地龙多肽类组分对SHR肾脏TLR4/NF-κB信号通路的影响

Effect of Lumbricus Peptides on TLR4/NF-κB Signaling Pathway in Spontaneous Hypertensive Rat Kidney

目的:基于Toll样受体4(TLR4)/核转录因子-κB(NF-κB)信号通路观察地龙多肽类组分对自发性高血压大鼠(SHR)早期肾损害的影响及作用机制。方法:将40只SHR随机分为模型组(等容量蒸馏水灌胃),地龙多肽类组分低、中、高剂量组(126,252,504 mg·kg^-1),贝那普利组(0. 9 mg·kg^-1),每组8只;同周龄雄性同源正常血压大鼠8只为正常组,给予等容量蒸馏水灌胃。各组连续给药60 d后,酶联免疫吸附测定(ELISA)法测定大鼠24 h尿液中微量白蛋白(mAlb),N-乙酰-β-D-氨基葡萄糖苷酶(NAG)及血清血管紧张素II(AngⅡ)的水平,透射电镜观察大鼠肾脏组织超微结构的变化,蛋白免疫印迹法(Western blot)检测肾脏TLR4,NF-κB p65蛋白水平,免疫组化检测肾脏肿瘤坏死因子-α(TNF-α),白细胞介素-10(IL-10)表达。结果:与正常组比较,模型组大鼠24 h尿mAlb,NAG及血清AngⅡ水平升高(P <0. 05),TLR4,NF-κB p65蛋白表达升高(P <0. 05),TNF-α表达增多(P <0. 05),IL-10表达减少(P <0. 05)。肾脏组织透射电镜显示,模型组大鼠肾小球大部分毛细血管节段足细胞足突融合、系膜细胞增多、系膜基质增生。与模型组比较,地龙多肽类组分各剂量组及贝那普利组大鼠尿mAlb,NAG及血清AngⅡ水平均降低(P <0. 05),TLR4,NF-κB p65蛋白表达均降低(P <0. 05),TNF-α肾脏阳性表达有不同程度减少(P <0. 05),IL-10表达增加(P <0. 05)。肾脏组织透射电镜显示,给予高剂量地龙多肽类组分及贝那普利后大鼠肾脏受损表现有不同程度改善。结论:地龙多肽类组分可以减轻自发性高血压大鼠早期肾损害,其机制可能通过调控AngⅡ-TLR4/NF-κB通路实现。

Objective: to observe the effect and mechanism of lumbricus peptides on early renal injury in spontaneous hypertensive rats( SHR) based on Toll-like receptors 4( TLR4)/nuclear factor-κB( NF-κB)signaling pathway. Method: The 40 SHRs were randomly divided into model group( equal volume of distilled water by intragastric administration), lumbricus peptides low, middle and high dose groups( 126, 252,504 mg·kg^-1,and Benazepril group( 0. 9 mg·kg^-1,n = 8 in each group. 8 male rats with normal blood pressure at the same age were set as the normal control group,with equal volume of distilled water. After treatment for 60 consecutive days,enzyme-linked immunosorbent assay( ELISA) was used to determine microalbumin( mAlb) and N-acetyl-β-D-glucosaminidase( NAG) content in 24 h urine as well as the level of serum angiotensinⅡ( AngⅡ). Ultrastructural changes of rat kidneys were observed by transmission electron microscope. Western blot was used to detect renal TLR4,NF-κB p65 protein levels. Immunohistochemical method was used to detect renal tumor necrosis factor-α( TNF-α) and interleukin 10( IL-10) expression. Result: As compared with the normal control group,the levels of urine mAlb,NAG and serum Ang Ⅱ were increased in the model group( P <0. 05);the expression of TLR4 and NF-κB p65 protein was increased( P < 0. 05);expression of TNF-α was increased( P < 0. 05),while IL-10 expression was decreased( P < 0. 05). Transmission electron microscopy of kidney tissues showed that most of the glomeruli of the model group had podocyte foot process fusion,mesangial cells and mesangial matrix hyperplasia. As compared with the model group,the levels of urine mAlb,NAG,and serum Ang Ⅱ were decreased in the rats in lumbricus peptides groups and benazepril group( P < 0. 05);the expression of TLR4 and NF-κB p65 protein was decreased( P < 0. 05);the positive expression of TNF-α in kidney was decreased to different extent( P < 0. 05),but the expression of IL-10 was increased( P < 0. 05). Transmission electron microscopy of kidney tissues showed that the damage of kidneys in rats after administration of high-dose lumbricus peptides and benazepril was improved in varying degrees. Conclusion: lumbricus peptides can reduce early renal damage in SHRs,and its mechanism may be achieved by regulating the AngⅡ-TLR4/NF-κB pathway.