WO2016150151A1 - 青蒿琥酯在制备治疗中晚期脓毒症药物中的运用 - Google Patents

青蒿琥酯在制备治疗中晚期脓毒症药物中的运用 Download PDF

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WO2016150151A1
WO2016150151A1 PCT/CN2015/092808 CN2015092808W WO2016150151A1 WO 2016150151 A1 WO2016150151 A1 WO 2016150151A1 CN 2015092808 W CN2015092808 W CN 2015092808W WO 2016150151 A1 WO2016150151 A1 WO 2016150151A1
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lps
group
sepsis
mice
artesunate
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周红
李小丽
李攀
郑江
潘夕春
邓冬梅
岑彦艳
李斌
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中国人民解放军第三军医大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel

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  • the invention belongs to the field of medicine, and particularly relates to the use of artesunate in the treatment of middle and late stage sepsis.
  • Sepsis is a systemic inflammatory response syndrome (SIRS) caused by infectious factors. It is a common complication of critically ill patients such as war wounds and burns. Severe cases of septic shock and multiple organ dysfunction syndrome (MODS) )even death. The rapid progress of sepsis and the high mortality rate are important causes of death in wartime or burn patients, timely diagnosis of the occurrence of sepsis and its progress, and effective treatment measures to improve the treatment of patients with sepsis. Survival rate is of great significance.
  • Lipopolysaccharide (LPS) present on Gram-negative bacteria membrane, peptidoglycan (PGN) of Gram-positive bacteria, Gram-negative bacteria and genomic DNA shared by positive bacteria (CpG DNA) are important pathogenic bacteria Factors, can induce the activation of the innate immune system, resulting in a large release of inflammatory mediators, the induction of inflammatory storms, and ultimately lead to septic shock and even death.
  • Clinical studies have shown that the inflammatory factors IL-6, IL-8, TNF- ⁇ , IL-1 are significantly elevated in the plasma of patients with sepsis, CD11b/CD18, HLA-DR, etc. are also significantly increased, is early in sepsis More sensitive indicators.
  • inflammatory mediators such as TNF- ⁇ , IL-1 and other monoclonal antibodies, glucocorticoids and other "immunosuppressive” and anti-inflammatory treatments can not improve the prognosis of patients with sepsis, and even increase the mortality rate.
  • inflammatory mediators such as TNF- ⁇ , IL-1 and other monoclonal antibodies, glucocorticoids and other "immunosuppressive” and anti-inflammatory treatments
  • the imbalance of the body causes immunosuppression of the body's immune system, such as neutrophil paralysis, transformation of Th1 cells into Th2 cells, and increased apoptosis of lymphocytes and dendritic cells.
  • the body can not effectively remove the primary pathogenic microorganisms, and is vulnerable to secondary attack by Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and other conditional pathogens. Further disorder of the body environment, severe septic shock, multiple organ failure eventually lead to death. Therefore, the immunosuppressive state of sepsis patients, in order to block the inflammatory response and suppress the immune treatment strategy, not only has no effect, but leads to the deterioration of the disease, the increase of mortality, so the accurate judgment of the condition of sepsis patients to improve pus The cure rate of toxic diseases is very important. Therefore, the development of new drugs based on advanced immune paralysis in patients with sepsis may be an effective strategy to effectively antagonize sepsis and reduce mortality.
  • Artemisinin and its derivatives have been used to treat malaria-related fever for more than a thousand years and are now clinically used primarily for drug-resistant falciparum malaria.
  • Efferth TM Wang X et al. describe in their article "Antiviral activity of artesunate towards wild-type, recombinant and ganciclovir-resistant human cytomegalovimses” (J Mol Med. 2002; 80(4): p223-224) that Artemisia annua also has Such as asthma, anti-cancer, anti-schistosomiasis and regulation of the immune system.
  • the research on artemisinin and its derivatives at home and abroad mainly focuses on the anti-malarial, anti-cancer, anti-schistosomiasis and its mechanism.
