Application of MG-132 in preparing medicine for resisting nervous necrosis virus

Abstract

The invention discloses an application of MG-132 in preparation of a medicine for resisting nervous necrosis virus. Researches show that the MG-132 can significantly reduce cytopathic effects caused by nervous necrosis virus infection, reduce the number of positive cells expressing RGNNV capsid proteins, and also can significantly reduce the mRNA transcriptional level and protein expression level of RGNNV key genes and the copy number of viral genome RNA; and along with the prolonging of RGNNV infection time, the MG-132 can also significantly inhibit virus gene transcription, protein synthesis and genome replication, and reduce virus titer. Therefore, the virulence of RGNNV on cells can be remarkably reduced through MG-132 treatment, the effect of resisting nervous necrosis virus is achieved, the cytotoxicity is low, and the safety is good. The invention provides a new drug intervention strategy for prevention and treatment of nervous necrosis virus infection, and has important application value for prevention and treatment of NNV related diseases in aquaculture.

Description

Technical Field

[0001] This invention relates to the field of aquatic animal pharmacology, specifically to the application of MG-132 in the preparation of drugs against nerve necrosis virus. Background Technology

[0002] Grouper belongs to the order Perciformes and the family Serranidae, and is widely distributed in tropical and subtropical waters. Currently, over 100 species of grouper have been recorded globally, with 67 species belonging to 10 genera found in my country's waters, geographically covering areas from Zhejiang to Hainan. Due to its tender flesh, delicious flavor, tolerance to short-term holding, and suitability for live transport, grouper plays a vital role in marine aquaculture. Statistics show that over 90% of global grouper production comes from Asia, where large-scale grouper farming is practiced.

[0003] In recent years, with the continuous progress and innovation of aquaculture technology, the grouper industry has developed rapidly. However, the frequent outbreaks of viral diseases pose a serious threat to the industry's development, among which the damage caused by Nervous Necrosis Virus (NNV) is the most significant. This virus can cause viral encephalopathy and retinopathy (VER), with a mortality rate of up to 100% in juvenile fish populations, making it one of the most destructive aquatic pathogens currently known. NNV belongs to the Nodaviridae family. Its virus particles have an icosahedral symmetry structure, a diameter of approximately 25-30 nm, and no envelope structure. The viral genome consists of two single-stranded RNAs: RNA1 (approximately 3.1 kb) encodes RNA-dependent RNA polymerase (RdRp); RNA2 (approximately 1.4 kb) encodes capsid protein (CP). Based on differences in molecular characteristics, NNV can currently be classified into four genotypes: Red Grouper Nervous Necrosis Virus (RGNNV), Striped Bass Nervous Necrosis Virus (SJNNV), Tiger Pufferfish Nervous Necrosis Virus (TPNNV), and Flounder Nervous Necrosis Virus (BFNNV). Among these, the RGNNV genotype is the most widespread, with a broad host range. RGNNV primarily attacks the central nervous system and retinal tissue of fish, and infected individuals typically exhibit abnormal swimming behavior, melanization, and other typical clinical symptoms. The pathogenic mechanism of this virus involves a complex network of interactions between viral proteins and various host proteins, and its specific infection mechanism is not yet fully understood. Due to the lack of effective control measures, NNV infection has caused enormous economic losses to the global grouper aquaculture industry, necessitating the development of new control technologies to ensure the sustainable development of the industry.

[0004] MG-132 (Z-Leu-Leu-Leu-al) is a potent proteasome and calpain inhibitor, with an IC50 value of [missing information]. 50 The concentrations were 100 nM and 1.2 μM, respectively. MG-132 effectively blocked the proteolytic activity of the 26S proteasome complex. MG-132 is a peptidaldehyde and an autophagy activator. MG-132 also induces apoptosis. The article "Targeting PTGS2 / NF-κB Pathway: MG-132's Role in Reducing Ischemic Stroke Injury" disclosed that the proteasome inhibitor MG-132 can significantly inhibit the PTGS2 / NF-κB pathway, reduce pro-inflammatory factors such as TNF-α and IL-1β, and reduce cerebral infarction volume and MDA levels. However, there are currently no reports on the use of the proteasome inhibitor MG-132 in the treatment of fish diseases caused by neuronecrosis virus. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of MG-132 in the preparation of drugs against nerve necrosis virus.

