WO2021088219A1 - Application of trpc1 peptide molecule in preparation of drug for treating inflammation caused by viral infection - Google Patents

Application of trpc1 peptide molecule in preparation of drug for treating inflammation caused by viral infection Download PDF

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WO2021088219A1
WO2021088219A1 PCT/CN2019/126575 CN2019126575W WO2021088219A1 WO 2021088219 A1 WO2021088219 A1 WO 2021088219A1 CN 2019126575 W CN2019126575 W CN 2019126575W WO 2021088219 A1 WO2021088219 A1 WO 2021088219A1
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trpc1
hsv
c1e3p
peptide molecule
mouse
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PCT/CN2019/126575
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Chinese (zh)
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马鑫
毛爱琴
何冬旭
蔡燕飞
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江南大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses

Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to the application of a TRPC1 peptide molecule in the preparation of a medicine for treating inflammation caused by viral infection.
  • Herpes simplex virus type 1 (Herpes simplex virus type 1, HSV-1) is an enveloped DNA virus that has long threatened human health. HSV-1 is ubiquitous in nature, and the human body is its main host. The virus has a wide range of infection sites, and it enters the human body mainly through direct contact or sexual contact of the skin and mucous membranes, and can establish a life-long latent infection in the trigeminal ganglion of the host, which is easy to relapse. In humans, the common symptoms of HSV-1 infection are pharyngitis and tonsillitis. It is still a huge global medical burden and has a potentially significant morbidity rate in humans. However, clinically, there is currently no one.
  • Effective drugs or methods can completely treat HSV-1 infection, or remove the latent HSV-1 in the human body. Only acyclovir and valacyclovir are prescription drugs used to treat HSV-1 infection, but their efficacy is in the current Mutations in the virus or delays in the first treatment will greatly decrease.
  • HSV-1 infects host cells, starting from the recognition and connection between glycoproteins on the surface of the viral particle envelope and receptors on the surface of the host cell membrane.
  • glycoprotein D glycoprotein D
  • HVEM herpes virus entry mediator A
  • HVEM Herpes virus entry mediator C
  • nectin-1 the membrane receptor of sulfated polysaccharides distributed on the surface of the host cell membrane.
  • TRP channel is a type of transmembrane non-selective cation channel with a tetramer structure, which is selectively permeable to Ca 2+ . It is a kind of calcium channel, which is widely distributed in many kinds of cells, mainly located in the cell membrane and endoplasmic reticulum. And the surface of mitochondrial membrane, participate in the regulation of a wide range of life activities such as vision, hearing, smell, blood pressure regulation, body fluid balance and so on. In the process of external calcium influx, the TRP channel located in the inner membrane of the cell can be transported to the surface of the cell membrane, and the TRP is activated and opened, which eventually causes Ca 2+ ions to enter the cell from outside the cell.
  • TRPC1 transient receptor potential cation channel subtype C1
  • the present application provides the application of a TRPC1 peptide molecule in the preparation of a medicine for treating inflammation caused by viral infection.
  • the present invention found through research that the polypeptide C1E3p can significantly inhibit TRPC1 and HSV-1
  • the co-localization of gD significantly reduces the degree of HSV-1 entry into the cell.
  • a mouse stromal keratitis (HSK) model was constructed through HSV-1 infection. After C1E3p treatment, the HSK of the mice was significantly reduced. H&E staining also showed that C1E3p can alleviate the inflammatory symptoms in the sclera, choroid, and retina, as well as the detachment of the retina. And the survival rate of mice is also significantly improved, which is of great significance to the research of drugs for the treatment of inflammation caused by HSV-1 infection.
  • TRPC1 peptide molecule An application of a TRPC1 peptide molecule in the preparation of a medicine for treating inflammation caused by viral infection.
  • the TRPC1 peptide molecule is a peptide molecule C1E3p of the third extracellular domain of TRPC1, and the amino acid sequence of the peptide molecule C1E3p is shown in Seq ID No.1.
  • the virus is HSV-1.
  • a medicament contains functional ingredients containing TRPC1 peptide molecule C1E3p.
  • a pharmaceutical composition which contains TRPC1 peptide molecule C1E3p.
  • the polypeptide C1E3p of the third extracellular domain of TRPC1 in the treatment of inflammation caused by viral infections at home and abroad.
  • the present invention found that the polypeptide C1E3p can significantly inhibit TRPC1 and HSV-1
  • the co-localization of gD significantly reduces the degree of HSV-1 entry into the cell.
  • a mouse stromal keratitis (HSK) model was constructed through HSV-1 infection. After C1E3p treatment, the HSK of the mice was significantly reduced. H&E staining also showed that C1E3p can alleviate the inflammatory symptoms in the sclera, choroid, and retina, as well as the detachment of the retina. And the survival rate of mice is also significantly improved. It shows that C1E3p containing the third extracellular domain of TRPC1 can effectively disrupt the interaction of TRPC1-gD and inhibit HSV-1 infection.
