Method for evaluating copper exposure hepatotoxicity based on endoplasmic reticulum stress-endoplasmic reticulum autophagy axis and application thereof

Abstract

This invention relates to the field of livestock and poultry breeding and food safety testing technology, and in particular to a method for assessing copper exposure hepatotoxicity based on the endoplasmic reticulum stress-endoplasmic reticulum autophagy axis and its application. This method obtains liver tissue samples or in vitro cultured hepatocyte samples from copper-exposed subjects, and jointly detects the expression levels of key markers of the endoplasmic reticulum stress pathway and characteristic markers of the endoplasmic reticulum autophagy pathway. Combined with two-way pharmacological validation, a grading assessment standard for copper exposure hepatotoxicity is established. This invention overcomes the shortcomings of traditional liver enzyme indicators, such as low sensitivity and strong lag, and has the advantages of early warning, high specificity, and reliable results. It can be widely applied to scenarios such as determining the copper safety threshold in livestock and poultry feed, early screening for copper exposure hepatotoxicity, assessment of copper pollution ecological risks, and screening of copper toxicity protectants, providing technical support for ensuring animal health and the safety of animal-derived food.

Description

Technical Field

[0001] This invention relates to the field of livestock and poultry breeding and food safety testing technology, and in particular to a method for assessing the hepatotoxicity of copper exposure based on the endoplasmic reticulum stress-endoplasmic reticulum autophagy axis and its application. Background Technology

[0002] Copper is an essential trace element for animals, but it is often excessively added to livestock feed in modern agricultural production (e.g., 100-250 mg / kg of copper is added to piglet diets) to promote growth and reduce diarrhea rates. Excessive copper exposure can lead to damage to organs such as the liver, and copper can migrate along the food chain, posing a dual threat to animal health and human food safety. Currently, countries have gradually tightened limits on copper addition to feed, but existing methods for assessing the hepatotoxicity of copper exposure still have significant shortcomings.

[0003] Traditional assessment methods primarily rely on serum ALT, AST, and other liver enzyme indicators, as well as observation of liver tissue pathological sections. However, these indicators have significant lag effects: elevated liver enzyme activity usually occurs after significant hepatocyte necrosis, failing to provide early warning; and pathological section observation is highly subjective, making it difficult to quantify mild toxic damage. Furthermore, existing molecular-level assessments often focus on single pathways (such as detecting only endoplasmic reticulum stress or single autophagy markers), neglecting the synergistic regulatory relationship of the "endoplasmic reticulum stress-endoplasmic reticulum autophagy axis" in copper toxicity. This results in insufficient accuracy and specificity of the assessment results, making them susceptible to interference from other stress factors.

[0004] Existing research has confirmed that copper overload can induce endoplasmic reticulum stress and autophagy, but a systematic assessment method based on their synergistic effects has not yet been established, and there is a lack of standardized procedures for verifying causality through pharmacological means. This fails to provide reliable technical support for setting dietary copper safety thresholds and early toxicity screening. Therefore, developing a copper exposure hepatotoxicity assessment method based on "axis" regulatory characteristics, possessing both high sensitivity and specificity, has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] To overcome the aforementioned deficiencies in the prior art, this invention provides a method for assessing the hepatotoxicity of copper exposure based on the endoplasmic reticulum stress-endoplasmic reticulum autophagy axis and its application, in order to solve the problems existing in the background art.

[0006] This invention provides the following technical solution: a method for assessing the hepatotoxicity of copper exposure based on the endoplasmic reticulum stress-endoplasmic reticulum autophagy axis, comprising the following steps: Step (1) Sample acquisition: Obtain liver tissue samples and serum samples from the subjects to be evaluated, or construct an in vitro copper-exposed porcine liver cell model and collect processed liver cell samples; Step (2) Biomarker detection: Detect the expression levels of endoplasmic reticulum stress pathway biomarkers and endoplasmic reticulum autophagy pathway biomarkers in the sample; the endoplasmic reticulum stress pathway biomarkers include at least three of GRP78, PERK, eIF2α, ATF6, and CHOP; the endoplasmic reticulum autophagy pathway biomarkers include at least three of FAM134B, CCPG1, SEC62, RTN3, and the LC3-II / LC3-I ratio; Step (3) Pharmacological verification: In vitro hepatocyte samples or in vivo models were grouped into control group, copper exposure group, copper + endoplasmic reticulum stress inhibitor (4-PBA) group, and copper + endoplasmic reticulum stress inducer (PR-619) group. The expression differences of the above markers in each group were detected to verify the causal relationship between endoplasmic reticulum stress and endoplasmic reticulum autophagy. Step (4) Hepatotoxicity determination: Based on the biomarker expression level in step (2) and the verification results in step (3), combined with the preset grading criteria, determine the degree of hepatotoxicity caused by copper exposure.

