Use of resveratrol in the preparation of a product for alleviating chromium picolinate-induced liver damage in fish

Abstract

The present application relates to the field of aquaculture, and in particular, to the application of resveratrol in the preparation of a product for alleviating fish liver damage induced by chromium picolinate. The present application provides the application of resveratrol in alleviating fish liver damage induced by chromium picolinate, to solve the problem that there is a lack of effective nutritional intervention means for fish liver damage caused by long-term exposure to chromium picolinate in the prior art, and further achieve multiple effects such as reducing liver chromium accumulation, inhibiting oxidative stress and inflammatory response, protecting liver and intestinal tissue structure, and improving growth performance.

Description

Technical Field

[0001] This invention relates to the field of aquaculture, and more specifically, to the application of resveratrol in the preparation of products for alleviating chromium pyridinecarboxylate-induced liver damage in fish. Background Technology

[0002] In aquaculture, appropriate doses of chromium picolinate (CrPic) can improve fish growth performance and glucose and lipid metabolism, and increase feed utilization efficiency, thus showing potential application. However, the toxicity characteristics of CrPic differ significantly from other heavy metals or hepatotoxic substances, making its safety assessment difficult to simply rely on existing experience. First, chromium in CrPic exists in a trivalent state, exhibiting different toxicity than hexavalent chromium. Furthermore, picolinic acid, as a ligand, can significantly alter the absorption pathway and tissue distribution of chromium, leading to specific accumulation of chromium in the liver. Second, CrPic can induce a damage pattern in cells different from general oxidative stress, manifesting as mitochondrial dysfunction, lipid peroxidation, and abnormal activation of inflammatory signaling pathways, with its toxic effects showing a clear dose- and time-dependent effect. More importantly, the toxicity of CrPic in aquatic animals exhibits significant strain differences; fish from different genetic backgrounds may have drastically different sensitivities, further increasing the complexity of risk assessment and intervention strategy development.

[0003] The liver, as the core organ for metal metabolism and detoxification, is a major target tissue for the toxic effects of CrPic. Long-term or high-dose CrPic exposure can lead to liver tissue damage, increased oxidative stress, and activated inflammatory responses in fish. However, systematic research on CrPic-induced liver injury in fish remains very limited, especially lacking in-depth analysis of its specific toxic mechanisms. More importantly, effective nutritional interventions for CrPic-induced liver injury have not yet been established in current technology. Although resveratrol (Res), as a natural polyphenol compound, has been reported to have antioxidant, anti-inflammatory, and hepatoprotective effects in various animal models, existing studies have mostly focused on liver injury induced by alcohol, high-fat diets, or other heavy metals (such as cadmium and lead). Whether it can alleviate liver injury in fish caused by CrPic, a specific compound, and whether its mechanism of action involves CrPic's unique toxic pathways, have not been publicly reported.

[0004] Furthermore, existing technologies lack a systematic review of the differential responses of different fish strains to CrPic exposure, and do not provide personalized protection strategies for highly sensitive strains. As an important freshwater aquaculture fish in my country, different strains of crucian carp exhibit differences in genetic background and stress response capabilities, but their tolerance to CrPic exposure and corresponding nutritional intervention programs have not yet been studied.

[0005] In summary, the existing technologies have the following shortcomings: (1) insufficient understanding of the specific toxicity mechanism of CrPic-induced liver injury in fish; (2) lack of specific nutritional intervention techniques targeting CrPic hepatotoxicity; and (3) failure to consider the differential responses of different fish strains to CrPic exposure. Therefore, developing a nutritional intervention strategy that can effectively alleviate CrPic-induced liver injury in fish, clarify its mechanism of action, and be applicable to different sensitive strains has significant academic value and application prospects.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide the application of resveratrol in alleviating chromium pyridinecarboxylate-induced liver damage in fish, in order to solve the problem of the lack of effective nutritional intervention methods for liver damage in fish caused by long-term exposure to chromium pyridinecarboxylate in the prior art, and to further achieve multiple effects such as reducing chromium accumulation in the liver, inhibiting oxidative stress and inflammatory response, protecting liver and intestinal tissue structure, and improving growth performance.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: Application of resveratrol in the preparation of products for alleviating chromium pyridinecarboxylate-induced liver injury in fish.

