Use of obeticholic acid in prevention and treatment of acute hepatopancreas necrosis disease in prawn

Abstract

The application provides application of obeticholic acid in prevention and treatment of acute hepatopancreas necrosis disease of shrimp, and belongs to the technical field of shrimp breeding disease prevention and control. The application adopts a kind of bile acid metabolism regulator, obeticholic acid (OCA), controls the metabolism of bile acid of diseased shrimp, reduces the accumulation of bile acid in the hepatopancreas of shrimp, and reduces the hepatopancreas damage caused by pathogenic vibrio infection. No matter injection or adding to feed, obeticholic acid significantly reduces the symptoms and mortality of shrimp after infection of acute hepatopancreas necrosis disease, which shows that obeticholic acid has certain application value in prevention and treatment of acute hepatopancreas necrosis disease of shrimp.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of disease prevention and control of prawn culture, and particularly relates to application of obeticholic acid in prevention and treatment of acute hepatopancreas necrosis disease of prawn. BACKGROUND

[0002] At present, the acute hepatopancreas necrosis disease of prawn is mainly prevented and treated by strengthening biological security measures and fine management, and comprehensive prevention and control measures such as adding probiotics, but the prevention and control effect is still difficult to meet the production demand.

[0003] The pathogenic bacteria of acute hepatopancreas necrosis disease is a type of Vibrio carrying pVA1 plasmid, the pVA1 plasmid carries gene sequences encoding PirA and PirB toxins, and the PirA / B toxin is the main factor causing damage to the hepatopancreas and intestinal tract of prawn. After the pathogenic Vibrio (VP AHPND ) infects prawn, it causes imbalance of cell metabolism and cell apoptosis of prawn. A large number of lipid droplets accumulate in the hepatopancreas of prawn infected with AHPND, bile acid accumulates abnormally, and lipid metabolism and bile acid metabolism are imbalanced, while the accumulation of bile acid can promote the secretion of pathogenic toxin PirA / B by pathogenic Vibrio and promote the formation of Vibrio biofilm, thereby enhancing the pathogenicity (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, DOI 10.1111 / cmi.13127). It is expected to reduce the damage of pathogenic Vibrio to prawn cells by adjusting the metabolic homeostasis of prawn, thereby reducing the damage loss.

[0004] Among the disclosed patent technologies, many technologies develop microbial preparations for preventing and controlling acute hepatopancreas necrosis disease by screening Vibrio antagonistic microorganisms. For example, CN202410427133.0 discloses a Pseudoalteromonas that has antagonistic effect on Vibrio, CN201880004885.9 discloses the application of Bacillus subtilis, Bacillus pumilus and Bacillus licheniformis in the prevention and control of acute hepatopancreas necrosis disease, and CN202410052733.3 discloses a bacteriophage that can kill Vibrio. Some patents use Chinese herbal medicine to prevent and control acute hepatopancreas necrosis disease. For example, CN202410713083.2 and CN202311498414.7 respectively disclose Chinese herbal medicine formulations that have preventive and control effects on acute hepatopancreas necrosis disease. New polypeptides or recombinant proteins targeting the interaction between Vibrio toxin and shrimp bodies have also been developed for the prevention and control of acute hepatopancreas necrosis disease. For example, CN202211357289.3 and CN202211599098.8 respectively disclose the application of a receptor protein LvFABP that can bind to PirB toxin and a polypeptide P2 that can bind to PirB toxin in disease control.

[0005] Among the above disclosed technologies, microbial preparations, Chinese herbal medicine, new polypeptides and recombinant proteins have significant effects in disease control. Among them, microbial preparations and Chinese herbal medicine have relatively broad-spectrum inhibitory effect on Vibrio, but cannot distinguish between pathogenic Vibrio and non-pathogenic Vibrio. New polypeptides and recombinant proteins such as LvFABP target Vibrio toxin PirB in pathogenic Vibrio, which can be targeted to prevent and control pathogenic Vibrio, but its main effect is in disease prevention, and no treatment effect has been reported. Therefore, there is still a lack of efficient prevention and control technology for acute hepatopancreas necrosis disease in shrimp. SUMMARY

[0006] The present application provides a use of obeticholic acid in the preparation of a medicament for improving the survival rate of shrimp caused by acute hepatopancreas necrosis disease caused by pathogenic Vibrio parahaemolyticus. By regulating the bile acid metabolism of diseased shrimp bodies, the symptoms and mortality of shrimp after infection with acute hepatopancreas necrosis disease are significantly reduced, and the prevention and control of acute hepatopancreas necrosis disease in shrimp has certain application value.

