虾青素通过CCN1调节肾间质纤维化的潜在分子作用机制

Potential molecular mechanism of astaxanthin regulating renal interstitial fibrosis through CCN1

目的探讨虾青素(AST)对肾间质纤维化的潜在分子作用机制,为临床防治肾脏纤维化提供数据。方法动物实验采用单侧输尿管结扎(UUO)法建立肾间质纤维化模型。60只雄性C57BL/6小鼠随机分为6组(10只/组):假手术组、假手术+AST(200 mg/kg)组、UUO模型组、UUO+AST(50 mg/kg)组、UUO+AST(100 mg/kg)组、UUO+AST(200 mg/kg)组。细胞实验采用TGF-β1诱导HK-2细胞株上皮间充质转化(EMT)模型。质粒转染实验将HK-2细胞分为7组:对照组、空白组、CCN1过表达组、CCN1敲低组、TGF-β1诱导组、TGF-β1+CCN1过表达组、TGF-β1+CCN1敲低组。AST处理实验将HK-2细胞分为5组:对照组、TGF-β1诱导组、TGF-β1+AST组、TGF-β1+CCN1敲低组、TGF-β1+CCN1敲低+AST组。实时定量荧光PCR法检测肾组织肾损伤生物标志物中性粒细胞明胶酶相关脂质运载蛋白(NGAL)、肾损伤分子-1(KIM-1)、血清胱抑素-C(Cys-C)和白介素-18(IL-18)。Western印迹检测肾组织和细胞的纤维化标志物Ⅰ型胶原蛋白(Col-Ⅰ)、纤连蛋白(FN)、α-平滑肌肌动蛋白(α-SMA)、波形蛋白和E-钙粘蛋白(E-cad),以及TGF-β1/Smad和Notch信号通路分子:pSmad3、Notch1、Notch3、Jagged-1的表达。结果与假手术组相比,UUO组小鼠肾损伤生物标志物NGAL、KIM-1、Cys-C和IL-18的水平显著升高;肾脏纤维化标志物蛋白表达水平显著升高,E-cad表达水平显著下降;pSmad3、Notch1、Notch3、Jagged-1的蛋白表达水平显著增加,Smad7表达显著减少。AST处理使肾损伤生物标志物(NGAL、KIM-1、Cys-C和IL-18)、纤维化标志物蛋白表达水平显著下降,E-cad蛋白表达显著上升且呈剂量依赖性。细胞实验结果显示:CCN1过表达可以抑制TGF-β1/Smad和Notch信号通路的激活,而敲低CCN1不仅增强了TGF-β1/Smad和Notch信号通路的激活,还减弱了AST在TGF-β1诱导的EMT模型中的保护作用。结论AST可能通过CCN1调节TGF-β1/Smad和Notch信号通路,从而可能具有减轻肾间质纤维化、保护肾脏的作用。

Objective To explore the potential molecular mechanism of astaxanthin(AST)in renal interstitial fibrosis and provide data for clinical prevention and treatment of renal fibrosis.MethodsIn animal experiment,unilateral ureteral ligation(UUO)was used to establish renal interstitial fibrosis model.60 male C57BL/6 mice were randomly divided into six groups with 10 mice each,including sham operation group,sham operation+AST 200mg/kg group,UUO group,UUO+AST 50,100,200 mg/kg groups.In cell experiments,the HK-2 cell line epithelial-mesenchymal transition(EMT)model was induced by TGF-β1.Plasmid transfection experiments divided HK-2 cells into 7 groups:control group,blank group,CCN1 overexpression group,CCN1 knockdown group,TGF-β1 group,TGF-β1+CCN1 overexpression group,and TGF-β1+CCN1 knockdown group.The AST treatment experiments divided the HK-2 cells into 5 groups:control group,TGF-β1 group,TGF-β1+AST group,TGF-β1+CCN1 knockdown group,and TGF-β1+CCN1 knockdown+AST group.Real time-PCR was used to detect the biomarkers of kidney injury in kidney tissue:neutrophil gelatinase-associated lipocalin(NGAL),kidney injury molecule-1(KIM-1),serum cystatin-C(Cys-C),and interleukin-18(IL-18).Western blot was used to determine expression of fibrosis markers:type I collagen(Col-Ⅰ),fibronectin(FN),α-smooth muscle actin(α-SMA),vimentin,and E-cadherin(E-cad)in the cells supernatant and kidney tissues,as well as expression of TGF-β1/Smad and Notch signaling pathway molecules including pSmad3,Notch1,Notch3,and Jagged-1.ResultsCompared with the sham operation group,the UUO group showed significant higher levels of kidney injury biomarkers as NGAL,KIM-1,Cys-C,and IL-18.The UUO group also displayed significant higher expression level of renal fibrosis marker proteins as well as lower expression level of E-cad.Besides,in the UUO group,the protein expression levels of pSmad3,Notch1,Notch3,and Jagged-1 also increased significantly,and the expression of Smad7 decreased significantly.AST treatment significantly decreased the expression of biomarkers of renal injury(NGAL,KIM-1,Cys-C and IL-18)and protein expression of fibrosis markers,but increased the expression of E-cad protein in a dose-dependent manner.The cell experiments showed that overexpression of CCN1 inhibited the activation of TGF-β1/Smad and Notch signaling pathways,while knocking down CCN1 not only intensified the activation of TGF-β1/Smad and Notch signaling pathways,but also weakened the protective effect of AST in the EMT model induced by TGF-β1.ConclusionAST may regulate TGF-β1/Smad and Notch signaling pathways through CCN1,so as to reduce renal interstitial fibrosis and play a role in renal protection.