Use of compound BMS 309403 in the preparation of a medicament for the control of acute hepatopancreas necrosis disease in prawns

Abstract

The application provides a use of a compound BMS309403 in preparation of a medicament for preventing and treating acute hepatopancreas necrosis disease of prawns, and belongs to the technical field of prawn disease prevention and treatment. It is unexpectedly found that an inhibitor of a fatty acid binding protein LvFABP of Litopenaeus vannamei, i.e. the compound BMS309403, can be effectively applied in the prevention and treatment of AHPND, which can not only reduce the disease of hepatopancreas and intestinal tract of prawns, but also effectively reduce the Vibrio virulence gene copy number of hepatopancreas of diseased prawns, so that the survival rate of prawns infected with acute hepatopancreas necrosis disease can be significantly improved. In addition, as a small molecule compound, the BMS309403 is more superior to small molecule double-stranded RNA in synthesis, preservation and use, and therefore can have a better prospect in the actual production application of AHPND prevention and treatment.

Description

Technical Field

[0001] This invention belongs to the field of shrimp disease prevention and control technology, and in particular relates to the use of a compound BMS309403 in the preparation of a drug for the prevention and control of acute hepatopancreatic necrosis disease in shrimp. Background Technology

[0002] Acute hepatopancreatic necrosis disease (AHPND) is caused by pathogenic Vibrio parahaemolyticus (Vp.) AHPND AHPND, a large-scale outbreak of disease in shrimp, has caused severe losses to the global shrimp farming industry. The main pathological feature of AHPND is extensive necrosis of hepatopancreatic cells, leading to high mortality rates in shrimp. Current prevention and control measures for AHPND primarily involve comprehensive approaches such as reducing stocking density, adding microbial preparations or traditional Chinese medicine, controlling pond water quality, and strengthening feeding management.

[0003] The pathogenesis of AHPND has been reported in some studies. Pathogenic Vibrio carries the pVA1 plasmid, which carries genes encoding the toxins PirA and PirB. The PirAB binary toxin encoded and secreted by these genes is the direct cause of AHPND. In our previous study (CN 202210222837.5), we found that Vp... AHPND Virulence factor PirAB vp PirB in vp By binding to the fatty acid-binding protein (LvFABP) of Litopenaeus vannamei, it exerts its pathogenic effect, inducing severe damage to the host's hepatopancreas. Interfering with LvFABP expression using small double-stranded RNA can slow the progression of AHPND and alleviate symptoms. However, the high difficulty in preparing and preserving small double-stranded RNA currently limits its application in production. Summary of the Invention

[0004] This invention provides the use of compound BMS309403 in the preparation of a drug for the prevention and treatment of acute hepatopancreatic necrosis disease (AHPND) in shrimp. BMS309403 not only significantly improves the survival rate of shrimp infected with AHPND, but as a small molecule compound, it is also significantly superior to small double-stranded RNA in terms of synthesis, preservation, and use, thus showing better prospects in practical production applications for AHPND prevention and treatment.

[0005] To achieve the above objectives, the present invention provides the use of compound BMS309403 in the preparation of a medicament for the prevention and treatment of acute hepatopancreatic necrosis in shrimp.

[0006] Preferably, the concentration of pathogenic Vibrio in the aquaculture water is 10. 7 When the concentration of CFU / ml was 15 mg / kg, BMS309403 was administered via intramuscular injection, with the injection dose being 15 mg / kg, injected once every 48 hours for a total of three injections.

[0007] Compared to the control group, which all died at 216h, the survival rate of the BMS309403 injection group at 216h was 72%.

[0008] The present invention also provides the use of BMS309403 in the preparation of a medicament for improving the survival rate of shrimp infected with acute hepatopancreatic necrosis disease.

[0009] The present invention also provides the use of BMS309403 in the preparation of a pharmaceutical agent for reducing the copy number of the vibrio virus gene in the hepatopancreas of shrimp infected with acute hepatopancreatic necrosis.

[0010] The present invention also provides the use of BMS309403 in the preparation of a medicament for alleviating hepatopancreatic and intestinal damage in shrimp infected with acute hepatopancreatic necrosis.

[0011] The present invention also provides the use of BMS309403 in the preparation of a pharmaceutical agent for reducing apoptosis in shrimp cells infected with acute hepatopancreatic necrosis.

[0012] As a preferred method, compound BMS309403 is administered via intramuscular injection at a dose of 15 mg / kg, once every 48 hours, for a total of three injections.

