CN111763706B

CN111763706B - Antiviral method using natural immune activator

Info

Publication number: CN111763706B
Application number: CN202010671915.0A
Authority: CN
Inventors: 周翔; 蔡苗苗; 王柯; 王晨光; 蒋争凡; 张同存
Original assignee: Wuhan University of Science and Engineering WUSE
Current assignee: Wuhan University of Science and Engineering WUSE
Priority date: 2020-07-14
Filing date: 2020-07-14
Publication date: 2023-08-18
Anticipated expiration: 2040-07-14
Also published as: CN111763706A

Abstract

The invention relates to an antiviral method using a small molecular compound chloroquinaldo, which comprises the steps of applying an effective concentration of chloroquinaldo to cells to be treated; the method can effectively activate the I-type interferon signal channel, express and secrete a large amount of interferon and interferon stimulating genes, thereby strongly inhibiting virus infection and replication.

Description

Antiviral method using natural immune activator

Technical Field

The invention relates to an antiviral method using a natural immune activator, in particular to a method for activating a natural immune I-type interferon signal channel by using a small molecular compound with high-efficiency antiviral capability.

Background

Natural immunity is the first line of defense of the body against viral infection. Infection with a virus can activate a natural immune antiviral response. There are a range of pattern recognition receptors (pattern recognition receptors, PRRs) in the natural immune system that recognize pathogen-associated molecular patterns of viral origin (pathen-associated molecular patterns, PAMPs) and recruit specific adaptor proteins to mediate downstream signaling pathways, leading to the expression and secretion of a large number of type I interferons and inflammatory factors. Type I interferons include single interferon beta (IFNbeta) and interferon alpha (IFNalpha) family proteins. The secreted I-type interferon is combined with an interferon receptor positioned on the surface of a cell membrane, activates a downstream JAK-STAT signal channel, starts the expression of a large amount of interferon stimulation genes ISGs, and plays roles in inhibiting the replication, proliferation and transmission of viruses. Thus, induction of type I interferon has a critical role in antiviral innate immune responses.

Many stimulus factors can induce the organism to produce I-type interferon and play an antiviral function. These stimuli include: viral infection, pathogen-associated molecular pattern stimulation of viral origin, overexpression of key protein molecules of the type I interferon signaling pathway, and the like. However, none of these stimuli is applicable to clinical antiviral therapy. Interferon itself has a limited role in that its function is affected by activity and half-life, although it has antiviral function, as a protein. How to find a substance which can activate immune cells to generate I-type interferon and is independent of virus, and at the same time, can meet the requirements of drug activity and stability, and can be effectively applied to clinical treatment of virus infection.

Disclosure of Invention

The invention aims to solve the defects of the prior antiviral clinical therapeutic drugs and provides a method for preparing a drug by applying a small molecular compound chloroquinaldol (C ₁₀ H ₇ C _l2 NO) antiviral methods, which are widely applicable, of controlled safety, strongly activate type I interferon signaling pathways in cells, producing antiviral natural immune responses.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a method of using the small molecule compound chloroquinaldo for antiviral therapy comprising applying an effective concentration of chloroquinaldo to cells to be treated.

Preferably, the final concentration of the chloroquinaldo in the cell culture medium is 20. Mu.M. More preferably, the chloroquinaldo is diluted with sterile DMSO liquid to give 20mM chloroquinaldo working solution according to the working solution: cell culture fluid = 1:1000 (while setting a concentration gradient in this range), chloroquinaldol working solution was added to the cell culture medium so that the final concentration of chloroquinaldol in the cell culture medium was 20. Mu.M (containing a concentration gradient of this concentration), the culture was shaken well, and the culture was allowed to stand at 37℃for 24 hours.

Further, the method comprises the following specific steps:

1. THP-1 cells were cultured (suspended) and brought into a stable exponentially dividing state.

Cell resuscitation: the cells were rapidly removed from the liquid nitrogen tank and then rapidly shaken in a 37℃water bath for about 1-2 minutes with the water surface below the freezing tube lid to prevent water from entering the freezing tube.

Transfer to plate: in an ultra-clean bench, a flat plate is taken, 12ml Gibco RPMI 1640 culture solution is firstly added, the bottom of the plate is fully paved, then cells in a freezing tube are all sucked into the flat plate, 8-shaped shaking is carried out, microscopic observation is carried out, and then culture is carried out at 37 ℃;

cell exchange liquid: after overnight recovery, the culture solution is observed to turn yellow (2-3 days), the solution is changed, the cell sap is firstly sucked into a 15ml centrifuge tube, and is centrifuged for 5min at 900-1000r/min (the rotating speed cannot be more than 1000), the upper culture solution is sucked, a new RPMI 1640 is added, the mixture is uniformly blown, and the whole mixture is sucked into a culture plate for culture at 37 ℃.

