Method for assessing metabolic toxicity of pollutants to mammals

Abstract

The application discloses a method for evaluating metabolic toxicity of pollutants on mammals, which comprises: feeding mammals in an exposure group and mammals in a control group respectively, the mammals in the exposure group are pre-administered with pollutants, and the mammals in the control group are not administered with the pollutants; obtaining tissue sections at the same position of the mammals in the exposure group and the control group respectively after the feeding; performing MALDI-TOF / TOF mass spectrum imaging analysis on the tissue sections to obtain tissue section mass spectrum imaging maps; screening characteristic differential substances between the exposure group and the control group according to the mass spectrum imaging maps; and performing metabolic pathway enrichment analysis on the characteristic differential substances. The method has the advantages of high throughput, high sensitivity and in-situ analysis, and provides a new technical means for evaluating health risks of emerging pollutants.

Description

Technical Field

[0001] This application relates to the fields of environmental toxicology and spatial metabolomics, specifically to a method for assessing the metabolic toxicity of pollutants to mammals. Background Technology

[0002] Emerging contaminants (ECs) are a class of chemical substances that are not yet widely included in environmental regulations but may pose a threat to ecosystems and human health. These pollutants mainly include pharmaceuticals and their metabolites, personal care products, industrial additives, agricultural chemicals and their degradation products. ECs are characterized by complexity, high risk at low concentrations, persistence, and bioaccumulation. While their concentrations in the environment are often low (ng / L to μg / L), they can have profound impacts on the metabolic function and health of mammals through the food chain, bioaccumulation, and long-term exposure.

[0003] Therefore, the metabolic toxicity of emerging pollutants to mammals still needs further investigation. Summary of the Invention

[0004] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a method for assessing the metabolic toxicity of pollutants to mammals. The method of this application combines matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) for in-situ metabolic analysis of mouse organ sections. Statistical processing of metabolite levels and enrichment analysis of metabolic pathways can clearly reflect the multi-organ metabolic toxicity of pollutants to mice at the molecular level. The method of this application has the advantages of high throughput, high sensitivity, and in-situ analysis capability, providing a new technical means for assessing the health risks of emerging pollutants.

[0005] In a first aspect of this application, a method for assessing the metabolic toxicity of a contaminant to mammals is proposed. The method includes: feeding mammals in an exposure group and a control group, respectively, wherein the mammals in the exposure group are pre-treated with the contaminant, and the mammals in the control group are not treated with the contaminant; obtaining tissue sections from the same location in both the exposed and control mammals after feeding; performing matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MADS) on the tissue sections to obtain tissue section mass spectrometry images; screening for characteristic differential substances between the exposed and control groups based on the mass spectrometry images; and performing metabolic pathway enrichment analysis on the characteristic differential substances.

[0006] Therefore, by using high-sensitivity and high-throughput mass spectrometry imaging technology, in-situ, label-free analysis of metabolite changes in mammalian tissues after pollutant exposure can be achieved, and metabolic pathway enrichment analysis can be performed. This allows for a direct reflection of the impact of pollutants on the metabolism of multiple organs in mammals at the molecular level, providing a new technical means for assessing the health risks of emerging pollutants.

[0007] According to embodiments of this application, the above method may also have at least one of the following additional technical features:

[0008] According to an embodiment of this application, the contaminant includes 6PPD-Q.

[0009] According to embodiments of this application, the mammals include mice, rats, guinea pigs, rabbits, and non-human primates.

[0010] According to embodiments of this application, the tissues include the heart, liver, spleen, lungs, kidneys, and testes.

[0011] According to an embodiment of this application, the thickness of the tissue section is 5–20 μm; the tissue section is frozen using liquid nitrogen.

[0012] According to an embodiment of this application, before performing the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry imaging analysis, the tissue sections are pre-treated with matrix spraying.

[0013] According to an embodiment of this application, the matrix solution for the matrix spraying treatment is a 1,5-diaminonaphthalene solution.

[0014] According to the embodiments of this application, the concentration of 1,5-diaminonaphthalene in the 1,5-diaminonaphthalene solution is 2 to 10 mg / mL.

[0015] According to an embodiment of this application, the 1,5-diaminonaphthalene solution comprises 1,5-diaminonaphthalene, hydrochloric acid, ethanol and water, wherein the volume ratio of hydrochloric acid, ethanol and water is 1:(2-6):(10-16).

[0016] According to an embodiment of this application, the detection mode used in the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry imaging analysis is negative ion mode.

