Application of compound BMS309403 in preparation of medicament for preventing and treating acute hepatopancreatic necrosis of prawns

Abstract

The invention provides application of a compound BMS309403 in preparation of a medicament for preventing and treating acute hepatopancreatic necrosis of prawns, and belongs to the technical field of prawn disease prevention and treatment. The invention accidentally finds that the inhibitor of Litopenaeus vannamei fatty acid binding protein LvFABP, namely the compound BMS309403, can be effectively applied to prevention and treatment of AHPND, not only can relieve diseases of hepatopancreas and intestinal tracts of prawns, but also can effectively reduce the copy number of vibrio virulence genes of hepatopancreas of infected prawns, so that the effect of preventing and treating AHPND is achieved. Therefore, the survival rate of prawns infected with acute hepatopancreatic necrosis can be remarkably improved. Besides, the BMS309403 serving as a small molecule compound is more remarkably superior to small molecule double-stranded RNA in the aspects of synthesis, preservation and use, so that the BMS309403 has a better prospect in actual production and application of AHPND prevention and treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of shrimp disease prevention and treatment, and in particular relates to use of a compound BMS309403 in preparing a medicament for preventing and treating acute hepatopancreatic necrosis of shrimp. Background Art

[0002] Acute Hepatopancreatic Necrosis Disease (AHPND) is caused by pathogenic Vibrio parahaemolyticus. AHPND ) has caused severe losses to the global shrimp farming industry. The main pathological feature of AHPND is the extensive necrosis of hepatopancreatic cells, which leads to high mortality of shrimp. The prevention and control of AHPND currently mainly adopts comprehensive prevention and control measures such as reducing the stocking density, adding microbial preparations or Chinese herbal medicines, regulating pond water quality, and strengthening feeding management.

[0003] At present, some studies have been reported on the pathogenesis of AHPND. Pathogenic Vibrio carries the pVA1 plasmid, which carries genes encoding toxins PirA and PirB. The PirAB binary toxin encoded and secreted by this gene is the direct cause of AHPND. In our previous study CN 202210222837.5, we found that Vp AHPND Virulence factor PirAB vp PirB vp By binding to the fatty acid binding protein (LvFABP) of Penaeus vannamei, it exerts its pathogenic effect and induces severe damage to the host's hepatopancreas. Using small molecule double-stranded RNA to interfere with the expression of LvFABP can slow down the onset of AHPND and alleviate symptoms. However, due to the high difficulty in the preparation and preservation of small molecule double-stranded RNA, its application in production is still limited. Summary of the invention

[0004] The present invention provides a use of a compound BMS309403 in preparing a medicament for preventing and treating acute hepatopancreatic necrosis of shrimp. BMS309403 can not only significantly improve the survival rate of shrimp infected with acute hepatopancreatic necrosis, but also, as a small molecule compound, is significantly superior to small molecule double-stranded RNA in terms of synthesis, storage and use, and therefore can show better prospects in the actual production application of AHPND prevention and treatment.

[0005] In order to achieve the above object, the present invention provides a use of a compound BMS309403 in preparing a medicament for preventing and treating acute hepatopancreatic necrosis of shrimp.

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

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

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

[0009] The present invention also provides a use of BMS309403 in preparing a medicament for reducing the number of copies of Vibrio toxic genes in the hepatopancreas of shrimp infected with acute hepatopancreatic necrosis.

[0010] The present invention also provides a use of BMS309403 in preparing a medicament for alleviating damage to the hepatopancreas and intestines of shrimp infected with acute hepatopancreatic necrosis disease.

[0011] The present invention also provides a use of BMS309403 in preparing a medicament for reducing apoptosis of shrimp cells infected with acute hepatopancreatic necrosis disease.

[0012] Preferably, the compound BMS309403 is administered by intramuscular injection at a dose of 15 mg / kg, once every 48 hours, for a total of three injections.

[0013] The invention provides a medicament for preventing and treating acute hepatopancreatic necrosis of shrimp, which takes compound BMS309403 as a main effective component.

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

[0015] The present invention unexpectedly found that the inhibitor of the fatty acid binding protein LvFABP of Litopenaeus vannamei - compound BMS309403 (purchased from Selleck, CAS: 300657-03-8. Reference: Lan H, et al. Small-molecule inhibitors of FABP4 / 5ameliorate dyslipidemia but not insulin resistance in mice with diet-induced obesity. J Lipid Res. 2011, 52(4): 646-56.) can be effectively used in the prevention and treatment of AHPND, which can not only alleviate the symptoms of the hepatopancreas and intestines of shrimp, but also effectively reduce the number of copies of Vibrio virulence genes in the hepatopancreas of the infected shrimp, thereby significantly improving the survival rate of shrimp infected with acute hepatopancreatic necrosis. In addition, as a small molecule compound, BMS309403 is significantly superior to small molecule double-stranded RNA in terms of synthesis, storage and use (dsRNA for RNA interference generally needs to rely on expensive in vitro transcription systems for synthesis, such as Promega's T7RiboMAXExpressRNAi System. The synthesized RNA is generally stored at -70°C and has no effect when taken orally. Small molecule compounds can be obtained through chemical synthesis, have relatively stable properties, and are generally stored at room temperature, 4°C or -20°C. They can generally be taken orally to exert their efficacy). Therefore, it can show better prospects in actual production applications for the prevention and treatment of AHPND. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0017] Figure 2 BMS309403 provided in the embodiment of the present invention can alleviate the hepatopancreatic and intestinal symptoms of shrimp infected with acute hepatopancreatic necrosis disease 48 hours later;

