雷公藤多苷对糖尿病肾病大鼠肾小管间质激活素A表达及转分化的研究机制

发布时间：2018-01-15 13:01

本文关键词：雷公藤多苷对糖尿病肾病大鼠肾小管间质激活素A表达及转分化的研究机制　出处：《山西医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：通过链脲佐菌素（STZ）诱导的糖尿病肾病（DN）实验大鼠动物模型的建立，观察高糖刺激下肾脏组织中激活素A(Act-A)的表达水平及肾小管间质上皮转分化相关蛋白α平滑肌肌动蛋白（α-SMA）、E-钙粘蛋白(E-cad）和肾脏纤维化的相关蛋白Ⅳ型胶原（Co-IV）的表达情况，用雷公藤多苷片对DN大鼠进行干预实验后，观察上述蛋白表达的变化情况，进一步探讨糖尿病肾病肾小管间质上皮转分化及肾脏纤维化的发病机制，以及雷公藤多苷片对这一病理过程的保护作用机制。方法：（1）选取清洁级Wistar雄性大鼠40只，体重180-200g，6周龄，随机分为正常对照组(A组)、糖尿病肾病模型组(B组)、厄贝沙坦治疗组（C组）、雷公藤多苷治疗组（D组），每组实验大鼠10只，正常对照组的实验大鼠给予腹腔注射等剂量的柠檬酸缓冲液，其余各组实验大鼠给予腹腔注射链脲佐菌素（STZ，55mg/kg），诱导成糖尿病模型，腹腔注射72小时后，尾静脉采血，以连续三次随机血糖（BG）≥16.7mmol/L,尿糖≥4+作为糖尿病大鼠模型建立成功的标准。糖尿病模型造模成功四周后检测24小时尿蛋白定量，测定24小时尿蛋白大于30mg时即认为糖尿病肾病的模型建模成功。（2）各组实验大鼠成模后，雷公藤多苷治疗组的实验大鼠给予雷公藤多苷混悬液0.5mg/100g.d灌胃治疗，厄贝沙坦阳性对照组的实验大鼠给予厄贝沙坦混悬液17.5mg/kg.d灌胃治疗，正常对照组和糖尿病肾病模型组的实验大鼠给予等量蒸馏水灌胃。（3）实验第4、8、12、16周末秤取大鼠体质量、尾静脉采血测定血糖，将代谢笼收集好的24小时尿液检测24h尿蛋白(24hPro)定量，第16周末腹腔麻醉处死大鼠，开胸后心尖部抽血，分离血清，检测血肌酐(Scr)和血糖，，切取肾脏组织并称重，计算肾脏肥大指数；取大鼠肾脏组织行HE、PAS染色，进行病理组织学观察。（4）用免疫组化法检测肾小管间质中激活素A（Act-A）、α平滑肌肌动蛋白(α-SMA）、Ⅳ型胶原（Co-IV）及E-钙粘蛋白(E-cad）的表达。（5）采用实时荧光定量PCR技术检测肾小管间质中激活素A（Act-A）、α平滑肌肌动蛋白(α-SMA）、Ⅳ型胶原（Co-IV）及E-钙粘蛋白(E-cad）蛋白mRNA的表达。结果：（1）16周后厄贝沙坦治疗组及雷公藤多苷治疗组的实验大鼠24h尿蛋白、血肌酐（Scr）和肾脏肥大指数均低于糖尿病肾病模型组(P均＜0.05)，其中厄贝沙坦治疗组和雷公藤多苷治疗组之间比较，差异无统计学意义。（2）正常对照组的实验大鼠肾小管形态、结构、分布以及肾小球结构，均未出现明显的病理改变。与糖尿病肾病模型组比较，厄贝沙坦治疗组和雷公藤多苷治疗组的实验大鼠体质量增加，病理切片均显示肾小管间质病变程度减轻，厄贝沙坦治疗组和雷公藤多苷治疗组之间比较，差异无统计学意义。（3）与糖尿病肾病模型组比较，免疫组化和实时荧光定量PCR均显示，厄贝沙坦治疗组和雷公藤多苷治疗组肾小管间质激活素A（Act-A）、α平滑肌肌动蛋白(α-SMA）和Ⅳ型胶原（Co-IV）的表达明显下降(P＜0.01)，E-钙粘蛋白(E-cad）的表达明显上升(P＜0.01)，但厄贝沙坦治疗组和雷公藤多苷治疗组之间比较，差异无统计学意义。结论：雷公藤多苷（TWP）可以降低糖尿病肾病24小时尿蛋白的排泄，改善肾小管间质的损伤，具有一定的肾脏保护作用，激活素A（Act-A）在糖尿病肾病中具有诱导肾小管间质上皮细胞转分化的作用，同时能促进肾脏发生纤维化，通过下调激活素A（Act-A）的表达，从而阻碍肾小管间质上皮细胞的转分化，延缓肾脏发生纤维化的进程。
[Abstract]:Objective: by streptozotocin (STZ) induced diabetic nephropathy (DN) to establish the experimental rat model of animal, were stimulated by high glucose in the kidney tissues of activin A (Act-A) and the expression level of renal tubular interstitial epithelial transdifferentiation of alpha smooth muscle actin related protein (-SMA alpha), E- cadherin (E-cad) and related proteins in renal fibrosis collagen type IV (Co-IV) expression, intervention experiment of DN rats with Tripterygium wilfordii tablets, observed the protein expression changes, further study of diabetic renal interstitial epithelial transdifferentiation and renal fibrosis in the pathogenesis and mechanism of Tripterygium the protective effect of the pathological process.
Methods: (1) select clean level 40 male Wistar rats, weight 180-200g, 6 weeks old, were randomly divided into normal control group (A group), diabetic nephropathy model group (group B), irbesartan treatment group (group C), Tripterygium wilfordii group (D group), each group of rats 10 rats, citric acid buffer in rats of normal control group received intraperitoneal dose, the rest rats were given intraperitoneal injection of streptozotocin (STZ, 55mg/kg), diabetic model induced by intraperitoneal injection, after 72 hours, the tailvein, with three consecutive random blood glucose (BG) is more than 16.7mmol/L, more than 4+ of urine as a diabetic rat model was established successfully. The standard of quantitative measurement of 24 hours urinary protein in diabetic model rats after four weeks, the determination of 24 hours urine protein greater than 30mg that the model of diabetic nephropathy.