[0009] The current application of chromium picolinate in aquatic feeds mainly focuses on its nutritional regulatory effects, such as promoting aquatic animal growth, improving glucose and lipid metabolism, and enhancing feed utilization. However, systematic evaluations of the chronic toxic effects of long-term, high-dose chromium picolinate exposure, including chromium accumulation in the liver, oxidative damage, and inflammatory responses, are lacking. Furthermore, existing technologies do not adequately consider the differences in tolerance to chromium picolinate exposure among heterozygous silver carp strains with different genetic backgrounds, and there is a lack of effective technical solutions to alleviate chromium picolinate-induced liver injury through nutritional interventions.

[0010] Therefore, this invention uses the hybrid silver carp 'Zhongke No. 3' and 'Zhongke No. 6' as research subjects, establishes a long-term feed exposure model by adding high doses of chromium pyridinecarboxate to the feed, and conducts nutritional intervention by adding resveratrol; by measuring growth performance, liver chromium content, antioxidant enzyme activity, lipid peroxidation level, inflammation-related indicators, and related gene expression, the liver damage response of different strains of hybrid silver carp to chromium pyridinecarboxate exposure and the alleviating effect of resveratrol are evaluated, thereby providing a technical solution for the safe application of chromium pyridinecarboxate in aquatic feed and the nutritional regulation of liver damage in fish.

[0011] This invention establishes a stable long-term dietary chromium pyridine formate exposure model by adding chromium pyridine formate to the feed of hybrid silver carp and feeding it continuously. It can be used to evaluate the effects of chromium pyridine formate on the growth performance, liver chromium accumulation, plasma biochemical indicators, liver and intestinal tissue structure, antioxidant capacity and inflammatory response of hybrid silver carp. It can also clarify the differences in sensitivity of different strains of hybrid silver carp to chromium pyridine formate exposure, among which "Zhongke No. 3" showed higher sensitivity.

[0012] Adding resveratrol to chromium pyridinecarboxate-exposed diets can improve chromium pyridinecarboxate-induced growth inhibition; reduce liver oxidative damage levels; enhance Nrf2-mediated antioxidant defense; inhibit NF-κB-mediated inflammatory responses; improve abnormal plasma biochemical indicators; and alleviate liver and intestinal tissue structural damage, thereby reducing the health risks posed by long-term high-dose chromium pyridinecarboxate exposure to hybrid silver carp. This method is simple to implement, can be achieved through conventional feed preparation and continuous feeding, and is suitable for safety evaluation of chromium pyridinecarboxate applications in aquaculture and the development of damage mitigation technologies.

[0013] Furthermore, the fish species mentioned include, but are not limited to, the heterotrophic silver carp.

[0014] Furthermore, the heterozygous silver carp includes, but is not limited to, the Zhongke No. 3 and / or Zhongke No. 6 strains.

[0015] Furthermore, the relief of chromium pyridinecarboxylate-induced liver injury in fish includes at least one of the following effects: (1) Reduces the accumulation level of chromium in the liver; Figure 1 A showed that, compared with the control group, the chromium content in the livers of both strains of fish in the CrPic group was significantly higher; while the chromium content in the livers of the CrPic+Res group was significantly lower than that of the CrPic group, indicating that resveratrol can effectively reduce the accumulation of chromium in the liver. (2) Reduce liver oxidative stress levels; Figure 3 As shown in A and 3B, the contents of malondialdehyde (MDA) and lipid peroxide (LPO) were significantly increased in the CrPic group, while both were significantly decreased in the CrPic+Res group. Figure 3 C showed that the content of reactive oxygen species (ROS) increased in the CrPic group and decreased significantly in the CrPic+Res group, indicating that resveratrol can reduce the level of oxidation products and alleviate oxidative stress. (3) Increase the activity of liver antioxidant enzymes; Figure 3 E showed that superoxide dismutase (SOD) activity was significantly reduced in the CrPic group, while SOD activity significantly increased in the CrPic+Res group; simultaneously Figure 4 and Figure 5 The results showed that resveratrol activates the NRF2 pathway and upregulates the expression of downstream antioxidant genes, confirming the increased activity of antioxidant enzymes at the molecular level.

