阅读说明：本技术 α-硫辛酸在抗鱼类病毒性出血性败血症病毒感染方面的应用 (Application of alpha-lipoic acid in resisting fish viral hemorrhagic septicemia virus infection ) 是由 易梅生 张弯弯 贾坤同 陈晓淇 于 2021-07-12 设计创作，主要内容包括：本发明涉及α-硫辛酸的新用途,具体涉及α-硫辛酸在抗鱼类病毒性出血性败血症病毒感染方面的应用,为寻找对病毒性出血性败血症病毒具有抗病毒活性的药物,本发明通过研究发现α-硫辛酸在病毒性出血性败血症病毒感染细胞过程中发挥显著的抗病毒功能,且在治疗病毒性出血败血症方面具有很好的效果,可用于鱼类感染病毒性出血性败血症病毒后的治疗。本发明不仅提供了α-硫辛酸的新用途,而且为鱼类病毒性出血性败血症的治疗提供了新的方向。(The invention relates to a new application of alpha-lipoic acid, in particular to an application of the alpha-lipoic acid in resisting fish viral hemorrhagic septicemia virus infection, and aims to find a medicine with antiviral activity to the viral hemorrhagic septicemia virus. The invention not only provides a new application of alpha-lipoic acid, but also provides a new direction for treating the fish viral hemorrhagic septicemia.)

1. Application of alpha-lipoic acid in preparing medicines for resisting fish viral hemorrhagic septicemia.

2. Application of alpha-lipoic acid in preparing medicines for resisting fish viral hemorrhagic septicemia virus infected cells.

3. The use according to claim 2, wherein the cell is infected with fish viral haemorrhagic septicaemia virus to inhibit replication of the fish viral haemorrhagic septicaemia virus.

4. The use of claim 2, wherein the cells include, but are not limited to, FHM cells.

5. A medicine for resisting fish viral hemorrhagic septicemia virus infected cells is characterized in that alpha-lipoic acid is taken as a main active ingredient.

6. The drug for resisting fish viral hemorrhagic septicemia virus infected cells according to claim 5, further comprising a pharmaceutically acceptable carrier and/or excipient.

7. The drug for treating fish viral hemorrhagic septicemia virus-infected cells according to claim 5, wherein the drug is formulated as a powder, a granule, or a liquid.

Technical Field

The invention relates to a new application of alpha-lipoic acid, in particular to an application of the alpha-lipoic acid in resisting fish viral hemorrhagic septicemia virus infection.

Background

Viral Hemorrhagic Septicemia (VHS) is an infectious fish viral disease caused by Viral Hemorrhagic Septicemia Virus (VHSV). VHSV can spread widely in fish populations with excreta of water, fish-feeding birds and infected fish as a transmission medium, causing explosive death. At present, VHS is mainly popular in Europe, North America and eastern Asia countries, can infect various sea fresh water economic fishes such as rainbow trout, salmon, largemouth bass and the like, and seriously jeopardizes the sustainable development of the aquaculture industry. Therefore, VHS has been listed in the second class of animal epidemic diseases and the second class of animal infectious epidemic disease which is prohibited from entering in China.

VHSV belongs to the order Mononegavirales (Mononegavirales), Rhabdoviridae (Rhabdoviridae), Rhabdoviridae (Novirrabovirus). The genome of VHSV is a single segment of negative strand RNA with a size of about 11kb (kilobase), and can replicate and translate 6 viral proteins in a host cell, wherein the arrangement sequence of the protein open reading frames in the genome is 3 '-N-P-M-G-NV-L-5'. The shape of the whole virus particle is similar to that of a bullet, the length is about 170-180nm, and the width is about 60-70 nm. VHSV can be divided into 4 major genotypes (I-IV) and subtypes (genotypes Ia-Ie, genotypes IVa-IVb) based on the difference between the N and G genes.

