WO2021052305A1 - Use of eight-treasure pill in preparation of medicine for preventing or treating diseases related to il-6 inflammatory cytokine storm - Google Patents

Use of eight-treasure pill in preparation of medicine for preventing or treating diseases related to il-6 inflammatory cytokine storm Download PDF

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WO2021052305A1
WO2021052305A1 PCT/CN2020/115246 CN2020115246W WO2021052305A1 WO 2021052305 A1 WO2021052305 A1 WO 2021052305A1 CN 2020115246 W CN2020115246 W CN 2020115246W WO 2021052305 A1 WO2021052305 A1 WO 2021052305A1
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use according
injection
medicine
babaodan
diseases related
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关斌
钱景
赖志成
王毅
程翼宇
南淑华
詹志学
墙世发
陈西翌
何敏婉
郑珊珊
许玉珍
陈晓琳
阳丽华
曾智发
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厦门中药厂有限公司
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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
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention belongs to the field of biomedicine. More specifically, the present invention relates to the use of Babaodan in the preparation of medicines for preventing or treating diseases related to IL-6 inflammatory factor storm.
  • Interleukin IL-6 is rapidly induced and expressed in large amounts in the case of infection, autoimmunity or cancer. IL-6 is also related to some mental illnesses. IL-6 is significantly elevated in patients with major depression. In tumor immunotherapy, the inflammatory factor storm caused by IL-6 is a common side effect.
  • Babao Dan is derived from ancient Chinese medicine and has the effects of clearing damp-heat, promoting blood circulation and detoxification, removing yellowness and relieving pain. In clinical practice, Babao Dan has a good therapeutic effect on viral infections, acute urinary system infections and other infectious diseases. The current pharmacological research results show that Babao Pill has good anti-inflammatory and anti-tumor effects.
  • TLRs agonists such as LPS, Pam3CKS4, Poly(I:C) and Pam3CKS4+Poly(I:C) were used to stimulate Raw 264.7 macrophages, and the secretion and expression of IL-6 inflammatory factors were detected by ELISA and RT-qPCR. , Evaluate the effect of Babaodan on IL-6 from the cellular and molecular level.
  • the research of the present invention found that Babao Pill can significantly inhibit the expression and release of IL-6 inflammatory factors.
  • the present invention provides the use of Babaodan in the preparation of medicines for preventing or treating diseases related to IL-6 inflammatory factor storm.
  • the diseases related to IL-6 inflammatory factor storm refer to various diseases related to IL-6 up-regulation, for example, including infectious diseases (including bacteria, viruses, parasites, mycoplasma, chlamydia, etc.). Diseases caused by pathogenic microorganisms), autoimmune diseases, cancer, and mental diseases, as well as immune checkpoint inhibitors (ICIs) (such as anti-PD-1 antibodies), chimeric antigen receptors (CAR) T Side effects caused by tumor immunotherapy such as cell (CAR-T).
  • infectious diseases including bacteria, viruses, parasites, mycoplasma, chlamydia, etc.
  • ICIs immune checkpoint inhibitors
  • CAR chimeric antigen receptors
  • the medicament of the present invention can be in various dosage forms conventional in the art, such as solid, semi-solid or liquid forms, and can be aqueous, non-aqueous, suspension, lozenges, capsules, tablets, granules, pills, and powders. Wait.
  • the route of administration of the drug can be injection or oral administration.
  • the injection administration may include intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.
  • the medicine may include 0.01-99.99% Babaodan or its extract, and 99.99-0.01% auxiliary components.
  • the medicine may include one or more pharmaceutically acceptable conventional excipients.
  • the pharmaceutically acceptable conventional excipients can be excipients, fillers or diluents and the like.
  • Figure 1 shows the dynamic changes of IL-6 in BALF.
  • Figure 2 shows the mRNA expression of the inflammatory factor IL-6 in lung homogenate.
  • Figure 3 shows the survival curve
  • Figure 4 shows a pathological section of lung tissue.
  • Figure 5 shows the effect of Babaodan on LPS stimulated macrophages to secrete IL-6. Compared with the model group, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Figure 6 shows the effect of Babaodan on Pam3CSK4 stimulated macrophages to secrete IL-6. Compared with the model group, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Figure 7 shows the effect of Babaodan on Poly(I:C)+Pam3CSK4 stimulated macrophages to secrete IL-6. Compared with the model group, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Drug preparation method for gavage Weigh the contents of the capsule, add an appropriate amount of MiliQ water to dissolve it to the desired concentration (40mg/mL), store it at 4°C after 30 minutes of ultrasound in an ice bath, and use it within 2 hours.
  • Preparation method of drug for cell experiment Weigh the contents of Babaodan capsules, add an appropriate amount of DMEM cell culture medium, sonicate for 30 minutes, filter with 0.22 ⁇ M membrane, add 10% inactivated FBS and 100X penicillin, add DMEM Make up the volume to obtain a 2mg/mL Babaodan solution. Store at 4°C and use within 24 hours.
  • Mouse IL-6 ELISA detection kit (eBioscience); ultrapure RNA extraction kit (Beijing Kangwei Century Biotechnology Co., Ltd.), RNase-Free DNase Set (QIAGEN), HiFiScript cDNA first-strand synthesis kit (Beijing Kang It is Century Biotechnology Co., Ltd.), UltraSYBR Mixture (Beijing Kangwei Century Biotechnology Co., Ltd.), the primer sequences are shown in Table 1 below, and the primers are all synthesized by Beijing Kinco Xinye Biotechnology Co., Ltd.
  • M1000 PRO multifunctional microplate reader (Switzerland Tecan company), Mastercycler gradient PCR machine (Eppendorf company), CFX96 TM Touch real-time quantitative PCR system (BIO-RAD company), chemiluminescence imager (BIO-RAD company).
