CN115025188B - 调节炎症和/或纤维化相关信号通路的中药及其应用 - Google Patents
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Abstract
本发明涉及中药技术领域，尤其涉及调节炎症和/或纤维化相关信号通路的中药及其应用。本发明提供了连翘、葛根、当归、生地黄、桃仁、红花、枳壳、甘草、法半夏、陈皮、党参、炙黄芪、茯苓、广藿香、砂仁中至少一种在制备调节炎症和/或纤维化相关信号通路的药物中的应用；其中任意一种单一药材都能够作用于调节炎症和/或纤维化相关信号通路中的至少一条。以上述15种药材组成的组合物，能够作用于8条与炎症和/或纤维化相关的信号通路，各组分缺一不可，相互配合产生了增效协同作用。
Description
本申请是申请日为2020年06月24日、发明名称为“调节炎症和/或纤维化相关信号通路的中药及其应用”、申请号为202010588510.0的发明的分案申请。
技术领域
本发明涉及中药技术领域，尤其涉及调节炎症和/或纤维化相关信号通路的中药及其应用。
背景技术
炎症与组织纤维化与多种疾病密切相关，例如：炎症细胞在糖尿病肾病的发生、发展中发挥着重要的作用。肾间质的主要炎症细胞巨噬细胞及T淋巴细胞，同样参与糖尿病肾病肾组织的慢性炎症及间质纤维化。同时，中性粒细胞可能也参与糖尿病肾病肾间质纤维化的发生。
炎症细胞的浸润、活化所致细胞因子的反常表达及细胞因子分泌的不平衡，在间质性肺炎的发生和发展过程中发挥着重要作用。例如，肺损伤中起重要作用的细胞因子有TFG-β、TNF-α、IFN-γ、IL及单核细胞趋化蛋白I、MCP-1等。
心肌纤维化在冠心病、高血压、肥厚性心肌病、心力衰竭等多种心血管疾病中普遍存在，现认为其与心律失常、心功能障碍甚至心脏猝死密切相关，众多炎症因子在心肌纤维化的进展过程汇总起到重要作用，例如：血管紧张素II与醛固酮、转化生长因子β、结缔组织生长因子、单核细胞趋化蛋白-1、肿瘤坏死因子-α、白介素-1、基质金属蛋白酶、一氧化氮和内皮素。
肝纤维化是多种慢性肝病进展过程中的重要病理阶段，其本质是肝脏对各种慢性损伤后持续进行伤口愈合反应的结果，主要表现为细胞外基质异常增生与沉积，伴随肝实质细胞的坏死和凋亡。肝纤维化持续发展会形成肝硬化，甚至进展为肝癌和肝衰竭。
炎症和/或纤维化的发生、发展与多条信号通路密切相关，在药物的研发过程中，针对这些信号通路进行有针对性的研究有利于药物的筛选。但目前对于中药材对炎症和/或纤维化的治疗作用的研究仍不足。
发明内容
有鉴于此，本发明要解决的技术问题在于提供调节炎症和/或纤维化相关信号通路的中药及其应用。
本发明提供了中药在制备调节炎症和/或纤维化相关信号通路的药物中的应用；
所述中药选自连翘、葛根、当归、生地黄、桃仁、红花、枳壳、甘草、法半夏、陈皮、党参、炙黄芪、茯苓、广藿香、砂仁中至少一种。
所述炎症和/或纤维化相关信号通路包括：
细胞因子-细胞因子受体相互作用、紧密连接、抗利尿激素调节的水重吸收、醛固酮调节的钠重吸收、近曲小管碳酸氢盐重吸收、HIF-1信号通路、钙离子信号通路、神经活性物质和受体的相互作用通路。
本发明中，所述炎症为由细菌、病毒等病原微生物感染导致的炎症；所述纤维化为感染导致的纤维化；所述纤维化为肾纤维化、肝纤维化肺纤维化或心肌纤维化。
所述病毒包括：冠状病毒、腺病毒、疱疹病毒、肠道病毒、麻疹病毒、乙型肝炎病毒、丙型肝炎病毒、人乳头瘤病毒、甲型H1N1流感病毒、人类T细胞白血病病毒。
