CN114617872A - Sil用于制备抗败血症及其诱发的心肌损伤药物的应用 - Google Patents
Sil用于制备抗败血症及其诱发的心肌损伤药物的应用 Download PDFInfo
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Abstract
本发明公开了SIL用于制备抗败血症及其诱发的心肌损伤药物的应用。本发明研究发现:SIL可提高败血症损伤后的小鼠生存率,改善小鼠败血症评分及肛温,升高白细胞、淋巴细胞、单核细胞、中性粒细胞的数量以及降低红细胞的数量,降低乳酸脱氢酶、肌酸激酶、谷草转氨酶、尿素氮的水平,升高白蛋白的水平;SIL具有改善心脏功能及维持心肌组织正常形态的作用;此外,SIL也能减少由败血症引起的超氧化物阴离子累积,并降低败血症诱发的心肌损伤后肿瘤坏死因子α、F4/80、Ly6G的表达,降低炎症相关分子TNF‑α、白细胞介素‑6、IL‑8、NOD样受体家族3、IL‑1β、Caspase1的mRNA水平,从而发挥抗氧化应激和抗炎作用。
Description
技术领域
本发明涉及水飞蓟宾(silibinin,SIL)的新适应症,具体涉及SIL用于抗败血症及其诱发的心肌损伤药物的应用。
背景技术
SIL结构式如式所示,其具有多种药理活性,包括抗肿瘤、保肝、护心、抑菌等作用。
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,观察小鼠生存率、败血症评分、肛温、血常规相关指标、血生化相关指标、心功能相关指标、组织病理学染色以及炎症相关mRNA水平发现:
SIL可提高败血症生存率,改善败血症评分及肛温,升高白细胞(White BloodCell,WBC)、淋巴细胞(Lymphocyte,LYM)、单核细胞(Monocytes,MON)、中性粒细胞(Granulocyte,GRA)的数量、降低红细胞(Red Blood Cell,RBC)的数量,降低乳酸脱氢酶(Lactate Dehydrogenase,LDH)、肌酸激酶(Creatine Kinase,CK)、谷草转氨酶(AspartateAminotransferase,AST)、尿素氮(Blood Urea Nitrogen,BUN)的水平以及升高白蛋白(Albumin,ALB)的水平。
SIL能改善由败血症引起的心脏功能障碍,表现为提高每搏输出量(StrokeVolume,SV)、心输出量(Cardiac Output,CO)、左心室收缩末期容积(Left VentricularEnd Systolic Volume,LVESV)、左心室舒张末期容积(Left Ventricular End DiastolicVolume,LVEDV),降低左心室收缩末期后壁厚度(Left Ventricular End SystolicPosterior Wall,LVPWs)和左心室舒张末期后壁厚度(Left Ventricular End DiastolicPisterior Wall,LVPWd);同时,SIL可改善由败血症引起的心肌组织形态紊乱,发挥减轻心肌损伤的作用。
SIL也能减少由败血症引起的超氧化物阴离子累积,并降低败血症诱发的心肌组织损伤后TNF-α、F4/80、Ly6G的表达,降低TNF-α、IL-6、IL-8、NLRP3、IL-1β、Caspasel的mRNA水平,从而发挥抗氧化应激和抗炎作用。
基于上述发现,本发明提供了SIL用于制备治疗和/或预防败血症及其诱发的心肌损伤药物的应用。
同时提供一种治疗和/或预防败血症药物,所述药物由SIL和药物辅料制备而成,所述药物为静脉注射和/或口服给药制剂,所述药物的给药剂量为每千克体重50mg-150mgSIL。
附图说明:
图1为SIL对CLP损伤后小鼠生存率的影响,观察CLP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.Sham组,*P<0.05,#vs.CLP组,#P<0.05;
图2为SIL对CLP损伤8h后小鼠败血症评分及肛温的影响,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图为败血症评分统计分析图,C图为小鼠肛温统计分析图,结果以“均数±标准差”表示,n=6;*vs.CLP组,*P<0.05;
图3为SIL对CLP损伤8h后小鼠血常规各项指标的影响,于小鼠术后8h测定血常规,WBC:白细胞;LYM:淋巴细胞;MON:单核细胞;GRA:中性粒细胞;RBC:红细胞;结果以“均数±标准差”表示,n=6;*vs.CLP组,*P<0.05;
图4为SIL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,LDH:乳酸脱氢酶;CK:肌酸激酶;AST:谷草转氨酶;BUN:尿素氮;ALB:白蛋白;结果以“均数±标准差”表示,n=6;*vs.CLP组,*P<0.05;