  • Figure 1 is a schematic diagram of the result of C1E3p inhibiting the co-localization of TRPC1 and HSV-1 gD of the present invention
  • Fig. 2 is a schematic diagram of the result of C1E3p effectively blocking the interaction of TRPC1-gD and inhibiting HSV-1 infection.
  • HEp-2 Human laryngeal carcinoma epithelial cells
  • ATCC American Type Culture Collection
  • ATCC ATCC®CCL-23 TM
  • the HSV-1 virus and its two commonly used in vivo strains, 17DeltaSty (parent strain, 17syn+) HSV-1 and HSV-1 dLAT371 (parent strain, McKrae) were kindly provided by Kunming Institute of Zoology, China.
  • TRPC1 wild-type (TRPC1 WT) mice were raised from the Animal Experiment Center of Jiangnan University.
  • C1E3p is a short peptide with amino acid sequence of the third extracellular domain (AA608-616) of TRPC1, synthesized by Suzhou Hongxun Biotechnology Co., Ltd.
  • Leica laser confocal microscope Leica laser confocal microscope, biological tissue embedding machine, embedding machine freezing table, rotary microtome, dryer, CO2 incubator.
  • Example 1 C1E3p can significantly inhibit the colocalization of TRPC1 and HSV-1 gD
  • TRPC1 short peptide (C1E3p, AA608-616 referred to as C1E3p) for fluorescent staining of HEp-2 cells: Plant HEp-2 cells in a confocal culture dish, and divide them into a control group and an experimental group. When the confluence is about 80%, the control group is directly infected with 0.5 MOI HSV-1 at 37°C for 3 hours, while the experimental group is incubated with the drug C1E3p (10 ⁇ M) and HSV-1 for 30 minutes, and then the cells are infected at 37°C.
  • a short peptide selected from the amino acid sequence of the third extracellular domain C1E3p of TRPC1 was used to hinder the interaction between TRPC1 and HSV-1 gD, so as to study the effect of TRPC1 on HSV-1 infection of HEp-2.
  • the results are shown in Figure 1.
  • HSV-1 and C1E3p at a concentration of 10 ⁇ M were co-incubated and then infected with HEp-2 cells, the Fret results showed that TRPC1 and HSV-1
  • the co-localization efficiency of gD has reached the lowest, indicating that C1E3p can significantly inhibit the co-localization of TRPC1 and HSV-1 gD, thereby significantly reducing the degree of HSV-1 entering the cell.
  • Example 2 C1E3p can effectively block the interaction of TRPC1-gD and inhibit HSV-1 infection
  • mice were divided into 4 groups A1, A2, B1, and B2, with 5 in each group.
  • A1 and B1 are TRPC1 WT control groups
  • A2 and B2 are C1E3p treatment groups. All mice were anesthetized with 20% urethane solution, the anesthesia dose was 0.1 mL/10g, and the right cornea of the mouse was scratched with a syringe needle.
  • the normal saline was incubated with 10 4 pfu/mouse HSV-1 17syn+ for 30 min, and dropped into the right cornea of the A1 group of mice.
  • Drug effect 5 ⁇ M C1E3p and the same volume of normal saline were instilled into the right cornea of mice in groups A2, B2, A1, and B1, once every 2 days, for a total of 15 days; observe and record the changes in the mouse cornea .
  • Paraffin section Fixation: The mice infected with HSV-1 were sacrificed by carbon dioxide euthanasia. The infected right eyeball was taken out under an operating microscope, and the surrounding tissues of the eyeball were cut off with scleral scissors and fixed in 4% paraformaldehyde. Medium, the time is 24 h.
  • Dehydration Take out the eyeballs from the fixative and dehydrate at room temperature. The volume fractions are 50% ethanol for 2 h ⁇ 70% ethanol for 1.5 h ⁇ 80% ethanol for 1 h ⁇ 90% ethanol for 1 h ⁇ 95% ethanol for 30 min ⁇ 95% ethanol for 30 min ⁇ 100% ethanol for 15 min ⁇ 100% ethanol for 15 min.
  • Transparent xylene 15 min ⁇ xylene 15 min.
  • Wax immersion the transparent tissue is immersed in wax I (56-58°C) for 1 h ⁇ wax II (58-60°C) is immersed for 1 hour.
  • Paraffin embedding Embed with paraffin at 56-58°C. Place the eyeball specimen at the level of embedding. After the wax block is fully cooled and solidified on the freezing table of the embedding machine, prepare for sectioning. Sections dewaxing: use a rotary microtome to slice, 4 ⁇ m thick, 42°C-46°C water bath, bake the slices in a 60°C incubator for 1 h, and dewax to water.