[0007] In order to achieve the above purpose, the present application provides a use of obeticholic acid in the preparation of a medicament for improving the survival rate of shrimp caused by acute hepatopancreas necrosis disease caused by pathogenic Vibrio parahaemolyticus.

[0008] As a preferred, when the concentration of pathogenic Vibrio parahaemolyticus in the breeding water is 10 7 The obeticholic acid is administered by intramuscular injection, the injection dose is 20 mg / kg, and the injection is performed once every 48 hours, for a total of three times;

[0009] Compared with the control group, the survival rate of the obeticholic acid injection group at 216h was 57%.

[0010] The present application also provides a use of obeticholic acid in the preparation of a medicament for reducing the copy number of Vibrio toxic plasmid in the hepatopancreas of shrimp infected with acute hepatopancreas necrosis disease.

[0011] The present application also provides a use of obeticholic acid in the preparation of a medicament for reducing the damage to the hepatopancreas of shrimp infected with acute hepatopancreas necrosis disease.

[0012] Preferably, obeticholic acid reduces the damage to the hepatopancreas of shrimp by promoting bile acid efflux, inhibiting bile acid reflux, and inhibiting the expression of bile acid synthesis-related genes.

[0013] Preferably, obeticholic acid promotes bile acid efflux by up-regulating the expression of bile acid efflux-related genes MRP2, MRP3, and MRP4 genes.

[0014] Preferably, obeticholic acid reduces the reflux of bile acid from the intestine to the hepatopancreas by down-regulating the expression of bile acid reflux-related genes OATP and NTCP genes.

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

[0016] The present application also provides a feed for preventing and treating acute hepatopancreas necrosis disease in shrimp, wherein the obeticholic acid described in the above technical solution is added to the ordinary feed for shrimp.

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

[0018] Compared with the prior art, the present application has the following advantages and positive effects:

[0019] The present application uses a bile acid metabolism regulator, obeticholic acid (OCA), to regulate the bile acid metabolism of diseased shrimp, reduce the accumulation of bile acid in the hepatopancreas of shrimp, and reduce the hepatopancreas damage caused by pathogenic Vibrio infection. Whether injected or added to feed, obeticholic acid significantly reduces the symptoms and mortality rate of shrimp after infection with acute hepatopancreas necrosis disease, indicating that obeticholic acid has certain application value in the prevention and treatment of acute hepatopancreas necrosis disease in shrimp. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The injection of OCA provided in the embodiments of the present application can significantly reduce the mortality rate of shrimp after infection with AHPND;

[0021] Figure 2The schematic diagram of the tissue section microscopic observation provided by the embodiment of the present application shows that OCA can reduce the pathological changes of hepatopancreas tissue of shrimp caused by AHPND infection;

[0022] Figure 3 The schematic diagram of the injection of OCA provided by the embodiment of the present application can significantly reduce the toxic plasmid copy number of pathogenic Vibrio in the hepatopancreas of diseased shrimp;

[0023] Figure 4A The schematic diagram of the injection of OCA provided by the embodiment of the present application can reduce the damage of AHPND infection by maintaining the homeostasis of bile acid metabolism;

[0024] Figure 4B The schematic diagram of the injection of OCA provided by the embodiment of the present application can reduce the damage of AHPND infection by promoting the expression of genes such as bile acid efflux, inhibiting bile acid reflux, and inhibiting bile acid synthesis. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0026] Example 1: Injection of OCA can significantly improve the survival rate of AHPND infected shrimp and reduce the symptoms of hepatopancreas and intestinal tract

