木犀草素通过抑制URAT1与调节Nrf2/HO-1通路改善尿酸诱导的肾脏损伤

Luteolin Alleviates Hyperuricemic Nephropathy by Inhibiting URAT1 and Regulating Nrf2/HO-1 Pathway

为了探讨木犀草素改善尿酸性肾病(hyperuricemic nephropathy,HN)的可能机制,通过14C尿酸摄取实验测定木犀草素对尿酸转运体1(uric acid transporter 1,URAT1)尿酸转运活性的影响;采用MTT法评价木犀草素对高尿酸诱导的小鼠肾小管上皮细胞损伤的保护作用;采用氧嗪酸钾联合腺嘌呤制备小鼠HN模型,测定血清尿酸、肌酐(creatinine,CR)、尿素氮(blood urea nitrogen,BUN)、丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)与谷胱甘肽(glutathione,GSH)的水平;借助H&E染色与Masson染色分别观察小鼠肾组织的病理学变化与纤维化情况;通过qRT-PCR检测肾脏URAT1、转化生长因子-β(transforming growth factor-β,TGF-β)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、E-cadherin、核因子E2-相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)和血红素加氧酶-1(heme oxygenase-1,HO-1)的mRNA表达水平;通过蛋白质免疫印迹法检测肾脏URAT1、Nrf2和HO-1的蛋白质表达水平。^(14)C尿酸摄取实验结果显示,木犀草素能剂量依赖性地抑制URAT1转运14C尿酸,其IC50值为23.42μmol/L;MTT实验结果显示,木犀草素能明显改善高尿酸诱导的细胞损伤;体内实验中,与模型组相比,木犀草素能降低模型小鼠血清尿酸、CR、BUN水平,下调肾脏URAT1的mRNA与蛋白质表达水平,并通过影响TGF-β、α-SMA、E-cadherin改善肾脏纤维化,通过上调Nrf2/HO-1通路降低肾脏氧化应激水平。总之,木犀草素可通过抑制URAT1降低血清尿酸,并通过Nrf2/HO-1通路依赖的抗氧化应激反应显著改善肾损伤和纤维化。

To investigate the mechanisms of luteolin on improving hyperuricemic nephropathy(HN),14C uric acid(UA)uptake assay was conducted to test the effect of luteolin on uric acid transporter 1(URAT1).MTT assay was carried out to evaluate the protective effect of luteolin on renal tubular epithelial cell injury induced by high UA levels.Based on the established HN mouse model,the changes of serum biochemical indexes in mice,including UA,serum creatinine(CR),urea nitrogen(BUN),malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH),were detected before and after luteolin treatment.H&E staining and Masson’s trichrome staining were employed to detect the pathological changes and renal fibrosis in mice.The mRNA expression levels of URAT1,transforming growth factor-β(TGF-β),α-smooth muscle actin(α-SMA),E-cadherin,nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1),and the protein expression levels of URAT1,Nrf2 and HO-1 were separately detected by qRT-PCR and Westernblot.The results showed that luteolin inhibited URAT1-mediated transport of 14C UA in a dose-dependent manner,with an IC50 value of 23.42μmol/L.MTT assay showed that luteolin significantly improved the cell damage induced by high UA levels.Compared with the HN group,luteolin reduced the serum UA,CR and BUN levels,down-regulated the mRNA and protein expression levels of renal URAT1,improved renal fibrosis and affected the mRNA levels of TGF-β,α-SMA and E-cadherin.Besides,oxidative stress in kidney was alleviated by up-regulating Nrf2/HO-1 pathway.In conclusion,luteolin can reduce serum UA by inhibiting URAT1,and significantly ameliorate renal injury and fibrosis due to Nrf2/HO-1 pathway-dependent antioxidant stress response.