  • Staining conventional H&E staining, hematoxylin staining for 1 min, tap water to remove floating color, hydrochloric acid ethanol (volume fraction of 1%) for 1 min, then alkalization with tap water for 10 min, eosin (concentration of 10 g/L) staining for 30 s, gradient ethanol dehydration, the volume fractions were 70% ethanol 5 min ⁇ 80% ethanol 5 min ⁇ 95% ethanol 5 min ⁇ 100% ethanol 5 min ⁇ 100% ethanol 5 min, xylene 5 min ⁇ xylene 5 min Transparent, neutral gum mount, observation under a microscope, taking pictures, statistical analysis.
  • HSV-1 mainly infects the facial area. HSV-1 infects the eyes and can cause stromal keratitis (Herpes simplex heratitis, HSK), blepharitis and retinitis. Therefore, we constructed a mouse eye infection disease model to study the effect of TRPC1 on HSV-1 infection and treated it with C1E3p. The results are shown in Figure 2.
  • HSK simplex heratitis
  • TRPC1 peptide molecule Application of a TRPC1 peptide molecule in the preparation of drugs for the treatment of inflammation caused by viral infections

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Abstract

Provided is an application of a TRPC1 peptide molecule in preparation of a drug for treating inflammation caused by viral infection, relating to the technical field of biomedicine. Studies show that a third extracellular domain polypeptide C1E3p of TRPC1 can significantly inhibit co-localization of RPC1 and HSV-1gD, and therefore, the degree of HSV-1 entering cells can be reduced significantly. The herpetic stromal keratitis (HSK) of a mouse can be reduced obviously after the mouse is treated by C1E3p in a mouse HSK model constructed by HSV-1 infection. H&E dyeing also shows that C1E3p can relieve symptoms of inflammation in sclera, choroid, and retina and detachment of retina, and the survival rate of the mouse is also improved significantly.

Description

一种TRPC1肽类分子在制备治疗病毒感染引起的炎症的药物中的应用Application of a TRPC1 peptide molecule in the preparation of a medicine for treating inflammation caused by viral infection 技术领域Technical field
本发明属于生物药物技术领域，尤其是涉及一种TRPC1肽类分子在制备治疗病毒感染引起的炎症的药物中的应用。The invention belongs to the technical field of biomedicine, and in particular relates to the application of a TRPC1 peptide molecule in the preparation of a medicine for treating inflammation caused by viral infection.
背景技术Background technique
单纯疱疹病毒Ⅰ型（Herpes simplex virus type 1, HSV-1）是长期威胁人类健康的一种具有包膜的DNA病毒。HSV-1在自然界普遍存在，人体是其最主要的宿主。该病毒感染部位广泛，主要通过皮肤、粘膜的直接接触或性接触进入人体，并可在宿主的三叉神经节建立终生潜伏感染，容易复发。在人类中，HSV-1感染的常见症状是咽炎，扁桃体炎，现今它仍然是一个巨大的全球医疗负担，并在人类中具有潜在的显著发病率，然而，在临床上，目前并没有一种有效的药品或方法可以彻底治疗HSV-1感染，或清除HSV-1在人体内的潜伏，只有阿昔洛韦和伐昔洛韦作为处方药用来治疗HSV-1感染，但其药效在当病毒发生突变或首次治疗受到拖延时，都将大幅度下降。Herpes simplex virus type 1 (Herpes simplex virus type 1, HSV-1) is an enveloped DNA virus that has long threatened human health. HSV-1 is ubiquitous in nature, and the human body is its main host. The virus has a wide range of infection sites, and it enters the human body mainly through direct contact or sexual contact of the skin and mucous membranes, and can establish a life-long latent infection in the trigeminal ganglion of the host, which is easy to relapse. In humans, the common symptoms of HSV-1 infection are pharyngitis and tonsillitis. It is still a huge global medical burden and has a potentially significant morbidity rate in humans. However, clinically, there is currently no one. Effective drugs or methods can completely treat HSV-1 infection, or remove the latent HSV-1 in the human body. Only acyclovir and valacyclovir are prescription drugs used to treat HSV-1 infection, but their efficacy is in the current Mutations in the virus or delays in the first treatment will greatly decrease.
