CN114569640A - 植物乳杆菌用于制备抗败血症药物的应用 - Google Patents
植物乳杆菌用于制备抗败血症药物的应用 Download PDFInfo
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Abstract
本发明公开了植物乳杆菌用于制备抗败血症药物的应用。本发明研究发现植物乳杆菌可提高败血症生存率、改善败血症评分、增加WBC、LYM、MID、GRA降低RBC数量以及减少LDH、BUN、CK的水平；同时，植物乳杆菌能改善由败血症引起的心肌组织损伤；可改善由败血症引起的每搏输出量、心输出量、左心室收缩末期容积、左心室舒张末期容积的降低；改善左心室收缩末期后壁厚度、左心室舒张末期后壁厚度、心率的增加，从而达到改善心脏收缩功能的作用；并且植物乳杆菌也能抑制败血症诱发的心肌组织损伤后MPO、IL‑1β、Ly6g和NOX2的高表达，发挥抗炎和抗氧化应激的作用。
Description
技术领域
本发明涉及G11的新适应症，具体涉及G11的多种益生功能和用于抗败血症及其诱发的心肌损伤益生菌的应用。
背景技术
保藏编号为CGMCC No.16937的植物乳杆菌(本文编号为G11)是从西北地区特殊食品浆水中分离的益生菌，其主要益生功能包括降解亚硝酸盐、降解胆固醇、抑菌、产γ-氨基丁酸(GABA)、产EPS、胃肠道定殖。
发明内容
发明人通过构建败血症及其诱发的心肌损伤动物模型，观察给模型植物乳杆菌后其生存率、血常规、血生化、炎症、氧化应激及心肌损伤各项指标发现：
G11可提高败血症生存率、改善败血症评分、增加WBC、LYM、MID、GRA降低RBC数量以及减少LDH、BUN、CK的水平；
同时，G11能改善由败血症引起的心肌组织损伤；可改善由败血症引起的每搏输出量(SV)、心输出量(CO)、左心室收缩末期容积(Volume；s)、左心室舒张末期容积(Volume；d)的降低；改善左心室收缩末期后壁厚度(LVPW；s)、左心室舒张末期后壁厚度(LVPW；d)、心率(HR)的增加，从而达到改善心脏收缩功能的作用；
G11也能抑制败血症诱发的心肌组织损伤后MPO、IL-1β、Ly6g和NOX2的高表达，发挥抗炎和抗氧化应激的作用。
基于上述发现，本发明提供了植物乳杆菌用于制备治疗和/或预防败血症及其诱发的心肌损伤益生菌的应用,所述植物乳杆菌的保藏编号为CGMCC No.16937。
本发明还提供了一种治疗和/或预防败血症的药物。所述药物包括植物乳杆菌和药物辅料，所述植物乳杆菌的保藏编号为CGMCC No.16937。
进一步，所述药物为口服液剂给药制剂。
更进一步，所述药物的给药剂量为0.01ml((1×106CFU/mL)-(1X108CFU/mL))/g体重。
附图说明
图1为菌株G11的溶血性；
图2为菌株G11的耐药性活性；
图3为G11对CLP损伤后小鼠生存率的影响，观察CLP术后72h内各组小鼠的生存情况，A图为小鼠生存率造模图片，B图为小鼠生存率曲线，结果以均数±标准差表示，n＝12。*vs.CLP组，P<0.05；
图4为G11对正常小鼠和给药小鼠体重的影响，观察给药后12天内给小鼠体重的变化，*vs.Control组，P＞0.05；
图5为G11对CLP损伤后小鼠败血症评分的影响，于小鼠术后8h，按照败血症评分相关指标对小鼠损伤后状态进行评分，A图为小鼠功能学检测造模图片，B图为败血症评分结果，结果以均数±标准差表示，n＝8；*vs.CLP组，P<0.05；
图6为G11对CLP损伤后小鼠血常规各项指标的影响，于小鼠术后8h测定血常规，结果以均数±标准差表示，n＝6；*vs.CLP组，P<0.05；
图7为G11对CLP损伤8h后小鼠血生化各项指标的影响，于小鼠术后8h测定血生化，结果以均数±标准差表示，n＝6；*vs.CLP组，P<0.05；
