摘要
目的 研究慢性肾病(chronic kidney disease, CKD)微炎症大鼠肠源性外泌体携带miR-146a的表达特性。方法 健康SPF级Wistar雄性大鼠16只,随机分为2组,分别为正常组、模型组。采用腺嘌呤诱导CKD大鼠模型。适应性喂养3 d后,大鼠灌胃腺嘌呤(冲击量220 mg/(kg·d),4周;维持量100 mg/kg, 3周,共7周)。实验终点,大鼠经麻醉后,腹中动脉取血,留取血清和EDTA抗凝血浆各一份。测定血清中的尿素氮(BUN)、肌酐(Cr);酶联免疫吸附测定法(ELISA)检测血清白介素-1β(IL-1β)、白介素-6(IL-6)、肿瘤坏死因子α(TNF-α)含量;制备肾组织病理切片,苏木-伊红染色(HE)观察肾组织形态,马松(Masson)染色观察肾纤维化程度;透射电镜观察肠组织超微结构和外泌体分泌情况;试剂盒法提取血浆外泌体,超高速离心法提取肠道组织中外泌体;负染电镜技术鉴定外泌体形态;NTA技术测定外泌体粒径;免疫蛋白印迹法(Western blot)测定外泌体表面生物标记物;反转录聚合酶链反应(RT-PCR)技术检测血浆外泌体中携带的微小RNA-146a(miR-146a)表达水平;Western blot法测定肠道组织肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6,TRAF6)、核因子-κB(nuclear factor-κB signalling pathway, NF-κB)、Toll样受体4(TLR4)炎性信号通路蛋白表达。结果 与正常组比较,模型组灌胃腺嘌呤7周后,构建CKD大鼠模型,模型大鼠的血清BUN、Cr水平显著增加(P<0.01)。模型组血清和回肠组织IL-1β、IL-6、TNF-α水平明显高于正常组(P<0.01)。模型组肾病理显示肾小管明显萎缩、扩张,部分呈囊性病变,肾小管内大量褐色结晶,可见管型和再生;肾小球硬化,肾小球萎缩,鲍曼氏囊腔扩张,数目明显减少并大小不一;间质纤维化及炎性细胞浸润明显。模型组大鼠血浆和肠道组织中均可见粒径在155 nm的外泌体,透射电镜可见模型组大鼠的肠道组织释放出大量外泌体。模型组血浆外泌体携带miR-146a的表达显著高于正常组(P<0.01),而肠道组织外泌体携带miR-146a的表达却显著低于正常组(P<0.01)。结论 腺嘌呤导致大鼠肾功能损伤,肾组织形成病理性改变,伴发全身微炎症。同时,CKD大鼠肠道组织分泌并释放外泌体,使血浆中外泌体携带miR-146a被激活增加。
Objective To study the expression characteristics of intestinal exosomes carrying miR-146a in rats with chronic kidney disease(CKD)micro-inflammation.Methods Sixteen healthy SPF-grade Wistar male rats were randomly divided into two groups:the normol group and the model group.An adenine-induced CKD rat model was used.After 3 days of adaptive feeding,rats were gavaged with a gastric adenine shock dose of 220 mg/(kg·d)for 4 weeks and a maintenance dose of 100 mg/kg for 3 weeks for a total of 7 weeks.At the end of the experiment,after anesthesia,blood was taken from the mid-abdominal artery of the rats.Serum and anticoagulant plasma with EDTA are left behind.For the determination of blood urea nitrogen(BUN)and creatinine(Cr)in serum,By enzyme-linked immunosorbent assay(ELISA),we determined levels of serum interleukin-1,interleukin-6 and tumor necrosis factor.Histopathological observation of kidney sections was performed,including an examination of kidney histomorphology by hematoxylin and eosin staining and kidney fibrosis by Masson staining.Transmission electron microscopy was used to observe the intestinal tissue ultrastructure and exosome secretion.Plasma exosomes were extracted from intestinal tissues via a reagent kit method and ultra-high speed centrifugation.Negative-stain electron microscopy was used in the identification of exosome morphology,and the NTA technique was used to determine exosome particle size.Western blot assays were employed to examine exosome surface biomarkers.Reverse transcription-polymerase chain reaction was used to detect the expression level of miR-146a in plasma exosomes.Western blot was used to determine the presence of tumor necrosis factor receptor-associated factor 6(TRAF6),nuclear factor-κB(NF-κB),and Toll-like receptor 4(TLR4)inflammatory signaling pathway proteins in the intestinal tissues.Results A CKD rat model was formed after 7 weeks of adenine gavage,and the model was characterized by a significant increase in serum BUN and Cr levels compared with the control group(P<0.01).Serum and ileal tissue levels of IL-1β,IL-6,and TNF-αwere significantly higher in the model group than the control group(P<0.01).Pathology of the model group revealed atrophied and expanded renal tubules,cystic lesions,and many brown crystals found in the renal cortex glomerulosclerosis,glomerular atrophy,Bowman’s cystic cavity dilatation.Clearly there is a reduction in the number of Bowman’s capsules and a difference in the size of the capsule cavity.The model grouped showed obvious interstitial fibrosis and inflammatory cell infiltration.Exosomes of 155 nm were found in the plasma and intestinal tissues of rats in the model group,and transmission electron microscopy revealed that a large number of exosomes were released from the intestinal tissues.The number of plasma exosomes carrying miR-146a in the model group was significantly higher than that in the control group(P<0.01),whereas the number of intestinal exosomes carrying miR-146a was significantly lower than that in the control group(P<0.01).Conclusions Adenine causes chronic renal failure with impaired kidney function and morphological pathological changes with systemic microinflammation in rats.Exosomes are secreted and released from the intestinal tissues of CKD rats,Resultsing in the increased activation of plasma exosomes carrying miR-146a.
作者
刘琳娜
王宏
江茜
袁玲
崔晓雪
徐娟
潘钰榕
王蕾
李燕林
LIU Linna;WANG Hong;JIANG Qian;YUAN Ling;CUI Xiaoxue;XU Juan;PAN Yurong;WANG Lei;LI Yanlin(Zhongshan Hospital of Traditional Chinese Medicine,Zhongshan 528400,China;Tianjin Institute of Pharmaceutical Sciences,Tianjin 300020;Guangzhou University of Traditional Chinese Medicine,Guangzhou 510006)
出处
《中国比较医学杂志》
CAS
北大核心
2023年第4期83-89,108,共8页
Chinese Journal of Comparative Medicine
基金
中山市社会公益与基础研究项目(2020B3005)
中山市第三批社会公益与基础研究项目(2021B3006)
天津市卫生健康委员会面上项目(MS20023)。
