CN113368219B - Globulol的应用 - Google Patents
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- A61K38/19—Cytokines; Lymphokines; Interferons
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Abstract
本发明提供了一种细胞因子组合物及其应用，所述细胞因子组合物包括γ‑IFN、TNF‑α和IL‑6。本发明还提供了Globulol的应用。Globulol可以明显降低由γ‑IFN，TNF‑α和IL‑6诱导的肺上皮细胞BEAS‑2B的焦亡和死亡。
Description
技术领域
本发明涉及一种细胞因子组合物及其应用，还涉及Globulol的应用。
背景技术
新型冠状病毒肺炎(COVID-19)，是由一种新型的冠状病毒感染而导致的急性呼吸道传染病，主要表现为发热、干咳、乏力等临床表现。COVID-19重症或危重症患者的免疫系统异常激活，免疫细胞释放大量细胞因子造成严重的全身炎症反应，激发细胞因子风暴,导致严重的呼吸综合征和急性肺损伤，甚至致人死亡。细胞因子风暴相关的细胞因子是免疫细胞在被激活、与靶细胞相互作用的时候释放的，相关的细胞因子(γ-IFN，TNF-α和IL-6等)主要来源于激活的免疫细胞，而且又重新作用于免疫细胞的激活(包括T-细胞，单核细胞和巨噬细胞等)，触发急性炎症反应诱导肺部上皮及组织损伤，导致微血管渗漏、心衰甚至死亡。多种细胞因子迅速大量产生的现象，也是引起急性呼吸窘迫综合症和多器官衰竭的重要原因。γ-IFN，TNF-α和IL-6可以激活细胞膜上相关受体的活化(例如Toll受体等等)，从而激活相关信号通路(例如MAPK信号通路、NF-κB信号通路等等)，最终引起细胞内更多促炎细胞因子的释放，从而引起更加严重的炎症反应。
细胞焦亡是一种程序性细胞死亡方式，被认为是针对细胞内病原体的重要的先天免疫反应，以半胱氨酸蛋白酶-1(caspase-1)介导、质膜破坏及细胞成分和促炎性介质释放为特征。caspase-1在体内无活性，需经炎症小体NLRP3将其激活为具有两个p20和两个p10亚基的异四聚体，激活后的caspase-1紧接着将白细胞介素(IL)-1β前体和IL-18前体裂解为成熟的活性IL-1β和IL-18。此外，caspase-1可裂解GSDMD形成其N末端结构域片段，其N末端片段富集促进膜孔形成，从而导致炎性因子释放，引起细胞焦亡。
将细胞因子风暴与器官损伤联系起来的可能是一种引起细胞焦亡和死亡的机制，在COVID-19的体内都存在着细胞因子介导的炎症以及急性肺损伤的表现，其中人类肺上皮细胞BEAS-2B细胞是从人正常支气管上皮提取分离的细胞株，并且具备正常人支气管上皮细胞的基本生理生化特征，已经被应用在关于气道纤维化重塑及气道炎症反应的实验研究中，BEAS-2B细胞在LPS的刺激下合成并分泌过多的γ-IFN、IL-6、IL-1β和TNF-α等细胞因子，这些细胞因子会刺激肺部产生严重的炎症反应，这种炎症的结果会导致严重的肺部功能障碍和肺部上皮细胞和内皮细胞的损伤，从而引起肺部细胞的焦亡和死亡，这种肺部细胞的焦亡和死亡会引起急性肺损伤或者更为严重的急性呼吸窘迫综合症。COVID-19会对患者的呼吸系统尤其是肺组织造成严重的影响，而且大大降低了患者的生活质量，严重的甚至威胁生命，因此，急需开发出一种抑制细胞因子引起的急性肺损伤的一种抗炎药物。
发明内容
本发明的目的在于提供的一种细胞因子组合物及其应用，本发明还提供了Globulol的应用。Globulol可以明显降低由γ-IFN，TNF-α和IL-6诱导的肺上皮细胞BEAS-2B的焦亡和死亡。
为实现上述目的，所采取的技术方案：一种细胞因子组合物，包括γ-IFN、TNF-α和IL-6。