HSV-1感染宿主细胞，起始于病毒颗粒包膜表面的糖蛋白与宿主细胞膜表面的受体之间的识别和连接。而在所有的HSV-1包膜糖蛋白之中，糖蛋白D(glycoprotein D, gD)是最重要的组成者之一，控制病毒包膜对宿主细胞膜的吸附和病毒DNA的穿膜，对HSV-1感染宿主细胞起到决定性的作用。目前已知gD可以与分布于宿主细胞膜表面的疱疹病毒进入介质(Herpes virus entry mediator A , HVEM、Herpes virus entry mediator C , nectin-1)蛋白及硫酸酯化多糖的膜受体相互作用。以往研究显示，gD介导和调控HSV-1感染的一个重要特征，就是激活宿主细胞的钙离子（Ca 2+）依赖的多种信号通路。该钙信号的活化，有效地辅助了HSV-1其它包膜蛋白与细胞膜受体结合、病毒DNA进入宿主及后期病毒挟持细胞代谢以促进子代病毒复制等多个感染、复制的过程。 HSV-1 infects host cells, starting from the recognition and connection between glycoproteins on the surface of the viral particle envelope and receptors on the surface of the host cell membrane. Among all HSV-1 envelope glycoproteins, glycoprotein D (glycoprotein D, gD) is one of the most important constituents. It controls the adsorption of the virus envelope to the host cell membrane and the penetration of viral DNA. -1 infection of host cells plays a decisive role. It is currently known that gD can interact with the herpes virus entry mediator A (Herpes virus entry mediator A, HVEM, Herpes virus entry mediator C, nectin-1) protein and the membrane receptor of sulfated polysaccharides distributed on the surface of the host cell membrane. Previous studies have shown that an important feature of gD-mediated and regulated HSV-1 infection is the activation of multiple signal pathways dependent on calcium ions (Ca 2+) in host cells. The activation of the calcium signal effectively assists the binding of other envelope proteins of HSV-1 with cell membrane receptors, the entry of viral DNA into the host, and the later virus holding cell metabolism to promote the replication of progeny viruses and other processes of infection and replication.
技术问题technical problem
TRP通道是一类具有四聚体结构的跨膜非选择性阳离子通道，对Ca 2+选择性通透，属于钙通道的一种，广泛分布于多种细胞内，主要位于细胞膜、内质网及线粒体膜表面，参与如视觉、听觉、嗅觉、血压调节、体液平衡等种类繁多的生命活动的调节。在外钙内流的过程中，位于细胞内膜的TRP通道可转运到细胞膜表面，TRP被激活开放，最终引起Ca 2+离子从细胞外进入细胞。而我们的前期研究发现，当HSV-1感染宿主细胞Hep-2时，瞬时感受器电位阳离子通道亚型C1（TRPC1）能够与HSV-1 gD在细胞膜上发生共定位，形成复合体，促进TRPC1从细胞质易位至细胞膜，并且发现复合体的结合域在TRPC1的第三胞外结构域，并将其命名为C1E3p，从而促进HSV-1 gD进入细胞进行感染、复制过程。 TRP channel is a type of transmembrane non-selective cation channel with a tetramer structure, which is selectively permeable to Ca 2+ . It is a kind of calcium channel, which is widely distributed in many kinds of cells, mainly located in the cell membrane and endoplasmic reticulum. And the surface of mitochondrial membrane, participate in the regulation of a wide range of life activities such as vision, hearing, smell, blood pressure regulation, body fluid balance and so on. In the process of external calcium influx, the TRP channel located in the inner membrane of the cell can be transported to the surface of the cell membrane, and the TRP is activated and opened, which eventually causes Ca 2+ ions to enter the cell from outside the cell. Our previous studies have found that when HSV-1 infects host cells Hep-2, transient receptor potential cation channel subtype C1 (TRPC1) can co-localize with HSV-1 gD on the cell membrane to form a complex and promote TRPC1 from The cytoplasm is translocated to the cell membrane, and the binding domain of the complex is found in the third extracellular domain of TRPC1, which is named C1E3p, which promotes HSV-1 gD to enter the cell for infection and replication.
目前，国内外未见以TRPC1的第三胞外域的肽类分子C1E3p在治疗病毒感染引起的炎症中药物研究的应用。At present, there is no application of the peptide molecule C1E3p of the third extracellular domain of TRPC1 in the treatment of inflammation caused by viral infections at home and abroad.
技术解决方案Technical solutions
本申请针对现有技术的不足，本发明提供了一种TRPC1肽类分子在制备治疗病毒感染引起的炎症的药物中的应用。本发明通过研究发现，多肽C1E3p能够显著抑制TRPC1和HSV-1 gD的共定位，从而使HSV-1进入细胞的程度显著降低。通过HSV-1感染构建小鼠基质性角膜炎（HSK）模型，用C1E3p作用后小鼠的HSK明显减轻，H&E染色也表明C1E3p能够缓解在巩膜、脉络膜和视网膜的炎症症状以及视网膜的脱落情况，并且小鼠的生存率也显著提高，对治疗HSV-1感染引起的炎症的药物研究具有重要意义。In view of the shortcomings of the prior art, the present application provides the application of a TRPC1 peptide molecule in the preparation of a medicine for treating inflammation caused by viral infection. The present invention found through research that the polypeptide C1E3p can significantly inhibit TRPC1 and HSV-1 The co-localization of gD significantly reduces the degree of HSV-1 entry into the cell. A mouse stromal keratitis (HSK) model was constructed through HSV-1 infection. After C1E3p treatment, the HSK of the mice was significantly reduced. H&E staining also showed that C1E3p can alleviate the inflammatory symptoms in the sclera, choroid, and retina, as well as the detachment of the retina. And the survival rate of mice is also significantly improved, which is of great significance to the research of drugs for the treatment of inflammation caused by HSV-1 infection.
