CN118021923A - 小柴胡汤在制备用于减轻脓毒血症心肌炎的产品中的应用 - Google Patents
小柴胡汤在制备用于减轻脓毒血症心肌炎的产品中的应用 Download PDFInfo
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Abstract
本发明公开了小柴胡汤在制备用于减轻脓毒血症心肌炎的产品中的应用，涉及医药技术领域，其技术方案要点是：小柴胡汤中的活性成分能够通过调节一些关键炎症信号通路，抑制炎症介质TNF‑、IL‑1、IL‑6的过度释放，从而减轻心肌细胞的炎症反应，保护心肌，改善脓毒血症心肌炎所致的心脏损伤。研究结果表明，XCHD对于改善LPS诱导的脓毒症小鼠的心功能障碍具有显著效果。XCHD不仅能够提升心功能参数如LVEF和LVFS，还能够修复由LPS引起的心肌细胞损伤。此外，XCHD还能够有效减轻脓毒症引起的心脏炎症，通过降低炎症因子TNF‑α、IL‑6和IL‑1β在mRNA和蛋白水平的表达来实现。
Description
技术领域
本发明涉及医药技术领域，更具体地说，它涉及小柴胡汤在制备用于减轻脓毒血症心肌炎的产品中的应用。
背景技术
脓毒血症心肌炎是一种严重的心脏疾病，其发病机制复杂，治疗效果往往不尽人意。现有的治疗方法主要集中在控制感染和炎症，且疗效有限，对于疾病的根本性治疗仍有待进一步提高。
发明内容
本发明的目的是为了解决上述问题，提供小柴胡汤在制备用于减轻脓毒血症心肌炎的产品中的应用，小柴胡汤通过其独特的作用机理，为脓毒血症心肌炎的治疗提供了一种新的、有效的选择。其相比其他药物的显著优势，使其在临床实践中具有广泛的应用前景和重要的治疗价值。
脓毒血症心肌炎是一种严重的临床病症，其中心肌细胞受到炎症和感染的多重打击，导致心功能受损。小柴胡汤，作为一种经典的中药复方，其多成分、多靶点的药理作用为这一复杂病症提供了全面的治疗策略。具体来说，小柴胡汤中的活性成分能够通过调节一些关键炎症信号通路，抑制炎症介质TNF-、IL-1、IL-6的过度释放，从而减轻心肌细胞的炎症反应，保护心肌，改善脓毒血症心肌炎所致的心脏损伤。
本发明具有以下有益效果：
1、XCHD不仅能够提升心功能参数如LVEF和LVFS，还能够修复由LPS引起的心肌细胞损伤。此外，XCHD还能够有效减轻脓毒症引起的心脏炎症，通过降低炎症因子TNF-α、IL-6和IL-1β在mRNA和蛋白水平的表达来实现；
2、与其他治疗脓毒血症心肌炎的药物相比，小柴胡汤具有整体调节和个体化治疗的特点。许多现代药物往往针对单一的病理环节，而小柴胡汤则能够同时作用于多个治疗靶点，提供更为全面的治疗效果；
3、由于小柴胡汤的配方来源于天然草药，安全性较高，长期使用的毒副作用较小，更适合于患者的长期管理和治疗。
附图说明
图1是本发明实施例中小柴胡汤提取物和小柴胡汤含药血清在不同条件下的基峰图；
图2是本发明实施例中根据代谢物数量绘制的代谢物分类饼状图；
图3是本发明实施例中展示空白血清、含药血清、小柴胡汤提取物中的共有有效成分的韦恩图；
图4是本发明实施例中CCK-8筛选含药血清不抑制细胞生长的浓度；
图5是本发明实施例中不同浓度的含药血清抑制炎症因子IL-1β、IL-6、TNF-α的释放的效果；
图6是本发明实施例中不同浓度的含药血清抑制炎症因子IL-1β、IL-6、TNF-α的蛋白水平的效果；
图7是本发明实施例中不同分组小鼠的M型超声心电图；
图8是本发明实施例中与对照组相比，LPS组的LVIDs值以及XCHD治疗后，LPS组的LVIDs值；
