调控Nrf2/GPX4信号通路抑制铁死亡并缓解UUO小鼠肾纤维化

Inhibitory effect of modulating Nrf2/GPX4 signaling pathway on ferrop⁃tosis and renal fibrosis in unilateral ureteral obstruction mice

目的:评估单侧输尿管梗阻(UUO)小鼠模型中调控核因子E2相关因子2(Nrf2)/谷胱甘肽过氧化物酶4(GPX4)信号通路抑制铁死亡及肾脏纤维化的作用,检测慢性肾脏病(CKD)患者肾脏组织相关铁死亡及纤维化指标的表达情况。方法:收集9例CKD患者及9例非CKD患者肾穿标本石蜡切片,免疫组化法检测GPX4、Nrf2和α-平滑肌肌动蛋白(α-SMA)的表达水平。动物实验中,25只6~8周龄雄性C57BL/6小鼠采用简单随机抽样方法分为5组:假手术组、UUO模型组、UUO+liproxstatin-1(Lip-1;铁死亡抑制剂)组、UUO+萝卜硫素(SFN;Nrf2激动剂)组、UUO+厄贝沙坦(IRB)组,每组5只。正常饲料喂养1周,后3组分别腹腔注射Lip-1(10 mg·kg^(−1)·d^(−1))、SFN(25 mg·kg^(−1)·d^(−1))和IRB(20 mg·kg^(−1)·d^(−1))。取血清标本测定血清肌酐和尿素氮;肾脏组织石蜡包埋后行HE、Masson和Sirius red染色观察肾脏病理改变;Western blot、实时荧光定量PCR和免疫组化法检测小鼠肾脏组织Nrf2、GPX4、溶质载体家族7成员11(SLC7A11)、纤连蛋白(Fn)、I型胶原(Col-I)、α-SMA和NADPH氧化酶4(NOX4)的表达。结果:免疫组化结果显示,CKD患者肾脏组织石蜡切片GPX4和Nrf2表达低于非CKD患者,而α-SMA表达高于非CKD患者。动物实验中,与UUO组相比,3组用药组肾功能有改善,肾脏纤维化程度及相关指标表达减少;Lip-1和SFN用药组肾脏组织中Nrf2、GPX4和SLC7A11表达水平高于UUO组(P<0.05),NOX4表达水平低于UUO组(P<0.05)。结论:CKD患者及UUO小鼠肾脏纤维化及铁死亡显著增加;调控Nrf2/GPX4信号通路可抑制铁死亡并有效减轻UUO小鼠肾脏纤维化。

AIM:To investigate the inhibitory effect of modulating nuclear factor E2-related factor 2(Nrf2)/glutathione peroxidase 4(GPX4)signaling pathway on ferroptosis and renal fibrosis in unilateral ureteral obstruction(UUO)mice.METHODS:The expression levels of GPX4,Nrf2 andα-smooth muscle actin(α-SMA)were measured by immunohistochemistry in paraffin sections of kidney puncture specimens from 9 chronic kidney disease(CKD)patients and 9 non-CKD patients.In animal experiments,25 male C57BL/6 mice(6 to 8 weeks old)were randomly divided into 5 groups:sham group,UUO group,UUO+liproxstatin-1(Lip-1;ferroptosis inhibitor)group,UUO+sulforaphane(SFN;Nrf2 agonist)group,and UUO+irbesartan(IRB)group.The mice in each group were fed with normal chow for 1 week,and those in the latter 3 groups were intraperitoneally injected with Lip-1(10 mg·kg^(−1)·d^(−1)),SFN(25 mg·kg^(−1)·d^(−1))and IRB(20 mg·kg^(−1)·d^(−1))respectively.Blood serum samples were collected to determine the concentration of creatinine and urea nitrogen.Kidney tissues were embedded in paraffin and stained with HE,Masson and Sirius red to observe the renal pathological changes.Western blot,RT-qPCR and immunohistochemistry were applied to determine the expression levels of Nrf2,GPX4,solute carrier family 7 member 11(SLC7A11),fibronectin(Fn),collagen type I(Col-I),α-SMA,and NADPH oxidase 4(NOX4)in mouse kidney tissues.RESULTS:Immunohistochemical results showed that the expres⁃sion levels of GPX4 and Nrf2 were lower in CKD patients than those in non-CKD patients,while the expression ofα-SMA was higher than that in non-CKD patients.In animal experiments,the mice in the 3 treatment groups had improved renal function,reduced fibrosis,and decreased expression of renal fibrosis-related indexes compared with UUO group.The ex⁃pression levels of Nrf2,GPX4 and SLC7A11 in renal tissues of Lip-1 and SFN treatment groups were higher than those of UUO group(P<0.05),while the expression of NOX4 was lower than that of UUO group(P<0.05).CONCLUSION:Fi⁃brosis and ferroptosis were significantly increased in the kidneys of CKD patients and UUO mice.Regulation of Nrf2/GPX4 signaling pathway could effectively inhibit ferroptosis and attenuate kidney fibrosis in UUO mice.