[0013] This invention provides a drug for the prevention and treatment of acute hepatopancreatic necrosis disease in shrimp, with compound BMS309403 as the main active ingredient.

[0014] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0015] This invention unexpectedly discovered that the inhibitor of fatty acid-binding protein LvFABP in Litopenaeus vannamei—compound BMS309403 (purchased from Selleck, CAS: 300657-03-8. Reference: Lan H, et al. Small-molecule inhibitors of FABP4 / 5 ameliorate dyslipidemia but not insulin resistance in mice with diet-induced obesity. J Lipid Res. 2011, 52(4): 646-56.)—can be effectively applied to the prevention and treatment of AHPND. It can not only alleviate the symptoms in the hepatopancreas and intestines of shrimp, but also effectively reduce the copy number of Vibrio virulence genes in the hepatopancreas of infected shrimp, thereby significantly improving the survival rate of shrimp infected with acute hepatopancreatic necrosis disease. Furthermore, as a small molecule compound, BMS309403 is significantly superior to small double-stranded RNA in terms of synthesis, storage, and use (dsRNA for RNA interference generally requires expensive in vitro transcription systems for synthesis, such as Promega's T7 RiboMAX ExpressRNAi System; the synthesized RNA typically needs to be stored at -70°C and has no oral effect. Small molecule compounds, on the other hand, can be obtained through chemical synthesis, are more stable, and can generally be stored at room temperature, 4°C, or -20°C; they are generally effective when ingested orally). Therefore, it shows better promise in the practical production and application of AHPND prevention and control. Attached Figure Description

[0016] Figure 1 The BMS309403 provided in this embodiment of the invention can significantly improve the survival rate of shrimp infected with acute hepatopancreatic necrosis disease;

[0017] Figure 2 The BMS309403 provided in this embodiment of the invention can alleviate hepatopancreatic and intestinal symptoms in shrimp 48 hours after infection with acute hepatopancreatic necrosis.

[0018] Figure 3 BMS309403 provided in this embodiment of the invention has a significant protective effect on the hepatopancreatic tissue of shrimp infected with acute hepatopancreatic necrosis disease;

[0019] Figure 4 BMS309403 provided in this embodiment of the invention has a significant protective effect on the hepatopancreatic tissue of shrimp infected with acute hepatopancreatic necrosis disease;

[0020] Figure 5 BMS309403 provided in this embodiment of the invention can reduce the copy number of Vibrio virulence genes in the hepatopancreas of diseased shrimp;

[0021] Figure 6 The BMS309403 provided in this embodiment of the invention can significantly inhibit AHPND-induced apoptosis of liver and pancreatic cells. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: BMS309403 can significantly improve the survival rate of shrimp infected with acute hepatopancreatic necrosis virus.

[0024] Compound BMS309403 (purchased from Selleck, CAS: 300657-03-8) was dissolved in a mixture of DMSO and PBS and injected into the third and fourth segments of shrimp at a dose of 15 mg / kg body weight. Injections were given three times, every 48 hours, with three replicates per group and each replicate containing 50 shrimp. Forty-eight hours after the last injection, the challenge group underwent Vp... AHPND Immersion therapy for poisoning, immersion concentration is 10. 7 CFU / mL, and the shrimp were continuously observed for two weeks after challenge to record their vital activity status.

[0025] Experimental results show that, at Vp AHPND In the challenge experiment, the survival rates of different treatment groups showed significant differences. Twelve hours after challenge, shrimp in the control group (PBS and PBS+DMSO) began to die, while shrimp injected with the FABP inhibitor BMS309403 did not die until 24 hours later, indicating that the inhibitor could delay early mortality.

[0026] As the challenge continued, the mortality rate gradually increased in all groups. By 72 hours, the survival rate of the control group had dropped to approximately 50%, while the survival rate of the BMS309403 group reached 88%. Between 72 and 120 hours, the rate of mortality slowed in all groups, particularly in the BMS309403 group, where no significant change in mortality was observed. By 216 hours, all shrimp in the control group had died, while the final survival rate of the BMS309403 group was 72%. Figure 1 This result indicates that BMS309403 can effectively delay the mortality process of AHPND-infected shrimp and increase the survival rate after infection.

[0027] Example 2: BMS309403 can alleviate symptoms in the hepatopancreas and intestines of shrimp.

