益气活血通络方调控NR4A1/NF-κB减轻UUO大鼠炎性损伤的机制研究

Study on the Mechanism of Yiqi huoxue tongluo Recipe Regulating NR4A1/NF-κB to Reduce Inflammatory Injury in UUO Rats

目的观察益气活血通络方通过调控孤核受体4A1(Nuclear receptor subfamily 4 group A member 1,NR4A1)对单侧输尿管结扎(Unilateral uretera obstruction,UUO)大鼠炎性损伤的影响。方法健康雄性Wistar大鼠随机分为假手术组(Sham)、UUO组(UUO)、氯沙坦组(LOS)、益气活血通络方组(TCM),每组12只。UUO组、LOS组、TCM组采用单侧输尿管结扎建立肾纤维化模型。TCM组给予益气活血通络方1.92 g·kg^(-1)·d^(-1)灌胃,LOS组给予氯沙坦溶液20 mg·kg^(-1)·d^(-1)灌胃,其余两组给予生理盐水灌胃,每日1次。术后第14天,处死大鼠,收集梗阻侧肾脏,检测各组大鼠肾功能,免疫组化、蛋白印迹法(Western BLOT)检测NR4A1、核因子κB(Nuclear factor-kappa B,NF-κB)及炎症因子白介素-6(Interleukin-6,Il-6),单核细胞趋化蛋白-1(Monocyte chemoattractant protein-1,MCP-1)的表达,实时荧光定量PCR(qRT-PCR)检测NR4A1、NF-κB mRNA的表达。结果与Sham组比较,UUO组大鼠血清肌酐(Scr)、尿素氮(BUN)水平显著升高(P<0.05);经治疗,LOS组、TCM组Scr、BUN水平较UUO组显著降低(P<0.05)。免疫组化结果显示,与Sham组比较,UUO组NF-κB、IL-6、MCP-1表达显著升高(P<0.05),NR4A1表达显著降低(P<0.05);与UUO组比较,LOS组、TCM组大鼠肾组织中NF-κB、IL-6、MCP-1表达显著降低(P<0.05),NR4A1表达明显升高(P<0.05)。Western blot结果显示,与Sham组比较,UUO组NF-κB、IL-6、MCP-1蛋白表达显著升高(P<0.05),NR4A1蛋白表达显著降低(P<0.05);与UUO组比较,LOS组、TCM组大鼠肾组织中NF-κB、IL-6、MCP-1表达显著降低(P<0.05),NR4A1表达显著升高(P<0.05)。qRT-PCR结果显示,与Sham组比较,UUO组NR4A1 mRNA显著降低(P<0.05),NF-κB mRNA显著升高(P<0.05);与UUO组比较,LOS组、TCM组NR4A1 mRNA水平显著升高(P<0.05),NF-κB mRNA显著降低(P<0.05)。结论益气活血通络方可通过调控NR4A1,下调NF-κB、MCP-1与IL-6的表达,减轻UUO大鼠肾脏炎性损伤。

Objective To observe the effect of Yiqi huoxue tongluo Recipe on inflammatory injury in unilateral ureteral ligation(UUO) rats by Nuclear receptor subfamily 4 group A member 1(NR4A1).Methods Healthy male Wistar rats were randomly divided into sham operation group(Sham),UUO group(UUO),losartan group(LOS),and Yiqi huoxue tongluo Recipe group(TCM),with 12 rats in each group.The renal fibrosis model was established by unilateral ureteral ligation in UUO group,LOS group and TCM group.The TCM group was given Yiqi huoxue tongluo Recipe 1.92 g·kg^(-1)·d^(-1) by gavage,the LOS group was given 20 mg·kg^(-1)·d^(-1) of losartan solution by gavage,and the other two groups were given normal saline by gavage,once a day.On the 14th day after the operation,the rats were sacrificed,and the kidneys on the obstructed side were collected to detect the renal function of the rats in each group.Immunohistochemistry and Western blot to detect NR4A1,Nuclear factor-kappa B(NF-κB)and inflammatory factor interleukin-6(IL-6),monocyte chemotactic protein-1(MCP-1),Real-time fluorescence quantitative PCR(qRT-PCR) detection of NR4A1,NF-κB mRNA expression.Results Compared with the Sham group,the levels of serum creatinine(Scr) and blood urea nitrogen(BUN) in the UUO group were significantly increased(P0.05);after treatment,the levels of Scr and BUN in the LOS group and the TCM group were significantly lower than those in the UUO group(P0.05).The results of immunohistochemistry showed that compared with the Sham group,the expressions of NF-κB,IL-6 and MCP-1 in the UUO group were significantly increased(P0.05),and the expression of NR4A1 was significantly decreased(P0.05);compared with the UUO group,The expression of NF-κB,IL-6 and MCP-1 in kidney tissue of rats in LOS group and TCM group were significantly decreased(P0.05),and the expression of NR4A1 was significantly increased(P0.05).Western blot results showed that compared with the Sham group,the protein expressions of NF-κB,IL-6 and MCP-1 in the UUO group were significantly increased(P0.05),and the protein expression of NR4A1 was significantly decreased(P0.05).Compared with the UUO group the expressions of NF-κB,IL-6 and MCP-1 in kidney tissue of LOS group and TCM group were significantly decreased(P0.05),and the expression of NR4A1 was significantly increased(P0.05).The results of qRT-PCR showed that compared with the Sham group,the NR4A1 mRNA in the UUO group was significantly decreased(P0.05),NF-κB mRNA was significantly increased(P0.05) Compared with the UUO group,the NR4A1 m RNA level in the LOS group and the TCM group was significantly increased(P0.05),and the NF-κB m RNA was significantly decreased(P0.05).Conclusion Yiqi Huoxue tongluo Recipe can down-regulate the expression of MCP-1 and IL-6 by regulating NR4A1/NF-κB,and reduce the kidney inflammation injury in UUO rats.