所述细菌包括：嗜血杆菌(Hemophilus)、假单胞菌(Pseudomxonas)、链球菌(Streptococcus)、葡萄球菌(Staphylococcus)、奈瑟菌(Neisseria)、韦荣球菌(Veillonella)等。
传统中医理论认为：陈皮，理气健脾，燥湿化痰；甘草，补脾益气，清热解毒，祛痰止咳，缓急止痛，调和诸药，用于脾胃虚弱；党参，健脾益肺，养血生津，可用于脾肺气虚。砂仁，化湿开胃，温脾理气；枳壳，理气宽中，行滞消胀；炙黄芪，益气补中，用于气虚乏力。生地黄，清热凉血，养阴生津；法半夏，燥湿化痰，用于痰多咳喘；广藿香，芳香化浊，和中止呕，发表解暑。连翘，清热解毒，消肿散结，疏散风热；桃仁，活血祛瘀，润肠通便，止咳平喘。葛根，解肌退热，生津止渴，透疹，升阳止泻，通经活络，可解酒毒；当归，补血活血，调经止痛，润肠通便；茯苓，利水渗湿，健脾，宁心。
本发明通过对8个信号通路中，3000余个基因的表达情况，分析8个信号通路受中药材的影响，其中，细胞因子-细胞因子受体相互作用、紧密连接与炎症及纤维化改变相关，抗利尿激素调节的水重吸收、醛固酮调节的钠重吸收参与调控水钠的重吸收，近曲小管碳酸氢盐重吸收调控酸碱平衡，HIF-1信号通路参与氧化应激保护过程，钙离子信号通路影响心肌收缩力，神经活性配体-受体相互作用决定了神经活性物质和受体的相互作用。对这些信号通路的调控可以改善病毒感染合并纤维化的各种病理过程，阻止病情的发展。
具体的，本发明实验表明：
红花能够上调钙离子信号通路、神经活性配体-受体相互作用；
砂仁能够上调近曲小管碳酸氢盐重吸收、钙离子信号通路、神经活性配体-受体相互作用，而对紧密连接、抗利尿激素调节的水重吸收存在抑制作用；
甘草能够上调钙离子信号通路、神经活性配体-受体相互作用，而对紧密连接、醛固酮调节的钠重吸收、抗利尿激素调节的水重吸收存在抑制作用；
法半夏能够上调HIF-1信号通路，而对紧密连接存在抑制作用；
广藿香能够上调近曲小管碳酸氢盐重吸收；
当归能够上调HIF-1信号通路、钙离子信号通路，而对细胞因子-细胞因子受体相互作用、紧密连接存在抑制作用；
炙黄芪能够上调HIF-1信号通路，而对细胞因子-细胞因子受体相互作用存在抑制作用；
桃仁能够上调HIF-1信号通路，而对细胞因子-细胞因子受体相互作用存在抑制作用；
陈皮能够上调钙离子信号通路，而对细胞因子-细胞因子受体相互作用存在抑制作用；
连翘能够上调钙离子信号通路，而对细胞因子-细胞因子受体相互作用存在抑制作用；
地黄对细胞因子-细胞因子受体相互作用、紧密连接、醛固酮调节的钠重吸收存在抑制作用；
枳壳、茯苓、党参、葛根对细胞因子-细胞因子受体相互作用存在抑制作用；
本发明还提供了一种中药组合物，其由连翘、葛根、当归、生地黄、桃仁、红花、枳壳、甘草、法半夏、陈皮、党参、炙黄芪、茯苓、广藿香、砂仁组成
本发明所述的中药组合物由如下质量份的组分组成：连翘5～30份、葛根6～30份、当归6～15份、生地黄10～20份、桃仁5～24份、红花3～15份、枳壳3～15份、甘草2～15份、法半夏3～15份、陈皮2～15份、党参9～24份、炙黄芪9～30份、茯苓6～30份、广藿香3～20份、砂仁3～6份组成。
一些具体实施例中，所述中药组合物由如下质量份的组分组成：连翘10份、葛根10份、当归10份、生地黄10份、桃仁10份、红花10份、枳壳10份、甘草10份、法半夏10份、陈皮10份、党参10份、炙黄芪10份、茯苓10份、广藿香10份、砂仁10份。