[0016] (4) Inhibits liver inflammatory response; Figure 6 AC showed that phosphorylated NF-κBp65 and TNF-α protein expression was increased in the CrPic group and significantly decreased in the CrPic+Res group. Figure 6 DH showed that the gene expression of pro-inflammatory factors (il-1β, il-6, il-12) was upregulated in the CrPic group, and the expression of anti-inflammatory factors (tgf-β, il-4) was upregulated in the CrPic+Res group; Figure 7 AC showed that plasma TNF-α, IL-6, and IL-1β levels were elevated in the CrPic group and significantly decreased in the CrPic+Res group, demonstrating at multiple levels that resveratrol inhibits the inflammatory response. (5) Reduces damage to liver tissue structure; Figure 2 Liver H&E staining showed that the CrPic group exhibited significant pathological changes such as vacuolation, deep staining of the nuclei, and nuclear pyknosis in hepatocytes; the CrPic+Res group showed a significant reduction in the above-mentioned damage, and the liver tissue structure tended to be intact, directly proving that resveratrol reduced liver tissue damage.

[0017] Furthermore, the reduction of liver oxidative stress level includes reducing malondialdehyde, lipid peroxide and / or reactive oxygen species content; the enhancement of liver antioxidant enzyme activity includes enhancing superoxide dismutase activity; the inhibition of liver inflammatory response includes inhibiting NF-κB pathway activation, reducing phosphorylated NF-κB p65 protein expression and / or reducing inflammatory factor expression.

[0018] Furthermore, the relief of chromium pyridinecarboxylate-induced liver injury in fish is achieved by activating the NRF2 antioxidant pathway and / or inhibiting the NF-κB inflammatory pathway.

[0019] Furthermore, the resveratrol content in the product is 0.1wt%~0.2wt%.

[0020] Furthermore, the products include, but are not limited to, feed additives.

[0021] Application of resveratrol in the preparation of formulations for alleviating chromium pyridinecarboxylate-induced intestinal tissue structure damage in fish.

[0022] Figure 2 H&E staining results of B showed that the intestines of heterotrophic silver carp in the CrPic group had obvious pathological damage, including disordered arrangement of intestinal villi, destruction of structural integrity, reduction of goblet cells, and epithelial cell shedding; while the morphology of intestinal villi in the CrPic+Res group was significantly improved, the arrangement was more regular, and the degree of epithelial shedding was significantly reduced, approaching the level of the control group. Figure 2 The results showed that the intestinal villus height of both strains in the CrPic group was significantly reduced; while the intestinal villus height of the CrPic+Res group was significantly increased, indicating that resveratrol has a protective effect on intestinal structure.

[0023] A feed for alleviating chromium pyridinecarboxylate-induced liver damage in fish, the feed containing resveratrol.

[0024] Application of resveratrol in the preparation of feed additives for improving chromium pyridinecarboxylate-induced growth inhibition in fish and / or improving feed efficiency.

