芪苓通络方抑制TLR4/MyD88/NF-κB信号通路改善特发性膜性肾病大鼠肾损伤

Qiling Tongluo Prescription Improves Renal Injury in Rats with Idiopathic Membranous Nephropathy by Inhibiting TLR4/MyD88/NF-κB Signaling Pathway

目的:探讨芪苓通络方基于Toll样受体4/髓样分化因子88/核转录因子-κB(TLR4/MyD88/NF-κB)信号通路对特发性膜性肾病(IMN)大鼠的治疗效果及作用机制。方法:60只雄性SD大鼠随机分为正常组、模型组、盐酸贝那普利组(10 mg·kg^(-1)),芪苓通络方低、中、高剂量组(6.48,12.95,25.9 g·kg^(-1)),通过尾静脉注射阳离子化牛血清白蛋白(C-BSA)复制IMN大鼠模型。模型制备成功后进行药物灌胃,每日1次,连续给药4周。治疗结束后,苏木素-伊红(HE)染色、马松(Masson)染色和碘酸六胺银(PASM)染色观察大鼠肾脏病理学形态变化;检测24 h尿蛋白(24 h UTP),血浆白蛋白(ALB),血清总蛋白(TP),血肌酐(SCr),尿素氮(BUN),尿酸(UA)含量水平;酶联免疫吸附测定试验(ELISA)检测白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)含量水平;实时荧光定量聚合酶链式反应(Real-time PCR),免疫组化法(IHC)和蛋白免疫印迹法(Western blot)检测大鼠肾组织中TLR4,MyD88,NF-κB p65 mRNA和蛋白表达水平。结果:与正常组相比,模型组大鼠24 h UTP水平和血清中SCr,BUN,UA,IL-1β,IL-6的含量明显升高(P<0.05,P<0.01),ALB,TP水平显著降低(P<0.01);肾组织中TLR4,MyD88,NF-κB p65 mRNA及蛋白表达量明显升高(P<0.05,P<0.01);模型组大鼠炎症反应明显,肾小球结构紊乱,体积增大,基底膜呈不规则增厚,肾间质可见炎性细胞浸润,肾小管上皮细胞数量减少、脱落和凋亡,部分呈现空泡样变性。与模型组相比,盐酸贝那普利组、芪苓通络方高剂量组大鼠血清中SCr,UA的含量明显降低(P<0.05),ALB,TP水平明显升高(P<0.05);盐酸贝那普利组和芪苓通络方低、中、高剂量组大鼠24 h UTP水平、血清中IL-1β,IL-6含量水平及肾组织中TLR4,MyD88,NF-κB p65mRNA和蛋白表达水平均有不同程度的降低(P<0.05,P<0.01);盐酸贝那普利组和芪苓通络方各剂量组能够减少IMN炎症反应,大鼠肾小球肿胀、体积增大程度减轻,肾小球毛细血管轻微扩张,系膜基质少量增生,肾间质偶见炎性细胞浸润,大鼠肾脏病理形态学损伤有不同程度的减轻。结论:芪苓通络方可能通过调控TLR4/MyD88/NF-κB信号通路,减少炎症因子的释放和表达,抑制IMN大鼠炎症反应,减少蛋白尿,减轻肾损伤,保护肾脏功能。

Objective:To explore the therapeutic effect and mechanism of Qiling Tongluo prescription against idiopathic membranous nephropathy(IMN)in rats based on Toll-like receptor 4/myeloid differentiation factor 88/nuclear transcription factor-κB(TLR4/MyD88/NF-κB)signaling pathway.Method:Sixty male SD rats were randomly divided into the normal group,model group,benazepril hydrochloride(10 mg·kg^(-1))group,and low-,medium-,and high-dose(6.48,12.95,and 25.9 g·kg^(-1))Qiling Tongluo prescription groups.The IMN rat model was established by injection of cationized bovine serum albumin(C-BSA)into the tail vein.After the model was successfully prepared,the rats were gavaged with the corresponding drugs,once a day,for four consecutive weeks.After the treatment,the pathological changes in rat kidneys were observed by hematoxylineosin(HE)staining,Masson staining,and periodic acid-silver metheramine(PASM)staining,followed by the detection of 24 h urinary total protein(24 h UTP),plasma albumin(ALB),total serum protein(TP),serum creatinine(SCr),urea nitrogen(BUN),and uric acid(UA)levels.The levels of interleukin-1β(IL-1β)and interleukin-6(IL-6)were determined by enzyme-linked immunosorbent assay(ELISA),and the mRNA and protein expression levels of TLR4,MyD88,and NF-κB in the kidney tissue were assayed by real-time fluorescent quantitative polymerase chain reaction(Real-time PCR),immunohistochemistry(IHC),and Western blot.Result:Compared with the normal group,the model group exhibited elevated 24 h UTP and serum SCr,BUN,UA,IL-1β,and IL-6(P<0.05,P<0.01),decreased ALB and TP(P<0.01),up-regulated TLR4,MyD88,and NF-κB p65 mRNA and protein expression in kidney tissue(P<0.05,P<0.01),obvious inflammation,disordered glomerular structure with enlarged volume,irregularly thickened basement membrane,inflammatory cell infiltration in the renal interstitium,reduced renal tubular epithelial cells due to shedding and apoptosis,and some vacuolar degeneration.Compared with the model group,benazepril hydrochloride and Qiling Tongluo prescription at the high dose remarkably lowered the serum SCr and UA(P<0.05)and increased ALB and TP(P<0.05).Benazepril hydrochloride and Qiling Tongluo prescription at the low,medium,and high doses down-regulated the 24 h UTP,serum IL-1βand IL-6 levels,and renal TLR4,MyD88,and NF-κB p65 mRNA and protein expression to varying degrees(P<0.05,P<0.01),alleviated IMN inflammatory reaction,glomerular swelling,and volume increase,slightly dilated glomerular capillaries,proliferated mesangial matrix,and relieved pathological and morphological damages in rat kidney,with inflammatory cell infiltration occasionally observed.Conclusion:Qiling Tongluo prescription may reduce the release and expression of inflammatory factors by regulating the TLR4/MyD88/NF-κB signaling pathway to inhibit the inflammatory response in IMN rats,ameliorate proteinuria and kidney damage,and protect kidney function.