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CN113209062A - Study on carp liver injury protection effect of curcumin through activation of activity of Nrf2 - Google Patents

Study on carp liver injury protection effect of curcumin through activation of activity of Nrf2 Download PDF

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CN113209062A
CN113209062A CN202110514461.0A CN202110514461A CN113209062A CN 113209062 A CN113209062 A CN 113209062A CN 202110514461 A CN202110514461 A CN 202110514461A CN 113209062 A CN113209062 A CN 113209062A
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liver
carp
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张媛媛
姚芳斌
成慧中
王晓丽
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Shandong Freshwater Fisheries Research Institute
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Abstract

The invention discloses a study on the protective effect of curcumin on carp liver injury by activating activity of Nrf2, which comprises three tests, wherein the first test is to study the effect of curcumin on carp (namely that curcumin with different addition amounts on carp: (A)Cyprinus carpio) The second test is the effect of curcumin on carbon tetrachloride (CCl)4) And thirdly, the influence of liver injury of the carp body is induced, and the research on the recovery effect of curcumin on the carp acute liver injury induced by carbon tetrachloride is tested. The research of the invention proves that the addition of 120mg/kg curcumin in the feed can promote the Nrf2 to transfer into the nucleus and improve the antioxidant state of the carp by improving the activity of the Nrf2, thereby improving the activity of antioxidant enzyme and finally protecting the carp from CCl4Liver damage ofIn addition, research results also show that the addition of curcumin in the feed has a recovery effect on the carp to treat acute liver injury.

Description

Study on carp liver injury protection effect of curcumin through activation of activity of Nrf2
Technical Field
The invention relates to a study on carp liver injury protection effect of curcumin, in particular to a study on carp liver injury protection effect of curcumin by activating activity of Nrf2, and belongs to the field of freshwater biological application.
Background
In recent years, the development of aquaculture industry in China gradually draws close to a high-density intensive culture mode, the culture density is rapidly improved, a large amount of artificial compound feed is fed, medicines and hormones are not used in a standardized way, the environment of culture water is increasingly worsened, harmful substances exist in the feed, the nutrient content of the feed is lost and unbalanced, and other adverse factors are increased, so that great pressure is caused on the fish body, the metabolic system of the fish is unbalanced, particularly the damage to the liver function is great, the large-scale outbreak of the liver-gall syndrome of the fish is finally caused, and the development of the aquaculture industry is limited unprecedentedly. When the medicine is taken, if the disease is not symptomatic, a large amount of fish bodies can die rapidly, and the death rate reaches 60-80 percent. Therefore, the medicine has the advantages of solving the pathogenesis of the fish liver and gall syndrome and timely taking medicine according to symptoms when the fish liver and gall syndrome occurs, and has important significance for preventing and treating the fish liver and gall syndrome.
Many studies now show that some chemicals such as ethanol, carbon tetrachloride (CCl)4) And chromated, among others, can cause liver damage, among which CCl4Is a classical liver poison which can damage liver cell membranes and organelle membranes, so that lipid peroxidation occurs on the membranes, and finally, liver function is reduced, liver cells are killed, and the like. In mammals, CCl4As a model poison, the compound is widely used for constructing a liver (cell) injury model, screening liver-protecting medicaments, researching liver mechanisms and the like.Similar to mammals, fish liver cells are also paired with CCl4Is sensitive and is widely used for constructing a fish liver injury model in recent years.
For the treatment of fish liver and gall syndrome, no specific medicine is found, and western medicine antibiotics are mainly used at present, however, many researches find that the fish oral administration antibiotics not only cause the reduction of the digestive tract function of the fish body, but also cause damage to organs of the body such as liver, gall, spleen and kidney, and in addition, a large amount of pathogenic bacteria can be remained in the body to cause certain drug resistance to the medicines, and finally cause harm to human health. Therefore, the development of a green Chinese patent fishery medicine which has no toxic or side effect and has the effects of protecting and treating liver injury of fish bodies becomes vital. Curcumin (A)Curcumin) Is a polyphenol substance with small molecular weight extracted from Zingiberaceae plant. A large number of researches show that curcumin has the effects of resisting oxidation, sterilizing, diminishing inflammation, removing free radicals, resisting cancer and the like, and the curcumin can be widely applied to the treatment of aspects of cardiovascular systems, digestive systems and the like as a specific medicine in clinic. At present, the researches on curcumin oxidation resistance and liver injury protection are mostly found in mice (A)Mus musculus) And livestock and poultry, and the research results show that curcumin can be used for mice or broilers (Gallus gallus) Liver damage in these animals is protective, but fish have been rarely studied, and rare cases are well known for grass carp(s) ((R))Ctenopharynodon idellus) The research on the protection effect of acute liver injury, however, curcumin is a gastric lavage administration method in the research, the method is difficult to operate, has certain damage to the digestive tract function of a fish body, and is not suitable for a large-scale culture process. In this study CCl is used4The induction carp liver acute injury model is based on, curcumin is added into feed in an additive form, and the protective effect of curcumin in feed on fish body growth and acute liver injury is researched, so that a theoretical basis is provided for further development of liver protection medicines for fish.
Disclosure of Invention
The invention aims to solve the problems and provide a research on the protective effect of curcumin on carp liver injury by activating activity of Nrf 2.