优选地，所述γ-IFN、TNF-α和IL-6的浓度比为100：60：10。
本发明提供了γ-IFN、TNF-α和IL-6的联合使用在制备诱导肺上皮细胞BEAS-2B细胞焦亡、死亡的制剂中的应用。
本发明提供了γ-IFN、TNF-α和IL-6的联合使用在制备抑制肺上皮细胞BEAS-2细胞的细胞活力的制剂中的应用。
本发明提供了γ-IFN、TNF-α和IL-6的联合使用在制备肺上皮细胞BEAS-2B细胞炎症反应模型中的应用。
优选地，所述γ-IFN的使用浓度为100ng/mL，所述TNF-α的使用浓度为60ng/mL，所述IL-6的使用浓度为10ng/mL。
本发明提供了Globulol在制备抑制由细胞因子诱导的肺上皮细胞BEAS-2B损伤、焦亡或死亡的药物中的应用。
优选地，所述细胞因子包括上述所述的细胞因子组合物。
本发明提供了Globulol在制备治疗急性肺损伤或急性呼吸窘迫综合症的药物中的应用。
优选地，所述急性肺损伤或急性呼吸窘迫综合症是由细胞因子引起的。
优选地，所述细胞因子包括上述所述的细胞因子组合物。
有益效果：
本发明提供了Globulol的应用，实验结果表明，γ-IFN，TNF-α和IL-6的联合使用能够引起肺上皮细胞BEAS-2B细胞的焦亡和死亡，Globulol能够有效地减轻细胞因子风暴引起的肺上皮细胞BEAS-2B细胞的细胞焦亡和死亡，因此，Globulol有望成为一种治疗急性肺损伤和急性呼吸窘迫综合症的一种天然药物，具有很好的应用价值和开发前景。
附图说明
图1为细胞因子γ-IFN，TNF-α和IL-6联合使用对BEAS-2B细胞的MTT实验结果图。
图2为细胞因子γ-IFN，TNF-α和IL-6联合使用对BEAS-2B细胞的台盼蓝实验和活死细胞计数实验结果图。
图3为细胞因子γ-IFN，TNF-α和IL-6联合使用对BEAS-2B细胞的caspase-1活性检测和ELISA实验结果图。
图4为Globulol对BEAS-2B细胞的MTT实验结果图。
图5为Globulol与γ-IFN，TNF-α和IL-6联合使用对BEAS-2B细胞的MTT实验结果图。
图6为Globulol与γ-IFN，TNF-α和IL-6联合使用对BEAS-2B细胞的台盼蓝实验实验结果图。
图7为Globulol与γ-IFN，TNF-α和IL-6联合使用对BEAS-2B细胞的caspase-1活性检测和ELISA实验结果图。
具体实施方式
下面结合具体实施例进一步说明本发明的内容，但不应理解为对本发明的限制。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。Globulol：兰桉醇(C15H26O)，结构式如下：
具体实施例中，使用的原料及试剂均可由市场购得。
实施例1MTT实验
用MTT实验测定细胞因子γ-IFN、TNF-α和IL-6联合实验以及Globulol对BEAS-2B细胞的生长影响，具体实施方案如下：
1.取生长状态良好的BEAS-2B细胞，吹打下来后用细胞计数板计数，调整细胞终浓度为5×104个/ml。在96孔板中每孔加入100μl细胞混悬液，置于37℃和5％CO2培养箱中培养24h后，弃去旧培养基；
2.分别设置三组实验，分别是(1)设置γ-IFN、TNF-α和IL-6浓度分别为100ng/ml、60ng/ml和10ng/ml，(2)设置Globulol的浓度为2μM、5μM，10μM，20μM，(3)设置细胞因子组合(γ-IFN 100ng/ml，TNF-α60ng/ml，IL-6 10ng/ml)与不同浓度Globulol(2μM、5μM，10μM，20μM)联合使用。每个浓度设置4个复孔，同时设置调零孔。5％CO2，37℃培养72h，然后弃去上清液，每孔加入100μl浓度为0.5mg/ml的MTT溶液，继续培养4h，弃上清液，每孔加入150μl分析纯DMSO溶液，在摇床上低速震荡10min使结晶完全溶解。将96孔板置于酶标仪上于570nm处测量各孔的吸光值。