[0006] A second objective of this invention is to provide the use of MG-132 in the preparation of medicaments for the prevention or treatment of viral encephalopathy and retinopathy in fish caused by neuronecrosis virus infection.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: Cellular experiments of this invention showed that MG-132 can significantly alleviate the cytopathic effect (CPE) induced by neuronecrosis virus (RNNV) infection. Fluorescence detection revealed a significant reduction in the number of positive cells expressing the RGNNV capsid protein (CP) in the MG-132-treated group. Quantitative results showed that MG-132 downregulated the mRNA transcription levels of key RGNNV genes (CP, RdRp) and significantly reduced the copy number of viral genomic RNA (including +ssRNA and -ssRNA). Western blot analysis confirmed that MG-132 effectively inhibited the expression level of the capsid protein CP. With prolonged RGNNV infection time, MG-132 also significantly inhibited viral gene transcription, protein synthesis, and genome replication, reducing viral titer. Therefore, MG-132 treatment can significantly reduce the virulence of RGNNV on cells, exhibiting anti-RNNV efficacy with low cytotoxicity and good safety.

[0008] Therefore, the present invention provides the use of MG-132 in the preparation of a drug for treating nerve necrosis virus.

[0009] The present invention also provides the use of MG-132 in the preparation of medicaments for the prevention or treatment of viral encephalopathy and retinopathy in fish caused by neuronecrosis virus infection.

[0010] Furthermore, the fish in question is a grouper.

[0011] Furthermore, the nerve necrosis virus is the nerve necrosis virus of the red-spotted grouper.

[0012] Furthermore, the drug achieves its therapeutic effect by reducing cytopathic effects and inhibiting the expression of key genes in neuronecrosis virus.

[0013] Furthermore, the key genes of the neural necrosis virus are the capsid protein CP and the virus-dependent RNA polymerase RDRP.

[0014] Furthermore, the concentration of MG-132 in the drug is 0.1–50 μM.

[0015] Furthermore, the concentration of MG-132 in the drug is 1 μM.

[0016] Furthermore, the drug also contains pharmaceutically acceptable excipients.

[0017] Furthermore, the excipients are selected from pharmaceutically acceptable carriers and excipients.

[0018] Furthermore, the drug is formulated as an injection, powder, capsule, or granule.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of MG-132 in the preparation of drugs against neuronecrosis virus (RNNV). Cellular experiments of this invention show that MG-132 can significantly alleviate the cytopathic effect (CPE) induced by RNNV infection. Fluorescence detection revealed a significant reduction in the number of positive cells expressing the RGNNV capsid protein (CP) in the MG-132-treated group. Quantitative results showed that MG-132 downregulated the mRNA transcription levels of key RGNNV genes (CP, RdRp) and significantly reduced the copy number of viral genomic RNA (including +ssRNA and -ssRNA). Western blot analysis confirmed that MG-132 effectively inhibited the expression level of the capsid protein CP. With prolonged RGNNV infection time, MG-132 also significantly inhibited viral gene transcription, protein synthesis, and genome replication, reducing viral titer. Therefore, MG-132 treatment can significantly reduce the virulence of RGNNV on cells, exhibiting anti-RNNV efficacy with low cytotoxicity and good safety. This invention provides a new drug intervention strategy for the prevention and treatment of nerve necrosis virus infection, and has important application value for the prevention and control of NNV-related diseases in aquaculture. Attached Figure Description

[0020] Figure 1 This is a graph showing the results of detecting the viability of GS cells treated with different concentrations of MG-132 using the CCK-8 assay in Example 2 of the present invention.

[0021] Figure 2 The graph shows the results of viral infection levels after treating GS cells with different concentrations of MG-132, as provided in Example 3 of this invention.

[0022] Figure 3 This is a graph showing the results of detecting the transcriptional and protein levels of viral genes after treating GS cells with different concentrations of MG-132, as provided in Example 3 of this invention.

[0023] Figure 4 This is a graph showing the detection results of the fluorescence expression level of the viral CP protein in GS cells treated with MG-132 after infection with RGNNV in Example 4 of the present invention.

[0024] Figure 5 The figure shows the results of detecting the transcriptional levels of CP and RDRP genes, and the expression levels of CP protein and β-tubulin in MG-132-treated GS cells infected with RGNNV at different times in Example 5 of this invention.

[0025] Figure 6 This figure shows the results of detecting the expression levels of viral genomic RNA +ssRNA and -ssRNA at different time points in GS cells treated with MG-132 and infected with RGNNV in Example 5 of this invention.

