Construction and evaluation method and application of thioacetamide-induced hepatopancreas injury model of procambarus clarkia

Abstract

The invention belongs to the technical field of aquaculture, and relates to a thioacetamide-induced procambarus clarkii hepatopancreas injury model construction and evaluation method and application thereof, and the method comprises the following steps: selecting healthy and temporarily cultured procambarus clarkii with the weight of 10.0 + / -1.45 g as an object for establishing the hepatopancreas injury model; fasting the procambarus clarkii for 12 hours, and then giving a thioacetamide solution, wherein the application dosage is 0.4-0.8 mg / g body weight; the thioacetamide is a thioacetamide solution which is prepared from a 0.9 wt% NaCl solution and has the concentration of 40 to 80 mg / mL; the mode of administration of the thioacetamide solution is intramuscular injection. The TAA is applied to construction of the hepatopancreas injury model of the procambarus clarkia for the first time, individual death is not induced in the model construction process, and an ideal experimental model capable of researching pathogenesis of hepatopancreas diseases and screening related therapeutic drugs is obtained. The method has the advantages of being short in period, low in cost, easy to operate, good in repeatability and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture and relates to a construction method and application of a thioacetamide-induced hepatopancreas injury model of Procambarus clarkii. Background Art

[0002] Procambarus clarkii Procambarus clarkia ), also known as crayfish, with the expansion of breeding scale and deterioration of breeding environment, various diseases of Procambarus clarkii continue to appear, causing serious economic losses to the breeding industry. Surveys show that most of the diseased Procambarus clarkii have abnormalities in hepatopancreas pathological characteristics and hepatopancreas-related injury evaluation indicators. The hepatopancreas, also known as the midgut gland or hepatopancreas, is a protruding part differentiated from the midgut. It is an important metabolic and immune organ of crustaceans and plays a key role in the growth and disease resistance of crustaceans. The state of the hepatopancreas can usually be used as a sign of health status. The experimental liver injury model is of great significance in studying the pathogenesis of animal liver disease, screening liver-protecting drugs, and exploring the liver-protecting effects of drugs.

[0003] In mammals, the research methods of liver injury models are relatively mature, but there are few reports on shrimp liver injury models. Thioacetamide (TAA) is a typical hepatotoxic compound that can induce oxidative stress and inflammatory response to damage liver cells and trigger fibrosis to cause liver injury. TAA usually induces hepatotoxicity through oral and intraperitoneal administration, and can also induce acute toxic effects in rat livers through intravenous administration without anesthesia. TAA can be added to drinking water to chronically induce liver fibrosis in mice. Acute exposure to TAA can cause liver necrosis in rats, while chronic exposure can cause apoptosis of rat liver cells. In recent years, the TAA-induced liver injury model has been successfully used to evaluate and screen a variety of liver protection drugs. At present, there is no research on the use of TAA-induced liver injury model in shrimp. The establishment of a hepatopancreatic injury model of Procambarus clarkii using TAA is not only helpful for studying the pathogenesis of hepatopancreatic diseases, but also provides an ideal experimental model for screening related therapeutic drugs. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a method for constructing and evaluating a thioacetamide-induced hepatopancreatic injury model of Procambarus clarkii and its application. The model construction method has the advantages of short cycle, low cost, easy operation, good repeatability, etc.

[0005] The technical solution provided by the present invention is as follows:

[0006] The present invention provides a method for constructing a hepatopancreas injury model of Procambarus clarkii induced by thioacetamide, comprising the following steps: The healthy and temporarily cultured Procambarus clarkii weighing 10.0±1.45 g were selected as the subjects for establishing the hepatopancreas injury model. The above-mentioned Procambarus clarkii was fasted for 12 hours and then treated with thioacetamide solution, with an application dose of 0.4-0.8 mg / g body weight; the thioacetamide was a thioacetamide solution with a concentration of 40-80 mg / mL prepared with 0.9wt% NaCl solution; the thioacetamide solution was administered by intramuscular injection.

