CN116077528A - Application of gallic acid carbon dots in resisting pseudorabies virus - Google Patents

Abstract

The invention provides an application of gallic acid carbon dots in resisting pseudorabies viruses. In the invention, a gallic acid carbon dot is prepared by taking a plant natural product gallic acid as a precursor carbon source, compared with GA, GACDs show more remarkable antiviral effect on PRV, and the GACDs have remarkable antiviral effect before and during the early period of virus infection, and in order to further explore the antiviral mechanism of the GACDs, proteomic analysis is carried out on Vero cells treated by the GACDs, and the result shows that the GACDs treatment can cause the increase of the expression quantity of interferon-related antiviral proteins such as MAVS, OAS1, ISG-15 and the like in the cells, and meanwhile, the expression quantity of key molecules TYK2 and STAT1 of a downstream signal path of the interferon also shows remarkable change, so that the carbon dot can regulate the interferon-related signal path, thereby playing the antiviral effect. The research can provide theoretical basis for biological application of carbon dots, and can also provide basis and thought for prevention and treatment of PRV and research and development of novel antiviral preparations.

Description

Application of gallic acid carbon dots in resisting pseudorabies virus

Technical Field

The invention belongs to the field of preparation of antiviral materials, and particularly relates to application of gallic acid carbon dots in resisting pseudorabies viruses.

Background

Pseudorabies virus (Pseudorabies Virus, PRV) is equivalent to human herpes simplex virus (herpes simplex virus type, HSV-1) as herpes zoster virus (VZV). PRV has very high homology with the alpha herpesvirus of the above several people, the host range is also very wide, and is a kind of multiple zoonosis virus, and pigs are natural infection hosts and infectious sources of the virus, can infect multiple animals such as pigs, cows, sheep, dogs, cats, foxes, raccoons and the like, and has been found to be widely popular worldwide, and the harm is extremely serious. In particular, there have been reports of human infection of PRV in recent years, which indicate that PRV also has a potential threat to human health.

Pseudorabies is also one of the new representatives of the old diseases. Prior to 2011, pseudorabies epidemic is well controlled due to effective vaccine immunization. However, since 2011, epidemic situations of suspected PR are outbreaked in a plurality of provincial immune pig farms in China, and pathogen separation and sequence analysis show that the pseudorabies epidemic situations of the large-area outbreak of the immune pig farm are caused by variant PRV. Molecular epidemiological investigation shows that PRV variant strains have become the dominant strains prevalent in our country, and that more and more studies have shown that vaccine strains have recombined with wild strains. Existing vaccines do not provide complete protection due to strain variation. Virus variation has prompted the vaccine to be updated and replaced continuously, so research into effective antiviral agents is also attracting more and more attention.

Because of the easy variation of viruses, the traditional antiviral drugs often face tolerance problems, and the research of 'nano antibiotics' is promoted. The nano material shows remarkable antiviral property due to the unique physicochemical property. The carbon dots have better application prospect in the aspect of biological medicine due to the advantages of wide raw materials, low cost, simple and convenient preparation, good biocompatibility, easy functionalization and the like. Carbon Dots (CDs) are a broad term as a novel fluorescent nanomaterial and generally refer to fluorescent carbon nanoparticles having at least one dimension below 10nm in size, chemical structures often being sp2 and sp3 hybridized carbon structures, single-or multi-layer graphitic carbon structures, and polymeric aggregate structures. Studies by Iannazzo et al show that graphene quantum dots can inhibit HIV and that the inhibition of HIV is significantly synergistically enhanced after binding to the antiretroviral drug CHI 499. Lin et al prepared curcumin as a carbon spot by one-step heating, had significantly higher antiviral effect on EV71 than curcumin itself, and had significantly lower cytotoxicity than curcumin. The glycyrrhizic acid is used as a raw material for preparing carbon dots by Tong and the like, so that the replication of PRRSV can be obviously inhibited, and the carbon dots can obviously improve the expression levels of interferon stimulating genes ISG-54, ISG-56, ISG-60 and ZAP. These studies indicate that carbonization of plant natural products to carbon dots using appropriate conditions greatly enhances the biocompatibility of the drug and has better biological activity, but there are relatively few studies on carbon dots in the antiviral field.

