Method for protecting liver from programmed necrosis caused by aniline by using melatonin

Abstract

The invention relates to a method for protecting liver from programmed necrosis caused by aniline by melatonin, and belongs to the technical field of melatonin liver protection, the method comprises the following steps: S1, establishing an aniline exposure model, randomly dividing experimental animals into a control group and an aniline exposure group, and applying aniline to the aniline exposure group through intragastric administration for 14 days; s2, melatonin is given to the intervention group through intragastric administration for 14 days; s3, liver function detection: after the experiment is finished, collecting a blood sample; s4, histopathologic analysis: after the animals are killed, taking liver tissues for HE staining, and observing the morphological change of liver cells; s5, observing a mitochondrial structure; s6, programmed necrosis protein detection; s7, intestinal barrier function analysis; and S8, FXR activation verification is carried out. The melatonin is used for improving the barrier function of the intestinal tract, regulating the composition of intestinal flora and cutting off damage signal transmission of the intestinal liver axis to the liver, so that the liver is protected from programmed necrosis damage caused by aniline in multiple dimensions.

Description

Technical Field

[0001] The present invention relates to the technical field of melatonin protecting the liver, and particularly relates to a method for protecting the liver from programmed necrosis caused by aniline using melatonin. Background Art

[0002] As an important industrial raw material, aniline is widely used in industries such as dyes, pharmaceuticals, and rubber. However, aniline has obvious hepatotoxicity, and long-term exposure can lead to hepatocyte damage and necrosis, thereby affecting the normal function of the liver. Programmed necrosis, as a regulated form of cell death, plays an important role in aniline-induced liver injury. Research has shown that aniline can activate the programmed necrosis signaling pathway, resulting in hepatocyte death and disrupting the liver tissue structure and function.

[0003] Melatonin is an endogenous bioactive substance with various biological functions such as antioxidant, anti-inflammatory, and regulation of cell death. It has been found that melatonin can protect the liver from aniline injury by activating FXR to regulate mitochondrial dynamics and programmed necrosis. Summary of the Invention

[0004] The object of the present invention is to address the problems in the background art and propose a method for protecting the liver from programmed necrosis caused by aniline using melatonin, which improves the intestinal barrier function through melatonin, regulates the intestinal flora composition, cuts off the damage signal transmission of the gut-liver axis to the liver, and thus protects the liver from programmed necrosis damage caused by aniline in multiple dimensions.

[0005] The technical solution of the present invention: A method for protecting the liver from programmed necrosis caused by aniline using melatonin, the method comprising the following steps:

[0006] S1. Establish an aniline exposure model, randomly divide experimental animals into a control group and an aniline exposure group, and the aniline exposure group is given aniline by gavage for 14 consecutive days;

[0007] S2. The intervention group is given melatonin by gavage for 14 consecutive days;

[0008] S3. Liver function detection, after the experiment ends, collect blood samples;

[0009] S4. Histopathological analysis, after sacrificing the animals, take liver tissues for HE staining to observe the morphological changes of hepatocytes;

[0010] S5. Observation of mitochondrial structure, detect the morphology and structural integrity of liver mitochondria by transmission electron microscopy, evaluate mitochondrial function, use JC-1 probe to detect mitochondrial membrane potential, and mitoSOX probe to detect the reactive oxygen species (ROS) level;

[0011] S6. Detection of necroptosis proteins. Detect the expression of RIPK1, RIPK3, MLKL and their phosphorylated forms in liver tissues by Western blot and immunohistochemistry;

[0012] S7. Intestinal barrier function analysis: Detect the expression of Claudin, Occludin, and ZO-1 in intestinal tissues, and analyze the intestinal flora composition by 16S rRNA sequencing;

[0013] S8. Verification of FXR activation: Detect the expression of intestinal farnesoid X receptor (FXR) by Western blot and PCR, and verify its correlation with necroptosis and mitochondrial dynamics.

[0014] Preferably, the experimental animals are SPF-grade male C57BL / 6 mice.

[0015] Preferably, the liver function detection in step S3 includes detecting the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), detecting the levels of albumin (ALB), total protein (TP) and lactate dehydrogenase (LDH).

[0016] Preferably, the mitochondrial structure observation includes evaluating the integrity of mitochondrial cristae and matrix density.

[0017] Preferably, the detection of mitochondrial dynamics proteins is carried out after the detection of necroptosis proteins: Detect the expression levels of mitochondrial fusion proteins (Mfn1, Mfn2, OPA1) and fission proteins (DRP1, FIS1).

