E show significantly descending tendency (P<0.01). B. Protein expression of MCP-1:Result of group B is significantly higher than group A, significant difference shown (P<0.01). Compared to group B, results of group C, D1, D2, E show significantly decreased (P<0.01) 11.Influence to TNF-α:A. m-RNA content: Result of group B is significantly higher than group A, significant difference shown (P<0.01). Compared to group B, results of group C, D1, D2, E show significantly descending tendency (P<0.01)B. Protein expression of TNF-α:Result of group B is significantly higher than group A, significant difference shown (P<0.01). Compared to group B, results of group C, D1, D2, E show significantly decreased (P<0.01).Conclusion:1.Pescription of invigorate vital energy, detoxicating and activating collaterals (IDAP) can obviously regulate and amend the pharmacodynamics figure of DN rats, including general condition, weight, blood glucose, glycosylated hemoglobin, 24h-urina mALB,β2- microglobulin in serum and patho-change in nephridial tissue, meanwhile IDAP can control the BG and protect nephridial tissue.2.Pescription of invigorate vital energy, detoxicating and activating collaterals (IDAP) can obviously regulate and amend the figure of drug action as ADPN, hs-CRP, MCP-1 and TNF-α. IDAP can control the inflammatory reaction during the occurrence and development of DN and its mechanism maybe take the way of regulating the above figures.3.Decrease in ADPN expression, increase in hs-CRP content and increase in expression of MCP-1 and TNF-αcan be checked in the DN rat, which indicates that the mechanism in the occurrence and development of DN in early period maybe related to the abnormal change of the above figures.更多还原