[0017] According to an embodiment of this application, the molecular weight range of the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry imaging analysis is 0–1000 Da.

[0018] According to an embodiment of this application, the metabolic pathway enrichment analysis is performed using the KEGG database.

[0019] In a second aspect of this application, a method is proposed for constructing an animal model of abnormal metabolic pathways, wherein the metabolic pathways include at least one of the following: the ascorbic acid synthesis pathway in the liver; the tricarboxylic acid cycle pathway, the aspartate shuttle pathway, and the glutamate metabolism pathway in the liver; the glutathione metabolism pathway in the red pulp of the spleen; and the glutathione metabolism pathway in the testes. The method comprises administering 6PPD-Q to the test animal.

[0020] According to embodiments of this application, the abnormal metabolic pathway includes at least one of the following: upregulation of the ascorbic acid synthesis pathway in the liver; upregulation of the tricarboxylic acid cycle pathway, aspartate shuttle pathway, and glutamate metabolism pathway in the liver; upregulation of the glutathione metabolism pathway in the red pulp of the spleen; and upregulation of the glutathione metabolism pathway in the testes.

[0021] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0022] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0023] Figure 1 The mass spectrometry imaging results of glutathione in the liver and testes obtained in Example 2 of the present invention;

[0024] Figure 2 The above describes the spleen mass spectrometry imaging results obtained in Example 2 of the present invention. Figure 2 (a) shows the division between the white pith and red pith regions; Figure 2 (b) shows the mass spectrometry signal intensity of the metabolite in 2(a);

[0025] Figure 3 The results of metabolic pathway enrichment in the liver obtained in Example 3 of the present invention;

[0026] Figure 4 This refers to the changes and distribution of metabolites in important metabolic pathways in the liver obtained in Example 3 of the present invention, wherein... Figure 4 (a) is the ascorbic acid synthesis pathway; Figure 4 (b) represents the TCA cycle-related pathway;

[0027] Figure 5 The results of metabolic pathway enrichment in the testes obtained in Example 3 of this invention;

[0028] Figure 6 This is the result of metabolic pathway enrichment in the white and red pulp regions of the spleen obtained in Example 3 of the present invention. Detailed Implementation

[0029] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0030] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0031] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0032] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0033] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0034] Terms and Definitions

[0035] In this article, the term "emerging pollutants" refers to chemical substances that have not yet been widely incorporated into environmental regulations but have attracted attention and may pose a potential threat to ecosystems and human health. These pollutants are typically characterized by complexity, high risk at low concentrations, persistence, and bioaccumulation, and commonly include pharmaceuticals and their metabolites, personal care products, industrial additives, agricultural chemicals and their degradation products, etc.

[0036] In this paper, the term "metabolic pathway enrichment analysis" is a bioinformatics analysis method used to identify the biochemical pathways involved in metabolites that change significantly under specific conditions.

[0037] In this paper, the term "MALDI-TOF / TOF" refers to matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, a high-resolution mass spectrometry technique that uses a laser to desorb and ionize molecules in a sample from a matrix, and then uses a time-of-flight mass spectrometer to measure the flight time of the ions to determine their mass.

[0038] In this article, the term "KEGG database" refers to a comprehensive bioinformatics database designed to integrate information on genomes, biological pathways, diseases, chemicals, and chemical substances.

[0039] In this paper, the term "1,5-DAN" refers to 1,5-diaminonaphthalene, an organic compound used as a matrix material in matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / TOF) analysis.

[0040] Methods for assessing the metabolic toxicity of pollutants to mammals

[0041] In a first aspect of this application, a method for assessing the metabolic toxicity of a contaminant to mammals is proposed. The method includes: feeding mammals in an exposure group and a control group, respectively, wherein the mammals in the exposure group are pre-treated with the contaminant, and the mammals in the control group are not treated with the contaminant; obtaining tissue sections from the same location in both the exposed and control mammals after feeding; performing matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MADS) on the tissue sections to obtain tissue section mass spectrometry images; screening for characteristic differential substances between the exposed and control groups based on the mass spectrometry images; and performing metabolic pathway enrichment analysis on the characteristic differential substances.

[0042] According to embodiments of this application, the contaminant includes 6PPD-Q. Therefore, 6PPD-Q is an emerging contaminant whose metabolic toxicity to mammals has not been fully studied. Using 6PPD-Q as a contaminant, its specific effects on mouse metabolism can be assessed through mass spectrometry imaging analysis and metabolic pathway enrichment analysis, thereby revealing the potential toxic mechanism of 6PPD-Q on mammalian metabolism.

