CN108578420B - 木犀草苷在制备防治脓毒血症药物中的应用 - Google Patents
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Abstract
本发明公开了木犀草苷在制备防治脓毒血症药物中的应用。本发明通过动物实验证明在盲肠结扎穿孔术(cecal ligation puncture,CLP)诱导的脓毒血症小鼠模型上,木犀草苷能显著降低脓毒血症小鼠的死亡率,提高存活率,显著减少脓毒血症小鼠血清中早期及晚期炎症因子TNF‑α、IL‑6、HMGB1的表达水平,减轻靶器官肺、肾、肝的损伤,具有明显的防治脓毒血症的作用,且木犀草苷抗脓毒血症的作用可能与抑制HMGB1介导的MAPK和NF‑κB信号通路密切相关,表明该化合物可以进一步开发为治疗或预防脓毒血症的药物。
Description
技术领域
本发明涉及医药技术领域,尤其涉及木犀草苷在制备防治脓毒血症药物中的应用。
背景技术
脓毒血症是细菌等严重感染所致的全身炎症反应综合征,迄今尚无特效药物或疗法,致死率高达20%至45%,被视为人类健康的巨大威胁。随着抗生素耐药性的不断增加,研发确实有疗效的抗脓毒血症药物已经成为医药领域的重大命题。
木犀草苷是天然黄酮类化合物,其分子式为C21H20O11,CAS号5373-11-5,结构式如式(I)所示。
木犀草苷是多种抗病毒、清热中药及中成药中的活性成分,如金银花、热毒宁注射液、痰热清注射液等,具有抑菌、抗炎、抗过敏、抗肿瘤、抗病毒等功效。
申请公布号为CN107412242A的发明专利申请公开了木犀草苷在制备预防和治疗阿尔茨海默病药物中的应用;该发明发现木犀草苷在细胞及5xFAD小鼠模型中可显著加ApoE、ABCA1、ABCG1蛋白水平及大颗粒ApoE脂化蛋白水平,在5xFAD小鼠模型中木犀草苷显著降低大脑皮层中Aβ水平及Aβ斑块的数量。故,木犀草苷可以用于治疗ApoE蛋白水平或大颗粒ApoE脂化蛋白水平降低引起的机能障碍或者疾病的治疗用途的化合物。
申请公布号为CN105920026A的发明专利申请公开了木犀草苷在制备预防或治疗手足口病药物中的应用,其中,所述的手足口病是由肠道病毒71型、柯萨奇病毒A组16型、4型、5型、7型、9型和10型、柯萨奇病毒B组2型、5型或埃可病毒引起的手足口病。
然而,关于木犀草苷在脓毒血症治疗和/或预防方面的作用未见任何报道。
发明内容
本发明提供了木犀草苷在防治脓毒血症中的新用途,可以作为新的防治脓毒血症有效药物。
具体内容如下:
本发明提供了木犀草苷在制备防治脓毒血症药物中的新用途。
申请人通过大量动物实验发现,在盲肠结扎穿孔术(cecal ligation puncture,CLP)诱导的脓毒血症小鼠模型上,即目前国际上被认为最接近脓毒血症临床情况的动物模型上,确证了木犀草苷能够显著降低脓毒血症小鼠的死亡率,提高存活率。
故,本发明提供了木犀草苷在制备降低脓毒血症死亡率的药物中的应用。
此外,本发明还提供了木犀草苷在制备用于防治脓毒血症引起的靶器官损伤药物中的应用。
具体地,所述的靶器官为肺脏、肝脏或肾脏。
申请人通过动物实验发现,在盲肠结扎穿孔术(cecal ligation puncture,CLP)诱导的脓毒血症小鼠模型上,木犀草苷能够明显减轻脓毒血症引起的靶器官损伤,尤其是减轻肺、肾、肝的损伤。
本发明还提供了木犀草苷在制备用于控制脓毒血症引起的炎症反应药物中的应用。
具体地,所述的炎症反应由促炎细胞因子TNF-α、促炎细胞因子IL-6或炎症介质HMGB1的分泌所引起。
申请人通过动物实验发现,在盲肠结扎穿孔术(cecal ligation puncture,CLP)诱导的脓毒血症小鼠模型上,木犀草苷能够显著减少脓毒血症小鼠血清中早期及晚期炎症因子TNF-α、IL-6、HMGB1的表达水平。
本发明所述的木犀草苷无来源上的限制,只要保证纯度大于99%即可。
申请人还通过实验发现,木犀草苷可能通过抑制HMGB1介导的MAPK和NF-κB信号通路发挥抗脓毒血症的作用。
与现有技术相比,本发明具有以下有益效果:
本发明通过动物实验证明在盲肠结扎穿孔术(cecal ligation puncture,CLP)诱导的脓毒血症小鼠模型上,木犀草苷能显著降低脓毒血症小鼠的死亡率,提高存活率,显著减少脓毒血症小鼠血清中早期及晚期炎症因子TNF-α、IL-6、HMGB1的表达水平,减轻靶器官肺、肾、肝的损伤,具有明显的防治脓毒血症的作用,表明该化合物可以进一步开发为治疗或预防脓毒血症的药物。