  • Example 1 Babaodan inhibits the expression and release of IL-6 inflammatory factors in a model of acute lung injury caused by SA infection of influenza virus PR8
  • H1N1 influenza virus strain PR8 Puerto-Rico/8
  • SA Staphylococcus aureus
  • the PR8+SA model group, BBD TR group, and BBD RG group mice were infected with PR8.
  • the infection method was intranasal infection (1 ⁇ 10 4 PFU/mL, 20 ⁇ L). After the infection is complete, observe its recovery. The day of infection is day 0.
  • SA infection was performed on the 5th day after infection.
  • the BBD TR group and BBD RG group were administered intragastrically at a dose of 1.0 g crude drug/kg (approximately 0.4 to 0.5 mL of liquid medicine) 2 hours before SA infection.
  • the control group (Control) and model group (Model) were given corresponding volumes of solvent (MiliQ water) according to body weight.
  • mice in the model group, BBD TR group, and BBD RG group were infected by instilling 50 ⁇ L of Staphylococcus aureus suspension through the trachea guided by a laryngoscope.
  • the survival rate and lung pathological examination test bacteria count was 5 ⁇ 10 7 CFU, and the others were 2.5 ⁇ 10 7 CFU.
  • BALF alveolar lavage fluid
  • mice After SA infection for 12h and 24h, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (1.5%), and the mouse's chest cavity was opened to expose the trachea and lungs.
  • Make a T-shaped incision at the epiglottis cartilage insert a cannula to fix it, use a 1mL syringe to perform bronchoalveolar lavage on the mouse’s whole lung, withdraw 0.8mL PBS each time, and repeat the injection twice. The recovery rate is above 80%.
  • the obtained alveolar lavage fluid was immediately placed on ice, centrifuged at 1000 rpm at 4°C for 5 minutes, and the supernatant was transferred to a clean EP tube. After aliquoting, it was immediately stored at -80°C for inflammatory factor detection. Then take the alveolar lavage fluid (BALF) according to the ELISA kit operating instructions to detect the IL-6 cytokines in each sample.
  • the IL-6 concentration in BALF increased significantly at 12h and 24h in the PR8+SA model group (P ⁇ 0.05), indicating that the expression and release of IL-6 inflammatory factors in the PR8+SA model increased significantly.
  • the inflammatory factor IL-6 in the Baodan intervention group was significantly down-regulated (P ⁇ 0.05) ( Figure 1), indicating that Babaodan can inhibit the expression and release of IL-6 inflammatory factors.
  • Figure 1 the dynamic change detection results showed that cytokines increased at 2h after SA infection, and IL-6 levels reached their peaks at 6h and 12h after SA infection. Babaodan significantly inhibited the expression of IL-6 at 6h and 12h (P ⁇ 0.05).
  • mice After SA infection for 12h and 24h, the mice were sacrificed by cervical dislocation. Open the chest cavity, take the whole lung and place it in a homogenization tube containing 1.0mL PBS, homogenize (Tissuelyser-48 automatic sample rapid grinder), and record the volume of the homogenate as 1.0mL. Take 100 ⁇ L and place it in an EP tube containing 0.9 mL of lysis solution TRIzol (Beijing Kangwei Century Biotechnology Co., Ltd.) for RNA extraction, and then use RT-qPCR to detect the expression level of cytokine mRNA.
  • TRIzol Beijing Kangwei Century Biotechnology Co., Ltd.
  • RNA was extracted according to the instructions of the ultra-pure RNA extraction kit (Beijing Kangwei Century Biotechnology Co., Ltd.), after DNase treatment, the RNA concentration was measured with NanoDrop 2000, and 2 ⁇ g RNA was taken according to the HiFiScript cDNA first-strand synthesis kit (Beijing Kangwei Century Biotechnology Co., Ltd.) Reverse transcription to obtain cDNA.
  • RNA is stored at -80°C
  • cDNA is stored at 4°C for short-term storage.
  • Use UltraSYBR Mixture (Beijing Kangwei Century Biotechnology Co., Ltd.) kit for fluorescent quantitative PCR.
  • the primer sequences are shown in Table 1 above.
  • the fluorescence quantitative PCR baseline was automatically set by Bio-Rad's CFX manager V3.0 software, ⁇ -actin was used as the internal reference gene and the relative gene expression was calculated by the 2- ⁇ CT method.
  • the above-mentioned Babaodan inhibits the expression and release of IL-6 inflammatory factors for the treatment of diseases related to IL-6 inflammatory factor storms, which can be confirmed by observation of general and survival conditions of mice and lung pathological examination.
  • mice that awakened within 2 hours after SA infection were included in the general and survival observation.
  • General observation indicators include: activity, mental state, breathing, response to external stimuli, etc.
  • the survival observation frequency is: within 24h of infection, record once every 2 or 4h, then every 6 or 12h and record to 72h.
  • mice in the model group showed symptoms such as severe malaise, shortness of breath, and indifference to external stimuli. Most of the mice died 24 hours after infection. Compared with the model group, the general condition of mice in the BBD TR and BBD RG groups has improved.
  • mice Twelve hours after SA infection, the mice were sacrificed by cervical dislocation. The chest cavity was opened, the whole lung was taken, and the lung consolidation and congestion were observed and immediately fixed with paraformaldehyde, followed by routine paraffin embedding, sectioning and HE staining (Sevier (Google) Biotechnology Co., Ltd.). Observe the slices under a microscope, record the structural integrity of alveoli, inflammatory cell infiltration and other lung pathological changes.
  • the general observation results show that the mouse model group has blood red lesions on the surface of the lungs, swelling and increased volume of the lung lobes; compared with the model group, Babao Dan can improve lung injury, reducing the blood red lesions on the lung surface, swelling and congestion. Lighten up.
