Wnt/β-catenin通路在肾衰康灌肠液抑制HK-2细胞缺氧/复氧损伤的作用  

Effects of Shenshuaikang Enema Liquid on the Wnt/β-catenin pathway for inhibiting the hypoxia reoxygenation injury in HK-2 cells

作　　者：邓荣荣[1] 罗芯怡[1] 叶乃菁 吴新萍[1] 王意君[2] 李明权[3] DENG Rong-rong;LUO Xin-yi;YE Nai-jing;WU Xin-ping;WANG Yi-jun;LI Ming-quan(Chengdu University of Traditional Chinese Medicine, Chengdu 610075, China;The First People ＇s Hospital of Chengdu, Chengdu 610000,China;The Hospital Affiliated to Chengdu University of Traditional Chinese Medicine,Chengdu 610075, China)

机构地区：[1]成都中医药大学,四川成都610075 [2]成都市第一人民医院内分泌科,四川成都610000 [3]成都中医药大学附属医院,四川成都610075

出　　处：《中成药》2018年第5期1036-1042,共7页Chinese Traditional Patent Medicine

基　　金：国家中医药管理局国家中医临床研究基地业务建设科研专项(JDZX2012127)

摘　　要：目的观察肾衰康灌肠液(大黄、丹参、红花、黄芪)含药血清对缺氧/复氧损伤人肾小管上皮细胞的保护作用,并探讨其对Wnt/β-catenin通路的影响。方法将新西兰大白兔随机分为对照组、肾衰康高剂量组、PBS组,分别予相应药物灌肠3 d后取血清。以对照组、PBS组、肾衰康高剂量组、肾衰康中剂量组、肾衰康低剂量组兔血清分别处理缺氧/复氧HK-2细胞。以ROS荧光探针检测H/R损伤发生。CFSE/PI法、流式细胞分析联合Annexin V-FITC/PI法分析细胞的损伤/凋亡情况。荧光定量PCR技术考察药物对Wnt4、β-catenin mRNA表达的影响。结果与造模前相比,造模后细胞内ROS表达的荧光强度明显增高。CFSE/PI双染显示,与对照组和PBS组相比,肾衰康高、中、低剂量组细胞死亡率明显降低,且以肾衰康高剂量组死亡率最低。Annexin V-FITC/PI联合流式细胞术检测显示,与对照组[12 h,(45.6±2.2)%]和PBS组[12 h,(41.6±0.7)%]相比,肾衰康低、中、高剂量组均有明显的凋亡/死亡抑制[12 h,低剂量组(14.8±0.3)%,中剂量组(10.3±0.6)%,高剂量组(12.9±0.9)%]。荧光定量PCR结果显示,肾衰康灌肠液含药血清对Wnt4 mRNA表达有显著上调作用,对β-caterin mRNA表达有显著双向调节作用(P<0.05)。结论肾衰康灌肠液含药血清对缺氧/复氧损伤HK-2细胞的凋亡/死亡有明显的抑制效果,其作用可能与激活Wnt/β-catenin通路有关。AIM To investigate the protective effect of serum containing Shenshuaikang Enema Liquid（ Rhei Radix et Rhizoma,Salviae miltiorrhizae Radix et Rhizoma,Carthami Flos,Astragali Radix） on HK-2 cells injured by hypoxia/reoxygenation（ H/R） and its effect on Wnt/β-catenin pathway. MEHTODS Six New Zealand white rabbits randomly divided into control group,Shenshuaikang high dose group and PBS group（ n = 2） were treated accordingly for serum collection after 3 days ＇enema. The HK-2 cells injured by hypoxia/reoxygenation were then administered with serum of rabbits from the control group,PBS group,Shenshuaikang groups（ high dose,middle dose and low dose groups due to the differently diluted concentrations） respectively. The H/R damage was determined by ROS fluorescence probe,the cellular damage/apoptosis were analyzed by CFSE/PI method and flow cytometry combined with Annexin V-FITC/PI,and the investigation on effects of drugs on expression of Wnt4 mRNA and β-catenin mRNA were accomplished by fluorescence quantitative PCR. RESULTS The fluorescence intensity of intracellular ROS expression was significantly increased after modeling. CFSE/PI double staining showed that the variant Shenshuaikang dose groups displayed obvious proportional mortality superiority to either the control group or PBS group,and the high dose group achieved the lowest mortality. Annexin V-FITC/PI and flow cytometry showed that,at 12 h,compared with the control group（ 45. 6 ± 2. 2） % and PBS group（ 41. 6 ±0. 7） %,Shenshuaikang groups [low dose group（14. 8 ± 0. 3） %,middle dose group（10. 3 ± 0. 6） %,high dose group（12. 9 ± 0. 9） %] obviously inhibited apoptosis/death. Shenshuaikang Enema Liquid medicated serum demonstrated its significant effect on the increase of the Wnt4 mRNA expression and a dual-directional regulation on the expression of β-caterin mRNA by quantitative PCR（ P〈0. 05）. CONCLUSION Inhibition of the apoptosis/death of HK-2 cells with hypoxia/reoxygenation injury due to the serum contain

关 键 词：肾衰康灌肠液 缺氧/复氧 H/R HK-2细胞 WNT/Β-CATENIN 

分 类 号：R285.5[医药卫生—中药学]