一些具体实施例中，所述中药组合物由如下质量份的组分组成：连翘5份、葛根6份、当归6份、生地黄10份、桃仁5份、红花3份、枳壳3份、甘草2份、法半夏3份、陈皮2份、党参9份、炙黄芪9份、茯苓6份、广藿香3份、砂仁3份。
一些具体实施例中，所述中药组合物由如下质量份的组分组成：连翘30份、葛根30份、当归15份、生地黄20份、桃仁24份、红花15份、枳壳15份、甘草15份、法半夏15份、陈皮15份、党参30份、炙黄芪30份、茯苓30份、广藿香20份、砂仁6份。
一些具体实施例中，所述中药组合物由如下质量份的组分组成：连翘9份、葛根12份、当归9份、生地黄12份、桃仁6份、红花6份、枳壳6份、甘草9份、法半夏6份、陈皮6份、党参15份、炙黄芪15份、茯苓12份、广藿香9份、砂仁6份。
本发明还提供了一种中药提取物，其由本发明所述的中药组合物提取获得。
所述中药提取物的制备方法包括：将连翘、葛根、当归、生地黄、桃仁、红花、枳壳、甘草、法半夏、陈皮、党参、炙黄芪、茯苓、广藿香、砂仁以90vol％乙醇水溶液进行标准索氏回流提取，所得提取液干燥，制得提取物。
所述90vol％乙醇水溶液的标准索氏回流提取的时间为3h，每10g药材以150mL90vol％乙醇水溶液提取。所述水的标准索氏回流提取的时间为2h，每10g药材以150mL水提取。
本发明所述中药提取物的制备方法中，各组分可混合提取物也可先后分别提取后混合，本发明对此不做限定。
本发明所述的中药组合物、中药提取物或所述制备方法制得的提取物，在制备治疗炎症和/或纤维化的药物中的应用。
所述防治包括：调节炎症和/或纤维化相关信号通路；
所述炎症和/或纤维化相关信号通路包括：
细胞因子-细胞因子受体相互作用、紧密连接、抗利尿激素调节的水重吸收、醛固酮调节的钠重吸收、近曲小管碳酸氢盐重吸收、HIF-1信号通路、钙离子信号通路、神经活性物质和受体的相互作用通路。
所述调节包括上调HIF-1信号通路、神经活性配体-受体相互作用、钙离子信号通路、近曲小管碳酸氢盐重吸收；下调细胞因子-细胞因子受体相互作用、醛固酮调节的钠重吸收、紧密连接、抗利尿激素调节的水重吸收。
本发明中，所述炎症为由细菌、病毒等病原微生物感染导致的炎症；所述纤维化为感染导致的纤维化；所述纤维化为肾纤维化、肝纤维化、肺纤维化或心肌纤维化。肺纤维化的发生会影响呼吸，引起呼吸困难，严重时出现呼吸衰竭，所述治疗还包括对肺纤维化导致的呼吸衰竭的治疗。肾纤维化的发生会影响机体对水、电解质和酸碱的调节，严重时出现肾功能衰竭，所述治疗还包括对肾纤维化导致的肾功能衰竭的治疗。
所述病毒包括：冠状病毒、腺病毒、疱疹病毒、肠道病毒、麻疹病毒、乙型肝炎病毒、丙型肝炎病毒、人乳头瘤病毒、甲型H1N1流感病毒、人类T细胞白血病病毒。
所述细菌包括：嗜血杆菌(Hemophilus)、假单胞菌(Pseudomxonas)、链球菌(Streptococcus)、葡萄球菌(Staphylococcus)、奈瑟菌(Neisseria)、韦荣球菌(Veillonella)等。
本发明还提供了一种药物，其包括本发明所述的中药组合物、中药提取物或所述制备方法制得的提取物。
所述药物中还包括药学上可接受的辅料；
所述药学上可接受的辅料为矫味剂、填充剂、润滑剂、防腐剂、助悬剂、食用色素、稀释剂、乳化剂、崩解剂或增塑剂中的一种或两者以上的混合物；
所述药物的剂型为注射剂、片剂、丸剂、口服液剂、胶囊剂、糖浆剂、滴丸剂或颗粒剂。
本发明还提供了一种治疗治疗炎症和/或纤维化的方法，其为给予本发明所述的药物。所述给予的方式为注射或口服。
