Application of naringenin in resisting siniperca chuatsi and frog iridovirus

Abstract

The invention belongs to the technical field of biology, discloses application of naringenin in resisting siniperca chuatsi and frog iridovirus, and discovers the antiviral activity of the naringenin to the siniperca chuatsi and frog iridovirus, the naringenin can down-regulate the expression level of NF-kB and MAPK pathway genes triggered by MRV, effectively inhibit MRV-induced excessive inflammatory response and inhibit MRV proliferation, and the application of the naringenin in resisting the siniperca chuatsi and frog iridovirus. In addition, tissue damage caused by MRV infection can be relieved, and the survival rate of MRV infected fishes is effectively increased; a foundation is provided for preparing a product for resisting the siniperca chuatsi and frog iridovirus by adopting naringenin; meanwhile, the invention also discloses a drug containing naringenin, a fish feed and a fish feed additive.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of naringenin in resisting Rana chuatsi iridovirus. Background Art

[0002] Mandarin fish iridovirus (MRV) belongs to the genus Ranavirus in the family Iridoviridae. It is an icosahedral cytoplasmic DNA virus that can infect mandarin fish (Siniperca chuatsi) and largemouth bass (Micropterus salmoides), causing a mortality rate exceeding 80%. MRV induces excessive inflammatory responses and promotes viral replication by activating the host NF-κB signaling pathway. In addition, excessive inflammatory responses can cause inflammatory damage to the host. Naringenin, a dihydroflavonoid compound found primarily in citrus fruits and tomatoes, has high intestinal absorption efficiency and exhibits anti-inflammatory and antiviral activities. It has shown efficacy in treating inflammation and infections caused by porcine epidemic diarrhea virus (PEDV), Zika virus (ZIKV), and novel coronavirus type 2 (SARS-CoV-2).

[0003] Porcine epidemic diarrhea virus (PEDV) is a single-stranded RNA virus that infects pigs, causing intestinal villi to atrophy and fuse, reducing absorptive surface area and triggering osmotic diarrhea. It also inhibits the function of the sodium-glucose cotransporter (SGLT1), exacerbating water and electrolyte imbalances. Zika virus, a member of the Flaviviridae family and the genus Flavivirus, is a single-stranded positive-strand RNA virus transmitted by mosquitoes, primarily in wild primates and tree-dwelling mosquitoes. Existing research only supports the claim that naringenin has limited antiviral activity against the aforementioned viruses. Summary of the Invention

[0004] The object of the present invention is to provide a use of naringenin in preparing a product for preventing and / or treating fish infected with Rana chuatsi iridovirus.

[0005] At the same time, the present invention also provides medicine, fish feed and fish feed additive containing the naringenin.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] Use of naringenin in preparing a product for preventing and / or treating fish infected with Rana chuatsi iridescent virus.

[0008] The viral genome of the mandarin frog iridovirus is double-stranded DNA. The mandarin frog iridovirus infects fish such as mandarin fish and largemouth bass, causing systemic inflammation, multi-organ inflammatory damage, ascites, and surface ulcers in fish.

[0009] Based on existing research on the antiviral mechanism of naringenin, it has been found that the Siniperca chuatsi iridovirus and related viruses that traditional naringenin can be effective against have different viral genome types, significantly different species, and completely different viral action locations and symptoms.

[0010] Through research, the present invention discovered the antiviral activity of naringenin against MRV. Naringenin can downregulate the expression levels of NF-κB and MAPK pathway genes triggered by MRV, effectively inhibit the excessive inflammatory response induced by MRV, inhibit the proliferation of MRV, and it can reduce tissue damage caused by MRV infection and effectively improve the survival rate of MRV-infected fish.

[0011] This discovery provides a certain basis for using naringenin to prepare products against mandarin frog iridovirus.

[0012] Preferably, the chemical formula of naringenin is C 15 H 12 O5, molecular weight is 272.256.

[0013] Preferably, the product is a drug whose active ingredient is naringenin.

[0014] More preferably, the dosage form of the drug is an oral preparation or an injection.

[0015] Preferably, the product is fish feed or fish feed additive containing naringenin.

[0016] In addition, the present invention discloses a drug for resisting Rana chuatsi iridovirus, wherein the drug contains naringenin.

[0017] In addition, the invention also discloses a fish feed resistant to Rana chuatsi iridovirus, wherein the fish feed contains naringenin.

