摘要
目的观察活血解毒方对糖尿病大鼠视网膜中转化生长因子-β2(TGF-β2)、Smad2及基质金属蛋白酶9(MMP-9)、基质金属蛋白酶2(MMP-2)、Ⅳ型胶原表达的影响,从TGF-β/Smads信号转导通路调控基质代谢的角度探讨活血解毒方改善糖尿病大鼠视网膜病变的可能作用机制。方法 SPF级雄性SD大鼠12只,大鼠适应性喂养1周后,按照随机数字表法选取8只大鼠以65 mg/kg的剂量一次性腹腔注射链脲佐菌素(STZ),诱导糖尿病模型。剩余4只作为正常组注射等量的柠檬酸钠溶液。1周后尾静脉采血检测血糖,以禁食12 h血糖≥16.7 mmol/L为糖尿病模型造模成功。将8只糖尿病模型大鼠按照拉丁方设计,分为模型组和活血解毒方组,各4只。活血解毒方组给予活血解毒方7.70 g/kg,1次/d,正常组和模型组予以等体积纯净水,持续16周。采用Real-time PCR法检测大鼠视网膜中TGF-β2mRNA及Smad2 mRNA的表达;Western blotting检测MMP-9、MMP-2蛋白的表达;免疫组织化学法检测Ⅳ型胶原的表达。结果 3组大鼠视网膜中TGF-β2mRNA、Smad2 mRNA表达比较,差异均有统计学意义(F=6.232,P=0.034;F=9.518,P=0.014),其中模型组和活血解毒方组视网膜中TGF-β2mRNA、Smad2 mRNA表达水平均高于正常组;活血解毒方组视网膜中TGF-β2mRNA、Smad2 mRNA表达水平均低于模型组(P<0.05)。3组大鼠视网膜中MMP-9、MMP-2蛋白表达比较,差异均有统计学意义(F=17.387,P=0.001;F=8.593,P=0.008),其中模型组和活血解毒方组视网膜中MMP-9、MMP-2蛋白表达均高于正常组(P<0.05);活血解毒方组视网膜中MMP-9、MMP-2蛋白表达均低于模型组(P<0.05)。正常组大鼠视网膜中Ⅳ型胶原表达低,呈淡棕色;模型组:视网膜Ⅳ型胶原表达升高,呈深棕褐色;活血解毒方组:视网膜Ⅳ型胶原表达呈淡褐色,较模型组表达降低。结论活血解毒方可能通过下调TGF-β2mRNA及Smad2 mRNA的表达,干预TGF-β/Smads信号转导通路调控基质代谢,使MMP-9、MMP-2、Ⅳ型胶原的表达下调,从而改善糖尿病视网膜病变。
Objective To observe the effects of Huoxue Jiedu Recipe（ HJR） on TGF- β2,Smad2,MMP- 9,MMP- 2,and collagen Ⅳ in diabetic rats with retina to explore possible mechanism of HJR in improving retinopathy of diabetic rats through TGF- β / Smads signal transduction pathways regulating matrix metabolism. Methods 12 SPF male SD rats were enrolled in this study. Eight rats,chosen randomly after 1 week of adaptive feeding,were given single intraperitoneal injection of streptozotocin（ STZ） in a dose of 65 mg / kg,to induce diabetic models. The rest 4 rats,as control group,were given equal amount of sodium citrate. The 8 diabetic models were divided,according to latin square design,into groups model,HJR,4 in each. HJR group were given HJR,7. 70 g / kg,1 time / d,groups control and model given equal volume of purified water,16 weeks. Real- time PCR method was used to determine the expressions of TGF- β2mRNA and Smad2 mRNA, the Western blotting used to detect the expressions of MMP- 9,MMP- 2 proteins,immunohistochemistry used to measure the expression of collagen Ⅳ. Results There was significant difference in the expressions of TGF- β2mRNA and Smad2 mRNA among 3 groups（ F = 6. 232,P = 0. 034; F = 9. 518,P = 0. 014）,thereinto higher in groups model and HJR than in control group,lower in HJR group than in model group（ P〈0. 05）. There was difference in MMP- 9,MMP- 2 expressions among 3 groups（ F =17. 387,P = 0. 001; F = 8. 593,P = 0. 008）,thereinto higher in groups model and HJR than in control group（ P〈0. 05）,lower in HJR group than in model group（ P〈0. 05）. The expression of collagen Ⅳ was low,assuming hazel in control group;high,assuming dark brown in model group; lower in HJR group than in model,assuming pale brown. Conclusion HJR can intervene,by down- regulating the expressions of TGF- β2mRNA and Smad2 mRNA, TGF- β / Smads signal transduction pathways regulating matrix metabolism to down- regulate the expressions of MMP- 9,MMP- 2,collagen Ⅳ to improve diabetic retinopathy.
出处
《中国全科医学》
CAS
CSCD
北大核心
2015年第5期507-512,共6页
Chinese General Practice
基金
国家科技部“十二五”支撑计划项目(2012BAI29B07)
北京中医药大学在读研究生项目(2014-JYBZZ-XS-024)