The invention realizes the aim through the following technical scheme, and the research on the protective effect of curcumin on carp liver injury by activating activity of Nrf2 comprises the step of researching curcumin with different addition amounts on carp: (Cyprinus carpio) Growth performance impact of curcumin on carbon tetrachloride (CCl)4) The research on the influence of the induced fish liver injury and the recovery effect of curcumin on the carp acute liver injury induced by carbon tetrachloride comprises the following steps,
first, experimental design
The test is divided into 6 groups, wherein the P1 group is a control group, the P2 group is a blank control group, the P3 group, the P4 group, the P5 group and the P6 group are test groups, and the P1 group and the P2 group are fed with basic feed; feeding test feeds to P3, P4, P5 and P6 groups, wherein curcumin is not added to the basic feed, and the addition levels of curcumin in the test feeds are respectively 30, 60, 120 and 240 mg/kg;
test one: after continuously feeding for 10 weeks, P1, P3, P4, P5 and P6 groups were sampled, and different added amounts of curcumin were analyzed for carp: (A), (B), (C), (D) and D) in aCyprinus carpio) The effect of growth performance;
and (2) test II: after continuously feeding for 10 weeks, 30% olive oil-soluble CCl is intraperitoneally injected into fish bodies of P1, P3, P4, P5 and P6 groups at a ratio of 0.5mL/100g of body weight4A solution; carp of P2 group was not subjected to CCl after continuously feeding for 10 weeks4Solution injection, but CCl in the same ratio4The solution is 0.5mL/100g body weight, injected with olive oil, and sampled 72h later, to study curcumin to CCl4Inducing the influence of liver injury of fish bodies;
and (3) test III: according to the second test, groups P1 (negative control group), P2 (positive control group), P5 and P6 were selected, and after the culture test was completed, fish bodies of groups P1, P5(120mg/kg) and P6(240mg/kg) were intraperitoneally injected with 30% olive oil-dissolved CCl at a ratio of 0.5mL/100g body weight4Solution, P2 group not subjected to CCl4Injecting solution, namely injecting olive oil according to the same proportion, namely 0.5mL/100g of body weight, and sampling test fishes at the 2 nd, 3 rd, 5 th and 7 th days after injection so as to discuss the recovery effect of curcumin on the carbon tetrachloride-induced carp liver acute injury;
selection and breeding management of test objects
Randomly selecting 540 carp fries with basically consistent sizes, specifications and constitutions, dividing the carp fries into 6 groups, putting 3 parallel carps in each group, putting 30 parallel carps in 18 circulating water culture barrels for culture, feeding three times after each day of full feeding, and keeping each feeding for more than 30 min;
third, sampling and processing
After the experiment is finished, 3 fish tails are randomly taken from each barrel, rapid deep anesthesia is carried out by using MS-222 with the concentration of 100mg/L, tail venous blood and liver are collected, blood samples are centrifuged for 10min in an anticoagulation tube at the temperature of 4 ℃ and the speed of 6000r/min, and prepared blood plasma is frozen and stored for standby at the temperature of ‒ 80 ℃; dividing the collected liver into two parts, one part being stored in a Boehn's stationary liquid for slicing, and the other part being stored in a refrigerator at ‒ 80 ℃ for further analysis;
fourth, determination of growth and body index
After the test is finished, stopping feeding for 24 hours, respectively weighing each barrel, measuring the total weight and the tail number of each barrel of fish, and calculating the specific growth rate, the weight gain rate and the feed coefficient; then randomly extracting 3 fishes from each culture barrel to measure the weight, the body length, the visceral weight and the liver weight, and using the fish to measure the fullness, the visceral ratio and the liver-body ratio, wherein the calculation method is as follows:
weight gain ratio (weight gain, WG/%) = (Wt-W)0)×100%/W0
Specific growth rate (SGR/(%/d)) = (LnWt-LnW)0)×100%/T
Visceral body ratio (VSI/%) = visceral weight × 100%/Wt
Hepatosomal ratio (HSI/%) = liver weight × 100%/Wt =
Fullness factor (CF/%) = Wt × 100%/L3
Feed Conversion Ratio (FCR) = F/(Wt-W)0)
In the formula W0Weight (g) of fish at the start of the test; wt is the weight (g) of the fish at the end of the test; l is the body length (cm) of the fish at the end of the test; t is the fish culture days (d); f is the amount of feed ingested (g);
fifth, plasma liver function index determination
Determining Total Protein (TP), glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT) and total antioxidant capacity (T-AOC) by using the kit;
sixthly, measuring anti-oxidation indexes of liver and analyzing tissue slices
Taking out the liver stored in the refrigerator of ‒ 80 ℃ in the third step, placing the liver in the refrigerator of ‒ ℃ for dissolving, washing with normal saline to remove blood, then sucking the liver dry with filter paper, preparing homogenate with normal saline as a medium according to the proportion of 1:9, then centrifuging at 3000r/min for 10min, and taking the supernatant for measuring glutathione peroxidase (GSH-Px), Glutathione (GSH), superoxide dismutase (SOD) and Malondialdehyde (MDA);
the liver slices stored in the Boenh's stationary liquid in the third step are observed by adopting an optical microscope under the condition of an oil microscope multiplied by 1000;
seventhly, liver Nrf2mRNA measurement
Extracting the total RNA of the carp liver tissue in each time period by adopting a Trizol method, and determining the purity of the total RNA; detecting the total RNA quality by gel electrophoresis; the Sybergreen method is used for detecting the amplification of a target gene, and the Nrf2mRNA level adopts 2-ΔΔCtCalculating a method;
eighthly, monitoring the content of Nrf2 in a nucleus
Detecting the content of Nrf2 in the liver cell nucleus by adopting a western blot technology;
data statistics and analysis
SPSS17.0 software is adopted to carry out one-factor variance analysis on test data, when the data difference is obvious, a Duncan's test method is adopted to carry out multiple comparisons, and the difference level is determined asP<0.05The results are expressed as Mean ± standard error (Mean ± s.e.m).
Preferably, the curcumin is pure>95% of curcumin and CCl4Provided by seirada biotechnology limited and tezhou runxin laboratory instruments limited, respectively.
Preferably, the carp fry is provided by a high-quality breeding farm of fresh water fishery research institute in Shandong province, before the test is started, temporary rearing is carried out for 21 days by sinking feed provided by Tongwei feed company, and then the selection operation of the test object is carried out.
Preferably, the volume of water in the culture barrels is 250L, the culture barrels used in each group of tests are randomly distributed in spatial positions, the feeding time is 8:30, 12:30 and 4:30 each day, and the water quality conditions during the whole test period are as follows: the water temperature is 24.5-27.5 ℃, the dissolved oxygen is above 6mg/L, the ammonia nitrogen is below 0.1mg/L, the nitrite is below 0.1mg/L, and the pH is 6.8-7.0.
Preferably, the kits adopted in the plasma liver function index and liver antioxidant index mapping are purchased from Nanjing institute of bioengineering; the preparation components of the Boenshi stationary liquid are as follows: 75mL of 1.22% saturated picric acid, 25mL of 40% formalin solution, 5mL of glacial acetic acid, an optical microscope model of NIKON DS-Fi1, and a microtome model of Leica RM-2255 (Germany).
Preferably, the kits used in step seven are all purchased from Tiangen Biotechnology Ltd.
Preferably, 1.2 ml of blood is collected in each anticoagulation tube during the sampling process, and the anticoagulation tube is a heparin sodium anticoagulation tube purchased from Nantong medical Co., Ltd, and the model is 1.5 ml.
The invention has the beneficial effects that:
1. the growth performance of the carp can be improved by adding 60mg/kg and 120mg/kg of curcumin in the feed, and the excessive addition of curcumin can cause the growth performance of the carp to be reduced along with the gradual increase of the curcumin;
2. the curcumin added into the feed does not cause carp liver damage in a breeding period; after the fish body is induced by carbon tetrachloride, a certain level of curcumin is added into the basic feed, so that the activity of GPT and GOT in blood can be obviously reduced, and when the curcumin is added at the level of 120mg/kg, the activity of GPT is obviously different from that of a control group, which shows that the curcumin added at a certain level in the feed has a protective effect on the liver of the carp;
3. the addition of 120mg/kg curcumin in the feed can improve the antioxidant state of the carp by promoting the release of Nrf2, thereby improving the activity of antioxidant enzyme, and finally protecting the carp from CCl4Liver damage of (4);
4. the addition of curcumin in the feed has a recovery effect on the carp from acute liver injury, and compared with a high-dose (240mg/kg) group, the recovery capability of the 120mg/kg group is stronger.
Drawings
FIG. 1A shows the effect of curcumin on the activity of superoxide dismutase (SOD) in carp liver in different treatment groups according to the present invention.
FIG. 1B shows the effect of curcumin in different treatment groups of the present invention on Malondialdehyde (MDA) content in carp liver.
FIG. 2A shows the effect of curcumin on glutathione peroxidase (GSH-Px) activity in carp liver in different treatment groups according to the present invention.
FIG. 2B shows the effect of curcumin on Glutathione (GSH) content in carp liver in different treatment groups according to the present invention.
FIG. 3 shows CCl in different processing groups according to the present invention4The influence of curcumin on carp liver tissue structure after induction for 72 h.