图8为G11对CLP损伤8h后心脏功能各项指标的影响，于小鼠术后8h进行超声心动图检测，A图为超声心动图长轴切面、M模典型图片以及各项心脏功能指标的统计分析图，B图为超声心动图短轴切面、M模典型图片以及各项心脏功能指标的统计分析图，结果以均数±标准差表示，n＝6，*vs.CLP组，P<0.05；每搏输出量；心输出量；左心室收缩末期容积；左心室舒张末期容积；左心室收缩末期后壁厚度；左心室舒张末期后壁厚度；心率；
图9为G11对CLP损伤8h后心肌组织形态的影响，心肌组织切片HE染色结果；HE，苏木精-伊红；
图10为G11对CLP损伤8h后心肌炎症相关指标的影响，炎症相关典型指标MPO、IL-1β、Ly6g、TNF-α的免疫组织化学染色结果及统计分析图，结果以均数±标准差表示，n＝6，*vs.CLP组，P<0.05；
图11为G11对CLP损伤8h后心肌氧化应激相关指标的影响，氧化应激标志物NOX2的免疫组织化学染色结果及统计分析图，结果以均数±标准差表示，n＝6。*vs.CLP组，P<0.05。
具体实施方式
除非有特殊说明，本文中的术语或方法根据相关领域普通技术人员的常规认识理解或采用已有方法实现。
败血症是指宿主对感染的反应失调导致危及生命的器官功能障碍。败血症加重可引起感染性休克、弥散性血管内凝血和器官功能受损，严重威胁患者生命。心脏是败血症损伤最为严重的器官之一。临床研究表明，伴有严重心肌损伤的败血症患者死亡率高达80％。若心肌损伤出现在败血症早期，可加速败血症病情恶化。因此，对败血症诱发的心肌损伤进行早期干预和治疗具有十分重要的临床意义。
盲肠结扎穿孔(Cecal Ligation and Puncture，CLP)动物模型是经典的败血症模型，本发明采用CLP模型为研究对象，根据现有技术研究，该动物模型可造成急性心肌损伤，如败血症诱发的心肌损伤。
败血症诱发的心肌损伤属于感染性心肌损伤的一种。感染性心肌损伤指微生物(细菌、病毒等)感染过程中或恢复期中出现的如心脏扩大、心力衰竭、心源性休克或心律异常等反应，典型的症状为疲乏无力、食欲不振、恶心、呕吐、呼吸困难、面色苍白，发热。
本发明所述的感染性心肌损伤，特别是指细菌性心内膜炎、系统性或者其他脏器感染所致菌血症导致的心肌炎性病变和系统性或者其他脏器感染所致脓毒血症导致的心肌炎性病变。临床上多用肾上腺皮质激素和广谱抗生素，防止菌群失调。在应用过程中应严加观察，特别注意有无消化道、泌尿道和呼吸道的真菌感染。
以下通过实施例对本发明做进一步的阐述。实施例是对本发明进行详细说明，但本发明并不受这些的任何限定。以下实施例所用的G11(CGMCC No.16937的植物乳杆菌)是由西北大学生命科学学院张珺老师实验室馈赠。所用动物购自空军军医大学实验动物中心，所用试剂为市场采购。如无特殊说明，以下实施例中所采用的实验方法或相关检测方法采用本领域已知方法。
实施例1:G11安全性评估
步骤：
(1)测定G11菌株溶血性检测
将菌株以1％的接种量活化24h，取0.5μLG11菌株培养于哥伦比亚血(羊血)琼脂平板，30℃培养48h，以金黄色葡萄球菌(α-溶血)和铜绿假单胞菌(β-溶血)为阳性对照，观察G11菌株的溶血性。
(2)G11菌株抗生素耐药性检测
将菌株以1％的接种量活化24h，用PBS稀释100倍，取0.5mL的稀释菌液均匀的涂布在MRS固体培养基上，吹干40min，在平皿中放入含抗生素滤纸片，以不含抗生素的滤纸片为空白对照，30℃培养48h，记录抑制圈直径；使用卡那霉素(30μg)、庆大霉素(10μg)、万古霉素(30μg)、四环素(30μg)、克林霉素(20μg)、链霉素(10μg)、氨苄西林(10μg)、红霉素(15μg)、青霉素(10μg)抗生素进行检测。