[0025] Table 2 shows that, compared with the CrPic group, the CrPic+Res group exhibited significantly higher final body weight, specific growth rate, and weight gain rate in the hybrid silver carp, while feed efficiency was significantly improved and feed intake was significantly reduced. This indicates that resveratrol does not promote growth by increasing feed intake, but rather effectively reverses the growth inhibition caused by CrPic by improving feed utilization efficiency, and the effect exceeds that of the control group.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, through the addition of resveratrol to feed containing chromium picolinate, significantly reduces pathological damage to the liver and intestines of crucian carp after continuous feeding, decreases hepatic chromium accumulation, inhibits oxidative stress and inflammatory responses, and simultaneously improves growth inhibition and feed efficiency decline caused by chromium picolinate exposure. Resveratrol achieves these protective effects by activating the NRF2 antioxidant pathway and inhibiting the NF-κB inflammatory pathway, with particularly pronounced effects in the more sensitive "Zhongke No. 3" strain. This method is simple to operate and applicable to feed additives or veterinary formulations, providing an effective nutritional intervention for the safe application of chromium picolinate in aquatic feed. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 The effects of resveratrol on liver chromium accumulation and plasma biochemical parameters induced by chromium pyridinecarboxate in two strains of heterotrophic silver carp; Figure 2 The effect of resveratrol on liver and intestinal pathological damage induced by chromium pyridinecarboxate in two strains of heterotrophic silver carp; Figure 3 The effect of resveratrol on liver oxidative stress-related indicators induced by chromium pyridinecarboxate in two strains of heterotrophic silver carp; Figure 4 The effect of resveratrol on the NRF2 pathway in the liver of two strains of heterotrophic silver carp induced by chromium pyridinecarboxate; Figure 5 The effect of resveratrol on NRF2 pathway-related genes in the livers of two strains of heterotrophic silver carp induced by chromium pyridinecarboxate. Figure 6 The effects of resveratrol on the liver NF-κB pathway and inflammatory response induced by chromium pyridinecarboxate in two strains of heterotrophic silver carp; Figure 7 The effect of resveratrol on plasma inflammatory markers induced by chromium pyridinecarboxate in two strains of heterotrophic silver carp. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0030] Example 1. Experimental fish and aquaculture conditions The hybrid silver carp used in the experiment, 'Zhongke No. 3' (initial weight: 5.78±0.01g) and 'Zhongke No. 6' (initial weight: 5.76±0.01g), were purchased from Huangshi Fu'er Aquatic Seedling Co., Ltd. (Huangshi, Hubei). Before the experiment, all experimental fish were temporarily held in net cages for two weeks to acclimatize to the culture environment. They were fasted for 24 hours before the formal experiment, and then individuals of uniform size and good health were randomly assigned to the experimental net cages. Two strains, 'Zhongke No. 3' and 'Zhongke No. 6', were used in the experiment, each strain was fed three different experimental feeds, and each treatment group had three replicates. The culture experiment was conducted in pond net cages at the Laohe Yangtze River Four Major Freshwater Fish Breeding Farm in Shishou City, Hubei Province. The net cages were 2m×2m×2m in size, with a water depth of approximately 1.7m.

[0031] The experiment lasted for 8 weeks. During the rearing period, the fish were fed to their full capacity at 8:30, 13:30, and 18:30 daily. The aquatic environment was kept relatively stable during the experiment, with a water temperature of 27.7±0.5℃, dissolved oxygen >7mg / L, ammonia nitrogen of 0.27±0.02mg / L, and a pH range of 6.2~7.2.

[0032] 2. Experimental feed Chromium picolinate (CrPic; CAS No. 14639-25-9, purity ≥98%) and resveratrol (Res; CAS No. 501-36-0, purity ≥98%) were added to the basal diet to prepare experimental feeds. Three treatment groups were set up: a control group (Con), a CrPic group (200 mg / kg, calculated as Cr), and a CrPic+Res group (CrPic 200 mg / kg + Res 160 mg / kg). The total chromium content in the feeds was determined according to GB / T13088-2006 and detected using an atomic absorption spectrometer (CONTAA700). The measured values ​​were 1.50, 210.40, and 208.30 mg / kg, respectively. The composition and nutrient levels of the experimental feeds are shown in Table 1.