[0043] According to embodiments of this application, the mammals include mice, rats, guinea pigs, rabbits, and non-human primates.

[0044] According to embodiments of this application, the tissues include the heart, liver, spleen, lungs, kidneys, and testes. Therefore, by analyzing these vital tissues, the effects of pollutants on the metabolism of multiple organs in mammals can be comprehensively assessed, thereby revealing the toxicity and potential health risks of the pollutants more accurately.

[0045] According to an embodiment of this application, the thickness of the tissue section is 5–20 μm; the tissue section is frozen using liquid nitrogen. Therefore, a thickness range of 5–20 μm is chosen to ensure that the section is thin enough for high-resolution mass spectrometry imaging while avoiding the fragility and handling difficulties caused by excessively thin sections.

[0046] According to an embodiment of this application, before performing the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry imaging analysis, the tissue sections are pre-treated with a matrix coating. This matrix coating treatment enhances the signal intensity of metabolites, improves the sensitivity and resolution of mass spectrometry imaging, and thus ensures the accuracy and reliability of subsequent analyses.

[0047] According to embodiments of this application, the matrix solution used for matrix spraying is a 1,5-diaminonaphthalene solution. This improves the sensitivity and resolution of detection in mass spectrometry imaging analysis. Furthermore, 1,5-diaminonaphthalene does not chemically react with the target pollutant, thus avoiding interference with detection.

[0048] According to embodiments of this application, the concentration of 1,5-diaminonaphthalene in the 1,5-diaminonaphthalene solution is 2–10 mg / mL. This further improves the sensitivity and resolution of detection in mass spectrometry imaging analysis.

[0049] According to an embodiment of this application, the 1,5-diaminonaphthalene solution comprises 1,5-diaminonaphthalene, hydrochloric acid, ethanol, and water, wherein the volume ratio of hydrochloric acid, ethanol, and water is 1:(2-6):(10-16). Thus, hydrochloric acid facilitates the dissolution and stability of 1,5-diaminonaphthalene, ethanol regulates the volatility of the solution and the uniformity of matrix distribution, and water serves as the base solvent. By precisely controlling the proportions of each component, the performance of the matrix solution is optimized to improve the sensitivity and resolution of mass spectrometry imaging analysis.

[0050] According to embodiments of this application, the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry imaging analysis employs a negative ion mode. This improves the sensitivity and accuracy of detecting specific metabolites.

[0051] According to embodiments of this application, the molecular weight range of the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry imaging analysis is 0–1000 Da. This molecular weight range thus covers most metabolites associated with pollutant exposure, enabling a comprehensive analysis of the effects of pollutants on mammalian metabolism.

[0052] According to embodiments of this application, the metabolic pathway enrichment analysis is performed using the KEGG database. Therefore, by utilizing the rich biological metabolic pathway information in the KEGG database, a systematic analysis and classification of the screened differentially characterized substances can reveal how pollutants interfere with normal metabolic processes, leading to metabolic pathway dysregulation.

[0053] Methods for constructing animal models of metabolic pathway abnormalities

[0054] In a second aspect, this application proposes a method for constructing an animal model of abnormal metabolic pathways, wherein the metabolic pathways include at least one of the following: the ascorbic acid synthesis pathway in the liver; the tricarboxylic acid cycle pathway, the aspartate shuttle pathway, and the glutamate metabolism pathway in the liver; the glutathione metabolism pathway in the red pulp of the spleen; and the glutathione metabolism pathway in the testes. The method comprises administering 6PPD-Q to the test animal. Thus,

[0055] According to embodiments of this application, the abnormal metabolic pathway includes at least one of the following: upregulation of the ascorbic acid synthesis pathway in the liver; upregulation of the tricarboxylic acid cycle pathway, aspartate shuttle pathway, and glutamate metabolism pathway in the liver; upregulation of the glutathione metabolism pathway in the red pulp of the spleen; and upregulation of the glutathione metabolism pathway in the testes.

[0056] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0057] Example 1

[0058] A 6PPD-Q exposure mouse model was established using 10 six-week-old Balb / c male mice, which were randomly divided into two groups. Animal experiments were conducted according to the NIH guidelines for the care and use of laboratory animals. For the exposure group, 6PPD-Q was dispersed in olive oil and administered intraperitoneally at a dose of 4 mg / kg (400 μL) every 4 days for 28 days. For the control group, 400 μL of olive oil was injected intraperitoneally at the same frequency. The mice were euthanized by cervical dislocation, and their spleen, testes, and liver were dissected and collected, and then rapidly frozen using liquid nitrogen.