[0007] Furthermore, in step (1), the object to be evaluated is livestock and poultry, preferably weaned piglets; the copper exposure concentration of the in vitro copper-exposed pig liver cell model is 180~360μM, and the exposure time is 24h; the concentration of the endoplasmic reticulum stress inhibitor 4-PBA is 4mM, and the concentration of the endoplasmic reticulum stress inducer PR-619 is 3μM.

[0008] Furthermore, in step (2), the method for detecting the expression level of the biomarker is selected from at least one of Western blot, reverse transcription quantitative PCR (RT-qPCR), immunohistochemistry (IHC), and immunofluorescence (IF); wherein, the protein level is detected by Western blot or immunofluorescence, and the mRNA level is detected by RT-qPCR.

[0009] Furthermore, the grading criteria in step (4) are as follows: No hepatotoxicity: The expression levels of endoplasmic reticulum stress markers and endoplasmic reticulum autophagy markers were not significantly different from those in the control group (P≥0.05), and the markers showed no significant changes after pharmacological verification; Mild hepatotoxicity: The expression levels of endoplasmic reticulum stress markers (GRP78, PERK) and autophagy markers (FAM134B, LC3-II / LC3-I) were 1.5 to 2.5 times higher than those in the control group (P<0.05), and the expression of these markers decreased by ≥30% after 4-PBA treatment; Moderate hepatotoxicity: The expression levels of endoplasmic reticulum stress markers (GRP78, PERK, ATF6, CHOP) and autophagy markers (FAM134B, CCPG1, LC3-II / LC3-I) were 2.5 to 4 times higher than those in the control group (P<0.01). After PR-619 treatment, the expression of markers increased by ≥50%, and mild inflammatory infiltration was observed in the liver tissue. Severe hepatotoxicity: The expression levels of all detected biomarkers were ≥4 times higher than those in the control group (P<0.001), liver tissue showed fibrosis and petechial hemorrhage, and serum ALT and AST enzyme activities were significantly increased.

[0010] Application of any of the assessment methods described above in early screening for hepatotoxicity caused by copper exposure in livestock and poultry.

[0011] The application of any of the above-described assessment methods in determining the safe threshold for copper addition in livestock and poultry feed involves detecting the activation threshold of "endoplasmic reticulum stress-endoplasmic reticulum autophagy axis" markers in liver tissue under different copper addition doses to determine the maximum safe amount of copper added to the feed.

[0012] The application of any of the above-described assessment methods in the risk assessment of hepatotoxicity from copper pollution in agricultural ecosystems involves assessing the toxic risk of copper migration along the soil-crop-animal food chain by detecting the expression levels of liver tissue markers in livestock and poultry in copper-polluted areas.

[0013] According to the application of any of the above-described evaluation methods in the screening of copper toxicity protectants, candidate protectants are added to copper-exposed hepatocyte models or in vivo models, and protectants that can inhibit the abnormal activation of the "endoplasmic reticulum stress-endoplasmic reticulum autophagy axis" markers are screened by detecting changes in the expression of markers of the "endoplasmic reticulum stress-endoplasmic reticulum autophagy axis".