  • Example 2 Evaluation of the pharmaceutical efficacy of Babaodan on the expression and release of IL-6 inflammatory factors based on the macrophage TLRs activation model
  • Mouse mononuclear macrophage cell line Raw 264.7 (ATCC TIB-71), the culture medium is DMEM high glucose medium containing 10% inactivated FBS, penicillin and streptomycin, and the culture conditions are 37°C and 5% CO 2 .
  • the cells in the logarithmic growth phase were used for experiments.
  • TLRs agonists The list of TLRs agonists and working concentrations is shown in Table 2 below.
  • the experimental protocol is as follows: Raw 264.7 cells are seeded in a 12-well plate at 1 ⁇ 10 5 /well. After 24h, change the culture medium containing Babaodan. After 22h, the culture medium containing the medicine was changed again, TLRs agonist was added after 2h, and the cell supernatant was collected after 24h. IL-6 was detected by ELISA.
  • LPS (100ng/mL) stimulation for 24h can significantly cause Raw 264.7 cells to secrete IL-6; as shown in Figure 6, Pam3CSK4 (1 ⁇ g/mL) stimulation for 24h can significantly cause Raw 264.7 cells to secrete IL- 6; As shown in Figure 7, Poly(I:C)+Pam3CSK4 stimulation for 24h can significantly cause Raw 264.7 cells to secrete IL-6.
  • Babao Pill has a significant inhibitory effect on the secretion of IL-6 caused by LPS, Pam3CSK4 and Poly(I:C)+Pam3CSK4 stimulation, and presents a dose-effect relationship.
  • Babaodan has a significant inhibitory effect on the release of cytokine IL-6 caused by activation of different TLRs receptors, and can be used to prevent or treat diseases related to IL-6 inflammatory factor storm.

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Abstract

Provided is a use of eight-treasure pill in the preparation of medicine for preventing or treating diseases related to IL-6 inflammatory cytokine storm.

Description

八宝丹在制备用于预防或治疗与IL-6炎症因子风暴相关的疾病的药物中的用途Use of Babaodan in the preparation of medicines for preventing or treating diseases related to IL-6 inflammatory factor storm 技术领域Technical field
本发明属于生物医药领域，更具体而言，本发明涉及八宝丹在制备用于预防或治疗与IL-6炎症因子风暴相关的疾病的药物中的用途。The present invention belongs to the field of biomedicine. More specifically, the present invention relates to the use of Babaodan in the preparation of medicines for preventing or treating diseases related to IL-6 inflammatory factor storm.
背景技术Background technique
白介素IL-6在感染、自身免疫或癌症的情况下会快速诱导，大量表达。IL-6也与一些精神疾病有关。在重度抑郁症患者中IL-6明显升高。在肿瘤免疫治疗中，IL-6引起的炎症因子风暴是常见副作用。Interleukin IL-6 is rapidly induced and expressed in large amounts in the case of infection, autoimmunity or cancer. IL-6 is also related to some mental illnesses. IL-6 is significantly elevated in patients with major depression. In tumor immunotherapy, the inflammatory factor storm caused by IL-6 is a common side effect.
八宝丹源自中药古方，具有清利湿热、活血解毒、去黄止痛的功效。在临床实践中，八宝丹对病毒性感染、急性泌尿***感染等感染性疾病等具有良好的治疗效果。现在药理学研究结果表明八宝丹具有良好的抗炎、抗肿瘤效应。Babao Dan is derived from ancient Chinese medicine and has the effects of clearing damp-heat, promoting blood circulation and detoxification, removing yellowness and relieving pain. In clinical practice, Babao Dan has a good therapeutic effect on viral infections, acute urinary system infections and other infectious diseases. The current pharmacological research results show that Babao Pill has good anti-inflammatory and anti-tumor effects.
目前，尚未有研究表明八宝丹用于预防或治疗与IL-6炎症因子风暴相关的疾病的应用。At present, there is no research showing the application of Babao Pill for preventing or treating diseases related to IL-6 inflammatory factor storm.
发明内容Summary of the invention
本发明分别以LPS、Pam3CKS4、Poly(I:C)以及Pam3CKS4+Poly(I:C)等TLRs激动剂刺激Raw 264.7巨噬细胞，以ELISA和RT-qPCR检测IL-6炎症因子的分泌和表达，从细胞和分子水平评价八宝丹对IL-6的效应。In the present invention, TLRs agonists such as LPS, Pam3CKS4, Poly(I:C) and Pam3CKS4+Poly(I:C) were used to stimulate Raw 264.7 macrophages, and the secretion and expression of IL-6 inflammatory factors were detected by ELISA and RT-qPCR. , Evaluate the effect of Babaodan on IL-6 from the cellular and molecular level.
本发明研究发现八宝丹可显著抑制IL-6炎症因子的表达和释放。The research of the present invention found that Babao Pill can significantly inhibit the expression and release of IL-6 inflammatory factors.
因此，本发明提供八宝丹在制备用于预防或治疗与IL-6炎症因子风暴相关的疾病的药物中的用途。Therefore, the present invention provides the use of Babaodan in the preparation of medicines for preventing or treating diseases related to IL-6 inflammatory factor storm.
本发明中，所述与IL-6炎症因子风暴相关的疾病指的是与IL-6上调相关的各种疾病，例如，包括感染性疾病(包括由细菌、病毒、寄生虫、支原体、衣原体等致病微生物引发的疾病)、自身免疫性疾病、癌症、精神疾病，还包括免疫检查点抑制剂(ICIs)(如抗PD-1抗体)、嵌合抗原受体(chimeric  antigen receptor,CAR)T细胞(CAR-T)等肿瘤免疫治疗手段所引起的副作用。In the present invention, the diseases related to IL-6 inflammatory factor storm refer to various diseases related to IL-6 up-regulation, for example, including infectious diseases (including bacteria, viruses, parasites, mycoplasma, chlamydia, etc.). Diseases caused by pathogenic microorganisms), autoimmune diseases, cancer, and mental diseases, as well as immune checkpoint inhibitors (ICIs) (such as anti-PD-1 antibodies), chimeric antigen receptors (CAR) T Side effects caused by tumor immunotherapy such as cell (CAR-T).