[0033] Table 1. Experimental feed formulation and biochemical composition (% dry matter)

[0034] 3. Grouping and Feeding Management After the experiment, the total weight of the fish in each tank was weighed and recorded. Three whole fish samples were weighed and bagged. Blood was then drawn, centrifuged at 3000 rpm for 15 minutes, and the supernatant plasma was collected and stored at -80℃. Liver tissue was then collected and stored at -80℃. Tissue sections from the liver and intestines were fixed in fixative.

[0035] 4. Nutritional composition and chromium content determination The chemical composition (crude protein, crude fat, moisture, and ash) of feed and fish samples was determined using the AOAC method. Crude protein content was determined by Kjeldahl nitrogen determination. Crude fat content was determined using a Soxhlet extractor. Chromium content in liver tissue was determined by atomic absorption spectrometry according to GB5009.123-2014. Samples were fully mineralized by acid digestion before analysis, and quantitative analysis was performed using a blank control and a standard curve to evaluate the chromium accumulation level in liver tissue.

[0036] 5. Measurement of biochemical and liver oxidative stress related indicators The levels of glucose, triglycerides, acid phosphatase, alkaline phosphatase, creatinine, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in plasma were measured using commercially available kits manufactured by Nanjing Jiancheng Biotechnology Institute (Nanjing, China). The specific procedures were performed according to the kit instructions. The corresponding kit catalog numbers are A154-1-1, A110-1-1, A060-2-1, A059-2-2, C011-2-1, C010-2-1, and C009-2-1, respectively.

[0037] Liver malondialdehyde (MDA), lipid peroxides, superoxide dismutase (SOD) activity, total antioxidant capacity, and nitric oxide (NO) content were measured using corresponding kits from Nanjing Jiancheng Biotechnology Institute (Nanjing, China). The assay procedures were performed according to the manufacturer's instructions. The kit catalog numbers are A003-1-2, A106-1-2, A001-3-2, A015-2-1, and A013-2-1, respectively. Liver reactive oxygen species (ROS) levels were measured using a fish ROS enzyme-linked immunosorbent assay (ELISA) kit (catalog number: KT-86537), purchased from Merck Biotechnology Co., Ltd. (Wuhan, China).

[0038] 6. Liver and intestinal tissue sections Liver and intestinal tissues were fixed in 4% paraformaldehyde for more than 24 hours, and then histologically observed using paraffin embedding and hematoxylin-eosin staining (H&E). After graded ethanol dehydration, xylene clearing, and paraffin embedding, sections with a thickness of 4 μm were prepared. After spreading, mounting, and drying at 60℃, the sections were sequentially dewaxed, rehydrated, stained with hematoxylin for 3–8 min, and stained with eosin for 1–3 min. Following dehydration, clearing, and mounting, the sections were observed under an optical microscope, and six fields of view were randomly selected from each section for image acquisition and analysis.

[0039] 7. Real-time quantitative PCR analysis Total RNA was extracted from the intestinal tissue of crucian carp using TRIzol reagent, and RNA integrity was detected by agarose gel electrophoresis. RNA concentration and purity were determined using a NanoDrop® ND-2000 UV-Vis spectrophotometer. Reverse transcription was then performed using the M-MLV first-strand cDNA synthesis kit (Invitrogen, Shanghai), followed by real-time quantitative PCR on a LightCycle480II system (Roche, Basel, Switzerland). Amplification efficiency was calculated based on a standard curve, and primer amplification efficiency was confirmed to be approximately 100%. Three replicates were performed for each sample. The relative expression levels of target genes were calculated using the Pfaffl (2001) method, and normalization analysis was performed using β-actin as an internal reference gene.

