CN109303776B - 邪蒿素在制备治疗炎症性疾病药物中的应用 - Google Patents
邪蒿素在制备治疗炎症性疾病药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种天然香豆素化合物邪蒿素作为活性成分应用于治疗炎症性疾病的用途。邪蒿素作为抗炎活性成分，具有毒性小、疗效好的特点，对炎症因子尤其是巨噬细胞因子有显著抑制作用，因此可作为抗炎制剂用于其它多种巨噬细胞参与的炎症性疾病，如脓毒症、关节炎、免疫型肠炎、糖尿病等，特别对难以治愈的脓毒症具有良好的治疗作用。
Description
技术领域
本发明属于生物制药技术领域，具体涉及一种香豆素化合物邪蒿素Seselin作为活性成分治疗炎症性疾病中的应用，主要应用于多种巨噬细胞参与的炎症性疾病，如关节炎、免疫型肠炎、糖尿病等，特别对难以治愈的脓毒症具有良好的治疗作用。
背景技术
巨噬细胞做为天然免疫系统的忠告组成成分，参与多种严正性疾病的病理过程，包括脓毒症，关节炎、免疫型肠炎、糖尿病等。其中，脓毒症在世界范围仍然是一个巨大的医疗难题，伴随有严重的全身性炎症综合征，导致多器官衰竭，并造成极高的死亡率。在北美国家，推测脓毒症发病率每年超过600000例，死亡率高达30%-50%。目前临床上对脓毒症的治疗尚缺乏有效的治愈手段。巨噬细胞参与的炎症反应被认为是脓毒症发病过程极为重要的因素，抑制巨噬细胞炎症反应能够有效的缓解脓毒症症状，因此靶向巨噬细胞介导的炎症可以作为脓毒症治疗药物的筛选指标。另外，类风湿性关节炎患者滑膜组织处存在巨噬细胞，分泌促炎因子，生长因子，趋化因子等，诱发滑膜炎症，造成关节损伤，所以调控巨噬细胞炎症反应也可以作为抗关节炎药物的评价指标。
邪蒿素，英文名称Seselin，分子式：C14H12O3，分子量: 228.24300， CAS No：523-59-1，该化合物溶于甲醇、乙醇、丙酮、乙酸乙酯、氯仿等有机溶剂。对其抗炎活性研究较少，至今没有作为活性成分治疗炎症性疾病中的应用。
发明内容
本发明的目的是探索邪蒿素作为抗炎药物在脓毒症及相关炎症性疾病中的应用。
本发明通过研究证明，邪蒿素主要抗炎机制为调整巨噬细胞极化，可用于治疗脓毒症等炎症性疾病。
邪蒿素是从天然植物中分离纯化得到或合成可得。邪蒿素可抑制其炎症因子的表达，表明邪蒿素具有较好的抗炎作用。随后我们进行体内实验进行检测其抗炎功效。与对照组相比，邪蒿素在30 mg/kg 的剂量给药时能够显著提高小鼠的存活率，改善肺脏损伤，同时对血清炎症因子水平IL-1β, IL-6, TNF-α有显著抑制作用。
与现有技术比较本发明的有益效果：邪蒿素来源于天然植物的提取物中，作为抗炎活性成分，具有毒性小、疗效好的特点，对炎症因子尤其是巨噬细胞因子有显著抑制作用，因此可作为抗炎制剂用于其它多种巨噬细胞参与的炎症性疾病，如脓毒症、关节炎、免疫型肠炎、糖尿病等，特别对难以治愈的脓毒症具有良好的治疗作用。
附图说明
图1：邪蒿素的分子结构
图2：邪蒿素不影响巨噬细胞的存活率，同时抑制巨噬细胞分泌IL-1β, IL-6细胞因子。
（A，B）Raw 264.7 细胞以1*106个细胞/孔种在6孔板中，同时加入邪蒿素、10 ng/ml LPS 和10 ng/ml IFN-γ, 37℃孵育6小时后，QPCR 法检测IL-1β, IL-6 mRNA 水平的变化。结果以mean ± s.e.m表示。*P<0.05, **P<0.01, vs LPS & IFN-γ组。
图3：邪蒿素改善小鼠脓毒症的存活率。
小鼠腹腔注射不同剂量的邪蒿素（10mg/kg, 30mg/kg）2小时后盲肠穿刺结扎（CLP）。观察并记录小鼠存活率。n=10，*P<0.05, **P<0.01, vs CLP组。
图4: 邪蒿素降低脓毒症小鼠血清炎症因子IL-1β, IL-6, TNF-α水平。
小鼠腹腔注射不同剂量的邪蒿素（10mg/kg, 30mg/kg）2小时后盲肠穿刺结扎（CLP）。八小时后将小鼠处死，摘眼球取血，ELISA法检测血清炎症因子IL-1β, IL-6, TNF-α水平。n=6，*P<0.05, **P<0.01, vs CLP组。
图5：邪蒿素改善脓毒症小鼠肺脏病变。
小鼠腹腔注射不同剂量的邪蒿素（10mg/kg, 30mg/kg）2小时后盲肠穿刺结扎（CLP）。四小时后将小鼠处死，一部分肺脏组织10％福尔马林溶液固定，石蜡包埋，作4-5微米厚的冠状切片，并用苏木精－曙红染色。观察组织病变以及炎症细胞浸润。
图6: 邪蒿素降低脓毒症小鼠肺脏组织炎症因子水平。
