本研究从上皮间质转化(epithelial-mesenchymal transition,EMT)和NLRP3通路角度研究藏红花素(crocin,CRO)治疗糖尿病肾病(diabetic kidney disease,DKD)的效果及作用机制.将60只C57BL/6J小鼠适应性喂养1周,随机选取10只作为正常组,采取正常饲养.其余小鼠接受高脂饮食继续喂养8周,之后链脲佐菌素造模并随机平均分为模型组、MCC950组(10 mg/kg MCC950,腹腔注射)、低剂量CRO组(5 mg/kg,灌胃)、中剂量CRO组(10 mg/kg,灌胃)、高剂量CRO组(20 mg/kg,灌胃).治疗8周后,收集小鼠24 h尿液样本、血液样本、肾脏进行分析测定.采用试剂盒测定小鼠24 h尿蛋白定量(24 h-urine total protein,24 h-UTP)、血清肌酐(serum creatinine,Scr)、血尿素氮(blood urea nitrogen,BUN)评估其肾功能.对肾脏同一部位进行苏木素-伊红(hematoxylin-eosin,HE)、马松(Masson)染色,观察其病理学变化.采用实时荧光定量聚合酶链反应(real-time quantitative polymerasechain reaction,RT-qPCR)以及蛋白质印迹(Western blotting)检测EMT相关因子[上皮细胞钙黏蛋白(E-cadherin,E-cad)、波形蛋白(vimentin,VIM)、平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)、转化生长因子-β(transforming growth factor beta,TGF-β)]表达情况以评估CRO对EMT的影响,检测NOD样受体热蛋白结构域相关蛋白(NOD-like receptor thermal protein do-main associated protein 3,NLRP3)相关因子[NLRP3、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein con-taining a CARD,ASC)、半胱氨酸天冬氨酸酶 1 剪切体(cleaved-caspase-1)、白介素 1β 成熟体(mature-interleukin-1 be-ta,mature-IL-1β)、白介素18成熟体(mature-IL-18)]表达情况以评估CRO对NLRP3通路的影响.结果表明,与模型组相比,CRO治疗后,DKD小鼠Scr、BUN、24 h-UTP有不同程度降低,肾组织病理损伤有不同程度的好转,E-cad表达升高,VIM、α-SMA、TGF-β1 表达降低,NLRP3、ASC、cleaved-caspase-1、mature-IL-1β、mature-IL-18 表达下调.以上结果提示CRO可以通过抑制NLRP3通路抑制上皮细胞-间充质细胞转换EMT最终缓解DKD.
Mechanism of crocin in the treatment of diabetic kidney disease by inhibiting epithelial-mesenchymal transition through regulating NLRP3 pathway
This study aims to investigate the effect and mechanism of crocin(CRO)on diabetic kidney disease(DKD)from the perspective of epithelial-mesenchymal transition(EMT)and NLRP3 pathway.Sixty C57BL/6J mice were adaptively fed for one week and ten mice were selective as normal control group randomly.The remain mice received high-fat diet for eight weeks followed by streptozotocin(STZ)injection.After STZ modeling,the modeled mice were randomly divided into model group,NLRP3 inhibitor group(10 mg/kg,intraperitoneal injection),low-dose CRO group(5 mg/kg,gavage),middle-dose CRO group(10 mg/kg,gavage),high-dose CRO group(20 mg/kg,gavage).After eight weeks of treatment,24 h urine sam-ples,blood samples and kidneys were collected for analysis and determination.The 24 h-urine total protein(24 h-UTP),ser-um creatinine(Scr)and blood urea nitrogen(BUN)were measured by the kit to evaluate the renal function of the mice.The pathological changes of the kidney were observed by hematoxylin-eosin(HE)and Masson staining.Real-time quantitative polymerasechain reaction(RT-qPCR)and Western blotting were used to detect the expression of EMT-related factors[E-cadherin(E-cad),vimentin(VIM),alpha-smooth muscle actin(α-SMA),transforming growth factor beta(TGF-β)]to e-valuate the effect of CRO on EMT.The expression of NOD-like receptor thermal protein domain associated protein 3(NLRP3)related factors[NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),cleaved-caspase-1,mature-interleukin-1 beta(mature-IL-1β),mature-IL-18]was detected to evaluate the effect of CRO on NLRP3 pathway.The results showed that Scr,BUN,and 24 h-UTP in DKD mice treated with CRO decreased to varying degrees,and the pathologi-cal damage of renal tissue was improved to varying degrees.The expression of E-cad increased,while the expression of VIM,α-SMA,and TGF-β1 decreased.The expression of NLRP3,ASC,cleaved-caspase-1,mature-IL-1β and mature-IL-18 were down-regulated.These results suggest that CRO inhibits EMT by inhibiting NLRP3 pathway to alleviate DKD.
万方数据
维普
