摘要
目的:研究大黄-丹参药对对慢性肾衰竭(CRF)模型大鼠肠源性尿毒素含量和肠道屏障功能的影响。方法:将55只雄性Wistar大鼠随机分为假手术组(10只)和造模组(45只),假手术组大鼠分离肾组织但不切除,造模组大鼠采用5/6肾切除法复制CRF模型。造模成功后(剔除死亡和不成模大鼠5只),将模型大鼠分为模型组(水)、尿毒清颗粒组(2.5 g/kg)和大黄-丹参药对高、低剂量组(6、3 g/kg,以生药量计),每组10只。假手术组和模型组大鼠灌胃等体积水,各给药组灌胃相应药物,每天1次,连续12周。末次给药后,采用全自动生化分析仪检测各组大鼠血清中肌酐(Scr)、尿素氮(BUN)含量和24 h尿液中尿肌酐(Ucr)含量,并计算肌酐清除率(Ccr);采用超高效液相色谱-电喷雾串联三重四极杆质谱法同时测定大鼠血清中尿毒素[氧化三甲胺(TMAO)、硫酸吲哚酚(IS)、硫酸对甲酚(PCS)]的含量;采用实时荧光定量聚合酶链式反应(RT-PCR)法、免疫荧光法分别检测大鼠回肠组织中闭合蛋白(Occludin)、紧密连接蛋白1(ZO-1)的m RNA和蛋白表达水平;采用苏木素-伊红染色法、Masson染色法观察大鼠肾组织的病理学变化;采用透射电镜法观察大鼠结肠组织的超微结构变化。结果:与假手术组比较,模型组大鼠血清中Scr、BUN、TMAO、PCS、IS含量均显著升高(P<0.01),尿液中Ucr含量和Ccr及回肠组织中Occludin、ZO-1的mRNA及蛋白表达水平均显著降低(P<0.01);肾组织中肾小球硬化、肾小管扩张,肾间质可见炎性细胞浸润和纤维化;结肠上皮屏障结构重度损伤。与模型组比较,各给药组大鼠血清中Scr、BUN、TMAO、PCS、IS含量均显著降低(P<0.05或P<0.01);尿液中Ucr含量和回肠组织中Occludin、ZO-1的mRNA及蛋白表达水平均显著升高(大黄-丹参药对低剂量组的ZO-1 mRNA除外,P<0.05或P<0.01);肾间质炎性细胞浸润、纤维化程度和结肠上皮屏障结构损伤程度均减轻。结论:大黄-丹参药对能有效保护CRF模型大鼠残存的肾功能,其作用机制可能与降低大鼠血清中肠源性尿毒素含量,上调回肠组织中Occludin、ZO-1的mRNA及蛋白表达水平从而改善肠道屏障功能有关。
OBJECTIVE:To study the effects of couplet medicine of Rheum palmatum-Salvia miltiorrhiza on the contents of enterogenous urotoxin and intestinal barrier function in chronic renal failure(CRF)model rats.METHODS:Totally 55 male Wistar rats were randomly divided into sham operation group(10 rats)and modeling group(45 rats).In sham operation group,the kidneys were isolated but not removed;CRF model was reproduced by 5/6 nephrectomy in modeling group.After modeling(excluding 5 dead and non-modeling rats),modeling rats were divided into model group(water),Niaoduqing granules group(2.5 g/kg),couplet medicine of R.palmatum-S.miltiorrhiza groups(6,3 g/kg,by crude drug),with 10 rats in each group.Sham operation group and model group were given constant volume of water intragastrically.Administration groups were given relevant medicine intragastrically,once a day,for consecutive 12 weeks.After last administration,the contents of creatinine(Scr)and urea nitrogen(BUN)in serum,the content of urinary creatinine(Ucr)in urine were determined by automatic biochemical analyzer;creatinine clearance rate(Ccr)was calculated.The contents of enterogenous urotoxin[trimethylamine-N-oxide(TMAO),indoxyl sulfate(IS)and p-cresyl sulphate(PCS)]were determined by UPLC-ESI-MS/MS.Real-time RT-PCR and immunofluorescence assay were used to detect the mRNA and protein expression of Occludin and ZO-1 in the ileum tissue.HE staining and Masson staining were used to observe the pathological changes of renal tissue.The ultrastructural changes of rat colon were observed by transmission electron microscope.RESULTS:Compared with sham operation group,serum contents of Scr,BUN,TMAO,PCS and IS were increased significantly in model group(P<0.01),while urine content of Ucr,Ccr,mRNA and protein expression of Occludin and ZO-1 in ileum tissue were decreased significantly(P<0.01);renal glomerulosclerosis,renal tubules dilation and inflammatory invasion and fibrosisin the interstitium were all found;the intestinal epithelial barrier structure of colon tissue was severely damaged.Compared with model group,serum contents of Scr,BUN,TMAO,PCS and IS were decreased significantly in administration groups(P<0.05 or P<0.01);the mRNA and protein expression of Occludin and ZO-1 in the ileum tissue were increased significantly(except for mRNA expression of ZO-1 in R.palmatum-S.miltiorrhiza low-dose group(P<0.05 or P<0.01);the infiltration of inflammatory cells in renal interstitium,the degree of fibrosis and the damage of intestinal epithelial barrier structure in colon tissue were reduced.CONCLUSIONS:Couplet medicine of R.palmatum-S.miltiorrhiza can effectively protect the residual renal function of CRF model rats,the mechanism of which may be associated with reducing the serum contents of enterogenous urotoxin,up-regulating mRNA and protein expresssion of Occludin and ZO-1 in the ileum tissue so as to improve intestinal barrier function.
作者
王宏
徐娟
江茜
康利
刘伟伟
袁玲
崔晓雪
刘琳娜
冯仁蕊
王蕾
李燕林
WANG Hong;XU Juan;JIANG Qian;KANG Li;LIU Weiwei;YUAN Ling;CUI Xiaoxue;LIU Linna;FENG Renrui;WANG Lei;LI Yanlin(Cardiovascular and Cerebrovascular Drug Research Laboratory,Tianjin Institute of Medical&Pharmaceutical Sciences,Tianjin 300020,China;Dept.Nephrology,Zhongshan Hospital of TCM,Guangdong Zhongshan 528401,China)
出处
《中国药房》
CAS
北大核心
2021年第7期825-831,共7页
China Pharmacy
基金
广东省自然科学基金资助项目(No.2017A030313720)
中山市社会公益科技研究项目(No.2018B1008)
2020年度中山市第三批社会公益与基础研究专项(No.2020B3005)
天津市卫生健康委员会科技人才培育项目(No.KJ20018)。
关键词
大黄
丹参
慢性肾衰竭
肠源性尿毒素
肠道屏障
大鼠
Rheum palmatum
Salvia miltiorrhiza
Chronic renal failure
Enterogenous urotoxins
Intestinal barrier
Rats