[0018] Preferably, the detection of necroptosis proteins also includes detecting cytochrome c release and caspase-3 activity.

[0019] Preferably, the intestinal barrier function analysis also includes detecting the levels of diamine oxidase (DAO) and D-lactic acid in serum by ELISA.

[0020] Preferably, the verification of FXR activation includes constructing an AAV-FXR overexpression model and observing its effects on necroptosis and mitochondrial dynamics.

[0021] Preferably, 2 hours before aniline gavage, melatonin is gavaged to the animals in the aniline exposure group.

[0022] Preferably, SPF-grade male 7-week-old mice are selected for adaptive cultivation and pre-experiment, and the formal experiment starts at 8 weeks of age.

[0023] Compared with the prior art, the present invention has the following beneficial technical effects:

[0024] In the present invention, melatonin has a powerful antioxidant capacity, can scavenge free radicals generated by aniline metabolism, and reduce oxidative stress. The present invention confirms that melatonin can activate intestinal FXR, and FXR inhibits the activation of the NF-κB signaling pathway and reduces the release of inflammatory factors by regulating the expression of downstream target genes. At the same time, the activated FXR improves mitochondrial dynamics, upregulates the expression of fusion proteins such as Mfn1, Mfn2, and OPA1, inhibits the activities of fission proteins such as DRP1 and FIS1, restores mitochondrial structure and function, and reduces the ROS level. In addition, melatonin reduces MLKL phosphorylation and translocation by inhibiting the RIPK1 / RIPK3 / MLKL signaling pathway, blocking the process of programmed necrosis. At the intestinal level, melatonin repairs intestinal tight junction proteins Claudin, Occludin, and ZO-1, improves intestinal barrier function, regulates the composition of the intestinal flora, and cuts off the damage signal transmission from the gut-liver axis to the liver, thereby protecting the liver from programmed necrosis damage caused by aniline in multiple dimensions.

[0025] AML12 cells were exposed to aniline for 24 h, and it was found that aniline could significantly induce the death of AML12 cells, reduce cell viability, and there was a dose-response relationship. In vivo experiments found that aniline could induce an inflammatory response in the livers of mice.

[0026] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it in accordance with the content of the specification, the following describes in detail with preferred embodiments of the present invention and accompanying drawings. The specific implementation manners of the present invention are given in detail by the following embodiments and their accompanying drawings. Brief Description of the Drawings

[0027] The drawings described herein are used to provide a further understanding of the present invention, form a part of this application, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0028] Figure 1 It is the experimental flow chart of the embodiment in the present invention. Detailed Description of the Invention

[0029] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. In the following paragraphs, the present invention is described more specifically by way of example with reference to the accompanying drawings. It should be noted that the drawings are all in a very simplified form and use non-precise scales, only for the purpose of facilitating and clearly assisting in explaining the objectives of the embodiments of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0031] Example 1

[0032] As Figure 1 shown, a method for protecting the liver from aniline-induced necroptosis proposed by the present invention includes the following steps:

[0033] S1. Establish an aniline exposure model, randomly divide experimental animals into a control group and an aniline exposure group, and the aniline exposure group is given aniline by gavage for 14 consecutive days;

[0034] S2. The aniline exposure group is given melatonin by gavage for 14 consecutive days;

[0035] S3. Liver function detection, after the experiment ends, collect blood samples;

[0036] S4. Histopathological analysis, after sacrificing the animals, take liver tissues for HE staining to observe the morphological changes of hepatocytes;

[0037] S5. Observation of mitochondrial structure, detect the morphology and structural integrity of liver mitochondria by transmission electron microscopy, mitochondrial function assessment, use JC-1 probe to detect mitochondrial membrane potential, and mitoSOX probe to detect the level of reactive oxygen species ROS;

[0038] S6. Detection of necroptosis proteins, detect the expression of RIPK1, RIPK3, MLKL and their phosphorylated forms in liver tissues by Western blot and immunohistochemistry;

[0039] S7. Intestinal barrier function analysis: detect the expression of Claudin, Occludin, and ZO-1 in intestinal tissues, and analyze the intestinal flora composition by 16S rRNA sequencing;

[0040] S8. Verification of FXR activation: detect the expression of intestinal farnesoid X receptor (FXR) by Western blot and PCR, and verify its correlation with necroptosis and mitochondrial dynamics.