[0059] Mass spectrometry imaging tissue sections were prepared. Frozen organs were fixed onto tissue scaffolds and cryosectioned to a thickness of 10 μm using a Leica CM1950 cryostat. For each organ, all sections from the control and exposed groups were gently transferred to the same ITO glass slide and dried under vacuum for 40 min. 39.5 mg of 1,5-DAN was added to a mixed solvent of 500 μL hydrochloric acid (1 mmol / L) and 6.5 mL deionized water, dissolved by sonication, and then 2 mL of ethanol was added. The slides were then sealed and stored in the dark to obtain a matrix solution. The matrix was sprayed onto the tissue sections using a custom-made SoniCoat matrix spraying instrument. The matrix solution flow rate was 10 μL / min, the nitrogen supply flow rate was 0.85 L / min, the ultrasonic power was 0.8 W, the line spacing was 1.5 mm, the nozzle movement speed was 1.0 mm / s, and the nozzle height was 60 mm. Spraying was performed three times, and the slides were dried under vacuum for 40 min.

[0060] A MALDI-TOF / TOF-MSI analysis method was established for in-situ analysis of metabolites in tissue sections. Mass spectrometry imaging analysis of spleen sections was performed using an Ultraflextreme MALDI TOF / TOF MS system equipped with a smart beam laser (Nd:YAG 355nm). Signals in the mass range of 0 to 1000 Da were captured in negative ion mode. Spatial resolution was set as needed: 100 μm for the testes, 110 μm for the liver and lungs, and 120 μm for the spleen. Each spectrum was obtained from 200 laser irradiations at 1000 Hz. Mass spectrometry images of tissue sections from each organ were obtained.

[0061] Example 2

[0062] The mass spectrometry imaging results from Example 1 were divided into regions and data extracted. The MALDI-TOF / TOF-MSI data were imported into flexImaging 4.0 software for visualization and analysis. Regions of interest (ROIs) were delineated, and average mass spectra were extracted. For homogeneous organs with no complex tissue structures, such as the liver and testis, a complete tissue slice was used as a unit for data extraction and analysis. Figure 1 The distribution of glutathione in the liver and testes obtained in this embodiment showed that upregulation of glutathione was observed in both the liver and testes of the exposed group. For the spleen, which has a complex tissue structure, due to the specific distribution of heme chloride in the red pulp region, the sub-organ tissue regions of red pulp and white pulp were divided according to the distribution of heme chloride, and data extraction and analysis were carried out on a sub-organ basis. Figure 2 This is the mass spectrometry imaging result of the spleen obtained in this embodiment. Figure 2 (a) shows the distribution of heme chloride in the red pulp region of the spleen tissue, and the distribution of glutathione, taurine, and ascorbic acid in the red and white pulp regions. Figure 2(b) is a box plot of the metabolite intensities in (a), showing significant upregulation of the antioxidants glutathione and ascorbic acid in the red pulp region of the spleen, and significant upregulation of glutathione and taurine in the white pulp region.

[0063] Example 3

[0064] Metabolite data from organ sections of the exposed and control groups were analyzed. Statistical analysis was performed on the online database https: / / www.metaboanalyst.ca / to process the metabolite data of the exposed and control groups respectively, and metabolites with significant changes after pollutant exposure (FC value > 1.5 or < 0.66, and P < 0.05 in one-way ANOVA) were identified. To further observe the metabolic toxicity of pollutants to multiple organs, metabolic pathway enrichment analysis was performed in the KEGG database.

[0065] Figure 3 The enrichment ratio for this example is calculated by dividing the number of observed dysregulated metabolites by the expected number of metabolites in the pathway. The P-value represents the significance of the difference between the exposed group and the control group; the smaller the P-value, the greater the impact of 6PPD-Q on this metabolic pathway.

[0066] Figure 4 This example illustrates the changes and distribution of metabolite levels in important liver metabolic pathways. Figure 4 (a) shows that ascorbic acid levels were elevated and L-gulonic acid, an intermediate in its synthesis pathway, was reduced in the exposed group, demonstrating that 6PPD-Q exposure upregulates the ascorbic acid synthesis pathway in the liver in response to oxidative stress caused by pollutant exposure. Figure 4 (b) showed elevated levels of citrate, aspartate, glutamine, and glutamate in the exposed group. These metabolites are associated with dysregulation of metabolic pathways related to energy metabolism in the liver, including the tricarboxylic acid cycle, aspartate shuttle, and glutamate metabolism pathway. This demonstrates that 6PPD-Q exposure causes dysregulation of hepatic energy metabolism.