本发明的药物可以为本领域常规的各种剂型，例如固体、半固体或液体的形式，可以为水溶液、非水溶液、混悬液、锭剂、胶囊剂、片剂、颗粒剂、丸剂和散剂等。所述药物的给药途径可以为注射给药或口服给药。所述注射给药可以包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。The medicament of the present invention can be in various dosage forms conventional in the art, such as solid, semi-solid or liquid forms, and can be aqueous, non-aqueous, suspension, lozenges, capsules, tablets, granules, pills, and powders. Wait. The route of administration of the drug can be injection or oral administration. The injection administration may include intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.
基于质量百分比，所述药物可以包括0.01～99.99％八宝丹或其提取物，以及99.99～0.01％的辅助成分。Based on mass percentage, the medicine may include 0.01-99.99% Babaodan or its extract, and 99.99-0.01% auxiliary components.
所述药物可以包括一种或多种药学上可接受的常规辅料。所述药学上可接受的常规辅料可以为赋形剂、填充剂或稀释剂等。The medicine may include one or more pharmaceutically acceptable conventional excipients. The pharmaceutically acceptable conventional excipients can be excipients, fillers or diluents and the like.
附图说明Description of the drawings
图1显示BALF中IL-6的动态变化。Figure 1 shows the dynamic changes of IL-6 in BALF.
图2显示肺匀浆中炎性因子IL-6的mRNA表达。Figure 2 shows the mRNA expression of the inflammatory factor IL-6 in lung homogenate.
图3显示生存曲线。Figure 3 shows the survival curve.
图4显示肺组织病理切片。Figure 4 shows a pathological section of lung tissue.
图5显示八宝丹对LPS刺激巨噬细胞分泌IL-6的作用。和模型组比较，*P<0.05，**P<0.01，***P<0.001。Figure 5 shows the effect of Babaodan on LPS stimulated macrophages to secrete IL-6. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.
图6显示八宝丹对Pam3CSK4刺激巨噬细胞分泌IL-6的作用。和模型组比较，*P<0.05，**P<0.01，***P<0.001。Figure 6 shows the effect of Babaodan on Pam3CSK4 stimulated macrophages to secrete IL-6. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.
图7显示八宝丹对Poly(I:C)+Pam3CSK4刺激巨噬细胞分泌IL-6的作用。和模型组比较，*P<0.05，**P<0.01，***P<0.001。Figure 7 shows the effect of Babaodan on Poly(I:C)+Pam3CSK4 stimulated macrophages to secrete IL-6. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.
具体实施方式detailed description
在采用流感病毒和金黄色葡萄球菌序贯感染小鼠急性肺损伤模型中发现IL-6炎症因子的表达和释放显著增加，而八宝丹可抑制IL-6炎症因子的表达和释放，显著改善感染小鼠肺部炎症反应；在巨噬细胞Toll样受体(TLRs)活化模型中，不同TLRs受体被激活后会引起细胞因子IL-6的释放显著增加，而八宝丹可显著抑制免疫细胞过度活化所致IL-6炎症因子的分泌。由此，证实了八宝丹具有预防或治疗与IL-6炎症因子风暴相关的疾病的作用。In the acute lung injury model of mice infected with influenza virus and Staphylococcus aureus sequentially, it was found that the expression and release of IL-6 inflammatory factors increased significantly, and Babaodan could inhibit the expression and release of IL-6 inflammatory factors and significantly improved Inflammatory response in the lungs of infected mice; in the activation model of macrophage Toll-like receptors (TLRs), activation of different TLRs receptors will cause a significant increase in the release of cytokine IL-6, and Babaodan can significantly inhibit immunity The secretion of IL-6 inflammatory factors caused by excessive cell activation. Thus, it was confirmed that Babao Pill has the effect of preventing or treating diseases related to IL-6 inflammatory factor storm.
实验药品及配制方法Experimental drugs and preparation methods
八宝丹胶囊性状：胶囊内容物为黄褐色或灰棕褐色的内容物，给药组1批号：150332020，给药组2批号：140103067，提供单位：厦门中药厂有限公司，贮存方法：阴凉、干燥、室温保存。Properties of Babaodan Capsules: The contents of the capsules are yellow-brown or gray-brown, batch number of administration group 1: 150332020, batch number of administration group 2: 140103067, provider: Xiamen Traditional Chinese Medicine Factory Co., Ltd., storage method: cool, Store in a dry place at room temperature.
灌胃用药物配制方法：称取胶囊内容物，加适量MiliQ水溶解至所需浓度(40mg/mL)，冰浴超声30分钟后4℃保存，2小时内使用。Drug preparation method for gavage: Weigh the contents of the capsule, add an appropriate amount of MiliQ water to dissolve it to the desired concentration (40mg/mL), store it at 4°C after 30 minutes of ultrasound in an ice bath, and use it within 2 hours.
细胞实验用药物配制方法：称取八宝丹胶囊内容物，加入适量DMEM细胞培养液超声30分钟，用0.22μM滤膜过滤后，加入10％的灭活FBS和100X青链霉素，加入DMEM补体积，得到2mg/mL八宝丹溶液。4℃保存，24小时内使用。Preparation method of drug for cell experiment: Weigh the contents of Babaodan capsules, add an appropriate amount of DMEM cell culture medium, sonicate for 30 minutes, filter with 0.22μM membrane, add 10% inactivated FBS and 100X penicillin, add DMEM Make up the volume to obtain a 2mg/mL Babaodan solution. Store at 4°C and use within 24 hours.
