Application of chlorpromazine hydrochloride in preparation of medicine for resisting acute hepatopancreatic necrosis

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of chlorpromazine hydrochloride in preparation of medicine for resisting acute hepatopancreatic necrosis disease, the way for PirABVp to enter prawn cells is defined through tests, an endocytosis inhibitor CPZ is screened out, the endocytosis inhibitor CPZ remarkably inhibits PirABVp from entering prawn cells by inhibiting gridding protein-dependent endocytosis, and the effect of resisting acute hepatopancreatic necrosis disease is achieved. The content of PirABVp in target organs is reduced, the cell viability of the prawns is improved, and finally the survival rate of the prawns is improved. This shows that CPZ has potential in the prevention and treatment of AHPND.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic animal disease prevention and treatment, and specifically relates to the application of chlorpromazine hydrochloride (CPZ) in the preparation of drugs for resisting acute hepatopancreatic necrosis. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Shrimp farming is an important pillar industry of my country's aquaculture, but diseases are the main bottleneck restricting the sustainable development of shrimp farming. Among them, acute hepatopancreatic necrosis disease (AHPND) has the characteristics of rapid onset and high mortality, causing huge economic losses every year.

[0004] The original pathogen of AHPND has been identified as a specific Vibrio parahaemolyticus ( Virbio parahaemolyticus ), which carries a pVA1 virulence plasmid. This plasmid expresses PirAB Vp The toxin protein complex is the main pathogenic factor. The pVA1 plasmid can be horizontally transferred between species. After Vibrio campbellii, Vibrio harveyi and Vibrio erwinia acquire the plasmid, they can cause shrimp to suffer from AHPND, further accelerating the spread of the disease.

[0005] The clinical symptoms of AHPND are mainly: whitening and atrophy of the hepatopancreas, empty stomach and jejunum, softening of shrimp shells, weakening of vitality and even lethargy. Histopathological characteristics include: epithelial cells of the hepatopancreatic tubules fall off into the lumen, obvious blood cell infiltration and secondary bacterial infection appear in and between the hepatopancreatic tubules in the late infection period, and large-scale necrosis of the hepatopancreas. Recent studies have found that shrimp blood cells are also an important target tissue of AHPND. PirAB Vp After being synthesized by bacteria and released, it can enter the shrimp's blood cells, causing them to lyse and eventually lead to the shrimp's death.

[0006] At present, there is no very effective treatment for AHPND. Chemical disinfection and antibiotics are often used in aquaculture. However, these methods can cause environmental pollution, drug residues in food, and drug resistance. Therefore, it is urgent to develop new effective and safe drugs and prevention methods. Summary of the invention

[0007] The present invention aims to solve the problem that there is currently a lack of effective treatment methods for shrimp AHPND and to provide the use of chlorpromazine hydrochloride (CPZ) in the preparation of drugs against acute hepatopancreatic necrosis. VpThe pathway of entering shrimp cells was screened out as an endocytosis inhibitor CPZ, which significantly inhibited PirAB by inhibiting clathrin-dependent endocytosis. Vp Enters shrimp cells and reduces PirAB in target organs Vp The content can improve the cell vitality of shrimp and ultimately increase the survival rate of shrimp.

[0008] In order to achieve the above object, the present invention is implemented through the following technical solutions: In a first aspect, the present invention provides the use of chlorpromazine hydrochloride (CPZ) in the preparation of a drug for preventing acute hepatopancreatic necrosis.

[0009] The chlorpromazine hydrochloride (CAS: 69-09-0) is also known as 2-chloro-10-(3-dimethylaminopropyl) phenothiazine hydrochloride, CPZ, chlorpromazine, and its molecular formula is C 17 H 19 ClN2S•HCl, molecular weight is 355.33.

[0010] In some embodiments, the anti-acute hepatopancreatic necrosis drug has the following effects: (1) Inhibition of PirAB Vp It enters shrimp cells via clathrin-dependent endocytosis; (2) Reduce PirAB in target organs Vp Content; (3) Improve PirAB Vp Resulting changes in the morphology of hepatopancreatic tissue; (4) Improve the cell viability of shrimp.

[0011] In some embodiments, the chlorpromazine hydrochloride is a pure product, a solvate or an isomer thereof.

[0012] Preferably, the prawn is Penaeus japonicus.

[0013] In some embodiments, the effective concentration of chlorpromazine hydrochloride is 0.5-4 μg / μL.