本发明的技术方案如下：The technical scheme of the present invention is as follows:
一种TRPC1肽类分子在制备治疗病毒感染引起的炎症的药物中的应用。An application of a TRPC1 peptide molecule in the preparation of a medicine for treating inflammation caused by viral infection.
所述TRPC1肽类分子为TRPC1的第三胞外域的肽类分子C1E3p，所述肽类分子C1E3p的氨基酸序列如Seq IDNo.1所示。The TRPC1 peptide molecule is a peptide molecule C1E3p of the third extracellular domain of TRPC1, and the amino acid sequence of the peptide molecule C1E3p is shown in Seq ID No.1.
所述病毒为HSV-1。The virus is HSV-1.
一种药物，所述药物含有功效成分含有TRPC1肽类分子C1E3p。A medicament, the medicament contains functional ingredients containing TRPC1 peptide molecule C1E3p.
一种药物组合物，所述药物中含有TRPC1肽类分子C1E3p。A pharmaceutical composition, which contains TRPC1 peptide molecule C1E3p.
有益效果Beneficial effect
本发明有益的技术效果在于：The beneficial technical effects of the present invention are:
目前，国内外未见以TRPC1的第三胞外域的肽类分子C1E3p在治疗病毒感染引起的炎症中的应用。本发明发现，多肽C1E3p能够显著抑制TRPC1和HSV-1 gD的共定位，从而使HSV-1进入细胞的程度显著降低。通过HSV-1感染构建小鼠基质性角膜炎（HSK）模型，用C1E3p作用后小鼠的HSK明显减轻，H&E染色也表明C1E3p能够缓解在巩膜、脉络膜和视网膜的炎症症状以及视网膜的脱落情况，并且小鼠的生存率也显著提高。表明含有TRPC1的第3胞外域的C1E3p能有效扰乱TRPC1-gD的相互作用并抑制HSV-1感染。At present, there is no application of the peptide molecule C1E3p of the third extracellular domain of TRPC1 in the treatment of inflammation caused by viral infections at home and abroad. The present invention found that the polypeptide C1E3p can significantly inhibit TRPC1 and HSV-1 The co-localization of gD significantly reduces the degree of HSV-1 entry into the cell. A mouse stromal keratitis (HSK) model was constructed through HSV-1 infection. After C1E3p treatment, the HSK of the mice was significantly reduced. H&E staining also showed that C1E3p can alleviate the inflammatory symptoms in the sclera, choroid, and retina, as well as the detachment of the retina. And the survival rate of mice is also significantly improved. It shows that C1E3p containing the third extracellular domain of TRPC1 can effectively disrupt the interaction of TRPC1-gD and inhibit HSV-1 infection.
附图说明Description of the drawings
图1为本发明C1E3p抑制TRPC1和HSV-1 gD共定位的结果示意图；Figure 1 is a schematic diagram of the result of C1E3p inhibiting the co-localization of TRPC1 and HSV-1 gD of the present invention;
图2为本发明C1E3p有效阻断TRPC1-gD的相互作用并抑制HSV-1感染的结果示意图。Fig. 2 is a schematic diagram of the result of C1E3p effectively blocking the interaction of TRPC1-gD and inhibiting HSV-1 infection.
本发明的实施方式Embodiments of the present invention
下面结合附图和实施例，对本发明进行具体描述。In the following, the present invention will be described in detail with reference to the drawings and embodiments.
实施例所用实验材料如下：The experimental materials used in the examples are as follows:
人喉癌上皮细胞（HEp-2）购自美国菌种保藏中心（ATCC，ATCC®CCL-23 TM）。HSV-1病毒及其两种常用在体毒株17DeltaSty (parent strain, 17syn+)HSV-1 和HSV-1 dLAT371 (parent strain, McKrae)由中国昆明动物研究所惠赠。TRPC1野生型（TRPC1 WT）小鼠养自江南大学动物实验中心。C1E3p是TRPC1的第三胞外结构域（AA608-616）氨基酸序列短肽，是由苏州泓迅生物科技有限公司合成所得。 Human laryngeal carcinoma epithelial cells (HEp-2) were purchased from the American Type Culture Collection (ATCC, ATCC®CCL-23 TM ). The HSV-1 virus and its two commonly used in vivo strains, 17DeltaSty (parent strain, 17syn+) HSV-1 and HSV-1 dLAT371 (parent strain, McKrae), were kindly provided by Kunming Institute of Zoology, China. TRPC1 wild-type (TRPC1 WT) mice were raised from the Animal Experiment Center of Jiangnan University. C1E3p is a short peptide with amino acid sequence of the third extracellular domain (AA608-616) of TRPC1, synthesized by Suzhou Hongxun Biotechnology Co., Ltd.
