【作者】 王大鹏；

【导师】 谢春光；

【作者基本信息】 成都中医药大学 ， 中医内科学， 2009， 博士

【摘要】 目的：初步观察川芎嗪联合贝那普利对GK大鼠并发肾病的大鼠肾组织TGFβ1,MCP-1、TGFβ1 mRNA、PKC活性、PKCβ等表达水平的影响及其调控作用的可能机制；探讨DN病变程度与信号传导及细胞因子表达的关系,并进一步探讨探讨川芎嗪对糖尿病肾病肾小球硬化及肾间质纤维化的保护作用和防治机制,寻找早期治疗DN病变的中医药新方法。方法：将40只雄性GK大鼠随机分为模型组、贝那普利组、川芎嗪组、川芎嗪+贝那普利组；每组10只。模型组,按5ml·kg-1·d-1灌服生理盐水；贝那普利组,按1.6mg·kg-1·d-1灌服贝那普利悬液；川芎嗪组,按150mg·kg-1·d-1灌胃(其浓度为20mg/ml)；川芎嗪+贝那普利组；另设10只同周龄WISTER大鼠作为(空白对照组),共五组动物。川芎嗪按150mg·kg-1·d-1灌服,加灌贝那普利悬液(按1.6mg·kg-1·d-1)；共16周。实验第8周、第12周和第16周将大鼠放入代谢笼中,收集24h尿液,测定24h尿蛋白定量；治疗16周后,将大鼠用3%戊巴比妥钠腹腔麻醉后剖腹,摘取肾脏。反转录聚合酶链反应法(RT-PCR)测肾组织TGFβ1 mRNA含量,免疫组化检测肾脏MCP-1,TGFβ1和PKCβⅠ、PKCβⅡ的表达,并用[3H]PDBu结合法测定肾皮质的PKC的活性,同时测定各组大鼠体重、血糖、血脂、Scr、BUN及Ccr变化。结果：与其它组比较,模型组大鼠血糖明显增高(P<0.05),24h尿蛋白定量明显升高(P<0.05),24h尿蛋白等指标增加(P<0.05,或p<0.01)。免疫组化及RT-PCR显示模型组大鼠肾组织MCP-1、TGFβ1、TGFβ1 mRNA表达明显增加(P<0.05,或p<0.01)；模型组和治疗各组PKCm活性均较正常组明显升高(p<0.05),而PKCc活性较正常组明显降低(p<0.05)。治疗各组PKCm活性较模型组明显降低(p<0.05),PKCc活性较模型组明显升高(p<0.05)。治疗各组组间比较,PKCm活性和PKCc活性均无统计学差异(p>0.05)。模型组肾小球内PKCβⅠ表达明显高于正常组(P<0.01)：治疗组肾小球内PKCβⅠ表达明显低于DM组(P<0.01),治疗组组间肾小球内PKCβⅡ表达无明显差异(P>0.05)。结论：川芎嗪联合贝那普利能降低糖尿病血糖、TG和TC；下调肾组织TGFβ1、MCP-1、TGFβ1 mRNA表达水平,降低血清肌酐、尿素氮水平,提高内生肌酐清除率,减少尿白蛋白的排泄,改善肾组织炎症病变和抗凝作用,从而改善DN大鼠的肾功能。抑制肾皮质中PKC转位激活及PKCβⅠ、PKCβⅡ亚型的表达,抑制细胞外基质蓄积。川芎嗪联合贝那普利对实验性糖尿病大鼠具有肾保护作用,其作用机制与其抑制肾皮质PKC激活和抑制PKCβ亚型的表达和降低肾组织TGFβ1、MCP-1水平有关。更多还原

【Abstract】 Objective: To observe ligustrazine combined with benazepil tablets _prevention and cure in expression level of TGFβ1、MCP-1、TGFβ1mRNA in GK rats nephridial tissue tentatively and influences renal protein kinase C(PKC) activity and expression of PKCβisofoims in diabetic rat. To evalute the possible mechanisms of effects on diabetic neuropathy (DN) on earlier period. To probe into the connection between diabetic neuropathy and inflammatory to open out the possible mechanisms of DN for appraising and diagnosing new quideline on diabetic neuropathy on earlier period.Methods:Forty GK male rats were randomly divided into four groups:10 every groups, respectively for model group, ligustrazine group, benazepil group and ligustrazine combined with benazepil group. Drench with NS 5ml·kg-1·d-1、benazepill.6mg·kg-1·d-1、ligustrazine 150mg·kg-1·d-1、ligustrazine combined with benazepil (150mg·kg-1·d-1+1.6mg·kg-1·d-1) to rats respectively, according to the group in 16 weeks. Puting big mouses into metabolism cages in the 8th week, the 12th week and the 16th week, collecting urine to determine quantitation of Urine protein in 24 hours. Treat for 16 weeks, anaesthetize the big mouse by 3% pentobarbital,then cut open the belly of rats and pick its kidney, The mRNA levels of TGF-β1 in renal cortex were measured by reverse transcription polymerase chain reaction(RT-PCR),examine TGF-β1、MCP-1 content in renal organize by means of by means of immunohistochemistry, The PKC activity was measured by[3H] phorbol 12,13-dibutyrate([3H] PDBu) binding assay. The expression of PKCI andⅡisoforms in glomerulus was assessed by immunohistochemistry. Result:Compared with other group, the blood glucose of model group rats increase obviously (P<0.05), quantitation of 24h Urine protein rise obviously (P<0.05), degrade the express level of TGF-β1、MCP-1、TGFβ1mRNA and CPKβin renal organizes increase obviously (P<0.05,或p<0.01); ligustrazine combined with benazepil tablets can protect the kidney for lowerring blood glucose、TG and TC, degrading TGFβ-1、MCP-1、TGFβ1 mRNA express level in renal organize, decreasing serum albumin、blaood urea mitrogen and increasing creatinine clearance,at the same time, reducing Urine protein level.[3H] PDBu binding assay of group model showed a significant increase in memberane associated protein Icinase C (mPKC) activity and a significant decrease in cytosol associated protein kinase C (cPKC) activity in venal cortex compared with group normal(P<0.01); control group showed asignificant increase in cPKC activity and a significant decrease in mPKC activity compared with group DM (P<0.01); No significant difference was found between control group in renal cortical PKC activity (P>0.05). Immunohistochemistry showed a significant increase in PKCβ1 expression in glomerulus in group model,compared with in control group (P<0.01); PKCβⅠexpression in glomerulus in control group was significantly lower than in group model (P<0.01).The control group showed nodifference in glomemlar expression of PKCβⅠand renal cortical expression of PKCβⅠand CPKβⅡisoforms (P>0.05).Conclusion:ligustrazine combined with benazepil can reduce great mouse blood glucose level of diabetic rats and improve nephric tubule pathological change, moreover; it can play its roles depend on resisting inflammation and anticoagulatory function.One of the possible mechanisms of DN is to degrade TGF-β1、MCP-1 and TGFβ1mRNA and at least partially via the inhibition of PKC activation and PKCβisoform expression in renal cortex.更多还原