[0014] Preferably, the effective concentration of chlorpromazine hydrochloride is 1-4 μg / μL.

[0015] In a second aspect, the present invention provides a drug for preventing acute hepatopancreatic necrosis, the drug comprising chlorpromazine hydrochloride and a pharmaceutical carrier or excipient.

[0016] Preferably, the pharmaceutical carrier or excipient is selected from one or more pharmaceutically acceptable dispersants, wetting agents, disintegrants, binders, lubricants, regulators, defoamers, antifreeze agents, thickeners or fillers.

[0017] In some embodiments, the dosage form of the anti-acute hepatopancreatic necrosis drug is a wettable powder, a water dispersible granule, an aqueous suspension or a dispersible oil suspension.

[0018] In some embodiments, the anti-acute hepatopancreatic necrosis drug is administered by feeding or injection.

[0019] The beneficial effects achieved by one or more embodiments of the present invention are as follows: The present invention is to treat PirAB in shrimp blood cells Vp Cytosolic Amount; PirAB in Target Tissues and Cells such as Hepatopancreas, Intestine and Blood Cells Vp The content of CPZ, the activity of blood cells and the survival rate of shrimps were tested, and it was confirmed that the use of CPZ in shrimps can effectively inhibit the virulence factor PirAB of the pathogen of acute hepatopancreatic necrosis. Vp Enters shrimp cells via clathrin-dependent endocytosis and reduces PirAB in target organs Vp content, avoid PirAB Vp Damage to cells, improve PirAB Vp The morphological changes in the hepatopancreas caused by CPZ are beneficial to improving the cell viability of shrimp and significantly increasing the survival rate of shrimp. This shows that CPZ has potential in preventing and treating AHPND in shrimp. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 is the immunofluorescence image of the control group of Example 1 of the present invention; Figure 2 is the immunofluorescence image of the CPZ group in Example 1 of the present invention; Figure 3 This is a blood cell activity diagram provided in Example 2 of the present invention.

[0022] Figure 4 This is a diagram of the immunoblotting results provided in Example 3 of the present invention.

[0023] Figure 5 This is a diagram of the H&E results of the hepatopancreas of shrimp provided in Example 4 of the present invention.

[0024] Figure 6 This is a graph showing the survival rate of shrimps provided in Example 5 of the present invention. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0026] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0027] Example 1 Preparation of each solution: PBS solution: 140mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4, pH7.4.

[0028] CPZ solution: CPZ (Shanghai, China, A506232) was dissolved in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH7.4) to a final concentration of 2 μg / μL.

[0029] PyR Vp Protein solution: Purified PirA was assayed using the BCA protein concentration assay kit (enhanced, Shanghai Bio-Tech Biotechnology Co., Ltd., catalog number P0010). Vp The protein concentration was measured and PirA was lysed with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH7.4). Vp The protein was diluted to a final concentration of 25 μM; PirB Vp Protein solution: Purified PirB was assayed using the BCA protein concentration assay kit (enhanced, Shanghai Bio-Tech Biotechnology Co., Ltd., catalog number P0010). Vp The protein concentration was measured and PirA was lysed with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH7.4). Vp The protein was diluted to a final concentration of 25 μM; PirAB Vp Protein solution: Purified PirAB was quantified using the BCA protein concentration assay kit (enhanced, Shanghai Bio-Tech Biotechnology Co., Ltd., catalog number P0010). VpThe protein concentration was measured and PirA was lysed with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH7.4). Vp The protein was diluted to a final concentration of 25 μM.

[0030] Non-virulence protein (empty protein expressed by pET30a plasmid) solution: The concentration of purified non-virulence protein (empty protein expressed by pET30a plasmid) was measured using the BCA protein concentration assay kit (Enhanced, Shanghai Biotime Biotechnology Co., Ltd., Catalog No. P0010), and the non-virulence protein was diluted with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH7.4) to a final concentration of 25 μM.

[0031] Experimental animal groups: Divided into control group and CPZ group, the control group was divided into control group 1, control group 2, control group 3, control group 4 and control group 5, each group had 20 Japanese marsupials, and the weight of each Japanese marsupial was 10 ± 2g; The CPZ groups were divided into CPZ group 1, CPZ group 2, CPZ group 3, CPZ group 4 and CPZ group 5, with 20 Japanese marsupials in each group and the weight of each Japanese marsupial was 10 ± 2g.