[0018] Finally, the invention also discloses a fish feed additive for resisting Rana chuatsi iridovirus, wherein the fish feed additive contains naringenin.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This study discovered the antiviral activity of naringenin against Rana chuatsi iridovirus. Naringenin can downregulate the expression of genes in the NF-κB and MAPK pathways triggered by MRV, effectively inhibiting MRV-induced excessive inflammatory responses and proliferation. It can also reduce tissue damage caused by MRV infection and effectively improve the survival rate of MRV-infected fish. This discovery provides a foundation for the use of naringenin in the preparation of products that protect against Rana chuatsi iridovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the result diagram of the effect of naringenin on cell morphology;

[0022] Figure 2 This is the result diagram of the effect of different concentrations of naringenin on cell viability;

[0023] Figure 3 This figure shows the effect of different concentrations of naringenin on the CPE of MRV-infected cells;

[0024] Figure 4 This is the result diagram of the inhibitory effect of naringenin on MRV replication;

[0025] Figure 5 EC of naringenin 50 Analysis chart;

[0026] Figure 6 This is the survival curve analysis diagram;

[0027] Figure 7 This is the result of naringenin inhibiting MRV proliferation;

[0028] Figure 8 This is the result of naringenin inhibiting the inflammatory response induced by MRV infection;

[0029] Figure 9 The figure shows the results of naringenin inhibiting the expression of genes related to MAPK and NF-κB pathways;

[0030] Figure 10 This is the result of MAPK P38 protein expression analysis;

[0031] Figure 11 The figure shows the expression results of NF-κB P65 protein;

[0032] Figure 12 H&E staining analysis results of mandarin fish spleen sections. DETAILED DESCRIPTION

[0033] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] Materials and product information:

[0035] Mandarin fish iridovirus (MRV) was isolated and preserved by our laboratory from mandarin fish infected with MRV;

[0036] Naringenin was purchased from Chengdu Munster Biotechnology Co., Ltd. It was dissolved in DMSO and stored in an ultra-low temperature refrigerator. It was diluted to a safe concentration with L-15 culture medium before use.

[0037] Siniperca chuatsi cell line (SCB3): cells were cultured in L-15 medium at 25°C.

[0038] Part I: Cell Assays

[0039] 1. Determination of safe concentration of naringenin

[0040] SCB3 cells in the logarithmic growth phase were plated at a density of approximately 5,000 cells / well in a 96-well cell culture plate, and 100 μL of cell suspension was added to each well. The culture plate was then placed in a 25°C incubator and cultured for 24 hours until the cells were completely attached. The culture medium in the 96-well plate was removed, and 100 μL of L-15 medium (containing 5% fetal bovine serum) containing a gradient dilution of naringenin solution (final concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.125 μM) was added to each well of the experimental group. 100 μL of L-15 medium (containing 5% FBS) containing 1% DMSO was added to the control group. Three replicate wells were set up for each group. The treated cells were placed back in a 25°C incubator and continued to be cultured. After 96 hours, the changes in cell morphology were observed using an optical microscope. Subsequently, the culture medium was discarded and the cells were gently washed once with PBS buffer. 100 μL of diluted CCK-8 solution (CCK-8 and PBS were diluted in a ratio of 1:10) was added to each well and incubated at 28°C in the dark for 6 h. After incubation, the absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader. The cell viability was calculated by the following formula: Cell viability (%) = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%. Finally, the graph was drawn using GraphPad Prism9.5 software; the test results were referenced. Figure 1 、 Figure 2 .

[0041] like Figure 1 As shown in Figure 2, when the concentration of naringenin was in the range of 6.125 to 200 μM, the cell morphology was similar to that of the untreated control group, and no significant morphological changes were observed. Figure 2 ) further supported the above observations. Within the concentration range of 6.125-200 μM, there was no significant difference in cell viability between the drug-treated groups and the untreated control group (p>0.05).

[0042] Figure 1 This is the result diagram of the effect of naringenin on cell morphology;

[0043] Figure 2 The figure shows the effect of different concentrations of naringenin on cell viability. The same letters in the figure indicate no significant difference among the groups, and different letters indicate significant difference, p<0.05.