[0026] Figure 7 This is a graph showing the results of detecting the viral titer of MG-132-treated GS cells infected with RGNNV at different times in Example 5 of the present invention. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0028] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0029] Example 1: Preparation and Identification of RGNNV Preparation of RGNNV: Grouper spleen (GS) cells were seeded in 25cm... 2 The cells were cultured in culture flasks at 28°C in a cell culture incubator until they adhered and reached the logarithmic growth phase. Red-spotted grouper nervous necrosis virus (RGNNV) was added to the culture system at a multiplicity of infection (MOI) of 2, and the cells were cultured for 48-72 h to observe the cytopathic effect. Once more than 80% of the cells showed typical vacuolation lesions, the cell culture was collected, frozen at -80°C for 30 min, and then thawed at room temperature. This freeze-thaw cycle was repeated three times to fully lyse the cells and release the virus particles.

[0030] Virus aliquoting and storage: Centrifuge at 12,000×g for 10 min at 4℃ to remove cell debris, collect the supernatant, aliquot and store at -80℃ for long-term storage.

[0031] Virus identification: RT-PCR, electron microscopy, and TCID were used. 50 Methods such as assays are used to identify the virus's molecular biology and biological activity, ensuring that the virus titer and purity meet experimental requirements.

[0032] Example 2: Drug toxicity test of MG-132 I. Experimental Methods Logarithmic growth phase GS cells were seeded at 100 μL per well in 96-well plates using Leibovitz's L-15 complete medium containing 10% fetal bovine serum (FBS) and cultured overnight at 28°C to allow cell adhesion. MG-132 cells were serially diluted with L-15 medium containing 10% FBS to final concentrations of 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 50 μM, at 100 μL per well, with 6 replicates per group. A blank control group containing 0.1% DMSO was also included. After culturing the cells at 28°C for 24 h, 10 μL of CCK-8 solution was added to each well, gently vortexed, and incubated at 28°C in the dark for 1–4 h. The absorbance (OD value) of each well at a wavelength of 450 nm was measured using a multi-functional microplate reader. The relative cell viability (%) was calculated as follows: (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0033] II. Experimental Results The results are as follows Figure 1 As shown, compared with the DMSO control group, GS cell viability remained above 80% after 24 h of treatment with MG-132 at concentrations ranging from 0.02 to 50 μM (P>0.05), with no significant difference in cell viability between the 0.02–1 μM concentration groups and the control group. This indicates that the MG-132 concentration used in this invention has no significant toxicity to GS cells and is suitable for subsequent viral infection experiments.

[0034] Example 3: Antiviral activity detection experiment of MG-132 I. Experimental Methods GS cells in logarithmic growth phase were seeded into 24-well culture plates and cultured at 28°C until a monolayer of adherent cells formed. The original culture medium was discarded, and fresh culture medium with different concentrations of MG-132 was added. A DMSO solvent control group was also included, with three replicates per group. After 2 hours of pretreatment, RGNNV virus solution prepared in Example 1 was inoculated at an MOI of 2. 24 hours after infection, the cytopathic effects of each group were observed and recorded using an inverted optical microscope. Cell samples from different groups were collected, and total RNA was extracted using the Ce1l Total RNA Isolation Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., product number RC112). cDNA synthesis was performed using the reverse transcription system (10 μL reaction volume) in Table 1 and the procedure in Table 2. The viral gene transcription level was detected using the quantitative fluorescence reaction system in Table 3 and the quantitative fluorescence amplification procedure in Table 4, and viral protein expression was analyzed by Western blot.

[0035] 1. The Cell Total RNA Isolation Kit is operated as follows: (1) Add 250 μL of Buffer cRL1 lysis buffer to the collected cell sample, shake well and incubate on ice for 30 min to allow the cells to lyse completely; (2) Transfer the lysis buffer to a DNA-Cleaning Column, centrifuge at 12,000 rpm (=13,400×g) for 2 min, discard the purification column, and retain the supernatant in the collection tube; (3) Add 1.6 times the volume of Buffer cRL2 (about 400 μL) to the supernatant obtained in step (2) and mix gently; (4) Transfer the entire mixture to an RNA-only column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid; (5) Add 500uL Buffer RW1 to the purification column of step (4), centrifuge at 12,000rpm for 1 min, and discard the waste liquid; (6) Add 700uL of Buffer RW2 to the above purification column, centrifuge at 12,000rpm for 1min, and discard the waste liquid; (7) Repeat the Buffer RW2 washing step once; (8) Centrifuge the empty column at 12,000 rpm for 2 minutes to remove residual ethanol; (9) Transfer the purification column to a new 1.5 mL RNase-free EP tube, add 30 μL of RNase-Free ddH2O preheated at 65 °C to the center of the membrane of the purification column, and let stand at room temperature for 2 minutes; (10) Collect the RNA solution by centrifugation at 12,000 rpm for 1 minute; (11) Take 4 μL of RNA sample for 1.5% agarose gel electrophoresis detection; (12) Take 1 μL of RNA sample and use Nanodrop to determine its concentration and purity; (13) Heat the remaining RNA at 65°C for 5 min, then immediately place it on ice for 5 min, and briefly centrifuge to collect the liquid from the tube wall.