图9是本发明实施例中与对照组相比，LPS组的LVFS值以及XCHD治疗后，LPS组的LVFS值；
图10是本发明实施例中与对照组相比，LPS组的LVEF值以及XCHD治疗后，LPS组的LVEF值；
图11是本发明实施例中不同分组小鼠心肌组织的HE染色图；
图12是本发明实施例中各组小鼠血清的CK-MB水平；
图13是本发明实施例中各组小鼠血清的LDH水平；
图14是本发明实施例中各组小鼠血清的cTn-I水平；
图15是本发明实施例中各组小鼠血清的炎症因子水平；
图16是本发明实施例中各组小鼠心脏组织的炎症因子相关蛋白的表达；
图17是本发明实施例中各组小鼠心脏组织的炎症因子相关mRNA的表达。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明的实施例及附图，对本发明的技术方案进行进一步详细地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
实施例：
一、实验材料
小柴胡汤(XCHD)的组成和数量如下：
柴胡24g，黄芩9g，人参9g，甘草9g，松果9g，生姜9g，红枣12g。
二、实验方法
1、XCHD的制备
XCHD的成分和用量如下：柴胡24克，黄芩9克，人参9克，甘草9克，菠萝9克，生姜9克，大枣12克。如上所述，从江西省肿瘤医院获得7种中草药，重81克。将草药粉碎，在810毫升蒸馏水中浸泡2小时，然后煎煮1小时。过滤所得混合物以收集滤液。随后，加入486ml蒸馏水，然后再进行一小时的煎煮和过滤以获得第二滤液。将两种滤液合并，并使用旋转蒸发器进行浓缩，以达到2g/ml的浓度(基于原始药物量)。。
2、制备XCHD的含药血清
将20只体重为200±10g的雄性Sprague-Dawley(SD)无特定病原体(SPF)级大鼠随机分为对照组和XCHD组，每组10只。实验组每天口服2.4g/ml XCHD，对照组每天口服相同剂量的生理盐水，连续7天。在最后一次给药后两小时采集血液样本。将血液在室温下静置两小时，离心分离血清上层。然后将血清在56℃下灭活30分钟，通过0.22-μm滤膜消毒，并储存在-80℃。
3、LC-MS/MS分析
数据采集仪器系统主要由超高效液相色谱仪(Vanquish，UPLC，赛默飞，美国)和高分辨质谱仪(Q Exactive HFX，赛默飞，美国)组成。样品在4℃下缓慢解冻后，取适量样品(0.1-0.2ml)，加入800ul提取甲醇乙腈溶液(1:1，v/v)，涡旋60s，低温超声30min，重复两次，置于-20℃下1h沉淀蛋白质，然后在4℃下以12000rpm离心20min。将上清液冷冻干燥，重新溶解于200ul 30％ACN中，涡旋，并在4℃下以14000g离心15min。
4、动物与实验方案
选取8-10周龄的雄性C57/BL6J小鼠，体重25-30g，购自南昌大学实验动物科学部(中国南昌)。
在正式实验前，小鼠在标准条件下预适应7天：温度为24±2℃，12小时光/暗循环，自由获取标准食物和水。共有50只小鼠随机分为五组(每组n＝10)：对照组(CON)、LPS组(LPS)、LPS+地塞米松组(LPS+DEX，10mg/kg)、LPS+低剂量XCHD组(LPS+XCHD(L)，5.7g/kg)、LPS+中剂量XCHD组(LPS+XCHD(M)，11.57g/kg)和LPS+高剂量XCHD组(LPS+XCHD(H)，23.14g/kg)。除CON和LPS组外，所有组均通过每日灌胃连续七天给予相应剂量。CON和LPS组每日灌胃给予等量的生理盐水。第8天，除CON组外，所有小鼠均腹腔注射LPS(10mg/kg)；CON组注射等量的PBS。诱导12小时后，对小鼠进行M型超声心动图检查。第8天，处死所有小鼠，收集血清和组织进行后续实验分析。