[0014] The technical effects and advantages of this invention are as follows: The copper exposure hepatotoxicity assessment method based on the endoplasmic reticulum stress-endoplasmic reticulum autophagy axis described in this patent effectively overcomes many shortcomings of traditional assessment methods. Firstly, it uses the endoplasmic reticulum stress-endoplasmic reticulum autophagy axis as the core assessment target. The activation of molecular markers at this target precedes abnormal liver enzyme levels and significant pathological damage to liver tissue, enabling early identification of copper exposure hepatotoxicity risk and solving the problems of lagging and inability to warn of mild toxicity in traditional methods. Secondly, by jointly detecting multiple key markers of the two pathways and combining endoplasmic reticulum stress inhibitors and inducers for bidirectional pharmacological validation, the causal regulatory relationship between the two is clarified, effectively eliminating interference from non-copper exposure-related stress factors and avoiding the one-sidedness of single indicator or pathway detection, significantly improving the specificity and accuracy of the assessment results. Thirdly, relying on Western spectroscopy... Mature quantitative detection technologies such as blot and RT-qPCR, combined with quantitative indicators such as biomarker expression fold and ratio, establish standardized hepatotoxicity grading standards, replacing the subjective qualitative analysis of traditional pathological observation. This achieves objective quantitative determination of the degree of copper exposure hepatotoxicity, making the assessment results more comparable. Fourth, the sample acquisition, biomarker detection, and pharmacological verification involved in the method are all routine and mature technologies in the fields of biodetection and livestock physiological research. No special equipment is required, the process can be standardized, and it is highly practical, making it easy for those skilled in the art to master and promote. Fifth, this method also expands the application scenarios of copper exposure-related assessments. It can not only be used for early screening of hepatotoxicity from copper exposure in livestock and poultry, but also provide reliable technical basis for the scientific determination of safe thresholds for copper addition in livestock and poultry feed, the risk assessment of hepatotoxicity from copper migration along the food chain in agricultural ecosystems, and the efficient screening of copper toxicity protectants. This provides practical technical support for the scientific management of copper addition in livestock and poultry farming, the safety assurance of animal-derived food, and the risk prevention and control of copper pollution in agriculture. Attached Figure Description

[0015] Figure 1 This is a flowchart of the method. Detailed Implementation

[0016] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0017] Example 1: Construction and biomarker detection of an in vitro copper-exposed porcine hepatocyte model 1. Cell Culture and Processing Primary hepatocytes from healthy weaned piglets or immortalized porcine hepatocyte cell lines (such as PICM-19, hTERT-PNH, or HepLiu) were selected and seeded into 6-well plates. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5%... Incubate in an incubator until 80% confluence is achieved.

[0018] A control group (without copper), and low, medium, and high dose copper exposure groups (final concentrations of 180, 270, and 360 μM, respectively) were set up, with 3 replicates in each group and a treatment time of 24 h.

[0019] 2. Biomarker detection Cells were collected, and total protein and total RNA were extracted. Western blot analysis: Protein was extracted using RIPA lysis buffer, and protein concentration was determined by the BCA method. 30 μg of protein was loaded into each well, separated by SDS-PAGE electrophoresis, transferred to a membrane, blocked, and incubated overnight at 4°C with primary antibodies (GRP78, PERK, eIF2α, ATF6, CHOP, FAM134B, CCPG1, SEC62, RTN3, and LC3-II / I, diluted 1:1000). The next day, the membrane was incubated with secondary antibody, ECL was applied, and the band grayscale values ​​were analyzed using ImageJ software, with GAPDH as an internal control.

[0020] RT-qPCR assay: Total RNA was extracted using the Trizol method, reverse transcribed into cDNA, and the mRNA expression levels of the above biomarkers were detected using the SYBR Green assay, with β-actin as an internal control. The relative expression level is calculated using this method.

[0021] 3. Results Analysis The results showed that, compared with the control group, the levels of GRP78, PERK, ATF6, CHOP, FAM134B, CCPG1, and LC3-II / I ratio were significantly increased in the copper-exposed group (P<0.05), and this increase was dose-dependent, indicating that copper exposure activated the endoplasmic reticulum stress and autophagy pathways. Specific data are shown in Table 1.

[0022]

[0023] Table 1. Relative expression levels of biomarker proteins after treatment with different copper concentrations (fold change, Mean ± SD, n=3) Example 2: Two-way pharmacological validation experiment. To further verify the causal regulatory relationship between endoplasmic reticulum stress and endoplasmic reticulum autophagy, the following groups were set up: Control group, copper exposure group (270 μM), copper + endoplasmic reticulum stress inhibitor group (4-PBA, 4 mM), copper + endoplasmic reticulum stress inducer group (PR-619, 3 μM) Changes in biomarker expression were detected 24 hours after treatment in each group.