[0036] (14) Using ReverTraAce ® The qPCR RT Kit (purchased from Shanghai Xinrui Biotechnology Co., Ltd., product number FSQ-101B) was used to prepare a 10 μL reverse transcription reaction system according to Table 1; the reverse transcription program of the PCR instrument was set according to Table 2; the product was used immediately or stored at 4℃ for short-term storage and at -20℃ for long-term storage.

[0037] Table 1 Reverse transcription system

[0038] Table 2 Reverse Transcription Procedure

[0039] 2. The experimental procedures for real-time PCR are as follows: A 10 μL reaction mixture was prepared using 2×SYBR Green Real-time PCR Master Mix, as shown in Table 3, and the reaction program was set according to Table 4. Each sample was tested in triplicate, with β-Actin used as an internal control gene. -ΔΔCt The method calculates the relative expression level of the target gene.

[0040] Table 3. Fluorescence Quantitative System

[0041] Table 4. Quantitative Fluorescence Procedure

[0042] 3. The experimental procedure for Western blot is as follows: (1) Sample preparation: Collect cell samples from 6-well plates into 1.5 mL centrifuge tubes, add 40 μL Pierce IP lysis buffer, and lyse on ice for 30 min; add 10 μL 5× protein loading buffer and mix well; heat in a boiling water bath at 100℃ for 5 min to denature the protein; centrifuge the sample at 12,000×g for 5 min and collect the supernatant; load the supernatant immediately or store it at -20℃ for later use; (2) Preparation of separating and stacking gels: Prepare 10% separating gel and 5% stacking gel using the SDS-PAGE gel preparation kit (KeyGen BioTECH); (3) Sample loading: Load the prepared gel into the vertical electrophoresis tank, add 1×SDS-PAGE electrophoresis buffer, load 20 μg of protein sample into each well, and add pre-stained protein marker at the same time; (4) Electrophoretic separation: stacking gel electrophoresis at low pressure 60V for 30min, separating gel electrophoresis at high pressure 120V for 60min; (5) Transfer: The PVDF membrane (Milipore) was activated with methanol for 30 seconds before use. After electrophoresis, the protein gel was removed and the transfer clip was assembled in the order of "sponge-filter paper-gel-membrane-filter paper-sponge". The transfer was carried out at a constant current of 100mA for 60 minutes. (6) Blocking: After the transfer, block the membrane in 5% skim milk prepared with PBST on a shaker at room temperature for 2 hours; (7) Primary antibody incubation: Dilute the primary antibody according to the instructions and incubate at room temperature on a horizontal shaker for 2 hours or incubate overnight in a refrigerator at 4°C; (8) Secondary antibody incubation: Wash the membrane twice with PBST, 5 min each time, and put the membrane into HRP-labeled secondary antibody (1:5000) and incubate at room temperature for 45 min; (9) Color development: Wash the membrane 3 times with PBST for 10 min each time; perform color development using the enhanced HRP-DAB substrate color development kit (TIANGEN); (10) Photographing and analysis: The signal was collected by exposure using a chemiluminescence imaging system and photographed. The grayscale value was analyzed using ImageJ software.

[0043] II. Experimental Results Depend on Figure 2 It can be seen that, compared with the DMSO control group, GS cells pretreated with different concentrations of MG-132 maintained good cell monolayer integrity 24 h after RGNNV (MOI=2) infection. The degree of cell vacuolation in the MG-132 treatment group was lower than that in the control group. The above results indicate that MG-132 has anti-RGNNV efficacy.

[0044] Depend on Figure 3 It can be seen that after 24 hours of infection with RGNNV (MOI=2), GS cells pretreated with different concentrations of MG-132 showed a significant decrease in the transcriptional levels of the RGNNV capsid protein (CP) gene and the transcriptional levels of virus-dependent RNA polymerase (RDRP). Furthermore, the synthesis of the CP protein was inhibited, and the inhibitory effect was concentration-dependent.

