Use of mg-132 in the preparation of a drug for resisting neurodegenerative virus

Abstract

The application discloses application of MG-132 in preparation of a drug for resisting nerve necrosis virus. Researches of the application show that MG-132 can significantly reduce the cytopathic effect caused by nerve necrosis virus infection, reduce the number of positive cells expressing RGNNV capsid protein, and also can significantly reduce the mRNA transcription level of a key gene of RGNNV, the protein expression level and the copy number of virus genomic RNA; and with the extension of RGNNV infection time, MG-132 can also significantly inhibit virus gene transcription, protein synthesis and genome replication, and reduce virus titer. Therefore, MG-132 treatment can significantly reduce the virulence of RGNNV to cells, has the efficacy of resisting nerve necrosis virus, and has low cytotoxicity and good safety. The application provides a new drug intervention strategy for prevention and treatment of nerve necrosis virus infection, and has important application value for prevention and control of NNV related diseases in aquaculture.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of aquatic animal pharmacology, in particular, to application of MG-132 in preparation of a drug for resisting nervous necrosis virus. BACKGROUND

[0002] Epinephelus belongs to Perciformes and Serranidae, and has a wide distribution in tropical and subtropical sea areas. At present, more than 100 species of Epinephelus have been recorded in the world, 67 species of which are distributed in China's sea areas, and their geographical range covers sea areas from Zhejiang to Hainan. Due to the excellent characteristics of Epinephelus such as tender meat, delicious taste, tolerance to temporary breeding, and suitability for live transportation, Epinephelus occupies an important position in the marine aquaculture industry. According to statistics, more than 90% of the global Epinephelus production comes from Asia, and large-scale breeding of Epinephelus is realized.

[0003] In recent years, with the continuous progress and innovation of breeding technology, the Epinephelus industry has developed rapidly. However, the frequent outbreak of viral diseases poses a serious threat to the development of the industry, among which the damage caused by nervous necrosis virus (NNV) is the most significant. This virus can cause viral encephalopathy and retinopathy (VER), and the mortality rate in juvenile fish populations can reach 100%, which has become one of the most destructive aquatic pathogens. NNV belongs to the family Nodaviridae, and its virion has a icosahedral symmetry structure with a diameter of about 25-30 nm and no envelope structure. The viral genome consists of two single-stranded RNAs: RNA1 (about 3.1 kb) encodes RNA-dependent RNA polymerase (RdRp); RNA2 (about 1.4 kb) encodes capsid protein (CP). According to the differences in molecular characteristics, NNV can be divided into four genotypes: RGNNV, SJNNV, TPNNV, and BFNNV. Among them, RGNNV is the most widely distributed and has a wide host range. RGNNV mainly attacks the central nervous system and retinal tissue of fish, and infected individuals usually exhibit typical clinical symptoms such as abnormal swimming behavior and body color darkening. The pathogenic mechanism of this virus involves a complex network of interactions between viral proteins and various host proteins, and the specific infection mechanism has not been fully elucidated. Due to the lack of effective prevention and control measures, NNV infection has caused huge economic losses to the global Epinephelus breeding industry, and it is urgent to develop new prevention and 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 IC 50 100 nM and 1.2 μM, respectively. MG-132 can effectively block the proteolytic activity of the 26S proteasome complex. MG-132 is a peptide aldehyde and an autophagy activator. MG-132 also induces apoptosis. It is disclosed in the article “Targeting PTGS2 / NF-κB Pathway: MG-132’s Role in Reducing Ischemic Stroke Injury” 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 the volume of cerebral infarction and the level of MDA. However, there is no relevant report on the use of the proteasome inhibitor MG-132 in the treatment of fish diseases caused by nervous necrosis virus. SUMMARY

[0005] The present application aims to overcome the above-mentioned defects and deficiencies in the prior art and provide the use of MG-132 in the preparation of a drug for resisting nervous necrosis virus.

[0006] A second object of the present application is to provide the use of MG-132 in the preparation of a drug for preventing or treating viral encephalopathy and retinopathy in fish caused by nervous necrosis virus infection.

