CN110664990B

CN110664990B - Application of TRPC1 peptide molecule in preparation of medicine for treating inflammation caused by virus infection

Info

Publication number: CN110664990B
Application number: CN201911081938.XA
Authority: CN
Inventors: 马鑫; 毛爱琴; 何冬旭; 蔡燕飞
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-11-07
Filing date: 2019-11-07
Publication date: 2021-03-16
Anticipated expiration: 2039-11-07
Also published as: CN110664990A; WO2021088219A1

Abstract

The invention provides an application of a TRPC1 peptide molecule in preparing a medicament for treating inflammation caused by virus infection, belonging to the technical field of biological medicaments. Through research, the third extracellular domain polypeptide C1E3p of TRPC1 can obviously inhibit the co-localization of TRPC1 and HSV-1gD, so that the degree of entering the HSV-1 into cells is obviously reduced. A mouse basal keratitis (HSK) model is constructed through HSV-1 infection, HSK of a mouse is obviously relieved after the effect of C1E3p, H & E staining also shows that C1E3p can relieve inflammation symptoms of sclera, choroid and retina and retinal detachment, and the survival rate of the mouse is also obviously improved. Therefore, the polypeptide C1E3p has important significance for the research of medicaments for treating inflammations caused by HSV-1 and other virus infections of the same type.

Description

Application of TRPC1 peptide molecule in preparation of medicine for treating inflammation caused by virus infection

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an application of a TRPC1 peptide molecule in preparation of a medicine for treating inflammation caused by virus infection.

Background

Herpes simplex virus type 1 (HSV-1) is a DNA virus with an envelope which threatens human health for a long time. HSV-1 is ubiquitous in nature, and the human body is the most prominent host. The virus has wide infection parts, mainly enters a human body through direct contact or sexual contact of skin and mucosa, can establish lifetime latent infection in trigeminal ganglia of a host, and is easy to relapse. In human, the common symptoms of HSV-1 infection are pharyngitis and tonsillitis, which is still a huge global medical burden nowadays and has a potential significant morbidity in human, however, clinically, no effective medicine or method can completely treat HSV-1 infection or eliminate the latent HSV-1 in human body, only acyclovir and valacyclovir are used as prescription medicines for treating HSV-1 infection, but the efficacy of the medicine is greatly reduced when the virus is mutated or the first treatment is delayed.

HSV-1 infects host cells, initiating recognition and ligation 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 (gD) isOne of the most important components, controlling the adsorption of the viral envelope to the host cell membrane and the penetration of viral DNA, plays a crucial role in HSV-1 infection of host cells. It is now known that gD can interact with membrane receptors for Herpes virus entry mediator (HVEM, Herpes virus entry mediator C, nectin-1) proteins and sulfated polysaccharides that are distributed on the surface of host cell membranes. Previous studies have shown that an important feature of gD in mediating and regulating HSV-1 infection is the activation of calcium ions (Ca) in host cells²⁺) A variety of signal paths are relied upon. The activation of the calcium signal effectively assists the combination of other envelope proteins of HSV-1 and cell membrane receptors, the entry of virus DNA into a host and the later-stage virus-holding cell metabolism to promote the replication of progeny viruses and other multiple infection and replication processes.

TRP channels are a class of transmembrane nonselective cation channels with tetramer structure, and are Ca-resistant²⁺Is selectively permeable, belongs to one of calcium channels, is widely distributed in various cells, is mainly positioned on the surfaces of cell membranes, endoplasmic reticulum and mitochondrial membranes, and participates in the regulation of various vital activities such as vision, hearing, smell, blood pressure regulation, body fluid balance and the like. During the process of outer calcium influx, TRP channel located in the inner cell membrane can be transported to the cell membrane surface, and TRP is activated to open, finally causing Ca²⁺Ions enter the cell from outside the cell. However, our previous studies found that when HSV-1 infects host cell Hep-2, transient receptor potential cation channel subtype C1(TRPC1) can co-localize with HSV-1gD on cell membrane, form complex, promote translocation of TRPC1 from cytoplasm to cell membrane, and found that binding domain of complex is in the third extracellular domain of TRPC1 and named C1E3p, thus promoting entry of HSV-1gD into cell for infection and replication process.

At present, no application of peptide molecule C1E3p of the third ectodomain of TRPC1 in drug research for treating inflammation caused by virus infection is found at home and abroad.