[0032] The treatment methods of the Japanese shrimp in the control group and the CPZ group were as follows: Control group Control group: 50 μL PBS solution was injected into the body of Japanese marsupial, and then 50 μL PBS solution was injected into the body of Japanese marsupial; Control group 2: 50 μL PBS solution was injected into the body of Japanese marsupial Penaeus japonicus, followed by 50 μL non-toxic protein solution (empty protein expressed by pET30a plasmid, concentration of 25 μM) injected into the body of Japanese marsupial Penaeus japonicus; Control group 3: 50 μL PBS solution was injected into the body of Japanese shrimp, followed by 50 μL PirA Vp The solution (concentration of 25 μM) was injected into the Japanese shrimp M. japonicus; Control group 4: 50 μL PBS solution was injected into the body of Japanese shrimp, followed by 50 μL PirB Vp The solution (concentration of 25 μM) was injected into the Japanese shrimp M. japonicus; Control group 5: 50 μL PBS solution was injected into the body of Japanese shrimp, followed by 50 μL PirABVp The solution (concentration of 25 μM) was injected into the body of Japanese shrimp.

[0033] CPZ group CPZ group: 50 μL CPZ solution (2 μg / μL) was injected into the body of Japanese marsupials to inhibit clathrin-mediated endocytosis; then 50 μL PBS solution was injected into the body of Japanese marsupials; CPZ group II: 50 μL CPZ solution (2 μg / μL) was injected into the body of Japanese marsupials to inhibit clathrin-mediated endocytosis; then 50 μL non-toxic protein solution (empty protein expressed by pET30a plasmid, concentration of 25 μM) was injected into the body of Japanese marsupials; CPZ group III: 50 μL CPZ solution (2 μg / μL) was injected into the body of Japanese shrimp to inhibit clathrin-mediated endocytosis; then 50 μL PirA Vp The solution (concentration of 25 μM) was injected into the Japanese shrimp M. japonicus; CPZ group 4: 50 μL CPZ solution (2 μg / μL) was injected into the body of Japanese shrimp to inhibit clathrin-mediated endocytosis; then 50 μL PirB Vp The solution (concentration of 25 μM) was injected into the Japanese shrimp M. japonicus; CPZ group 5: 50 μL CPZ solution (2 μg / μL) was injected into the body of Japanese shrimp to inhibit clathrin-mediated endocytosis; then 50 μL PirAB Vp The solution (concentration of 25 μM) was injected into the body of Japanese shrimp.

[0034] After 6 hours, hemolymph of P. japonicus in the control group and CPZ group was extracted and blood cells were collected by centrifugation. Blood cells were resuspended in 200 μL 4% paraformaldehyde and allowed to stand at room temperature for 30 min. 60 μL of cell suspension was dropped on a glass slide and the following treatments were performed in sequence: 1) After standing at room temperature, place the cells in a clean bench to dry them, and wash the blood cells three times with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH7.4). After permeabilization of the cells, wash the blood cells three times with PBS; 2) Block with 3% bovine serum albumin (BSA) (solvent: PBS) at room temperature for 1 h; 3) Dilute the his-tagged mouse primary antibody with 3% BSA (the concentration of the diluted his-tagged primary antibody is 2.5 μg / mL), add the diluted primary antibody to the blood cells on the blocked slide at room temperature, incubate at 4°C overnight, and then wash the blood cells with PBS; 4) After the blood cells on the slide were blocked again at room temperature for 1 h, goat anti-mouse secondary antibody diluted with 3% BSA was added (the concentration of the diluted secondary antibody was 2.5 μg / mL). After incubation at room temperature for 2 h, the blood cells were washed with PBS to obtain the treated slide.

[0035] Dilute the 4',6-diamidino-2-phenylindole (DAPI) stock solution at 1:1000 with PBS to obtain DAPI dilution solution. Add 1 mL of DAPI dilution solution to each treated slide, incubate at room temperature in the dark for 10 min, and then wash with PBS before sealing.

[0036] The prepared slide was placed on a laser confocal microscope to observe PirA Vp 、PirB Vp and PirAB Vp Whether the toxin protein enters the blood cells.

[0037] The results are as follows Figure 1 and Figure 2 As shown, in the control group, PirA Vp 、PirB Vp and PirAB Vp Both can enter shrimp hemocytes; in the CPZ group, after CPZ injection, PirA Vp 、PirB Vp and PirAB Vp The above results show that CPZ can effectively inhibit PirA Vp 、PirB Vp and PirAB Vp Enters shrimp hemocytes via clathrin-mediated endocytosis.