[0007] The above-mentioned objects of the present application are achieved by the following technical solutions:

[0008] The cell experiments of the present application show that MG-132 can significantly reduce the cytopathic effect (CPE) caused by nervous necrosis virus infection; it is found through fluorescence detection that the number of positive cells expressing RGNNV capsid protein (CP) in the MG-132 treatment group is significantly reduced; it is found through quantitative results that MG-132 can down-regulate the mRNA transcription level of RGNNV key genes (CP, RdRp) and significantly reduce the copy number of viral genomic RNA (including +ssRNA and -ssRNA); Western blot analysis confirms that MG-132 can effectively inhibit the expression level of capsid protein CP. With the extension of the RGNNV infection time, MG-132 can also significantly inhibit viral gene transcription, protein synthesis and genome replication, and reduce the viral titer. Therefore, MG-132 treatment can significantly reduce the viral force of RGNNV on cells, has the effect of resisting nervous necrosis virus, and has low cytotoxicity and good safety.

[0009] Therefore, the present application provides the use of MG-132 in the preparation of a drug for resisting nervous necrosis virus.

[0010] The application also provides the use of MG-132 in the preparation of a medicine for preventing or treating viral encephalopathy and retinopathy of fish caused by nervous necrosis virus infection.

[0011] Further, the fish is Epinephelus.

[0012] Further, the nervous necrosis virus is Epinephelus akaara nervous necrosis virus.

[0013] Further, the medicine achieves the treatment by reducing cytopathic effect and inhibiting the expression of key genes of nervous necrosis virus.

[0014] Further, the key genes of nervous necrosis virus are capsid protein CP and virus-dependent RNA-dependent RNA polymerase RDRP.

[0015] Further, the concentration of MG-132 in the medicine is 0.1-50 μM.

[0016] Further, the concentration of MG-132 in the medicine is 1 μM.

[0017] Further, the medicine further contains pharmaceutically acceptable excipients.

[0018] Further, the excipients are selected from pharmaceutically acceptable carriers and excipients.

[0019] Further, the preparation of the medicine is injection, powder, capsule and granule.

[0020] Compared with the prior art, the application has the following beneficial effects:

[0021] The application provides application of MG-132 in preparation of a medicine for resisting a nerve necrosis virus. Cell experiments show that MG-132 can significantly reduce a cytopathic effect (CPE) caused by a nerve necrosis virus infection; it is found through fluorescence detection that the number of positive cells expressing a RGNNV capsid protein (CP) in an MG-132 treatment group is obviously reduced; it is found through quantitative results that the MG-132 can down-regulate mRNA transcription levels of key genes (CP and RdRp) of the RGNNV and significantly reduce copy numbers of viral genomic RNA (including +ssRNA and -ssRNA); Western blot analysis proves that the MG-132 can effectively inhibit expression levels of the capsid protein CP. With the extension of an RGNNV infection time, the MG-132 can also significantly inhibit viral gene transcription, protein synthesis and genomic replication, and reduce a viral titer. Therefore, the MG-132 treatment can significantly reduce a viral force of the RGNNV on cells, has an anti-nerve necrosis virus effect, and has low cytotoxicity and good safety. The application provides a new medicine intervention strategy for prevention and treatment of a nerve necrosis virus infection, and has important application value for prevention and control of NNV-related diseases in aquaculture. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A GS cell activity result graph treated by different concentrations of MG-132 in example 2 of the application is shown.

[0023] Figure 2 A virus infection degree result graph of GS cells treated by different concentrations of MG-132 in example 3 of the application is shown.

[0024] Figure 3 A viral gene transcription level and protein level detection result graph of GS cells treated by different concentrations of MG-132 in example 3 of the application is shown.

[0025] Figure 4 A virus CP protein fluorescence expression level detection result graph of GS cells treated by MG-132 and infected by RGNNV in example 4 of the application is shown.

[0026] Figure 5 A CP gene and RDRP gene transcription level, CP protein and beta-tubulin expression level detection result graph of GS cells treated by MG-132 and infected by RGNNV for different times in example 5 of the application is shown.

[0027] Figure 6 A viral genomic RNA +ssRNA and -ssRNA expression level detection result graph of GS cells treated by MG-132 and infected by RGNNV for different times in example 5 of the application is shown.

[0028] Figure 7 The results of detecting the virus titers of the GS cells treated with MG-132 in Example 5 of the present application at different times of infection with RGNNV are shown in the graph. DETAILED DESCRIPTION

[0029] The present application is further illustrated by the following description with reference to the accompanying drawings and specific examples. The reagents, methods and apparatuses used in the present application are conventional in the art unless otherwise specified. The examples do not in any way limit the present application.