[0067] Figure 5In this embodiment, the metabolic pathways dysregulated in the red and white pulp of the spleen by the pollutant 6PPD-Q, identified through metabolic pathway enrichment analysis, include the β-pyruvate metabolic pathway, the nicotinic acid and nicotinamide metabolic pathway, and the pantothenic acid and acetyl-CoA biosynthesis pathway. These pathways are disrupted by 6PPD-Q in both the white and red pulp. Unlike the white pulp region, the glutathione metabolic pathway is specifically dysregulated in the red pulp due to functional differences between the two. The white pulp is a structured lymphoid region responsible for initiating adaptive immune responses, while the main function of the red pulp is to filter and store blood, interacting directly with circulating blood, and therefore may be more susceptible to oxidative stress.

[0068] Figure 6 In this embodiment, metabolic pathway enrichment analysis identified 6PPD-Q as the pollutant causing dysregulated metabolic pathways in the testes, including the glutathione metabolic pathway, glycerophospholipid metabolic pathway, and glycerol ester metabolic pathway. Among these, 6PPD-Q had the greatest impact on the glutathione metabolic pathway in the testes. Figure 1 The upregulation of glutathione suggests that 6PPD-Q induces oxidative stress in the testes.

[0069] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0070] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A method for assessing the metabolic toxicity of pollutants to mammals, characterized in that, include: Mammals in an exposure group and a control group were fed separately. The mammals in the exposure group were given the contaminant in advance, while the mammals in the control group were not given the contaminant. Tissue sections were obtained from the same location in the exposed group and the control group of mammals after feeding. The tissue sections were subjected to matrix-assisted laser desorption / ionization time-of-flight mass spectrometry imaging analysis to obtain mass spectrometry images of the tissue sections. Based on mass spectrometry images, characteristic substances that differed between the exposed group and the control group were screened out; Metabolic pathway enrichment analysis was performed on the substances with the aforementioned distinctive differences.

2. The method according to claim 1, characterized in that, The pollutant includes 6PPD-Q.

3. The method according to claim 1, characterized in that, The mammals mentioned include mice, rats, guinea pigs, rabbits, and non-human primates.

4. The method according to claim 1, characterized in that, The organs include the heart, liver, spleen, lungs, kidneys, and testes.

5. The method according to claim 1, characterized in that, The thickness of the tissue sections is 5–20 μm; the tissue sections are frozen using liquid nitrogen.

6. The method according to claim 1, characterized in that, Before performing the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry imaging analysis, the tissue sections are pre-treated with matrix spraying. Optionally, the matrix solution for the matrix spraying treatment is a 1,5-diaminonaphthalene solution; Optionally, the concentration of 1,5-diaminonaphthalene in the 1,5-diaminonaphthalene solution is 2 to 10 mg / mL; Optionally, the 1,5-diaminonaphthalene solution comprises 1,5-diaminonaphthalene, hydrochloric acid, ethanol, and water, wherein the volume ratio of hydrochloric acid, ethanol, and water is 1:(2-6):(10-16).

7. The method according to claim 1, characterized in that, The matrix-assisted laser desorption / ionization time-of-flight mass spectrometry imaging analysis uses a negative ion mode for detection. Optionally, the molecular weight range of the mass spectrometry imaging analysis is 0–1000 Da.

8. The method according to claim 1, characterized in that, The metabolic pathway enrichment analysis was performed using the KEGG database.

9. A method for constructing an animal model of abnormal metabolic pathways, wherein the metabolic pathways include at least one of the following: The ascorbic acid synthesis pathway in the liver; The tricarboxylic acid cycle pathway, aspartate shuttle pathway, and glutamate metabolism pathway in the liver; Glutathione metabolic pathway in spleen red pulp; Glutathione metabolic pathway in the testes; The method is characterized by administering 6PPD-Q to the test animal.

10. The method according to claim 9, characterized in that, The abnormal metabolic pathway includes at least one of the following: Upregulates the ascorbic acid synthesis pathway in the liver; Upregulates the tricarboxylic acid cycle pathway, aspartate shuttle pathway, and glutamate metabolism pathway in the liver; Upregulates the glutathione metabolic pathway in the red pulp of the spleen; Upregulates the glutathione metabolism pathway in the testes.

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