Use of ursodeoxycholic acid in preparing a medicament for improving the survival rate of shrimp with acute hepatopancreatic necrosis

Abstract

The present invention provides a method for preparing a medicament for improving the survival rate of shrimp infected with acute hepatopancreatic necrosis, belonging to the technical field of shrimp aquaculture disease prevention and control. The present invention utilizes ursodeoxycholic acid (UDCA), a lipid metabolism regulator, to regulate the metabolic pathways of infected shrimp, significantly reducing the damage caused by pathogenic Vibrio infection to shrimp. Experiments have shown that UDCA, whether injected or added to feed, significantly reduces symptoms and mortality in shrimp infected with acute hepatopancreatic necrosis, demonstrating its potential for preventing and treating acute hepatopancreatic necrosis in shrimp.

Description

Technical Field

[0001] The invention belongs to the technical field of shrimp aquaculture disease prevention and control, and in particular relates to use of ursodeoxycholic acid in preparing a medicament for improving the survival rate of shrimp with acute hepatopancreatic necrosis. Background Art

[0002] my country is the world's leading shrimp aquaculture country, with marine and freshwater shrimp production reaching 2.57 million tons in 2023 (China Fisheries Statistical Yearbook, 2024). However, Acute Hepatopancreas Necrosis Disease (AHPND), caused by pathogenic Vibrio spp., poses a serious threat to my country's shrimp aquaculture industry. AHPND primarily occurs in the early stages of shrimp farming and has a high mortality rate, resulting in significant losses to the shrimp aquaculture industry each year. Currently, AHPND is primarily controlled through comprehensive prevention and control measures, such as strengthening aquaculture management and administering probiotics. However, these measures are generally ineffective and fail to meet production needs.

[0003] In fact, not all Vibrio are pathogens of acute hepatopancreatic necrosis. Studies have shown that Vibrio in aquaculture water can participate in denitrification to improve aquaculture water quality (Li, Y. et al., 2017. Aerobic-heterotrophic nitrogen removal through nitrate reduction and ammonium assimilation by marine bacterium Vibrio sp. Y1-5. BIORE SOURCE TECHNOLOGY, 230, 103-111.). Only Vibrio carrying the pVA1 toxic plasmid can cause acute hepatopancreatic necrosis. The pVA1 plasmid carries the gene sequences encoding PirA and PirB. The toxin is the main factor causing damage to the hepatopancreas and intestines of shrimp. Pathogenic Vibrio (VP AHPND) after infecting shrimp, leading to metabolic imbalance and apoptosis in shrimp cells. After infection with AHPND, a large amount of lipid droplets accumulate in the hepatopancreas of shrimp, bile acid accumulates abnormally, and lipid metabolism and bile acid metabolism are unbalanced (Kumar, R. et al., 2020. Bile acid and bile acid transporters are involved in the pathogenesis of acute hepatopancreatic necrosis disease in white shrimp Litopenaeus vannamei. CELLULAR MICROBIOLOGY, 22, DOI10.1111 / cmi.13127). By adjusting the metabolic homeostasis of shrimp bodies, it is hoped that the damage caused by pathogenic Vibrio to shrimp cells will be reduced, thereby reducing disease losses.

[0004] Among the disclosed patent technologies, many technologies develop microbial preparations to prevent and control acute hepatopancreatic necrosis by screening Vibrio antagonistic microorganisms. For example, CN202410427133.0 discloses a Pseudoalteromonas that has an antagonistic effect on Vibrio, CN201880004885.9 discloses the use of Bacillus subtilis, Bacillus pumilus, and Bacillus licheniformis in the prevention and treatment of acute hepatopancreatic necrosis, and CN202410052733.3 discloses a bacteriophage that can kill Vibrio. Some patents also use Chinese herbal medicine to prevent and treat acute hepatopancreatic necrosis, such as CN202410713083.2 and CN202311498414.7, which respectively disclose Chinese herbal medicine formulas that have preventive and therapeutic effects on acute hepatopancreatic necrosis. There are also corresponding technologies for the prevention and treatment of acute hepatopancreatic necrosis disease by developing new polypeptides or recombinant proteins targeting the interaction between Vibrio toxins and shrimp bodies. For example, CN202211357289.3 and CN202211599098.8 respectively disclose the use of a receptor protein LvFABP that can bind to PirB toxin and a polypeptide P2 that can bind to PirB toxin in disease prevention and control.