[0007] Furthermore, the temporary rearing conditions are: rearing in tap water that has been fully aerated for 72 h, water temperature 20-25°C, dissolved oxygen>6 mg / L, pH 7.2-7.9, ammonia nitrogen content<0.2 mg / L, and nitrite content<0.2 mg / L.

[0008] Furthermore, the temporary rearing time is 7 days, during which crayfish feed is fed twice a day, at 9:00 a.m. and 16:00 p.m., each feeding amount is 2% of the shrimp body weight, and 1 / 3 of the water is changed every 3 days.

[0009] Furthermore, the thioacetamide solution treatment time is 24h~48h.

[0010] The present invention also provides a method for evaluating the hepatopancreas injury model of Procambarus clarkii obtained according to the above-mentioned construction method, comprising any one or more of (a)-(c): (a) Determine the hepatopancreatic injury indexes in the hemolymph and hepatopancreatic tissues of Procambarus clarkii and the oxidative stress indexes in the hepatopancreatic tissues using relevant enzyme detection kits; (b) The pathological condition of the hepatopancreas tissue of Procambarus clarkii was observed under a microscope after hematoxylin and eosin staining; (c) Microscope photography was used to detect hepatopancreatic cell apoptosis and hepatopancreatic tissue fibrosis using a cell apoptosis detection kit and a modified Sirius red staining kit, respectively.

[0011] Furthermore, the hepatopancreatic damage indicators in the hemolymph and hepatopancreatic tissue of Procambarus clarkii are aspartate aminotransferase and alanine aminotransferase, and the oxidative stress indicators in the hepatopancreatic tissue are MDA content, SOD activity and CAT activity.

[0012] The present invention also provides the application of the Procambarus clarkii hepatopancreatic injury model obtained by the construction method in the research of hepatopancreatic injury diseases, wherein the research is aimed at non-disease diagnosis or treatment.

[0013] Furthermore, the hepatopancreatic injury disease research is a study on screening shrimp liver-protecting drugs.

[0014] Beneficial Effects

[0015] The present invention obtains a hepatopancreatic injury model of Procambarus clarkii by acutely inducing thioacetamide TAA, and can study the pathogenesis of TAA-induced hepatopancreatic injury, the metabolic changes of the hepatopancreas, and further understand the mechanism of hepatopancreatic injury, so as to take some new intervention and treatment measures to promote the hepatopancreatic health of Procambarus clarkii, and screen relevant therapeutic drugs for liver protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Effects of different concentrations of TAA on the activities of GOT and GPT in the hepatopancreas and hemolymph (A: hepatopancreas GPT; B: hemolymph GPT; C: hepatopancreas GOT; D: hemolymph GOT).

[0017] Figure 2 Effects of different concentrations of TAA on the MDA content, SOD and CAT activities in the hepatopancreas of Procambarus clarkii (A: MDA; B: SOD; C: CAT).

[0018] Figure 3 This is the HE staining image of the hepatopancreas of Procambarus clarkii (ad: control group; eh: TAA 0.2; il: TAA 0.4; mp: TAA 0.8; HC: hepatic tubules; HL: hepatic cavity; BC: alveolar cells; AC: absorptive cells; HLD: hepatic cavity deformation; HCD: hepatic tubule damage; HCDD: disordered distribution of hepatic tubules; VA: abnormal vacuoles; ACS: swollen absorptive cells).

[0019] Figure 4 Figure 2 TUNEL staining images of the hepatopancreas of the control group and TAA 0.8 group (ad: control group; eh: TAA 0.8 group; red arrows indicate cell apoptosis).

[0020] Figure 5 Sirius Red staining images of the hepatopancreas of the control group and TAA 0.8 group (ad: control group; eh: TAA 0.8 group; red arrows indicate fibrotic tissue). DETAILED DESCRIPTION

[0021] The embodiment of the present invention provides a method for constructing a thioacetamide-induced hepatopancreas injury model of Procambarus clarkii, comprising the following steps: The healthy and temporarily cultured Procambarus clarkii weighing 10.0±1.45 g were selected as the subjects for establishing the hepatopancreas injury model. The above-mentioned Procambarus clarkii was fasted for 12 hours and then treated with thioacetamide solution, with an application dose of 0.4-0.8 mg / g body weight; the thioacetamide was a thioacetamide solution with a concentration of 40-80 mg / mL prepared with 0.9wt% NaCl solution; the thioacetamide solution was administered by intramuscular injection.