Based on this, it is necessary to develop a carbon dot applicable to the antiviral field, for widening the application range of the carbon dot and for selecting an antiviral treatment mode.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention aims to provide an application of gallic acid carbon dots in resisting pseudorabies viruses.

In order to achieve the above object, the present invention adopts the technical scheme that:

the invention discloses an application of gallic acid carbon dots in resisting pseudorabies viruses.

In the invention, gallic Acid Carbon Dots (GACDs) are prepared by taking plant natural product gallic acid as a precursor carbon source, compared with Gallic Acid (GA), the GACDs have more remarkable antiviral effect on PRV, and the GACDs are added before and during the early period of virus infection, so that proteomic analysis is carried out on the Vero cells treated by the GACDs for further exploring the antiviral mechanism of the GACDs, and the result shows that the GACDs treatment can cause the increase of the expression quantity of interferon-related antiviral proteins such as MAVS, OAS1, ISG-15 and the like in the cells, meanwhile, the expression quantity of key molecules TYK2 and STAT1 of a downstream signal path of the interferon also has remarkable change, and the carbon dots are suggested to regulate the interferon-related signal path, thereby playing antiviral effect. The research can provide theoretical basis for biological application of carbon dots, and can also provide basis and thought for prevention and treatment of PRV and research and development of novel antiviral preparations.

Furthermore, the gallic acid carbon dot realizes the effect of resisting pseudorabies virus by improving the expression quantity of the interferon related antiviral protein and the expression quantity of key molecules in an interferon downstream signal path.

Further, the interferon-related antiviral proteins include, but are not limited to, one or more of MAVS, OAS1, or ISG-15.

Further, the key molecules include, but are not limited to, one or both of TYK2 or STAT 1.

Further, the average particle size of the gallic acid carbon dots is 2-20nm.

Further, the gallic acid carbon dots are prepared according to the following steps:

weighing gallic acid powder, adding absolute ethyl alcohol, transferring into a reaction kettle with a polytetrafluoroethylene lining for hydrothermal reaction after complete dissolution, transferring the reaction solution into a dialysis bag for dialysis after the reaction is finished, and freeze-drying after the dialysis is finished.

Further, the hydrothermal reaction temperature is 160 ℃, and the hydrothermal reaction time is 4-6h.

Further, the molecular weight cut-off of the dialysis bag is 1kDa.

Further, the dialysis time is 6-8 hours.

The invention has the beneficial effects that:

the invention provides an application of gallic acid carbon dots in resisting pseudorabies viruses. In the invention, a mild and simple reaction method is adopted, and the plant natural product gallic acid is used as a precursor to prepare the GACDs with uniform size and good biocompatibility, compared with GA, the GACDs have more remarkable antiviral effect on PRV at the cellular level and in vivo, and the antiviral effect of the GACDs is remarkably superior to that of the precursor GA. GA can inhibit the reproduction process of PRV proliferation, and GACDs after carbon-dotation can inhibit adsorption, invasion and reproduction processes in the PRV proliferation process, so that GACDs have remarkable antiviral effect before and during the pre-infection addition of viruses. In order to further explore the antiviral mechanism of GACDs, proteomics analysis is carried out on the Vero cells treated by GACDs, and the result shows that the GACDs treatment can cause the increase of the expression level of interferon related antiviral proteins such as MAVS, OAS1, ISG-15 and the like in the cells, and meanwhile, the expression level of key molecules TYK2 and STAT1 of a downstream signal path of the interferon also has obvious change, so that a carbon point can regulate and control the interferon related signal path, thereby playing an antiviral role. Animal experiments showed that GACDs provided 30% protection to PRV infected mice and that virus content in the tissues of the diseased mice was also significantly lower than in PRV infected and GA treated groups, whereas GA did not provide protection. In addition to its better water solubility and biocompatibility, the enhancement of antiviral activity of GACDs can also better stimulate the natural immune response of cells. The research can provide theoretical basis for biological application of carbon dots, and can also provide basis and thought for prevention and treatment of PRV and research and development of novel antiviral preparations.