[0027] Obecholic acid OCA was dissolved in dimethyl sulfoxide (DMSO) and injected into the muscle of shrimp at a dose of 20 mg / kg, with an injection interval of 48 hours, and a total of three injections. Phosphate buffer (PBS) and PBS+DMSO were injected as control treatment. During this process, the number of surviving shrimp in each group was recorded to detect the effect of OCA injection on the survival rate of shrimp. 48 hours after the last injection, the challenge test was performed on each treatment group, which was divided into VP AHPND infected group (challenge group) and uninfected group (control group), each containing 200 shrimp. The challenge test used the immersion challenge method, and the pathogenic Vibrio VP AHPND was cultured in large quantities and added to the shrimp culture water to a final concentration of 10 7 CFU / ml.

[0028] The results show that injection of OCA has no effect on the survival and health status of shrimp, and the survival rate of shrimp in each group after injection is 100%, and the injection of OCA does not affect the health status of shrimp. In the VP AHPNDIn the challenge experiment, the survival rate of non-challenged control shrimp in each group remained 100% throughout the experiment. In contrast, death occurred in all groups, including the control groups (PBS and DMSO injection) and the experimental groups (OCA injection). In the control groups, death began to occur at 12 h, while death in the OCA-injected groups began to occur only after 24 h. As the infection continued, the mortality rate of shrimp in each group gradually increased. At 72 h, the mortality rate of shrimp in the control groups reached 50%, and all shrimp died at 216 h. In contrast, the survival rate of shrimp in the OCA-injected groups was 57% at 216 h. These results indicate that, although OCA injection before Vibrio challenge did not completely prevent the death of shrimp due to acute hepatopancreas necrosis disease caused by infection, it significantly delayed the death process caused by infection and ultimately improved the survival rate of shrimp AHPND ( Figure 1 ).

[0029] The HE staining of the hepatopancreas tissue sections of challenged shrimp showed that, under uninfected conditions, the hepatopancreas tissue structure of shrimp in the four groups remained intact. HE staining showed that the B and R cell types in the hepatopancreas were clearly distinguishable, and the star-shaped lumen structure in the hepatopancreatic tubule was clear, indicating that the hepatopancreas had a good tissue morphology under uninfected conditions and no pathological changes were observed. After AHPND infection, the damage to the hepatopancreas gradually worsened over time. At 24 h after infection, although the hepatopancreas cell structure remained intact, the B and R cells in each group were still clearly distinguishable, but the hepatopancreatic tubule epithelium had already begun to extend into the lumen, and the star-shaped lumen structure began to deform. By 48 h, the pathological damage to the hepatopancreas was significantly aggravated, and the hepatopancreatic tubule epithelial cells in the PBS and PBS+DMSO groups retracted in large numbers, the lumen structure gradually disappeared, and a large number of blood cells infiltrated, reflecting severe tissue damage.

[0030] In the OCA group, although the extension of the lumen epithelium was observed, the star-shaped lumen structure remained, and the blood cell infiltration was relatively less. By 72 h after infection, the pathological damage further worsened. In the PBS and PBS+DMSO groups, the star-shaped lumen structure of the hepatopancreas completely disappeared, and the blood cell infiltration was significant, indicating that the hepatopancreas had suffered severe damage. Although the hepatopancreas in the OCA group was also damaged to some extent, with some epithelial cells extending into the lumen, the star-shaped tubular structure was still visible, the cell types were still clearly distinguishable, and the blood cell infiltration was light, indicating that OCA could alleviate the damage to the hepatopancreas caused by infection Figure 2 ).

[0031] Example 2 Injection of OCA can significantly reduce the copy number of toxic plasmids in the hepatopancreas of shrimp infected with AHPND

[0032] The OCA injection and Vibrio challenge steps were as described above. The copy number of the virulence factor pirA​vp To evaluate VP by copy number AHPND The colonization status was assessed. The absolute quantitative detection method of real-time PCR (qRT-PCR) was used to detect PirA in the hepatopancreas of shrimp. vp Gene copy number. The experimental method followed the approach 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). 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 value. vp The copy number of a gene.

