糖尿病大鼠肾脏中vWF的表达及丹红注射液的干预研究

Expression of vWF in Kidney of diabetic rats and effeet of danhong

目的 观察丹红注射液干预前后糖尿病大鼠血清血管性假血友病因子（vWF）水平及肾组织中vWF的表达变化,探讨糖尿病肾病发病机制及丹红的干预效果.方法 36只雄性SD大鼠随机分为3组：正常对照组10只、糖尿病组14只、丹红干预组12只.除正常对照组外,其余各组均按55mg/kg剂量一次性腹腔注射1％链脲佐菌素（STZ）建立糖尿病大鼠模型.72h后采尾静脉血测血糖,以血糖值＞16.7mmol/L作为糖尿病大鼠模型建立的标准.造模成功后,丹红干预组按2mL/kg.d剂量腹腔注射丹红注射液.干预为期8周.于第8周末,收集各组大鼠血液、尿液及肾组织标本.然后检测血糖（BG）、总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）、血肌酐（Scr）、尿素氮（BUN）.放射免疫法检测24h尿白蛋白定量.酶联免疫分析法（ELISA）测定各组大鼠血清vWF的水平.肾组织进行HE染色在光镜观察肾脏形态学变化.用免疫组化的方法检测各组大鼠肾脏vWF的表达情况.应用实时荧光定量PCR技术检测肾组织vWF的表达情况.结果 糖尿病组大鼠血糖、胆固醇、甘油三酯、低密度脂蛋白胆固醇、血肌酐、尿素氮、24h尿白蛋白定量、肾脏肥大指数、血vWF水平明显高于正常对照组（P＜0.01）,糖尿病大鼠肾脏出现病理改变,糖尿病大鼠肾组织vWF的表达较正常对照组大鼠明显增加;应用丹红干预8周后,干预组大鼠胆固醇、甘油三酯、低密度脂蛋白胆固醇、血肌酐、尿素氮、24h尿白蛋白定量、肾脏肥大指数、血清vWF水平较糖尿病组明显减低（P＜0.05）,肾组织病理损伤减轻,肾脏vWF表达较糖尿病组明显降低.结论 糖尿病大鼠血清vWF水平及肾组织vWF表达增加,提示vWF与糖尿病肾病关系密切,可能参与了糖尿病肾病的发生、发展;而丹红能通过调节血脂、降低尿白蛋白、降低vWF的表达来发挥肾脏保护作用.

Objectives To observe the level of serum yon Willebrand factor（vWF） and expression of vWF in Kidney of Diabetic Rats to study the change of vWF before and after the interventionof Danhong.The pathogenesis of diabetic nephropathy and renoprotective effects of Danhong are specially studied.Methods 36 SD rats were randomly divided into three groups as follows：10 normol rats,14 diabetic rats,11 Danhong intervention ratsand 11 Metformin intervention rats. （Two died in diabetic group,one died in Danhong intervention group） . Except the normal control group,all the rats in the other groups were established an animal model of diabetes by a single intraperitoneal injection of streptozotocin （55mg/kg）.After72 hours,get blood from caudal vein to test blood sugar,those higher than 16.7mmol/L regard as succeed in diabetic models.Then Danhong （2ml/kg.d） was given by intraperitoneal injection to Danhong intervention group.The same amount of water was given by intraperitoneal injection to normal control group and diabetic group.The experiment lasted for eight weeks.At the end of eight weeks,blood,urine and renal tissue samples were collected in four groups of rats.Then blood sugar,cholesterol （TC）,triglycerides （TG）,Low density lipoprotein cholesterol （LDL-C）,Serum creatinine （Scr）,Urea nitrogen （BUN） were tested.24h Urinary albuminwere measured by radioimmunoassay.Serum vWF was determined by Enzyme-linked immunoassay （ELISA）.We used HE staining to observe the renal pathological changes under optical microscope. Expression of vWF in renal tissue was measured by immunohistochemistry.Renal vWFmRNA expression was measured by Real-time fluorescent quantitative PCR.Results After eight weeks,Compared with normal control group,blood sugar,TC,TG,LDL-C,Scr,BUN,Kidney hypertrophy index,24h Urinary albumin,Serum vWF were all inereased significantlyin diabetic group（P ＜0.01）.Kidneys of Diabetic rats have obvious pathological changes.The expression of vWF in Diabetic rat kidneywas obviously increased compared with normal control group.After treated by Danhong,TC,TG,LDL-C,Scr,BUN,Kidney hypertrophy index,24h Urinary albumin,Serum vWFwerereduced significantly （P ＜ 0.05） and there was a mitigated pathomorphology change in renal tissues.The expression of vWFin rat kidney was decreased significantly.Conclusions The Serum vWF level and the expression of vWFin diabetie rats were increased significantly,which may be involved in the occurence and development of diabetic nephropathy.Danhong have renoprotective effect on diabetic nephropathy byregulating thelevels of serum lipids,reducing the level of albuminuria and suppressing vWF expression.