Disclosure of Invention

The invention provides an application of a TRPC1 peptide molecule in preparing a medicament for treating inflammation caused by virus infection, aiming at overcoming the defects of the prior art. According to the invention, researches show that the polypeptide C1E3p can obviously inhibit the co-localization of TRPC1 and HSV-1gD, so that the degree of HSV-1 entering cells is obviously reduced. A mouse basal keratitis (HSK) model is constructed through HSV-1 infection, HSK of a mouse is obviously relieved after C1E3p acts, H & E staining also shows that C1E3p can relieve inflammation symptoms of sclera, choroid and retina and retinal detachment, the survival rate of the mouse is also obviously improved, and the method has important significance for research of medicaments for treating inflammation caused by HSV-1 infection.

The technical scheme of the invention is as follows:

an application of TRPC1 peptide molecule in preparing the medicines for treating the inflammation caused by virus infection is disclosed.

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.

The virus is HSV-1.

A medicine contains an effective component containing TRPC1 peptide molecule C1E3 p.

A pharmaceutical composition, wherein the pharmaceutical composition comprises TRPC1 peptide molecule C1E3 p.

The beneficial technical effects of the invention are as follows:

at present, the application of peptide molecule C1E3p of the third ectodomain of TRPC1 in treating inflammation caused by virus infection is not seen at home and abroad. The invention discovers that the polypeptide C1E3p can obviously inhibit the co-localization of TRPC1 and HSV-1gD, thereby obviously reducing the degree of HSV-1 entering cells. A mouse basal keratitis (HSK) model is constructed through HSV-1 infection, HSK of a mouse is obviously relieved after the effect of C1E3p, H & E staining also shows that C1E3p can relieve inflammation symptoms of sclera, choroid and retina and retinal detachment, and the survival rate of the mouse is also obviously improved. It was shown that C1E3p, which contains the 3 rd extracellular domain of TRPC1, effectively disrupts the TRPC1-gD interaction and inhibits HSV-1 infection.

Drawings

FIG. 1 is a graph showing the results of C1E3p of the present invention in inhibiting co-localization of TRPC1 and HSV-1 gD;

FIG. 2 is a graph showing the results of C1E3p of the present invention effectively blocking the TRPC1-gD interaction and inhibiting HSV-1 infection.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings and examples.

The experimental materials used in the examples are as follows:

human laryngeal carcinoma epithelial cells (HEp-2) were purchased from american type culture collection (ATCC,

CCL-23^TM). HSV-1 virus and its two commonly used in vivo strains, 17DeltaSty (parent strain,17syn +) HSV-1 and HSV-1dLAT371(parent strain, McKrae), were gifted by the Kunming animal institute, China. TRPC1 wild-type (TRPC1 WT) mice were maintained at the animal experimental center of south of the river university. C1E3p is the amino acid sequence short peptide of the third extracellular domain (AA608-616) of TRPC1, which was synthesized by Suzhou Hongxin Biotech, Inc.

An experimental instrument:

leica laser confocal microscope, biological tissue embedding machine, embedding machine freezing table, rotary microtome, chip baking machine and CO₂An incubator.

Example 1C 1E3p significantly inhibited TRPC1 and HSV-1gD co-localization

The experimental method comprises the following steps: TRPC1 short peptide (C1E3p, AA608-616 is called C1E3p) acts on the fluorescent staining of HEp-2 cells: planting HEp-2 cells in a confocal culture dish, dividing the cells into a control group and an experimental group, directly infecting the control group with 0.5MOI HSV-1 when the cells grow to the fusion degree of about 80%, and incubating the control group with HSV-1 and medicine C1E3p (10 mu M) for 30min, then infecting the cells, and performing incubation for 3h at37 ℃; primary antibody for cell fluorescent staining: Anti-TRPC1 rabbitpolyclone antibody and Anti-HSV gD mouse polyclone antibody were mixed in a 5% BSA solution at a dilution ratio of 1: 200 of a carrier; secondary antibody: AF 488Donkey anti Mouse antibody and AF 546Donkey anti Rabbit antibody were mixed in 5% BSA solution at a dilution ratio of 1: 200. fret images were taken under a confocal microscope.

The experimental results are as follows:

a short peptide selected from the amino acid sequence of the third extracellular domain C1E3p of TRPC1 was used to block the interaction between TRPC1 and HSV-1gD, thereby investigating the effect of TRPC1 on HSV-1 infected HEp-2. As shown in the figure 1, when HEp-2 cells are infected after HSV-1 and C1E3p (with the concentration of 10 mu M) are incubated together, Fret results show that the co-localization efficiency of TRPC1 and HSV-1gD is the lowest, which indicates that C1E3p can obviously inhibit the co-localization of TRPC1 and HSV-1gD, and therefore, the degree of HSV-1 entering the cells is obviously reduced.