[0005] Among the previously disclosed technologies, microbial preparations, Chinese herbal medicines, novel peptides, and recombinant proteins have demonstrated significant efficacy in disease control. While microbial preparations and Chinese herbal medicines have a relatively broad-spectrum inhibitory effect against Vibrio, they cannot distinguish between pathogenic and non-pathogenic Vibrio. While novel peptides and recombinant proteins such as LvFABP, which target the Vibrio toxin PirB in pathogenic Vibrio, can provide targeted control of pathogenic Vibrio, their primary efficacy is in disease prevention, with no reported therapeutic effects. Consequently, there is currently a lack of effective control technologies for acute hepatopancreatic necrosis in shrimp. Summary of the Invention

[0006] The present invention provides the use of ursodeoxycholic acid in the preparation of a medicament for improving the survival rate of shrimp infected with acute hepatopancreatic necrosis. Experiments have demonstrated that ursodeoxycholic acid significantly reduces symptoms and mortality in shrimp infected with acute hepatopancreatic necrosis by regulating metabolic pathways in the diseased shrimp, demonstrating its application value in the prevention and treatment of acute hepatopancreatic necrosis in shrimp.

[0007] In order to achieve the above object, the present invention provides a use of ursodeoxycholic acid in the preparation of a medicament for improving the survival rate of shrimp with acute hepatopancreatic necrosis disease caused by pathogenic Vibrio parahaemolyticus.

[0008] As a preferred method, the concentration of pathogenic Vibrio parahaemolyticus in aquaculture water is 10 7 CFU / ml, ursodeoxycholic acid was administered intramuscularly at a dose of 20 mg / kg, once every 48 hours for a total of three injections;

[0009] Compared with the control group in which all the mice died at 216 hours, the survival rate of the ursodeoxycholic acid injection group was 43% at 216 hours.

[0010] The present invention also provides a use of ursodeoxycholic acid in preparing a medicament for reducing the copy number of toxic plasmids in the hepatopancreas of shrimp infected with acute hepatopancreatic necrosis disease.

[0011] The present invention also provides a use of ursodeoxycholic acid in preparing a medicament for alleviating hepatopancreatic damage of shrimp infected with acute hepatopancreatic necrosis disease.

[0012] Preferably, ursodeoxycholic acid alleviates the damage to the hepatopancreas of shrimp by maintaining normal bile acid efflux, inhibiting excessive bile acid reflux and inhibiting the expression of genes related to bile acid synthesis.

[0013] Preferably, ursodeoxycholic acid maintains normal bile acid efflux by upregulating the expression of bile acid efflux-related genes MRP3 and MRP4.

[0014] Preferably, ursodeoxycholic acid alleviates excessive bile acid reflux by maintaining the expression homeostasis of bile acid reflux-related genes OATP and NTCP.

[0015] Preferably, ursodeoxycholic acid inhibits the synthesis of bile acid by down-regulating the expression of CYP7A1 gene, a gene related to bile acid synthesis.

[0016] The present invention also provides a feed for preventing and treating acute hepatopancreatic necrosis of shrimp, wherein the ursodeoxycholic acid described in the above technical solution is added to ordinary shrimp feed.

[0017] Preferably, the added amount of ursodeoxycholic acid is 150-200 mg / Kg.