FIG. 4 shows CCl in different processing groups according to the present invention4Influence of curcumin on expression level of Nrf2mRNA in carp liver cells after 72h of induction.
FIG. 5A shows CCl in different treatment groups according to the present invention4And (3) developing graph of curcumin on carp liver cell nucleus Nrf2 content after 72h of induction.
FIG. 5B shows CCl in different processing groups according to the present invention4And (3) a gray level analysis graph of curcumin on carp liver cell nucleus Nrf2 content after 72h induction.
Fig. 6A shows the effect of curcumin on glutamic-pyruvic transaminase (GPT) in carp plasma liver function index at different time points after carbon tetrachloride induction.
Fig. 6B shows the effect of curcumin on glutamic-oxaloacetic transaminase (GOT) in carp plasma liver function index at different time points after carbon tetrachloride induction according to the present invention.
Fig. 7A is a graph showing the effect of curcumin on superoxide dismutase (SOD) activity in carp liver function at different time points after carbon tetrachloride induction according to the present invention.
Fig. 7B shows the effect of curcumin on Malondialdehyde (MDA) content in carp liver function at different time points after induction by carbon tetrachloride according to the invention.
FIG. 8A is the effect of curcumin on glutathione peroxidase (GSH-Px) activity in carp liver function at different time points after induction by carbon tetrachloride according to the invention.
Fig. 8B shows the effect of curcumin on reduced Glutathione (GSH) content in carp liver function at different time points after induction by carbon tetrachloride according to the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A study on the protective effect of curcumin on carp liver injury by activating activity of Nrf2 comprises two tests, wherein the first test is to study the effect of curcumin on carp with different addition amountsCyprinus carpio) The second test is the effect of curcumin on carbon tetrachloride (CCl)4) And (3) inducing the influence of liver injury of the fish body, wherein the third test is the research on the recovery effect of curcumin on the carp acute liver injury induced by carbon tetrachloride, and the test design is as follows:
the test is divided into 6 groups, wherein the P1 group is a control group, the P2 group is a blank control group, the P3 group, the P4 group, the P5 group and the P6 group are test groups, and the P1 group and the P2 group are fed with basic feed; feeding test feeds to P3, P4, P5 and P6 groups, wherein curcumin is not added to the basic feed, and the addition levels of curcumin in the test feeds are respectively 30, 60, 120 and 240 mg/kg;
test one: after continuously feeding for 10 weeks, P1, P3, P4, P5 and P6 groups were sampled, and different added amounts of curcumin were analyzed for carp: (A), (B), (C), (D) and D) in aCyprinus carpio) The effect of growth performance;
and (2) test II: after continuously feeding for 10 weeks, 30% olive oil-soluble CCl is intraperitoneally injected into fish bodies of P1, P3, P4, P5 and P6 groups at a ratio of 0.5mL/100g of body weight4A solution; carp of P2 group was not subjected to CCl after continuously feeding for 10 weeks4Solution injection, but CCl in the same ratio4The solution is 0.5mL/100g body weight, injected with olive oil, and sampled 72h later, to study curcumin to CCl4Inducing the influence of liver injury of fish bodies;
and (3) test III: selecting P1 (negative control group), P2 (positive control group), P5 and P6, and injecting 30% olive oil-soluble CCl into abdominal cavity of fish of P1, P5(120mg/kg) and P6(240mg/kg) at a ratio of 0.5mL/100g body weight4Solution, P2 group not subjected to CCl4Solution injection, namely, olive oil is injected according to the same proportion, namely 0.5mL/100g of body weight, and the test fish is sampled at the 2 nd, 3 rd, 5 th and 7 th days after injection, so as to investigate the recovery effect of curcumin on carbon tetrachloride-induced acute injury of carp liver.
In the following examples, SPSS17.0 software is used for single-factor analysis of variance, when the data difference is significant, Duncan's test method is used for multiple comparisons, and the difference level is determined asP<0.05The results are expressed as Mean ± standard error (Mean ± s.e.m).
Example A different amount of curcumin added to carp: (Cyprinus carpio) Effect of growth Performance
Ⓐ selection of test subjects and management of breeding
The carp fry is provided by a fine variety farm of fresh water fishery research institute in Shandong province.
Before the test is started, temporary rearing is carried out for 21d by sinking feed provided by Tongwei feed company, 540 fries with basically consistent size, specification and constitution are randomly selected and divided into 6 groups of 3 parallel fries, and 30 parallel fries are respectively placed into 18 circulating water cultivation barrels, wherein the water volume of the cultivation barrels is 250L.
The breeding barrels used in each group of tests are randomly distributed in a spatial position, so that errors caused by factors such as light among the test groups are reduced.
Feeding 3 times per day with satiation, wherein the feeding time is 8:30, 12:30 and 4:30 respectively, and each feeding lasts for more than 30 min.
The breeding barrel is decontaminated every day to ensure the water quality, and the breeding barrel is continuously aerated for increasing oxygen day and night.
The water quality conditions during the whole test were: the water temperature is 24.5-27.5 ℃, the dissolved oxygen is above 6mg/L, the ammonia nitrogen is below 0.1mg/L, the nitrite is below 0.1mg/L, and the pH is 6.8-7.0.
In the above-mentioned breeding testCurcumin purity>95% of curcumin and CCl4Provided by seirada biotechnology limited and tezhou runxin laboratory instruments limited, respectively.
The test feed was prepared in the nutrition and feed processing research laboratory at the fresh water fishery research institute of Shandong province.
The feed is prepared by pulverizing feed raw materials, sieving with 40 mesh sieve, mixing the raw materials step by step, adding water, and making into granule with diameter of 2mm with small-sized granulator (SLP-45), wherein the small-sized granulator is purchased from fishery machinery research institute of Chinese aquatic product science research institute, and the basic feed formula and nutrition level are shown in Table 1.
Table 1 test basal feed formula and nutrient levels
Table 1 Formulation and nutrient contents of the experimental diets %
Figure 426144DEST_PATH_IMAGE001
Note: 1) each kilogram of vitamin premix comprises the following vitamins: VA, 900000 IU; VD, 200000 IU; VE, 4500 mg; VK3, 220 mg; VB1, 320 mg; VB2, 1090 mg; nicotinic acid, 2800 mg; VB5, 2000 mg; VB6, 500 mg; VB12, 1.6 mg; VC, 5000 mg; pantothenic acid, 1000 mg; folic acid, 165 mg; choline, 60000 mg;
2) each kilogram of mineral premix contains the following minerals: FeSO4 & 7H2O, 25 g; CuSO4 & 5H2O, 2.0 g; ZnSO4 & 7H2O, 22 g; na2SeO3, 0.04 g; KI, 0.026 g; MnSO4 & 4H2O, 7 g; CoCl2 & 6H2O, 0.1 g.
Ⓑ sampling and processing
After the experiment is finished, randomly taking 3 fish tails from each barrel, quickly and deeply anaesthetizing the fish by using MS-222 of 100mg/L, collecting tail venous blood and liver, centrifuging the blood sample in an anticoagulation tube for 10min at the temperature of 4 ℃ and 6000r/min, taking supernatant fluid, namely blood plasma, and freezing the prepared blood plasma at the temperature of ‒ 80 ℃ for later use; the harvested liver was divided into two portions, one portion was stored in a Boehringer's fixative for slicing and the other portion was stored in a refrigerator at ‒ 80 ℃ for further analysis.