附图说明
图1为木犀草苷对CLP引起的小鼠脓毒血症生存曲线的影响;其中,n=10,与假手术组比,*p<0.05;与模型组比,#p<0.05。
图2为木犀草苷对脓毒血症小鼠血清中TNF-α表达水平的影响;其中,与假手术组比,*p<0.05,**p<0.01,***p<0.001;与模型组比,#p<0.05,##p<0.01。
图3为木犀草苷对脓毒血症小鼠血清中IL-6表达水平的影响;其中,与假手术组比,***p<0.001;与模型组比,###p<0.001。
图4为木犀草苷对脓毒血症小鼠血清中HMGB1表达水平的影响;其中,与假手术组比,*p<0.05,***p<0.001;与模型组比,#p<0.05,##p<0.01。
图5为木犀草苷对脓毒血症小鼠靶器官(肺/肝/肾)损伤的影响。
图6为木犀草苷在LPS诱导的巨噬细胞炎症反应模型中对MAPK通路中p-ERK信号的影响。与对照组相比,***p<0.001;与模型组相比,###p<0.001。
图7为木犀草苷在LPS诱导的巨噬细胞炎症反应模型中对MAPK通路中p-JNK信号的影响。与对照组相比,***p<0.001;与模型组相比,##p<0.01。
图8为木犀草苷在LPS诱导的巨噬细胞炎症反应模型中对MAPK通路中p-p38信号的影响。与对照组相比,***p<0.001;与模型组相比,###p<0.001。
图9为木犀草苷在LPS诱导的巨噬细胞炎症反应模型中对NF-κB通路中p-IκBα信号的影响。与模型组相比,#p<0.05。
图10为木犀草苷在LPS诱导的巨噬细胞炎症反应模型中对NF-κB通路中p-p65入核的影响。与对照组相比,**p<0.01;与模型组相比,##p<0.01。
图11为木犀草苷在LPS诱导的巨噬细胞炎症反应模型中对细胞浆中HMGB1的影响。与对照组相比,*p<0.05;与模型组相比,##p<0.01。
图12为木犀草苷在LPS诱导的巨噬细胞炎症反应模型中对HMGB1入核的影响。与对照组相比,***p<0.001;与模型组相比,###p<0.001。
具体实施方式
下面结合具体实施例对本发明作进一步描述,以下列举的仅是本发明的具体实施例,但本发明的保护范围不仅限于此。
实施例1木犀草苷对CLP诱导的脓毒血症小鼠生存期的影响
体重18~22g的雌性C57BL/6小鼠适应性饲养3天后,根据体重随机分为4组,每组10只,分别设为假手术组,模型组,木犀草苷组(购自成都曼思特生物科技有限公司,纯度≥99%)及阳性药组(头孢他啶,购自武汉远成共创科技有限公司,纯度>99%)。除假手术组外,其余小鼠进行盲肠结扎穿孔术诱导脓毒血症。假手术组小鼠只将盲肠提出后放回腹腔,不进行结扎和穿孔。
木犀草苷组在造模麻醉前2h以15mg/kg的剂量灌胃给药,阳性药在造模2h后尾静脉注射头孢他啶溶液1.23g/kg,连续给药5天。每日观察小鼠的情况,记录死亡时间,直至造模后第10天。
采用GraphPad Prism 6软件中Survival分析绘制小鼠生存曲线,采用SPSS 20软件的Life Tables进行统计分析。
结果如图1所示,假手术组小鼠在10天内均未死亡;模型组小鼠于造模后第2天出现死亡,造模后第10天小鼠生存率为30%;木犀草苷组小鼠于造模后第4天出现死亡,造模后第10天小鼠生存率为70%,与模型组相比,木犀草苷组生存率显著提高;阳性药组小鼠于造模后第5天出现死亡,造模后第10天小鼠生存率为90%,与模型组相比,阳性药组生存率显著提高。
该结果表明,木犀草苷能显著降低CLP诱导的脓毒血症小鼠的死亡率,并且,虽然阳性药组生存率略高于木犀草苷组,但木犀草苷的作用剂量仅为15mg/kg,远低于阳性药头炮他啶给药剂量1.23g/kg,而且前者给药方式采用口服。
实施例2木犀草苷对脓毒血症小鼠血清中炎症因子表达水平的影响
小鼠如实例1进行造模和给药(n=5)。造模24h后,麻醉小鼠,打开腹腔,进行下腔静脉取血。在室温静置2h后,4℃,4000rpm,离心10min,收集血清,分装后冻存于-80℃。根据ELISA试剂盒的操作步骤对血清中的TNF-α、IL-6、HMGB1炎症因子的含量进行测定。