  • CN1833644 discloses that artemisinin and its derivatives such as artesunate can reduce the mortality of inactivated E. coli, bacteria (including Gram-negative, positive bacteria) attack mice, and can significantly inhibit mouse serum cells.
  • the release of factor TNF- ⁇ and IL-6 has significant protective effects on the main organs (heart, liver, intestine, lung, kidney) of mice inactivated by E. coli and bacteria.
  • the study is still based on the excessive release phase of inflammatory mediators in the early and middle stages of sepsis.
  • Artesunate mainly exerts therapeutic effects by inhibiting the release of inflammatory factors.
  • inflammatory factors such as TNF- ⁇ , IL-6, IL-1 and ⁇ -IFN are significantly reduced, while IL-10 and other anti-inflammatory factors are observed.
  • the factor is significantly higher.
  • the body can not effectively remove the primary pathogenic microorganisms, and is vulnerable to secondary attack by Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and other conditional pathogens. Further disorder of the body environment, severe septic shock, multiple organ failure eventually lead to death.
  • immunosuppression is the main cause of death of sepsis patients after receiving "second strike” such as bacteria and fungi. Therefore, the development of new drugs based on advanced immune paralysis in patients with sepsis may be an effective strategy to effectively antagonize advanced sepsis and reduce mortality.
  • the object of the present invention is the use of artesunate for the treatment of intermediate or advanced sepsis, in particular for the treatment of advanced sepsis.
  • the sepsis is preferably a patient with advanced pus.
  • the intermediate or late stage sepsis refers to a state in which the secretion of inflammatory factors in the body of a sepsis patient is significantly reduced, or the immunity of the patient suffering from sepsis is suppressed or the immunity is paralyzed.
  • the present inventors have surprisingly found that artesunate no longer inhibits the release of inflammatory factors when the immune function of the body is inhibited or the immune is paralyzed, but rather promotes the release of inflammatory factors, restores or enhances the body's immune function, thereby improving
  • the body's resistance to Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus can effectively prevent the body from being hit by bacteria, fungi, etc., and reduce the mortality of patients with sepsis.
  • Figure 1 shows the content of inflammatory factors in each control group of Example 2.
  • Figure 4 The amount of bacterial PA in the lung, spleen, and serum of LPS-tolerant mice in Example 6 after suffering a second round of bacterial attack.
  • mice 120 mice were divided into 6 groups, 20 in each group. Each group was intravenously injected with LPS at 10, 20, 30, 40, 50 and 80 mg/kg. The survival of each group was observed in 7 days. Screening of LPS resulted in 100% death of normal mice. The lowest dose. The results are shown in Table 1. The results of the experiment show that 50 mg/kg of LPS can cause 100% death in normal mice.
  • mice were divided into 3 groups, 6 mice in each group, one group was given daily (intraperitoneal injection) 0.3 mg/kg LPS, one group was given daily (intraperitoneal injection) 1.0 mg/kg LPS, and one group was given daily (intraperitoneal injection).
  • the LPS of 3.0 mg/kg was given to the pre-LPS for 3 days.
  • each group was given (intraperitoneal injection) 50 mg/kg of LPS, and the number of mice in each group was observed within 7 days after LPS injection. The results are shown in Table 2.
  • mice 24 mice were divided into 4 groups, 6 mice in each group, normal group (NS), given the same volume of normal saline; LPS (0.3) group: 0.3 mg /kg injection of LPS for 3 consecutive days, the fourth day of intraperitoneal injection of 50mg / kg LPS; LPS (50) group: the same volume of normal saline was given for 3 consecutive days, the fourth day of intraperitoneal injection of 50mg / kg LPS.
  • the lung (A), spleen (B) and serum (C) of the mice were taken to detect the expression of inflammatory factors IL-1 ⁇ , IL-6 and TNF- ⁇ .