1.2 ml of blood is collected in each anticoagulation tube in the sampling process, and the anticoagulation tube is a heparin sodium anticoagulation tube purchased from Nantong medical Co., Ltd, and the model is 1.5 ml.
Ⓒ study on the influence of curcumin with different addition levels on carp growth performance and body index
After the test is finished, stopping feeding for 24 hours, weighing each barrel, measuring the total weight and the tail number of each barrel of fish, and calculating the specific growth rate, the weight gain rate, the feed coefficient and the protein efficiency; then randomly extracting 3 fishes from each culture barrel to measure the weight, the body length, the visceral weight and the liver weight, and using the fish to measure the fullness, the visceral ratio and the liver-body ratio, wherein the calculation method is as follows:
weight gain ratio (weight gain, WG/%) = (Wt-W)0)×100%/W0
Specific growth rate (SGR/(%/d)) = (LnWt-LnW)0)×100%/T
Visceral body ratio (VSI/%) = visceral weight × 100%/Wt
Hepatosomal ratio (HSI/%) = liver weight × 100%/Wt =
Fullness factor (CF/%) = Wt × 100%/L3
Feed Conversion Ratio (FCR) = F/(Wt-W)0)
In the formula W0Weight (g) of fish at the start of the test; wt is the weight (g) of the fish at the end of the test; l is the body length (cm) of the fish at the end of the test; t is the fish culture days (d); f is the amount of feed ingested (g);
TABLE 2 influence of curcumin addition in daily ration on carp growth performance and body index
Table2. Growth performance of Cyprinus carpiofed diets containing different levels curcumin
n=9;
Figure 15388DEST_PATH_IMAGE002
Figure 60705DEST_PATH_IMAGE003
Figure 672558DEST_PATH_IMAGE004
Note that the co-current data shoulder marks are significantly different between the two sets of mean values without the same letters (1)P<0.05)。
As can be seen from Table 2, the addition of curcumin at various levels to the diet significantly affected the rate of weight gain and specific growth rate of carp (seeP<0.05) The weight gain rate and specific growth rate were significantly increased in the 60(P4) and 120mg/kg (P5) curcumin groups compared to the control group (P1) and the 30mg/kg curcumin group (P3), without significant difference from the 240mg/kg (P6) group; curcumin with different addition levels has no obvious influence on liver body ratio, viscera body ratio, feed coefficient, fullness and the like.
Improving the growth and disease resistance of fish is the most concerned problem. Antibiotics are currently widely used in terrestrial animal farming to promote growth and immunity, resulting in pathogens that develop a degree of resistance that ultimately endangers human health. Therefore, other environmentally friendly methods of preventing and treating fish diseases are emphasized. In aquaculture, Chinese herbal medicines have received much attention as potential substitutes for antibiotics. Previous researches show that curcumin can promote gastrointestinal tract movement and improve the activities of intestinal tract and pancreatic digestive enzymes of rats (brown rats), and curcumin is reported to increase the activities of intestinal protease and amylase of tilapia mossambica and improve the growth performance of tilapia mossambica. In the research, 60mg and 120mg of curcumin kg-1 are respectively added into the diet for 10 weeks, so that the weight gain rate (WG) and the Specific Growth Rate (SGR) of juvenile carp are obviously improved, which is similar to the research result of the previous people, and a reasonable explanation on the result is that the addition of curcumin enhances the immune response of the carp, thereby improving the growth performance. However, compared with 60 and 120mg of curcumin kg-1, addition of 240mg of curcumin kg-1 significantly reduces the weight gain rate (WG) and Specific Growth Rate (SGR), and improves the feed Factor (FCR), which may be caused by imbalance of intestinal flora due to excessive use of curcumin, and disturbs intestinal digestion and absorption.
Therefore, the growth performance of the carp can be improved by adding 60mg/kg and 120mg/kg of curcumin into the feed, and when the addition amount of the curcumin is 240mg/kg, the growth performance of the carp is obviously reduced compared with that of the groups of 60 and 120mg/kg, so that the excessive addition of the curcumin can or causes the growth performance of the carp to be reduced along with the continuous increase of the addition amount of the curcumin.
Example Dicurcumin to carbon tetrachloride (CCl)4) Influence research of induced fish liver injury
Ⓐ research on influence of curcumin on blood and liver function index of carp after liver injury induced by carbon tetrachloride
The kits used in this study were purchased from Nanjing, a institute for bioengineering.
After plasma stored in the ‒ 80 ℃ refrigerator in example one was taken out and dissolved in a 4 ℃ refrigerator, Total Protein (TP), glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT) contents, and total antioxidant capacity (T-AOC) were measured in the plasma.
TABLE 3CCl4Influence of curcumin on carp plasma liver function index before induction
Table3. Effects of dietary curcumin supplementation on the plasma physiological indexes of Cyprinus carpio before CCl4-challenged
n=9;
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Note that the co-current data shoulder marks are significantly different between the two sets of mean values without the same letters (1)P<0.05)。
As can be seen from Table 3, the fish body did not pass CCl4When inducing liver damage, the addition level of curcumin in the diet did not significantly affect the activity of GPT and GOT in plasma (b)P>0.05); the total protein content in plasma increases and then decreases with the increase of curcumin addition, and when the curcumin addition in the feed is 60 and 120mg/kg, the total protein content in blood is obviously higher than (A)P< 0.05) Control groups (P1 and P2), but no significant difference with each other curcumin group (A)P>0.05); total antioxidant capacity in plasmaThe force was significantly affected by the added level of curcumin in the ration, and the T-AOC was significantly increased in the P3 (30mg/kg), P4(60 mg/kg) and P5(120mg/kg) groups compared to the control groups (P1 and P2) (B)P<0.05), T-AOC of group P5 was significantly higher than (A)P<0.05) P3 group, the difference between the other groups was not significant (P>0.05), group P6(240mg/kg) had significantly lower T-AOC than group P5.
TABLE 4CCl4Influence of curcumin on carp plasma liver function index after induction for 72h
Table 4. Effects of dietary curcumin supplementation on the plasma physiological indexes of Cyprinus carpioafter 72 h CCl4-challenged.
n=9;
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Note that the co-current data shoulder marks are significantly different between the two sets of mean values without the same letters (1)P<0.05)。
As shown in Table 4, carp is processed by CCl4After induction treatment, the GPT and GOT activity in the blood of the control group is obviously improved, the TP and ALB content and the T-AOC are obviously reduced, and the difference is very obvious compared with that of a blank control group (P2) ((P<0.05). With the gradual increase of the added amount of the curcumin in the daily ration, the GPT activity and the GOT activity are both reduced and then increased, and when the added amount is 120mg/kg (P5), the GPT activity in blood is obviously lower than that in (A), (B), (C), (D) and D), (D) and D), (D) and D), (D) andP<0.05) control group (P1), no significant difference from the other groups; the GOT viability of the P4, P5 and P6 groups was all significantly lower than (P<0.05) group P1, and the effect is best when curcumin is added at a level of 120 mg/kg. Compared with P1 group, the addition of curcumin at a certain level in daily ration can make fish body resist CCl to a certain extent4Invasion, with elevated TP, ALB and T-AOC expression, and optimal effect with 120mg/kg curcumin.