And (3) passage: the method adopts a direct passage method: (1) naturally precipitating the suspended cells at the bottom of the bottle; (2) sucking the supernatant by a suction pipe for 1/2 to 2/3; (3) gently beating the cell suspension; (4) aliquots were placed into several flasks (dishes). Note that the density of cells must be high, between 5X 10 ⁵ ～10 ⁶ between/ML, the value-added is fast.

2. HeLa cells (adherent) were cultured and brought into a stable exponentially dividing state.

Transfer to plate: in an ultra-clean bench, a flat plate is taken, 12ml of Gibco DMEM culture solution is firstly added, the bottom of the plate is fully paved, then cells in a freezing tube are all sucked into the flat plate, 8-shaped shaking is carried out uniformly, the condition is observed microscopically, and then the culture is carried out at 37 ℃;

cell exchange liquid: after overnight recovery, the culture medium was observed to turn yellow, the medium was changed at this time, the upper layer of the culture medium was sucked off, new DMEM was added, and the culture was performed at 37 ℃.

And (3) passage: the pancreatin digestion method is adopted: (1) sucking the supernatant with a pipette; (2) adding 1ml of pancreatin and incubating for 2-4min; (3) adding new DMEM culture solution, gently blowing into a cell suspension; (4) according to 1: 4-1: 6 aliquots were placed into several new dishes. Note that the cell density is appropriate, typically for 2-3 days, once.

3. Mouse peritoneal macrophages (adherent) were collected for antiviral experiments.

BALBc mice were intraperitoneally injected with 5ml of 4℃pre-chilled PBS. Gentle mouse abdomen 2-3min, standing for 5-7min, killing cervical dislocation, placing on dissecting plate, opening abdominal cavity under aseptic condition, extracting abdominal cavity liquid with syringe, centrifuging for 5min (1000 rpm/min), discarding supernatant, and washing with PBS for 2 times. Then the mixture was adjusted to 2X 10 with RPMI-1640 medium (0.1% double antibody solution) containing 10% fetal bovine serum ⁶ Inoculating/ml into 6-well plate, placing at 37deg.C, 5% CO ₂ Culturing in an incubator.

4. A chloroquinaldo working solution is prepared and applied to the cells to be treated.

Preparing a working solution: according to the 10-fold dilution method, the chloroquinaldo stock solution is diluted with sterile DMSO liquid to reach working concentration: 20mM.

According to the working solution: cell culture fluid = 1:1000 (while setting a concentration gradient in this range), a chloroquinaldol working solution was added to the cell culture medium so that the final concentration of chloroquinaldol in the cell culture medium was 20 μm (containing a concentration gradient of this concentration). Shaking the culture evenly in 8-word, standing at 37 ℃ for culturing for 24 hours, and fully entering cells to activate the signal path of the intracellular type I interferon to express the type I interferon in a large amount to generate antiviral effect.

5. Various biochemical, molecular biological, and bioimaging methods identify the antiviral response of cells.

(1) Immunofluorescence imaging and flow cytometry demonstrated that chloroquinaldo strongly inhibited replication and amplification of viral particles with green fluorescent protein in cells.

Recombinant viruses stably expressing green fluorescent protein, after a certain period of infection, the virus particles entering the cells express GFP, and the infected cells can be clearly seen to be green under a 488nm channel by a fluorescent microscope.

In order to prove that the chlorquinaldol can effectively inhibit the infection and replication of viruses, preparing a chlorquinaldol working solution with a certain concentration gradient, adding the chlorquinaldol working solution into a cell culture medium growing in an exponential phase, incubating for 24 hours at 37 ℃, adding different types of virus-infected cells expressing green fluorescent protein, taking cells which are not incubated by the chlorquinaldol as a control, and infecting for 24-48 hours:

(1) observing the number of cells expressing the green fluorescent protein of the virus and the number of cells infected with the virus in the cells without chloroquinaldo after co-incubation with chloroquinaldo under a fluorescence microscope;

(2) cells were analyzed on a flow cytometer using GFP green fluorescent protein channels. Thus, it was semi-quantitatively determined whether chloroquinaldo could cause significant differences in the number of cells infected with the virus.

(2) RT-PCR method verifies that chlorquinaldol activates up-regulation of the transcription level of type I interferon gene in cells.

The chloroquinaldo exerts an antiviral immune response in cells, relying on its strong activation of the intracellular type I interferon signaling pathway. Thus, chloroquinaldo is capable of stimulating up-regulation of the intracellular type I interferon gene at the transcriptional level.