5、超声心动图评估
使用超声心动图(产品型号)测量小鼠的心脏功能变化，小鼠通过吸入1.5-2％的异氟烷进行麻醉，并保持在稳定的体温约37℃±0.5℃。从短轴视图中的乳头肌水平获得至少三个心搏的二维引导M型左心室(LV)内径测量值，并取平均值。在LPS注射后计算左心室射血分数(LVEF)和左心室缩短分数(LVFS)。
6、苏木精和伊红(HE)染色
实验结束后处死小鼠，将小鼠心脏组织固定在4％的多聚甲醛中至少3天，然后将样品进行石蜡包埋，切成5μm的切片，并分别用苏木精和伊红(HE)染色液进行形态学评估。使用显微镜(产品型号)观察形态学变化，并在每组中随机选择3-5个视野以观察形态学变化。
7、血清IL-1β、IL-6和TNF-α水平的测定
小鼠血清短暂地储存在4℃的冰箱中。使用ELISA试剂盒评估IL-1β(cat.#ED-20174；LunChangShuoBiotech)、IL-6(cat.#ED-20188；LunChangShuoBiotech)和TNF-α(cat.#ED-20852；LunChangShuoBiotech)的浓度，遵循生产商的协议。数值以pg/mg总蛋白表示。
8、蛋白质印迹分析
收集心脏组织或心肌细胞，在4℃下裂解，离心收集上清液，并使用BCA方法测量上清液中的总蛋白质。蛋白质样品(每孔30μg蛋白质)在10％的SDS-PAGE凝胶上进行电泳，并转移到PVDF膜上，在4℃下与一抗孵育过夜，然后在室温下与辣根过氧化物酶标记的二抗孵育2小时。使用ECL发光溶液检测条带，并使用ImageJ 1.8.0软件对蛋白质条带进行灰度分析。
9.测定IL-6、IL-1β和TNF-α的相对mRNA水平
从动物组织中分离出RNA，并评估其浓度、纯度和质量。使用提取的RNA作为模板进行逆转录，在20μL反应体积中于25℃下进行10分钟，50℃下进行30分钟，以及85℃下进行5分钟，以合成互补DNA(cDNA)。使用Primer Premier 6.0和Beacon Designer 7.8软件程序设计PCR引物，引物序列详见表1。
表1PCR引物序列
实时PCR系统扩增IL-6、IL-1β和TNF-α基因，以甘油醛-3-磷酸脱氢酶(GAPDH)作为内参基因。实时PCR扩增条件包括在95℃下进行1分钟的初始变性步骤，随后进行40个循环，每个循环包括在95℃下变性15秒，在63℃下退火25秒，以及荧光收集。随后，通过逐渐将温度从55℃升高到95℃来进行熔解曲线分析。每个样品进行三次重复分析，并量化每个基因的相对表达量。
三、实验结果
1、含XCHD药物的血清LC-MS/MS分析
分别对XCHD草药提取物、空白血清和含药血清进行了LC-MS/MS分析，得到基峰色谱图(见图1)，并将结果与MS数据库进行了比较，以选择得分70或更高的结果，之后，我们鉴定了含药血清样品的代谢物，根据文献ClassyFire中的分类方法对代谢物进行了分类，并根据代谢物的数量绘制了分类饼图(见图2)。空白组、草药组和给药组的代谢物结果以维恩图的形式呈现(见图3)。结果表明，草药提取物和含药血清中都存在60种成分，另外三种成分是含药血清特有的。这60种成分是根据与数据库相比得分大于70而筛选出来的，如下表所示。
表2与数据库相比得分大于70而筛选出来的60种成分
2、XCHD含药血清对LPS诱导的H9c2细胞炎症反应的影响