  • Figure 1A inflammation of inflammatory cytokines in the lungs
  • Figure 1B expression of inflammatory cytokines in the spleen
  • Figure 1C involved expression of inflammatory factors in the blood
  • * p ⁇ 0.05 and ** , p ⁇ 0.01 vs NS; p ⁇ 0.05and p ⁇ 0.01 vs LPS (50).
  • LPS0.3+LPS50 tolerance group mice have a mortality rate of 0% compared with the LPS50 group, and the inflammatory factors IL-1 ⁇ , IL- in the lung, spleen and blood. The expression of 6and TNF- ⁇ was significantly reduced.
  • the results showed that the LPS tolerance model was successfully established by continuous intraperitoneal injection of 0.3 mg/kg/day LPS for 3 days and the lethal dose of 50 mg/kg LPS on the fourth day. Therefore, the LPS0.3+LPS50 group was selected as the LPS tolerant mouse model.
  • the LPS (0.3+50) group was determined to be an LPS-tolerant mouse model, and the LPS-tolerant mouse model was hereinafter referred to as "LPS-tolerant mouse".
  • PA P. aeruginosa Bacillus
  • Example 4 Effect of artesunate (abbreviated as AS) on the expression of inflammatory factors in lung, spleen and serum of LPS-resistant mice
  • mice 42 mice were divided into 7 groups on average:
  • the normal group (NS group) was given the same volume of physiological saline;
  • LPS (0.3) group LPS was injected into the tail vein at 0.3 mg/kg;
  • LPS 50 mg/kg
  • LPS (0.3) + LPS (50) group ie, LPS-tolerant mice of Example 2;
  • LPS-resistant mice were intramuscularly injected with 5 mg/kg artesunate at 0 hours and 4 hours after the last LPS induction in LPS-tolerant mice. AS);
  • LPS-resistant mice were intramuscularly injected with 10 mg/kg artesunate at 0 hours and 4 hours after the last LPS induction in LPS-tolerant mice. AS).
  • Fig. 2A inflammation factor expression in the lung
  • Fig. 2B inflammation factor expression in the spleen
  • Fig. 2C inflammation factor expression in the blood
  • the data in Fig. 2 is the mean + standard deviation.
  • Example 5 AS can reduce mortality after LPS-tolerant mice suffer secondary bacterial attack
  • mice were divided into 4 groups on average, with 17 mice in each group:
  • PA group normal mice were injected with PA1.0 ⁇ 10 7 CFU/kg;
  • LPS (0.3) + LPS (50) + PA + AS (5) group LPS-tolerant mice were infused with PA 1.0 ⁇ 10 7 CFU / kg 6 hours after the last LPS induction, respectively, infusion of PA Intramuscular injection of 5 mg/kg artesunate (AS) for the first 2 hours and 6 hours;
  • AS artesunate
  • LPS (0.3) + LPS (50) + AS (10) group LPS-tolerant mice were infused with PA 1.0 ⁇ 10 7 CFU/kg 6 hours after the last LPS induction, respectively before infusion of PA 2 10 mg/kg artesunate (AS) intramuscularly at 6 hours and 6 hours;
  • mice in each group were observed within 7 days.
  • the results are shown in Fig. 3, ** in Fig. 3, p ⁇ 0.01 vs PA; p ⁇ 0.01 vs LPS (0.3) + LPS (50) + PA. From the experimental results in Fig. 3, it can be seen that, compared with LPS (0.3) + LPS (50) + PA, 5, 10 mg / kg of AS can reduce the mortality of LPS-resistant mice after the second round of bacterial attack.
  • mice were divided into 4 groups, with 6 mice in each group:
  • mice In the NS group, normal mice were injected with the same volume of physiological saline as the LPS group.
