论文部分内容阅读
目的:评价含十字形结构域蛋白3(JMJD3)在顺铂致急性肾损伤小鼠肾纤维化中的作用。方法:健康C57BL/6雄性小鼠48只,8~10周龄,体重20~30 g,采用随机数字表法分为4组(n n=12):对照组(CON组)、对照+ JMJD3抑制剂组(CON-A组)、顺铂组(CIS组)和顺铂+ JMJD3抑制剂组(CIS-A组)。CIS组和CIS-A组分别于第1和14天时腹腔注射顺铂15 mg/kg,制备急性肾损伤小鼠肾纤维化模型;第4天时分别腹腔注射JMJD3抑制剂GSKJ4 10 mg/kg和等容量PBS,间隔3 d注射1次,共注射6次。CON组和CON-A组分别在相应时点腹腔注射等容量PBS和GSKJ4 10 mg/kg。每组取6只小鼠,于第1次注射顺铂后第3天,采集眼眶血检测血清Cr和BUN的浓度,然后处死取肾组织,行HE和PAS染色,光镜下进行观察并行肾损伤评分。第1次注射顺铂后第28天处死6只小鼠,取肾组织,行天狼星红和Masson染色,测定肾纤维化面积;采用免疫荧光法测定纤维连接蛋白、Ⅰ型胶原蛋白和α-平滑肌肌动蛋白(α-SMA)表达水平,免疫组化法行F4/80n +细胞和CD3n +细胞计数,RT-PCR法检测IL-6、TNF-α、趋化因子CXC配体16(CXCL16)和单核细胞趋化蛋白1(MCP-1)的mRNA表达水平。n 结果:与CON组比较,CIS组血清BUN、Cr浓度和肾损伤评分升高,肾纤维化面积增加,肾组织纤维连接蛋白、Ⅰ型胶原蛋白和α-SMA表达上调,CD3n +细胞和F4/80n +细胞计数升高,IL-6、CXCL16、TNF-α、MCP-1的mRNA表达上调(n P0.05);与CON-A组比较,CIS-A组BUN、Cr浓度和肾小管评分升高,肾纤维化面积增加,肾组织纤维连接蛋白、Ⅰ型胶原蛋白和α-SMA表达上调,CD3n +细胞和F4/80n +细胞计数升高,IL-6、CXCL16、TNF-α、MCP-1的mRNA表达上调(n P<0.05) ;与CIS组比较,CIS-A组血清BUN、Cr浓度和肾损伤评分降低,肾纤维化面积减少,肾组织纤维连接蛋白、Ⅰ型胶原蛋白和α-SMA表达下调,CD3n +细胞和F4/80n +细胞计数降低,IL-6、CXCL16、TNF-α、MCP-1的mRNA表达下调(n P<0.05)。n 结论:JMJD3参与了急性肾损伤小鼠肾纤维化的过程,其机制可能与促进炎症反应有关。“,”Objective:To evaluate the role of Jumonji domain-containing 3 (JMJD3) in cisplatin-induced renal fibrosis following acute kidney injury in mice.Methods:Forty-eight healthy C57BL/6 male mice, aged 8-10 weeks, weighing 20-30 g, were divided into 4 groups (n n=12 each) using a random number table method: control group (group CON), control plus JMJD3 inhibitor group (group CON-A), cisplatin group (group CIS), and cisplatin plus JMJD3 inhibitor group (group CIS-A). In group CIS and group CIS-A, cisplatin was intraperitoneally administered on 1st and 14th days, respectively, to develop a renal fibrosis model in mice with acute kidney injury, and the JMJD3 inhibitor GSKJ4 10 mg/kg and equal volume of PBS were intraperitoneally injected on 4th day, respectively, once every 3 days, 6 injections in total.The equal volume of PBS and GSKJ4 10 mg/kg were intraperitoneally injected at the corresponding time points in group CON and group CON-A, respectively.Six mice in each group were selected, and orbital blood samples were collected on 3rd day after the first injection of cisplatin to determine the concentrations of serum creatinine (Cr) and blood urea nitrogen (BUN), then the animals were sacrificed, and kidney tissues were obtained for microscopic examination of pathological changes after HE and PAS staining (with a light microscope), and the damage to kidneys was assessed and scored.Six mice were sacrificed on 28th day after the first injection of cisplatin, and kidney tissues were removed for determination of the area of renal fibrosis (n via Sirius red and Masson staining), expression of fibronectin (Fn), collagen type Ⅰ (Col Ⅰ) and α-smooth muscle actin (α-SMA) (by immunofluorescence), F4/80 n + cell and CD3n + cell count (using immunohistochemical method), and expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), CXC chemokine ligand 16 (CXCL16), and monocyte chemoattractant protein1 (MCP-1) mRNA (by real-time polymerase chain reaction).n Results:Compared with group CON, the serum BUN and Cr concentrations, renal injury scores, and area of renal fibrosis were significantly increased, the expression of Fn, Col Ⅰ and α-SMA was up-regulated, the F4/80 n + cell and CD3n + cell count was increased, and the expression of IL-6, CXCL16, TNF-α and MCP-1 mRNA was up-regulated in group CIS (n P0.05). Compared with group CON-A, the serum BUN and Cr concentrations, renal injury scores, and area of renal fibrosis were significantly increased, the expression of Fn, Col Ⅰ and α-SMA was up-regulated, the F4/80n + cell and CD3n + cell count was increased, and the expression of IL-6, CXCL16, TNF-α and MCP-1 mRNA was up-regulated in group CIS-A (n P<0.05). Compared with group CIS, the serum BUN and Cr concentrations, renal injury scores, and area of renal fibrosis were significantly decreased, the expression of Fn, Col Ⅰ and α-SMA was down-regulated, the F4/80n + cell and CD3n + cell count was decreased, and the expression of IL-6, CXCL16, TNF-α and MCP-1 mRNA was down-regulated in group CIS-A (n P<0.05).n Conclusions:JMJD3 is involved in the process of renal fibrosis following acute kidney injury in mice, and the mechanism may be related to promotion of inflammatory responses.