Method for alleviating aniline liver injury by activating FXR with melatonin

Abstract

The invention discloses a method for relieving aniline liver injury by activating FXR with melatonin, and belongs to the technical field of biomedicines.The method comprises the steps that S1, an effective dose of melatonin is applied to an aniline-exposed individual or animal model; and S2, verifying the activation effect of the melatonin on FXR (farnesoid receptor) by detecting the expression or activity level of the FXR in the intestinal tract. In the invention, an intestinal FXR-mitochondrial cross-organ regulation network is disclosed, by specifically targeting intestinal FXR and RIPK1 / MLKL phosphorylation pathway, the stable state of mitochondria can be effectively regulated, the ultrastructure and gene time sequence map of the mitochondria are ingeniously integrated, the dynamic change of the mitochondria is deeply analyzed from the microscopic level, the mechanism deficiency of the traditional research is made up, and the research on the mitochondria is facilitated. Meanwhile, it is clear that 20 mg / kg is the optimal dosage of the melatonin, the ALT / AST level is remarkably reduced, the fuzzy dilemma of the traditional clinical medication dosage is broken through, a melatonin and antibiotic synergistic treatment scheme is further developed, the treatment effect is greatly improved, and intestinal microenvironment factors are fully considered.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for melatonin to activate FXR to reduce aniline-induced liver injury. Background Art

[0002] Aniline, also known as aminobenzene, is an important organic compound with a chemical structure formed by the combination of a benzene ring and an amino group. Due to its diverse chemical properties, aniline is widely used in the production of dyes (such as fabrics, papers, and plastics), drugs (such as antibiotics, painkillers, and anticancer drugs), and pesticides (such as nitrofen, propachlor, etc.). Therefore, pesticide residues in fruits and vegetables can lead to an increased intake of aniline by humans. Currently, aniline has been classified by the International Agency for Research on Cancer (IARC) from a Group 3 carcinogen to a Group 2A carcinogen. The use of aniline also poses certain safety hazards, and attention should be paid to protection and reasonable use during production and use to reduce potential harm to the environment and the human body;

[0003] The liver is one of the largest internal organs in the human body and can regulate the metabolism of sugars, fats, and proteins, maintaining the balance of blood sugar and blood lipids. Research shows that the liver plays a key role in the metabolic process and has multiple important functions, including metabolic regulation, toxin clearance, protein synthesis, bile production, energy storage, etc. As the body's main detoxification organ, the liver can convert harmful substances in the body into harmless substances and excrete them through bile. During liver injury caused by various reasons, hepatocyte death is the "terminal" event and is closely related to the onset of the disease. The research group's previous investigation found that aniline workers had an increased incidence of symptoms such as nausea, vomiting, and aversion to greasy foods, elevated transaminases, and a high prevalence of liver injury, but its mechanism of action has not been clarified and further research is needed.

[0004] Traditional research often focuses on the local injury mechanism of the liver, only concentrating on the direct effects of oxidative stress and inflammatory responses, but ignoring the systemic regulatory function of the gut-liver axis in toxic metabolism. Currently, treatment methods such as N-acetylcysteine mainly rely on broad-spectrum antioxidant and anti-inflammatory effects. Due to the lack of specific targeting of key signaling pathways, off-target effects are easily triggered. In terms of efficacy evaluation, over-reliance on non-specific biochemical indicators such as ALT / AST cannot accurately reflect mitochondrial ultrastructural damage and the dynamic changes of genes. During clinical drug use, there are also problems such as vague dose definition (the common dose range is 5 - 50 mg / kg) and ignoring the interference of the intestinal microenvironment, ultimately resulting in unstable treatment efficacy.

[0005] Based on this, the present invention designs a method for melatonin to activate FXR to reduce aniline-induced liver injury to solve the above problems. Summary of the Invention

[0006] The object of the present invention is to propose a method for melatonin to activate FXR and reduce aniline-induced liver injury in order to solve the above problems.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for melatonin to activate FXR and reduce aniline-induced liver injury, comprising:

[0009] S1. Administer an effective dose of melatonin to an individual or animal model exposed to aniline;

[0010] S2. Verify the activation of FXR by melatonin by detecting the expression or activity level of intestinal farnesoid X receptor (FXR);

[0011] S3. Evaluate the improvement effect of melatonin on aniline-induced hepatic programmed necrosis, mitochondrial dynamics disorder and liver injury;

[0012] S4. Combine intestinal flora regulation and mitochondrial function repair to achieve synergistic treatment of liver injury.

[0013] As a further description of the above technical solution:

[0014] The effective dose in step S1 is 0.1 - 50 mg / kg body weight per day, preferably 20 mg / kg, and the administration route is oral or intraperitoneal injection.

[0015] As a further description of the above technical solution:

[0016] In step S2, the activation of FXR is verified by the following methods:

[0017] 2.1. Detect the expression level of FXR protein in intestinal tissues by Western Blot;

[0018] 2.2. Detect the transcriptional activity of FXR by luciferase reporter gene assay;

[0019] 2.3. Analyze the localization of FXR in intestinal epithelial cells by immunohistochemistry.

[0020] As a further description of the above technical solution:

[0021] The evaluation in step S3 includes:

[0022] 3.1. Detect the levels of serum ALT, AST and LDH;

[0023] 3.2. Observe the morphological changes of hepatocyte mitochondria and the changes of membrane potential by transmission electron microscopy;

[0024] 3.3. Detect the expression levels of programmed necrosis marker proteins (RIPK1, RIPK3, MLKL and their phosphorylated forms);

[0025] 3.4. Analyze the mRNA and protein expressions of mitochondrial dynamics-related proteins (Mfn1, Mfn2, OPA1, DRP1, FIS1).