Drawings

Fig. 1 is a TEM, HRTEM, AFM, XPS characterization of the gallic acid carbon dot material prepared in example 1.

FIG. 2 is a comparison of cytotoxicity data of GACDs at gallic acid carbon point and GA prepared in example 1.

FIG. 3 is a comparison of the effect of GACDs on inhibition of PRV of gallic acid carbon dots prepared in example 1 with GA.

FIG. 4 is an analysis of the effect of GACDs and GA on the PRV replication phase of the gallic acid carbon dots prepared in example 1.

FIG. 5 is an analysis of the effect of GACDs and GA on PRV infected mice prepared in example 1.

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments and the accompanying drawings. It should be understood that the described embodiments are merely some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1: preparation of carbon dots

Taking 85mg of gallic acid powder (Ala-dine) powder, putting the gallic acid powder (Ala-dine) powder into a glass bottle, adding 10mL of absolute ethyl alcohol, transferring the absolute ethyl alcohol into a polytetrafluoroethylene-lined hydrothermal reaction kettle after complete dissolution, and reacting for 6h at 160 ℃. After the reaction solution is cooled, the reaction solution is transferred into a dialysis bag (1 kDa) for dialysis for 8 hours. And freeze-drying the dialyzate for standby.

The invention adopts a mild and simple method to synthesize Gallic Acid Carbon Dots (GACDs) by taking gallic acid as a precursor carbon source. The GACDs were observed by Transmission Electron Microscopy (TEM) as uniformly sized dispersed spherical particles (A in FIG. 1) having an average particle size of 3.2.+ -. 0.6nm. HRTEM analysis showed that the lattice spacing of GACDs was 0.22nm (B in fig. 1), consistent with the (100) in-plane spacing of sp2 carbon. Atomic Force Microscopy (AFM) also showed that GACDs were particles smaller than 10nm (fig. 1C). Notably, AFM results showed that the GACDs thickness was around 1nm (C in fig. 1) and thus can be defined as graphene quantum dots.

Analysis of the surface element state of gadds by XPS showed that peaks at 290eV and 529eV, respectively, were present at 62.37% and 37.63%, respectively, corresponding to C, O elements.

The gallic acid carbon dots prepared above were then tested and analyzed as follows:

1) GACDs cytotoxicity

Vero cells were seeded in 96-well plates at 37℃in 5% CO ₂ After culturing for 24 hours in a cell culture box, discarding the old culture solution, adding materials to be tested with different concentrations, incubating for 48 hours in the cell culture box, adding 810 mu L/hole of CCK, incubating for 2 hours in the culture box, measuring absorbance at 450nm by using a microplate reader, and calculating cell activity.

2) Plaque formation assay

Vero cells were plated into 12-well plates and after the cells had grown to a monolayer, washed twice with serum-free DMEM medium. Then with serum-free DMThe virus was diluted in EM medium, 400. Mu.L of diluted virus solution was added to each well, and three replicates were made for each concentration gradient. After incubation at 37℃for 1h, the incubation was discarded, washed once with serum-free DMEM medium, then covered with 1.5% sodium carboxymethylcellulose in DMEM, 1 mL/well, 37℃5% CO ₂ The cells were placed in the incubator for 5d. And 5d, fixing for 12 hours by using 10% formaldehyde fixing solution, adding crystal violet staining solution for staining, and counting the number of plaques.

3) GACDs affecting PRV replication phase analysis

Adsorption: and (3) pre-cooling the PK-15 cells full of the monolayer at 4 ℃ for 30min, simultaneously adding the PRV and carbon dots into the cells, incubating for 2h at 4 ℃, discarding the supernatant after the adsorption is finished, washing for 2 times by using DMEM, and carrying out a plaque test.