[0030] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0031] Example 1 Preparation and identification of RGNNV

[0032] Preparation of RGNNV: Grouper spleen (GS) cells were inoculated into 25 cm 2 culture flasks and incubated in a 28℃ cell incubator until the cells reached the logarithmic growth phase. RGNNV was added to the culture system at a multiplicity of infection (MOI) of 2, and the cells were incubated for 48-72 h. The cytopathic effect was observed. When more than 80% of the cells showed typical vacuolation, the cell culture was collected and frozen at -80℃ for 30 min. The sample was then thawed at room temperature and subjected to repeated freeze-thawing for 3 times to fully lyse the cells and release the virus particles.

[0033] Virus sub-packaging and storage: The cell debris was removed by centrifugation at 12,000 x g for 10 min at 4℃, and the supernatant was collected and stored at -80℃ for long-term storage.

[0034] Virus identification: The virus was identified by molecular biology and biological activity using RT-PCR, electron microscopy and TCID 50 determination, to ensure that the virus titer and purity meet the experimental requirements.

[0035] Example 2 Drug toxicity detection experiment of MG-132

[0036] I. Experimental methods

[0037] The GS cells in logarithmic growth phase were inoculated in 96-well culture plates at 100 μL per well, and the culture medium was Leibovitz's L-15 complete medium containing 10% fetal bovine serum (FBS), and the cells were cultured in an incubator at 28°C overnight to adhere to the wall. MG-132 was gradiently diluted with L-15 medium containing 10% FBS to a final concentration of 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50 μM, 100 μL per well, and 6 replicate wells were set for each group, and a blank control group containing 0.1% DMSO was also set. After the cells were continuously cultured at 28°C for 24 h, 10 μL of CCK-8 solution was added to each well, and the cells were slightly shaken and mixed, and then incubated at 28°C for 1-4 h in the dark. The absorbance (OD value) of each well at 450 nm was measured by a multifunctional enzyme label instrument, and the relative cell viability (%) was calculated as follows: (OD value of the experimental group-OD value of the blank group) / (OD value of the control group-OD value of the blank group) x 100%.

[0038] II. Experimental results

[0039] The results are shown in Table 1. Figure 1 As shown in Table 1, compared with the DMSO control group, the cell viability of GS cells treated with MG-132 at a concentration ranging from 0.02 to 50 μM for 24 h was maintained at more than 80% (P>0.05), and the cell viability of the groups at a concentration ranging from 0.02 to 1 μM had no significant difference from the control group. It is indicated that the concentration of MG-132 used in the present application has no significant toxicity to GS cells, and is suitable for subsequent virus infection experiments.

[0040] Example 3. Detection of the antiviral activity of MG-132

[0041] I. Experimental method

[0042] The GS cells in logarithmic growth phase were inoculated in 96-well culture plates at 100 μL per well, and the culture medium was Leibovitz's L-15 complete medium containing 10% fetal bovine serum (FBS), and the cells were cultured in an incubator at 28°C overnight to adhere to the wall. MG-132 was gradiently diluted with L-15 medium containing 10% FBS to a final concentration of 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50 μM, 100 μL per well, and 6 replicate wells were set for each group, and a blank control group containing 0.1% DMSO was also set. After the cells were continuously cultured at 28°C for 24 h, 10 μL of CCK-8 solution was added to each well, and the cells were slightly shaken and mixed, and then incubated at 28°C for 1-4 h in the dark. The absorbance (OD value) of each well at 450 nm was measured by a multifunctional enzyme label instrument, and the relative cell viability (%) was calculated as follows: (OD value of the experimental group-OD value of the blank group) / (OD value of the control group-OD value of the blank group) x 100%.