Plasma GOT and GPT are widely recognized as indicators of liver damage.
In this study, the glutamic-pyruvic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activities in the feed supplemented with curcumin were lower than those of the control group, but the difference was not significant. This phenomenon indicates that the liver of fish is not damaged by the addition of a certain amount of curcumin in this experiment. CCl4Plasma GOT and GPT activities were significantly increased in the P1 group compared to the P2 group 72h after injection, suggesting CCl4Can cause carp liver injury. The activity of GPT in the feed added with 120 (P5) mg/kg of curcumin is obviously lower than that of the P1 group, and the GOT activity of the P4, P5 and P6 treated groups is obviously lower than that of the P1 group. Therefore, the carp liver is protected by adding a certain amount of curcumin into the feed.
The total phosphorus content of blood plasma is an important index reflecting the nutrition and metabolic conditions of an organism and also indirectly reflects the antioxidant capacity of the organism. Protein metabolism is liver-dominant, and plasma TP levels decrease when the liver is damaged. Thus, TP can also be clinically identified as an index for detecting liver function. Researches suggest that the traditional Chinese medicine can not only improve the antioxidant function of sturgeon, but also obviously improve the TP content in blood plasma, which is consistent with the research results of the people. Plasma TP levels were significantly higher in the P4 and P5 groups than in the control group. However, CCl4After 72h of toxic attack, the TP content of the plasma in the P1 group is obviously lower than that in the P2 group, and the TP content is in a trend of increasing firstly and then decreasing with the increase of the added amount of the curcumin. Therefore, the addition of a certain amount of curcumin in the feed can promote protein synthesis and protect the liver. This is probably because curcumin has an immune-activating function, and can resist the damage of diseases and promote the growth performance and protein metabolism of fish.
The results show that the carp liver cannot be damaged in a breeding period by adding curcumin into the feed, after the carp body is induced by carbon tetrachloride, the activity of GPT and GOT in blood can be obviously reduced when curcumin with a certain level is added into the basic feed, and the activity of GPT is obviously different from that of a control group when curcumin with a certain level is added at the level of 120mg/kg, which indicates that the carp liver can be protected by adding curcumin with a certain level in the feed.
Ⓑ research on influence of curcumin on carp liver antioxidant enzyme after carbon tetrachloride induction
The kits used in this study were purchased from Nanjing, a institute for bioengineering.
The liver stored in the refrigerator of ‒ 80 ℃ in the first example is taken out and placed in the refrigerator of 4 ℃ for dissolving, then the blood is washed by normal saline to remove, then the liver is sucked dry by filter paper, homogenate is prepared by taking normal saline as a medium according to the proportion of 1:9, then the liver is centrifuged at 3000r/min for 10min, and the supernatant is taken for the measurement of glutathione peroxidase (GSH-Px), Glutathione (GSH), superoxide dismutase (SOD) and Malondialdehyde (MDA).
As shown in figure 1, adding curcumin to the daily ration can improve liver SOD activity and reduce MDA content to a certain extent, while the fish body passes through CCl4After induction treatment, compared with a blank control group, the liver SOD activity of the P1 group is obviously reduced, and the MDA content is obviously increased (P<0.05); however, with the gradual increase of the curcumin addition level in the daily ration, the SOD activity tends to increase firstly and then decrease, and the effect is best in the curcumin group of 120mg/kg (1)P<0.05) (fig. 1A); the MDA content is in the opposite trend, and when the added amount of the curcumin is 120mg/kg, the MDA content is remarkably lower than that of (A)P<0.05) group P1 (FIG. 1B).
As can be seen from FIG. 2, CCl4Before induction, GSH-Px and GSH activity in fish liver are increased and then decreased with the increase of curcumin addition amount in the feed, but GSH-Px activity among groups has no significant difference, and when the curcumin addition amount is 120mg/kg, the liver GSH is significantly higher than that of (A), (B), (C), (D) and D), (D) and (D), (D) and (D) and D)P<0.05) control group (P1) and high dose P6 group (240 mg/kg); when CCl4After 72h induction, the GSH-Px activity of the control group is remarkably reduced, the GSH-Px activity of the control group is improved in the curcumin group, and the GSH-Px activity of the curcumin group is remarkably higher than that of the P4(60 mg/kg), P5(120mg/kg) and P6(240mg/kg) groupsP<0.05) control group (P1), and the GSH activity was highest in the 120mg/kg group.
The results show that the anti-oxidation function of the fish body is improved to a certain extent by 120mg/kg of curcumin, and the liver is protected from being damaged. This is probably because curcumin can inhibit CCl by inhibiting CCl4The expression quantity of NF-kB/c-Rel, IL-1 beta, TNF-alpha mRNA and the protein level of NF-kB/c-Rel are up-regulated to regulate the activity of partial antioxidase and finally protect liverIs not damaged.
Ⓒ research on influence of curcumin on carp liver tissue structure after carbon tetrachloride induction
In the research, the preparation components of the Boenh fixing solution are 75mL of 1.22% saturated picric acid, 25mL of 40% formalin solution and 5mL of glacial acetic acid.
The microtome used was Leica RM-2255 (Germany) and the optical microscope used was NIKON DS-Fi 1.
The liver stored in the first example in the Bonn's fixative for tissue fixation was removed, and the fixed liver tissue was washed with water, dehydrated, cleared, waxed, embedded and sliced () according to the operating specification. After the wax chip was prepared, it was attached to a glass slide treated with protein glycerol for HE staining and then mounted with neutral gum. After the mounting, the tissue sections were observed under an optical microscope under an oil microscope (x 1000).
Carp meridian CCl4Pathological sections of liver tissue after induction are shown in FIG. 3.
72h after induction, a sample is observed under an oil microscope (multiplied by 1000), and the result shows that compared with a blank control group (P2), carbon tetrachloride can cause serious damage to the liver of a fish body, the liver cells have serious vacuolation and cell expansion, the cell nucleuses of most liver cells shift, and the vacuolation and expansion of the liver cells of each test group are reduced to different degrees along with the increase of the addition amount of curcumin compared with a P1 group, wherein the shapes of the liver cells of the P5(120mg/kg) and the P6(240mg/kg) groups are normal, the cell expansion is not shown, obvious fat vacuolation is not shown, the cell nucleuses are moderate in size and are distributed in the middle of the cells.
Previous studies demonstrated that degeneration of vacuoles, necrosis, and infiltration of inflammatory cells within the liver are biomarkers of liver injury. However, the study shows that the addition of curcumin to the feed can reduce liver inflammatory cell infiltration and vacuole degeneration, especially in the P5 group. Illustrating curcumin (120mg/kg feed) to CCl4The induced carp liver injury has a certain protection effect. Similar results were also observed in humans and mice.
The result directly verifies the liver protection effect of curcumin in the carp, and shows that curcumin has the function of protecting the liver from being damaged on the carp body, and the effect is best at the addition level of 120 mg/kg. This is probably because curcumin can induce the body to produce ROS, which can damage DNA molecules of damaged cells, and repair liver function by regulating the expression of apoptosis-related genes, but either under or over dose may not achieve optimal results.