[0024] Results: After 4-PBA treatment, the levels of GRP78, PERK, CHOP, FAM134B, and the LC3-II / I ratio were significantly lower than those in the copper exposure group (reduction ≥30%), indicating that inhibiting endoplasmic reticulum stress can attenuate endoplasmic reticulum autophagy. After PR-619 treatment, the expression of the above markers further increased (increase ≥50%), indicating that inducing endoplasmic reticulum stress can enhance endoplasmic reticulum autophagy. These results confirm that copper exposure activates endoplasmic reticulum autophagy through endoplasmic reticulum stress, constituting an "endoplasmic reticulum stress-endoplasmic reticulum autophagy axis" regulatory mechanism. Specific data are shown in Table 2.

[0025]

[0026] Table 2. Relative expression levels of biomarker proteins after pharmacological intervention (fold change, Mean ± SD, n=3) Example 3: Construction of an in vivo copper exposure model and hepatotoxicity grading Weaned piglets aged 28 days were randomly divided into 4 groups of 6 piglets each. They were fed a basal diet (copper content 10 mg / kg) and diets with different amounts of copper (100, 200, 300 mg / kg) for 28 consecutive days.

[0027] After the experiment, serum was collected to detect ALT and AST activities, liver tissue was taken for HE staining to observe pathological changes, and the expression of the above markers was detected.

[0028] Grading criteria: No hepatotoxicity: Marker expression was not significantly different from the control group (P ≥ 0.05), liver tissue was normal, and ALT / AST was normal. Mild hepatotoxicity: GRP78, PERK, FAM134B, and LC3-II / I increased by 1.5 to 2.5 times (P<0.05), and markers decreased by ≥30% after 4-PBA treatment, with no obvious liver lesions. Moderate hepatotoxicity: GRP78, PERK, ATF6, CHOP, FAM134B, CCPG1, and LC3-II / I increased by 2.5 to 4 times (P<0.01), and increased by ≥50% after PR-619 treatment, with mild inflammatory infiltration in liver tissue. Severe hepatotoxicity: All markers increased by ≥4 times (P<0.001), liver tissue showed fibrosis and petechial hemorrhage, and ALT / AST was significantly elevated.

[0029] See Tables 3 and 4 for specific data.

[0030]

[0031] Table 3. Relative expression levels of biomarker proteins in piglet liver tissue under different copper addition levels.

[0032] Table 4. Serum liver enzyme activity and pathological scores of piglets under different copper addition levels. Example 4: Application of determining the safe copper threshold in feed Using the method described in Example 3, multiple copper supplementation doses (0, 50, 100, 150, 200, 250, 300 mg / kg) were set, and changes in biomarker expression in liver tissue were detected. The copper supplementation level corresponding to the first significant increase in biomarkers (P<0.05) was used as the "activation threshold," and the maximum safe supplementation level was determined in conjunction with pathological changes. The results of this experiment show that when the copper supplementation level is ≥150 mg / kg, biomarkers such as GRP78 and FAM134B are significantly increased, suggesting that the safe threshold for copper in feed should be ≤150 mg / kg.

[0033]

[0034] Table 5. Dose points at which biomarkers first showed significant increases under different copper addition levels. Example 5: Risk assessment of hepatotoxicity in copper-contaminated areas Liver tissue was collected from pigs in a copper-contaminated area, and the expression of the aforementioned biomarkers was detected and compared with that of pigs in a clean area. The results showed that the expression of biomarkers in pigs in the contaminated area was significantly increased (P<0.01), suggesting that copper migration along the "soil-crop-animal" food chain has posed a toxic risk to animal livers.

[0035]

[0036] Table 6 Comparison of biomarker expression in liver tissue of pigs from clean and copper-contaminated areas. (Multiple change, Mean ± SD, n=10) Example 6 Application of copper toxicity protectants in screening Candidate protective agents (such as NAC, vitamin E, curcumin, etc.) were added to an in vitro copper exposure model (270 μM), and changes in biomarker expression were detected.

[0037] Screening criteria: Patients with a ≥40% reduction in biomarker expression compared to the copper-exposed group and significant restoration of cell viability are considered to have protective potential.

[0038] Experimental results showed that curcumin (20 μM) could significantly inhibit copper-induced increases in GRP78, FAM134B, and LC3-II / I, suggesting that it may exert a protective effect by regulating the endoplasmic reticulum stress-endoplasmic reticulum autophagy axis.