Example 2C 1E3p was effective in blocking the TRPC1-gD interaction and inhibiting HSV-1 infection

The experimental method comprises the following steps: construction of mouse eye infection virus model: the 20 mice were divided into groups a1, a2, B1, B24, 5 mice each. Wherein A1 and B1 are TRPC1 WT control group, and A2 and B2 are C1E3p treatment group. All mice were anesthetized with 20% urethane solution at a dose of 0.1mL/10g, and the right cornea of the mice was scratched with a syringe needle. Mixing normal saline with 10⁴pfu/mouse HSV-117syn + was incubated for 30min and dropped onto the right cornea of mice in group A1. Mixing 10 μ M of C1E3p and 10⁴pfu/mouse HSV-117syn + was incubated for 30min and dropped onto the right cornea of mice in group A2. Mixing normal saline with 10³pfu/mouse HSV-1McKrae was co-incubated for 30min and dropped onto the right cornea of mice in group B1. Mixing 10 μ M of C1E3p and 10³pfu/mouse HSV-1McKrae was co-incubated for 30min and dropped onto the right cornea of mice in group B2. The medicine has the following functions: dripping 5 μ M C1E3p and physiological saline with the same volume to the right cornea of mice in A2, B2, A1 and B1 groups, respectively, for 2 days and 1 time, wherein the total time is 15 days; changes in the mouse cornea were observed and recorded.

Paraffin section: fixing: HSV-1 infected mice were euthanized by carbon dioxide euthanasia, the infected right eye ball was removed under a surgical microscope, the tissue surrounding the eye ball was cut off with sclera and fixed in 4% paraformaldehyde for 24 hours. And (3) dehydrating: the eyeball is taken out of the fixative and dehydrated at room temperature, and the volume fractions are respectively 50% ethanol 2h → 70% ethanol 1.5h → 80% ethanol 1h → 90% ethanol 1h → 95% ethanol 30min → 100% ethanol 15 min. And (3) transparency: xylene 15min → xylene 15 min. Wax dipping: the transparent tissue is soaked in wax I (56-58 ℃) for 1h → wax II (58-60 ℃) for 1 h. Paraffin embedding: embedding with paraffin of 56-58 deg.C, placing eyeball specimen in horizontal position during embedding, and cooling and solidifying the paraffin block on the freezing table of embedding machine. Slicing and dewaxing: slicing with a rotary slicer, spreading in 42-46 deg.C water bath with a thickness of 4 μm, baking in 60 deg.C oven for 1 hr, and dewaxing to water. Dyeing: conventional H & E staining, hematoxylin staining for 1min, tap water washing for floating color, hydrochloric acid ethanol (volume fraction is 1%) differentiation for 1min, then tap water alkalization for 10min, eosin (concentration is 10g/L) staining for 30s, gradient ethanol dehydration, volume fractions are respectively 70% ethanol 5min → 80% ethanol 5min → 95% ethanol 5min → 100% ethanol 5min, xylene 5min → xylene 5min for transparency, neutral gum sealing piece, observing under microscope, taking picture, and performing statistical analysis.

The experimental results are as follows:

HSV-1 primarily infects the facial area, and HSV-1 infects the eye and causes stromal keratitis (HSK), blepharitis and retinitis. Then, we constructed a mouse eye infection disease model to investigate the effect of TRPC1 on HSV-1 infection effect and treated with C1E3p, and as a result, as shown in fig. 2, when mouse eyeballs were infected with HSV-1, mouse corneas exhibited varying degrees of clouding and eyeballs exhibited edema, i.e., HSK, and at day 5, the mouse eyeballs exhibited HSK statistically, and as a result, it was found that mice treated with C1E3p exhibited lower HSK scores than control mice (fig. a), and most mice treated with C1E3p did not exhibit high HSK after day 15 of examination (fig. B), and it was shown that C1E3p could alleviate inflammatory symptoms at sclera, choroid and retina and retinal detachment by the method of H & E staining (fig. C), and survival rate of mice was also significantly improved (fig. D). Thus, these results all indicate that C1E3p, which contains the 3 rd extracellular domain of TRPC1, is effective in disrupting TRPC1-gD interactions and inhibiting HSV-1 infection. The magnification is 40X in the figure.

SEQUENCE LISTING

<110> university of south of the Yangtze river

Application of TRPC1 peptide molecule in preparing medicine for treating inflammation caused by virus infection

<130> 1

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 9

<212> PRT

<213> C1E3p

<400> 1

Phe Ser Asn Asn Glu Glu Leu Gln Ser

1 5

Claims

1. An application of TRPC1 peptide molecule in preparing medicine for treating inflammation caused by virus infection;

the TRPC1 peptide molecule is a peptide molecule C1E3p of a third extracellular domain of TRPC1, and the amino acid sequence of the peptide molecule C1E3p is shown as Seq ID No. 1;

the virus is HSV-1.