摘要
目的研究氧化苦参碱(OMT)对微小隐孢子虫(Cryptosporidium parvum)感染小鼠肠黏膜Toll样受体2 (TLR2)和TLR4的调节作用,探讨OMT治疗隐孢子虫病的分子机制。方法30只雄性健康BALB/c小鼠随机分为感染组、感染后OMT治疗组(简称OMT治疗组)和未感染组,每组10只。感染组和OMT治疗组小鼠经口灌胃微小隐孢子虫卵囊(1×10~5个/只),建立微小隐孢子虫肠道感染小鼠模型,未感染组小鼠正常饮食、饮水。OMT治疗组小鼠建模成功后经口灌胃OMT (50 mg/kg),每日1次,连续2周。采用金胺酚-改良抗酸染色法镜检计数小鼠克粪便卵囊数。治疗2周后剖杀各组小鼠, HE染色镜下观察小鼠肠黏膜的病理改变,测量肠绒毛高度、隐窝深度。抽提小鼠肠黏膜组织总RNA,逆转录成cDNA,实时荧光定量PCR (qRT-PCR)检测TLR2和TLR4的mRNA相对表达量。蛋白质印迹(Western blotting)检测小鼠肠黏膜组织中TLR2和TLR4的相对表达量。结果金胺酚-改良抗酸染色镜检结果显示,感染组小鼠克粪便卵囊数自建模成功后逐渐增加,至第7天达到最高,为(179.24±21.12)个/克,感染强度为4级,然后趋于平衡。OMT治疗组小鼠克粪便卵囊数在治疗第5天开始下降,治疗2周后,感染强度趋近于0级。HE染色镜下观察结果显示,感染组小鼠肠绒毛明显萎缩变短、脱落,黏膜下层结构严重水肿,与肌层间形成明显的间隙; OMT治疗组小鼠肠绒毛基本修复,结构趋于完整,排列趋于整齐;未感染组小鼠肠绒毛结构完整。OMT治疗组小鼠肠绒毛高度为(346.1±19.2)μm,高于感染组[(278.0±52.9)μm](P <0.01),与未感染组[(348.1±18.2)μm]差异无统计学意义(P> 0.05);隐窝深度为(155.4±5.3)μm,低于感染组[(173.1±11.1)μm](P <0.05),与未感染组[(149.9±27.4)μm]差异无统计学意义(P> 0.05);绒毛高度/隐窝深度为2.2±0.2,高于感染组(1.6±0.3)(P <0.01),与未感染组(2.4±0.6)差异无统计学意义(P> 0.05)。qRT-PCR结果显示, OMT治疗
Objective To study the effects of oxymatrine（OMT） on Toll-like receptors 2（TLR2） and Tolllike receptors 4（TLR4） on intestinal mucosa of mice infected with Cryptosporidium parvum and investigate the molecular mechanisms of cryptosporidiosis treatment by OMT.Methods Thirty healthy BALB/c male mice were randomly divided into the infection group, OMT treatment group and non-infection group（n = 10 in each group）.Mice in the infection group and OMT treatment group received intragastrical inoculation of C.parvum oocysts（1 ×10~5/gram） to establish the model of C.parvum intestinal infection, while mice in the non-infection group were fed with normal diet and water.Mice in the OMT treatment group received daily intragastrical inoculation of 50 mg/kg for 2 weeks.The numbers of mean oocysts per gram of mouse feces were counted using the auramine-phenol modified anti-acid staining method.All mice were sacrificed after 2 weeks of treatment.The pathological changes of intestinal mucosa were observed by HE staining, and the height of intestinal villi and the depth of crypt were measured.RNA was extracted from intestinal mucosa, and transcribed into cDNA.The relative expression of TLR2 mRNA and TLR4 mRNA in intestinal mucosa was assessed by real-time fluorescence PCR.The relative expression of TLR2 and TLR4 in intestinal mucosa was assessed by Western blotting.Result The auramine-phenol modified antiacid staining revealed increased numbers of mean oocysts in mouse feces in the infection group, peaking at day 7 with（179.24 ± 21.12） oocysts/gram, infection intensity was level 4, and reaching a plateau afterwards.In the OMT treatment group, the numbers decreased from day 5, and infection intensity was approaching level 0 after 2 weeks of treatment.HE staining showed atrophy and shedding of intestinal villi in the infection group, with submucosal edema and a significant gap between muscle layers, while the intestinal villi were intact in the non-infection group.The intestinal villi were restored in the OMT t
作者
汲蕊
梁瑞文
管志玉
李瑞芳
付玉荣
王红艳
JI Rui;LIANG Rui-wen;GUAN Zhi-yu;LI Rui-fang;FU Yu-rong;WANG Hong-yan(Department of Pathogen Biology,Weifang Medical College,Weifang 261053,China)
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2018年第5期455-459,482,共6页
Chinese Journal of Parasitology and Parasitic Diseases
基金
山东省自然科学基金(No.ZR2015HQ030)
山东省高等学校科技计划项目(No.J13LK04)
山东省中医药管理局项目(No.2015-238)~~