[0040] 8. Western blot analysis Approximately 0.04 g of liver tissue was collected and lysed with RIPA lysis buffer containing protease and phosphatase inhibitors at a ratio of 1:10 (w / v). After homogenization on ice, the mixture was centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was collected. Protein concentration was determined using the BCA method. An equal volume of protein was mixed with 5× loading buffer, boiled for denaturation, separated by SDS-PAGE, and transferred to a PVDF membrane (100 V, 60 min). The PVDF membrane was blocked with TBST containing 5% skim milk powder at room temperature for 1 h, and then Nrf2, p-NF-κB, NF-κB, and TNF-α primary antibodies were added, and the membrane was incubated overnight at 4 °C. The next day, after washing, HRP-labeled secondary antibody was added, and the membrane was incubated at room temperature for 1 h. ECL imaging was used, and images were acquired using a ChemiDocMP imaging system. The gray values ​​of protein bands were analyzed using ImageJ, normalized with β-actin as an internal control, and phosphorylated proteins were corrected for corresponding total protein levels.

[0041] 9. Statistical Analysis SPSS 19.0 was used for statistical analysis, and data are expressed as mean ± standard error (mean ± SEM). Normality and homogeneity of variance were tested before analysis. Two-way ANOVA was used to analyze the effects of strains (CASIII, CASVI), feed treatments (Con, CrPic, CrPic+Res), and their interactions. When interactions were significant, one-way ANOVA combined with Duncan's multiple comparisons was used for inter-group comparisons; when interactions were not significant, only main effect differences were analyzed. The significance level was set at [value missing]. P <0.05, highly significant level is P <0.001.

[0042] 10. Results Analysis After 56 days of continuous feeding with the experimental diet, the diet treatment significantly affected the growth performance of the two strains of hybrid silver carp (Table 2). Compared with the Con group, the specific growth rate and weight gain rate of both strains in the CrPic group were significantly reduced; in the CrPic+Res group, the final body weight, feed efficiency, specific growth rate, and weight gain rate of both strains were significantly increased, while the feed intake rate was significantly reduced. These results indicate that Res supplementation can partially improve the growth decline caused by CrPic treatment. Regarding body morphology, the diet treatment had no significant effect on condition factor and visceral ratio, but the CrPic group significantly reduced the hepatobiliary ratio of both strains. Inter-strain comparison showed that the condition factor and hepatobiliary ratio of "Zhongke No. 3" were significantly higher than those of "Zhongke No. 6", suggesting differences in body morphology between the two strains. Body composition analysis showed that the interaction between strain and treatment affected the crude fat content of the whole fish. The CrPic group significantly increased the crude fat content of "Zhongke No. 3", while the crude fat content of the CrPic+Res group significantly decreased. Regarding strain effects, the crude fat and crude protein content of "Zhongke No. 3" was significantly higher than that of "Zhongke No. 6," while the moisture content was significantly lower. The results indicate that long-term exposure to CrPic in the diet inhibited the growth of hybrid silver carp and affected their nutritional composition; significant differences existed in the responses to CrPic exposure among different strains.

[0043] Table 2. Effects of dietary chromium pyridinecarboxate exposure on growth performance, morphological indicators, and body composition of two strains of hybrid silver carp.

[0044] To assess the impact of CrPic exposure on the metabolic state of the organism, the liver chromium content and plasma biochemical indicators of two strains of hybrid silver carp were measured. Figure 1 The results showed a significant interaction between strain and feed treatment. Compared with the Con group, the CrPic group significantly increased the Cr content in the liver of both strains, indicating that chromium from CrPic accumulates in the liver; while the CrPic+Res group significantly decreased the Cr content in the liver. Figure 1 A). Strain comparisons showed that the liver Cr content of "Zhongke No. 3" was significantly higher than that of "Zhongke No. 6," suggesting a higher accumulation level of CrPic-derived chromium. Plasma biochemistry results showed that in "Zhongke No. 3," compared with the Con group, the CrPic group significantly reduced glucose (GLU) levels and significantly increased creatinine (CRE) content and aspartate aminotransferase (AST) activity; the CrPic+Res group significantly reduced CRE content (…). Figure 1B, D, E). In "Zhongke 6", compared with the Con group, the CrPic group significantly increased GLU levels and decreased AST activity, while the above indicators did not change significantly after CrPic+Res treatment. Triglyceride (TG) level analysis showed that the TG level of "Zhongke 3" was significantly lower than that of "Zhongke 6", and the TG level of the "Zhongke 6" CrPic+Res group was significantly lower than that of the CrPic group (B, D, E). Figure 1 C). Furthermore, the alanine aminotransferase (ALT) activities in both CrPic+Res groups were significantly lower than those in other treatment groups (C). Figure 1 F).

