Application of Gallic Acid Carbon Dots in Anti-Pseudorabies Virus

Abstract

The present invention provides an application of gallic acid carbon dots in anti-pseudorabies virus. In the present invention, gallic acid carbon dots are prepared using the plant natural product gallic acid as a precursor carbon source. Compared with GA, GACDs exhibit a more significant antiviral effect against PRV, and GACDs have a significant antiviral effect when added before and in the early stage of virus infection. To further explore the antiviral mechanism of GACDs, proteomic analysis was performed on Vero cells treated with GACDs. The results showed that the treatment with GACDs led to an increase in the expression levels of interferon-related antiviral proteins such as MAVS, OAS1, and ISG-15 in the cells. At the same time, significant changes also occurred in the expression levels of the key molecules TYK2 and STAT1 in the interferon downstream signaling pathway, suggesting that carbon dots can regulate the interferon-related signaling pathway and thus play an antiviral role. This study can provide a theoretical basis for the biological application of carbon dots and also provide a basis and ideas for the prevention and treatment of PRV and the development of new antiviral preparations.

Description

Technical Field

[0001] The present invention belongs to the field of preparation of antiviral materials, and specifically includes the application of gallic acid carbon dots in anti-pseudorabies virus. Background Art

[0002] Pseudorabies virus (PRV) belongs to the same genus as human herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV), etc., namely alpha herpesvirus. PRV has a very high homology with the above-mentioned several human alpha herpesviruses and a wide host range. It is a zoonotic virus that can infect various animals such as pigs, cattle, sheep, dogs, cats, foxes, and raccoons. Since its discovery, it has been widely prevalent globally and its harm is extremely serious. In particular, in recent years, there have been successive reports of human infection with PRV, indicating that PRV also poses a potential threat to human health.

[0003] Pseudorabies is also one of the representatives of old diseases with new outbreaks. Before 2011, the effective vaccination well controlled the pseudorabies epidemic. However, since the end of 2011, suspected PR epidemics have broken out in vaccinated pig farms in many provinces in China. Pathogen isolation and sequence analysis showed that the large-scale outbreak of pseudorabies in the vaccinated pig population was caused by mutant PRV. Molecular epidemiological investigations showed that the mutant strains of PRV have become the main epidemic strains in China, and more and more studies have shown that the vaccine strains have recombined with the wild strains. Due to the strain mutation, the existing vaccines cannot provide complete protection. The mutation of the virus has prompted the continuous update of vaccines, so the research on effective antiviral agents has received increasing attention.

[0004] Due to the easy mutation of viruses, traditional antiviral drugs often face tolerance problems, which has given rise to the research on "nanoantibiotics". Due to their unique physical and chemical properties, nanomaterials exhibit remarkable antiviral properties. Among them, carbon dots have good application prospects in biomedicine due to their wide range of raw materials, low cost, simple preparation, good biocompatibility and easy functionalization. As a new type of fluorescent nanomaterial, carbon dots (CDs) is a general term, generally referring to fluorescent carbon nanoparticles with at least one dimension less than 10 nm in size, and their chemical structures are often sp2 and sp3 hybrid carbon structures, single-layer or multi-layer graphite carbon structures, and polymer-like aggregated structures. Studies by Iannazzo et al. have shown that graphene quantum dots can inhibit HIV, and the inhibitory effect on HIV is significantly synergistically enhanced after binding with the antiretroviral drug CHI499. Lin et al. prepared curcumin into carbon dots by one-step heating, and its antiviral effect against EV71 was significantly higher than that of curcumin itself, and its cytotoxicity was significantly lower than that of curcumin. Tong et al. prepared carbon dots from glycyrrhizic acid as raw materials, which could significantly inhibit the replication of PRRSV, and the carbon dots could significantly increase the expression levels of interferon-stimulated genes ISG-54, ISG-56, ISG-60 and ZAP. These studies have shown that carbonizing plant natural products into carbon dots under appropriate conditions can greatly enhance the biocompatibility of drugs and have good biological activity. However, the current research on carbon dots in the field of antiviral is still relatively scarce.