[0043] 1. Cell Total RNA Isolation Kit kit is operated as follows:

[0044] (1) Add 250 μL Buffer cRL1 to the collected cell sample, shake well and stand on ice for 30 min to completely lyse the cells;

[0045] (2) Transfer the lysate to the DNA-Cleaning Column, centrifuge at 12,000 rpm (= 13,400 x g) for 2 min, discard the purification column, and retain the supernatant in the collection tube;

[0046] (3) Add 1.6 times the volume of Buffer cRL2 (about 400 μL) to the supernatant obtained in step (2), and mix gently;

[0047] (4) Transfer the mixture to the RNA-only Column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid;

[0048] (5) Add 500 uL Buffer RW1 to the purification column in step (4), centrifuge at 12,000 rpm for 1 min, and discard the waste liquid;

[0049] (6) Add 700 uL Buffer RW2 to the above purification column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid;

[0050] (7) Repeat the Buffer RW2 washing step once;

[0051] (8) Centrifuge the empty column at 12,000 rpm for 2 min to remove residual ethanol;

[0052] (9) Transfer the purification column to a new 1.5 mL RNase-free EP tube, and add 30 μL of 65°C preheated RNase-Free ddH2O to the center of the membrane of the purification column, and stand at room temperature for 2 min;

[0053] (10) Centrifuge at 12,000 rpm for 1 min to collect the RNA solution;

[0054] (11) Take 4 μL of the RNA sample for 1.5% agarose gel electrophoresis detection;

[0055] (12) Take 1 μL of the RNA sample to determine the concentration and purity with a Nanodrop;

[0056] (13) Heat the remaining RNA at 65°C for 5 min, then immediately ice bath for 5 min, and briefly centrifuge to collect the liquid on the tube wall.

[0057] (14) Use ReverTraAce ® qPCR RT Kit kit (purchased from Shanghai Xinrui Biotechnology Co., Ltd., product number FSQ-101B), prepare 10 μL reverse transcription reaction system according to Table 1; set the reverse transcription program of the PCR instrument according to Table 2; the product is immediately used or stored at 4℃ for short-term storage, and stored at -20℃ for long-term storage.

[0058] Table 1 Reverse transcription system

[0059]

[0060] Table 2 Reverse transcription program

[0061]

[0062] 2、The steps of the fluorescence quantitative PCR experiment are as follows:

[0063] Prepare 10 μL reaction system using 2×SYBR Green Real-time PCR Master Mix, prepare according to Table 3, and set the reaction program according to Table 4; set 3 duplicate holes for each sample, and use β-Actin as the internal reference gene; use 2 -ΔΔCt method to calculate the relative expression amount of the target gene.

[0064] Table 3 Fluorescence quantification system

[0065]

[0066] Table 4 Fluorescence quantification program

[0067]

[0068] 3、The steps of the Western blot experiment are as follows:

[0069] (1) Sample preparation: collect the cell samples in the 6-well plate into a 1.5 mL centrifuge tube, add 40 μL Pierce IP lysis buffer, and lyse on ice for 30 min; add 10 μL 5× protein loading buffer, mix well; heat at 100℃ in a boiling water bath for 5 min to denature the protein; centrifuge the sample at 12,000×g for 5 min, and take the supernatant; immediately load the supernatant or store it at -20℃ for standby;

[0070] (2) Prepare the separation gel and the concentration gel: use the SDS-PAGE gel preparation kit (KeyGen BioTECH) to prepare 10% separation gel and 5% concentration gel;

[0071] (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;

[0072] (4) Electrophoretic separation: stacking gel electrophoresis at low pressure 60V for 30min, separating gel electrophoresis at high pressure 120V for 60min;

[0073] (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.

[0074] (6) Blocking: After the transfer, block the membrane in 5% skim milk prepared with PBST on a shaker at room temperature for 2 hours;

[0075] (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;

[0076] (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;

[0077] (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);

[0078] (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.

[0079] II. Experimental Results

[0080] 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.

[0081] Depend on Figure 3It can be seen that after the GS cells pretreated by different concentrations of MG-132 were infected by RGNNV (MOI=2) for 24 h, the transcription levels of coat protein (CP) gene and RNA-dependent RNA polymerase (RDRP) gene of RGNNV were significantly decreased, and the synthesis of CP protein was inhibited, and the inhibitory effect was concentration-dependent.