主要试剂Main reagent
小鼠IL-6 ELISA检测试剂盒(eBioscience)；超纯RNA提取试剂盒(北京康为世纪生物科技有限公司)，RNase-Free DNase Set(QIAGEN)，HiFiScript cDNA第一链合成试剂盒(北京康为世纪生物科技有限公司)，UltraSYBR Mixture(北京康为世纪生物科技有限公司)，引物序列见下表1，引物均由北京擎科新业生物技术有限公司合成。Mouse IL-6 ELISA detection kit (eBioscience); ultrapure RNA extraction kit (Beijing Kangwei Century Biotechnology Co., Ltd.), RNase-Free DNase Set (QIAGEN), HiFiScript cDNA first-strand synthesis kit (Beijing Kang It is Century Biotechnology Co., Ltd.), UltraSYBR Mixture (Beijing Kangwei Century Biotechnology Co., Ltd.), the primer sequences are shown in Table 1 below, and the primers are all synthesized by Beijing Kinco Xinye Biotechnology Co., Ltd.
主要仪器Main instrument
Figure PCTCN2020115246-appb-000001
M1000 PRO多功能酶标仪(瑞士Tecan公司)，Mastercycler gradient PCR仪(Eppendorf公司)，CFX96 TM Touch实时定量PCR***(BIO-RAD公司)，化学发光成像仪(BIO-RAD公司)。
Figure PCTCN2020115246-appb-000001
M1000 PRO multifunctional microplate reader (Switzerland Tecan company), Mastercycler gradient PCR machine (Eppendorf company), CFX96 TM Touch real-time quantitative PCR system (BIO-RAD company), chemiluminescence imager (BIO-RAD company).
[表1][Table 1]
Figure PCTCN2020115246-appb-000002
Figure PCTCN2020115246-appb-000002
数据处理及统计学分析Data processing and statistical analysis
使用GraphPad v6.01软件进行数据处理及分析。数据以Mean±SEM表示，生存率采用Log-rank(Mantel-Cox)检验进行分析，其他均采用方差分析(ANOVA)并进行两两比较，P<0.05为有统计学差异。Use GraphPad v6.01 software for data processing and analysis. The data are expressed as Mean±SEM, the survival rate was analyzed by Log-rank (Mantel-Cox) test, and the others were analyzed by analysis of variance (ANOVA) and pairwise comparisons were performed. P<0.05 was considered statistically different.
实施例1：八宝丹对流感病毒PR8继发SA感染所致急性肺损伤模型中 对IL-6炎症因子的表达和释放的抑制Example 1: Babaodan inhibits the expression and release of IL-6 inflammatory factors in a model of acute lung injury caused by SA infection of influenza virus PR8
实验动物Experimental animal
SPF级C57BL/6小鼠，雌性，6～8周龄，体重16～21g。浙江大学动物实验中心提供，购自上海斯莱克实验动物有限责任公司。实验动物质量合格证：SCXK(沪)2007-0005。实验动物于浙江大学动物中心清洁级饲养，室温20～22℃，光照为12h照明，12h黑暗，给予充足的标准饲料和饮用水。实验操作规程严格遵循浙江大学《实验动物管理条例》。SPF C57BL/6 mice, female, 6-8 weeks old, weighing 16-21g. Provided by Animal Experiment Center of Zhejiang University, purchased from Shanghai Slack Laboratory Animal Co., Ltd. Laboratory animal quality certificate: SCXK (Shanghai) 2007-0005. The experimental animals were bred at a clean level in the Animal Center of Zhejiang University, at a room temperature of 20-22°C, with 12 hours of illumination and 12 hours of darkness, and adequate standard feed and drinking water were provided. The experimental operating procedures strictly follow the "Regulations on Laboratory Animal Management" of Zhejiang University.
实验细菌及病毒Experimental bacteria and viruses
H1N1流感病毒毒株PR8(Puerto-Rico/8)以及金黄色葡萄球菌(SA)由本实验室保存。常规培养、扩增、滴定，保存于-80℃。H1N1 influenza virus strain PR8 (Puerto-Rico/8) and Staphylococcus aureus (SA) are preserved in our laboratory. Routine culture, expansion, titration, and storage at -80°C.
造模及给药Modeling and drug delivery
将小鼠随机分为PR8+SA模型组、八宝丹给药组1(BBD TR)、八宝丹给药组2(BBD RG)以及空白对照组(生存率实验n＝8～16只/组，其余每项检测分别n＝3～5只/组)。The mice were randomly divided into PR8+SA model group, Babaodan administration group 1 (BBD TR), Babaodan administration group 2 (BBD RG), and blank control group (survival rate experiment n=8-16 mice/ Group, each of the remaining tests was n=3~5 per group).
以异戊巴比妥钠腹腔注射麻醉后，以PR8感染PR8+SA模型组、BBD TR组、BBD RG组小鼠，感染方式为滴鼻感染(1×10 4PFU/mL，20μL)。感染完成后，观察其恢复情况。感染病毒当天为第0天。感染病毒后第5天进行SA感染。BBD TR组、BBD RG组于SA感染前2小时按剂量1.0g生药/kg灌胃给药(约0.4～0.5mL药液)。对照组(Control)和模型组(Model)按照体重给予相应体积的溶剂(MiliQ水)。以异戊巴比妥钠腹腔注射进行麻醉后，模型组、BBD TR组、BBD RG组小鼠经喉镜引导气管滴注金黄色葡萄球菌悬液50μL进行感染。生存率和肺病理检查实验菌量为5×10 7CFU，其他为2.5×10 7CFU。 After anesthesia by intraperitoneal injection of isoamylbarbital sodium, the PR8+SA model group, BBD TR group, and BBD RG group mice were infected with PR8. The infection method was intranasal infection (1×10 4 PFU/mL, 20 μL). After the infection is complete, observe its recovery. The day of infection is day 0. SA infection was performed on the 5th day after infection. The BBD TR group and BBD RG group were administered intragastrically at a dose of 1.0 g crude drug/kg (approximately 0.4 to 0.5 mL of liquid medicine) 2 hours before SA infection. The control group (Control) and model group (Model) were given corresponding volumes of solvent (MiliQ water) according to body weight. After intraperitoneal injection of sodium isoamylbarbital for anesthesia, mice in the model group, BBD TR group, and BBD RG group were infected by instilling 50 μL of Staphylococcus aureus suspension through the trachea guided by a laryngoscope. The survival rate and lung pathological examination test bacteria count was 5×10 7 CFU, and the others were 2.5×10 7 CFU.