[0005] Based on this, it is very necessary to study a kind of carbon dots that can be applied in the field of antiviral to broaden the application scope of carbon dots and the choice of antiviral treatment methods. Summary of the Invention

[0006] Aiming at the above problems existing in the prior art, the purpose of the present invention is to provide an application of gallic acid carbon dots in anti-pseudorabies virus.

[0007] To achieve the above purpose, the technical solutions adopted by the present invention include:

[0008] The present invention discloses an application of gallic acid carbon dots in anti-pseudorabies virus.

[0009] In the present invention, gallic acid carbon dots (GACDs) were prepared using plant natural product gallic acid as the precursor carbon source. Compared with gallic acid (GA), GACDs exhibited a more significant antiviral effect against PRV, and significant antiviral effects were observed when GACDs were added before and in the early stage of virus infection. To further explore the antiviral mechanism of GACDs, proteomic analysis was performed on Vero cells treated with GACDs. The results showed that treatment with GACDs led to an increase in the expression levels of interferon-related antiviral proteins such as MAVS, OAS1, and ISG-15 in the cells. At the same time, significant changes were also observed in the expression levels of the key molecules TYK2 and STAT1 in the downstream interferon signaling pathway, suggesting that carbon dots can regulate the interferon-related signaling pathway and thus exert an antiviral effect. This study can provide a theoretical basis for the biological application of carbon dots and also provide a basis and ideas for the prevention and treatment of PRV and the development of new antiviral preparations.

[0010] Furthermore, the gallic acid carbon dots achieve the anti-pseudorabies virus effect by increasing the expression levels of interferon-related antiviral proteins and the key molecules in the downstream interferon signaling pathway.

[0011] Furthermore, the interferon-related antiviral proteins include, but are not limited to, one or more of MAVS, OAS1, or ISG-15.

[0012] Furthermore, the key molecules include, but are not limited to, one or two of TYK2 or STAT1.

[0013] Furthermore, the average particle size of the gallic acid carbon dots is 2 - 20 nm.

[0014] Furthermore, the gallic acid carbon dots are prepared according to the following steps:

[0015] Weigh gallic acid powder, add absolute ethanol, and transfer it to a reaction kettle with a polytetrafluoroethylene inner lining after complete dissolution for hydrothermal reaction. After the reaction is completed, transfer the reaction solution into a dialysis bag for dialysis, and freeze-dry it after dialysis to obtain the product.

[0016] Furthermore, the hydrothermal reaction temperature is 160 °C, and the hydrothermal reaction time is 4 - 6 h.

[0017] Furthermore, the cut-off molecular weight of the dialysis bag is 1 KDa.

[0018] Furthermore, the dialysis time is 6 - 8 h.

[0019] Advantages of the present invention:

[0020] The present invention provides an application of gallic acid carbon dots in anti-pseudorabies virus. In the present invention, a mild and simple reaction method is adopted to prepare GACDs with uniform size and good biocompatibility using the plant natural product gallic acid as a precursor. Compared with GA, GACDs have a more significant antiviral effect on PRV at the cellular level and in vivo, and the antiviral effect of GACDs is significantly better than that of its precursor GA. GA can inhibit the replication process of PRV proliferation, while carbonized GACDs can inhibit multiple processes such as adsorption, invasion, and replication during PRV proliferation. Therefore, GACDs have a significant antiviral effect when added before and in the early stage of virus infection. To further explore the antiviral mechanism of GACDs, proteomic analysis was performed on Vero cells treated with GACDs. The results showed that the treatment with GACDs led to an increase in the expression levels of interferon-related antiviral proteins such as MAVS, OAS1, and ISG-15 in the cells. At the same time, significant changes also occurred in the expression levels of the key molecules TYK2 and STAT1 in the interferon downstream signaling pathway, suggesting that carbon dots can regulate the interferon-related signaling pathway to exert an antiviral effect. The results of animal experiments showed that GACDs could provide 30% protection against PRV-infected mice, and the virus content in the tissues of the diseased mice was also significantly lower than that in the PRV-infected group and the GA treatment group, while GA could not provide protective efficacy. The improvement of the antiviral activity of GACDs is related not only to its better water solubility and biocompatibility and other properties, but also GACDs can better stimulate the natural immune response of cells. This study can provide a theoretical basis for the biological application of carbon dots, and also provide a basis and ideas for the prevention and treatment of PRV and the research and development of new antiviral preparations. Description of the Drawings

[0021] Figure 1 TEM, HRTEM, AFM, and XPS characterizations of the gallic acid carbon dot material prepared in Example 1.