To verify that chlorquinaldol activates up-regulation of transcription level of type I interferon gene in cells, chlorquinaldol working solution containing a certain concentration gradient is prepared, the working solution is added into a cell culture medium growing in exponential phase, after incubation is carried out at 37 ℃ for 24 hours, the culture medium is removed to collect cells, the Trizol method is used for extracting total RNA in the cells, the oligo-dT primer is used for obtaining cDNA template, and then the primer of type I interferon gene is used for amplification. The RT-PCR method can intuitively obtain the transcription up-regulation of the I-type interferon gene in the chlorquinaldol-stimulated cells.

(3) Western blotting confirmed that chlorquinaldo strongly inhibited the expression of intracellular viral proteins.

The intracellular antiviral response is accomplished by activation of the natural immune signaling pathway type I interferon. Thus, by detecting the activation state of the type I interferon signaling pathway in cells and the expression of the interferon stimulating gene, the effect of chloroquinaldo on activating the type I interferon signaling pathway can be reflected.

In order to verify that the chloroquinaldol can efficiently activate the I-type interferon signal channel in cells, a chloroquinaldol working solution with a certain concentration gradient is prepared, the chloroquinaldol working solution is added into a cell culture medium which grows in an exponential phase, after the cell culture medium is incubated for 24 hours at 37 ℃, the culture medium is removed to collect cells, the cells are lysed by using RIPA lysate, and a protein immunoblotting Western blot method is adopted to detect the phosphorylation level of a key transcription factor IRF3 which characterizes the activation of the I-type interferon in the cells and the expression quantity change of an interferon stimulation gene.

The invention has the advantages that:

1. the core reagent of the invention, namely the small molecular chlorquinaldo, is a natural immune high-efficiency activator, and can remarkably produce the effect of resisting virus infection.

2. The small molecular compound chloroquinaldo is a safe antiviral agent. In the experimental process, no matter the observation of a common microscope or a fluorescence microscope, no change of the cell state after chlorquinate multiprocessing is found, and the small molecular compound is proved to be a safe antiviral reagent without obvious toxic or side effect to a great extent.

3. The small molecule immune activator can rapidly penetrate through the cell surface to enter cells, is very simple and easy to use, and can induce the cells to generate antiviral response by direct co-incubation. The limitations of limited activity, short half-life, need for blood injection administration, etc. of other types of interferon agonists are avoided.

4. The small molecular compound chloroquinaldo can be suitable for a plurality of different kinds of cultured cells, has wide application range and is beneficial to the use of a plurality of different antiviral stress occasions.

Description of the drawings:

figure 1 is a chloroquinate multi-structure diagram,

FIG. 2 shows that chloroquinaldo inhibits replication and proliferation of DNA viruses,

FIG. 3 shows that chlorquinaldol inhibits the replication and proliferation of viruses in THP-1 cells,

FIG. 4 shows that chloroquinaldol stimulates the production of type I interferon in mouse cells,

FIG. 5 shows that chlorquinaldol activates human THP-1 cells to produce type I interferon,

figure 6 shows the antiviral effect of chloroquinaldo at various concentrations.

Detailed Description

In the following examples, the structures of chloroquinaldol (chloroquinaldol) used are shown in fig. 1, and the chloroquinaldol stock solution was prepared: the dry powder in the EP tube was centrifuged at 4 ℃ at high speed, and sterile DMSO was added in the amounts shown in table 1 to be sufficiently dissolved to prepare a stock solution of the corresponding concentration. After the powder is completely dissolved, the EP tube is inverted and uniformly mixed and centrifuged briefly at 4 ℃, the mixture is packed in sterile EP tubes and can be stored for 2 years at-80 ℃.

TABLE 1 preparation of chloroquinaldol stock solution

Preparing a chloroquinaldo working solution:

taking out the sub-packaged chlorquinaldo stock solution by a refrigerator at the temperature of minus 80 ℃, fully melting at normal temperature, and diluting the chlorquinaldo stock solution into sterile DMSO liquid according to the use concentration for subsequent use. The chlorquinaldol working solution is prepared and used at present.

Example 1:20 mu M chloroquinaldo inhibits replication and proliferation of viruses

1. After 20. Mu.M of chloroquinaldo was added to THP-1 cells and the cells were pretreated for 12 hours, the cells were infected with DNA viruses HSV-1 and VacV labeled with green fluorescent protein, and the cells (Mock) without chloroquinaldo pretreatment were used as a control.

The results show that: the DNA virus can carry out high-level infection, replication and proliferation in cells without chlorquinate multiprocessing; whereas the expression of active viral green fluorescent protein was almost completely not observed in chloroquinaldo pretreated cells (fig. 2).