实验仪器：laboratory apparatus:
Leica激光共聚焦显微镜，生物组织包埋机，包埋机冷冻台，轮转式切片机，烘片机，CO2培养箱。Leica laser confocal microscope, biological tissue embedding machine, embedding machine freezing table, rotary microtome, dryer, CO2 incubator.
实施例1 C1E3p可显著抑制TRPC1和HSV-1 gD共定位Example 1 C1E3p can significantly inhibit the colocalization of TRPC1 and HSV-1 gD
实验方法：TRPC1短肽（C1E3p，AA608-616称之为C1E3p）作用于HEp-2细胞的荧光染色：将HEp-2细胞种于共聚焦培养皿中，分为对照组和实验组，待细胞长至融合度为80%左右时，对照组直接感染0.5 MOI HSV-1，37℃，3 h，实验组则将药物C1E3p（10μM）与HSV-1先共同孵育30min，再感染细胞，37℃，3 h；细胞荧光染色所用一抗：Anti-TRPC1 rabbit polyclonal antibody和Anti-HSV gD mouse polyclonal antibody混合于5% BSA溶液中，稀释比例为1：200；二抗：AF 488 Donkey anti Mouse antibody和AF 546 Donkey anti Rabbit antibody混合于5% BSA溶液中，稀释比例为1：200。在共聚焦显微镜下进行Fret拍摄。Experimental method: TRPC1 short peptide (C1E3p, AA608-616 referred to as C1E3p) for fluorescent staining of HEp-2 cells: Plant HEp-2 cells in a confocal culture dish, and divide them into a control group and an experimental group. When the confluence is about 80%, the control group is directly infected with 0.5 MOI HSV-1 at 37°C for 3 hours, while the experimental group is incubated with the drug C1E3p (10μM) and HSV-1 for 30 minutes, and then the cells are infected at 37°C. , 3 h; Primary antibody used for cell fluorescence staining: Anti-TRPC1 rabbit The polyclonal antibody and Anti-HSV gD mouse polyclonal antibody are mixed in 5% BSA solution at a dilution ratio of 1:200; secondary antibody: AF 488 Donkey anti Mouse antibody and AF 546 Donkey anti Rabbit antibody is mixed in 5% BSA solution at a dilution ratio of 1:200. Fret shooting under a confocal microscope.
实验结果：Experimental results:
将选自TRPC1的第三胞外结构域C1E3p的氨基酸序列的短肽来阻碍TRPC1与HSV-1 gD之间的相互作用，从而研究TRPC1对HSV-1感染HEp-2的影响。结果如图1所示，当HSV-1与C1E3p（浓度为10μM）共孵育后再感染HEp-2细胞时， Fret结果显示TRPC1与HSV-1 gD的共定位效率已达到最低，说明C1E3p能够显著抑制TRPC1和HSV-1 gD的共定位，从而使HSV-1进入细胞的程度显著降低。A short peptide selected from the amino acid sequence of the third extracellular domain C1E3p of TRPC1 was used to hinder the interaction between TRPC1 and HSV-1 gD, so as to study the effect of TRPC1 on HSV-1 infection of HEp-2. The results are shown in Figure 1. When HSV-1 and C1E3p (at a concentration of 10μM) were co-incubated and then infected with HEp-2 cells, the Fret results showed that TRPC1 and HSV-1 The co-localization efficiency of gD has reached the lowest, indicating that C1E3p can significantly inhibit the co-localization of TRPC1 and HSV-1 gD, thereby significantly reducing the degree of HSV-1 entering the cell.