本发明中，所述炎症为由细菌、病毒等病原微生物感染导致的炎症；所述纤维化为感染导致的纤维化；所述纤维化为肾纤维化、肝纤维化、肺纤维化或心肌纤维化。肺纤维化的发生会影响呼吸，引起呼吸困难，严重时出现呼吸衰竭，所述治疗还包括对肺纤维化导致的呼吸衰竭的治疗。肾纤维化的发生会影响机体对水、电解质和酸碱的调节，严重时出现肾功能衰竭，所述治疗还包括对肾纤维化导致的肾功能衰竭的治疗。
本发明提供了连翘、葛根、当归、生地黄、桃仁、红花、枳壳、甘草、法半夏、陈皮、党参、炙黄芪、茯苓、广藿香、砂仁中至少一种在制备调节炎症和/或纤维化相关信号通路的药物中的应用；其中任意一种单一药材都能够作用于调节炎症和/或纤维化相关信号通路中的至少一条。以上述15种药材组成的组合物，能够作用于8条与炎症和/或纤维化相关的信号通路，各组分缺一不可，相互配合产生了增效协同作用。
附图说明
图1示15种中药在肾小管上皮细胞种对8条相关通路的富集情况；
图2示实施例2～5的药物对8条相关通路的影响；
图3示实施例2(组方)和对比例1～4的药物对8条相关通路的影响。
具体实施方式
本发明提供了调节炎症和/或纤维化相关信号通路的中药及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明检测涉及8条信号通路，共检测三千余种基因，其中各信号通路中具有代表性的基因如表1：
表1信号通路及代表性基因
本发明采用的试材皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1
中药提取物的制备，具体过程为：
1、分别称取中药连翘、葛根、当归、生地黄、桃仁、红花、枳壳、甘草、法半夏、陈皮、党参、炙黄芪、茯苓、广藿香、砂仁。
2、制备方法：上述药物粉碎后，分别称取10g，各药物分别用150mL的90％乙醇，标准索氏回流提取3h，提取液浓缩20min至体积为2mL，利用冻干机减压冻干24h，分别制成上述13种各种中药提取物的冻干粉。
称取一定质量冻干粉，溶于DMSO中制成提取物母液，母液浓度是20mg/mL，制备终浓度100μg/mL的中药提取物，-20℃保存。
实施例2～5
中药组合物的制备，具体过程为：
1、分别称取中药连翘、葛根、当归、生地黄、桃仁、红花、枳壳、甘草、法半夏、陈皮、党参、炙黄芪、茯苓、广藿香、砂仁。
2、制备方法：上述药物均单独制备。
分别称取各药物10g，用150mL的90％乙醇，标准索氏回流提取3h，提取液浓缩20min至体积为2mL，利用冻干机减压冻干24h，分别制成上述13种各种中药提取物的冻干粉。按照表1所示配比称取各提取物的冻干粉，溶于DMSO中制成各中药提取物母液，母液浓度是20mg/mL，制备终浓度100μg/mL的中药提取物，-20℃保存。
表2实施例2～5的组合物的配比(g)
| 实施例2 | 实施例3 | 实施例4 | 实施例5 | |
| 连翘 | 10 | 5 | 30 | 9 |
| 葛根 | 10 | 6 | 30 | 12 |
| 当归 | 10 | 6 | 15 | 9 |
| 生地黄 | 10 | 10 | 20 | 12 |
| 桃仁 | 10 | 5 | 24 | 6 |
| 红花 | 10 | 3 | 15 | 6 |
| 枳壳 | 10 | 3 | 15 | 6 |
| 甘草 | 10 | 2 | 15 | 9 |
| 法半夏 | 10 | 3 | 15 | 6 |
