氧化苦参碱对微小隐孢子虫感染小鼠肠黏膜Toll样受体的影响

摘要/Abstract

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 × 10⁵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治疗组小鼠肠黏膜组织中TLR2 mRNA的相对表达量为1.4 ± 0.3,低于感染组（3.0 ± 0.1）（P < 0.01）,与未感染组（1.0 ± 0.0）差异无统计学意义（P > 0.05）;TLR4 mRNA的相对表达量为1.5 ± 0.1,低于感染组（3.1 ± 0.3）（P < 0.01）,与未感染组（1.0 ± 0.0）差异无统计学意义（P > 0.05）。Western blotting检测结果显示,OMT治疗组小鼠肠黏膜组织中TLR2的相对表达量为0.2 ± 0.0,低于感染组（0.6 ± 0.1）（P < 0.01）,与未感染组（0.2 ± 0.1）差异无统计学意义（P > 0.05）;TLR4的相对表达量为0.3 ± 0.1,低于感染组（0.6 ± 0.0）（P < 0.01）,与未感染组（0.2 ± 0.1）差异无统计学意义（P > 0.05）。结论 OMT可通过下调微小隐孢子虫感染小鼠肠黏膜组织中TLR2和TLR4的表达,可促进小鼠受损肠黏膜的修复。

关键词: 微小隐孢子虫, Toll样受体, 氧化苦参碱

Abstract:

Objective To study the effects of oxymatrine (OMT) on Toll-like receptors 2 (TLR2) and Toll-like 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 × 10⁵/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 anti-acid 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 treatment group. The villi heights in the non-infection group, infection group and OMT treatment group were (348.1 ± 18.2), (278.0 ± 52.9) and (346.1 ± 19.2) μm, respectively, OMT treatment group was higher than infection group (P < 0.01), and there was no significant difference between OMT treatment group and non-infection group (P > 0.05). The crypt depths in the non-infection group, infection group and OMT treatment group were (149.9 ± 27.4), (173.1 ± 11.1) and (155.4 ± 5.3) μm, OMT treatment group was lower than infection group (P < 0.05), and there was no significant difference between OMT treatment group and non-infection group (P > 0.05). The ratios of villi height/crypt depth in the non-infection group, infection group and OMT treatment group were 2.4 ± 0.6, 1.6 ± 0.3 and 2.2 ± 0.2, OMT treatment group was higher than infection group (P < 0.01), and there was no significant difference between OMT treatment group and non-infection group (P > 0.05). qRT-PCR showed that the relative expression of TLR2 mRNA in intestinal mucosa were 1.0 ± 0.0, 3.0 ± 0.1 and 1.4 ± 0.3 in the non-infection group, infection group and OMT treatment group, and that of TLR4 mRNA were 1.0 ± 0.0, 3.1 ± 0.3 and 1.5 ± 0.1, respectively. That of OMT treatment group was lower than infection group (P < 0.01), and there was no significant difference between OMT treatment group and non-infection group (P > 0.05). Western blotting showed that the relative expression of TLR2 in the intestinal mucosa were 0.2 ± 0.1, 0.6 ± 0.1 and 0.2 ± 0.0, and that of TLR4 were 0.2 ± 0.1, 0.6 ± 0.0 and 0.3 ± 0.1 in the non-infection group, infection group and OMT treatment group respectively. That of OMT treatment group was lower than that of infection group (P < 0.01), and there was no significant difference between OMT treatment group and non-infection group (P > 0.05). Conclusion OMT improves intestinal mucosal repair through down-regulating TLR2 and TLR4 expression in mice infected with C. parvum.

Key words: Cryptosiporidium parvum, Toll-like receptors, Oxymatrine

中图分类号:

R382.3

汲蕊, 梁瑞文, 管志玉, 李瑞芳, 付玉荣, 王红艳. 氧化苦参碱对微小隐孢子虫感染小鼠肠黏膜Toll样受体的影响[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(5): 455-460.

Rui JI, Rui-wen LIANG, Zhi-yu GUAN, Rui-fang LI, Yu-rong FU, Hong-yan WANG. Effect of oxymatrine on Toll-like receptors on intestinal mucosa of mice infected by Cryptosporidium parvum[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2018, 36(5): 455-460.