[0022] Figure 2 Comparison of cytotoxicity data between the gallic acid carbon dots GACDs and GA prepared in Example 1.

[0023] Figure 3 Comparison of the effects between the gallic acid carbon dots GACDs and GA prepared in Example 1 in inhibiting PRV.

[0024] Figure 4 Analysis of the effects of the gallic acid carbon dots GACDs and GA prepared in Example 1 on the PRV replication stage.

[0025] Figure 5 Analysis of the effects of the gallic acid carbon dots GACDs and GA prepared in Example 1 on PRV-infected mice. Detailed Embodiments

[0026] To more clearly illustrate the present invention, the present invention will be further described below in conjunction with preferred embodiments and the accompanying drawings. It should be clear that the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0027] Example 1: Preparation of carbon dots

[0028] Take 85 mg of gallic acid powder (Aladdin) in a glass bottle, add 10 mL of absolute ethanol, and after complete dissolution, transfer it to a hydrothermal reaction kettle lined with polytetrafluoroethylene, and react at 160 °C for 6 h. After the reaction solution is cooled, transfer it to a dialysis bag (1 KDa) and dialyze for 8 h. The dialysate is freeze-dried for later use.

[0029] The present invention adopts a mild and simple method to synthesize gallic acid carbon dots (GACDs) using gallic acid as a precursor carbon source. Through transmission electron microscopy (TEM), it can be observed that GACDs are uniformly dispersed spherical particles ( Figure 1 in A), with an average particle size of 3.2 ± 0.6 nm. HRTEM analysis shows that the lattice spacing of GACDs is 0.22 nm ( Figure 1 in B), which is consistent with the (100) plane spacing of sp2 carbon. Atomic force microscopy (AFM) also shows that GACDs are particles smaller than 10 nm ( Figure 1 C). It is worth noting that the AFM results show that the thickness of GACDs is about 1 nm ( Figure 1 in C), and thus can be defined as graphene quantum dots.

[0030] XPS is used to analyze the surface element state of GACDs. Figure 1 The results shown in D indicate that peaks appear at 290 eV and 529 eV respectively, corresponding to C and O elements, with contents of 62.37% and 37.63% respectively.

[0031] Then, the following tests and analyses are carried out on the prepared gallic acid carbon dots:

[0032] 1) Cytotoxicity of GACDs

[0033] Vero cells are seeded in a 96-well plate and cultured in a 5% CO2 cell incubator at 37 °C for 24 h. Then, the old culture medium is discarded, and different concentrations of the test materials are added. After co-incubating with the cells for 48 h, 10 μL / well of CCK8 is added, and the incubation is continued in the incubator for 2 h. The absorbance at 450 nm is measured with an enzyme-labeled instrument, and the cell viability is calculated.

[0034] 2) Plaque formation assay

[0035] Vero cells were seeded into 12-well plates. After the cells grew to confluence, they were washed twice with serum-free DMEM medium. Then, the virus was diluted with serum-free DMEM medium, and 400 μL of the diluted virus solution was added to each well. Three replicates were set for each concentration gradient. After incubation at 37 °C for 1 h, the incubation solution was discarded, and the cells were washed once with serum-free DMEM medium. Then, DMEM containing 1.5% sodium carboxymethylcellulose was added to cover the cells, 1 mL per well, and the cells were placed in a 37 °C, 5% CO2 cell culture incubator for 5 days. On the 5th day, the cells were fixed with 10% formaldehyde fixative for 12 h, then stained with crystal violet staining solution, and the number of plaques was counted.

[0036] 3) Analysis of the effect of GACDs on the replication stage of PRV

[0037] Adsorption: The confluent monolayer PK-15 cells were pre-cooled at 4 °C for 30 min. PRV and carbon dots were added to the cells simultaneously, and the cells were incubated at 4 °C for 2 h. After adsorption, the supernatant was discarded, and the cells were washed twice with DMEM for plaque assay.