alpha-Lipoic Acid (LA) is a octanoic Acid derivative containing two sulfur atoms and has the chemical formula C₈H₁₄O₂S₂. LA has two enantiomers, dextro (R) and levo (S), endogenous LA often exists in the form of R-LA, and synthetic LA is a mixture of the two enantiomers. LA is known as the "most functional and most active" antioxidant. LA in cells can reduce the oxidative damage to molecules such as lipid, DNA and amino acid and relieve the oxidative stress reaction of organisms by eliminating peroxidized molecules and superoxide molecules generated by cell metabolism; also can promote the regeneration of antioxidant molecules such as intracellular glutathione, vitamin E, vitamin C and the like to play the antioxidant function. Therefore, LA has been used as a feed additive in the aquaculture industry to improve the antioxidant capacity of fish and to protect against damage to the fish body from oxidative stress. In addition, LA can also be used as an antiviral drug against a variety of viral diseasesThe viruses have inhibitory effect, and include human immunodeficiency virus (HIV-1), vaccinia virus (VACV), coronavirus, etc. By inhibiting the activation of NF- κ B-dependent long terminal repeats, LA is able to prevent HIV-1 gene integration into the host genome, thereby significantly inhibiting HIV-1 replication. In several cell lines, such as human epithelial cells, fibroblasts and macrophages, LA shows significant inhibition of later-stage gene expression of VACV, thereby exerting an anti-VACV function. LA also inhibits the invasion process of SARS-CoV-2 by increasing the intracellular pH, thereby enhancing the human defense against new coronavirus. However, at present, there is no relevant literature demonstrating that LA has activity against VHSV.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides the application of the alpha-lipoic acid in the aspect of resisting the viral hemorrhagic septicemia virus infection of fish.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides application of alpha-lipoic acid in preparing a medicament for resisting fish viral hemorrhagic septicemia.

The invention also provides application of the alpha-lipoic acid in preparing a medicine for resisting fish viral hemorrhagic septicemia virus infected cells.

Preferably, the fish viral hemorrhagic septicemia virus infection resisting cell inhibits replication of fish viral hemorrhagic septicemia virus.

Preferably, the cells include, but are not limited to, FHM cells.

The results of in vitro cytotoxicity experiments show that the half lethal concentration CC of LA to FHM cells₅₀472.6 μ M; the invention detects that LA has protective effect on FHM cells under VHSV infection, and finds that the half maximum effect concentration of LA is 42.7 mu M; the invention detects the capability of LA in resisting VHSV infection, and the medicine is before virus infection and before virus infectionDuring infection, the medicine is added into cells after virus infection, and the CPE is obviously reduced after 24 hours and 36 hours, and the replication of virus nucleic acid in the cells is obviously reduced; the invention detects the process of LA resisting VHSV infection, and finds that LA treatment is added in the process of VHSV replication, so that the method can generate obvious inhibition effect on the replication of VHSV G genes in cells.

The invention also provides a medicine for resisting fish viral hemorrhagic septicemia virus infected cells, which takes alpha-lipoic acid as a main active ingredient.

Preferably, the medicament further comprises a pharmaceutically acceptable carrier and/or excipient. Namely, the medicine can be mixed with a pharmaceutically acceptable carrier or excipient to prepare a composition.

The excipient refers to diluents, binders, lubricants, disintegrants, cosolvents, stabilizers and the like which can be used in the pharmaceutical field, and some medicinal matrixes. The carrier is functional pharmaceutical adjuvant available in the pharmaceutical field, and comprises surfactant, suspending agent, emulsifier and some novel medicinal polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid polylactic acid copolymer (PLGA), hyaluronic acid, etc.

Preferably, the medicament is prepared into powder, granules or liquid medicine. In addition, the medicine can also be prepared into other clinically acceptable dosage forms.

Compared with the prior art, the invention has the beneficial effects that:

in order to find a medicine with antiviral activity to the viral hemorrhagic septicemia virus, the research discovers that the alpha-lipoic acid plays a remarkable antiviral function in the process of infecting cells by the viral hemorrhagic septicemia virus, has a good effect on treating the viral hemorrhagic septicemia, and can be used for treating the fish infected with the viral hemorrhagic septicemia virus. The invention not only provides a new application of alpha-lipoic acid, but also provides a new direction for treating the fish viral hemorrhagic septicemia.