[0044] 2. Naringenin EC against MRV 50 calculate

[0045] Take SCB3 cells in the logarithmic growth phase, adjust to an appropriate cell density, and then inoculate 200 μL per well into a 48-well plate and continue to culture in a constant temperature incubator at 25°C until the cells are completely attached to the bottom of the culture plate. The experimental group was added with 200 μL of naringenin solution (concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.125 μM) diluted in L-15 medium containing 5% FBS and incubated at 25°C for 4 hours. The drug solution was then removed and 100 TCID 50 The MRV virus liquid was cultured at 25°C for 4 hours. After removing the virus liquid, it was washed once with PBS, and then 200 μL of L-15 medium containing 5% FBS was added and cultured for another 96 hours. At the same time, two control groups were set up: a virus infection control group (only inoculated with virus, without adding drugs) and a cell control group (neither inoculated with virus nor added drugs). The culture plate was returned to the incubator and cultured for another 96 hours to observe the development of cell pathology. Subsequently, the cells were repeatedly frozen and thawed three times to release the virus particles in the cells. 400 μL of the frozen and thawed cell fluid was taken for nucleic acid extraction and virus copy number determination. The inhibition rate calculation formula is: Inhibition rate (%) = (1-virus copy number of the experimental group / virus copy number of the virus infection control group) × 100%. GraphPad Prism 9.5 was used to calculate the median effective concentration (EC) of naringenin. 50 );Result reference Figure 3 、 Figure 4 、 Figure 5 .

[0046] Optical microscopy results showed that the pathological changes of MRV-infected cells gradually decreased with the increase of naringenin concentration ( Figure 3 ). The results of qPCR detection of viral copy number showed that as the concentration of naringenin gradually increased, the viral copy number of MRV in the cells gradually decreased ( Figure 4 ), showing dose dependence. Based on the viral load inhibition rate data of different concentrations of naringenin, combined with fitting curve analysis, the EC of naringenin was calculated. 50 33.69μM( Figure 5 ).

[0047] Figure 3 This figure shows the effect of different concentrations of naringenin on the CPE of MRV-infected cells;

[0048] Figure 4The figure shows the inhibitory effect of naringenin on MRV replication; in the figure, ns represents no significant difference, and ** represents p < 0.01;

[0049] Figure 5 EC of naringenin 50 Analysis chart.

[0050] Part II: Naringenin protection test against Siniperca chuatsi

[0051] The Siniperca chuatsi (40 g ± 5 g) were divided into 3 groups, with 20 fish in each group. (1) MRV infection group: Siniperca chuatsi were injected intraperitoneally with 10 7 TCID 50 The fish were challenged with 100 μL of MRV virus culture solution and then gavaged with 100 μL of PBS at 0 h and 72 h. (2) Naringenin administration group: The fish were intraperitoneally injected with 10 7 TCID 50 The fish were challenged with 100 μL of MRV virus culture medium. At 0 h and 72 h after the challenge, 30 mg of naringenin (100 μL / time) was administered orally per kg of fish body weight. (3) Simulated infection group: The fish were simulated infected by intraperitoneal injection of 100 μL of cell culture medium. At 0 h and 72 h after the injection, 100 μL / time of PBS was administered orally. The challenge experiment was analyzed as follows:

[0052] (1) Statistical analysis of the survival rate of virus challenge

[0053] During the experiment, the survival of fish in each group was recorded every day until no death occurred for 7 consecutive days. The experiment was terminated and the survival rate was analyzed using Kaplan-Meier.

[0054] (2) After the experiment, three fish were randomly selected from each of the three groups of surviving fish and subjected to the following tests and analyses:

[0055] ① Collect part of the spleen tissue, extract the total DNA of the tissue, and use the qPCR method to detect the MRV viral load of the tissue.

[0056] ② Collect part of the spleen tissue and perform indirect immunofluorescence test to analyze the expression of MRV major capsid protein (MCP):

[0057] The tissues were fixed with 4% paraformaldehyde for 24 hours and dehydrated in a series of ethanol solutions (70%, 80%, 90%, and 100%) for 2 hours. After dehydration, the tissue samples were transparentized in xylene, embedded, and cut into 4-μm thick sections. The sections were baked, deparaffinized in xylene, and rehydrated in a series of ethanol solutions (100%, 90%, 80%, 70%, and 50%, followed by deionized water) before microwave repair. The sections were permeabilized with 0.3% Triton X-100 and blocked with 5% BSA for 60 minutes at room temperature. The sections were incubated with rabbit anti-MRV-MCP (primary antibody) and goat anti-rabbit IgG Alexa Fluor 647 (secondary antibody). Laser confocal microscopy was performed after DAPI staining.

[0058] ③ Collect some spleen tissue and detect tissue gene expression:

[0059] Total RNA was extracted by Trizol method and reverse transcribed into cDNA. RPL13a was used as internal reference. -ΔΔCt The relative quantitative method was used to detect and analyze the inflammation-related factors IL-1β, IL-8, TNF-α, MPO, INOS and the genes related to the MAPK and NF-κB signaling pathways: JNK, ERK, p38, MYD88, TRAF6, and p65.