[0018] Compared with the prior art, the advantages and positive effects of the present invention are:

[0019] This invention utilizes ursodeoxycholic acid (UDCA), a lipid metabolism regulator, to regulate metabolic pathways in infected shrimp, significantly mitigating the damage caused by pathogenic Vibrio bacteria. Experimental studies have shown that UDCA, whether injected or added to feed, significantly reduces symptoms and mortality in shrimp infected with acute hepatopancreatic necrosis (AHPN), demonstrating its potential application in the prevention and treatment of AHPN in shrimp. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The UDCA injection provided in the embodiment of the present invention can significantly reduce the mortality of shrimp infected with AHPND;

[0021] Figure 2 Schematic diagram showing that UDCA injection can alleviate hepatopancreas and intestinal symptoms in shrimp infected with AHPND according to an embodiment of the present invention;

[0022] Figure 3 Schematic diagram showing that injection of ursodeoxycholic acid (UDCA) can significantly reduce the number of virulence plasmid copies of pathogenic Vibrio in the hepatopancreas of diseased shrimps provided by an embodiment of the present invention;

[0023] Figure 4A Schematic diagram of the method provided by the embodiment of the present invention for injecting UDCA to reduce AHPND infection damage by maintaining bile acid metabolic homeostasis;

[0024] Figure 4B Schematic diagram of how UDCA injection reduces AHPND infection damage by maintaining normal bile acid efflux, inhibiting excessive bile acid reflux, and inhibiting the expression of genes such as bile acid synthesis, provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0025] The following is a clear and complete description of the technical solutions in 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 any creative efforts are within the scope of protection of the present invention.

[0026] Example 1: UDCA injection significantly improves the survival rate of shrimp infected with AHPND and alleviates hepatopancreatic and intestinal symptoms

[0027] Ursodeoxycholic acid (UDCA) was dissolved in dimethyl sulfoxide (DMSO) and injected into the shrimp muscle at a dose of 20 mg / kg, once every 48 hours, for a total of three injections. Phosphate buffered saline (PBS) and PBS+DMSO were injected at the same time as controls. During this process, the number of surviving shrimp in each group was recorded to detect the effect of UDCA injection on the survival rate of shrimp. 48 hours after the last injection, the challenge experiment of each treatment group was carried out. The treatment group was divided into VP AHPND The infection group (challenge group) and the uninfected group (control group) each contained 200 shrimps. The challenge experiment used the immersion challenge method to inoculate the shrimps with pathogenic Vibrio parahaemolyticus VP. AHPND To expand the culture, add Vibrio solution to the shrimp culture water to a final concentration of 10 7 CFU / ml.

[0028] The results showed that the injection of UDCA had no effect on the survival and health of the shrimp. The survival rate of the shrimp in each group after injection was 100%. The injection of UDCA did not affect the health of the shrimp. AHPND In the challenge experiment, the survival rate of shrimp in the non-challenged control group remained 100% throughout the experiment. In contrast, the challenged shrimp died in all groups, including the control group (PBS and DMSO injection) and the experimental group (UDCA injection). In the control group, death began to appear at 12h, while the shrimp in the UDCA injection group began to die after 24h. As the infection continued, the mortality rate of shrimp in each group gradually increased. At 72h, the mortality rate of shrimp in the control group had reached 50%, and all died at 216h. In contrast, at 216h, the survival rate of the UDCA injection group was 43%. These results show that although UDCA injection before Vibrio challenge cannot completely prevent the death of shrimp, they significantly delay VP AHPND infection-induced mortality process and ultimately increased shrimp survival rate ( Figure 1 ). After dissecting the hepatopancreas and intestines of the shrimp after the poison challenge, it was found that the shrimp in the control experimental group (PBS and DMSO injection group) had obvious hepatopancreatic lesions after the poison challenge, with the hepatopancreas atrophied and whitened, and the jejunum and stomach empty, while the hepatopancreas and intestines of the shrimp in the UDCA injection group remained relatively healthy ( Figure 2 ).

[0029] Example 2: Injection of UDCA can significantly reduce the number of hepatopancreatic toxic plasmid copies in shrimp infected with AHPND

[0030] The UDCA injection and Vibrio challenge steps were the same as above. vp The copy number of pathogenic Vibrio virulence plasmids in the hepatopancreas was assessed by the copy number of the virulence plasmids.

[0031] Detection of PirA in shrimp hepatopancreas by qRT-PCR vp Gene copy number. The experimental method was based on the method reported by Han et al. (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.). Absolute quantification was performed using a 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 the gene.