1.1、肺泡灌洗液(BALF)中炎症因子的表达1.1. Expression of inflammatory factors in alveolar lavage fluid (BALF)
SA感染12h、24h后，戊巴比妥钠(1.5％)腹腔注射麻醉小鼠，打开小鼠胸腔，暴露气管及肺。在会厌软骨处做一T形切口，***套管固定，使用1mL注射器对小鼠全肺进行支气管肺泡灌洗术，每次抽取0.8mL PBS，反复抽注2次，回收率在80％以上，约1.6mL。得到的肺泡灌洗液立即置于冰 上，4℃1000rpm离心5分钟，转移上清至干净的EP管中，分装后立即于-80℃保存，用于炎性因子检测。然后取肺泡灌洗液(BALF)按照ELISA试剂盒操作说明书检测各样本中的IL-6细胞因子。After SA infection for 12h and 24h, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (1.5%), and the mouse's chest cavity was opened to expose the trachea and lungs. Make a T-shaped incision at the epiglottis cartilage, insert a cannula to fix it, use a 1mL syringe to perform bronchoalveolar lavage on the mouse’s whole lung, withdraw 0.8mL PBS each time, and repeat the injection twice. The recovery rate is above 80%. About 1.6mL. The obtained alveolar lavage fluid was immediately placed on ice, centrifuged at 1000 rpm at 4°C for 5 minutes, and the supernatant was transferred to a clean EP tube. After aliquoting, it was immediately stored at -80°C for inflammatory factor detection. Then take the alveolar lavage fluid (BALF) according to the ELISA kit operating instructions to detect the IL-6 cytokines in each sample.
结果：result:
与正常对照组相比，PR8+SA模型组12h和24h BALF中IL-6浓度显著增加(P<0.05)，表明在PR8+SA模型中IL-6炎症因子的表达和释放显著增加，而八宝丹干预组炎症因子IL-6呈显著下调趋势(P<0.05)(图1)，表明八宝丹可抑制IL-6炎症因子的表达和释放。如图1所示，动态变化检测结果显示SA感染后2h细胞因子即出现升高，IL-6在SA感染后6h和12h的水平达到顶峰。八宝丹显著地抑制6h和12h IL-6的表达(P<0.05)。Compared with the normal control group, the IL-6 concentration in BALF increased significantly at 12h and 24h in the PR8+SA model group (P<0.05), indicating that the expression and release of IL-6 inflammatory factors in the PR8+SA model increased significantly. The inflammatory factor IL-6 in the Baodan intervention group was significantly down-regulated (P<0.05) (Figure 1), indicating that Babaodan can inhibit the expression and release of IL-6 inflammatory factors. As shown in Figure 1, the dynamic change detection results showed that cytokines increased at 2h after SA infection, and IL-6 levels reached their peaks at 6h and 12h after SA infection. Babaodan significantly inhibited the expression of IL-6 at 6h and 12h (P<0.05).
1.2、肺组织中炎症因子mRNA的表达1.2. Expression of inflammatory factors mRNA in lung tissue
SA感染12h、24h后，颈椎脱臼处死小鼠。打开胸腔，取全肺置于含1.0mL PBS的匀浆管中，匀浆(Tissuelyser-48全自动样品快速研磨仪)，匀浆液体积记为1.0mL。取100μL置于含0.9mL裂解液TRIzol(北京康为世纪生物科技有限公司)的EP管中用于RNA提取，然后，以RT-qPCR检测细胞因子mRNA的表达水平。具体而言，按照超纯RNA提取试剂盒(北京康为世纪生物科技有限公司)操作说明提取RNA，经DNase处理后，用NanoDrop 2000测定RNA浓度，取2μg RNA按照HiFiScript cDNA第一链合成试剂盒(北京康为世纪生物科技有限公司)的操作步骤逆转录得cDNA。RNA于-80℃保存，cDNA于4℃短期保存。使用UltraSYBR Mixture(北京康为世纪生物科技有限公司)试剂盒进行荧光定量PCR。引物序列见上表1。荧光定量PCR基线采用Bio-Rad公司CFX manager V3.0软件的自动设定，以β-actin为内参基因并用2 -△△CT法计算基因相对表达量。 After SA infection for 12h and 24h, the mice were sacrificed by cervical dislocation. Open the chest cavity, take the whole lung and place it in a homogenization tube containing 1.0mL PBS, homogenize (Tissuelyser-48 automatic sample rapid grinder), and record the volume of the homogenate as 1.0mL. Take 100 μL and place it in an EP tube containing 0.9 mL of lysis solution TRIzol (Beijing Kangwei Century Biotechnology Co., Ltd.) for RNA extraction, and then use RT-qPCR to detect the expression level of cytokine mRNA. Specifically, the RNA was extracted according to the instructions of the ultra-pure RNA extraction kit (Beijing Kangwei Century Biotechnology Co., Ltd.), after DNase treatment, the RNA concentration was measured with NanoDrop 2000, and 2 μg RNA was taken according to the HiFiScript cDNA first-strand synthesis kit (Beijing Kangwei Century Biotechnology Co., Ltd.) Reverse transcription to obtain cDNA. RNA is stored at -80°C, and cDNA is stored at 4°C for short-term storage. Use UltraSYBR Mixture (Beijing Kangwei Century Biotechnology Co., Ltd.) kit for fluorescent quantitative PCR. The primer sequences are shown in Table 1 above. The fluorescence quantitative PCR baseline was automatically set by Bio-Rad's CFX manager V3.0 software, β-actin was used as the internal reference gene and the relative gene expression was calculated by the 2-△△CT method.