结果：
结果如图1和2所示，G11菌株不具有溶血性，并对万古霉素、卡那霉素、庆大霉素和链霉素具有耐药性，平板上没有明显的抑菌圈，而对红霉素、克林霉素、氨苄西林、青霉素和四环素具有不同的敏感性。
实施例2：发明人研究发现G11能够改善败血症引起的小鼠死亡
步骤：
使用野生型BALB/c小鼠作为研究对象，按照研究设计，用随机数字表法进行分组，小鼠感染性心肌损伤模型按照Rittirsch D等发表的CLP实验方法，复制重度感染性心肌损伤模型。具体实验步骤如下：
(1)分组：生存率实验：将BALB/c小鼠分为假手术组、CLP组、G11组(1x106和1x108CFU/mL)，每组10只；
制药：G11液体培养48小时后，数量达到108CFU/mL，将菌液8000r，5min离心收集菌体沉淀，再用同体积的PBS缓冲液重悬，制成108CFU/mL菌悬液，在将得到的108CFU/mL的菌悬液稀释100倍，即得到106CFU/ml的菌悬液；
(2)给药：手术前14天进行预保护处理(灌胃)，按照0.01ml/g(体重)的浓度给假手术组、CLP组PBS(磷酸缓冲盐)，保护组G11(PBS溶解)，每天给药1次，共14次，并记录体重；确保给药的时间段和手术时间相同；
(3)造模：①第15天造模：采用小动物吸入麻醉系统对小鼠进行麻醉：小鼠吸入含异氟烷2％(体积分数vol/vol)的氧气，麻醉流量为3L/min，造模过程中流量为1.5L/min；麻醉程度监测标准为肢体的撤回反射消失，将小鼠固定并持续吸入含异氟烷2％的氧气维持麻醉；②小鼠腹正中区域备皮，75％乙醇消毒皮肤两次，延中下腹部正中行纵切口1cm，逐层切开分离皮肤、皮下组织，见腹白线，延腹白线切开腹直肌及腹膜，切口两侧用0.9％生理盐水湿润，用弯镊进腹，找到盲肠后轻柔牵出，将靠近回盲瓣处的粪便轻柔挤向盲肠末端(避免空气残留)，在盲肠末端至回盲瓣连线上2/3处(生存率实验为CLP损伤加重模型，功能学检测采用盲肠末端至回盲瓣连线上1/3处结扎，其余步骤与加重模型相同)用4号无菌手术缝线结扎盲肠，在结扎线与盲肠末端中点处用2ml注射器针头对穿已结扎肠(避开血管)，穿孔后轻轻挤压盲肠，可见结扎段盲肠内容物质顺穿刺孔流出，将盲肠连同周围所有肠管还纳入腹腔。用4号无菌手术缝线逐层间断缝合腹膜及皮肤。③术毕，所有实验小鼠均于术后立即背部皮下注射37℃生理盐水(0.1ml/g体重)进行液体复苏，标记后放回鼠笼，等待苏醒。假手术组除不进行盲肠结扎穿孔外，其余步骤与实验组相同；
(4)CLP手术后开始计时，每1h观察一次，记录72h内各个分组小鼠死亡数量，统计并分析生存率；
(5)根据各组生存率结果和小鼠体重的变化确定G11最佳保护浓度，筛选确定出最佳保护浓度后，同上进行动物实验，所取得的标本进行后续检测。
结果：
小鼠生存率造模如图3A所示，生存率曲线所图3B所示，与对照组相比，CLP处理后，小鼠约在48h全部死亡；值得注意的是，G11治疗提高了小鼠生存率(与CLP组相比，P<0.05)。G11(1×108和1×106)具有明显的保护作用，使小鼠在CLP后72h内的存活率提高了约30％。此外，与对照组相比，G11(1×108)处理对正常小鼠体重无显著影响(图4)，因此后续功能学检测将采用该浓度的G11进一步研究。
实施例3:发明人研究发现G11能改善败血症评分及败血症引起的血常规变化。
方案：
采用CLP手术，于在体水平上构建败血症及其诱发的心肌损伤模型(后续所有功能学检测采用盲肠末端至回盲瓣连线上1/3处结扎，其余步骤与加重模型相同，见图5A)，给与G11处理。
步骤：
(1)根据败血症评分表对CLP后8h的小鼠进行打分，评分表根据文献“A robustscoring system to evaluate sepsis severity in an animal model”中公开的败血症评分评分标准。