[0038] Invasion: The confluent monolayer PK-15 cells were pre-cooled at 4 °C for 30 min. The cells were infected with PRV and incubated at 4 °C for 2 h. After adsorption, the supernatant was discarded, and the cells were washed twice with pre-cooled DMEM. Then, the medium containing carbon dots was added, and the cells were incubated at 37 °C for 3 h. The supernatant was discarded, and the cells were washed twice with DMEM for plaque assay.

[0039] Replication: After the cells grew to confluence, the supernatant was discarded. The cells were infected with PRV and incubated at 37 °C for 1 h. The incubation solution was discarded, and the cells were washed twice with DMEM to remove the unadsorbed virus. Then, the medium containing carbon dots was added and the cells were cultured for another 24 h. The virus solution was taken for fluorescence quantitative analysis and TCID50 detection of the virus content.

[0040] Release: After the cells grew to confluence, the supernatant was discarded. The cells were infected with PRV and incubated at 37 °C for 1 h. The incubation solution was discarded, and the cells were washed twice with DMEM to remove the unadsorbed virus. Then, the medium containing DMEM was added and the cells were cultured for 18 h. The supernatant was discarded, and the cells were washed twice with DMEM. Then, the medium containing carbon dots was added and the cells were cultured for 2 h. The virus contents in the supernatant and cells were collected and detected respectively.

[0041] 4) Analysis of the effect of GACDs on PRV-infected mice

[0042] Six-week-old female Balb / c mice were selected and divided into four groups: PRV+DMEM group (inoculation group), PRV+GACDs group (carbon dot treatment group), PRV+GA group (gallic acid treatment group), and DMEM group (blank control group), with 10 mice in each group.

[0043] Dilute PRV to 103 TCID50 / 100 μL with DMEM. Except for the blank control (injecting an equal amount of DMEM), inject 100 μL of PRV into the plantar surface of each mouse. In the GACDs group and the GA group, GACDs or GA was intraperitoneally injected at a dose of 50 mg / kg body weight every day 1 day before infection and 1 h after infection. Observe and record the clinical manifestations and the number of deaths of the mice every day. Take the parenchymal tissues of the dead mice and detect the virus content by fluorescence quantitative method. Fix the brain tissues of each group and perform immunohistochemical analysis.

[0044] 5) Statistical analysis

[0045] Use the Graphpad Prism 7 statistical analysis software to calculate the means and standard errors of the data of each group in the experiment, and express them as means ± SEM. Use the t-test to analyze the significance of the difference in the means of the data of each group. Among them, ns means no significant difference; *, p < 0.05 means significant difference; **, P < 0.01 means extremely significant difference; ***, P < 0.001 means very significant difference.

[0046] Experimental example 1: Cytotoxicity test of GACDs

[0047] GACDs was serially diluted 2-fold from 640 to 5 μg / mL and added to Vero cells respectively. After co-incubation for 48 h, the cell viability was detected. The results are as Figure 2 shown. The cytotoxicity of GACDs after carbon dot modification is significantly less than that of GA, and the water solubility and stability of GACDs are significantly enhanced compared with GA. When the concentration of GACDs is 160 μg / mL, the survival rate of Vero cells is still greater than 80%, while the survival rate of cells treated with GA at this concentration is only 45.63%. When the concentration is 80 μg / mL, the effect on cell viability is small. Therefore, we selected 80 μg / mL for subsequent research.

[0048] Experimental example 2: Analysis of the inhibition of PRV by GACDs

[0049] Dilute the PRV virus, mix it with GACDs or GA respectively, incubate at 37 °C for 1 h, and then infect Vero cells with the mixture to determine the number of plaque formations of the virus. As Figure 3 shown in A, GACDs can significantly reduce the number of PRV plaques, and its inhibitory activity against PRV is significantly better than that of GA. We also explored the inhibitory effect of traditional citric acid carbon dots (cCDs, as Figure 3 shown in A) on PRV. The results showed that cCDs did not show obvious antiviral effects. These results indicate that the antiviral effect of carbon dots is closely related to their synthetic precursors. The inhibitory effect of GACDs on PRV has an obvious dose-dependent effect ( Figure 3 shown in B and C).