  • PA group was injected with normal cells of PA1.0 ⁇ 10 7 CFU/kg;
  • LPS (0.3) + LPS (50) + PA + AS (10) group LPS-tolerant mice were infused with PA 1.0 ⁇ 10 7 CFU/kg 6 hours after the last LPS induction, respectively, infusion of PA Intramuscular injection of 10 mg/kg artesunate (AS) for the first 2 hours and 6 hours;
  • mice in each group were taken 2 hours and 6 hours after the injection of PA, and the blood, lung and spleen of the mice were taken, and the lung and spleen tissues and the PA in the blood were collected, and the CFU value of the PA was measured.
  • the results are shown in Figure 4, Figure 4, **, p ⁇ 0.01 vs NS; p ⁇ 0.01vs PA; p ⁇ 0.01vs LPS(0.3)+LPS(50)+PA
  • artesunate animal experiments show that artesunate can promote the release of inflammatory factors in the middle and late stage of sepsis patients, activate the body immunity, enhance the patient's ability to resist bacteria, and effectively reduce the middle and late stage sepsis. Mortality caused by a second attack on bacteria.

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Abstract

本发明公开了一种青蒿琥酯治疗中、晚期脓毒症的用途。青蒿琥酯可提升促炎症因子，激活免疫功能，有效降低中、晚期脓毒症患者遭受细菌二次打击导致的死亡率。

Description

青蒿琥酯在制备治疗中晚期脓毒症药物中的运用 技术领域
本发明属于医药领域，具体涉及青蒿琥酯治疗中、晚期脓毒症的用途。
背景技术
脓毒症(sepsis)是由感染因素引起的全身炎症反应综合征(SIRS)，是战创伤、烧伤等急危重患者常见的并发症，严重者出现脓毒性休克、多器官功能障碍综合征(MODS)甚至死亡。脓毒症病情进展迅速、病死率高，是导致战时减员或烧伤病人死亡的重要原因，及时诊断脓毒症的发生及其病情进展，并采取有效的救治措施，对提高脓毒症患者的生存率具有重要意义。
早期对脓毒症发病机制的研究表明,脓毒症的发生与感染因素或者严重组织损伤诱导的先天免疫系统活化从而导致炎症介质过度释放有关，补体系统的活化和细胞免疫的过度活化是脓毒症的特征。其中存在于革兰阴性菌膜上的脂多糖(LPS)、革兰阳性菌的肽聚糖(PGN)、革兰阴性菌和阳性菌共有的基因组DNA(CpG DNA)都是细菌重要的致病因子，均能诱导先天免疫系统活化，从而导致炎症介质的大量释放，诱导炎症风暴的产生，最终引起脓毒症休克甚至死亡。临床研究表明，炎性因子IL-6、IL-8、TNF-α、IL-1在脓毒症患者血浆中明显升高，CD11b/CD18、HLA-DR等也明显增高，是脓毒症早期的较敏感指标。基于“抗炎理论”的炎症介质TNF-α、IL-1等单克隆抗体、糖皮质激素等“免疫抑制”及抗炎治疗措施并不能改善脓毒症患者的预后，甚至使病死率增加。上述情况说明，仅过度炎症并不是脓毒症的本质和全部过程。