Intrusion: pre-cooling the PK-15 cells full of the monolayer at 4 ℃ for 30min, incubating the PRV infected cells at 4 ℃ for 2h, discarding the supernatant after the adsorption is finished, washing the pre-cooled DMEM for 2 times, adding a carbon dot-containing culture medium for incubation at 37 ℃ for 3h, discarding the supernatant, washing the DMEM for 2 times, and carrying out a plaque test.

Replication: removing the supernatant after the cells grow to a monolayer, infecting PRV, incubating for 1h at 37 ℃, removing the incubation liquid, washing with DMEM for 2 times to remove unadsorbed virus, adding a carbon dot-containing culture medium for continuous culture for 24h, taking a virus liquid, and detecting the virus content by fluorescence quantification and TCID 50.

Releasing: removing the supernatant after the cells grow to a monolayer, infecting PRV, incubating for 1h at 37 ℃, removing the incubation liquid, washing with DMEM for 2 times to remove unadsorbed virus, adding a DMEM-containing culture medium for further culturing for 18h, removing the supernatant, washing with DMEM for 2 times, adding a carbon dot-containing culture medium for further culturing for 2h, and respectively collecting and detecting the virus content in the supernatant and the cells.

4) Analysis of influence of GACDs on PRV infected mice

Female Balb/c mice of 6 weeks of age were selected into four groups: prv+dmem group (challenge group), prv+gacds group (carbon dot treatment group), prv+ga group (gallic acid treatment group), DMEM group (blank control group), 10 mice each.

PRV was diluted to 103TCID50/100 μl with DMEM, and PRV was plantar injected 100 μl per mouse except for the blank (equivalent DMEM injection). GACDs and GA groups were intraperitoneally injected with GACDs or GA at 50mg/kg body weight daily 1d before infection and 1h after infection. Mice were observed daily and the number of clinical manifestations and deaths recorded. Taking the parenchymal tissue of the dead mice, quantitatively detecting the virus content by fluorescence, and performing immunohistochemical analysis after fixing the brain tissues of each group.

5) Statistical analysis

The mean and standard deviation (standard error) of each group of data in the statistical test were analyzed by means of GraphpadPrism7 statistical analysis software and expressed as means±sem, and the significance of the mean difference of each group of data was analyzed by means of t-test. Wherein ns is the difference is not significant; * P <0.05 is significant difference; * P <0.01 is very significant; * P <0.001 is very significant.

Test example 1: cytotoxicity test of GACDs

The GACDs are diluted continuously by 2 times from 640 to 5 mug/mL, and are respectively added into Vero cells for co-incubation for 48 hours to detect the cell activity, and the result is shown in figure 2, the cytotoxicity of the GACDs after carbonization is obviously less than that of GA, and the water solubility and stability of the GACDs are obviously enhanced compared with those of GA. Vero cell viability was still greater than 80% at a GACDs concentration of 160. Mu.g/mL, whereas GA was only 45.63% at this concentration. At a concentration of 80. Mu.g/mL, there was less impact on cell activity, so we chose 80. Mu.g/mL for the test in subsequent studies.

Test example 2: inhibition PRV analysis of GACDs

PRV virus was diluted, mixed with GACDs or GA, respectively, and incubated at 37℃for 1h, and the mixture was then infected with Vero cells to determine the amount of plaque formation of the virus, as shown in FIG. 3A, GACDs significantly reduced the amount of PRV plaque, which inhibited PRV activity significantly better than GA. We have also explored the inhibitory effect of traditional citric acid carbon dots (cds, as shown in figure 3 a) on PRV, and the results show that cds do not exhibit significant antiviral effects. These results indicate that the antiviral effect of carbon dots is closely related to its synthetic precursors. GACDs had a pronounced dose-dependent effect on PRV inhibition (B, C in FIG. 3).