| 陈皮 | 10 | 2 | 15 | 6 |
| 党参 | 10 | 9 | 30 | 15 |
| 炙黄芪 | 10 | 9 | 30 | 15 |
| 茯苓 | 10 | 6 | 30 | 12 |
| 广藿香 | 10 | 3 | 20 | 9 |
| 砂仁 | 10 | 3 | 6 | 6 |
3、上述中药组合物如应用于病人，则采用水煎剂的制备方法，煎煮二次，各30min，合并煎液服用。
对比例1
以现有技术中的六味地黄汤作为阳性对照，制备方法为：
熟地、山茱萸、山药、牡丹皮、泽泻、茯苓。各药物分别称取10g，分别用150mL的90％乙醇，标准索氏回流提取3h，提取液浓缩20min至体积为2mL，利用冻干机减压冻干24h，分别制成上述5种各种中药提取物的冻干粉。称取一定质量冻干粉，溶于DMSO中制成提取物母液，母液浓度是20mg/mL，各组分按照等比例配比，以终浓度100μg/mL中药提取物处理细胞。
对比例2
以现有技术中的参芪地黄汤作为阳性对照，制备方法为：
人参、黄芪、熟地、山药、茯苓、丹皮、山茱萸。各药物分别称取10g，分别用150mL的90％乙醇，标准索氏回流提取3h，提取液浓缩20min至体积为2mL，利用冻干机减压冻干24h，分别制成上述5种各种中药提取物的冻干粉。称取一定质量冻干粉，溶于DMSO中制成提取物母液，母液浓度是20mg/mL，各组分按照等比例配比，以终浓度100μg/mL中药提取物处理细胞。
对比例3
以现有技术中的四君子汤作为阳性对照，制备方法为：
人参、白术、茯苓、甘草。各药物分别称取10g，分别用150mL的90％乙醇，标准索氏回流提取3h，提取液浓缩20min至体积为2mL，利用冻干机减压冻干24h，分别制成上述5种各种中药提取物的冻干粉。称取一定质量冻干粉，溶于DMSO中制成提取物母液，母液浓度是20mg/mL，各组分按照等比例配比，以终浓度100μg/mL中药提取物处理细胞。
对比例4
以现有技术中的金匮肾气丸作为阳性对照，制备方法为：
熟地、山茱萸、山药、牡丹皮、泽泻、茯苓、桂枝、附子、牛膝、车前子。各药物分别称取10g，分别用150mL的90％乙醇，标准索氏回流提取3h，提取液浓缩20min至体积为2mL，利用冻干机减压冻干24h，分别制成上述5种各种中药提取物的冻干粉。称取一定质量冻干粉，溶于DMSO中制成提取物母液，母液浓度是20mg/mL，各组分按照等比例配比，以终浓度100μg/mL中药提取物处理细胞。
功效验证
一、单味药物对关键通路的影响
利用HTS2(基于高通量测序的高通量筛选)技术，HK2人肾小管上皮细胞上对3000余个基因的表达情况，继而对和炎症和纤维化相关的多条通路进行富集分析，具体实验过程为：
1.分别利用实施例1中制备的中药提取物即连翘、葛根、当归、生地黄、桃仁、红花、枳壳、甘草、法半夏、陈皮、党参、炙黄芪、茯苓、广藿香、砂仁的提取物，100μg/mL浓度处理诱导的HK2人肾小管上皮细胞24小时。
2.细胞裂解，加入基因特异性探针结合mRNA，并利用DNA连接酶将结合成功的一对探针连接。
3.将连接成功的探针洗脱，利用PCR技术进行扩增和建库测序。
4.数据处理：以溶剂对照DMSO处理组为对照组，药物处理组与对照组比较，计算相关通路基因及背景基因的Fold change。
5.进行GSEA(Gene set enrichment analysis)信号通路富集分析，FDR<0.25，认为通路被显著调控。