近年来研究证实，在脓毒症的发生发展过程中，致炎与抗炎网络因子处于此消彼长的过程中，因此机体并非一直处于过度炎症状态。早在90年代，有临床研究表明，脓毒症患者可在过度炎症状态后进入免疫抑制状态。随着研究的深入，脓毒症患者的免疫抑制状态越来越受到重视：免疫抑制是导致接受诸如细菌、真菌等“二次打击”后的脓毒症患者死亡的主要原因。
研究发现，炎症介质的瀑布效应，将促发机体正常的应激反应，即导致机体抗炎机制的激活，导致抗炎因子显著升高，促炎因子明显降低，使抗炎/促炎因 子失衡，使机体免疫系统随之出现免疫抑制，如中性粒细胞麻痹、Th1细胞向Th2细胞的转化、淋巴细胞和树突状细胞凋亡增加。近年来对死亡的脓毒症患者进行尸检后发现，脓毒症后期表现为严重的免疫麻痹：患者脾细胞和肺上皮细胞表面CD4、CD8、协同刺激分子CD28和HLA-DR的表达明显减少甚至缺乏；患者中、晚期时，TNF-α、IL-6、IL-1以及γ-IFN等炎性因子明显减少，而IL-10等抗炎因子明显升高。在免疫抑制或免疫麻痹时，机体对原发性致病微生物不能进行有效的清除，容易遭受铜绿假单胞菌、大肠埃希菌、金黄色葡萄球菌等条件致病菌的二次打击，引起机体内环境进一步紊乱、严重的将导致脓毒症休克、多器官衰竭最终导致患者死亡。因此，针对脓毒症患者免疫抑制状态，以阻断炎症反应和抑制免疫的治疗策略不仅没有效果，反而导致病情恶化、死亡率的增加，故而对脓毒症患者病情进行准确的判断对提高脓毒症的治愈率具有非常重要的意义。因此，基于脓毒症患者晚期免疫麻痹状态研发新药，有可能成为有效拮抗脓毒症并降低死亡率的有效策略。
青蒿素及其衍生物如青蒿琥酯用来治疗疟疾相关的发热已经有一千多年的历史，现在临床上主要用于耐药恶性疟疾。Efferth TM，Wang X等在其文章《Antiviral activity of artesunate towards wild-type，recombinantandganciclovir-resistant human cytomegalovimses》(J Mol Med.2002；80(4)：p223-224)中描述了青蒿类物质还具有如平喘、抗癌、抗血吸虫及对免疫系统的调节等作用。国内外有关青蒿素及其衍生物的研究主要集中在抗疟、抗癌、抗血吸虫的作用及其机制上。
李春英等在其文章《青蒿琥酯对内毒素诱导的一氧化氮合成的抑制作用》(中国中药杂志.2001；26(11)：p770-773.)中描述了青蒿素衍生物青蒿琥酯对LPS及合并干扰素刺激小鼠腹腔巨噬细胞NO的合成有明显的抑制作用，对LPS刺激的小鼠腹腔巨噬细胞RAW264.7也具有相似的保护作用，而且随着青蒿琥酯浓度的增加青蒿琥酯对NO合成的抑制作用也增强。梁爱华、薛宝云、王金华等在其文章《青蒿琥酯对内毒素诱导的炎症因子合成抑制作用的研究》(中国中西医结合急救杂志.2001；8(5)：p262.-265)中描述了青蒿琥酯在25～100mg/L对LPS诱导的TNF-α产生具有明显的抑制作用，与LPS单独应用比较抑制率为43％～58％。
CN1833644公开青蒿素及其衍生物如青蒿琥酯对脓毒辣都能降低灭活大肠杆菌、细菌(包括革兰阴性、阳性菌)攻击小鼠的死亡率，都可以显著抑制小鼠血清细胞因子TNF-α与IL-6释放，并对灭活大肠杆菌、细菌攻击小鼠的主要脏器(心、肝、肠、肺、肾)具有显著保护作用。但该研究仍基于脓毒症早中期炎症介质过度释放阶段，青蒿琥酯主要通过抑制炎症因子的释放而起治疗效果。