(2)检测CLP手术8h后小鼠血常规各项指标的变化：损伤8h后，采用摘眼球取血法取血，采用全自动血常规仪进行血常规检测。
结果：
于小鼠CLP处理8h进行败血症评分，结果如图5B所示，与对照组相比，CLP组败血症评分显著升高，给与G11保护后，败血症评分显著降低；
于小鼠CLP处理8h后检测了血常规相关指标的变化，如图7所示，与对照组相比，白细胞(White Blood Cell，WBC)、淋巴细胞(Lymphocyte，LYM)、中间细胞(Middle Cells，MID)、粒细胞(Granulocytes，GRA)和血小板(Platelets，PLT)显著下降，红细胞(Red bloodcells,RBC)显著增加(P<0.05)，而G11处理后可逆转这些变化(P<0.05)，除了PLT(P>0.05)；
实施例4:发明人研究发现G11能改善败血症引起的血生化变化
方案：
采用CLP手术，于在体水平上构建败血症及其诱发的心肌损伤模型，给与G11处理。
步骤：
检测CLP手术8h后小鼠血生化各项指标的变化：损伤8h后，采用摘眼球取血法取血，收集每组全血，3000r/min，离心10min，吸取血清，随后使用全自动血生化分析仪进行检测。
结果：
于小鼠CLP处理8h后检测了血生化相关指标的变化，如图6所示，与对照组相比较，CLP损伤8h后，血清中乳酸脱氢酶(LDH)、尿素氮(BUN)和谷草转氨酶(AST)水平均显著上升，ALB显著下降(P<0.05)，而给与G11处理后，LDH、BUN和AST水平出现明显下降(P<0.05)，而ALB无显著性差异(P＞0.05)。
实施例5：发明人研究发现G11可以改善败血症引发的心肌功能损伤方案：
采用CLP手术，于在体水平上构建败血症及其诱发的心肌损伤模型，给与G11处理。
步骤：
小动物超声检测CLP手术8h后小鼠心脏功能：各组动物于超声检测前一天脱去小鼠左胸区域被毛，小鼠经2％异氟烷麻醉，麻醉气体流量为1L/min，异氟烷吸入麻醉后固定于37℃恒温板上，充分暴露左侧胸廓，采用30MHz探头，选取标准胸骨旁左室长轴切面及标准左心室乳头肌短轴切面，记录M模心脏超声切面图像，测量指标包括：心输出量、每搏输出量、左室收缩末期容积、左室舒张末期容积、左室收缩末期后壁厚度和左室舒张末期后壁厚度。
在检测过程中注意以下几点可能影响检测结果的细节：第一，麻醉状态不可以过深，否则会影响小鼠的心率和收缩功能；第二，小鼠的体位要摆好，四肢不可固定地过伸过紧，否则会压迫小鼠心脏，最终影响心功能检测的准确性；第三，小鼠靠近心脏部分至少提前一天进行脱毛处理，脱毛太早会导致检测时生出新毛发，成像时生成伪影，影响超声结果，太晚则使小鼠处于应激状态干扰心功能结果。
结果：
小动物超声检测CLP手术8h后小鼠心肌收缩功能，结果如图8A(左室长轴超声结果),图8B(左室短轴超声结果)所示，与对照组相比，小鼠心脏的每搏输出量、心输出量、左室收缩末期容积、左室舒张末期容积显著下降(P<0.05)，给与G11保护后，心功能明显改善(P<0.05)。以及与对照组相比，小鼠左室收缩末期后壁厚度、左室舒张末期后壁厚度明显变厚，心率明显加快(P<0.05)，给与G11保护后，心功能明显改善(P<0.05)。
实施例6:发明人研究发现G11能改善败血症引起的心肌组织损伤
方案：
采用CLP手术，于在体水平上构建败血症及其诱发的心肌损伤模型，给与G11处理。
步骤：
(1)心肌组织HE染色：