统计方法采用GraphPad Prism 6软件中的单因素方差(one-wayANOVA)及Dunnett后验分析,当p<0.05时,表明差异显著。
结果如图2-4所示,与假手术组相比,模型组小鼠血清中的TNF-α、IL-6、HMGB1炎症因子水平均显著上升;而与模型组相比,木犀草苷组均能显著降低这些早期或晚期炎症因子在血清中的表达水平,表明木犀草苷治疗能明显地控制脓毒血症引起的炎症反应,治疗脓毒血症。
实施例3木犀草苷对脓毒血症小鼠靶器官损伤的影响
由于病理切片能直观地反应机体组织的病变程度。我们进一步考察了木犀草苷对脓毒血症小鼠靶器官损伤的影响。
小鼠同实例1进行造模和给药。造模24h后,剖取小鼠的肺、肝、肾,采用10%的中性福尔马林溶液固定组织,48h后,进行石蜡包埋,以4μm厚度切片,二甲苯脱蜡后,进行苏木精-伊红染色。在倒置显微镜下观察各组织样本的病理学变化,并拍摄照片。
结果如图6所示。在肺组织上,假手术组小鼠的肺组织无明显的病理变化;脓毒血症模型组小鼠的肺泡增厚,肺泡空间严重缩小,血管充血严重;阳性药组和木犀草苷组小鼠的肺损伤情况比模型组轻,均表现为肺泡壁少部分增厚,肺泡空间轻微缩小,伴有少量血管充血。
在肝组织上,假手术组小鼠的肝组织无明显的病理变化;模型组小鼠的肝细胞严重坏死,脂肪性变性严重,伴有大量的炎症细胞浸润;阳性药组和木犀草苷组小鼠的肝损伤情况比模型组轻,均表现为肝细胞部分坏死,脂肪性变性较轻微,伴有少量炎症细胞浸润。
在肾组织上,假手术组小鼠的肾组织无明显的病理变化;模型组小鼠的肾组织水肿很严重,伴有大量充血;阳性药组和木犀草苷组小鼠的肾损伤情况比模型组轻,均表现为水肿和充血情况较轻微。
上述结果表明:木犀草苷能明显减轻脓毒血症引起的靶器官损伤。
实施例4木犀草苷抗脓毒血症的作用机制研究
针对参与脓毒血症的主要炎性信号通路,采用LPS诱导的RAW264.7巨噬细胞炎症反应模型,研究了木犀草苷抗脓毒血症的作用机制。
取对数生长期的RAW264.7细胞,接种于6孔板,每孔6×105细胞。24h后,弃去原培养液。模型组以2mL/孔加入含有200ng/mL LPS的DMEM培养液,空白对照组以2mL/孔加入不含LPS的DMEM培养液,木犀草苷组以2mL/孔加入含有200ng/mL LPS和50μg/mL木犀草苷的DMEM培养液。孵育24h后,去除原培养液,用预冷的PBS清洗细胞,弃去PBS,加入80μL含1%PMSF的蛋白裂解液,提取蛋白,进行MAPK通路的western blot检测。当提取p-IκBα蛋白时,RAW264.7细胞接种于6孔板,每孔8×105细胞,给药浓度同上,孵育30min后提取蛋白,进行western blot检测。
取对数生长期的RAW264.7细胞,接种于10cm培养皿,接种密度为3×105/mL,10mL/孔。24h后,弃去原培养液。模型组以10mL/孔加入含有200ng/mL LPS的DMEM培养液,空白对照组以10mL/孔加入不含LPS的DMEM培养液,木犀草苷组以10mL/孔加入含有200ng/mL LPS和50μg/mL木犀草苷的DMEM培养液,孵育24h后,提取细胞核蛋白与细胞浆蛋白,进行p-p65和HMGB1蛋白的western blot检测。
研究结果表明:在LPS诱导的RAW264.7巨噬细胞炎症反应模型中,与对照组相比,模型组能促进HMGB1从细胞核进入细胞浆;显著升高MAPK通路中ERK1/2、JNK及p38的磷酸化水平;提高NF-κB通路中IκBα的磷酸化水平,促进p-p65从细胞浆进入细胞核;而与模型组相比,木犀草苷能阻滞HMGB1从细胞核进入细胞浆;显著降低MAPK通路中ERK1/2、JNK及p38的磷酸化水平,降低NF-κB通路中IκBα的磷酸化水平,抑制p-p65从细胞浆进入细胞核,表明木犀草苷可能通过抑制HMGB1介导的MAPK和NF-κB信号通路发挥抗脓毒血症的作用。
Claims (3)
1.木犀草苷作为唯一的活性成分在制备防治脓毒血症药物中的应用,所述的木犀草苷通过抑制HMGB1介导的MAPK和NF-κB信号通路发挥抗脓毒血症的作用。
2.木犀草苷作为唯一的活性成分在制备用于防治脓毒血症引起的靶器官损伤药物中的应用。
3.如权利要求2所述的应用,其特征在于,所述的靶器官为肺脏、肝脏或肾脏。
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