图5为SIL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功能指标的统计分析图:SV:每搏输出量;CO:心输出量;LVESV:左心室收缩末期容积;LVEDV:左心室舒张末期容积;LVPWs:左心室收缩末期后壁厚度;LVPWd:左心室舒张末期后壁厚度;B图为超声心动图短轴切面、M模典型图片以及各项心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;
图6为SIL对CLP损伤8h后心肌组织形态的影响,各组心肌组织切片HE染色结果;HE:苏木精-伊红;
图7为SIL对CLP损伤8h后心肌组织中超氧化物阴离子水平的影响,各组心肌组织切片DHE染色结果;DHE:超氧化物阴离子荧光探针;
图8为SIL对CLP损伤8h后心肌组织炎症相关指标的影响,A图为炎症相关典型指标TNF-α、F4/80、Ly6G的免疫组织化学染色照片和统计分析图;B图为炎症相关分子TNF-α、IL-6、IL-8、NLRP3、IL-1β、Caspasel的mRNA表达水平的统计分析图,结果以“均数±标准差”表示,n=6,*vs.CLP组,*P<0.05。
具体实施方式
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50%。在败血症早期,心肌损伤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关键研究意义。
盲肠结扎穿孔(Cecal Ligation and Puncture,CLP)动物模型是经典的败血症动物模型,本发明采用CLP动物模型为研究对象,根据现有技术研究,该动物模型可造成急性心肌损伤,即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、面色苍白以及发热等。
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。
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。
需要说明的是,以下实施例所用的SIL纯度为HPLC≥98%。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:发明人研究发现SIL能够提高败血症损伤后的小鼠生存率
方案:
采用CLP手术,于在体水平上构建败血症模型,给予SIL预保护处理。
步骤:
使用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(Sham)、CLP组、SIL+CLP组(50、100、150mg/kg剂量),共五组,每组12只;
(2)制药:使用DMSO(二甲亚砜)溶解SIL,制成剂量分别为50mg/kg、100mg/kg、150mg/kg的SIL溶液;
(3)给药:CLP造模前6天对各组小鼠进行预保护处理(腹腔注射),按照1ml/kg(体重)的浓度给予Sham组、CLP组DMSO(腹腔注射);SIL+CLP组:注射50mg/kg、100mg/kg、150mg/kg的SIL溶液,腹腔注射,每2天给药1次,共3次,确保每次给药的时间段和手术时间相同;
(4)CLP造模:①第7天造模,采用小动物吸入麻醉系统对小鼠进行麻醉:小鼠吸入浓度为3%的异氟烷(空气流量为1L/min),造模过程中持续麻醉的异氟烷浓度为1.5%(空气流量为1L/min),麻醉程度监测标准为肢体的撤回反射消失;②小鼠腹正中区域备皮,用75%乙醇消毒皮肤两次,沿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,沿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4-0无菌手术缝线结扎盲肠,在结扎线与盲肠末端中点处用25G注射器针头对穿已结扎盲肠(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4-0无菌手术缝线逐层间断缝合腹膜及皮肤,造模过程如图2A;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(10ml/kg体重)进行液体复苏,妥善标记后放回鼠笼,等待苏醒;Sham组除不进行盲肠结扎穿孔外,其余步骤与CLP组造模相同。
(5)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。
结果:
小鼠生存率曲线如图1B所示,与Sham组相比,CLP损伤后,小鼠72h内生存率为25%,SIL(50mg/kg)处理后小鼠生存率约为58%(P<0.05),SIL(100mg/kg)处理后小鼠生存率约为91%(P<0.05),SIL(150mg/kg)处理后,小鼠生存率约为91%(P<0.05),提示SIL可以提高小鼠CLP后的生存率,并选取100mg/kg为SIL的最佳保护浓度(后续功能学检测将采用此浓度)。
实施例2:发明人研究发现SIL能改善败血症评分及小鼠肛温
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,如图2B),给予SIL预保护处理。
步骤:
(1)分组:功能学检测:将BALB/c小鼠分为Sham组、CLP组、SIL+CLP组(100mg/kg剂量,腹腔注射),每组6只,给药方式同实施例1;
