Application of ursodesoxycholic acid in preparation of medicament for improving survival rate of acute hepatopancreatic necrosis disease of prawns

Abstract

The invention provides application of ursodesoxycholic acid in preparation of a medicament for improving the survival rate of acute hepatopancreatic necrosis of prawns, and belongs to the technical field of disease prevention and control in prawn culture. According to the invention, a lipid metabolism regulator ursodesoxycholic acid (UDCA) is adopted, and the damage of pathogenic vibrio infection to prawns is obviously reduced by regulating and controlling the metabolic pathway of infected prawn bodies. Tests prove that no matter the ursodesoxycholic acid is injected or added into the feed for feeding, the symptoms and the death rate of the prawns infected with the acute hepatopancreatic necrosis disease are remarkably reduced, and the ursodesoxycholic acid has a certain prevention and treatment effect in the prevention and treatment of the acute hepatopancreatic necrosis disease of the prawns.

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 acute hepatopancreatic necrosis disease. Background Art

[0002] my country is the world's largest shrimp farming country, with the output of marine and freshwater shrimp farming reaching 2.57 million tons in 2023 (China Fisheries Statistical Yearbook, 2024). However, Acute Hepatopancreas Necrosis Disease (AHPND) caused by pathogenic Vibrio spp. seriously harms my country's shrimp farming industry. Acute Hepatopancreas Necrosis Disease mainly occurs in the early stages of shrimp farming, with a high mortality rate, and the shrimp farming industry suffers huge losses every year. At present, Acute Hepatopancreas Necrosis Disease of shrimp is mainly prevented and controlled by comprehensive prevention and control measures such as strengthening farming management and releasing probiotics, but the prevention and control effect is general and it is difficult to meet production needs.

[0003] In fact, not all Vibrio are pathogenic bacteria 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 gene sequences encoding PirA and PirB, which are the main factors causing hepatopancreatic and intestinal damage to shrimp. Pathogenic Vibrio (VP AHPND) After infecting shrimp, it leads to metabolic imbalance and apoptosis of shrimp cells. After infection with AHPND, a large amount of fat 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, it is expected to reduce the damage of pathogenic Vibrio to shrimp cells, thereby reducing disease losses.

[0004] Among the disclosed patent technologies, many technologies develop microbial preparations to prevent and control acute hepatopancreatic necrosis by screening antagonistic microorganisms of Vibrio. For example, CN202410427133.0 discloses a pseudoalteromonas that has an antagonistic effect on Vibrio, CN201880004885.9 discloses the application 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 in new polypeptides or recombinant proteins developed 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 above-mentioned disclosed technologies, microbial preparations, Chinese herbal medicines, novel polypeptides and recombinant proteins have significant effects in disease prevention and control. Among them, microbial preparations and Chinese herbal medicines have a relatively broad spectrum of inhibitory effects on Vibrio, but cannot distinguish between pathogenic Vibrio and non-pathogenic Vibrio. Although novel polypeptides and recombinant proteins such as LvFABP target the Vibrio toxin PirB in pathogenic Vibrio, they can prevent and control pathogenic Vibrio in a targeted manner, but their main effect is in the prevention of the disease, and no treatment effect has been reported. Therefore, there is currently a lack of efficient prevention and control technology for acute hepatopancreatic necrosis of shrimp. Summary of the invention

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

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

[0008] Preferably, the concentration of pathogenic Vibrio parahaemolyticus in the 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 216h, the survival rate of the ursodeoxycholic acid injection group was 43% at 216h.

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

[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 of shrimp hepatopancreas by maintaining normal bile acid efflux, inhibiting excessive bile acid reflux and inhibiting the expression of bile acid synthesis related genes.

[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 genes.

[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 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] The present invention adopts a lipid metabolism regulator, ursodeoxycholic acid (UDCA), to regulate the metabolic pathway of the infected shrimp, thereby significantly reducing the damage caused by pathogenic Vibrio infection to the shrimp. Experiments have shown that ursodeoxycholic acid, whether injected or added to feed, significantly reduces the symptoms and mortality of shrimp infected with acute hepatopancreatic necrosis, indicating that ursodeoxycholic acid has certain application value in the prevention and treatment of acute hepatopancreatic necrosis of shrimp. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0023] Figure 4A A schematic diagram of reducing AHPND infection damage by injecting UDCA provided by an embodiment of the present invention by maintaining bile acid metabolic homeostasis;

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

[0025] 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.

[0026] Example 1 Injection of UDCA can significantly improve the survival rate of shrimp infected with AHPND and alleviate 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 buffer (PBS) and PBS+DMSO were injected at the same time as control treatments. 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 groups were VP AHPND The infected group (challenge group) and the uninfected group (control group) each contained 200 shrimps. The challenge experiment used the immersion challenge method to inoculate the pathogenic Vibrio parahaemolyticus VP AHPND For expanded 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 occur from 12 hours, while the shrimp in the UDCA injection group began to die after 24 hours. As the infection continued, the mortality rate of shrimp in each group gradually increased. At 72 hours, the mortality rate of shrimp in the control group had reached 50%, and all died at 216 hours. In contrast, at 216 hours, the survival rate of the UDCA injection group was 43%. These results show that although the injection of UDCA 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 challenge, it can be seen that the shrimp in the control experimental group (PBS and DMSO injection groups) had obvious hepatopancreatic lesions after the 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 copies of the hepatopancreatic toxic plasmid in shrimp infected with AHPND

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

[0031] Detection of PirA in shrimp hepatopancreas by qRT-PCR vp 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.), and the standard curve was used for absolute quantification. 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.