Ⓓ study on influence of curcumin on Nrf2mRNA expression level in carp liver after carbon tetrachloride induction
The kits used in this example were all purchased from Tiangen Biotech Ltd.
Extracting total RNA of carp liver tissue by using a kit and adopting a Trizol method, determining the purity of the total RNA to enable OD260/OD280 to be 1.8-2.0, and detecting the quality of the total RNA by gel electrophoresis; reverse transcribing the RNA to cDNA using a reverse transcription kit;
designing primers according to the complete gene sequence of carp Nrf2mRNA (see Table 5 in detail) and carrying out real-time fluorescent quantitative PCR reaction under the following reaction conditions: 15 min at 95 ℃; the temperature is 95 ℃ for 10 s, the temperature is 55 ℃ for 30 s, and the temperature is 72 ℃ for 32 s, and the circulation is carried out for 40 times.
The amplification of a target gene is detected by a Sybergreen method, beta-actin is an internal reference gene, and 3 repeats are set in all reactions. Nrf2mRNA levels using 2-ΔΔCtAnd (4) calculating.
TABLE 5Nrf2Gene fluorescent quantitative PCR primer
Table5 Primers for real time PCR analysis of Nrf2 genes
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As shown in fig. 4, CCl4Before induction, the expression level of Nrf2mRNA in the liver of the fish body of the P5 group is obviously higher than that of (A)P<0.05) control, placebo and P3 groups, which were not significantly different from the other two groups; when CCl4After 72h of induction, the expression level of Nrf2mRNA in the livers of the control group and the P3 group is obviously reduced, and the expression level of Nrf2mRNA in the livers of the P4 group and the P5 group is increased compared with that before the induction. Furthermore, via CCl4After induction, the amount of curcumin added in the liver is increasedA tendency of increasing first and then decreasing, wherein the Nrf2mRNA expression levels of the P4(60 mg/kg), P5(120mg/kg) and P6(240mg/kg) groups were significant (P<0.05) is higher than that of the control group, and the expression quantity of liver Nrf2mRNA is the highest in the group of 120 mg/kg.
Ⓔ study on influence of curcumin on carp liver cell nucleus Nrf2 protein level after carbon tetrachloride induction
In this study, Nrf2 primary antibody was purchased from Wuhan Sanying Biotechnology, Inc., and goat anti-rabbit secondary antibody was purchased from Jackson Immunoresearch.
And detecting the content of Nrf2 in the liver cell nucleus by adopting a western blot technology.
Taking 100mg of carp liver tissue, extracting total protein by a solution method, measuring protein concentration by a Bradford method, performing electrophoresis after denaturation, transferring the protein to a polyvinylidene fluoride (PVDF) membrane, sealing for 2h at room temperature by 50 g/L skimmed milk powder TBS solution, incubating for 18-24 h at 4 ℃ by Nrf2 primary antibody (1: 1000), washing the membrane for 3 times by washing membrane buffer TBST (Tris Buffered Saline Tween), 8 min each time, incubating for 2h at room temperature by using horseradish peroxidase (HRP) labeled goat anti-rabbit secondary antibody (1: 10000), washing the membrane for 3 times by using ST method, performing 2 min immunoblotting chemiluminescence (ECL) reaction, exposing, developing and fixing in a dark room. After incubation with HRP-coupled internal reference GAPDH primary antibody for 2h, the membrane was washed with TBST 4 times for 10min each, and 2 min ECL reaction, exposure, development and fixation were performed in the same manner.
The gray values of the bands of the target protein and the internal reference protein are measured by using QuantiScan 3.0 software, and the relative expression quantity is represented by the ratio of the Nrf2 gray value to the internal reference GAPDH gray value.
The results are shown in FIG. 5, in CCl4After 72h of induction, the protein level of Nrf2 in the liver cell nucleus of the fish body tends to increase and then decrease with the increase of the added amount of curcumin, and the protein content of the cell nucleus Nrf2 of groups of P3 (30mg/kg), P4(60 mg/kg), P5(120mg/kg) and P6(240mg/kg) is obvious (the content of the protein is shown in the specification) (the content of the protein in the cell nucleus Nrf2 of the fish body is shown in the specification)P<0.05) higher than the P1 group, the P5 group was significantly higher than the P6 group, and there was no significant difference from the P3 and P4 groups.
The following analyses were performed in combination with the results of example four, example six and example seven:
metabolism of chemicals in the liver depends primarily on first-stage metabolism (e.g., GSH) and second-stage enzymatic metabolism (e.g., GPX, SOD). There is increasing evidence that the Keap1-Nrf2 complex is a key molecular target for chemopreventive phase 2 enzyme inducers. Keap1 is a cytoplasmic actin-binding protein that is an inhibitor of Nrf2, and sequesters it in the cytoplasm under normal physiological conditions. However, the inducer promotes the release of Nrf2 by the cytosolic inhibitor Keap1 by altering the structural conformation of Keap 1. The transcription factor Nrf2 is a member of the basic leucine zipper NF-E2 family, which interacts with the Antioxidant Response Element (ARE) of the promoter region of the second-stage detoxification enzyme. Finally, the antioxidant capacity of the organism is improved by regulating the expression of the two-phase enzyme.
The research shows that appropriate dosage (60 and 120mg kg-1) of oral curcumin can obviously improve the antioxidant state of carp blood plasma, which is probably related to improving SOD and GPx activity, GSH content and MDA content. After CCl4 is detoxified for 72h, the mRNA level, SOD, GPX activity and GSH content of liver Nrf2 in the P1 group are all obviously lower than those in the P2 group, and the MDA content is obviously higher than that in the P2 group. 120mg kg-1 curcumin is added into the feed, so that the SOD activity, the GPX activity and the GSH content can be obviously improved, and the MDA content is reduced. Meanwhile, with the increase of the addition amount of curcumin, the protein level of Nrf2 in the carp liver cell nucleus tends to increase firstly and then decrease. The expression level of Nrf2 protein in nucleus is highest in P5 group. The Nrf2 protein level in the high dose group was significantly lower than in the P5 group. One reasonable explanation is that high doses of curcumin may disrupt the structure of hepatocytes, thereby inhibiting migration of Nrf 2. In addition, curcumin is a radical scavenger and hydrogen donor, having antioxidant and pro-oxidative activity, the latter predominating in high concentrations of curcumin. The results show that curcumin can promote a cytoplasmic inhibitor Keap1 to release Nrf2, and combine Nrf2 with SOD and a GPX promoter region ARE, so that the antioxidant state of carp is improved, and the carp liver is protected finally.
Example study on recovery effect of curcumin on carbon tetrachloride-induced acute liver injury of carp
Ⓐ study on influence of curcumin on carp plasma liver function index at different time points after carbon tetrachloride induction
The kits used in this study were purchased from Nanjing, a institute for bioengineering.
After the plasma stored in the ‒ 80 ℃ refrigerator in example one was taken out and dissolved in the 4 ℃ refrigerator, the contents of Glutamic Oxaloacetic Transaminase (GOT), Glutamic Pyruvic Transaminase (GPT) and total antioxidant capacity (T-AOC) were measured.