图/表 4

参考文献 20

[1]	汲蕊, 崔巍, 梁瑞文, 等. 苦参合剂对微小隐孢子虫感染小鼠肠黏膜的保护作用[J]. 中国寄生虫学与寄生虫病杂志, 2013, 31(4): 275-279.
[2]	崔巍, 梁瑞文, 辛玲, 等. 苦参合剂对隐孢子虫感染小鼠细胞免疫功能的保护作用[J]. 中国病原生物学杂志, 2007, 2(6): 445-447.
[3]	Guzman JR, Koo JS, Goldsmith JR, et al. Oxymatrine prevents NF-κB nuclear translocation and ameliorates acute intestinal inflammation[J]. Sci Rep, 2013, 3: 1629.
[4]	Yang Y, Xiu J, Zhang X, et al. Antiviral effect of matrine against human enterovirus 71[J]. Molecules, 2012, 17(9): 10370-10376.
[5]	Kan QC, Zhu L, Liu N, et al. Matrine suppresses expression of adhesion molecules and chemokines as a mechanism underlying its therapeutic effect in CNS autoimmunity[J]. Immunol Res, 2013, 56(1): 189-196.
[6]	陈甫, 黄克和. 3种微小隐孢子虫卵囊检测方法的比较研究[J]. 中国病原生物学杂志, 2006, 1(5): 352-355.
[7]	陈甫, 黄克和. 从鼠粪中分离纯化微小隐孢子虫卵囊方法的研究[J]. 中国寄生虫学与寄生虫病杂志, 2006, 24(3): 219-222.
[8]	章刚, 黄克和, 陈甫, 等. 苦参碱对微小隐孢子虫体内外感染的抑制作用[J]. 畜牧与兽医, 2009, 41(7): 67-69.
[9]	Casemore DP.ACP Broadsheet 128: June 1991. Laboratory methods for diagnosing cryptosporidiosis[J]. J Clin Pathol, 1991, 44(6): 445-451.
[10]	Laurent F, Lacroix-Lamandé S.Innate immune responses play a key role in controlling infection of the intestinal epithelium by Cryptosporidium[J]. Int J Parasitol, 2017, 47(12): 711-721.
[11]	潘卫庆, 汤林华. 分子寄生虫学[M]. 上海: 上海科学技术出版社, 2004.
[12]	Petry F, Jakobi V, Tessema TS.Host immune response to Cryptosporidium parvum infection[J]. Exp Parasitol, 2010, 126(3): 304-309.
[13]	鱼艳荣, 齐永芬. Toll样受体在蠕虫感染中的作用[J]. 中国寄生虫学与寄生虫病杂志, 2010, 28(5): 377-381.
[14]	Uematsu S, Akira S.Toll-like receptor and innate immunity[J]. Seikagaku, 2007, 79(8): 769-776.
[15]	Peterson CY, Costantini TW, Loomis WH, et al. Toll-like receptor-4 mediates intestinal barrier breakdown after thermal injury[J]. Surg Infect (Larchmt), 2010, 11(2): 137-144.
[16]	Yang Z, Fu Y, Gong P, et al. Bovine TLR2 and TLR4 mediate Cryptosporidium parvum recognition in bovine intestinal epithelial cells[J]. Microb Pathog, 2015, 85: 29-34.
[17]	Chen XM, O'Hara SP, Nelson JB, et al. Multiple TLRs are expressed in human cholangiocytes and mediate host epithelial defense responses to Cryptosporidium parvum via activation of NF-kappaB[J]. J Immunol, 2005, 175(11): 7447-7456.
[18]	余裕强, 宫鹏涛, 孙铭飞, 等. Caco-2细胞TLR2和TLR4对微小隐孢子虫刺激信号的识别[J]. 中国病原生物学杂志, 2015, 10(2): 148-151.
[19]	Fan H, Li L, Zhang X, et al. Oxymatrine downregulates TLR4, TLR2, MyD88, and NF-kappaB and protects rat brains against focal ischemia[J]. Mediators Inflamm, 2009, 2009: 704706.
[20]	Dong XQ, Yu WH, Hu YY, et al. Oxymatrine reduces neuronal cell apoptosis by inhibiting Toll-like receptor 4/nuclear factor kappa-B-dependent inflammatory responses in traumatic rat brain injury[J]. Inflamm Res, 2011, 60(6): 533-539.

引物Primer	序列(5′→3′) Sequence (5′→3′)
TLR2	F：GTGCCCTGTGCCACCATT
TLR2	R：GGAACGAAGTCCCGCTTGT
TLR4	F：GGGCTCATTCACTCACTAACGG
TLR4	R：CACAGGAGGGACCATCTTCATT
GAPDH	F：GAGCCAAAAGGGTCATCATCT
GAPDH	R：AGGGGCCATCCACAGTCTTC