健脾渗湿方对尿酸酶基因缺失高尿酸血症模式动物肾损伤及肠道微生态干预的研究  

Study on Effect of Jianpi Shenshi Formula on Kidney Damage and Intestinal Microecology Intervention in the Uricase Deficient Hyperuricemia Model Rats

作　　者：殷娜 李小丝 刘维超 祁燕 李兆福 段为钢 彭江云 万春平 YIN Na;LI Xiaosi;LIU Weichao;QI Yan;LI Zhaofu;DUAN Weigang;PENG Jiangyun;WAN Chunping(Yunnan University of Chinese Medicine,Kunming 650500,China)

机构地区：[1]云南中医药大学,云南昆明650500

出　　处：《云南中医学院学报》2021年第4期9-16,共8页Journal of Yunnan University of Traditional Chinese Medicine

基　　金：国家自然科学基金(30960476);云南省教育厅科学研究基金项目(2021Y463);国家中医药管理局国家中医(类风湿关节炎)临床研究基地建设项目(国中医药科技函[2018]131号);民族医药防治重大疾病诊疗方案及经典方剂安全性有效性评价研究(2017YFC1704005);云南省中医(风湿病)临床医学研究中心项目(202102AA310006)。

摘　　要：目的研究健脾渗湿方对尿酸酶基因缺失高尿酸血症模式动物肾损伤保护效应及肠道菌群的调控作用,为健脾渗湿方临床应用提供科学依据。方法采用CRISPR/Cas9技术获取尿酸酶基因缺失(Uox^(-/-))高尿酸血症模式动物模型,分为尿酸酶缺失(Uox^(-/-))高尿酸血症组和健脾渗湿方(JPSSF)给药组(12.50 g·kg^(-1)·d^(-1)),另设野生型SD大鼠(WT)对照组。药物干预后,全自动生化分析仪检测血清尿酸、尿素氮、肌酐含量;考马斯亮蓝法检测24 h尿中微量白蛋白含量;HE染色检测模式动物肾脏病理损伤程度;磷钨酸法测定肠组织尿酸含量;荧光定量PCR法检测肾脏组织尿酸盐转运蛋白基因mRNA的表达;16S rRNA V4区检测肠道菌群微生态结构多样性。结果健脾渗湿方能有效降低Uox^(-/-)高尿酸血症大鼠肾功能指标,包括尿酸、尿素氮、肌酐含量,减少Uox^(-/-)高尿酸血症大鼠肾小管及间质部位尿酸盐结晶沉积,改善肾脏组织炎细胞浸润,下调尿酸盐转运蛋白URAT1 mRNA表达(P<0.05);PCoA和NMDS分析及Shannon、Simpson指数结果表明,高尿酸血症给肠道菌群造成紊乱,而健脾渗湿方干预后对肠道菌群无明显重建作用(P>0.05);在属水平,与模型组比较,健脾渗湿方组大鼠肠道Lactobacillus,Proteus,Prevotellaceae NK3B31 group有一定增加,而Clostridium sensu stricto 1,Bifidobacterium具有减少趋势,但差异均无统计学意义(P>0.05)。结论健脾渗湿方对尿酸晶体沉积于肾组织引起肾脏损害具有较好的保护效应,对尿酸肠代谢及肠道微生态紊乱无明显的影响。Objective To explore the effect of Jianpi Shenshi Formula preotecting kidney damage and regulating intestinal flora in uricase-deficient(Uox^(-/-))hyperuricemia rats,and to provides a scientific basis for the clinical application for Jianpi Shenshi Formula.Methods An animal model of uricase-deficient(Uox^(-/-))hyperuricemia was established by using of CRISPR/Cas9 technique,dividing into uricase-deficient hyperuricemia group(Uox^(-/-))and JPSSF group(12.50 g·kg^(-1)·d^(-1)),wild-type SD rats(WT)were used as the control group.All animals were treated orally for 60 days.Renal function indexes(UA,UREA,CREA)from serum were detected by an automatic biochemical analyzer;Coomassie brilliant blue method was used to assess urinary protein for 24 h;HE staining was applied to assess kidney histopathological damage;Level of Uric acid from intestinal tissue was determined by phosphotungstic acid method;The qPCR was used to detect the expression levels of urate transporter gene;The microecological diversity of intestinal flora was determined by 16S rRNA V4 region sequencing.Results Here,we showed that Jianpi Shenshi Formula significantly decreased renal function indexes(UA,UREA,CREA)and alleviated renal lesions damage.However,no significant influence on uric acid from the colonic tissue(P>0.05).Down-regulated urate transporter URAT1 mRNA expression in JPSSF group.The PCoA and NMDS analysis have showed that the hyperuricemia causes disorders to the intestinal flora,while the Jianpi Shenshi Formula showed little effect on reconstruction of the intestinal flora(P>0.05).At the genus level,compared with the Uox^(-/-) group,there were certain increase in Lactobacillus,Proteus,Prevotellaceae NK3B31 group in JPSSF group,Clostridium sensu stricto 1 and Bifidobacterium were decreased in JPSSF group.However,there were no significant differences between Uox^(-/-) group and JPSSF group(P>0.05).Conclusion Our results indicated that Jianpi Shenshi Formula notably ameliorated hyperuricemia and related renal damage caused by the deposition of

关 键 词：健脾渗湿方 高尿酸血症 尿酸酶 肾损伤 肠道菌群 16S测序 

分 类 号：R285.5[医药卫生—中药学]