①石蜡包埋：心尖部缓慢注入含肝素生理盐水，右心耳流出的液体变为透明时，将灌注液体替换为4％多聚甲醛固定液；多聚甲醛组织固定成功后，沿心脏根部剪断各血管，完整取下心脏；心脏取出后，切下心脏左半边(左心房及左心室)放入4％多聚甲醛中，进行后固定至少24h；按照顺序依次在80％、95％、95％、100％乙醇中浸泡40min，再按照100％乙醇、100％乙醇∶二甲苯＝1∶1混合液、二甲苯浓度梯度浸泡30min进行组织脱水透明；然后于包埋机中浸蜡3h，最后进行滴蜡包埋。
②切片：设置切片厚度为5μm，使用捞片法将切片贴于多聚赖氨酸覆膜载玻片上，70℃烤片1h后，60℃烤片5h。
③染色：将切片置于二甲苯中浸泡10min，更换二甲苯再次浸泡10min，按照100％、100％、95％、95％、80％乙醇、去离子水的顺序依次浸泡2min脱蜡至水，以备染色；将切片浸入苏木素染液染色3min，使用自来水轻柔冲洗5min；浸入1％盐酸乙醇30s，1％氨水3min脱色，使用自来水轻柔冲洗3min；浸入伊红染液染色3min，使用自来水轻柔冲洗3min；按照70％、80％浓度乙醇30s，95％、95％、95％、100％、100％浓度梯度乙醇、二甲苯、二甲苯的顺序分别浸泡2min进行脱水透明处理；中性树胶封片。
结果：
小鼠心肌组织HE染色结果显示，如图9所示，与对照组相比，CLP损伤后心肌组织结构紊乱，间质水肿加重，细胞完整性破坏；与CLP组相比，G11显著减轻CLP引起的心肌形态损伤。
实施例7：发明人研究发现G11通过减轻炎症和氧化应激，改善感染性CLP引起的心肌损伤
方案：
采用CLP手术，于在体水平上构建败血症及其诱发的心肌损伤模型，给与G11处理。
步骤：
免疫组织化学检测：
①石蜡包埋、切片步骤同实施例6；
②染色：切片常规脱蜡至水：分别取各组小鼠心脏组织石蜡切片依次经二甲苯2次，每次10min，100％乙醇2次、每次10min；95％、90％、80％、70％乙醇各1次，每次5min，最后浸入蒸馏水中5min；抗原修复：柠檬酸钠缓冲液微波抗原修复20min，流水冲洗10min；阻断内源性过氧化物酶：3％双氧水，室温15min。PBS洗3次，每次5min；封闭：滴加5％正常山羊血清封闭液，室温孵育60min；滴加一抗：擦去多余血清，加一抗，4℃孵育过夜，PBS洗3次，每次5min；滴加二抗：滴加辣根过氧化物酶HRP标记的二抗(1∶5000，PBS配制)，37℃温箱内孵育1h，PBS洗3次，每次5min；DAB显色：滴加DAB 0.5-3min，镜下控制显色程度，流水冲洗10min，苏木素复染，1％盐酸酒精分化，1％氨水脱色，脱水，经二甲苯透明后用中性树胶封片。
③显微镜下观察并拍照：显微镜下观察并拍照，光镜下组织切片呈棕黄色颗粒性沉积区域为阳性染色部位，每张切片随机找出20-30个不重叠视野，采用医学图象分析软件Image J 5.0软件半定量计算。
结果：
小鼠心肌组织IHC染色结果显示，如图10和11所示，与对照组相比，CLP损伤后炎症相关指标(MPO、IL-1β、TNF-α和IL-6)与氧化应激标志物(NOX2)表达均显著升高(P<0.05)，给与G11处理后其表达量显著降低(P<0.05)。
Claims (5)
1.植物乳杆菌用于制备治疗和/或预防败血症药物的应用，所述植物乳杆菌的保藏编号为CGMCC No.16937。
2.植物乳杆菌用于制备治疗和/或预防败血症诱发的心肌损伤药物的应用，所述植物乳杆菌的保藏编号为CGMCC No.16937。
3.一种治疗和/或预防败血症的药物，其特征在于，所述药物包括植物乳杆菌和药物辅料，所述植物乳杆菌的保藏编号为CGMCC No.16937。
4.如权利要求3所述药物，其特征在于，所述药物为口服给药制剂。
5.如权利要求3或4所述药物，其特征在于，所述药物的给药剂量为0.01ml((1×106CFU/mL)-(1X108CFU/mL))/g体重。
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