[0033] The PCR primers used in the experiment were: PirA vp -F:5'-TTGGACTGTCGAACCAAACG-3',PirA vp -R: 5'-GCACCCCATTGGTATTGAATG-3'; The experimental probe is a TaqMan probe: 5'-AGACAGCAAACATACACCTATCATCCCGGA-3'.

[0034] 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: 95℃ pre-denaturation for 3 minutes; 95℃ for 15 seconds, 60℃ for 1 minute; 40 cycles. Each sample was tested three times to ensure data accuracy.

[0035] Depend on Figure 3 The test results showed that in the early stage of infection (within 24 hours), the pirA copy number in all groups showed a continuous upward trend, indicating a gradual increase in the copy number of the virulence plasmid carrying the pirA virulence gene in the hepatopancreas. In the early stage of infection (0-24 hours), there was a significant difference in pirA count between the OCA group and the control group (PBS and PBS+DMSO groups). vp Copy number did not show significant differences, and the infection process was basically the same in all groups. With prolonged infection time, at 48 and 72 hours after infection, the pirA count in the OCA group... vpCopy number began to be significantly lower than the PBS and PBS + DMSO groups, indicating that OCA injection reduced the copy number of pathogenic bacteria carrying toxic plasmids.

[0036] Example 3 Injection of OCA alleviated the damage of AHPND to hepatopancreas by increasing the hepatopancreatic bile acid efflux and inhibiting the metabolic processes such as bile acid synthesis

[0037] Bile acid accumulation is an important physiological change in AHPND infection, which is related to the differential expression of genes involved in bile acid synthesis and transport in infected shrimp. By detecting the accumulation of bile acids in the hepatopancreas of shrimp and the expression of genes related to bile acid metabolism, the alleviation of OCA on VP AHPND physiological and biochemical and molecular biological indicators of infection symptoms.

[0038] OCA injection and Vibrio challenge steps are as above. The total bile acid content in the hepatopancreas of shrimp was quantified using a total bile acid assay kit (Nanjing Jiancheng Biological Engineering Institute, China) according to the manufacturer's protocol. The expression of genes related to bile acid metabolism was detected by qRT-PCR, and the detected genes and corresponding primer sequences are shown in Table 1. The PCR reaction program is as follows: denaturation at 94°C for 2 min, followed by 40 cycles of 94°C denaturation for 15 s, 60°C annealing for 15 s, and 72°C extension for 30 s.

[0039] Table 1 Primer sequences for detecting genes related to bile acid metabolism

[0040]

[0041] Under non-infection conditions, the total bile acid level in the hepatopancreas of shrimp injected with OCA increased significantly, which was due to the fact that OCA itself belongs to a natural bile acid. However, after infection with AHPND, the hepatopancreatic bile acid content of the control groups injected with PBS and PBS + DMSO increased significantly, while the OCA injection group remained stable. It can be seen that AHPND can cause the accumulation of bile acids in the hepatopancreas of shrimp, but shrimp infected with AHPND after injection of OCA do not accumulate bile acids, showing that it has a significant regulatory effect on the bile acid metabolism of shrimp infected with AHPND Figure 4A

[0042] ​LvECR gene expression was significantly inhibited after shrimp infected with AHPND, but injection of OCA could significantly up-regulate the expression of LvECR gene. In addition, injection of OCA up-regulated the expression of MRP2, MRP3 and MRP4 genes, which have the effect of promoting the excretion of bile acids from the hepatopancreas. OCA significantly inhibited the expression of OATP and NTCP genes related to bile acid reflux and CYP7A1 gene related to bile acid synthesis, reducing the reflux of bile acids from the intestine to the hepatopancreas and the synthesis of bile acids. Therefore, OCA can reduce the accumulation of hepatopancreatic bile acids caused by AHPND infection in shrimp by promoting the expression of genes that promote bile acid excretion, inhibit bile acid reflux, and inhibit bile acid synthesis, thereby reducing the damage of AHPND Figure 4B