实施例2 C1E3p能有效阻断TRPC1-gD的相互作用并抑制HSV-1感染Example 2 C1E3p can effectively block the interaction of TRPC1-gD and inhibit HSV-1 infection
实验方法：小鼠眼部感染病毒模型的构建：将20只小鼠分为A1、A2、B1、B2 4组，每组5只。其中A1、B1为TRPC1 WT对照组，A2、B2为C1E3p处理组。所有小鼠均用20%的乌拉坦溶液将小鼠麻醉，麻醉剂量为0.1 mL/10g，用注射器针头在小鼠的右角膜上划痕。将生理盐水与10 4 pfu/mouse的HSV-1 17syn+共孵育30 min，滴入A1组小鼠的右角膜处。将10 μM的C1E3p与10 4 pfu/mouse的HSV-1 17syn+共孵育30 min，滴入A2组小鼠的右角膜处。将生理盐水与10 3 pfu/mouse的HSV-1 McKrae共孵育30 min，滴入B1组小鼠的右角膜处。将10 μM的C1E3p与10 3 pfu/mouse的HSV-1 McKrae共孵育30 min，滴入B2组小鼠的右角膜处。药物作用：将5 μM 的C1E3p和同体积的生理盐水分别滴入至A2、B2和A1、B1组小鼠右角膜处，2天1次，时间共为15天；观察记录小鼠角膜的变化。 Experimental method: Construction of mouse eye infection virus model: 20 mice were divided into 4 groups A1, A2, B1, and B2, with 5 in each group. Among them, A1 and B1 are TRPC1 WT control groups, and A2 and B2 are C1E3p treatment groups. All mice were anesthetized with 20% urethane solution, the anesthesia dose was 0.1 mL/10g, and the right cornea of the mouse was scratched with a syringe needle. The normal saline was incubated with 10 4 pfu/mouse HSV-1 17syn+ for 30 min, and dropped into the right cornea of the A1 group of mice. 10 μM C1E3p and 10 4 pfu/mouse HSV-1 17syn+ were incubated for 30 min, and then dropped into the right cornea of mice in group A2. The normal saline was incubated with 10 3 pfu/mouse HSV-1 McKrae for 30 min, and dropped into the right cornea of the B1 group of mice. C1E3p of 10 μM and HSV-1 McKrae of 10 3 pfu/mouse were incubated for 30 min, and dropped into the right cornea of mice in group B2. Drug effect: 5 μM C1E3p and the same volume of normal saline were instilled into the right cornea of mice in groups A2, B2, A1, and B1, once every 2 days, for a total of 15 days; observe and record the changes in the mouse cornea .
石蜡切片：固定：用二氧化碳安乐死法处死感染HSV-1的小鼠，在手术显微镜下取出感染之后的右眼球，将眼球的周围组织用巩膜剪剪去，将之固定在4%的多聚甲醛中，时间为24 h。脱水：从固定液中取出眼球，室温脱水，体积分数分别为50%乙醇2 h→70%乙醇1.5 h→80%乙醇1 h→90%乙醇1 h→95%乙醇30 min→95%乙醇30 min→100%乙醇15 min→100%乙醇15 min。透明：二甲苯15 min→二甲苯15 min。浸蜡：透明后的组织在蜡Ⅰ（56-58℃）浸1 h→蜡Ⅱ（58-60℃）浸1 h。石蜡包埋：用56-58℃的石蜡进行包埋，包埋时置眼球标本水平位，待蜡块放在包埋机冷冻台上充分冷却凝固后，准备切片。切片脱蜡：用轮转式切片机切片，厚4 μm，42℃-46℃水浴展片，在60℃的恒温箱中烤片1 h，脱蜡至水。染色：常规H&E染色，苏木素染色1 min，自来水洗去浮色，盐酸乙醇（体积分数为1%）分化1 min，再用自来水碱化10 min，伊红（浓度为10 g/L）染色30 s，梯度乙醇脱水，体积分数分别为70%乙醇5 min→80%乙醇5 min→95%乙醇5 min→100%乙醇5 min→100%乙醇5 min，二甲苯5 min→二甲苯5 min进行透明，中性树胶封片，显微镜下观察，拍摄图片，统计分析。Paraffin section: Fixation: The mice infected with HSV-1 were sacrificed by carbon dioxide euthanasia. The infected right eyeball was taken out under an operating microscope, and the surrounding tissues of the eyeball were cut off with scleral scissors and fixed in 4% paraformaldehyde. Medium, the time is 24 h. Dehydration: Take out the eyeballs from the fixative and dehydrate at room temperature. The volume fractions are 50% ethanol for 2 h → 70% ethanol for 1.5 h → 80% ethanol for 1 h → 90% ethanol for 1 h → 95% ethanol for 30 min → 95% ethanol for 30 min→100% ethanol for 15 min→100% ethanol for 15 min. Transparent: xylene 15 min→xylene 15 min. Wax immersion: the transparent tissue is immersed in wax I (56-58°C) for 1 h → wax II (58-60°C) is immersed for 1 hour. Paraffin embedding: Embed with paraffin at 56-58℃. Place the eyeball specimen at the level of embedding. After the wax block is fully cooled and solidified on the freezing table of the embedding machine, prepare for sectioning. Sections dewaxing: use a rotary microtome to slice, 4 μm thick, 42°C-46°C water bath, bake the slices in a 60°C incubator for 1 h, and dewax to water. Staining: conventional H&E staining, hematoxylin staining for 1 min, tap water to remove floating color, hydrochloric acid ethanol (volume fraction of 1%) for 1 min, then alkalization with tap water for 10 min, eosin (concentration of 10 g/L) staining for 30 s, gradient ethanol dehydration, the volume fractions were 70% ethanol 5 min→80% ethanol 5 min→95% ethanol 5 min→100% ethanol 5 min→100% ethanol 5 min, xylene 5 min→xylene 5 min Transparent, neutral gum mount, observation under a microscope, taking pictures, statistical analysis.