[0038] Example 2 Japanese shrimp blood cell processing: Grouping: The experiment was divided into a blank control group (without shrimp blood cells), an untreated group (with shrimp blood cells but without PirAB Vp Protein), phosphate buffer solution + PirAB Vp Chlorpromazine hydrochloride + PirAB Vp There were 10 groups, each group had 10 Japanese marsupials, and the body weight was 10 ± 2g.

[0039] The specific operations are as follows: The abdominal blood sinuses of each group of shrimp were disinfected with 75% alcohol cotton balls. The shrimp hemolymph was extracted with a syringe pre-filled with 5 mL of anticoagulant (the volume of the anticoagulant was 2 mL) (0.45 M NaCl, 10 mM KCl, 10 mM EDTA, 10 mM HEPES, pH 7.45) at a volume ratio of 1:1, and the blood was centrifuged at 800 × 4 °C. g Centrifuge for 10 min to collect blood cells. Remove the supernatant and resuspend the cells in culture medium (Insect-XPRESS basal medium supplemented with 1% penicillin-streptomycin). Adjust the cell density to 2 × 10 4 cells / mL, and inoculated into 96-well plates for culture. After shrimp blood cells were cultured for 12 h, the shrimp blood cells of each group were treated as follows: Blank control group: containing culture medium but without shrimp blood cells; Untreated group: containing shrimp blood cells and culture medium, but without PirAB Vp protein Phosphate buffer solution + PirAB Vp Group: 15 μL PBS was added to the 96-well plate to treat shrimp blood cells, followed by 15 μL PirAB Vp protein (concentration 25 μM); Chlorpromazine hydrochloride + PirAB Vp Group: 15 μL CPZ was added to the 96-well plate to treat shrimp blood cells, followed by 15 μL PirAB Vp Protein (concentration was 25 μM).

[0040] At specific time points after treatment (6 h, 12 h, 24 h, 48 h), the culture medium was removed and washed twice with culture medium, and new 100 μL culture medium (as described above) and 10 μL CCK-8 reagent were added to each well. The cells were cultured in the incubator for 1 h, and the OD value of each well of the 96-well plate at 450 nm was measured using a microplate reader.

[0041] The calculation formula of cell viability is: Cell viability = [OD (plus PirAB Vp group)-OD(blank control group)] / [OD(untreated group)-OD(blank control group)]×100.

[0042] The results are as follows Figure 3 As shown, pretreatment of Japanese shrimp with CPZ can significantly improve the cell viability of Japanese shrimp.

[0043] Example 3 Experimental animal treatment: Healthy Japanese shrimp were selected and randomly divided into six groups, 30 in each group. Five of the groups were randomly selected and injected with 0.25 μg / μl, 0.5 μg / μl, 1 μg / μl, 2 μg / μl, and 4 μg / μl of CPZ, respectively. The remaining group was not treated and served as the control group. After 12 hours, all shrimps were immersed in Vp AHPND (4 × 10 6 CFU / mL, 80 mL) of seawater (20 L).

[0044] After immersion for 24 h, shrimp hemolymph was extracted with a syringe pre-filled with 5 mL of anticoagulant (the volume of the anticoagulant was 2 mL) (the composition of the anticoagulant was: 0.45 MNaCl, 10 mM KCl, 10 mM EDTA, 10 mM HEPES, pH 7.45) at a volume ratio of 1:1 and then incubated at 4 °C at 12000 × g After centrifugation for 15 min, the shrimp blood cells were resuspended in RIPA lysis buffer (main components are 50 mM Tris (pH7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, and sodium orthovanadate, sodium fluoride, EDTA, leupeptin and other inhibitors) (Shanghai Biyuntian Biotechnology Co., Ltd., Catalog No. P0013D). In addition, the hepatopancreas and intestine of the shrimp were homogenized with RIPA lysis buffer to extract protein samples. 1 mg of the collected protein sample was mixed with 500 μL of protein loading buffer and heated at 100°C for 10 min. After cooling, it was stored at -20°C for subsequent experiments.

[0045] Western blotting was used to detect PirAB in protein samples. Vp The protein samples were separated by 12.5% ​​sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated gel was transferred to a nitrocellulose membrane using a semi-dry transfer device.

