沙凯辉 刘同刚 张晓丽
【摘要】 目的:探讨原花青素联合左氧氟沙星对尿路致病性大肠杆菌(UPEC)侵袭小鼠膀胱的抑制作用。方法:将雌性C57BL/6小鼠60只随机分为四组,分别为空白对照组、左氧氟沙星组、原花青素组、原花青素+左氧氟沙星组,每组15只。将UPEC J96稀释菌液注入各组小鼠膀胱内,感染2 h后在无菌条件下摘取膀胱,制备成组织匀浆,稀释后涂布于LB琼脂平板上,以37 ℃孵育过夜。采用平板菌落计数法检测各组UPEC J96相对侵袭率。结果:左氧氟沙星组、原花青素组、原花青素+左氧氟沙星组UPEC J96相对侵袭率低于空白对照组,原花青素+左氧氟沙星组UPEC J96相对侵袭率低于左氧氟沙星组、原花青素组,差异均有统计学意义(P<0.05);原花青素组与左氧氟沙星组UPEC J96相对侵袭率比较,差异无统计学意义(P>0.05)。结论:原花青素与左氧氟沙星联合应用更有利于抑制UPEC对小鼠膀胱的侵袭。
【关键词】 原花青素 左氧氟沙星 UPEC 侵袭 小鼠 膀胱 感染
doi:10.14033/j.cnki.cfmr.2020.24.073 文献标识码 B 文章编号 1674-6805(2020)24-0-02
[Abstract] Objective: To investigate the inhibitory effect of Proanthocyanidins and Levofloxacin on urinary tract pathogenic escherichia coli (UPEC) invading bladder of mice. Method: A total of 60 female C57BL/6 mice were randomly divided into four groups, including the blank control group, the Levofloxacin group, the Proanthocyanidins group, the Proanthocyanidins+Levofloxacin group, 15 mice in each group. UPEC J96 diluent was injected into the bladder of mice in each group, and the bladder was extracted under sterile conditions 2 h after infection to prepare tissue homogenate, which was diluted and coated on LB AGAR plate and incubated overnight at 37 ℃. The relative invasion rate of UPEC J96 was measured by plate colony counting method. Result: The relative invasion rate of UPEC J96 in the Levofloxacin group, the Proanthocyanidins group, the Proanthocyanidins+Levofloxacin group was significantly lower than that of the blank control group, and the relative invasion rate of UPEC J96 in the Proanthocyanidins+Levofloxacin group was significantly lower than that of the Levofloxacin group and the Proanthocyanidins group, and the differences were statistically significant (P<0.05). The relative invasion rate of UPEC J96 between the Proanthocyanidins group and the Levofloxacin group was compared, and the difference was not statistically significant (P>0.05). Conclusion: The combination of Proanthocyanidins and Levofloxacin is more beneficial to the inhibition of UPEC invasion on the bladder of mice.
[Key words] Proanthocyanidins Levofloxacin UPEC Invade Mice Bladder Infection