[0082] Example 4 Fluorescence detection analysis of the antiviral activity of MG-132

[0083] I. Experimental method

[0084] The effect of the anti-RGNNV activity of the cells treated by 1 μM MG-132 and infected by RGNNV was analyzed by fluorescence. Specifically, the GS cells were inoculated in a 24-well plate, and L-15 medium containing 10% FBS was used to culture the cells at 28°C until a monolayer of adherent cells was formed. The original culture medium was discarded, and fresh culture medium containing 1 μM MG-132 was added, and a DMSO solvent control group was set. After 2 h of pretreatment, the RGNNV virus liquid prepared in Example 1 was inoculated at MOI=2, and the culture was continued at 28°C. After 24 h, 4% paraformaldehyde was used for fixation, 0.1% Triton X-100 was used for permeabilization, and 0.2% bovine serum albumin (BSA) (Sigma) was used for blocking. Then, the cells were incubated with the primary antibody of RGNNV CP diluted in 0.2% BSA at room temperature for 2 h. After washing with PBS for 3 times, the secondary antibody Alexa Fluor 555-coupled anti-mouse IgG Fab2 (1:200) was added, and incubation was carried out at room temperature for 2 h.

[0085] II. Experimental results

[0086] The results observed under CLSM are shown in Figure 4 Compared with the control group, the number of cells showing RGNNV CP expression positive was significantly reduced, and the fluorescence intensity was reduced in the MG132-treated group after 24 h of RGNNV infection, indicating that MG132 can exert an antiviral effect by directly inhibiting the expression of viral structural protein CP.

[0087] Example 5 Detection analysis of the antiviral activity of MG-132 at different times

[0088] I. Experimental method

[0089] 1. The dynamic inhibitory effect of MG-132 on RGNNV infection was evaluated by time gradient experiment system. GS cells were inoculated in 24-well plates and cultured at 28°C until monolayer adherent cells were formed. The experimental group was pretreated with 1 μM MG-132 for 2 hours, then inoculated with RGNNV virus liquid at MOI=2, and RNA samples and protein samples were collected at 12h, 24h and 36h time points. Total RNA was extracted using CeiITotal RNA Isolation Kit kit, and the transcription level of CP and RDRP genes, the protein expression level of CP gene, and the expression level of viral genomic RNA+ssRNA and-ssRNA were detected; protein was extracted using Pierce IP lysis buffer to analyze CP protein expression.

[0090] 2. The viral titer in the cells collected from the above three time experiment groups was detected, and the specific operation steps were as follows:

[0091] (1) Cell suspension in logarithmic growth phase was inoculated in 96-well plates (100 μL / well);

[0092] (2) After the cells formed a monolayer, the virus liquid was diluted by ten times gradient in the medium containing 1% serum (10 -1 ~ 10 -10 );

[0093] (3) Eight duplicate wells were inoculated for each dilution (100 μL / well), and an uninfected control group was set up;

[0094] (4) Cell pathological changes (CPE) were observed daily, and TCID 50 was calculated by Reed-Muench method.

[0095] II. Experimental results

[0096] The results are shown in Figure 5 and Figure 6 , with the extension of RGNNV infection time, the transcription level of CP and RDRP genes was significantly reduced, and the synthesis of CP protein was significantly inhibited; viral genomic RNA replication was significantly inhibited. This experiment confirmed that MG-132 can long-term inhibit viral gene transcription, protein synthesis and genomic replication.

[0097] The results are shown in Figure 7 , which are consistent with the Western blot results: with the extension of infection time, the viral titer of 1 μM MG-132 treatment group showed a significant downward trend, indicating that treatment with MG-132 can significantly reduce the viral force of RGNNV on cells.

[0098] The above results show that MG-132 treatment can significantly reduce the viral force of RGNNV on cells, has the efficacy of anti-neurolytic necrosis virus, and has low cytotoxicity and good safety.

Claims

1. The application of MG-132 in the preparation of drugs against neuronecrosis virus, characterized in that, The nerve necrosis virus mentioned is the red-spotted grouper nerve necrosis virus.

2. The application of MG-132 in the preparation of drugs for treating viral encephalopathy and retinopathy in fish caused by neuronecrosis virus infection, characterized in that, The nerve necrosis virus mentioned is the red-spotted grouper nerve necrosis virus.

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 drug achieves its therapeutic effect by reducing cellular lesions and inhibiting the expression of key genes in neuronecrosis virus.

5. The application according to claim 4, characterized in that, The key genes of the neuronecrosis virus are the capsid protein CP and the virus-dependent RNA polymerase RDRP.

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

7. The application according to claim 6, characterized in that, The concentration of MG-132 in the drug is 1 μM.

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

9. 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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