图1为细胞因子γ-IFN，TNF-α和IL-6联合使用对BEAS-2B细胞的MTT实验结果图，从图1中可以看出，γ-IFN，TNF-α和IL-6的联合使用对BEAS-2B细胞的影响较大，其对BEAS-2B细胞的抑制率大于40％，可见γ-IFN，TNF-α和IL-6的联合使用可显著提高BEAS-2B细胞的死亡率。图4为Globulol对BEAS-2B细胞的MTT实验结果图，从图4中可以看出，Globulol对BEAS-2B细胞的生长影响较小，Globulol最高浓度为20μM时，其对BEAS-2B细胞的抑制率均小于10％。说明Globulol对BEAS-2B细胞的毒性不大。图5为细胞因子组合与不同浓度的Globulol联合作用BEAS-2B细胞的MTT实验结果图，从图5可以看出，细胞因子联合处理BEAS-2B细胞后，细胞活力明显下降，Globulol以剂量依赖性地减轻了细胞因子组合对细胞活力的抑制作用。与细胞因子联合处理组相比，在5-20μM剂量水平下，Globulol显著提高了BEAS-2B细胞的活力(图4)。与细胞因子组合处理的细胞相比，5μM Globulol处理的细胞(p<0.01)，10或20μM Globulol处理的细胞(p<0.001)可以显著降低BEAS-2B细胞的死亡率。这一结果表明，Globulol能保护肺上皮细胞免受细胞因子联合对细胞活力的损害。
实施例2台盼蓝实验
用台盼蓝实验测定细胞因子γ-IFN、TNF-α和IL-6联合使用对BEAS-2B细胞增值的影响，具体实施方案如下：
1.取生长状态良好的BEAS-2B细胞，吹打下来后用细胞计数板计数，调整细胞终浓度为0.2×105个/ml。在35mm细胞培养皿中每孔加入2ml细胞混悬液，置于37℃和5％CO2培养箱中培养24h后，弃去旧培养基。
2.分别设置二组实验，分别是(1)设置γ-IFN、TNF-α和IL-6浓度分别为100ng/ml、60ng/ml和10ng/ml，(2)设置细胞因子组合(γ-IFN 100ng/ml，TNF-α60ng/ml，IL-6 10ng/ml)与不同浓度Globulol(2μM、5μM，10μM，20μM)联合使用，每个浓度设置4个复孔，同时设置调零孔。5％CO2，37℃培养72h，处理后胰蛋白酶处理成单细胞悬液。将80μl的细胞悬液与20μl的0.4％台盼蓝溶液混合2min，用Countess自动细胞计数器计数活细胞和死细胞的数量.
图2为细胞因子γ-IFN，TNF-α和IL-6联合使用对BEAS-2B细胞的台盼蓝实验结果图，从图2可以看出，γ-IFN，TNF-α和IL-6的联合使用增加了死亡细胞的数量，说明细胞因子γ-IFN，TNF-α和IL-6的联合使用可以诱导BEAS-2B细胞的死亡。图6为细胞因子组合与不同浓度的Globulol联合作用BEAS-2B细胞的台盼蓝实验结果图，Globuol以剂量依赖性的方式降低了死亡细胞的百分比，统计分析显示，5μM(p<0.01)、10μM(p<0.001)和20μM(p<0.001)剂量下，Globuol显著降低了细胞的死亡百分比。实验结果表明，肾小球对细胞因子诱导的肺上皮细胞死亡具有保护作用。
实施例3Caspase-1活性实验
取生长状态良好的BEAS-2B细胞，吹打下来后用细胞计数板计数，调整细胞终浓度为0.2×105个/ml。在60mm细胞培养皿加入5ml细胞混悬液，置于37℃和5％CO2培养箱中培养24h后，弃去旧培养基。
分别设置二组实验，分别设置(1)γ-IFN、TNF-α和IL-6浓度分别为100ng/ml、60ng/ml和10ng/ml(2)设置细胞因子组合(γ-IFN 100ng/ml，TNF-α60ng/ml，IL-610ng/ml)与不同浓度Globulol(2μM、5μM，10μM，20μM)联合使用。细胞培养72h后，用胰酶消化贴壁细胞，600g4℃离心5分钟收集细胞，小心吸除上清，同时确保尽量没有细胞被吸除，PBS洗涤一次。同前吸尽上清后，按照每200万细胞加入100微升裂解液的比例加入裂解液，重悬沉淀，冰浴裂解15分钟。按照说明书进行Caspase-1Assay试剂盒检测Caspase-1活性。