[0043] Example 4 Feeding OCA can reduce the mortality of shrimp infected with AHPND

[0044] Shrimp feed containing OCA was prepared to verify its oral feeding effect on the prevention and treatment of AHPND. Shrimp compound feed containing OCA at 100 mg / Kg, 150 mg / Kg and 200 mg / Kg was prepared. Shrimp farming and challenge experiments were carried out, and shrimp with a body weight of about 10 g were cultured in 18 1m 3 buckets, 50 shrimp per bucket, and fed with control feed (without OCA). After 1 week of temporary culture, 12 buckets were randomly selected for VP AHPND (10 7 CFU / mL) bath challenge, 24 hours after challenge, 3 buckets were randomly selected to feed control feed, 3 buckets were randomly selected to feed 100 mg / Kg OCA compound feed, 3 buckets were randomly selected to feed 150 mg / Kg OCA compound feed, and 3 buckets were randomly selected to feed 200 mg / Kg OCA compound feed. Among the shrimp not challenged, 3 buckets continued to feed ordinary feed, and 3 buckets fed OCA feed. The survival rate of shrimp within 10 days was recorded to detect the prevention and treatment effect of OCA added in feed on AHPND. The results are shown in Table 2.

[0045] Table 2 Protective effect of OCA added in feed on acute hepatopancreatic necrosis disease of Litopenaeus vannamei

[0046]

[0047]

[0048] ​The experimental results show that in the 10-day culture test, the prawns fed with the normal feed and the OCA feed have almost no death without AHPND infection, which shows that the addition of OCA in the feed does not affect the health of the prawns. After AHPND infection, the survival rate of the prawns fed with the OCA feed is significantly higher than that of the control group. On the 10th day, the prawns fed with the normal feed almost all died, but the survival rate of the prawns fed with the 150mg / Kg or 200mg / Kg OCA feed is close to 60% (Table 2). It can be seen that the addition of OCA in the feed can significantly reduce the damage caused by AHPND to the prawns and improve the survival rate of the prawns infected with AHPND.

Claims

1. Use of obeticholic acid in the preparation of a medicament for increasing the survival rate of shrimp infected with acute hepatopancreas necrosis disease caused by pathogenic Vibrio parahaemolyticus.

2. Use according to claim 1, characterized in that, The concentration of pathogenic Vibrio parahaemolyticus in the aquaculture water was 10 7 CFU / ml, and obeticholic acid was administered by intramuscular injection at a dose of 20 mg / kg, once every 48 hours for a total of three injections. Compared with the control group, all shrimp died at 216h, and the survival rate of the obeticholic acid injection group was 57% at 216h.

3. Use according to claim 1, characterized in that, Obeticholic acid can reduce the copy number of Vibrio parahaemolyticus virulence plasmid in the hepatopancreas of shrimp infected with acute hepatopancreas necrosis disease.

4. Use of obeticholic acid in the preparation of a medicament for reducing the damage to the hepatopancreas of shrimp infected with acute hepatopancreas necrosis disease.

5. Use according to claim 4, characterized in that, Obeticholic acid can reduce the damage to the hepatopancreas of shrimp by promoting bile acid efflux, inhibiting bile acid reflux, and inhibiting the expression of bile acid synthesis-related genes.

6. Use according to claim 5, characterized in that, Ocalit promotes bile acid efflux by upregulating the expression of bile acid efflux-related genes MRP2 , MRP3 and MRP4 genes.

7. Use according to claim 5, characterized in that, Ocalit reduces the expression of genes associated with bile acid reflux OATP and NTCP reduces the reflux of bile acids from the intestine to the liver-pancreas.

8. Use according to claim 5, characterized in that, Ocalit reduces the expression of genes involved in the synthesis of bile acids CYP7A1 The expression of genes involved in the synthesis of bile acids is inhibited.

9. Use of obeticholic acid in the preparation of a feed for the prevention and treatment of acute hepatopancreas necrosis disease in shrimp.

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

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