[0045] Example 4: Fluorescence detection analysis of the antiviral activity of MG-132 I. Experimental Methods The effect of 1 μM MG-132 treatment on the anti-RGNNV activity of cells was analyzed by fluorescence analysis. Specifically, GS cells were seeded in 24-well plates and cultured in L-15 medium containing 10% FBS at 28°C until a monolayer of adherent cells formed. The original medium was discarded, and fresh medium containing 1 μM MG-132 was added, with a DMSO solvent control group included. After 2 h of pretreatment, RGNNV virus solution prepared in Example 1 was inoculated at MOI=2 and cultured at 28°C for another 24 h. After 24 h, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 0.2% bovine serum albumin (BSA) (Sigma). Then, the cells were incubated with primary antibody against RGNNV CP diluted in 0.2% BSA at room temperature for 2 h. After washing three times with PBS, secondary antibody Alexa Fluor 555 coupled with anti-mouse IgG Fab2 (1:200) was added and incubated at room temperature for 2 h.

[0046] II. Experimental Results The results observed under CLSM are as follows Figure 4 As shown, compared with the control group, 24 h after infection with RGNNV, the number of cells in the MG132-treated group that expressed RGNNV CP was significantly reduced, and the fluorescence intensity was decreased. The results indicate that MG132 can exert its antiviral effect by directly inhibiting the expression of the viral structural protein CP.

[0047] Example 5: Analysis of Antiviral Activity of MG-132 at Different Time Points I. Experimental Methods 1. The dynamic inhibitory effect of MG-132 on RGNNV infection was evaluated using a time-gradient experimental system. GS cells were seeded in 24-well plates and cultured at 28°C until a monolayer of adherent cells formed. After pretreatment with 1 μM MG-132 for 2 hours, the experimental group was inoculated with RGNNV virus at an MOI of 2. RNA and protein samples were collected at 12h, 24h, and 36h. Total RNA was extracted from the cells using the Ce1l Total RNA Isolation Kit to detect the transcriptional levels of CP and RDRP genes, the protein expression level of the CP gene, and the expression levels of viral genomic RNA +ssRNA and -ssRNA. Protein expression of CP was analyzed by extracting protein with Pierce IP lysis buffer.

[0048] 2. The viral titers in the cells collected from the above three time-limited experimental groups were detected. The specific operating steps are as follows: (1) Seed the cell suspension in the logarithmic growth phase into a 96-well plate (100 μL / well). (2) After the cells form a monolayer, the virus solution is serially diluted tenfold using a culture medium containing 1% serum (10... -1 ~10 -10 ); (3) Each dilution was inoculated in 8 replicates (100 μL / well), and a control group without virus inoculation was set up at the same time; (4) Observe the cytopathic effect (CPE) daily and calculate TCID using the Reed-Muench method. 50 .

[0049] II. Experimental Results The results are as follows Figure 5 and Figure 6 As shown, with prolonged RGNNV infection time, the transcriptional levels of CP and RDRP genes significantly decreased, CP protein synthesis was significantly inhibited, and viral genomic RNA replication was significantly inhibited. This experiment confirms that MG-132 can effectively inhibit viral gene transcription, protein synthesis, and genome replication over a long period.

[0050] The results are as follows Figure 7 As shown, this is consistent with the results of Western blot: as the infection time increases, the viral titer of the 1 μM MG-132 treatment group shows a significant decreasing trend, indicating that MG-132 treatment can significantly reduce the virulence of RGNNV on cells.

[0051] The above results indicate that MG-132 treatment can significantly reduce the virulence of RGNNV on cells, has anti-neural necrosis virus efficacy, and exhibits low cytotoxicity and good safety.

Claims

1. Application of MG-132 in the preparation of drugs against neuronecrosis virus.

2. Application of MG-132 in the preparation of drugs for the prevention or treatment of viral encephalopathy and retinopathy in fish caused by neuronecrosis virus infection.

3. The application according to claim 2, characterized in that, The fish in question is a grouper.

4. The application according to claim 1 or 2, characterized in that, The nerve necrosis virus mentioned is the red-spotted grouper nerve necrosis virus.

5. The application according to claim 1 or 2, characterized in that, The drug achieves its therapeutic effect by reducing cellular lesions and inhibiting the expression of key genes in neuronecrosis virus.

6. The application according to claim 5, characterized in that, The key genes of the neuronecrosis virus are the capsid protein CP and the virus-dependent RNA polymerase RDRP.

7. The application according to claim 1 or 2, characterized in that, The concentration of MG-132 in the drug is 0.1–50 μM.

8. The application according to claim 7, characterized in that, The concentration of MG-132 in the drug is 1 μM.

9. The application according to claim 1 or 2, characterized in that, The drug also contains pharmaceutically acceptable excipients.

10. The application according to claim 1 or 2, characterized in that, The drug is prepared in the form of injection, powder, capsule, or granule.

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