[0060] ④ Part of the spleen tissue was collected and 10% tissue homogenate (PBS) was prepared. Nitric oxide (NO) levels were detected using the nitrate reductase method. The specific operation was carried out according to the instructions of the NO determination kit of Nanjing Jiancheng Bioengineering Institute.

[0061] ⑤ Collect part of the spleen tissue for histopathological analysis:

[0062] The tissues were fixed with 4% paraformaldehyde for 24 hours and dehydrated in a gradient of ethanol solutions (70%, 80%, 90%, and 100%) for 2 hours. After dehydration, the tissue samples were immersed in xylene to make them transparent, embedded, and cut into 4-μm thick sections. The sections were baked, dewaxed in xylene, and rehydrated in a gradient of ethanol solutions (100%, 90%, 80%, 70%, and 50% and deionized water). After staining with hematoxylin, the sections were color-coded and blued, then dehydrated (50%, 70%, 80%, and 90% alcohol solutions), stained with eosin, color-coded, dehydrated, transparentized, and mounted for microscopic analysis.

[0063] Result Analysis

[0064] 1. Naringenin effectively improves the survival rate of MRV-infected fish

[0065] After MRV challenge, the survival and cumulative mortality of the mice were observed for 21 days. Figure 6) showed that oral administration of naringenin significantly improved the survival rate of mandarin fish following MRV infection (p < 0.05). The cumulative mortality rate of mandarin fish in the MRV-infected group was 80%, while that in the naringenin-treated group dropped to 40%. Throughout the observation period, the survival rate in the naringenin-treated group remained significantly higher than that in the MRV-treated group. Therapeutic administration of naringenin effectively delayed or prevented mortality caused by MRV infection, providing a protective effect on mandarin fish.

[0066] Figure 6 This is a survival curve analysis diagram.

[0067] 2. Naringenin inhibits MRV proliferation in mandarin fish

[0068] Viral load test revealed Figure 7 In a), the viral load in the mandarin fish treated with naringenin was significantly reduced by 99.9% compared with the MRV infection group (p<0.001), indicating that naringenin can effectively inhibit the replication of the virus in the host. The expression of MRV-MCP protein in spleen tissue sections was analyzed by IFA, and the results showed that ( Figure 7 In b), significant red fluorescence appeared in the cytoplasm of the MRV-infected group, demonstrating high-level translation and expression of the viral MCP protein. In contrast, the fluorescence signal intensity of the naringenin-treated group was only 73.0% of that of the infected group (p<0.01), demonstrating the inhibitory effect of naringenin on viral replication ( Figure 7 c) in the above example.

[0069] Figure 7 This is the result of naringenin inhibiting MRV proliferation, in which a: viral load, b: indirect immunofluorescence, c: relative fluorescence intensity.

[0070] 3. Naringenin inhibits MRV-induced inflammatory response in Siniperca chuatsi

[0071] Therapeutic administration of naringenin significantly reduced the expression of inflammatory factors (IL-1β, IL-8, TNF-α and MPO) in Siniperca chuatsi induced by MRV infection. Figure 8 Compared with the MRV-infected group, the mRNA expression of IL-1β, TNF-α, mpo, and inos in the naringenin-treated group was downregulated by 3.36-fold, 2.75-fold, 2.41-fold, and 2.74-fold, respectively. Measurements of inos expression and NO production showed that therapeutic administration of naringenin significantly reduced inos expression and significantly decreased NO concentration from 8.45 μmol / g prot to 3.91 μmol / g prot, a 53.7% decrease. These data, based on both the expression of inflammation-related genes and the levels of NO metabolites, confirm that naringenin effectively inhibits the excessive inflammatory response induced by MRV.

[0072] Figure 8 This figure shows the results of naringenin inhibiting the inflammatory response induced by MRV infection.

[0073] 4. Naringenin inhibits the transcriptional activation of key genes in the MAPK and NF-κB pathways

[0074] Therapeutic administration of naringenin can significantly inhibit MAPK and NF-κB signaling pathways. Figure 9 As shown, MRV infection activated the expression of genes involved in the NF-κB and MAPK signaling pathways. Compared with the mock-infected group, the mRNA levels of myd88, traf6, and p65 were upregulated by 4.45-fold, 7.78-fold, and 5.29-fold, respectively. Similarly, the mRNA levels of jnk, p38, and erk, core factors in the MAPK pathway, increased by 21.8-fold, 3.51-fold, and 2.18-fold, respectively. Oral administration of naringenin effectively inhibited the expression of these genes. Compared with the MRV-infected group, the mRNA levels of myd88, traf6, and p65 in the naringenin-treated group decreased by 1.68-fold, 1.83-fold, and 2.35-fold, respectively, while those of jnk, p38, and erk decreased by 1.41-fold, 1.43-fold, and 2.77-fold, respectively. These data indicate that naringenin can downregulate the expression of genes in the NF-κB and MAPK pathways triggered by MRV, effectively inhibiting the excessive inflammatory response induced by MRV.