脓毒症患者在过度炎症状态后的中晚期，进入免疫抑制状态，此时，TNF-α、IL-6、IL-1以及γ-IFN等炎性因子明显减少，而IL-10等抗炎因子明显升高。在免疫抑制或免疫麻痹时，机体对原发性致病微生物不能进行有效的清除，容易遭受铜绿假单胞菌、大肠埃希菌、金黄色葡萄球菌等条件致病菌的二次打击，引起机体内环境进一步紊乱、严重的将导致脓毒症休克、多器官衰竭最终导致患者死亡。随着研究的深入，脓毒症患者的免疫抑制状态越来越受到重视：免疫抑制是导致接受诸如细菌、真菌等“二次打击”后的脓毒症患者死亡的主要原因.。因此，基于脓毒症患者晚期免疫麻痹状态研发新药，有可能成为有效拮抗中晚期脓毒症并降低死亡率的有效策略。
发明内容
本发明的目的是提供一种青蒿琥酯在制备治疗中晚期脓毒症药物中的用途或运用。
具体来说，本发明的目的就是青蒿琥酯用于治疗中或晚期脓毒症的用途,特别是治疗晚期脓毒症的用途。
本发明的用途，青蒿琥酯在制造治疗中期或晚期脓毒症药物中的用途或运用。
上述本发明的用途，所述脓毒症优选为晚期脓症患者。
上述本发明的用途，所述中期或晚期脓毒症是指脓毒症患者机体中的炎症因子分泌明显减少的状态，或是指脓毒症患者的免疫受到抑制或免疫处于麻痹状态。
病理学研究发现中晚期脓毒症患者在炎症因子过度释放后，炎症因子明显减少，机身免疫功能受到抑制或免疫处于麻痹状态，此时，机体对原发性致病微生物不能进行有效的清除，容易遭受铜绿假单胞菌、大肠埃希菌、金黄色葡萄球菌等条件致病菌的二次打击，引起机体内环境进一步紊乱、严重的将导致脓毒症患 者休克、多器官衰竭最终导致死亡。本发明人惊奇的发现，青蒿琥酯在机身免疫功能受到抑制或免疫处于麻痹状态时，不再抑制炎症因子的释放，反而是促进炎症因子的释放，恢复或提升机体免疫功能，从而提高机体对铜绿假单胞菌、大肠埃希菌、金黄色葡萄球菌的抵抗，可有效预防机体遭受细菌、真菌等的二次打击，降低脓毒症患者死亡率。
附图说明
图1实施例2的各对照组的炎症因子的含量。
图2实施例4的LPS耐受小鼠的肺、脾和血清中炎性因子的含量。
图3实施例5的LPS耐受小鼠遭受细菌二次打击后的死亡率。
图4实施例6中LPS耐受小鼠遭受细菌二次打击后肺、脾、血清中细菌PA的数量。
具体实施方式
以下实施例用于进一步描述和理解本发明，而不限制本发明的范围。
实施例1  筛选LPS的致死剂量
120只小鼠分成6组，每组20只，每组分别按10,20,30,40,50and 80mg/kg静脉注射LPS，观察7天各组生存数量，筛选LPS导致正常小鼠100％死亡的最低剂量。结果见表1，实验结果可知：50mg/kg of LPS能导致正常小鼠100％死亡。
表1接受不同剂量LPS的小鼠在7天内每日的存活数量
Figure PCTCN2015092808-appb-000001
实施例2  建立LPS耐受小鼠模型
小鼠分成3组，每组小鼠6只，一组每天给予(腹腔注射)0.3mg/kg的LPS、一组每天给予(腹腔注射)1.0mg/kg的LPS、一组每天给予(腹腔注射)3.0mg/kg的LPS,连续给预LPS 3天，第4天各组均给予(腹腔注射)50mg/kg的LPS，观察注射LPS后7天内各组小鼠生存数量。结果见表2。
LPS耐受小鼠炎症因子的释放量：取小鼠24只，分成4组，每组小鼠6只，正常组(NS)，给予相同体积的生理盐水；LPS(0.3)组：按0.3mg/kg注射LPS,连续3天，第四天腹腔注射50mg/kg LPS；LPS(50)组：给予相同体积的生理盐水，连续3天，第四天腹腔注射50mg/kg LPS。各组在最后一次给予LPS后的第12小时，取小鼠的肺(A)、脾(B)和血清(C)，检测炎症因子IL-1β,IL-6和TNF-α的表达。结果见图1A(肺中炎症因子表达量)、图1B(脾中炎症因子表达量)、图1C(血液中炎症因子表达量)，图中*,p<0.05and**,p<0.01vs NS；