Drawings

FIG. 1 shows the cytotoxicity (half lethal concentration CC) of LA on FHM cells₅₀472.6 μ M);

FIG. 2 shows that LA protects FHM cells under VHSV infection (half maximal effect concentration is 42.7. mu.M);

FIG. 3 is a graph of the effect of LA on viral infection when added before, simultaneously with, or after infection of VHSV (A is added before infection; B is added simultaneously; C is added after infection;^*P<0.05，^**P<0.01)；

FIG. 4 is an electron microscope image of LA affecting viral infection when added before, simultaneously with or after VHSV infection;

FIG. 5 shows the effect of LA on the VHSV infection cycle (A is viral adsorption; B is viral endocytosis; C is viral gene replication; D, E is cell pretreatment;^**P<0.01)。

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

Example 1 cellular Activity assay for protection of cells by LA

(1) Cell lines and cell cultures

The cell line used in the experiment was fat head carp muscle cell line (FHM), the FHM cells were cultured in an M199 medium containing 10% Fetal Bovine Serum (FBS) at 28 ℃ in a constant temperature incubator, and the medium was changed to an M199 maintenance medium containing 2% FBS during the experiment.

(2) CCK-8 was used to test the cytotoxicity of LA on FHM

Cell Counting Kit-8(CCK-8) was used to test the toxicity of LA on FHM. FHM cells in the logarithmic growth phase are inoculated in a 96-well plate in advance, 100 mu L of cell suspension is added into each well, and the cells are cultured at 28 ℃ until the coverage density of the cells reaches 80-90%. The original medium was aspirated off, maintenance medium containing different final concentrations of LA (10. mu.M, 20. mu.M, 30. mu.M, 50. mu.M, 100. mu.M, 200. mu.M, 400. mu.M and 800. mu.M) was added, and the maintenance medium containing 0.1% DMSO was used as a negative control, and both the experimental and control groups were set in 4 duplicate wells and cultured at 28 ℃ for another 48 h. After the culture is finished, replacing 100 mu L of maintenance medium for each well, adding 10 mu L of CCK-8 reagent, placing the culture plate in a constant-temperature incubator at 28 ℃ for incubation for 2h, and then measuring the light absorption value of each well at 450nm by using a living cell imager, wherein the data come from three independent repeated experiments and an average value is taken. Cell viability per well was calculated as follows:

cell viability ═ 100% (experimental well absorbance-blank well absorbance)/(control well absorbance-blank well absorbance).

Plotting with GraphPad Prism 8.0.2 and calculating the median lethal concentration CC of LA₅₀。

As shown in FIG. 1, CC of LA under the experimental conditions₅₀At 472.6. mu.M, 50. mu.M LA had no significant effect on cell viability.

(3) CCK-8 was used to test the protective effect of LA on FHM

CCK-8 is adopted to detect the activity of FHM cells after LA treatment is carried out on VHSV infection. FHM cells in the logarithmic growth phase are inoculated in a 96-well plate in advance, 100 mu L of cell suspension is added into each well, and the cells are cultured at 28 ℃ until the coverage density of the cells reaches 80-90%. The original medium was aspirated off, and LA (0. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 40. mu.M and 50. mu.M) and 10. mu.M, which had contained different concentration gradients, were added^0.86TCID₅₀Maintenance medium for VHSV. And to contain 0.1% DMSO and 10^0.86TCID₅₀The maintenance medium of (2) was used as a negative control, and the experimental group and the control group were each provided with 4 duplicate wells and cultured at 28 ℃ for another 48 hours. After the culture is finished, replacing 100 mu L of maintenance medium for each well, adding 10 mu L of CCK-8 reagent, placing the culture plate in a constant-temperature incubator at 28 ℃ for incubation for 2h, and then measuring the light absorption value of each well at 450nm by using a living cell imager, wherein the data come from three independent repeated experiments and an average value is taken. Cell viability per well was calculated as follows:

cell viability ═ 100% (experimental well absorbance-blank well absorbance)/(control well absorbance-blank well absorbance).

Plotting with GraphPad Prism 8.0.2 and calculating the half maximal effect concentration EC of LA₅₀。

As shown in FIG. 2, EC of LA under the experimental conditions₅₀At 42.7 μ M, 50 μ M LA treatment was able to maintain cell viability in approximately 90% of VHSV infected cells.

Example 2qRT-PCR detection of anti-VHSV Activity of LA

1. Drug treatment

FHM cells in a logarithmic growth phase are inoculated in a 12-well plate in advance, 1mL of cell suspension is added into each well, the cells are cultured at 28 ℃ until the cell coverage density reaches 80-90%, and after a new maintenance culture medium is replaced, the VHSV resistance of LA is detected in 3 different drug treatment modes.

