贵州苗药刺梨调控SIRT1-TGFβ/Smads信号途径延缓肾纤维化的机制

Mechanism of Rosa roxbunghii,a Miao medicine,on delaying renal fibrosis by regulating SIRT1-TGFβ/Smads signal pathway

目的 探讨苗药刺梨延缓单侧输尿管、结扎术(UUO)模型大鼠肾间质纤维化的作用机制.方法 将50只SPF级雄性SD大鼠,适应性喂养1 w后,随机分为假手术组和手术组;手术组行UUO,假手术组只行输尿管分离,但不结扎.术后第2天,手术组随机分为模型组、SIRT1激动剂组、刺梨中剂量组、氯沙坦钾组,每组10只.各组于分组当天参照马氏定理设定干预剂量.氯沙坦组给予氯沙坦10 mg/(kg·d),浓度为20%;刺梨中剂量给予刺梨冻干粉3 g/(kg·d),浓度为3%;SIRT1激动剂组给予白藜芦醇25 mg/d.模型组和假手术组均同时给予同等容积无菌生理盐水灌胃.实验大鼠均每天干预1次,连续干预14 d后处死大鼠.取大鼠肾组织做苏木素-伊红(HE)、Masson染色以观察其病理变化;免疫组织化学法测定Western印迹检测肾组织转化生长因子(TGF)-β1、TGF-βⅠ型受体(TGF-βRⅠ)、Smad2、Smad3、Smad7的表达;用RT-PCR技术检测TGF-β1 mRNA、TGF-βRⅠmRNA、Smads2、Smad3、Smad7 mRNA的表达.结果 与假手术组比较,模型组病理损伤程度显著增加,丙二醛(MDA)含量增加、超氧化物歧化酶(SOD)含量降低(P<0.01),TGF-β1、TGF-βRⅠ的表达水平明显增加(P<0.05).与模型组比较各治疗组病理损伤减轻,TGF-β1、TGF-βRⅠ表达减少,Smda2、Smad3的表达降低,Smda7表达增加(均P<0.05).刺梨中剂量组与SIRT1激动剂组比较差异无显著性(P>0.05).结论 刺梨冻干粉与SIRT1激动剂(白藜芦醇)均可减轻单侧输尿管结扎肾间质纤维化大鼠的肾组织脂质过氧化物的产生,同时能增加抗氧化酶的含量,保护受损肾小管上皮细胞,有效改善肾脏纤维化病变.其机制可能与其富含维生素C、SOD、刺梨黄酮等多种抗氧化成分,激活体内SIRT1,从而活化Smda7以下调TGF-β1、TGF-βRⅠ及Smda2、Smad3的表达有关.

Objective To investigate the mechanism of Rosa roxbunghii,a Miao medicine,on delaying renal interstitial fibrosis in unilateral ureteric obstruction(UUO)rats model.Methods After one week of adaptive feeding,50 SPF male SD rats were randomly divided into sham group(10 rats)and operation group(40 rats).UUO was performed in the operation group,and ureteral separation was performed in the sham group without ligation.On the second day after operation,the operation group was randomly divided into model,SIRT1 agonist,middle dose Rosa roxburghii and losartan potassium groups,with 12 rats in each group.The intervention dose of each group was set according to Mahalanobis theorem on the day of grouping.In losartan group,losartan was given 10 mg/(kg·d)at a concentration of 20%;in the middle dose Rosa roxburghii group,Rosa roxburghii lyophilized powder was given 3 g/(kg·d)at a concentration of 3%;and in SIRT1 agonist group,resveratrol was given 25 mg/d.At the same time,the model group and sham group were given the same volume of sterile saline by irrigation.The rats were intervened once a day for 14 days and killed after 14 days.The renal tissues of rats were stained with HE and Masson to observe the pathological changes;the expressions of TGF-β1,TGF-βⅠtype receptor and Smad(2,3,7)in renal tissue were detected by Western blot;the expressions of TGF-β1 mRNA,TGF-βRⅠmRNA and Smad(2,3,7)mRNA were detected by RT-PCR.Results Compared with sham group,the pathological injury degree of model group was significantly worse,the content of MDA was higher,the content of SOD was lower(P<0.01),and the expression levels of TGF-β1 and TGF-βRⅠwere higher(P<0.05).Compared with model group,the pathological injury of each treatment group was alleviated,the expressions of TGF-β1 and TGF-βRⅠwere lower(P<0.05),the expression of Smad2/3 was lower(P<0.05),and the expression of Smad7 was higher(P<0.05).There was no significant difference between the middle dose Rosa roxbunghii lyophilized powder group and SIRT1 agonist group(P>0.05).Conclusions Both Rosa roxburghii lyophilized powder and SIRT1 agonist(resveratrol)could reduce the production of lipid peroxide in renal tissue of rats with unilateral ureteral ligation of renal interstitial fibrosis,increase the content of antioxidant enzymes,protect the damaged renal tubular epithelial cells,and effectively improve the renal fibrosis.Its mechanism might be related to its rich in vitamin C,SOD,Rosa roxburghii flavonoids and other antioxidants,activating SIRT1 in vivo,thus activating Smad7 to down regulate the expressions of TGF-β1,TGF-βRⅠand Smad2/3.