实验结果：Experimental results:
HSV-1主要感染面部区域，HSV-1感染眼睛会导致基质性角膜炎（Herpes simplex heratitis, HSK），睑缘炎和视网膜炎。于是，我们构建了小鼠眼部感染疾病模型来研究TRPC1对HSV-1感染效果的影响，并用C1E3p处理，结果如图2所示，当小鼠眼球感染HSV-1时，小鼠角膜呈现不同程度的混浊情况并且眼球出现水肿情况，即表现为HSK，统计第5天时，小鼠眼球表现出HSK的情况，结果发现用C1E3p处理的小鼠比对照小鼠的HSK分数相比则偏低（图 A），并且用C1E3p处理的大多数小鼠在检测的第15天后，没有表现出高HSK（图 B），通过H&E染色的方法表明C1E3p能够缓解在巩膜，脉络膜和视网膜的炎症症状以及视网膜的脱落情况（图 C），并且小鼠的生存率也显著提高（图D）。因此，这些结果都表明，含有TRPC1的第3胞外域的C1E3p能有效扰乱TRPC1-gD的相互作用并抑制HSV-1感染。图中放大倍数40X。HSV-1 mainly infects the facial area. HSV-1 infects the eyes and can cause stromal keratitis (Herpes simplex heratitis, HSK), blepharitis and retinitis. Therefore, we constructed a mouse eye infection disease model to study the effect of TRPC1 on HSV-1 infection and treated it with C1E3p. The results are shown in Figure 2. When the mouse eyeball is infected with HSV-1, the mouse cornea appears different Occurrence of turbidity and ocular edema is manifested as HSK. On the fifth day of statistics, the eyes of the mice showed HSK. It was found that the HSK scores of the mice treated with C1E3p were lower than those of the control mice ( Figure A), and most of the mice treated with C1E3p did not show high HSK after the 15th day of the test (Figure B). The H&E staining method showed that C1E3p can alleviate the inflammatory symptoms in the sclera, choroid and retina, as well as the retina The fall off situation (figure C), and the survival rate of mice is also significantly improved (Figure D). Therefore, these results indicate that C1E3p containing the third extracellular domain of TRPC1 can effectively disrupt the interaction of TRPC1-gD and inhibit HSV-1 infection. The magnification in the figure is 40X.
序列表自由内容Sequence Listing Free Content
SEQUENCE LISTINGSEQUENCE LISTING
<110>  江南大学<110> Jiangnan University
<120>  一种TRPC1肽类分子在制备治疗病毒感染引起的炎症的药物中的应用<120> Application of a TRPC1 peptide molecule in the preparation of drugs for the treatment of inflammation caused by viral infections
<130>  1<130> 1
<160>  1     <160> 1
<170>  PatentIn version 3.3<170> PatentIn version 3.3
<210>  1<210> 1
<211>  9<211> 9
<212>  PRT<212> PRT
<213>  C1E3p<213> C1E3p
<400>  1<400> 1
Phe Ser Asn Asn Glu Glu Leu Gln Ser Phe Ser Asn Asn Glu Glu Leu Gln Ser
1               5                   1 5

Claims (5)

  1. 一种TRPC1肽类分子在制备治疗病毒感染引起的炎症的药物中的应用。 An application of a TRPC1 peptide molecule in the preparation of a medicine for treating inflammation caused by viral infection.
  2. 根据权利要求1所述的应用，其特征在于，所述TRPC1肽类分子为TRPC1的第三胞外域的肽类分子C1E3p，所述肽类分子C1E3p的氨基酸序列如Seq IDNo.1所示。 The application according to claim 1, wherein the peptide molecule of TRPC1 is a peptide molecule C1E3p of the third extracellular domain of TRPC1, and the amino acid sequence of the peptide molecule C1E3p is shown in Seq ID No.1.
  3. 根据权利要求1所述的应用，其特征在于，所述病毒为HSV-1。 The application according to claim 1, wherein the virus is HSV-1.
  4. 一种药物，其特征在于，所述药物功效成分含有TRPC1肽类分子C1E3p。 A medicine, which is characterized in that the functional component of the medicine contains TRPC1 peptide molecule C1E3p.
  5. 一种药物组合物，其特征在于，所述药物中含有TRPC1肽类分子C1E3p。 A pharmaceutical composition, characterized in that the medicine contains TRPC1 peptide molecule C1E3p.
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