(1) LA addition to cells before VHSV infection

FHM cells were replaced with maintenance medium, LA was added to a final concentration of 50. mu.M, incubated at 28 ℃ for 4h, and then inoculated to a final concentration of 10^1.5TCID₅₀And culturing at 28 ℃ for 24h and 36h, respectively. After incubation, cells were digested with 0.25% trypsin and collected in rnase-free centrifuge tubes. The negative control was 0.1% DMSO and the positive control was 8 μ M Ribavirin (ribivirin).

(2) Co-addition of LA and VHSV to cells

FHM cells were replaced with maintenance medium, and LA and 10 were added to a final concentration of 50. mu.M^1.5TCID₅₀And (3) VHSV, co-cultured at 28 ℃ for 24h and 36 h. After incubation, cells were digested with 0.25% trypsin and collected in rnase-free centrifuge tubes. The negative control was 0.1% DMSO and the positive control was 8 μ M Ribavirin.

(3) The drug is added to the cells after viral infection

FHM cells were replaced with maintenance medium and inoculated 10^1.5TCID₅₀After VHSV, the cells were cultured at 28 ℃ for 4h, followed by addition of LA at a final concentration of 50. mu.M and co-culture at 28 ℃ for 24h and 36 h. After the culture was completed, cells were digested with 0.25% trypsin and collected in the absence of RNEnzyme A centrifuge tube. The negative control was 0.1% DMSO and the positive control was 8 μ M Ribavirin.

2. RNA extraction

After the drug treatment is finished, the TriZol method is used for extracting the total RNA of the cells in different samples. Centrifuging the collected cell suspension at 1000rpm at room temperature for 10min, removing the supernatant, adding 500. mu.L TriZol reagent, blowing, mixing until no obvious precipitate is formed, and standing at room temperature for 5 min. Then 100. mu.L of chloroform was added, vortexed, and allowed to stand at room temperature for 10min, followed by centrifugation at 13000rpm for 15min at 4 ℃. Dividing the liquid in the centrifugal tube into three layers, transferring the upper colorless transparent liquid into a new RNA-free enzyme centrifugal tube, adding equal amount of isopropanol, gently mixing, standing at room temperature for 10min, and centrifuging at 13000rpm for 15min at 4 ℃; removing liquid by suction, adding 800 μ L75% ethanol prepared with enucleated enzyme water, and centrifuging at 7500rpm at 4 deg.C for 5 min; sucking and removing the supernatant, and airing RNA precipitate for 10min at room temperature; and dissolving the RNA precipitate by 8 mu L of nucleic acid-free water to obtain the total RNA of the cells.

3. cDNA Synthesis

The resulting RNA was Reverse transcribed using the Reverse transcription Mix (Promega) Reverse transcription kit according to the instructions. The 10 μ L reaction was as follows: mu.L of Reaction Buffer and 1. mu.L of enzyme Mix were added to a 200. mu.L nuclease-free centrifuge tube, followed by addition of a total of no more than 5. mu.g of sample RNA template, made up to 10. mu.L with nuclease-free water. The reverse transcription reaction procedure was as follows: 42 ℃ for 20 min; at 85 deg.C, 5s, 16 deg.C, 2min to obtain cDNA, and storing at-20 deg.C.

4. Real-time fluorescent quantitative PCR

The Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR, qRT-PCR) adopts Promega GoTaq^TMqPCR kit, performed according to instructions. The 10 μ L reaction was as follows: mu.L of qPCR Mix (2X), 1. mu.L of cDNA template, 0.5. mu.L of forward primer (5'-AACTGTCTCCAAAGAAGTGTGT-3', 10. mu.M), 0.5. mu.L of reverse primer (5'-GCCATCAAGGAGATAATGTG-3', 10. mu.M) and 3. mu.L of water. The qRT-PCR reaction conditions were: 95 deg.C, 1min, 95 deg.C, 15s, 60 deg.C, 30s, 72 deg.C, 30s, 40 cycles. After the reaction is finished, the FHM beta-actin is used as an internal reference gene to carry out normalization processing on the sample. Use 2^{^-ΔΔCT}The method calculates the relative expression quantity of the target gene VHSV G.

As shown in fig. 3 and 4, in all of the 3 treatment modalities of LA, the expression of VHSV G gene was significantly suppressed, and the intracellular viral load was significantly reduced, indicating that LA had an anti-VHSV effect.