[0022] In this embodiment, the temporary culture conditions are: culture in tap water that has been fully aerated for 72 hours, water temperature 20-25°C, dissolved oxygen>6 mg / L, pH 7.2-7.9, ammonia nitrogen content<0.2 mg / L, and nitrite content<0.2 mg / L.

[0023] In this embodiment, the temporary culture period is 7 days, during which crayfish feed is fed twice a day, at 9:00 am and 16:00 pm, respectively, with each feeding amount being 2% of the shrimp body weight, and 1 / 3 of the water is changed every 3 days.

[0024] In this embodiment, the thioacetamide solution treatment time is 24h~48h.

[0025] The embodiment of the present invention further provides a method for evaluating the hepatopancreas injury model of Procambarus clarkii obtained according to the construction method described above, comprising any one or more of (a)-(c): (a) Determine the hepatopancreatic injury indexes in the hemolymph and hepatopancreatic tissues of Procambarus clarkii and the oxidative stress indexes in the hepatopancreatic tissues using relevant enzyme detection kits; (b) The pathological condition of the hepatopancreas tissue of Procambarus clarkii was observed under a microscope after hematoxylin and eosin staining; (c) Microscope photography was used to detect hepatopancreatic cell apoptosis and hepatopancreatic tissue fibrosis using a cell apoptosis detection kit and a modified Sirius red staining kit, respectively.

[0026] In this embodiment, the hepatopancreatic injury indicators in the hemolymph and hepatopancreatic tissue of Procambarus clarkii are aspartate aminotransferase and alanine aminotransferase, and the oxidative stress indicators in the hepatopancreatic tissue are MDA content, SOD activity and CAT activity.

[0027] The embodiment of the present invention also provides the application of the Procambarus clarkii hepatopancreatic injury model obtained by the construction method in the study of hepatopancreatic injury diseases, wherein the purpose of the study is non-disease diagnosis or treatment.

[0028] In this embodiment, the hepatopancreatic injury disease research is a research on screening shrimp liver-protecting drugs.

[0029] The present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] 1. Experimental animals and groups

[0032] Procambarus clarkii with complete appendages, strong vitality and similar size were selected for the experiment. The experimental shrimp weighed 10.5 ± 0.45 g. Before the start of the experiment, Procambarus clarkii was placed in a PVC plastic culture bucket with a radius of 1.0 m and a height of 1.5 m for 7 days. The culture water depth was 1.0 m. The experimental water was tap water that had been fully aerated for 72 hours. The water temperature was 20-25℃. Continuous aeration and oxygenation were used to ensure sufficient dissolved oxygen in the water. The dissolved oxygen was controlled to be >6 mg / L, pH 7.2-7.9, ammonia nitrogen content <0.2 mg / L, and nitrite content <0.2 mg / L. Crayfish feed was fed twice a day (9: 00 and 16: 00), and the feeding amount each time was 2% of the shrimp body weight. 1 / 3 of the water was changed every 3 days to ensure clean water quality.

[0033] 2. Establishment of TAA-induced model and sample collection

[0034] All shrimps were weighed after fasting for 12 hours, and the body weight was 10.0±0.4 g. According to the preliminary experimental results, each shrimp in the experimental group was injected intramuscularly with 100 μL of different concentrations of TAA saline solution (Macklin, China), so that the final drug injection volume reached 0.2 mg / g, 0.4 mg / g, and 0.8 mg / g (TAA / lobster weight), respectively. The blank control group was injected with 100 μL of saline, which was divided into four groups: Con, TAA 0.2, TAA 0.4, and TAA 0.8. Each group was set up with 3 replicates, each replicate had 20 shrimps, and a total of 240 shrimps were used in this experiment. No mortality occurred during the entire experimental period. At 6 h, 12 h, 24 h, and 48 h after injection, four shrimps were taken from each group and each replicate to extract crayfish hemolymph and prepare plasma. At the same time, the hepatopancreas tissue was separated. The hepatopancreas of two shrimps was frozen at -80°C for various biochemical oxidation indexes, transcriptome and metabolome detection, and the other two shrimps were fixed with Bouin's solution (Phygene, China) and stored at 4°C for paraffin sectioning.