[0041] The experimental animals are SPF-grade male C57BL / 6 mice, with no less than 8 mice in each group.

[0042] In step S3, liver function tests include detecting the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), albumin (ALB), total protein (TP), and lactate dehydrogenase (LDH), and evaluating the degree of liver injury through commonly used clinical liver function indicators (e.g., an elevated ALT indicates liver cell damage).

[0043] Observation of mitochondrial structure includes evaluating the integrity of mitochondrial cristae and matrix density.

[0044] After detecting necroptosis proteins, mitochondrial dynamics proteins are detected: detecting the expression levels of mitochondrial fusion proteins (Mfn1, Mfn2, OPA1) and fission proteins (DRP1, FIS1).

[0045] Detection of necroptosis proteins also includes detecting cytochrome c release and caspase-3 activity.

[0046] Analysis of intestinal barrier function also includes detecting the levels of diamine oxidase (DAO) and D-lactate in serum by ELISA.

[0047] The verification of FXR activation includes constructing an AAV-FXR overexpression model and observing its effects on necroptosis and mitochondrial dynamics.

[0048] Two hours before aniline gavage, melatonin is gavaged to the animals in the aniline exposure group.

[0049] SPF-grade male 7-week-old mice are selected for adaptive cultivation and pre-experiment, and the formal experiment starts at 8 weeks old.

[0050] In this example, according to their growth cycle and the characteristics of occupational populations exposed to aniline, SPF-grade male 7-week-old mice are selected for adaptive cultivation and pre-experiment. The formal experiment starts at 8 weeks old. In the formal experiment, power = 0.8, and the effect size is taken as 1 to obtain n = 7.727622, so 8 mice are taken in each group. 83 C57BL / 6J mice are ear-tagged and numbered, and randomly divided into a control group with 8 normal mice; an aniline exposure group with 8 mice in each of the high-dose group (10 mg / kg), medium-dose group (20 mg / kg), and low-dose group (40 mg / kg), a total of 24 mice; an intervention group with 8 mice in the aniline high-dose + melatonin group (20 mg / kg); 11 mice in the aniline high-dose + melatonin group of AAV-FXR, and 3 mice are randomly selected to test whether the AAV injection is successful; 8 mice in the aniline high-dose + melatonin + antibiotic group; 8 mice in the control group of fecal transplantation; 8 mice in the aniline high-dose group of fecal transplantation; 8 mice in the aniline high-dose + melatonin group of fecal transplantation.

[0051] Cage the mice by the random number method, raise them with standard diet in a barrier system, record the temperature, humidity, drinking water and diet every day, weigh them once a week, and intragastrically administer the drugs once a day. For the melatonin group, intragastric administration was carried out 2 hours before aniline intragastric administration, and the control group was intragastrically administered with an equal amount of normal saline for 14 consecutive days;

[0052] After the experiment, the mice were fasted for 12 hours, weighed, and then anesthetized by intraperitoneal injection of sodium pentobarbital (dose: 50 mg / kg body weight).

[0053] Blood was collected from the eyeballs, and the blood was collected in a centrifuge tube and centrifuged at 3000 rpm for 10 minutes to separate the serum, which was stored at -80 °C for liver function tests.

[0054] The mice were sacrificed, and the liver tissues were quickly removed. One part was fixed with 4% paraformaldehyde for histopathological analysis and immunohistochemistry; the other part was frozen in liquid nitrogen and stored at -80 °C for Western blot, PCR and other tests.

[0055] Take part of the intestinal tissue, fix it with 4% paraformaldehyde for detecting intestinal tight junction proteins, and freeze the other part in liquid nitrogen and store it at -80 °C for 16S rRNA sequencing.

[0056] Detect the levels of ALT, AST, ALP, GGT, ALB, TP, and LDH in the serum.

[0057] The liver tissues fixed with 4% paraformaldehyde were dehydrated, cleared, infiltrated with wax, embedded, and then 4-μm sections were made.

[0058] The sections were stained with HE as follows: The sections were dewaxed to water, stained with hematoxylin for 5 minutes, washed with water, differentiated with 1% hydrochloric acid alcohol for several seconds, washed with water, stained with eosin for 3 minutes, washed with water, dehydrated, cleared, and sealed.

[0059] Observation of mitochondrial structure: Take a small amount of liver tissue, fix it with 2.5% glutaraldehyde, after osmium acid fixation, dehydration, and embedding, make ultrathin sections, and observe the mitochondrial morphology under a transmission electron microscope to evaluate the integrity of mitochondrial cristae and matrix density.