[0032] The PCR primers used in the experiment are: PirA vp -F:5'-TTGGACTGTCGAACCAAACG-3',PirA vp -R: 5'-GCACCCCATTGGTATTGAATG-3'; the experimental probe was TaqMan probe: 5'-AGACAGCAAACATACACCTATCATCCCGGA-3'.

[0033] The experimental procedure involved adding 0.1 μM of each primer and 0.1 μM of the TaqMan probe to BlasTaq 2× qPCR MasterMix, resulting in a final reaction volume of 10 μL. The qPCR program was as follows: pre-denaturation at 95°C for 3 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Each sample was tested in triplicate to ensure data accuracy.

[0034] Depend on Figure 3 The test results showed that in the early stage of infection (within 24 hours of infection), the pirA copy number of all groups showed a continuous upward trend, indicating that the copy number of virulence plasmids carrying the pirA virulence gene in the hepatopancreas gradually increased. As the infection time prolonged, the pirA copy number of the UDCA group vp The copy number was significantly lower than that in the PBS and PBS+DMSO groups, indicating that UDCA injection reduced the copy number of virulence plasmids carried by pathogens.

[0035] Example 3: UDCA injection reduces AHPND-induced damage to the hepatopancreas by increasing hepatopancreatic bile acid pumping and inhibiting bile acid synthesis.

[0036] Bile acid accumulation is an important physiological change in AHPND infection, which is related to the differences in gene expression of bile acid synthesis and transport in infected shrimp. The effect of UDCA on alleviating VP was evaluated by detecting the accumulation of bile acids and the expression of genes related to bile acid metabolism in the hepatopancreas of shrimp. AHPND Changes in physiological, biochemical and molecular biological indicators of infection symptoms.

[0037] UDCA injection and Vibrio challenge procedures were the same as above. Total bile acid content in the hepatopancreas of shrimp was quantified using a total bile acid assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's protocol. The expression of genes involved in bile acid metabolism was detected using qRT-PCR. The genes tested and the corresponding primer sequences are shown in Table 1. The PCR reaction program was: denaturation at 94°C for 2 minutes, followed by 40 cycles of denaturation at 94°C for 15 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 30 seconds.

[0038] Table 1 Primer sequences for detecting expression of genes related to bile acid metabolism

[0039]

[0040]

[0041] Under non-infected conditions, after UDCA injection, the total bile acid level in the hepatopancreas of shrimp increased significantly. This is because UDCA itself is a natural bile acid. However, after infection with AHPND, the hepatopancreas bile acid content in the control group injected with PBS and PBS+DMSO increased significantly, while the UDCA injection group remained stable. This shows that AHPND can cause the accumulation of bile acid in the hepatopancreas of shrimp, but the shrimp injected with UDCA did not accumulate bile acid after infection with AHPND, indicating that it has a significant regulatory effect on the bile acid metabolism of shrimp infected with AHPND ( Figure 4A ).

[0042] After shrimp were infected with AHPND, the expression of the ecdysone receptor gene LvECR (GenBankACCESSIONNUMBER:XM_027356260), a key transcription factor in bile acid metabolism, was significantly inhibited. However, when UDCA was injected, the expression level of the LvECR gene remained stable, indicating that it plays a key role in maintaining the homeostasis of bile acid metabolism after shrimp were infected with AHPND.

[0043] In addition, UDCA's regulatory mechanism for bile acid metabolic homeostasis relies on improving bile acid excretion and inhibiting bile acid reflux and synthesis. Under AHPND infection, UDCA significantly upregulated the expression of the bile acid efflux-related genes MRP3 (GenBank ACCESSION NUMBER: XM_070115017) and MRP4 (GenBank ACCESSION NUMBER: XM_070131847), helping to alleviate bile acid accumulation. The bile acid reflux-related genes OATP (GenBank ACCESSION NUMBER: XM_070136381) and NTCP (GenBank ACCESSION NUMBER: XM_070134692) were significantly upregulated after AHPND infection, indicating that bile acid reflux is accelerated after AHPND infection, leading to bile acid accumulation in the hepatopancreas. However, UDCA administration maintained the expression homeostasis of these two genes, alleviating the accelerated bile acid reflux caused by AHPND infection. In addition, UDCA down-regulated the expression of CYP7A1 (GenBank ACCESSION NUMBER: XM_017440277), a gene related to bile acid synthesis, under infection conditions, reducing the production of bile acid. UDCA maintains the stability of bile acid metabolism after shrimp are infected with acute hepatopancreatic necrosis by maintaining normal bile acid excretion, inhibiting excessive bile acid reflux, and inhibiting the expression of genes such as bile acid synthesis, thereby playing a role in resisting AHPND infection ( Figure 4B ).

