过表达THEM4对糖尿病小鼠肾脏细胞外基质沉积的影响

Role of up-regulation of THEM4 on extracellular matrix accumulation in kidney of diabetic mice

目的观察过表达硫酯酶超家族成员4(thioesterase superfamily member 4,THEM4)对糖尿病(diabetes mellitus,DM)小鼠肾脏细胞外基质(extracellular matrix,ECM)沉积的影响。方法将♂CD1小鼠随机分为4组:分别为正常对照组(Control组)、糖尿病组(DM组)、糖尿病+THEM4质粒组(DM+THEM4)、糖尿病+空质粒组(DM+V)。后3组腹腔注射链脲佐菌素(streptozotocin,STZ)诱发糖尿病小鼠模型,质粒注射组小鼠在成模4周后,分别给予尾静脉快速注射p Yr-ads-4-THEM4质粒或p Yr-adshuttle-4质粒(1 mg·kg^(-1))。此后每隔7 d注射1次,共注射4次,之后处死小鼠并收集保存标本,Western blot检测THEM4、phospho-Akt(Ser 473)、TGF-β1、α-SMA的表达;免疫组化检测THEM4、phospho-Akt(Ser 473)、纤维黏连蛋白(fibronectin,FN)及Ⅲ型胶原(collagenⅢ,ColⅢ)蛋白表达和定位。结果DM小鼠肾脏THEM4 mRNA和蛋白均呈低表达,而phospho-Akt(Ser 473)、TGF-β1、α-SMA蛋白的表达明显增强,并出现ECM沉积。过表达的THEM4能够逆转DM小鼠肾脏phospho-Akt(Ser 473)蛋白活化,并下调α-SMA、TGF-β1的表达,减轻DM小鼠肾脏ECM的沉积,降低小鼠尿蛋白水平。结论过表达THEM4能够减轻DM小鼠肾脏ECM的沉积,可能是通过抑制phospho-Akt(Ser 473)蛋白的活化,下调α-SMA、TGF-β1表达而实现的。

AimTo observe the effect of thioesterase superfamily member 4(THEM4)expression on extracellular matrix(ECM)accumulation in the kidney of diabetic mice.MethodsFor in vivo vector delivery experiment,male CD1 mice were randomly divided into four groups:normal control mice(Control group),diabetic mice(DM group),diabetic mice receiving p Yr-ads-4-THEM4 vector(DM+THEM4 vector)and diabetic mice receiving p Yr-adshuttle-4 vector(DM+V vector).p Yr-ads-4-THEM4 vector or p Yr-adshuttle-4 vector(1 mg·kg^(-1))were mixed with Trans IT-EE Hydrodynamic Delivery Solution from Mirus Co.and injected into tail vein once a week for four weeks after STZ injection.Four weeks later,mice were sacrificed and Western blot,immunohistochemistry and real-time PCR were used to detect the expression of phospho-Akt(Ser 473),THEM4,TGF-β1,α-SMA,ColⅢ,FN proteins and THEM4 mRNA in kidneys respectively.Results THEM4 decreased in kidney of diabetes mellitus accompanied with increased phospho-Akt(Ser473),TGF-β1,α-SMA and ECM.The delivery of p Yr-ads-4-THEM4 vector increased THEM4 expression and decreased phospho-Akt(Ser 473),TGF-β1,α-SMA and ECM deposit in kidneys of diabetic mice.ConclusionThe up-regulation of THEM4 may prevent ECM deposit by inhibiting the phosphorylation of Akt and down-regulating the expression of TGF-β1 andα-SMA in kidneys of diabetic mice.