[0060] Detection of mitochondrial membrane potential: Prepare a homogenate from the liver tissue, centrifuge to obtain the precipitate, add the JC-1 probe working solution, incubate at 37 °C for 20 minutes, wash with PBS, and then observe the change of mitochondrial membrane potential under a fluorescence microscope. Red fluorescence (aggregated JC-1) indicates high membrane potential, and green fluorescence (monomeric JC-1) indicates low membrane potential.

[0061] Detection of ROS level: Take the liver tissue homogenate, add the mitoSOX probe working solution, incubate at 37 °C for 10 minutes, wash with PBS, and then observe the ROS level under a fluorescence microscope. The higher the fluorescence intensity, the higher the ROS content.

[0062] Western blot detection: Total proteins were extracted from liver tissues. After determining the protein concentration, SDS-PAGE electrophoresis was performed, followed by membrane transfer, blocking, and addition of RIPK1, RIPK3, MLKL, p-RIPK1, p-RIPK3, and p-MLKL antibodies (primary antibodies) respectively. Incubation was carried out overnight at 4°C. The next day, secondary antibody was added and incubated at room temperature for 1 hour. Development was performed using a chemiluminescence imaging system to analyze the protein expression levels.

[0063] Immunohistochemical detection: Liver tissue sections were dewaxed to water, antigen was repaired, blocked, and primary antibody was added. Incubation was carried out overnight at 4°C. The next day, secondary antibody was added and incubated at room temperature for 30 minutes. DAB was used for color development, hematoxylin was used for counterstaining, and the sections were sealed. The protein expression localization was observed under an optical microscope.

[0064] Cytochrome c release detection: Mitochondrial and cytoplasmic proteins were extracted, and Western blot was used to detect the expression of cytochrome c in the cytoplasm to judge the change in mitochondrial membrane permeability.

[0065] Caspase-3 activity detection: The operation was carried out according to the instructions of the caspase-3 activity detection kit to detect the caspase-3 activity in liver tissues.

[0066] Western blot was used to detect the protein expression levels of Mfn1, Mfn2, OPA1, DRP1, and FIS1 in liver tissues. The steps were the same as those for the detection of programmed necrosis proteins.

[0067] Intestinal tight junction protein detection: Western blot and immunohistochemistry were used to detect the protein expressions of Claudin, Occludin, and ZO-1 in intestinal tissues. The steps were the same as above.

[0068] Intestinal flora analysis: Total DNA was extracted from intestinal tissues, and the V3-V4 region of the 16S rRNA gene was amplified, followed by sequencing analysis of the intestinal flora composition and diversity.

[0069] Serum DAO and D-lactic acid detection: The operation was carried out according to the instructions of the ELISA kit to detect the levels of DAO and D-lactic acid in serum and evaluate intestinal permeability.

[0070] After aniline enters the body, it is metabolized by the liver to produce toxic metabolites, which can trigger oxidative stress and lead to mitochondrial dysfunction. The imbalance of mitochondrial dynamics, such as the excessive fission of mitochondria mediated by DRP1, destroys the structural integrity of mitochondria, reduces the mitochondrial membrane potential, and generates a large amount of ROS. At the same time, aniline activates the programmed necrosis signaling pathway. RIPK1 binds to RIPK3 to form necrosomes, recruits MLKL and phosphorylates it, resulting in the oligomerization and translocation of MLKL to the cell membrane, destroying the cell membrane integrity and triggering the programmed necrosis of hepatocytes. In addition, aniline damages the intestinal barrier function, causes intestinal flora dysbiosis, and endotoxins such as LPS enter the blood, aggravating liver inflammation and injury through the enterohepatic axis.

[0071] Melatonin has a strong antioxidant capacity, can scavenge free radicals generated by aniline metabolism, and reduce oxidative stress. This invention confirms that melatonin can activate intestinal FXR. FXR inhibits the activation of the NF-κB signaling pathway and reduces the release of inflammatory factors by regulating the expression of downstream target genes. At the same time, the activated FXR improves mitochondrial dynamics, upregulates the expression of fusion proteins such as Mfn1, Mfn2, and OPA1, inhibits the activities of fission proteins such as DRP1 and FIS1, restores the structure and function of mitochondria, and reduces the ROS level. In addition, melatonin inhibits the RIPK1 / RIPK3 / MLKL signaling pathway, reduces the phosphorylation and translocation of MLKL, and blocks the programmed necrosis process. At the intestinal level, melatonin repairs intestinal tight junction proteins (Claudin, Occludin, ZO-1), improves intestinal barrier function, regulates the composition of intestinal flora, and cuts off the transmission of liver injury signals through the enterohepatic axis, thereby protecting the liver from programmed necrosis damage caused by aniline in multiple dimensions.