[0028] BMS309403 injection and Vp AHPND As described above, in the challenge experiment, liver, pancreas and intestinal samples were collected 48 hours after infection, and the appearance changes of liver, pancreas and intestine were photographed and recorded. At 24h, 48h and 72h after infection, liver, pancreas and intestine were taken for tissue fixation, and the histomorphological changes of liver, pancreas and intestine were observed by tissue sectioning and HE staining.

[0029] The results showed that Vp AHPND Before the challenge, healthy shrimp had full, normal-colored hepatopancreas and distended intestines, indicating good feeding ability and health. As the infection progressed, shrimp in the control groups (PBS and DMSO injection groups) showed increased activity after infection with Vp. AHPND Subsequently, significant organ damage was observed. Specifically, the hepatopancreas showed marked atrophy and turned white, indicating severe necrosis. Simultaneously, the intestines were noticeably empty, indicating that the infection severely impaired the shrimp's feeding ability, thereby affecting overall digestion and nutrient absorption. In contrast, the treatment group injected with BMS309403 showed better results after infection with Vp. AHPND Subsequently, the condition of the liver, pancreas, and intestines showed significant improvement and protection. Figure 2 Observations showed that the hepatopancreas of shrimp in the inhibitor-treated group remained plump and normal in color, with no obvious signs of atrophy or necrosis, and the intestines remained full, with no significant impact on their feeding ability.

[0030] Figure 3 The tissue section results showed that without Vp AHPND At challenge, the hepatopancreatic tissue structure was clear in all treatment groups, cell types were easily identifiable, and the luminal structure was intact, displaying a typical stellate luminal structure. In Vp AHPND Within 24 hours of viral challenge, the hepatopancreas in the control group (PBS and PBS+DMSO) showed significant structural damage. Cell types were unidentifiable, the stellate tubular structure began to disappear, and hematocytic infiltration was observed. In contrast, the hepatopancreas structure in the BMS309403 group remained relatively intact within 24 hours. After 48 hours, the luminal structure of the hepatopancreas in the control group was completely destroyed, the stellate structure disappeared, and a large number of hematocytic infiltrations were observed, indicating severe inflammation and necrosis. In contrast, the pathological changes in the BMS309403 group were milder. The stellate tubular structure was partially damaged but still identifiable, the overall hepatopancreas tissue was relatively stable, and hematocytic infiltration was less. At 72 hours, the stellate tubular structure in the BMS309403 group was further destroyed but still identifiable, the cell types were still recognizable, and hematocytic infiltration was milder, showing stronger resistance to damage.

[0031] Figure 4 Intestinal tissue structure damage also showed a similar trend, in the absence of Vp AHPNDDuring challenge, the intestinal structure remained intact in all treatment groups. (Vp) AHPND Twenty-four hours after viral challenge, the intestinal structure of shrimp in all groups remained relatively intact. However, in the PBS and PBS+DMSO control groups, slight separation of the basement membrane from the epithelial layer began to appear, indicating that early infection had already caused local tissue damage. In contrast, the intestinal structure of the BMS309403 group showed no significant damage within 24 hours. At 48 hours, the intestinal lesions in all groups gradually worsened. In the PBS and PBS+DMSO control groups, the separation of the basement membrane from the epithelial layer was significantly aggravated, accompanied by the shedding of a small number of epithelial cells. While the BMS309403 group also showed separation of the basement membrane from the epithelial layer, it was less severe. By 72 hours, the intestinal damage had further deteriorated, with complete detachment of intestinal epithelial cells in the PBS and PBS+DMSO groups, indicating severe lesions. Although the BMS309403 experimental group also showed some degree of basement membrane-epithelial separation, the intestinal structure remained relatively intact, further demonstrating the protective effect of BMS309403 on intestinal tissue during later infection stages.

[0032] Example 3: BMS309403 can reduce the copy number of Vibrio virulence genes in the hepatopancreas of diseased shrimp.

[0033] BMS309403 injection and Vp AHPND As described above, in the challenge experiment, shrimp hepatopancreatic tissues were collected at 6, 12, 24, 48, and 72 hours post-infection, and DNA was extracted. PirA in the shrimp hepatopancreatic tissue was detected using quantitative real-time PCR (qRT-PCR) for absolute quantification. vp Gene copy number (Reference: Han et al., 2015, qPCR assay for detecting and quantifying a virulence plasmid in acute hepatopancreatic necrosis disease (AHPND) due to pathogenic Vibrio parahaemolyticus, AQUACULTURE, 442:12–15, https: / / doi.org / 10.1016 / j.aquaculture.). The PCR primers used in the experiment were:

[0034] PirAvp-F: 5'-TTGGACTGTCGAACCAAACG-3';

[0035] PirAvp-R: 5'-GACCCCATTGGTATTGAATG-3';

[0036] The probe used in the experiment was a TaqMan probe.