[0044] Example 4 Feeding UDCA can reduce the mortality of AHPND-infected shrimp

[0045] Prepare shrimp feed containing UDCA to verify its oral administration and preventive effect on AHPND. Prepare shrimp feed with UDCA content of 100mg / Kg, 150mg / Kg and 200mg / Kg. Carry out shrimp farming and challenge experiments. Vannamei shrimp weighing about 10g were cultured in 18 1m 3 In the bucket, 50 shrimps were placed in each bucket and fed with the control group compound feed (without UDCA). After 1 week of temporary cultivation, 12 buckets were randomly selected for VP AHPND (10 7 The shrimp were challenged with a 100 mg / kg UDCA (100 CFU / mL) immersion bath. Twenty-four hours after the challenge, three buckets were randomly selected and fed the control feed, three buckets were fed the 100 mg / kg UDCA-containing feed, three buckets were fed the 150 mg / kg UDCA-containing feed, and three buckets were fed the 200 mg / kg UDCA-containing feed. Among the unchallenged shrimp, three buckets continued to be fed the standard feed, while three buckets were fed the UDCA-containing feed. The cumulative mortality of the shrimp was recorded over 10 days to test the efficacy of UDCA in preventing and treating AHPND. The experimental results are shown in Table 2.

[0046] Table 2 Protective effect of in vitro recombinantly expressed LvECR against acute hepatopancreatic necrosis in Litopenaeus vannamei

[0047]

[0048] The experimental results showed that during a 10-day culture experiment, shrimp fed both standard and UDCA diets, without AHPND infection, experienced almost no mortality, demonstrating that the addition of UDCA to the diet did not affect the health of the shrimp. After infection with AHPND, the mortality rate of shrimp fed the UDCA diet was significantly reduced. By day 10, almost all shrimp fed the standard diet had died, while the mortality rate of shrimp fed the diet supplemented with 150-200 mg / kg of UDCA was approximately 70% (Table 2). This indicates that the addition of UDCA to the diet significantly mitigates AHPND-induced damage to shrimp and improves their survival rate after infection.

Claims

1. Use of ursodeoxycholic acid in the preparation of a medicament for improving the survival rate of shrimp suffering from acute hepatopancreatic necrosis caused by pathogenic Vibrio parahaemolyticus.

2. The use according to claim 1, characterized in that: The concentration of pathogenic Vibrio parahaemolyticus in aquaculture water is 10 7 CFU / ml, ursodeoxycholic acid was administered intramuscularly at a dose of 20 mg / kg, once every 48 hours for a total of three injections; Compared with the control group in which all the mice died at 216 hours, the survival rate of the ursodeoxycholic acid injection group was 43% at 216 hours.

3. Use of ursodeoxycholic acid in the preparation of a medicament for alleviating hepatopancreatic damage in shrimp infected with acute hepatopancreatic necrosis.

4. The use according to claim 3, characterized in that: Ursodeoxycholic acid alleviates shrimp hepatopancreas damage by maintaining normal bile acid efflux, inhibiting excessive bile acid reflux, and inhibiting the expression of genes related to bile acid synthesis.

5. The use according to claim 4, characterized in that: Ursodeoxycholic acid upregulates bile acid efflux-related genes MRP3 and MRP4 Gene expression maintains normal bile acid excretion.

6. The use according to claim 4, characterized in that: Ursodeoxycholic acid regulates bile acid reflux-related genes OATP and NTCP Gene expression homeostasis alleviates excessive bile acid reflux.

7. The use according to claim 4, characterized in that: Ursodeoxycholic acid downregulates bile acid synthesis-related genes CYP7A1 Gene expression, inhibiting bile acid synthesis.

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