As shown in fig. 6A and fig. 6B, curcumin added in the feed has a significant effect on carp acute liver injury induced by carbon tetrachloride stress, and GPT and GOT activities in fish plasma are different due to different curcumin addition amounts in a period of time. GPT and GOT of the negative control group (P1) were significant when induced by carbon tetrachloride for 2 and 3 days: (P<0.05) Compared with a positive control group (P2), the composition has no significant difference with a P5(120mg/kg) group and a P6(240mg/kg) group; the GOT activity of the P5(120mg/kg) group was significant when the cells were induced by carbon tetrachloride for 5 days (P<0.05) Lower than the negative control group (P1), and no significant difference from the positive control group (P2); the GPT and GOT activities of the P5(120mg/kg) and P6(240mg/kg) groups were not significantly different from the positive control group when induced with carbon tetrachloride for 7 days. In addition, after fish bodies are induced by carbon tetrachloride, the GPT and GOT activities of the P5(120mg/kg) and P6(240mg/kg) groups show a trend of increasing and then decreasing within a certain time, and the difference is significant (the activity of the GPT and the GOT activities show a trend of increasing and then decreasing) (the activity of the GPT and the GOT activities are different from each other)P<0.05). The activity of GPT and GOT in the P5(120mg/kg) group was further reduced to normal level than that in the P6(240mg/kg) group.
Ⓑ research on influence of curcumin on carp liver SOD activity and MDA content at different time points after carbon tetrachloride induction
The kits used in this study were purchased from Nanjing, a institute for bioengineering.
The liver stored in the refrigerator of ‒ 80 ℃ in the first example is taken out and placed in the refrigerator of 4 ℃ for dissolving, then the blood is washed by normal saline to remove, then the liver is sucked dry by filter paper, homogenate is prepared by taking normal saline as a medium according to the proportion of 1:9, then the liver is centrifuged at 3000r/min for 10min, and the supernatant is taken for the measurement of glutathione peroxidase (GSH-Px), Glutathione (GSH), superoxide dismutase (SOD) and Malondialdehyde (MDA).
As shown in FIG. 7A and FIG. 7B, the feed is added withThe curcumin is added for feeding for a period of time, the liver SOD activity and MDA content of carp induced by carbon tetrachloride after acute liver injury are obviously influenced, and the liver SOD activity and MDA content of the carp are different due to different curcumin adding amounts in a period of time. The SOD activity of the negative control (P1) was significantly reduced compared to the initial induction period when it was induced by carbon tetrachloride for 3 d: (P<0.05) And significantly lower than the P5(120mg/kg) and P6(240mg/kg) groups; the SOD activities of the negative control group (P1), the P5(120mg/kg) and the P6(240mg/kg) are all increased compared with the SOD activity of the former time point when the SOD is induced by carbon tetrachloride for 5 days, and the SOD activities of the P5(120mg/kg) group are obvious (the SOD activity of the SOD of the negative control group, the P5(120mg/kg) group is increased by the carbon tetrachloride for 5 days, the SOD of the negative control group is not increased by the carbon tetrachloride for the first time point, and the SOD of the negative control group is not increased by the second time point, and the SOD of the negative control group is not increased by the third time pointP<0.05) Higher than the positive control group, the difference between the positive control group and the negative control group and the P6(240mg/kg) group is not significant; SOD activity of P5(120mg/kg) group was significantly reduced compared to the previous time point after 7 days of carbon tetrachloride induction (P<0.05) No significant difference from the positive control group (P2), while the SOD activity of the P6(240mg/kg) group was no significant difference from the former time point, and (significant: (P2))P<0.05) Higher than the positive control group, there was no significant difference from the P5(120mg/kg) and the negative control group (P1). In addition, as shown in fig. 8A and 8B, the MDA content was changed in a trend opposite to SOD.
Ⓒ study on influence of curcumin on carp liver GPx activity and GSH content at different time points after carbon tetrachloride induction
As shown in fig. 8A and 8B, after being induced by carbon tetrachloride, the carp liver GPx activity and GSH content are also significantly affected by the addition amount of curcumin in the feed. When the GPX activity and the GSH content of the negative control group (P1) are obviously reduced compared with the GPX activity and the GSH content at the early stage of carbon tetrachloride induction when the GPX activity and the GSH content are induced by carbon tetrachloride for 3dP<0.05) And the activity of GPX in the negative control group (P1) is significant (P<0.05) The GPX activity of the P5(120mg/kg) group is also significantly higher than that of the positive control group (P2) and the P5(120mg/kg) group (P<0.05) Positive control group (P2) and P6(240mg/kg) groups, while negative control group (P1) had significant GSH content (P<0.05) Lower than the positive control group (P2), and has no significant difference with the P5(120mg/kg) and the P6(240 mg/kg); GPX activity of P5(120mg/kg) group was significant when induced by carbon tetrachloride for 5 days (P<0.05) Higher than the negative control group, with the positive control group and P6 (24)0mg/kg) group had no significant difference, while the negative control group (P1) had significant GSH content (GSH content) ((S)P<0.05) Compared with the positive control group (P2) and the P5(120mg/kg), the compound has no significant difference with the P6(240 mg/kg); the activity of GPX in a negative control group (P1) is obvious when 7 days are induced by carbon tetrachloride (P<0.05) Compared with the positive control group (P2) and the P5(120mg/kg), the GSH of the transgenic animals has no significant difference from the P6(240mg/kg) group and the GSH of the transgenic animals has no significant difference between the groups. In addition, after induction by carbon tetrachloride, the GPx activity and GSH content of fish livers of negative control groups (P1), P5(120mg/kg) and P6(240mg/kg) all showed a trend of decreasing first and increasing later with time, wherein the P5(120mg/kg) group recovered to normal levels more quickly than the P6(240mg/kg) group.