2. After 20. Mu.M of chlorquinate was added to THP-1 cells for 12 hours, cells were infected with GFP green fluorescent protein-labeled DNA virus HSV-1, and cells without drug treatment were used as a control.

The results show that: (A) Fluorescence imaging shows that chloroquinaldo almost completely inhibits viral replication and proliferation in cells; (B) Flow cytometric analysis showed that the number of cells expressing viral fluorescent protein decreased dramatically after chlorquinate multiprocessing (fig. 3).

Example 2:20 mu M chloroquinaldo induces up-regulation of type I interferon and interferon stimulation gene expression

BALBc mice were intraperitoneally injected with 2mL of 3% sodium thioglycolate for 5 days, macrophages in the abdominal cavity of the mice were treated with chlorquinate of different concentrations for 18 hours, and total RNA of the cells was extracted for RT-PCR experiments to detect the genes as shown in the figure.

The results show that: 20. Mu.M of chloroquinaldo was able to specifically stimulate up-regulation of the expression of Ifnβ and ISGs in mice, but did not induce the expression of inflammatory factor Il6 (FIG. 4), and the capacity of chloroquinaldo to induce interferon-related genes at concentrations above or below this was not as good as that at the optimal concentration of 20. Mu.M.

Example 3:20 mu M chloroquinaldo activated cells produced type I interferon (see FIG. 5)

Chlorquinate was added to THP-1 cells at various concentrations for 24 hours in a gradient manner, and cells infected with RNA virus SeV and DNA virus VacV were used as positive controls. (1) Cell culture supernatants were collected and the production of active type I interferon secreted into the supernatants was detected by type I interferon Bioassay assay (upper bar graph); (2) The lysed cells were subjected to Western blot experiments using whole protein to detect protein expression levels or phosphorylation levels as shown.

The results show that: 20. Mu.M of chloroquinaldo is effective to cause phosphorylation activation of the transcription factor IRF3 and induce up-regulation of the expression of the interferon-stimulated gene ISGs protein level (FIG. 5, panels); chlorquinaldo (e.g., 5 μm and 10 μm) at concentrations below this does not cause phosphorylation of transcription factor IRF3 and expression of interferon-stimulated genes (fig. 5, left hand sample of box), while chlorquinaldo (e.g., 50 μm and 100 μm) at concentrations above this causes IRF3 phosphorylation and expression of interferon-stimulated genes to be inhibited instead, unlike 20 μm which is significantly effective (fig. 5, right hand sample of box).

It follows that the key point of the present invention is that the solution of chloroquinaldol at a specific concentration, i.e. using chloroquinaldol at a final concentration of 20 μm, has the strongest effect of inducing the production of type I interferon and inhibiting the replication and propagation of viruses.

Example 4: the antiviral effect of chlorquinaldo at other concentrations is not as good as that of the optimum concentration of 20 mu M

After pretreatment for 12 hours with chloroquinaldo (10. Mu.M, 20. Mu.M, 50. Mu.M, 100. Mu.M) at various concentrations in THP-1 cells, cells were infected with RNA virus NDV labeled with green fluorescent protein, and cells not pretreated with chloroquinaldo (0. Mu.M) were used as a control.

The results show that: RNA virus was able to infect, replicate and proliferate at high levels (0. Mu.M) in cells without chlorquinate multiprocessing; replication and proliferation of the virus were observed to be inhibited to varying degrees in the chloroquinaldo pretreated cells, where the extent of inhibition of the virus was most thorough in the chloroquinaldo multiprocessed cells at the optimum concentration of 20 μm, whereas the antiviral effect of chloroquinaldo was rather reduced to varying degrees either below or above this concentration (fig. 6).

Claims

1. A method for applying a small molecule compound chloroquinaldo for non-therapeutic purposes, comprising applying an effective concentration of chloroquinaldo to a cell to be treated in a cell culture medium, wherein the effective concentration is 20 μm final concentration of chloroquinaldo in the cell culture medium, wherein the chloroquinaldo induces up-regulation of expression of a type I interferon stimulatory gene, and activates the cell to produce type I interferon; wherein, the chloroquinaldol is diluted to obtain 20mM chloroquinaldol working solution according to the working solution: cell culture fluid = 1:1000, adding chloroquinaldol working solution into cell culture medium to make final concentration of chloroquinaldol in cell culture medium be 20 μm, shaking culture, standing at 37deg.C for culturing for 24 hr, wherein the virus is DNA virus HSV-1 or VacV, or RNA virus NDV or SeV.

2. The method of claim 1, wherein the chloroquinaldo is diluted with a sterile DMSO liquid.