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

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

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

[0021] Figure 6 BMS309403 provided in the embodiment of the present invention can significantly inhibit the apoptosis of hepatopancreatic cells induced by AHPND. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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

[0024] The compound BMS309403 (purchased from Selleck, CAS: 300657-03-8) was dissolved in a mixture of DMSO and PBS and then injected between the third and fourth segments of the shrimp. The injection dose was 15 mg / kg body weight, once every 48 hours, for a total of three injections, with three replicates per group, each containing 50 shrimps. 48 hours after the last injection, the challenge group was tested for Vp AHPND Bath attack, bath concentration is 10 7 CFU / mL, and continued to observe for two weeks after the infection to record the vital activity status of the shrimp.

[0025] The experimental results show that at Vp AHPND In the challenge experiment, the survival rates of different treatment groups showed significant differences. After 12 hours of challenge, shrimps in the control group (PBS and PBS+DMSO) began to die, while shrimps injected with the FABP inhibitor BMS309403 did not die until 24 hours later, indicating that the inhibitor can delay the occurrence of early death.

[0026] As the challenge continued, the mortality rate of each group gradually increased. By 72 hours, the survival rate of the control group dropped to about 50%, while the survival rate of BMS309403 reached 88%. During the period from 72 hours to 120 hours, the mortality rate of each group slowed down, especially the group injected with BMS309403, which did not show significant changes in mortality. By 216 hours, all shrimps 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 death process of AHPND-infected shrimp and increase the survival rate after infection.

[0027] Example 2 BMS309403 can alleviate symptoms of shrimp hepatopancreas and intestines

[0028] BMS309403 Injection and Vp AHPND The challenge experiment was as described above. Hepatopancreas and intestinal samples were collected 48 hours after infection, and photos were taken to record the changes in the appearance of the hepatopancreas and intestine. The hepatopancreas and intestine were taken for tissue fixation 24h, 48h, and 72h after infection, and the tissue morphological changes of the hepatopancreas and intestine were observed by tissue sectioning and HE staining.

[0029] The results show that Vp AHPND Before infection, the hepatopancreas of healthy shrimp was full and normal in color, and the intestine was full, showing good feeding ability and health. As the infection progressed, the shrimp in the control group (PBS and DMSO injection groups) became AHPND The infection caused significant damage to the shrimp’s feeding ability, which in turn affected the overall digestion and nutrient absorption function. In contrast, the group injected with BMS309403 showed significant organ damage after infection with Vp AHPND After treatment, the status of the liver, pancreas and intestine showed significant improvement and protection. Figure 2 Observations showed that the hepatopancreas of shrimp in the inhibitor-treated group remained plump and had normal color, with no obvious atrophy or necrosis. The intestines were also still full, and the feeding ability was not significantly affected.

[0030] Figure 3 The tissue section results showed that there was no Vp AHPND During the challenge, the hepatopancreas tissue structure of all treatment groups was clear, the cell types were easy to identify, and the luminal structure was intact, showing a typical star-shaped luminal structure. AHPND Within 24 hours after the attack, the hepatopancreas of the control group (PBS and PBS+DMSO) showed significant structural damage. The cell type could not be identified, the star-shaped luminal structure began to disappear, and blood cell infiltration appeared. In contrast, the hepatopancreas structure of the BMS309403 group remained relatively intact within 24 hours. After 48 hours, the hepatopancreas luminal structure of the control group was completely destroyed, the star-shaped structure disappeared, accompanied by a large number of blood cell infiltrations, showing severe inflammation and necrosis. In contrast, the pathological changes in the BMS309403 group were milder. The star-shaped luminal structure was partially damaged, but still identifiable, and the hepatopancreas tissue was relatively stable as a whole, with less blood cell infiltration. At 72 hours, the star-shaped luminal structure of the BMS309403 group was further damaged, but still identifiable, the cell type was still identifiable, and the blood cell infiltration was relatively light, showing a strong ability to resist damage.