图3A为细胞因子γ-IFN，TNF-α和IL-6联合使用对BEAS-2B细胞的Caspase-1活性实验结果图，从图3A可以看出，细胞因子联合处理BEAS-2B细胞后，caspase-1的活性明显增强。统计分析显示，细胞因子联合处理的细胞caspase-1活性显著增加(p<0.001)。图7A为细胞因子组合与不同浓度的Globulol联合作用BEAS-2B细胞的Caspase-1活性实验结果图，从图7A可以看出，细胞因子组合诱导了caspase-1活性的升高，Globulol以剂量依赖性地降低了细胞因子联合诱导的细胞焦亡，统计学分析显示，5μM(p<0.05)、10μM(p<0.001)和20μM(p<0.001)剂量的Globulol均能显著降低细胞因子组合引起的焦亡。
实施例4ELISA实验
取生长状态良好的BEAS-2B细胞，吹打下来后用细胞计数板计数，调整细胞终浓度为0.2×105个/ml。在35mm细胞培养皿加入2ml细胞混悬液，置于37℃和5％CO2培养箱中培养24h后，弃去旧培养基。
分别设置二组实验，分别设置(1)γ-IFN、TNF-α和IL-6浓度分别为100ng/ml、60ng/ml和10ng/ml(2)设置细胞因子组合(γ-IFN 100ng/ml，TNF-α60ng/ml，IL-610ng/ml)与不同浓度Globulol(2μM、5μM，10μM，20μM)联合使用。细胞培养72h后，小心吸取细胞培养上清，按照说明书进行ELISA试剂盒检测IL-1β活性。
图3B为细胞因子γ-IFN，TNF-α和IL-6联合使用对BEAS-2B细胞的IL-1β含量实验结果图，从图3B可以看出，细胞因子联合处理BEAS-2B细胞后，IL-1β水平显著升高，IL-1β水平在对照组和细胞因子联合治疗组之间差异有统计学意义(p<0.001)。caspase-1(图3A)和IL-1β实验结果表明，细胞因子组合可诱导BEAS-2B细胞的焦亡。图7B为细胞因子组合与不同浓度的Globulol联合作用BEAS-2B细胞的IL-1β含量实验结果图，从图7B可以看出，细胞因子联合处理BEAS-2B细胞后，IL-1β水平显著升高，Globulol治疗导致IL-1β水平降低，呈剂量依赖性；与细胞因子联合治疗组相比，Globulol在5-20μM剂量水平显著降低IL-1β水平，与细胞因子组合处理的细胞相比，5μM Globulol处理的细胞(p<0.01，10或20μMGlobulol处理的细胞(p<0.001)具有明显的显著性差异。caspase-1(图7A)和IL-1β实验结果表明，Globuol对细胞因子联合诱导的BEAS-2B细胞焦亡有较强的抑制作用。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (3)
1.Globulol在制备治疗急性肺损伤或急性呼吸窘迫综合症的药物中的应用，其特征在于，所述急性肺损伤或急性呼吸窘迫综合症是由细胞因子诱导的肺上皮细胞损伤、焦亡或死亡引起的；所述细胞因子为γ-IFN、TNF-α和IL-6；所述Globulol的作用浓度为5-20μM。
2.根据权利要求1所述的应用，其特征在于，所述细胞因子中γ-IFN 、TNF-α和IL-6的浓度比为100：60：10。
3.根据权利要求1或2所述的应用，其特征在于，所述γ-IFN的使用浓度为100ng/ml，所述TNF-α的使用浓度为60ng/ml，所述IL-6的使用浓度为10ng/ml。
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