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

[0033] 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. Each sample was tested three times to ensure the accuracy of the data.

[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. vp The copy number began to be significantly lower than that of the PBS and PBS+DMSO groups, indicating that UDCA injection reduced the copy number of toxic plasmids carried by pathogens.

[0035] Example 3 Injection of UDCA reduces the damage of AHPND to the hepatopancreas by increasing hepatopancreatic bile acid pumping and inhibiting bile acid synthesis and other metabolic processes

[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 in the hepatopancreas of shrimp and the expression of genes related to bile acid metabolism. AHPND Changes in physiological, biochemical and molecular biological indicators of infection symptoms.

[0037] The steps of UDCA injection and Vibrio challenge were the same as above. The 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 related to bile acid metabolism was detected by qRT-PCR. The detected genes and 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 the following: 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 gene expression related to bile acid metabolism

[0039]

[0040]

[0041] Under non-infectious 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 was in a stable state. It can be seen that AHPND can cause the accumulation of bile acid in the hepatopancreas of shrimp, but the shrimp injected with UDCA will 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 (GenBankACCESSION NUMBER: 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, the regulatory mechanism of UDCA on bile acid metabolic homeostasis also depends on improving bile acid excretion and inhibiting bile acid reflux and synthesis. Under AHPND infection, UDCA significantly upregulated the expression of bile acid efflux-related genes MRP3 (GenBank ACCESSION NUMBER: XM_070115017) and MRP4 (GenBank ACCESSION NUMBER: XM_070131847), helping to relieve bile acid accumulation. Bile acid reflux-related genes OATP (GenBank ACCESSION NUMBER: XM_070136381) and NTCP (GenBank ACCESSION NUMBER: XM_070134692) were significantly upregulated after infection with AHPND, indicating that bile acid reflux was accelerated after infection with AHPND, leading to bile acid accumulation in the hepatopancreas. However, injection of UDCA can maintain the expression homeostasis of these two genes and alleviate the phenomenon of accelerated bile acid reflux caused by AHPND infection. In addition, UDCA down-regulated the expression of bile acid synthesis-related gene CYP7A1 (GenBank ACCESSION NUMBER: XM_017440277) under infection conditions, reducing the amount of bile acid produced. UDCA maintains the stability of bile acid metabolism after shrimp infection with acute hepatopancreatic necrosis by maintaining normal bile acid efflux, inhibiting excessive bile acid reflux, and inhibiting the expression of bile acid synthesis genes, thus 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 preventive and therapeutic effect on AHPND by oral feeding. 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 buckets, 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 CFU / mL) bath challenge, 24 hours after the challenge, randomly select 3 buckets to feed the control group feed, 3 buckets to feed 100mg / Kg UDCA compound feed, 3 buckets to feed 150mg / Kg UDCA compound feed, and 3 buckets to feed 200mg / Kg UDCA compound feed. Among the shrimps that were not challenged, 3 buckets continued to feed ordinary feed, and 3 buckets were fed with UDCA feed. The cumulative mortality of shrimp within 10 days was recorded to detect the prevention and control effect of adding UDCA to the feed on 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 in the 10-day breeding experiment, in the absence of AHPND infection, there was almost no death in the shrimp fed with ordinary feed and UDCA feed, indicating that the addition of UDCA in the feed did not affect the health of the shrimp. After infection with AHPND, the mortality rate of shrimp fed with UDCA feed was significantly reduced. On the 10th day, almost all shrimps fed with ordinary feed died, but the mortality rate of shrimps fed with 150-200 mg / Kg UDCA feed was about 70% (Table 2). It can be seen that the addition of UDCA in the feed can significantly reduce the damage caused by AHPND to shrimp and improve the survival rate of the virus.

Claims

1. Use of ursodeoxycholic acid in the preparation of a medicament for improving the survival rate of shrimps 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 216h, the survival rate of the ursodeoxycholic acid injection group was 43% at 216h.

3. Use of ursodeoxycholic acid in the preparation of a medicament for reducing the number of copies of Vibrio toxic plasmids in the hepatopancreas of shrimp infected with acute hepatopancreatic necrosis.

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

5. The use according to claim 4, 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.

6. The use according to claim 5, characterized in that: Ursodeoxycholic acid maintains normal bile acid efflux by upregulating the expression of bile acid efflux-related genes MRP3 and MRP4.

7. The use according to claim 5, characterized in that: Ursodeoxycholic acid alleviates excessive bile acid reflux by maintaining the expression homeostasis of bile acid reflux-related genes OATP and NTCP genes.

8. The use according to claim 5, characterized in that: Ursodeoxycholic acid inhibits the synthesis of bile acid by downregulating the expression of CYP7A1, a gene related to bile acid synthesis.

9. A feed for preventing and treating acute hepatopancreatic necrosis of shrimp, characterized in that: The ursodeoxycholic acid according to any one of claims 1 to 8 is added to the common feed for shrimp.

10. The feed according to claim 9, characterized in that: The added amount of ursodeoxycholic acid is 150-200 mg / Kg.

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