[0035] 3. Detection of hepatopancreatic injury indicators

[0036] Hepatopancreatic tissue was mixed with PBS solution in proportion, homogenized with a tissue homogenizer, and then centrifuged (4000 rpm, 4°C, 15 min). The supernatant was aspirated and poured into a centrifuge tube for subsequent determination. Hemolymph was directly sampled and measured. The GOT and GPT detection kits (Nanjing Jiancheng, China) of Nanjing Jiancheng Biological Company were used for processing. The OD values ​​were measured at a wavelength of 510 nm to detect the activities of aspartate aminotransferase (GOT) and alanine aminotransferase (GPT), and the protein concentration was determined using the Bradford protein assay kit (Nanjing Jiancheng, China) of Nanjing Jiancheng Biological Company. The results of each index were homogenized according to the amount of protein.

[0037] 4. Detection of hepatopancreas oxidative stress related indicators

[0038] Hepatopancreatic tissue was mixed with PBS solution in proportion, homogenized with a tissue homogenizer, and then centrifuged (4000 rpm, 4°C, 15 min). The supernatant was aspirated and poured into a centrifuge tube for subsequent determination. The MDA, SOD, and CAT assay kits (Nanjing Jiancheng, China) of Nanjing Jiancheng Biological Company were used for processing, and the OD values ​​were measured at wavelengths of 532 nm, 450 nm, and 405 nm, respectively, to detect the content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and catalase (CAT), and the Bradford protein assay kit (Nanjing Jiancheng, China) of Nanjing Jiancheng Biological Company was used to determine the protein concentration. The results of each index were homogenized according to the amount of protein.

[0039] 5. HE staining and apoptosis detection of hepatopancreas

[0040] The hepatopancreas fixed with Bouin's solution was dehydrated with graded ethanol and then transparentized with xylene. After washing, it was embedded in paraffin and cut into 8 μm thick paraffin sections using a paraffin slicer (Leica, Germany). The sections were dewaxed, rehydrated with graded ethanol, stained with hematoxylin for 5-8 min, differentiated with 1% hydrochloric acid alcohol, rinsed with running water, and then stained with eosin stain for 1-3 min, then dehydrated with graded ethanol, sealed with neutral resin, and finally observed and photographed under a microscope (Olympus, Japan).

[0041] TUNEL apoptosis detection kit (Beyotime, China) was used to detect apoptosis of hepatopancreatic cells. 20 μg / mL proteinase K working solution was dropped on paraffin sections for 15 min at 37°C, washed 3 times with PBS, added with biotin labeling solution (PBS for negative control) and incubated at 37°C in the dark for 60 min, then added with DAB colorimetric solution and incubated at room temperature for 10 min, stained with hematoxylin for 5-8 min, and finally observed and photographed under a microscope (Olympus, Japan).

[0042] 6. Hepatopancreatic fibrosis detection

[0043] The modified Sirius red staining kit (Solarbio, China) was used to detect hepatopancreatic tissue fibrosis. Paraffin sections were stained with iron hematoxylin for 8 min, washed with tap water for 10 min, then stained with Sirius red stain for 15 min, dehydrated with gradient alcohol, sealed with neutral resin, and finally observed and photographed under a microscope (Olympus, Japan).