(2) in experimental rats after modeling, rats GTW treatment group given Tripterygium wilfordii 0.5mg/100g.d gavage treatment, the rats of irbesartan in the positive control group given irbesartan 17.5mg/kg.d gavage treatment, the normal control group and diabetic nephropathy model group rats given the equal volume of distilled water.
(3) the 4,8,12,16 weekend weighed the body mass of rats, blood glucose of tail vein was 24 hours urine protein and 24h urine will collect good metabolic cages (24hPro) quantitative, sixteenth weeks were anesthetized rats were sacrificed after thoracotomy, apex blood, separation of serum, blood creatinine (Scr) and blood glucose, cut kidney tissue and weighed to calculate the renal hypertrophy index; rats kidney tissue HE, PAS staining, and histopathology.
(4) detected tubulointerstitial activin A immune group (Act-A), alpha smooth muscle actin (alpha -SMA), collagen type IV (Co-IV) and E- cadherin (E-cad) expression.
(5) using real-time fluorescence quantitative PCR detection of renal tubulointerstitial activin A (Act-A), alpha smooth muscle actin (alpha -SMA), collagen type IV (Co-IV) and E- cadherin (E-cad) expression of mRNA protein.
Results: (1) 16 weeks after irbesartan treatment group and Tripterygium wilfordii group rats, 24h urine protein, serum creatinine (Scr) and renal hypertrophy index were lower than those in diabetic nephropathy model group (P < 0.05), which between irbesartan treatment group and tripterygium glycosides group, no difference statistical significance.
(2) normal control group rat renal tubular morphology, structure, distribution and glomeruli, there were no significant pathological changes. Compared with diabetic nephropathy model group, irbesartan treatment group and tripterygium glycosides more body mass treatment group rats increased, pathological slices showed the degree of tubulointerstitial lesions were alleviated and the comparison between irbesartan group and twp treatment group, the difference was not statistically significant.
(3) compared with diabetic nephropathy model group, immunohistochemistry and real-time fluorescence quantitative PCR showed that irbesartan treatment group and Tripterygium wilfordii group tubulointerstitial activin A (Act-A), alpha smooth muscle actin (alpha -SMA) and collagen type IV (Co-IV) expression significantly decreased (P < 0.01) E- (E-cad), E-cadherin expression was significantly increased (P < 0.01), but between irbesartan treatment group and twp treatment group, the difference was not statistically significant.
Conclusion: tripterygium glycosides (TWP) can reduce diabetic nephropathy 24 hours urinary protein excretion, improve renal tubulointerstitial injury has some protective effect of kidney, activin A (Act-A) is induced by interstitial Epithelial Cells Transdifferentiation for renal tubule in diabetic nephropathy, and can promote kidney fibrosis. Through down-regulation of activin A (Act-A) expression, thus impeding the transdifferentiation of renal tubular epithelial cell mass, retard renal fibrosis process.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R692

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