[0050] Experimental Example 3: GACDs can inhibit the adsorption, invasion and replication of PRV

[0051] The analysis results of the influence of GACDs on the replication stage of PRV show that ( Figure 4 ), GACDs can inhibit the adsorption, invasion and replication processes during the proliferation process of PRV, have no effect on the release process, while GA only has a certain inhibitory effect on the replication of PRV and has no effect on the remaining stages. This result once again shows that the ability of gallic acid to inhibit PRV is significantly enhanced after carbon dot modification.

[0052] Experimental Example 4: GACDs can inhibit the infection of mice by PRV

[0053] After PRV infects the body, the virus will multiply in large numbers in the central nervous system, thereby causing neuroinflammation in the brain. To further evaluate whether GACDs can inhibit PRV infection in vivo, in this paper, mice were used as a model to analyze the effects of GACDs and GA treatments on PRV-infected mice. The mouse experiment was divided into 4 groups: blank control group, PRV infection group, GACDs treatment group and GA treatment group, with 10 mice in each group. The results are shown in the figure. 4 days after PRV infection, all mice in the PRV infection group and the GA treatment group showed obvious neurological symptoms, such as abnormal excitement, itching, and biting the inoculation site of the hind limbs (such as Figure 5 in A), until death. The number of mice showing symptoms in the GACDs treatment group was less, and the time of symptom appearance was about 24 h later. All mice in the PRV infection group died 5 days after infection, and all mice in the GA treatment group died 6 days after infection. Continuing to raise until the 10th day, 3 mice in the GACDs treatment group still survived and had no symptoms, and the protection rate was 30% (such as Figure 5 in C). Autopsy of the mice found that there were obvious infarcts in the spleens of the mice in the PRV infection group and the GA group, and congestion in the livers. There was no obvious difference in the GACDs group compared with the control (such as Figure 5 in B).

[0054] To explore the virus content in the tissues of mice in different treatment groups, after dissecting the mice in each group, the heart, liver, spleen, lung, kidney, brain and other parenchymal organs were taken, and the weights of the same organs in different treatment groups were ensured to be equal by weighing with a balance. After grinding, nucleic acids were extracted for fluorescence quantitative detection of the PRV virus content. The results showed that PRV was not detected in the organs of all the surviving mice without disease in the GACDs treatment group, and the tissue virus content of the diseased and dead mice in the GACDs treatment group was also significantly lower than that in the PRV infection group ( Figure 5 in D). The immunohistochemical results of the brain tissue showed obvious PRV positive signals in the mice in the PRV infection group and the GA group, while they were not obvious in the mice in the GACDs group. These results show that GACDs can significantly inhibit the infection of PRV in mice and can be used as a candidate preparation for antiviral treatment.

[0055] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is impossible to enumerate all the implementation manners here. Any obvious changes or variations derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. Application of gallic acid carbon dots in the preparation of anti-pseudorabies virus drugs, characterized in that, The gallic acid carbon dots are prepared according to the following steps: Weigh gallic acid powder, add anhydrous ethanol, transfer it to a reaction kettle with a polytetrafluoroethylene liner for hydrothermal reaction after complete dissolution, transfer the reaction solution to a dialysis bag for dialysis after the reaction ends, and freeze-dry it after dialysis to obtain the product; The hydrothermal reaction temperature is 160 °C, and the hydrothermal reaction time is 4-6 h; The cut-off molecular weight of the dialysis bag is 1 KDa; The average particle size of the gallic acid carbon dots is 2-20 nm.

2. The application according to claim 1, wherein The gallic acid carbon dots achieve the effect of anti-pseudorabies virus by increasing the expression levels of interferon-related antiviral proteins and key molecules in the interferon downstream signaling pathway.

3. The application according to claim 2, wherein The interferon-related antiviral proteins include one or more of MAVS, OAS1, or ISG-15.

4. The application according to claim 2, wherein The key molecules include one or two of TYK2 or STAT1.

5. The application according to claim 1, wherein The dialysis time is 6-8 h.

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