Figure PCTCN2015092808-appb-000002
p<0.05and
Figure PCTCN2015092808-appb-000003
p<0.01vs LPS(50)。
表2 LPS耐受小鼠组给药后7天内各组小鼠每日存活数
Figure PCTCN2015092808-appb-000004
从表2和图1的实验结果可知：与LPS50组比较，LPS0.3+LPS50(耐受组)小鼠死亡率为0％，且肺、脾、血液中炎性因子IL-1β,IL-6and TNF-α的表达明显降低。结果表明采用0.3mg/kg/day LPS连续腹腔注射3天，第四天采用致死剂量50mg/kg LPS可成功建立LPS耐受模型。因此,选择LPS0.3+LPS50组为LPS耐受小鼠模型。根据表2和图1的结果，确定LPS(0.3+50)组为LPS耐受小鼠模型，以下将LPS耐受小鼠模型简称为”LPS耐受小鼠”。
实施例3  LPS耐受小鼠细菌的敏感性
正常小鼠8组(NS1-8组)，每组6只，各组依次给予(NS1组)5.0×105 (NS2组)、1.0×106(NS2组)、5.0×106(NS3组)、1.0×107(NS4组)、5.0×107(NS5组)、1.0×108(NS6组)、5.0×108(NS7组)、1.0×109(NS8组)剂量的绿脓杆菌(简称PA)诱导，实施例2的LPS耐受小鼠8组(LPS1-8组)，每组6只，各组依次给与5.0×105(LPS1组)、1.0×106(LPS2组)、5.0×106(LPS3组)、1.0×107(LPS4组)、5.0×107(LPS5组)、1.0×108(LPS6组)、5.0×108(LPS7组)、1.0×109(LPS8组)剂量的绿脓杆菌(PA)诱导。观察诱导后第0.5天、1天、2天、3天、4天、5天、6天和第7天小鼠存活数量，结果见表3。
表3正常小鼠与LPS耐受上鼠细菌的敏感性试验结果
Figure PCTCN2015092808-appb-000005
由表3的实验结果可知：5.0×108CFU/kg的绿脓杆菌可导致正常小鼠100％死亡，而1.0×107CFU/kg的绿脓杆菌就可导致LPS耐受小鼠100％死亡，表明LPS 耐受小鼠对细菌的敏感性更高，LPS耐受小鼠更容易遭受细菌等条件致病菌的二次打击而死亡。
实施例4  青蒿琥酯(简称为AS)对LPS耐受小鼠肺、脾、血清中炎性因子表达的影响
42只小鼠分平均分成7组：
正常组(NS组)，给予相同体积的生理盐水；
LPS(0.3)组，按0.3mg/kg尾静脉注射LPS；
LPS(50)组，按50mg/kg尾静脉注射LPS；
LPS(0.3)+LPS(50)组，即实施例2的LPS耐受小鼠；
LPS(0.3)+LPS(50)+AS(2.5)组，在LPS耐受小鼠最后一次LPS诱导后的0小时和第4小时给LPS耐受小鼠肌注2.5mg/kg青蒿琥酯(AS)；
LPS(0.3)+LPS(50)+AS(5)组，在LPS耐受小鼠最后一次LPS诱导后的0小时和第4小时给LPS耐受小鼠肌注5mg/kg青蒿琥酯(AS)；
LPS(0.3)+LPS(50)+AS(10)组，在LPS耐受小鼠最后一次LPS诱导后的0小时和第4小时给LPS耐受小鼠肌注10mg/kg青蒿琥酯(AS)。
以上各组在最后一次LPS诱导后的12小时后，取小鼠的肺、脾、血液样品，检测IL-1β,IL-6and TNF-α的表达。结果见图2，图2A(肺中炎症因子表达量)、图2B(脾中炎症因子表达量)、图2C(血液中炎症因子表达量)，图2中的数据为平均值+标准偏差,其中，*,p<0.05和**,p<0.01vs NS；
Figure PCTCN2015092808-appb-000006
p<0.05和