Test example 3: GACDs can inhibit PRV adsorption, invasion and replication

Analysis of the effect of GACDs on PRV replication phase (FIG. 4) shows that GACDs inhibit the adsorption, invasion and replication processes of PRV proliferation, have no effect on release, and GA has only a certain inhibition on PRV replication, and have no effect on the rest of the phases. Again, this result demonstrates that the ability to inhibit PRV is significantly enhanced after the carbodotion of gallic acid.

Test example 4: GACDs can inhibit PRV infection in mice

After PRV infects the body, the virus can proliferate in large numbers in the central nervous system, causing neuroinflammation in the brain. To further evaluate whether gadds can inhibit PRV infection in vivo, mice were used herein as a model to analyze the effect of gadds and GA treatment on PRV infected mice. The mice experiments were divided into a blank control group, a PRV-infected group, a GACDs-treated group and a GA-treated group 4 groups of 10 mice each. The results are shown in figure, 4d after PRV infection, all mice in PRV-infected and GA-treated groups developed obvious neurological symptoms such as abnormal excitation, itching, biting hind limb inoculation sites (as in figure 5 a) until death. The number of mice in the GACDs-treated group developed symptoms was small, and the symptoms developed about 24 hours later. PRV infected mice died all 5d after infection, GA treated mice died all 6d after infection, and the gacds treated mice still had 3 mice survived without any symptoms, with a protection rate of 30% (as in fig. 5C). The mice were dissected and examined to find that there was significant infarction in the spleen of the PRV-infected and GA-infected mice, and that there was a congestion in the liver, and that there was no significant difference in the GACDs group from the control (as in FIG. 5B).

In order to explore the virus content in the tissues of mice in different treatment groups, the mice in each group are dissected and then the organs of the heart, liver, spleen, lung, kidney, brain and the like are taken, the weight of the same organs in different treatment groups is equal by weighing a balance, and nucleic acid is extracted after grinding to carry out fluorescence quantitative detection on the PRV virus content. The results showed that PRV was not detected in organs of all non-morbidity surviving mice in the GACDs treated group, while the tissue virus content of the morbidity-mortality mice in the GACDs treated group was also significantly lower than in the PRV infected group (D in fig. 5). Brain tissue immunohistochemical results showed significant PRV positive signal in PRV infected and GA mice, whereas no significant in GACDs mice. These results show that gadds can significantly inhibit PRV infection in mice and can be used as candidate agents for antiviral treatment.

It should be understood that the foregoing examples of the present invention are provided merely for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention, and that various other changes and modifications may be made therein by one skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims (9)

1. Application of gallic acid carbon dots in resisting pseudorabies virus is provided.

2. The use according to claim 1, wherein the carbon gallate point achieves an anti-pseudorabies virus effect by increasing the expression level of interferon-related antiviral proteins and the expression level of key molecules in the interferon downstream signal pathway.

3. The use of claim 2, wherein the interferon-related antiviral protein comprises one or more of MAVS, OAS1, or ISG-15.

4. The use of claim 2, wherein the key molecules comprise one or both of TYK2 or STAT 1.

5. The use according to claim 1, wherein the average particle size of the gallic acid carbon dots is 2-20nm.

6. The use according to claim 1, wherein the gallic acid carbon dots are prepared according to the following steps:

weighing gallic acid powder, adding absolute ethyl alcohol, transferring into a reaction kettle with a polytetrafluoroethylene lining for hydrothermal reaction after complete dissolution, transferring the reaction solution into a dialysis bag for dialysis after the reaction is finished, and freeze-drying after the dialysis is finished.

7. The use according to claim 6, wherein the hydrothermal reaction temperature is 160 ℃ and the hydrothermal reaction time is 4-6h.

8. The use according to claim 6, wherein the dialysis bag has a molecular weight cut-off of 1KDa.

9. The use according to claim 6, characterized in that the dialysis time is 6-8 hours.

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