结果如图1所示，其展示了15种中药在肾小管上皮细胞种对8条相关通路的富集情况：包括通路细胞因子-细胞因子受体相互作用、紧密连接、抗利尿激素调节的水重吸收、醛固酮调节的钠重吸收、近曲小管碳酸氢盐重吸收、HIF-1信号通路、钙离子信号通路和神经活性配体-受体相互作用。红色表示该通路显著上调，蓝色表示该通路显著下调，白色表示无影响。
结果显示，中药组合物中，当归、炙黄芪、桃仁、陈皮、连翘、地黄、枳壳、茯苓、党参、葛根均可调控炎症和纤维化相关通路：细胞因子-细胞因子受体相互作用，砂仁、甘草、法半夏、当归、地黄可调节纤维化相关通路紧密连接。砂仁、甘草、地黄可调控水钠的重吸收通路，包括抗利尿激素调节的水重吸收和醛固酮调节的钠重吸收，砂仁、广藿香可显著调节酸碱失衡相关通路，即近曲小管碳酸氢盐重吸收，法半夏、当归、炙黄芪和桃仁可显著调节应激相关通路，即HIF-1信号通路，红花、砂仁、甘草、当归、陈皮和连翘可以改善钙离子信号通路，红花、砂仁和甘草可以调节神经活性物质和受体的相互作用通路，即神经活性配体-受体相互作用。此中药组合物可显著调节多条与炎症和纤维化相关的通路，改善肾功能，保肾护肾。
二、实施例2～5对关键通路的影响
方法与(一)相同，实施例2、3、4、5的药物，分别按照表1组合物的配比，每种药物以终浓度100μg/mL处理诱导的THP1细胞24小时。结果如图2所示，红色表示该通路显著上调，蓝色表示该通路显著下调，白色表示无影响。结果表明：实施例2、3、4、5，均可显著调节炎症、纤维化相关通路、神经活性物质和受体的相互作用通路和应激的HIF-1信号通路，调控水、钠重吸收通路，改善酸碱失衡及钙离子信号通路，实施例2和4效果较强。
三、实施例2和对比例1～4的药物对关键通路的影响
方法与(一)相同，实施例2(组方)和对比例1～4的药物，分别以100μg/mL浓度处理诱导的THP1细胞24小时。结果如图3所示，红色表示该通路显著上调，蓝色表示该通路显著下调，白色表示无影响。结果表明：与实施例2所示的药物相比，对比例1～4的药物作用的靶点较少，例如，六味地黄丸对醛固酮调节的钠重吸收、紧密连接、近曲小管碳酸氢盐重吸收和HIF-1信号通路无调节作用；参芪地黄汤对醛固酮调节的钠重吸收、近曲小管碳酸氢盐重吸收和紧密连接无调节作用；四君子汤对近曲小管碳酸氢盐重吸收无调节作用；金匮肾气丸对醛固酮调节的钠重吸收、HIF-1信号通路和近曲小管碳酸氢盐重吸收无调节作用。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.一种调节炎症的中药组合物，其特征在于，如下质量份的原料制成：连翘5~30份、葛根6~30份、当归6~15份、生地黄10~20份、桃仁5~24份、红花3~15份、枳壳3~15份、甘草2~15份、法半夏3~15份、陈皮2~15份、党参9~24份、炙黄芪9~30份、茯苓6~30份、广藿香3~20份和砂仁3~6份。
2.一种调节炎症的中药提取物，其特征在于，由权利要求1所述的中药组合物提取获得。
3.权利要求2所述的中药提取物的制备方法，其特征在于，包括：将连翘、葛根、当归、生地黄、桃仁、红花、枳壳、甘草、法半夏、陈皮、党参、炙黄芪、茯苓、广藿香、砂仁以90vol%乙醇水溶液进行标准索氏回流提取，所得提取液干燥，制得提取物。
4.权利要求1所述的中药组合物、权利要求2所述的中药提取物或权利要求3所述制备方法制得的提取物在制备调节炎症的药物中的应用。
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