为了评估XCHD含药血清对H9c2细胞增殖的潜在抑制作用，本发明对从动物体内获取的含药血清和空白血清进行了检查。采用CCK-8法评估细胞活力。结果显示，最高浓度的空白血清对细胞活力无影响；然而，XCHD含药血清浓度超过4％时，对细胞活力产生显著影响(见图4)。因此，选择了4％、2％和1％作为XCHD含药血清的高、中、低浓度组。
研究结果表明，在空白血清和LPS组中，IL-6、IL-1β和TNF-α的表达均显著增加。相比之下，含药血清组显著抑制了IL-6、IL-1β和TNF-α的水平。此外，不同浓度的含药血清对不同炎症因子的抑制作用程度不同。值得注意的是，随着含药血清浓度的增加，其抑制TNF-α的能力也增强。然而，IL-1β和IL-6表现出相反的趋势，即在较低浓度的含药血清中观察到更强的抑制作用(见图5)。
如图6所示，模型组与空白对照组相比存在显著差异，显示出IL-6、IL-1β和TNF-α蛋白的表达增加。然而，经含药血清处理后的组，这些蛋白的表达显著降低。这些发现表明，XCHD含药血清能够影响IL-6、IL-1β和TNF-α蛋白的表达。
3、XCHD改善LPS诱导脓毒症小鼠的心功能障碍
本发明采用M型超声心动图评估小鼠心功能参数。与对照组相比，LPS组的LVIDs值升高。然而，XCHD治疗后，LVIDs有所降低；LPS组的LVFS和LVEF值降低。然而，XCHD治疗后，LVFS和LVEF值均有所升高(见图7—图10)。HE染色组织学检查显示，对照组小鼠心肌细胞排列整齐。相反，LPS组心肌细胞出现细胞膜破裂，细胞核暴露且排列不规则(见图11)。值得注意的是，给予XCHD可改善LPS诱导的小鼠心肌不规则破裂。此外，还评估了几种心肌损伤标志物，包括肌酸激酶MB(CK-MB)、乳酸脱氢酶(LDH)和心肌肌钙蛋白T(cTnI)，结果如图12—图14所示。值得注意的是，LPS组升高的LDH、心肌肌钙蛋白T和CK-MB水平在XCHD治疗后得到逆转。这些发现证实了XCHD能够减轻LPS诱导的心肌损伤。
4、XCHD抑制脓毒症诱导的心脏炎症因子生成
与对照组相比，LPS组的TNF-α、IL-6和IL-1β浓度升高，而XCHD治疗可有效抑制这种升高(见图15)。此外，LPS诱导后TNF-α、IL-6和IL-1β的mRNA表达也上调。然而，与模型组相比，XCHD治疗组TNF-α、IL-6和IL-1β的mRNA表达水平显著降低(见图16)。进一步地，XCHD治疗组TNF-α、IL-6和IL-1β的蛋白表达水平高于对照组。但与模型组相比，XCHD治疗组TNF-α、IL-6和IL-1β的蛋白表达水平明显降低(见图17)。这些综合发现支持XCHD能够通过减少炎症因子的生成，进而减轻脓毒症引起的心脏炎症。
四、结论：
本发明的结果表明，XCHD对于改善LPS诱导的脓毒症小鼠的心功能障碍具有显著效果。XCHD不仅能够提升心功能参数如LVEF和LVFS，还能够修复由LPS引起的心肌细胞损伤。此外，XCHD还能够有效减轻脓毒症引起的心脏炎症，通过降低炎症因子TNF-α、IL-6和IL-1β在mRNA和蛋白水平的表达来实现。这些发现共同支持XCHD在治疗脓毒血症心肌炎的潜在应用价值。
本具体实施例仅仅是对本发明的解释，其并不是对本发明的限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本发明的权利要求范围内都受到专利法的保护。
Claims (1)
1.小柴胡汤在制备用于减轻脓毒血症心肌炎的产品中的应用。
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