[0037] 5'-AGACAGCAAACATACACCTATCATCCCGGA-3'.

[0038] The experimental procedure involved adding 0.1 μM of each primer and 0.1 μM of the TaqMan probe to a BlasTaq 2×qPCR MasterMix, bringing the final reaction volume to 10 μL. The qPCR program was as follows: 95℃ pre-denaturation for 3 minutes; 95℃ for 15 seconds, 60℃ for 1 minute; 40 cycles. Absolute quantification was performed using a standard curve, with the regression equation being Ct = -3.235lg(N) + 37.555, where Ct is the cycle threshold and N is the PirA value. vp The copy number of a gene.

[0039] The results are as follows Figure 5 As shown, the copy number of Vibrio virulence factors in the late stage of infection (48h to 72h) was significantly lower in the BMS309403 injection group than in the PBS and DMSO injection groups, indicating that BMS309403 effectively reduced the replication of virulence factors. The virulence factor pirAB is Vp AHPND The copy number is a key factor in pathogenicity and better reflects the virulence intensity of the pathogen (because pirA and pirB toxins exert their virulence as heterotetramers, both genes are regulated by the same operon. Their coding genes exist in a tightly tandem configuration on the pVA1 plasmid. Therefore, the copy number of the pirA gene is essentially equal to the copy number of the pirB gene, reflecting the copy number of the coding gene for the pirAB binary toxin). The significant inhibitory effect of BMS309403 on the copy number of virulence factors in shrimp is one of the important reasons for its reduction of disease symptoms.

[0040] Example 4: BMS309403 can reduce shrimp cell apoptosis induced by AHPND infection.

[0041] BMS309403 injection and Vp AHPND The challenge experiment was conducted as described above. Three hepatopancreas tissue samples from each treatment group were preserved in 4% paraformaldehyde solution and subjected to TUNEL (TdT-mediated dUTP Nick-End Labeling) fluorescence staining for section analysis to detect apoptosis signals. Hepatopancreas tissue sections were dewaxed with xylene and hydrated with graded ethanol, then stained according to the TUNEL kit instructions. TUNEL-positive cells (apoptotic cells) and DAPI-stained nuclei were observed using a fluorescence microscope. Multiple fields of view were randomly selected, and the percentage of TUNEL-positive cells in each field was calculated to quantify the level of hepatopancreas cell apoptosis.

[0042] The results are as follows Figure 6As shown, under non-infected conditions, the apoptosis rate of hepatopancreatic cells was low in all groups, with no significant difference between groups. However, after infection with AHPND, the apoptosis-positive rate of hepatopancreatic cells in the control group was significantly increased, indicating that acute hepatopancreatic necrosis disease infection induced severe apoptosis in hepatopancreatic cells. In contrast, the apoptosis-positive rate in the BMS309403 injection group was not significantly different from that in the non-infected condition, demonstrating the significant inhibitory effect of BMS309403 on apoptosis.

Claims

1. Compound BMS309403 was used in the preparation of a drug for the prevention and control of pathogenic Vibrio v. p. AHPND Its use in pharmaceuticals for treating acute hepatopancreatic necrosis in shrimp.

2. The use according to claim 1, characterized in that, The concentration of pathogenic Vibrio in the aquaculture water was 10. 7 When the concentration of CFU / ml was 15 mg / kg, BMS309403 was administered via intramuscular injection, with the injection dose being 15 mg / kg, injected once every 48 hours for a total of three injections. Compared to the control group, which all died at 216h, the survival rate of the BMS309403 injection group at 216h was 72%.

3. The use according to claim 1, characterized in that, This is used to reduce the number of Vibrio virus gene copies in the hepatopancreas of shrimp infected with acute hepatopancreatic necrosis.

4. The use according to claim 1, characterized in that, Used to reduce hepatopancreatic and intestinal damage in shrimp infected with acute hepatopancreatic necrosis.

5. The use according to claim 1, characterized in that, Used to reduce apoptosis in shrimp cells infected with acute hepatopancreatic necrosis.

6. Use of BMS309403 in the preparation of agents for improving the survival rate of shrimp infected with acute hepatopancreatic necrosis disease.

7. The use according to claim 6, characterized in that, Compound BMS309403 was administered via intramuscular injection at a dose of 15 mg / kg, once every 48 hours, for a total of three injections.

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