[0045] Histological observations revealed a significant interaction between strain and feed treatment. Compared to the Con group, both strains of hybrid silver carp treated with CrPic showed significant pathological damage in their livers, manifested as hepatocyte vacuolation, deep nuclear staining, and nuclear pyknosis; while the CrPic+Res group exhibited reduced damage characteristics, with a relatively intact liver structure. Figure 2 A). Intestinal tissue analysis showed that, compared with the Con group, the CrPic group significantly reduced the intestinal villus height of both strains ( Figure 2 C), accompanied by disordered villus arrangement, decreased structural integrity, and a reduction in goblet cells and epithelial cell shedding; the morphology of the villus in the CrPic+Res group was significantly improved compared to the CrPic group, with villus arrangement becoming more regular and epithelial shedding reduced ( Figure 2 B). Strain comparisons show that the intestinal villus height of "Zhongke No. 3" is significantly higher than that of "Zhongke No. 6" (B). Figure 2 C) indicates that there are differences between the two strains in terms of intestinal tissue structure.

[0046] Analysis of oxidative stress indicators showed that the interaction between strain and feed treatment significantly affected the content of malondialdehyde (MDA), lipid peroxides (LPO), and superoxide dismutase (SOD) activity in the liver. Compared with the Con group, the CrPic group significantly increased the MDA and LPO content in the liver of "Zhongke No. 3" while significantly decreasing SOD activity; after CrPic+Res treatment, the MDA and LPO content decreased significantly, while the SOD activity increased significantly. Figure 3 A, B, E). In "Zhongke No. 6", no significant changes were observed in the above indicators. Furthermore, compared to the Con group, the CrPic group significantly increased the content of reactive oxygen species (ROS) and nitric oxide (NO) in the livers of both strains, and significantly decreased the total antioxidant capacity (T-AOC). The CrPic+Res group significantly decreased the ROS content and increased the T-AOC level (…). Figure 3C, D, F). Inter-strain comparisons showed that the liver MDA, ROS, and T-AOC levels of "Zhongke No. 3" were significantly higher than those of "Zhongke No. 6", indicating a significant difference in the response of the two strains to CrPic-induced oxidative stress.

[0047] To further analyze the molecular response to CrPic-induced oxidative stress, the expression levels of proteins and genes related to the NRF2 antioxidant pathway were examined. The results showed that the interaction between strain and feed treatment significantly affected... sod , ho-1 and gpx Gene expression. Compared with the Con group, the CrPic group significantly reduced the expression of NRF2 protein in the liver of both strains of hybrid silver carp, while the CrPic+Res group significantly increased its expression level. Figure 4 A, B). At the transcriptional level, compared with the Con group, the CrPic group significantly downregulated the liver function of "Zhongke No. 3". sod and gpx Gene expression, and the CrPic+Res group significantly upregulated it. sod , ho-1 and gpx Gene expression ( Figure 4 E; Figure 5 B, F). In contrast, no significant changes were observed in the aforementioned genes in "Zhongke 6". Furthermore, compared to the Con group, the CrPic group significantly downregulated the levels of these genes in the livers of both strains. nrf2 and bach1 Gene expression was significantly upregulated. keap1 Gene expression was significantly upregulated in the CrPic+Res group. nrf2 , nqo-1 and prx2 Gene expression and downregulation keap1 and bach1 Gene expression ( Figure 4 C,D; Figure 5 A, C, D). Inter-strain comparisons show that the liver of "Zhongke No. 3" contains... sod , ho-1 , bach1 and gpx The gene expression levels were significantly higher than those of "Zhongke 6". These results indicate that there are significant differences in the transcriptional response of the two lines to CrPic-induced oxidative stress.