[0039] Table 7 Effects of different protective agents on the expression of copper-exposed hepatocyte markers (fold change, Mean ± SD, n=3)

[0040] The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for assessing the hepatotoxicity of copper exposure based on the endoplasmic reticulum stress-endoplasmic reticulum autophagy axis, characterized in that, Includes the following steps: Step (1) Sample acquisition: Obtain liver tissue samples and serum samples from the subjects to be evaluated, or construct an in vitro copper-exposed porcine liver cell model and collect processed liver cell samples; Step (2) Biomarker detection: Detect the expression levels of endoplasmic reticulum stress pathway biomarkers and endoplasmic reticulum autophagy pathway biomarkers in the sample; the endoplasmic reticulum stress pathway biomarkers include at least three of GRP78, PERK, eIF2α, ATF6, and CHOP; the endoplasmic reticulum autophagy pathway biomarkers include at least three of FAM134B, CCPG1, SEC62, RTN3, and the LC3-II / LC3-I ratio; Step (3) Pharmacological verification: In vitro hepatocyte samples or in vivo models were grouped into control group, copper exposure group, copper + endoplasmic reticulum stress inhibitor group, and copper + endoplasmic reticulum stress inducer group. The expression differences of the above markers in each group were detected to verify the causal relationship between endoplasmic reticulum stress and endoplasmic reticulum autophagy. Step (4) Hepatotoxicity determination: Based on the biomarker expression level in step (2) and the verification results in step (3), combined with the preset grading criteria, determine the degree of hepatotoxicity caused by copper exposure.

2. The evaluation method according to claim 1, characterized in that, In step (1), the subject to be evaluated is livestock and poultry, preferably weaned piglets; the copper exposure concentration of the in vitro copper-exposed pig liver cell model is 180~360μM, and the exposure time is 24h; the concentration of the endoplasmic reticulum stress inhibitor 4-PBA is 4mM, and the concentration of the endoplasmic reticulum stress inducer PR-619 is 3μM.

3. The evaluation method according to claim 1, characterized in that, In step (2), the method for detecting the expression level of the biomarker is selected from at least one of Western blot, reverse transcription quantitative PCR, immunohistochemistry, and immunofluorescence; among which, the protein level is detected by Western blot or immunofluorescence, and the mRNA level is detected by RT-qPCR.

4. The evaluation method according to claim 1, characterized in that, The grading criteria in step (4) are: No hepatotoxicity: The expression levels of endoplasmic reticulum stress markers and endoplasmic reticulum autophagy markers were not significantly different from those in the control group, and the markers showed no significant changes after pharmacological verification; Mild hepatotoxicity: The expression levels of endoplasmic reticulum stress markers and autophagy markers were 1.5 to 2.5 times higher than those in the control group, and the expression of these markers decreased by ≥30% after 4-PBA treatment; Moderate hepatotoxicity: The expression levels of endoplasmic reticulum stress markers and autophagy markers were 2.5 to 4 times higher than those in the control group. After PR-619 treatment, the expression of markers increased by ≥50%, and mild inflammatory infiltration was observed in the liver tissue. Severe hepatotoxicity: The expression levels of all detected biomarkers were ≥4 times higher than those in the control group, liver tissue showed fibrosis and petechial hemorrhage, and serum ALT and AST enzyme activities were significantly increased.

5. The application of the assessment method according to any one of claims 1 to 4 in the early screening of liver toxicity caused by copper exposure in livestock and poultry.

6. The application of the evaluation method according to any one of claims 1 to 4 in determining the safety threshold for copper addition in livestock and poultry feed, characterized in that, The maximum safe amount of copper in feed was determined by detecting the activation threshold of "endoplasmic reticulum stress-endoplasmic reticulum autophagy axis" markers in liver tissue under different copper addition doses.

7. The application of the assessment method according to any one of claims 1 to 4 in the risk assessment of hepatotoxicity from copper pollution in agricultural ecosystems, characterized in that, By detecting the expression levels of liver tissue markers in livestock and poultry in copper-contaminated areas, the toxic risk of copper migration along the "soil-crop-animal" food chain can be assessed.

8. The application of the evaluation method according to any one of claims 1 to 4 in the screening of copper toxicity protective agents, characterized in that, Candidate protective agents were added to copper-exposed hepatocyte models or in vivo models, and protective agents that could inhibit the abnormal activation of the "endoplasmic reticulum stress-endoplasmic reticulum autophagy axis" were screened by detecting changes in the expression of markers of the "endoplasmic reticulum stress-endoplasmic reticulum autophagy axis".

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