[0044] 7. Test results

[0045] (1) Analysis of hepatopancreatic injury indicators

[0046] Aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) activities are typical biochemical indicators for detecting liver damage. When the hepatopancreas is damaged, the activities of GOT and GPT in the hepatopancreas will be significantly reduced, and the activities of GOT and GPT in the hemolymph will be significantly increased accordingly. During the experiment, the hepatopancreas GPT activities of the three treatment groups were significantly lower than those of the Con group. From 24 hours on, the activity of the TAA 0.8 group was significantly lower than that of TAA0.2 and 0.4 ( Figure 1 A); Figure 1 As shown in B, during the experiment, the GPT activities in the hemolymph of the three treatment groups were significantly higher than that of the Con group. At 24 h, the GPT activities in the TAA 0.4 and TAA 0.8 groups continued to increase. At 48 h, the activity in the TAA 0.8 group continued to increase, which was significantly higher than that in the other treatment groups ( Figure 1 B). The GOT activity of the hepatopancreas in the TAA 0.8 group continued to decrease as the treatment time increased ( Figure 1 C); TAA 0.8 showed a more sustained and stable induction effect on the activity of hemolymph GOT compared with TAA 0.2 and 0.4 during the entire treatment period ( Figure 1 D). During the experiment, the various indicators of the TAA 0.8 group were significantly different from those of the Con group and were basically time-dependent.

[0047] (2) Analysis of indicators related to hepatopancreas oxidative stress

[0048] TAA 0.2 and TAA 0.4 significantly induced an increase in MDA content in the hepatopancreas by 1.13 and 1.34 times, respectively, at 48 h after treatment. TAA 0.8 significantly induced an increase in MDA content in the hepatopancreas by 1.85 and 2.27 times, respectively, at 24 and 48 h after exposure ( Figure 2 A). TAA0.2 had no significant effect on the SOD activity of the hepatopancreas, while TAA 0.4 significantly inhibited the SOD activity by 1.25 and 1.11 times at 24 and 48 h after treatment, respectively. TAA 0.8 significantly inhibited the SOD activity by 1.11, 1.12, 1.31 and 1.33 times at 6, 12, 24 and 48 h after treatment, respectively ( Figure 2 B). In this experiment, the three concentrations of TAA had no significant effect on CAT activity ( Figure 2 C). During the experiment, the MDA content and SOD activity in the hepatopancreas of the TAA 0.8 group were significantly different from those of the Con group and were basically time-dependent.

[0049] (3) HE staining hepatopancreatic tissue pathological analysis

[0050] The hepatic ducts of the Con group were basically star-shaped, with complete structure, plump cells and uniform distribution ( Figure 3 ad). In the TAA 0.2 group, some hepatic ducts began to deform at 24 h, and at 48 h, a small number of vacuoles of alveolar cells (B cells) became larger, and a small number of hepatic ducts were deformed ( Figure 3 eh). In the TAA 0.4 group, the hepatic tubules were loosely distributed at 12 h, the hepatic tubule absorptive cells (R cells) were swollen, the hepatic duct lumen was reduced, and obvious small vacuoles appeared in B cells at 24 h. There was no significant change at 48 h ( Figure 3 jl). In the TAA0.8 group, the hepatic duct lumen was slightly deformed at 6 h, R cells began to swell at 12 h, and the hepatic ducts were chaotically distributed. At 24 h, the hepatic ducts were damaged and chaotically distributed, and a large number of hepatic ducts were interlaced and adhered. The hepatopancreas was severely damaged. At 48 h, the R cells were severely swollen, the hepatic ducts were damaged and adhered, and squeezed each other. A large number of obvious small vacuoles appeared in the B cells, and the hepatic duct lumen was squeezed into various irregular shapes, and the hepatopancreas damage was further aggravated ( Figure 3 mp).

[0051] (4) Analysis of hepatopancreatic cell apoptosis

[0052] Through the analysis of biochemical and oxidative indicators of liver injury, as well as histological analysis of HE staining, we found that the damaging effect of 0.8 mg / g TAA on the hepatopancreas of Procambarus clarkii was basically time-dependent. Therefore, we performed TUNEL staining analysis of cell apoptosis in the Con group and the TAA 0.8 group separately. The hepatopancreas of the Con group was basically normal, and no apoptosis was observed. In the TAA 0.8 group, apoptosis was observed between the hepatic ducts from 12 h to 48 h, and as the duration of action increased, apoptosis gradually intensified ( Figure 4 ).