[0048] On the basis of oxidative stress, CrPic exposure further induced a hepatic inflammatory response. Results showed that the interaction between strain and feed treatment significantly affected p-NF-κBp65 protein and... il-6 and il-4Gene expression. Compared with the Con group, the CrPic group significantly increased the expression of P-NF-κBp65 protein in the liver of both strains of hybrid silver carp, while the CrPic+Res group significantly decreased the expression of P-NF-κBp65 and TNF-α proteins. Figure 6 (AC). At the transcriptional level, compared with the Con group, the CrPic group significantly upregulated "Zhongke No. 3" liver enzymes. il-6 Gene expression, and the CrPic+Res group significantly upregulated it. il-4 Gene expression. In contrast, in "Zhongke 6", the CrPic group significantly downregulated [gene expression]. il-4 Gene expression was not significantly affected in the CrPic+Res group. Furthermore, compared to the Con group, the CrPic group significantly upregulated pro-inflammatory factors in the livers of both strains. il-1β and il-12 Gene expression, and the CrPic+Res group significantly increased anti-inflammatory factors. tgf-β Gene expression ( Figure 6 DH). Inter-strain comparisons showed that the P-NF-κBp65 protein in the liver of "Zhongke No. 3" and il-6 Gene expression levels in "Zhongke No. 3" were significantly higher than in "Zhongke No. 6", while TNF-α protein expression was significantly lower. Plasma inflammatory factor results showed that the interaction between strain and feed treatment significantly affected TNF-α, IL-6, and IL-1β levels. Compared with the Con group, the CrPic group significantly increased the plasma TNF-α, IL-6, and IL-1β levels in "Zhongke No. 3", while the CrPic+Res group significantly decreased these indicators; no significant changes were observed in "Zhongke No. 6". Furthermore, the plasma TNF-α, IL-6, and IL-1β levels in "Zhongke No. 3" were significantly higher than those in "Zhongke No. 6". Figure 7 AC).

[0049] Although the present invention has been illustrated and described with specific embodiments, it should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and scope of the present invention; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of resveratrol in the preparation of products for alleviating chromium pyridinecarboxylate-induced liver injury in fish.

2. The application according to claim 1, characterized in that, The fish species mentioned include: hybrid silver carp.

3. The application according to claim 2, characterized in that, The heterozygous silver carp includes: Zhongke No. 3 and / or Zhongke No. 6 strains.

4. The application according to claim 1, characterized in that, The relief of chromium pyridinecarboxylate-induced liver injury in fish includes at least one of the following effects: (1) Reduces the accumulation level of chromium in the liver; (2) Reduce liver oxidative stress levels; (3) Increase the activity of liver antioxidant enzymes; (4) Inhibits liver inflammatory response; (5) Reduce liver tissue structure damage.

5. The application according to claim 1, characterized in that, The relief of chromium pyridinecarboxylate-induced liver injury in fish is achieved by activating the NRF2 antioxidant pathway and / or inhibiting the NF-κB inflammatory pathway.

6. The application according to claim 1, characterized in that, The resveratrol content in the product is 0.1wt%~0.2wt%.

7. The application according to claim 1, characterized in that, The products include: feed additives.

8. Application of resveratrol in the preparation of formulations for alleviating chromium pyridinecarboxylate-induced intestinal tissue structure damage in fish.

9. A feed for alleviating chromium pyridinecarboxylate-induced liver damage in fish, characterized in that, The feed contains resveratrol.

10. Application of resveratrol in the preparation of feed additives for improving chromium pyridinecarboxylate-induced growth inhibition in fish and / or improving feed efficiency.

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