[0046] The nitrocellulose membrane loaded with proteins was blocked in TBST (150 mM NaCl, 10 mM Tris-HCl, 0.05% Tween-20, pH 8.0) containing 5% (% is mass percentage) skim milk powder, and then incubated with the primary antibody anti-PirAB. Vp Incubate overnight at 4 °C.

[0047] After washing with TBST for 3 times, the nitrocellulose membrane loaded with proteins was incubated with goat anti-rabbit secondary antibody coupled with horseradish peroxidase at room temperature for 3 h. After washing with TBST for 3 times, the immunoreactive protein bands were imaged and photographed using ECL immunoblotting solution and chemiluminescence imaging system.

[0048] The results are as follows Figure 4 As shown, after CPZ injection, PirAB in the hepatopancreas, intestine and blood cells of shrimp Vp The content of PirAB was significantly reduced, and the decrease was dose-dependent with CPZ. The above results show that CPZ can effectively inhibit PirAB Vp Enters the shrimp hepatopancreas, intestines and blood cells.

[0049] Example 4 Experimental animal treatment: Healthy Japanese marsupials were selected and randomly divided into two groups, 20 in each group. The first group was pretreated by injecting 50 μL phosphate buffer solution PBS (0.01M); the second group was pretreated by injecting 50 μL chlorpromazine hydrochloride CPZ (2 μg / μL).

[0050] After 12 h, the two groups of shrimp were immersed in Vp AHPND (4 × 10 6 CFU / mL, 80 mL) of seawater (20 L), and after immersion for 48 h, the hepatopancreas of shrimp were taken for paraffin sectioning and H&E staining to detect the morphological changes of hepatopancreas tissue.

[0051] The results are as follows Figure 5 As shown, pretreatment of shrimp with CPZ can significantly improve the morphological changes of shrimp hepatopancreas tissue.

[0052] Example 5 Experimental animal treatment: Healthy Japanese marsupials were selected and randomly divided into two groups, 20 in each group. The first group was pretreated by injecting 50 μL phosphate buffer solution PBS (0.01M); the second group was pretreated by injecting 50 μL chlorpromazine hydrochloride CPZ (2 μg / μL).

[0053] After 12 h, the two groups of shrimp were immersed in Vp AHPND (4 × 10 6 CFU / mL, 80 mL) of seawater (20 L), and the survival rate of shrimp was recorded over six days.

[0054] The results are as follows Figure 6 As shown, pretreatment of shrimp with CPZ can significantly increase the survival rate of shrimp.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of chlorpromazine hydrochloride in the preparation of drugs for treating acute hepatopancreatic necrosis.

2. The use according to claim 1, characterized in that: The effects of the anti-acute hepatopancreatic necrosis drug are any one or more of the following: (1) Inhibition of PirAB Vp It enters shrimp cells via clathrin-dependent endocytosis; (2) Reduce PirAB in target organs Vp Content; (3) Improve PirAB Vp Resulting changes in the morphology of hepatopancreatic tissue; (4) Improve the cell viability of shrimp.

3. The use according to claim 1, characterized in that: The chlorpromazine hydrochloride is a pure product, a solvent compound or an isomer thereof.

4. The use according to claim 2, characterized in that: The prawn is Penaeus japonicus.

5. The use according to claim 1, characterized in that: The effective concentration of chlorpromazine hydrochloride is 0.5-4 μg / μL.

6. The use according to claim 5, characterized in that: The effective concentration of chlorpromazine hydrochloride is 1-4 μg / μL.

7. A drug for treating acute hepatopancreatic necrosis, characterized in that: The medicine comprises chlorpromazine hydrochloride and a pharmaceutical carrier or auxiliary material.

8. The drug for preventing acute hepatopancreatic necrosis according to claim 7, characterized in that: The pharmaceutical carrier or excipient is selected from one or more pharmaceutically acceptable dispersants, wetting agents, disintegrants, binders, lubricants, regulators, defoamers, antifreeze agents, thickeners or fillers.

9. The drug for preventing acute hepatopancreatic necrosis according to claim 7, characterized in that: The dosage form of the drug for resisting acute hepatopancreatic necrosis is wettable powder, water dispersible granules, water suspension or dispersible oil suspension.

10. The drug for preventing acute hepatopancreatic necrosis according to claim 7, characterized in that: The administration method is feeding or injection.

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