[0053] (5) Detection and analysis of hepatopancreatic fibrosis

[0054] Through the analysis of biochemical and oxidative indicators of liver damage, as well as histological analysis of HE staining, we found that the damaging effect of 0.8 mg / g TAA on the hepatopancreas of Procambarus clarkii was basically time-dependent. Therefore, we performed Sirius red staining analysis on the Con group and the TAA 0.8 group separately. In the Con group, very little fibrosis occurred in the interstitial spaces of the hepatic tubules during the entire experiment. In the TAA 0.8 group, as the exposure time increased, more and more fibrosis occurred between the hepatic tubules. At 24 hours, a clear fibrotic tissue appeared along the hepatic ducts. At 48 hours, large areas of fibrotic tissue appeared, and the hepatopancreas was severely damaged ( Figure 5 ).

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing a hepatopancreas injury model of Procambarus clarkii induced by thioacetamide, characterized in that: The following steps are involved: The healthy and temporarily cultured Procambarus clarkii weighing 10.0±1.45 g were selected as the subjects for establishing the hepatopancreas injury model. The above-mentioned Procambarus clarkii was fasted for 12 hours and then treated with thioacetamide solution, with an application dose of 0.4-0.8 mg / g body weight; the thioacetamide was a thioacetamide solution with a concentration of 40-80 mg / mL prepared with 0.9wt% NaCl solution; the thioacetamide solution was administered by intramuscular injection.

2. The method for constructing a thioacetamide-induced hepatopancreas injury model of Procambarus clarkii according to claim 1, characterized in that: The temporary rearing conditions are: rearing in tap water that has been fully aerated for 72 hours, water temperature 20~25℃, dissolved oxygen>6mg / L, pH 7.2-7.9, ammonia nitrogen content <0.2 mg / L, nitrite content <0.2 mg / L.

3. The method for constructing the hepatopancreas injury model of Procambarus clarkii induced by thioacetamide according to claim 2, characterized in that: The temporary rearing period is 7 days, during which crayfish feed is fed twice a day, at 9:00 a.m. and 16:00 p.m., with each feeding amount being 2% of the shrimp's body weight, and 1 / 3 of the water is changed every 3 days.

4. The method for constructing a thioacetamide-induced hepatopancreas injury model of Procambarus clarkii according to claim 1, characterized in that: The duration of treatment with thioacetamide solution is 24h~48h.

5. A method for evaluating the hepatopancreas injury model of Procambarus clarkii obtained by the construction method according to any one of claims 1 to 4, characterized in that: Includes any one or more of (a)-(c): (a) Determine the hepatopancreatic injury indexes in the hemolymph and hepatopancreatic tissues of Procambarus clarkii and the oxidative stress indexes in the hepatopancreatic tissues using relevant enzyme detection kits; (b) The pathological condition of the hepatopancreas tissue of Procambarus clarkii was observed under a microscope after hematoxylin and eosin staining; (c) Microscope photography was used to detect hepatopancreatic cell apoptosis and hepatopancreatic tissue fibrosis using a cell apoptosis detection kit and a modified Sirius red staining kit, respectively.

6. The evaluation method according to claim 5, characterized in that: The hepatopancreatic damage indicators in the hemolymph and hepatopancreatic tissue of Procambarus clarkii are aspartate aminotransferase and alanine aminotransferase, and the oxidative stress indicators in the hepatopancreatic tissue are MDA content, SOD activity and CAT activity.

7. Use of the Procambarus clarkii hepatopancreatic injury model obtained by the construction method according to any one of claims 1 to 4 in the study of hepatopancreatic injury diseases, wherein the purpose of the study is non-disease diagnosis or treatment.

8. The use according to claim 7, characterized in that: The hepatopancreas injury disease research is a research for screening shrimp liver-protecting drugs.

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