(2)CLP造模:结扎位置采用盲肠末端至回盲瓣连线1/3处结扎,其余步骤同实施例1;
(3)根据败血症评分表(“A robust scoring system to evaluate sepsisseverity in an animal model”中公开的败血症评分评分标准)对CLP手术8h后的小鼠进行打分;
(4)检测CLP手术8h后小鼠肛温的变化:损伤8h后,将小鼠固定,用棉球将小鼠肛门清理干净,随后将温度检测探针轻轻插入小鼠肛门,待数据稳定后记录温度。
结果:
于小鼠CLP 8h后进行败血症评分,结果如图2B所示,与Sham组相比,CLP组败血症评分显著升高(P<0.05),给予SIL保护后,败血症评分显著降低(P<0.05);
于小鼠CLP 8h后进行肛温检测,结果如图2C所示,与Sham组相比,CLP组肛温显著降低(P<0.05),给予SIL保护后,肛温显著升高(P<0.05);
实施例3:发明人研究发现SIL能改善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,给予SIL预保护处理,具体步骤同实施例2。
步骤:
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 8h后检测血常规相关指标的变化,结果如图3所示,与Sham组相比,CLP损伤后WBC、LYM、MON、GRA显著降低(P<0.05),RBC显著升高(P<0.05),而SIL处理后WBC、LYM、MON、GRA均显著升高(P<0.05),RBC显著降低(P<0.05)。
实施例4:发明人研究发现SIL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,给予SIL预保护处理,具体步骤同实施例2。
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,3000rpm/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 8h后检测血生化相关指标的变化,结果如图4所示,与Sham组相比较,CLP损伤8h后,血清中LDH、CK、AST、BUN水平均显著上升(P<0.05),ALB显著下降(P<0.05),给予SIL处理后,LDH、CK、AST、BUN水平明显下降(P<0.05),而ALB无显著变化(P>0.05)。
实施例5:发明人研究发现SIL可以改善败血症引发的心功能障碍方案:
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,给予SIL预保护处理,具体步骤同实施例2。
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3%异氟烷麻醉,麻醉气体流量为1 L/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::SV、CO、LVESV、LVEDV、LVPWs、LVPWd。
在检测过程中应该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:
使用小动物超声检测CLP手术8h后小鼠心脏功能,结果如图5A(左室长轴超声指标)、5B(左室短轴超声指标)所示,与Sham组相比,CLP损伤后,小鼠心脏的SV、CO、LVESV、LVEDV显著下降(P<0.05),LVPWs、LVPWd明显增加(P<0.05),给予SIL保护后,小鼠心脏的SV、CO、LVESV、LVEDV显著升高(P<0.05),LVPWs、LVPWd明显减少(P<0.05),小鼠心脏功能得到明显改善。
实施例6:发明人研究发现SIL能改善败血症引起的心肌组织结构紊乱
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,给予SIL预保护处理,具体步骤同实施例2。
步骤:
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染色结果如图6所示,与Sham组相比,CLP损伤后心肌组织纤维断裂,间质水肿增加,细胞完整性破坏;给予SIL后,CLP引起的心肌组织结构损伤得到明显改善。
实施例7:发明人研究发现SIL能减少由败血症引起的超氧化物阴离子累积
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,给予SIL预保护处理,具体步骤同实施例2。
步骤:
DHE染色:
①石蜡包埋、切片步骤同实施例5。
②DHE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、去离子水的顺序浸泡2min脱蜡至水,以备染色;脱蜡结束后,使用DHE染液于37下孵育30min,经过适当洗涤后,荧光显微镜下观察并拍照,荧光显微镜下组织切片呈红色荧光阳性染色部位,每张切片随机找出20-30个不重叠视野,光镜下组织切片呈红色荧光区域为阳性染色部位。
结果:
小鼠心肌组织DHE染色结果如图7所示,与Sham组相比,CLP损伤后红色荧光部位显著增多,且红色荧光强度显著增强,SIL处理后其荧光部位显著减少,荧光强度明显下降,CLP引起的小鼠心肌组织氧化应激得到明显改善。
实施例8:发明人研究发现SIL能减轻由败血症引起的炎症反应
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,给予SIL预保护处理,具体步骤同实施例2。
步骤:
(1)免疫组织化学检测:
①石蜡包埋、切片步骤同实施例5;
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20min。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30min;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DAB0.5-3min,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,每张切片随机找出20-30个不重叠视野,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。
(2)实时定量荧光PCR
①样品总RNA提取:将装有小鼠心脏样本的离心管从液氮中取出后,加入1mLTrizol和2个研磨小珠,置于组织破碎仪上于60Hz破碎1min,轻轻摇晃使Trizol与样品混匀,置于冰上裂解5min;加入200μL氯仿,手动剧烈震荡管体15s,室温孵育15min后,置于4高速离心机离心15min,转速为12000rpm/min;此时RNA全部位于上层水相中,用移液器吸取400μL水相转移至干净的无RNA酶的离心管中,加入等体积的异丙醇进行混合,手动摇晃15s,室温孵育10min后,置于4高速离心机离心15min,转速为12000rpm/min;弃上清,加入75%乙醇(用DEPC水配制),手动摇晃使RNA沉淀悬浮,室温孵育5min后,4,8000rpm/min离心5min,重复该操作一次;弃去乙醇溶液,留下RNA沉淀,室温干燥5-10min;采用20μL DEPC水溶解RNA沉淀,打开DNA/RNA浓度测定仪,测定样品的浓度,放于-80冰箱进行保存。
②反转录:将提取的总RNA从-80冰箱中取出,加入反转录试剂后置于PCR仪器中进行反转录,反转录程序为:3715min;855s;反转后的cDNA放于-20保存,反转录体系如表1所示:
表1反转录体系
③qRT-PCR:将cDNA、试剂盒(购自湖南艾科瑞生物工程有限公司;货号:AG11409-S)及所需引物(购自苏州金唯智生物科技有限公司;TNF-α:正向引物5’-ACTGAACTTCGGGGTGATCG-3’;反向引物5’-TGGTGGTTTGCTACGACGTG-3’;IL-6:正向引物5’-TCCGGAGAGGAGACTTCACA-3’;反向引物5’-TGCCATTGCACAACTCTTTTCT-3’;IL-8:正向引物5’-ATGGCTGCTCAAGGCTGGTC-3’;反向引物5’-GACCAGCCTTGAGCAGCCAT-3’;NLRP3:正向引物:5’-TCTACTCTATCAAGGACAGGAACG-3’;反向引物5’-CCTTTCTCGGGCGGGTAAT-3’;IL-1β:正向引物5’-CCTTGTGCAAGTGTCTGAAGC-3’;反向引物5’-AAGGGCTTGGAAGCAATCCT-3’;Caspase1:正向引物5′-AGAACAGAACAAAGAAGATGGCACA-3′;反向引物5′-GTGCCATCTTCTTTGTTCTGTTCTT-3′)从-20冰箱中取出,准备好实验所需的器材;待样品和试剂盒融化后,按照以下比例配制溶液后,使用qRT-PCR仪器设置好的程序进行实验;反应条件为:95预变性10min;95变性15s;58退火延伸20s;72终末延伸30s,共40个循环,反应体系如表2所示:
表2 qRT-PCR反应体系
结果:
小鼠心肌组织IHC染色结果如图8A所示,与Sham组相比,CLP损伤后TNF-α、F4/80、Ly6G表达均显著升高(P<0.05),给予SIL处理后其表达显著降低(P<0.05);
小鼠心肌组织qRT-PCR结果如图8B所示,与Sham组相比,CLP损伤后炎症相关分子TNF-α、IL-6、IL-8、NLRP3、IL-1β、Caspase1的mRNA表达水平均显著升高(P<0.05),给予SIL处理后其表达水平显著降低(P<0.05),CLP引起的小鼠心肌组织炎症得到明显改善。
Claims (3)
1.SIL用于制备治疗和/或预防败血症药物的应用。
2.SIL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。
3.一种治疗和/或预防败血症及其诱发的心肌损伤药物,其特征在于,所述药物由SIL和药物辅料制备而成,所述药物为静脉注射和/或口服给药制剂,所述药物的给药剂量为每千克体重50mg-150mg SIL。
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