结果：result:
与空白对照组相比，PR8+SA模型组12h和24h肺组织匀浆中IL-6的表达显著增加(P<0.05)，表明在PR8+SA模型中IL-6炎症因子的表达和释放显著增加，而BBD TR组炎症因子IL-6呈下调趋势(P>0.05)(图2)，表明八宝丹可抑制IL-6炎症因子的表达和释放。Compared with the blank control group, the expression of IL-6 in the 12h and 24h lung tissue homogenates of the PR8+SA model group was significantly increased (P<0.05), indicating that the expression and release of IL-6 inflammatory factors in the PR8+SA model were significant However, the inflammatory factor IL-6 in the BBD TR group was down-regulated (P>0.05) (Figure 2), indicating that Babaodan can inhibit the expression and release of IL-6 inflammatory factors.
上述八宝丹抑制IL-6炎症因子的表达和释放而用于治疗与IL-6炎症因 子风暴相关的疾病的效果可以通过对于小鼠的一般及存活情况观察和肺病理检查而证实。The above-mentioned Babaodan inhibits the expression and release of IL-6 inflammatory factors for the treatment of diseases related to IL-6 inflammatory factor storms, which can be confirmed by observation of general and survival conditions of mice and lung pathological examination.
1.3、一般及存活情况观察1.3 Observation of general and survival conditions
SA感染后2h内苏醒的小鼠被纳入一般及存活情况观察。一般情况观察指标包括：活动度、精神状态、呼吸情况、对外界刺激反应等。存活情况观察频率为：在感染24h内，每隔2或4h记录1次，此后每隔6或12h观察并记录至72h。Mice that awakened within 2 hours after SA infection were included in the general and survival observation. General observation indicators include: activity, mental state, breathing, response to external stimuli, etc. The survival observation frequency is: within 24h of infection, record once every 2 or 4h, then every 6 or 12h and record to 72h.
结果：result:
一般情况观察：模型组小鼠出现严重精神萎靡不振、呼吸急促、对外界刺激反应淡漠等症状，大部分小鼠在感染24h后死亡。与模型组相比，BBD TR及BBD RG组小鼠的一般情况有所改善。General observation: The mice in the model group showed symptoms such as severe malaise, shortness of breath, and indifference to external stimuli. Most of the mice died 24 hours after infection. Compared with the model group, the general condition of mice in the BBD TR and BBD RG groups has improved.
生存率统计结果：与模型组相比，BBD TR及BBD RG组小鼠48和72小时存活率有提高趋势(P>0.05)(图3)。Survival rate statistics: Compared with the model group, the 48-hour and 72-hour survival rate of mice in the BBD TR and BBD RG groups showed an increasing trend (P>0.05) (Figure 3).
1.4、肺病理检查1.4, lung pathological examination
SA感染12h后，颈椎脱臼处死小鼠。打开胸腔，取全肺，观察肺实变和充血情况后立即用多聚甲醛固定，进行常规石蜡包埋、切片及HE染色(赛维尔(谷歌)生物科技有限公司)。显微镜下观察切片，记录肺泡结构完整性、炎症细胞浸润等肺部病理改变情况。Twelve hours after SA infection, the mice were sacrificed by cervical dislocation. The chest cavity was opened, the whole lung was taken, and the lung consolidation and congestion were observed and immediately fixed with paraformaldehyde, followed by routine paraffin embedding, sectioning and HE staining (Sevier (Google) Biotechnology Co., Ltd.). Observe the slices under a microscope, record the structural integrity of alveoli, inflammatory cell infiltration and other lung pathological changes.
结果：result:
大体观察结果显示：模型组小鼠肺表面有血红色病变区、肺叶肿胀且体积增大；与模型组相比，八宝丹能改善肺损伤，肺表面血红色病变区减少、肿胀和充血程度减轻。The general observation results show that the mouse model group has blood red lesions on the surface of the lungs, swelling and increased volume of the lung lobes; compared with the model group, Babao Dan can improve lung injury, reducing the blood red lesions on the lung surface, swelling and congestion. Lighten up.
HE染色显微镜观察结果显示(图4)：模型组肺泡腔部分消失，肺泡壁增厚，伴有大量中性粒细胞浸润；BBD TR组肺组织炎症细胞浸润缓解、部分肺泡结构恢复。The results of HE staining microscope observation showed (Figure 4): the alveolar cavity of the model group partially disappeared, the alveolar wall was thickened, accompanied by a large number of neutrophil infiltration; the lung tissue of the BBD TR group was relieved of inflammatory cell infiltration and some alveolar structure recovered.
上述对于小鼠的一般及存活情况观察和肺病理检查结果表明，八宝丹可以通过抑制IL-6炎症因子的表达和释放而用于预防或治疗与IL-6炎症因子风暴相关的疾病。The above observation of general and survival conditions of mice and lung pathological examination results show that Babaodan can be used to prevent or treat diseases related to IL-6 inflammatory factor storm by inhibiting the expression and release of IL-6 inflammatory factor.
实施例2：基于巨噬细胞TLRs活化模型的八宝丹对IL-6炎症因子的表达和释放的制药效评价研究Example 2: Evaluation of the pharmaceutical efficacy of Babaodan on the expression and release of IL-6 inflammatory factors based on the macrophage TLRs activation model
细胞培养Cell culture
小鼠单核巨噬细胞系Raw 264.7(ATCC TIB-71)，培养液为含有10％灭活FBS及青霉素和链霉素的DMEM高糖培养基，培养条件为37℃、5％CO 2。取对数生长期的细胞用于实验。 Mouse mononuclear macrophage cell line Raw 264.7 (ATCC TIB-71), the culture medium is DMEM high glucose medium containing 10% inactivated FBS, penicillin and streptomycin, and the culture conditions are 37°C and 5% CO 2 . The cells in the logarithmic growth phase were used for experiments.