[0072] AML12 cells were treated with aniline for 24 hours, and it was found that aniline could significantly induce the death of AML12 cells, reduce cell viability, and there was a dose-response relationship. In vivo experiments found that aniline could induce inflammatory reactions in the livers of mice.

[0073] As mentioned above, it is only the preferred embodiment of the present invention, and it does not impose any form of limitation on the present invention. Any ordinary technician in the industry can smoothly implement the present invention according to the instructions in the drawings and the above description. However, any equivalent changes such as slight modifications, evolutions made by those skilled in the art within the scope of the technical solution of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for protecting the liver from programmed necrosis caused by aniline using melatonin, characterized in that: The method comprises the following steps: S1. Establish an aniline exposure model, and randomly divide the experimental animals into a control group, an aniline exposure group, and an intervention group. The aniline exposure group was given aniline by oral gavage for 14 days. S2, intervention group was given melatonin by oral gavage for 14 days; S3, liver function test, after the experiment, blood samples were collected; S4. Histopathological analysis: After the animals were killed, liver tissues were obtained for HE staining to observe the morphological changes of hepatocytes; S5. Mitochondrial structure observation: the morphology and structural integrity of liver mitochondria were detected by transmission electron microscopy; mitochondrial function was evaluated by using JC-1 probe to detect mitochondrial membrane potential and mitoSOX probe to detect reactive oxygen species (ROS) levels; S6, programmed necrosis protein detection, the expression of RIPK1, RIPK3, MLKL and their phosphorylated forms in liver tissues were detected by Western blot and immunohistochemistry; S7. Intestinal barrier function analysis: Detect the expression of Claudin, Occludin, and ZO-1 in intestinal tissues, and analyze the composition of intestinal flora by 16S rRNA sequencing; S8. Verification of FXR activation: The expression of intestinal farnesoid receptor (FXR) was detected by Western blot and PCR, and its correlation with programmed necrosis and mitochondrial dynamics was verified.

2. A method for protecting the liver from programmed necrosis caused by aniline using melatonin according to claim 1, characterized in that: The experimental animals were SPF grade male C57BL / 6 mice.

3. A method for protecting the liver from programmed necrosis caused by aniline using melatonin according to claim 2, characterized in that: The liver function test in step S3 includes detecting the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), albumin (ALB), total protein (TP) and lactate dehydrogenase (LDH).

4. A method for protecting the liver from programmed necrosis caused by aniline using melatonin according to claim 3, characterized in that: Mitochondrial structural observations included assessment of mitochondrial cristae integrity and matrix density.

5. A method for protecting the liver from programmed necrosis caused by aniline using melatonin according to claim 4, characterized in that: After the programmed necrosis protein detection, mitochondrial dynamics protein detection was performed: the expression levels of mitochondrial fusion proteins (Mfn1, Mfn2, OPA1) and fission proteins (DRP1, FIS1) were detected.

6. A method for protecting the liver from programmed necrosis caused by aniline using melatonin according to claim 5, characterized in that: Necrotic protein testing also includes the detection of cytochrome c release and caspase-3 activity.

7. A method for protecting the liver from aniline-induced programmed necrosis using melatonin according to claim 6, characterized in that: The intestinal barrier function analysis also included the detection of serum diamine oxidase (DAO) and D-lactic acid levels by ELISA.

8. A method for protecting the liver from programmed necrosis caused by aniline using melatonin according to claim 7, characterized in that: The FXR activation validation includes constructing an AAV-FXR overexpression model to observe its effects on programmed necrosis and mitochondrial dynamics.

9. A method for protecting the liver from aniline-induced programmed necrosis using melatonin according to claim 8, characterized in that: Two hours before aniline gavage, animals in the aniline exposure group were gavaged with melatonin.

10. A method for protecting the liver from programmed necrosis caused by aniline using melatonin according to claim 2, characterized in that: SPF male mice aged 7 weeks were selected for adaptive training and preliminary experiments, and formal experiments began at 8 weeks of age.

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