First-authors address: School of Nursing, Binzhou Medical University, Binzhou 256600, China
尿路感染是一種十分常见的细菌感染性疾病,主要由尿路致病性大肠埃希菌(UPEC)和肺炎克雷伯杆菌等革兰阴性菌引发,约占所有感染病例的90%[1-2]。目前,抗生素疗法是治疗尿路感染的最有效手段,其中最常用的抗生素有阿莫西林、环丙沙星、左氧沙星等[3-5]。然而,由于抗生素的不合理使用,导致耐多药/泛耐药尿路致病菌出现,使得对于尿路感染的防治成为医学界有待解决的难题[6-7]。原花青素是一种复杂的黄酮类天然聚合物,存在于谷物、豆类种子中,尤其在一些水果和果汁中的含量特别丰富[8]。本研究中,笔者通过动物实验以验证原花青素与左氧氟沙星联合应用可以有效抑制UPEC侵袭膀胱,从而防治尿路感染。
1 材料与方法
1.1 一般材料
1.1.1 动物 选取滨州医学院动物实验中心的雌性C57BL/6小鼠60只。纳入标准:SPF级小鼠,饮食正常。排除标准:尿路畸形。随机分为四组,分别为空白对照组、左氧氟沙星组、原花青素组、原花青素+左氧氟沙星组,每组15只。体重18~20 g,平均(18.83±0.75)g。
1.1.2 主要药剂 原花青素(纯度>95%)购自长沙中仁生物科技有限公司。
1.1.3 UPEC UPEC J96(血清型O4:K6,ATCC 700336)购自ATCC,保存于-70 ℃冰箱中。于冰箱内取出细菌并置于室温下缓慢溶解,取50 ?l菌液接種于5 ml的LB肉汤液体培养基中,置于摇床中,以37 ℃、170 r/min振荡培养过夜;采用接菌环将细菌接种于TSA固体培养基中,37 ℃培养过夜形成单菌落,置于4 ℃冰箱备用,约可使用1周。
1.2 方法
1.2.1 四组小鼠膀胱给药 腹腔注射5%水合氯醛(0.1 ml/10 g)进行麻醉,仰卧位固定,挤出膀胱中的尿液,通过导尿管分别将蒸馏水、左氧氟沙星稀释液、原花青素稀释液及左氧氟沙星和原花青素混合稀释液注入四组小鼠膀胱内,作用2 h。随后,挤出膀胱中的液体,将UPEC J96稀释菌液以5×108 CFU/膀胱的数量注入小鼠膀胱内,感染2 h后挤出菌液,将100 ?g庆大霉素注入小鼠膀胱内,作用30 min以杀死胞外菌。
1.2.2 平板菌落计数检测侵袭率 以无菌磷酸缓冲盐溶液(PBS)灌洗小鼠膀胱,去除残留庆大霉素。在无菌条件下摘取膀胱,制备成组织匀浆,以PBS进行10倍梯度稀释。稀释100倍后,取100 ?l稀释液涂布于LB琼脂平板上,以37 ℃孵育过夜,菌落计数并计算相对侵袭率。
1.2.3 实验次数 实验重复3次。
1.3 观察指标
计算四组UPEC J96相对侵袭率。
1.4 统计学处理
数据采用SPSS 17.0统计软件进行分析。计量资料以(x±s)表示,采用t检验,多组间比较采用方差分析,P<0.05为差异有统计学意义。
2 结果
左氧氟沙星组、原花青素组、原花青素+左氧氟沙星组UPEC J96相对侵袭率均较空白对照组明显降低,差异均有统计学意义(P<0.05);原花青素组与左氧氟沙星组UPEC J96相对侵袭率比较,差异无统计学意义(P>0.05),见表1。
3 讨论
尿路感染是病原微生物在尿路中生长繁殖并入侵泌尿道黏膜或组织而引起的炎性反应[9]。最新的流行病学调查发现,全世界每年约有1.5亿人次因各种尿路感染问题就医或住院[10]。尿路感染具有易复发的特点,多见于女性,严重影响患者生活质量[5]。
原花青素是一类富含羟基的天然多酚化合物[11]。研究证明,原花青素具有抗菌、抗氧化、抗肿瘤的生物学活性[12-14]。UPEC主要导致急性膀胱感染(膀胱炎)和肾感染(肾盂肾炎),并可造成严重慢性肾功能衰竭或菌/败血症。尿道黏膜是病原微生物定殖最为棘手的部位之一,黏附和侵袭是UPEC等尿路致病菌逃避宿主免疫反应和抗生素治疗最重要的策略,可以促进病原体更有效地定殖于尿道并引发感染[15]。在最常见的膀胱感染中,最为关键的第一步即是UPEC等尿路致病菌利用其表达的Ⅰ型菌毛黏附素FimH与膀胱上皮细胞膜表面含有甘露糖残基的蛋白受体uroplakin Ia及Integrinα3/β1等结合,进而激活多条细胞内信号级联,介导UPEC等病原菌侵袭膀胱上皮细胞[16]。左氧氟沙星属于喹诺酮类抗菌药,能够有效抑制细胞内的菌落和大肠杆菌生物膜的形成,从而达到预防和治疗尿路感染的目的。本研究中,笔者发现,采用原花青素预处理小鼠膀胱后,UPEC J96对膀胱的侵袭明显减少,与左氧氟沙星的效果相比无差异,而原花青素与左氧氟沙星联合应用效果更显著,说明原花青素可以作为一种治疗尿路感染的替代物或辅助物与左氧氟沙星联合应用。可能机制是原花青素能够抑制UPEC与膀胱上皮细胞表面受体的结合,进而影响细胞内多条信号级联反应,达到抑制UPEC侵袭小鼠膀胱的目的。
综上所述,原花青素与左氧氟沙星可以有效抑制UPEC侵袭小鼠膀胱,为尿路感染的临床防治奠定一定基础。
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(收稿日期:2020-06-18) (本文编辑:李盈)