八宝丹对TLRs活化引起的IL-6炎症因子的表达和释放的作用Effects of Babaodan on the expression and release of IL-6 inflammatory factors caused by the activation of TLRs
TLRs激动剂及工作浓度清单见下表2。The list of TLRs agonists and working concentrations is shown in Table 2 below.
实验方案如下：Raw 264.7细胞以1×10 5/孔种于12孔板中。24h后换含八宝丹的培养液。22h后再次换含药培养液，2h后加入TLRs激动剂，24h后收取细胞上清液。以ELISA检测IL-6。 The experimental protocol is as follows: Raw 264.7 cells are seeded in a 12-well plate at 1×10 5 /well. After 24h, change the culture medium containing Babaodan. After 22h, the culture medium containing the medicine was changed again, TLRs agonist was added after 2h, and the cell supernatant was collected after 24h. IL-6 was detected by ELISA.
[表2]TLRs激动剂及工作浓度[Table 2] TLRs agonists and working concentrations
Figure PCTCN2020115246-appb-000003
Figure PCTCN2020115246-appb-000003
结果如图5、6、7所示。The results are shown in Figures 5, 6, and 7.
如图5所示，LPS(100ng/mL)刺激24h可以显著地引起Raw 264.7细胞分泌IL-6；如图6所示，Pam3CSK4(1μg/mL)刺激24h可以显著地引起Raw 264.7细胞分泌IL-6；如图7所示，Poly(I:C)+Pam3CSK4刺激24h可以显著地引起Raw 264.7细胞分泌IL-6。As shown in Figure 5, LPS (100ng/mL) stimulation for 24h can significantly cause Raw 264.7 cells to secrete IL-6; as shown in Figure 6, Pam3CSK4 (1μg/mL) stimulation for 24h can significantly cause Raw 264.7 cells to secrete IL- 6; As shown in Figure 7, Poly(I:C)+Pam3CSK4 stimulation for 24h can significantly cause Raw 264.7 cells to secrete IL-6.
如图5、6、7所示，八宝丹对LPS、Pam3CSK4和Poly(I:C)+Pam3CSK4刺激引起的IL-6的分泌具有显著的抑制作用，并呈现量效关系。As shown in Figures 5, 6, and 7, Babao Pill has a significant inhibitory effect on the secretion of IL-6 caused by LPS, Pam3CSK4 and Poly(I:C)+Pam3CSK4 stimulation, and presents a dose-effect relationship.
上述结果表明八宝丹对不同TLRs受体被激活后引起细胞因子IL-6的释放具有显著的抑制作用，从而可以用于预防或治疗与IL-6炎症因子风暴相关的疾病。The above results indicate that Babaodan has a significant inhibitory effect on the release of cytokine IL-6 caused by activation of different TLRs receptors, and can be used to prevent or treat diseases related to IL-6 inflammatory factor storm.

Claims (10)

  1. 八宝丹在制备用于预防或治疗与IL-6炎症因子风暴相关的疾病的药物中的用途。Use of Babaodan in preparing medicine for preventing or treating diseases related to IL-6 inflammatory factor storm.
  2. 根据权利要求1所述的用途，其中，所述与IL-6炎症因子风暴相关的疾病为与IL-6上调相关的疾病。The use according to claim 1, wherein the disease related to IL-6 inflammatory factor storm is a disease related to IL-6 up-regulation.
  3. 根据权利要求2所述的用途，其中，所述与IL-6上调相关的疾病包括肿瘤免疫治疗所引起的副作用、感染性疾病、自身免疫性疾病、癌症、精神疾病。The use according to claim 2, wherein the diseases related to the up-regulation of IL-6 include side effects caused by tumor immunotherapy, infectious diseases, autoimmune diseases, cancer, and mental diseases.
  4. 根据权利要求1所述的用途，其中，所述药物为固体、半固体或液体的形式。The use according to claim 1, wherein the medicine is in a solid, semi-solid or liquid form.
  5. 根据权利要求4所述的用途，其中，所述药物的制剂为水溶液、非水溶液、混悬液、锭剂、胶囊剂、片剂、颗粒剂、丸剂或散剂。The use according to claim 4, wherein the pharmaceutical preparation is an aqueous solution, non-aqueous solution, suspension, lozenge, capsule, tablet, granule, pill or powder.
  6. 根据权利要求1所述的用途，其中，所述药物的给药途径为注射给药或口服给药。The use according to claim 1, wherein the route of administration of the drug is injection or oral administration.
  7. 根据权利要求6所述的用途，其中，所述注射给药包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射。The use according to claim 6, wherein the injection administration comprises intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.
  8. 根据权利要求1所述的用途，其中，基于质量百分比，所述药物包括0.01～99.99％八宝丹或其提取物，以及99.99～0.01％的辅助成分。The use according to claim 1, wherein the medicine comprises 0.01-99.99% Babaodan or its extract, and 99.99-0.01% auxiliary components based on mass percentage.
  9. 根据权利要求1所述的用途，其中，所述药物包括一种或多种药学上可接受的常规辅料。The use according to claim 1, wherein the medicine comprises one or more pharmaceutically acceptable conventional excipients.
  10. 根据权利要求9所述的用途，其中，所述药学上可接受的常规辅料为赋形剂、填充剂或稀释剂。The use according to claim 9, wherein the pharmaceutically acceptable conventional excipients are excipients, fillers or diluents.
PCT/CN2020/115246 2019-09-18 2020-09-15 Use of eight-treasure pill in preparation of medicine for preventing or treating diseases related to il-6 inflammatory cytokine storm WO2021052305A1 (en)

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