Implementation of 3qRT-PCR to detect the influence of LA on VHSV infection process

1. Drug treatment

FHM cells in a logarithmic growth phase are inoculated in a 12-well plate in advance, 1mL of cell suspension is added into each well, the cells are cultured at 28 ℃ until the cell coverage density reaches 80-90%, a new maintenance culture medium is replaced, and then the influence of LA on the VHSV infection process is researched by adopting 5 different treatment modes for LA.

(1) Detection of the Effect of LA on VHSV adsorption

First, FHM was pre-cooled at 4 ℃ for 10min, the FHM cells were replaced with maintenance medium, LA was added to a final concentration of 50. mu.M and inoculated for 10min^1.8TCID₅₀VHSV of (1), cultured at 4 ℃ for 2 h. Subsequently, the culture medium was aspirated, washed 3 times with PBS, replaced with fresh maintenance medium, and placed in a 28 ℃ incubator for further 22 h. Cells were collected in rnase-free centrifuge tubes using 0.25% trypsin digestion. The negative control was 0.1% DMSO and the positive control was 8 μ M Ribavirin.

(2) Detecting the effect of LA on VHSV endocytosis

Precooling FHM at 4 deg.C for 10min, replacing FHM cells with maintenance medium and inoculating 10^1.8TCID₅₀Culturing the VHSV at 4 ℃ for 2 h; after 2h, the medium was aspirated, washed 3 times with PBS, replaced with a maintenance medium containing LA at a final concentration of 50. mu.M, and cultured at 28 ℃ for 2 h; the culture medium was then aspirated, washed 3 times with PBS, replaced with maintenance medium, and incubated at 28 ℃ for an additional 20 h. The cells were digested with 0.25% trypsin and collected in rnase-free centrifuge tubes. The negative control was 0.1% DMSO and the positive control was 8 μ M Ribavirin.

(3) Detection of the Effect of LA on VHSV replication

FHM cells were replaced with maintenance medium and inoculated 10^1.8TCID₅₀Culturing the VHSV at 28 ℃ for 4 h; subsequently, the original medium was aspirated, washed 3 times with PBS, and replaced with a solution already containing LA at a final concentration of 50. mu.MThe medium was maintained and incubation continued at 28 ℃ for 4 h. The cells were digested with 0.25% trypsin and collected in rnase-free centrifuge tubes. The negative control was 0.1% DMSO and the positive control was 8 μ M Ribavirin.

(4) Detection of the sensitive Effect of LA-pretreated FHM cells on VHSV

Replacing FHM cells with maintenance medium, adding LA with final concentration of 50 μ M, and culturing at 28 deg.C for 4 hr; the original medium was aspirated, washed 3 times with PBS and inoculated 10 times^1.8TCID₅₀Further culturing the VHSV of (1) at 28 ℃ for 4 h; the original medium was aspirated, washed 3 times with PBS, replaced with fresh maintenance medium, and incubated at 28 ℃ for an additional 20 h. The cells were digested with 0.25% trypsin and collected in rnase-free centrifuge tubes. The negative control was 0.1% DMSO and the positive control was 8 μ M Ribavirin.

(5) Testing the Effect of LA Pre-treated cells on VHSV infection

1.5mM LA was mixed with 10^1.8TCID₅₀The VHSV is mixed evenly and incubated for 4h at the temperature of 4 ℃, then the mixture is inoculated into cells, and the cells are placed in a constant temperature incubator at the temperature of 28 ℃ for culture for 4 h; the original medium was aspirated, washed 3 times with PBS, and replaced with fresh maintenance medium to remove unadsorbed VHSV virions and free LA, and the cells were placed in a 28 ℃ incubator for further 20 h. The cells were digested with 0.25% trypsin and collected in rnase-free centrifuge tubes. The negative control was 0.1% DMSO and the positive control was 8 μ M Ribavirin.

2. VHSV infection detection

RNA extraction, cDNA Synthesis, and real-time fluorescent quantitative PCR were performed as in example 2.

As shown in fig. 5, LA treatment was added during intracellular replication of VHSV, and expression of VHSV G gene could be significantly inhibited. LA is added in the processes of adsorption and endocytosis, after cells are pretreated by LA and inoculated with virus or after LA and virus are premixed, the LA has no obvious influence on the expression of VHSV G genes, and the LA can inhibit the replication of VHSV.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

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