JMJD3在小鼠药物相关性急性肾损伤中的作用

Role of JMJD3 in drug-associated acute kidney injury in mice

目的评价组蛋白去甲基化酶(JMJD3)在小鼠药物相关性急性肾损伤(AKI)中的作用。方法雄性C57BL/6小鼠24只,8~10周龄,体重20~30 g,采用随机数字表法分为4组(n=6):对照组(C组)、AKI组、JMJD3抑制剂GSKJ4对照组(GSKJ4组)和GSKJ4-AKI组。腹腔注射叶酸250 mg/kg制备药物性相关AKI模型。GSKJ4-AKI组于AKI建模前1 h、GSKJ4组于相应时点,腹腔注射GSKJ420 mg/kg。AKI建模后72 h时,取血液标本,测定血清BUN和Cr浓度;随后处死小鼠,取肾组织,并采用HE与PAS染色法,检测肾组织病理学形态,进行肾小管损伤评分;采用TUNEL法检测肾组织凋亡细胞数,蛋白免疫印迹法检测JMJD3、Bax和cleaved caspase-3的表达,免疫组织化学法检测JMJD3、髓过氧化物酶(MPO)、F4/80、CD3的阳性细胞数及cleaved caspase-3、Bax表达,RT-PCR法检测IL-1β、IL-6、TNF-α和单核细胞趋化蛋白-1(MCP-1)的mRNA表达。结果与C组比较,AKI组血清BUN和Cr浓度、肾小管损伤评分升高,肾组织JMJD3、MPO、F4/80和CD3的阳性细胞数增多,凋亡细胞数增多,Bax、cleaved caspase-3、JMJD3、IL-1βmRNA、TNF-αmRNA、IL-6 mRNA和MCP-1 mRNA表达上调(P<0.05),GSKJ4组上述指标差异无统计学意义(P>0.05);与AKI组比较,GSKJ4-AKI组血清BUN和Cr浓度、肾小管损伤评分降低,肾组织JMJD3、MPO、F4/80和CD3的阳性细胞数减少,凋亡细胞数减少,Bax、cleaved caspase-3、JMJD3、IL-1βmRNA、TNF-αmRNA、IL-6 mRNA和MCP-1 mRNA表达下调(P<0.05)。结论小鼠药物相关性AKI的发生机制可能与JMJD3表达上调,进而诱导细胞凋亡及炎症反应有关。

Objective To evaluate the role of histone demethylase(JMJD3)in drug-induced acute kidney injury(AKI)in mice.Methods Twenty-four male C57BL/6 mice,aged 8-10 weeks,weighing 20-30 g,were divided into 4 groups(n=6 each)using a random number table method:control group(C group),AKI group,a specific JMJD3 inhibitor GSKJ4+control group(GSKJ4 group),and GSKJ4-AKI group.Folic acid 250 mg/kg was injected intraperitoneally to develop AKI model.GSKJ420 mg/kg was intraperitoneally injected at 1 h before developing AKI model in GSKJ4-AKI group and at the corresponding time point in GSKJ4 group.Blood samples were collected at 72 h after development of AKI model for determination of serum BUN and Cr concentrations.The animals were then sacrificed and renal tissues were collected for microscopic examination of histopathological morphology(using HE and PAS staining)and for determination of cell apoptosis(by TUNEL)and expression of JMJD3,Bax and cleaved caspase-3(by Western blot),the number of JMJD3,myeloperoxidase(MPO),F4/80 and CD3 positive cells,expression of cleaved caspase-3 and Bax,and expression of IL-1β,IL-6,TNF-αand monocyte chemotactic protein 1(MCP-1)mRNA(by reverse transcription polymerase chain reaction).The damage to the renal tubules was scored.Results Compared with C group,the serum BUN and Cr concentrations and renal tubular damage score were significantly increased,the number of JMJD3,myeloperoxidase(MPO),F4/80 and CD3+positive cells was increased,the number of apoptotic cells was increased,and the expression of Bax,cleaved caspase-3,JMJD3 and IL-1β,TNF-α,IL-6 and MCP-1 mRNA was up-regulated in AKI group(P<0.05),and no significant change was found in the parameters mentioned above in GSKJ4 group(P>0.05).Compared with AKI group,the serum BUN and Cr concentrations and renal tubular damage score were significantly decreased,the number of JMJD3,myeloperoxidase(MPO),F4/80 and CD3 positive cells was decreased,the number of apoptotic cells was decreased,and the expression of Bax,cleaved caspase-3,JMJD3,and IL-1β,TNF-α,IL-6 and MCP-1 mRNA was down-regulated in GSKJ4-AKI group(P<0.05).Conclusions The mechanism of drug-associated AKI may be related to up-regulation of JMJD3 expression and thus induces cell apoptosis and inflammatory responses in mice.