In conclusion, the optimal curcumin (60 and 120mg/kg) added into the diet can improve the growth performance of the carps. The addition of 120mg/kg curcumin in the feed can improve the antioxidant state of the carp by promoting the release of Nrf2, thereby improving the activity of antioxidant enzyme, and finally protecting the carp from CCl4Liver damage of (1). The addition of curcumin in the feed has a recovery effect on the carp from acute liver injury, and compared with a high-dose (240mg/kg) group, the recovery capability of the 120mg/kg group is stronger. The research preliminarily establishes a liver protection mechanism of curcumin in the carp, and provides theoretical guidance for further developing fish liver protection medicines.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (7)

1. A study on the protective effect of curcumin on carp liver injury by activating activity of Nrf2 is characterized in that: comprises the study of curcumin in different addition amounts on carp(s) ((Cyprinus carpio) Growth performance impact of curcumin on carbon tetrachloride (CCl)4) The research on the influence of the induced fish liver injury and the recovery effect of curcumin on the carp acute liver injury induced by carbon tetrachloride comprises the following steps,
first, experimental design
The test is divided into 6 groups, wherein the P1 group is a control group, the P2 group is a blank control group, the P3 group, the P4 group, the P5 group and the P6 group are test groups, and the P1 group and the P2 group are fed with basic feed; feeding test feeds to P3, P4, P5 and P6 groups, wherein curcumin is not added to the basic feed, and the addition levels of curcumin in the test feeds are respectively 30, 60, 120 and 240 mg/kg;
test one: after continuously feeding for 10 weeks, P1, P3, P4, P5 and P6 groups were sampled, and different added amounts of curcumin were analyzed for carp: (A), (B), (C), (D) and D) in aCyprinus carpio) The effect of growth performance;
and (2) test II: on the basis of the test, 30 percent of CCl dissolved in olive oil is injected into the abdominal cavity of the fish bodies of P1, P3, P4, P5 and P6 groups respectively in a proportion of 0.5mL/100g of the body weight4A solution; carp of P2 group was not subjected to CCl after continuously feeding for 10 weeks4Solution injection, but CCl in the same ratio4The solution is 0.5mL/100g body weight, injected with olive oil, and sampled 72h later, to study curcumin to CCl4Inducing the influence of liver injury of fish bodies;
and (3) test III: selecting P1 (negative control group), P2 (positive control group), P5 (optimal protection group) and P6 (high dose group) according to the results of the second study, and injecting 30% olive oil-soluble CCl intraperitoneally into fish bodies of the P1, P5(120mg/kg) and P6(240mg/kg) groups at a ratio of 0.5mL/100g body weight after 10 weeks of breeding test4Solution, P2 group not subjected to CCl4Injecting solution, namely injecting olive oil according to the same proportion, namely 0.5mL/100g of body weight, and sampling test fishes at the 2 nd, 3 rd, 5 th and 7 th days after injection so as to discuss the recovery effect of curcumin on the carbon tetrachloride-induced carp liver acute injury;
selection and breeding management of test objects
Randomly selecting 540 carp fries with basically consistent sizes, specifications and constitutions, dividing the carp fries into 6 groups, putting 3 parallel carps in each group, putting 30 parallel carps in 18 circulating water culture barrels for culture, feeding three times after each day of full feeding, and keeping each feeding for more than 30 min;
third, sampling and processing
After the experiment is finished, randomly taking 3 fish tails from each barrel, quickly and deeply anaesthetizing the fish by using MS-222 of 100mg/L, collecting tail venous blood and liver, centrifuging the blood sample in an anticoagulation tube for 10min at the temperature of 4 ℃ and 6000r/min, taking supernatant fluid, namely blood plasma, and freezing the prepared blood plasma at the temperature of ‒ 80 ℃ for later use; dividing the collected liver into two parts, one part being stored in a Boehn's stationary liquid for slicing, and the other part being stored in a refrigerator at ‒ 80 ℃ for further analysis;
fourth, determination of growth and body index
After the test is finished, stopping feeding for 24 hours, respectively weighing each barrel, measuring the total weight and the tail number of each barrel of fish, and calculating the specific growth rate, the weight gain rate and the feed coefficient; then randomly extracting 3 fishes from each culture barrel to measure the weight, the body length, the visceral weight and the liver weight, and using the fish to measure the fullness, the visceral ratio and the liver-body ratio, wherein the calculation method is as follows:
weight gain ratio (weight gain, WG/%) = (Wt-W)0)×100%/W0
Specific growth rate (SGR/(%/d)) = (LnWt-LnW)0)×100%/T
Visceral body ratio (VSI/%) = visceral weight × 100%/Wt
Hepatosomal ratio (HSI/%) = liver weight × 100%/Wt =
Fullness factor (CF/%) = Wt × 100%/L3
Feed Conversion Ratio (FCR) = F/(Wt-W)0)
In the formula W0Weight (g) of fish at the start of the test; wt is the weight (g) of the fish at the end of the test; l is the body length (cm) of the fish at the end of the test; t is the fish culture days (d); f is the amount of feed ingested (g);
fifth, plasma liver function index determination
Determining Total Protein (TP), glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT) and total antioxidant capacity (T-AOC) by using the kit;
sixthly, measuring anti-oxidation indexes of liver and analyzing tissue slices
Taking out the liver stored in the refrigerator of ‒ 80 ℃ in the third step, placing the liver in the refrigerator of ‒ ℃ for dissolving, washing with normal saline to remove blood, then sucking the liver dry with filter paper, preparing homogenate with normal saline as a medium according to the proportion of 1:9, then centrifuging at 3000r/min for 10min, and taking the supernatant for measuring glutathione peroxidase (GSH-Px), Glutathione (GSH), superoxide dismutase (SOD) and Malondialdehyde (MDA);
the liver slices stored in the Boenh's stationary liquid in the third step are observed by adopting an optical microscope under the condition of an oil microscope multiplied by 1000;
seventhly, liver Nrf2mRNA measurement
Extracting the total RNA of the carp liver tissue in each time period by adopting a Trizol method, and determining the purity of the total RNA; detecting the total RNA quality by gel electrophoresis; the Sybergreen method is used for detecting the amplification of a target gene, and the Nrf2mRNA level adopts 2-ΔΔCtCalculating a method;
eighthly, monitoring the content of Nrf2 in a nucleus
Detecting the content of Nrf2 in the liver cell nucleus by adopting a western blot technology;
data statistics and analysis
SPSS17.0 software is adopted to carry out one-factor variance analysis on test data, when the data difference is obvious, a Duncan's test method is adopted to carry out multiple comparisons, and the difference level is determined asP<0.05The results are expressed as Mean ± standard error (Mean ± s.e.m).
2. The study on the protective effect of curcumin on carp liver injury by activating activity of Nrf2 according to claim 1,the method is characterized in that: the curcumin purity>95% of curcumin and CCl4Provided by seirada biotechnology limited and tezhou runxin laboratory instruments limited, respectively.
3. The study on carp liver injury protection effect of curcumin through activation of Nrf2 vitality according to claim 1 is characterized in that: the carp fry is provided by a good breeding field of fresh water fishery research institute in Shandong province, before the test is started, sinking feed provided by Tongwei feed company is adopted for temporary culture for 21 days, and then the selection operation of the test object is carried out.
4. The study on carp liver injury protection effect of curcumin through activation of Nrf2 vitality according to claim 1 is characterized in that: the volume of water in the breeding barrels is 250L, the breeding barrels used in each group of tests are randomly distributed in spatial positions, the feeding time is 8:30, 12:30 and 4:30 every day, and the water quality conditions during the whole test period are as follows: the water temperature is 24.5-27.5 ℃, the dissolved oxygen is above 6mg/L, the ammonia nitrogen is below 0.1mg/L, the nitrite is below 0.1mg/L, and the pH is 6.8-7.0.
5. The study on carp liver injury protection effect of curcumin through activation of Nrf2 vitality according to claim 1 is characterized in that: the kits adopted in the plasma liver function index and liver antioxidation index calibration are purchased from Nanjing institute of bioengineering; the preparation components of the Boenshi stationary liquid are as follows: 75mL of 1.22% saturated picric acid, 25mL of 40% formalin solution, 5mL of glacial acetic acid, an optical microscope model of NIKON DS-Fi1, and a microtome model of Leica RM-2255 (Germany).
6. The study on carp liver injury protection effect of curcumin through activation of Nrf2 vitality according to claim 1 is characterized in that: the kits adopted in the seventh step are all purchased from Tiangen Biotechnology Co.
7. The study on carp liver injury protection effect of curcumin through activation of Nrf2 vitality according to claim 1 is characterized in that: collect 1.2 ml blood in every anticoagulation pipe in the sampling process, anticoagulation pipe is the heparin sodium anticoagulation pipe of purchasing from south logical medical treatment limited company, and the model is 1.5 ml.
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