[0075] Figure 9 This figure shows the results of naringenin inhibiting the expression of genes related to the MAPK and NF-κB pathways.

[0076] 5. Naringenin inhibits the protein expression of MAPK P38

[0077] IFA analysis shows that ( Figure 10 In a), MRV infection can cause significant spleen tissue damage, manifested as extensive nuclear pyknosis (as indicated by the arrows). The red fluorescence signal indicates the expression of P38 protein. Compared with the mock infection group, the red fluorescence expression in the MRV group was significantly enhanced. Quantitative analysis by Image J software ( Figure 10 In Figure b), the P38 fluorescence intensity increased 2.79-fold in the MRV-infected group. Naringenin treatment effectively inhibited P38 protein expression: compared with the MRV-infected group, the P38 fluorescence intensity in the naringenin-treated group decreased 1.34-fold. This result, at the protein level, confirms that naringenin antagonizes the MRV-induced inflammatory response by inhibiting the abnormal activation of P38.

[0078] Figure 10 The results of the expression analysis of MAPK P38 protein are shown in the figure, a: indirect immunofluorescence, b: relative fluorescence intensity analysis.

[0079] 6. Naringenin inhibits the activation and nuclear translocation of NF-κB P65 protein

[0080] IFA analysis shows that ( Figure 11 In a), MRV infection significantly activated the expression of NF-κB P65 protein, and its fluorescence intensity increased by 3.13 times compared with the mock infection group. Fluorescence co-localization analysis showed that the P65 fluorescence signal accumulated from the cytoplasm to the nucleus, indicating that the P65 protein underwent nuclear translocation ( Figure 11 b), thereby initiating the transcription of downstream inflammatory genes. Therapeutic administration of naringenin effectively inhibited its activation. Compared with the MRV infection group, the fluorescence intensity of P65 protein in the naringenin-treated group decreased by 68% ( Figure 11 In (c), fluorescence colocalization observation showed that its nuclear translocation phenomenon was weakened. These results confirmed that naringenin can antagonize the MRV-induced inflammatory response by inhibiting P65 activation from the perspective of protein activation and subcellular localization.

[0081] Figure 11 The expression results of NF-κB P65 protein are shown in the figure, a: indirect immunofluorescence, b: fluorescence co-localization analysis, c: fluorescence intensity analysis.

[0082] 7. Naringenin significantly reduces tissue damage caused by MRV infection

[0083] MRV infection caused a significant increase in the spleen size of mandarin fish, accompanied by bleeding on the surface. Therapeutic administration of naringenin reduced the degree of spleen enlargement ( Figure 12 Further H&E staining analysis, such as Figure 12 As shown in Figures d, e, and f, pathological changes in spleen tissue structure occurred after MRV infection, including nuclear pyknosis and cell necrosis of splenocytes, as well as a significant expansion of the intercellular spaces between tissues, indicating that viral infection severely damaged the normal tissue structure of the spleen. Therapeutic administration of naringenin alleviated these pathological damages.

[0084] Figure 12 This is the result of H&E staining analysis of the spleen section of mandarin fish.

[0085] In summary, naringenin can downregulate the expression levels of NF-κB and MAPK pathway genes triggered by MRV, effectively inhibit the excessive inflammatory response induced by MRV, inhibit the proliferation of MRV, and it can reduce tissue damage caused by MRV infection and effectively improve the survival rate of MRV-infected fish.

[0086] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

Claims

1. Use of naringenin in preparing a product for preventing and / or treating fish infected with Rana chuatsi iridovirus.

2. The use according to claim 1, characterized in that The chemical formula of naringenin is C 15 H 12 O5, molecular weight is 272.

256.

3. The use according to claim 1, characterized in that The product is a medicine whose active ingredient is naringenin.

4. The use according to claim 3, characterized in that The dosage form of the medicine is oral preparation or injection.

5. The use according to claim 1, characterized in that The product is fish feed or fish feed additive containing naringenin.

6. A drug for resisting Rana chuatsi iridovirus, characterized in that: The medicine contains naringenin.

7. A fish feed resistant to Rana chuatsi iridovirus, characterized in that: Fish feed contains naringenin.

8. A fish feed additive for resisting Rana chuatsi iridovirus, characterized in that: Fish feed additives contain naringenin.

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