[0031] Figure 4 The intestinal tissue damage also showed a similar trend. AHPNDDuring the challenge, the intestinal structure of all treatment groups remained intact. AHPND 24 hours after the attack, the intestinal structure of each group of shrimp remained relatively intact, but in the PBS group and the PBS+DMSO control group, the basement membrane and the epithelial layer began to separate slightly, indicating that early infection had caused local tissue damage. However, the intestinal structure of the BMS309403 group did not show obvious damage within 24 hours. At 48h, the intestinal lesions of each group gradually worsened. In the PBS group and the PBS+DMSO control group, the separation of the basement membrane and the epithelial layer was significantly aggravated, accompanied by the shedding of a small amount of epithelial cells. Although the BMS309403 group also showed the separation of the basement membrane and the epithelial layer, the degree was milder. By 72h, the intestinal damage further worsened, and the intestinal epithelial cells of the PBS group and the PBS+DMSO group completely fell off, showing severe lesions. Although the BMS309403 experimental group also showed a certain degree of separation of the basement membrane and the epithelial layer, the intestinal structure remained relatively intact, further proving the protective effect of BMS309403 on intestinal tissue during the late infection process.

[0032] Example 3 BMS309403 can reduce the number of copies of Vibrio virulence genes in the hepatopancreas of infected shrimp

[0033] BMS309403 Injection and Vp AHPND As described above, the hepatopancreas tissues of shrimp were collected at 6, 12, 24, 48, and 72 h after infection, DNA was extracted, and the PirA in the hepatopancreas of shrimp was detected by fluorescence quantitative PCR (qRT-PCR) absolute quantitative detection method. 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 are:

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

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

[0036] The probe used in the experiment is TaqMan probe:

[0037] 5'-AGACAGCAAACATACACCTATCATCCCGGA-3'.

[0038] The experimental steps were to add 0.1 μM of each primer and 0.1 μM of TaqMan probe to BlasTaq 2×qPCR MasterMix, so that the final reaction system was 10 μL. The qPCR program was: 95°C pre-denaturation for 3 minutes; 95°C, 15 seconds, 60°C, 1 minute; 40 cycles. Absolute quantification was performed using the standard curve, and the regression equation was Ct = -3.235lg (N) + 37.555, where Ct is the cycle threshold and N is the PirA vp The copy number of a gene.

[0039] The results are as follows Figure 5 As shown in the figure, the copy number of Vibrio virulence factors in the late infection period (48h to 72h) was significantly lower in the BMS309403 injection group than in the PBS injection group and the DMSO injection group, indicating that BMS309403 effectively reduced the replication of virulence factors. AHPND The key factor of pathogenicity, its copy number can better reflect the virulence of pathogens (because pirA and pirB toxins exert their virulence in the form of heterotetramers, the two genes are regulated by the same operon. The coding genes of the two are closely connected in series on the pVA1 plasmid. Therefore, the copy number of the pirA gene is basically equivalent to the copy number of the pirB gene, and reflects the copy number of the coding gene of 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 caused by AHPND infection

[0041] BMS309403 Injection and Vp AHPND The challenge experiment was as described above. Three hepatopancreases from each treatment group were preserved in 4% paraformaldehyde solution, and TUNEL (TdT-mediated dUTP Nick-End Labeling) fluorescent staining section analysis was performed to detect cell apoptosis signals. After dewaxing of hepatopancreatic tissue sections with xylene and hydration with gradient ethanol, staining was performed according to the steps in the TUNEL kit instructions. TUNEL-positive cells (apoptotic cells) and DAPI-stained cell nuclei were observed using a fluorescence microscope. Multiple fields of view were randomly selected, and the ratio of TUNEL-positive cells in each field of view was calculated to quantify the level of hepatopancreatic cell apoptosis.

[0042] The results are as follows Figure 6As shown in the figure, in the non-infected case, the apoptosis rate of hepatopancreatic cells in all groups was low, and there was no significant difference between the groups. However, after infection with AHPND, the positive rate of apoptotic cells in the hepatopancreatic cells of shrimp in the control group increased significantly, indicating that the infection of acute hepatopancreatic necrosis disease triggered more serious apoptosis of hepatopancreatic cells. The apoptosis positive rate in the BMS309403 injection group was not significantly different from that in the non-infected case, and BMS309403 had a significant inhibitory effect on cell apoptosis.

Claims

1. Use of compound BMS309403 in the preparation of a medicament for preventing and treating acute hepatopancreatic necrosis of shrimp.

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

3. Use of BMS309403 in the preparation of a medicament for improving the survival rate of shrimp infected with acute hepatopancreatic necrosis.

4. Use of BMS309403 in the preparation of a medicament for reducing the number of copies of Vibrio toxic genes in the hepatopancreas of shrimp infected with acute hepatopancreatic necrosis.

5. Use of BMS309403 in the preparation of a medicament for alleviating damage to the hepatopancreas and intestines of shrimp infected with acute hepatopancreatic necrosis disease.

6. Use of BMS309403 in the preparation of a medicament for reducing apoptosis of shrimp cells infected with acute hepatopancreatic necrosis disease.

7. The use according to any one of claims 3 to 6, characterized in that: The compound BMS309403 was administered by intramuscular injection at a dose of 15 mg / kg, once every 48 hours, for a total of three injections.

8. A medicament for preventing and treating acute hepatopancreatic necrosis of shrimp, characterized in that: The compound BMS309403 is the main active ingredient.

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