（B）一部分肺脏组织加Trizol匀浆后提取总RNA，QPCR法检测IL-1β, IL-6 TNF-αmRNA水平的改变。n=6, 结果以mean ± s.e.m表示。*P<0.05, **P<0.01, vs CLP组。
图7: 各个细胞因子的参数
具体实施方式
以下通过具体实施方式对本发明效果做进一步说明：
实施例1. 邪蒿素抗炎活性检测
Raw 264.7 细胞以1*106个细胞/孔种在6孔板中，同时加入邪蒿素、10 ng/ml LPS和10 ng/ml IFN-γ, 37℃孵育6小时后，QPCR 法检测IL-1β, IL-6 mRNA 水平的变化。结果以mean ± s.e.m表示。*P<0.05, **P<0.01, vs LPS & IFN-γ组。
实验结果显示：LPS和IFN-Y刺激后，巨噬细胞被活化，细胞因子IL-1β, IL-6 mRNA水平升高，邪蒿素作用下，IL-1β, IL-6 mRNA水平显著降低（图2A和B））。揭示邪蒿素对炎症因子尤其是巨噬细胞因子有显著抑制作用，因此可作为抗炎制剂用于其它多种巨噬细胞参与的炎症性疾病，如脓毒症、关节炎、免疫型肠炎、糖尿病等。
实施例2. 邪蒿素对脓毒症的作用
1）盲肠穿刺结扎（CLP）诱导的小鼠sepsis 模型建立方法
取6-8周龄C57/BL6小鼠，实验前一天，小鼠腹部刮毛备用。手术前，小鼠1%戊巴比妥钠麻醉，仰位固定于手术板，腹部裸露皮肤用碘伏消毒。用手术刀在皮肤正中纵向切开小口，找到腹白线，随后用手术剪沿腹白线将切口开大。确定盲肠位置，一般在腹腔左侧，采用钝性解剖钳分离直肠并取出盲肠。用医用手术线结扎盲肠并根据所需疾病严重程度在盲肠不同位置穿刺。轻轻挤压穿刺位置，渗出少量粪便。将盲肠放回腹腔，缝合伤口，涂抹金霉素眼膏，用创可贴包裹伤口。将小鼠放置于温暖处苏醒。
2）小鼠存活率的观察
CLP前2小时，对小鼠腹腔注射橄榄油溶解的邪蒿素（10mg/kg, 30mg/kg），正常组和模型组腹腔注射同体积的橄榄油做为对照。每隔2个小时观察并记录小鼠存活率，直至小鼠存活率稳定不再发生变化。n=10, 结果以mean ± s.e.m表示。*P<0.05, **P<0.01, vsCLP组。
3）血清炎症因子IL-1β, IL-6, TNF-α的检测
另取一批小鼠，小鼠腹腔注射不同剂量的邪蒿素（10mg/kg, 30mg/kg）2小时后盲肠穿刺结扎（CLP），正常组和模型组腹腔注射橄榄油做为对照。八小时后将小鼠处死，摘眼球取血，37 °C 水浴30min 后3500 rpm离心15分钟，吸取血清ELISA法检测血清炎症因子IL-1β, IL-6, TNF-α水平。n=6, 结果以mean ± s.e.m表示。*P<0.05, **P<0.01, vs CLP组。
4）肺脏组织病理变化的观察
处死的小鼠同时取肺脏组织。一部分10％福尔马林溶液固定，石蜡包埋，作4-5微米厚的冠状切片，并用苏木精－曙红染色。观察组织病变以及炎症细胞浸润。
5）肺脏组织细胞因子mRNA水平的检测
处死的小鼠，取一部分肺脏组织加Trizol匀浆后提取总RNA，定量后取1μg RNA逆转录成cDNA，反应体系如下：1 μg 总RNA, 2000 pM Oligo dT, 1.0 mM dNTP, 200 U 逆转录酶, 5×转录缓冲液。全基因组cDNA 使用特异性引物进行扩增，反应体系如下：1 μlcDNA, 0.5μM 上下游引物，5μl iQ™ SYBR® Green permix。PCR引物及扩增参数见表1。扩增结果使用BioRad CFX manager software 进行分析。
结果显示：
小鼠腹腔注射不同剂量的邪蒿素（10mg/kg, 30mg/kg）2小时后盲肠穿刺结扎（CLP）。CLP诱发小鼠产生严重的急性炎症反应，血清炎症因子升高，多个器官发生病变，伴随较高的死亡率。与模型组相比，邪蒿素高剂量组（30 mg/kg）小鼠存活率显著升高（图3）。同时，短期实验结果表明，邪蒿素治疗组小鼠血清炎症因子IL-1β, IL-6, TNF-α水平被显著抑制（图4）。说明邪蒿素可显著改善脓毒症小鼠病情。
CLP作用下，小鼠多器官受损，其中肺脏最先受到损伤。CLP 作用4小时后，小鼠肺脏组织形态受到破坏，坏死细胞增多，并伴随明显的炎症细胞浸润。而邪蒿素治疗有效的保护了小鼠肺脏。其中，组织病变得到缓解，坏死细胞、浸润的炎症细胞减少（图5）。同时，肺脏组织细胞炎症因子IL-1β, IL-6, TNF-α mRNA 水平被抑制，抑制了炎症因子对脏器的破坏（图6）。说明邪蒿素可显著改善脓毒症小鼠肺脏病变。
Claims (1)
1.邪蒿素在制备用于治疗脓毒症药物中的用途。
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