Figure PCTCN2015092808-appb-000007
p<0.01vs LPS(50)；
Figure PCTCN2015092808-appb-000008
p<0.05和
Figure PCTCN2015092808-appb-000009
p<0.01vs LPS(0.3)+LPS(50)。
由图2的实验结果可知：与LPS0.3+LPS50(耐受组)比较，2.5，5，10mg/kg的AS能升高LPS耐受小鼠肺、脾、血清中炎性因子IL-1β,IL-6and TNF-α的表达。实验结果表明，AS能升高LPS耐受小鼠肺、脾、血清中炎性因子的表达。
实施例5  AS能降低LPS耐受小鼠遭受细菌二次打击后的死亡率
68只小鼠分平均分成4组，每组17只小鼠：
PA组 给正常小鼠注射PA1.0×107CFU/kg；
LPS(0.3)+LPS(50)+PA组，给LPS耐受小鼠注射PA1.0×107CFU/kg；
LPS(0.3)+LPS(50)+PA+AS(5)组，给LPS耐受小鼠在最后一次LPS诱导后的6小时输注PA1.0×107CFU/kg，分别在输注PA前2小时和6小时肌注5mg/kg青蒿琥酯(AS)；
LPS(0.3)+LPS(50)+AS(10)组，给LPS耐受小鼠在最后一次LPS诱导后的6小时输注PA1.0×107CFU/kg，分别在输注PA前2小时和6小时肌注10mg/kg青蒿琥酯(AS)；
观察7天内各组小鼠死亡率,结果见图3，图3中**,p<0.01vs PA；
Figure PCTCN2015092808-appb-000010
p<0.01vs LPS(0.3)+LPS(50)+PA。由图3实验结果可知：与LPS(0.3)+LPS(50)+PA比较，5，10mg/kg的AS能降低LPS耐受小鼠遭受细菌二次打击后的死亡率。
实施例6
AS能降低LPS耐受小鼠遭受细菌二次打击后肺、脾、血清中细菌的数量24只小鼠平均分成4组，每组6只小鼠:
NS组，给正常小鼠注射与给LPS组相同体积量的生理盐水。
PA组给正常小鼠注射PA1.0×107CFU/kg；
LPS(0.3)+LPS(50)+PA组，给LPS耐受小鼠注射PA1.0×107CFU/kg；
LPS(0.3)+LPS(50)+PA+AS(10)组，给LPS耐受小鼠在最后一次LPS诱导后的6小时输注PA1.0×107CFU/kg，分别在输注PA前2小时和6小时肌注10mg/kg青蒿琥酯(AS)；
每组各取3只小鼠分别在注射PA后2小时和6小时，取小鼠的血液、肺和脾，收集肺和脾组织及及血液中的PA，并检测PA的CFU值。结果见图4，图4中，**,p<0.01vs NS；
Figure PCTCN2015092808-appb-000011
p<0.01vs PA；
Figure PCTCN2015092808-appb-000012
p<0.01vs LPS(0.3)+LPS(50)+PA
由图4实验结果可知：与LPS(0.3)+LPS(50)+PA组比较，10mg/kg的AS能降低LPS耐受小鼠遭受细菌二次打击后肺、脾、血清中细菌的数量。
综上，青蒿琥酯动物试验表明，青蒿琥酯在脓毒症患者的中、晚期可促进炎症因子释放，激活机体免疫力，增强患者抵抗细菌的能力，可有效降低中、晚期脓毒症患者因细菌二次打击导致的死亡率。

Claims (4)

  1. 青蒿琥酯在制造治疗中期或晚期脓毒症药物中的用途。
  2. 如权利要求1所述的用途，所述脓毒症为晚期脓症患者。
  3. 如权利要求1所述的用途，所述中期或晚期脓毒症是指脓毒症患者机体中的炎症因子分泌较少的状态。
  4. 如权利要求1所述的用途，所述中期或晚期脓毒症是指脓毒症患者的免疫受到抑制或免疫处于麻痹状态。
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