[0026] As a further description of the above technical solution:

[0027] The intestinal flora regulation in step S4 includes:

[0028] 4.1. Restore the homeostasis of intestinal flora through fecal microbiota transplantation (FMT);

[0029] 4.2. Supplement probiotics (such as Lactobacillus and Bifidobacterium) to enhance the intestinal barrier function;

[0030] 4.3. Detect the expression levels of intestinal tight junction proteins (Claudin, Occludin, ZO-1).

[0031] As a further description of the above technical solution:

[0032] The animal model in step S1 is SPF-grade C57BL / 6 mice, and the aniline exposure dose is 10 - 40 mg / kg / day for 14 days.

[0033] As a further description of the above technical solution:

[0034] The method further includes an in vitro verification step:

[0035] 5.1. Extract primary mouse hepatocytes and detect the mitochondrial membrane potential with a JC-1 probe;

[0036] 5.2. Detect the level of mitochondrial reactive oxygen species (ROS) using a mitoSOX probe combined with flow cytometry;

[0037] 5.3. Through a RIPK1 gene intervention model, verify the regulatory effect of programmed necrosis on mitochondrial dynamics.

[0038] As a further description of the above technical solution:

[0039] The melatonin is used in combination with antibiotics to eliminate the interference of intestinal flora on the FXR signal. The specific scheme is as follows:

[0040] 6.1. Three days before melatonin administration, clear the intestinal flora with broad-spectrum antibiotics (such as ampicillin and vancomycin);

[0041] 6.2. Combine 16S rRNA sequencing to analyze the changes in the flora composition.

[0042] As a further description of the above technical solution:

[0043] The method further includes the following treatment optimization strategies:

[0044] 7.1 Dynamically adjust the melatonin dose according to the intestinal FXR expression level;

[0045] 7.2 Combine mitochondrial-targeted antioxidants (such as MitoQ) to enhance the protective effect;

[0046] 7.3 Verify the necessity of FXR in the protective mechanism of melatonin through the AAV-FXR gene overexpression model.

[0047] For any of the above methods, the application of the method includes the following steps:

[0048] 8.1 Prevent or treat chronic liver injury caused by occupational aniline exposure;

[0049] 8.2 As an adjuvant therapeutic drug for chemical liver injury;

[0050] 8.3 Used to improve mitochondrial dysfunction-related metabolic diseases (such as non-alcoholic fatty liver, liver fibrosis).

[0051] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows:

[0052] In the present invention, the "intestinal FXR-mitochondria" cross-organ regulatory network is revealed. By specifically targeting intestinal FXR and the RIPK1 / MLKL phosphorylation pathway, the mitochondrial homeostasis can be effectively regulated. The mitochondrial ultrastructure and gene time-course atlas are cleverly integrated, and the dynamic changes of mitochondria are deeply analyzed at the microscopic level, making up for the deficiencies in the mechanism of traditional research. At the same time, 20 mg / kg is determined as the optimal dose of melatonin, significantly reducing the ALT / AST level, breaking through the ambiguous dilemma of traditional clinical drug doses. A "melatonin + antibiotic" synergistic treatment plan is also developed, greatly improving the treatment effect, and fully considering the intestinal microenvironment factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is a schematic diagram of the experimental technical route of a method for melatonin to activate FXR to reduce aniline liver injury proposed by the present invention;

[0054] Figure 2 It is a schematic diagram of the step flow of a method for melatonin to activate FXR to reduce aniline liver injury proposed by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0055] Next, in combination with the accompanying drawings in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0056] Please refer to the attached Figure 1 - attached Figure 2 , the present invention provides a technical solution: a method for melatonin to activate FXR to reduce aniline-induced liver injury, including:

[0057] S1. Administer an effective dose of melatonin to an individual or animal model exposed to aniline;

[0058] S2. Verify the activation effect of melatonin on FXR by detecting the expression or activity level of intestinal farnesoid X receptor (FXR);

[0059] S3. Evaluate the improvement effect of melatonin on aniline-induced hepatic programmed necrosis, mitochondrial dynamics disorder and liver injury;

[0060] S4. Combine intestinal flora regulation and mitochondrial function repair to achieve synergistic treatment of liver injury.

[0061] The effective dose in step S1 is 0.1 - 50 mg / kg body weight per day, preferably 20 mg / kg, and the administration route is oral or intraperitoneal injection

[0062] In step S2, the activation of FXR is verified by the following methods:

[0063] 2.1. Detect the expression level of FXR protein in intestinal tissues by Western Blot;

[0064] 2.2. Detect the transcriptional activity of FXR by luciferase reporter gene assay;

[0065] 2.3. Combine immunohistochemistry to analyze the localization of FXR in intestinal epithelial cells.

[0066] The evaluation in step S3 includes:

[0067] 3.1. Detect the levels of serum ALT, AST, and LDH;

[0068] 3.2. Observe the morphology and membrane potential changes of hepatocyte mitochondria by transmission electron microscopy;

[0069] 3.3. Detect the expression levels of programmed necrosis marker proteins (RIPK1, RIPK3, MLKL and their phosphorylated forms);

[0070] 3.4. Analyze the mRNA and protein expression of mitochondrial dynamics-related proteins (Mfn1, Mfn2, OPA1, DRP1, FIS1).

[0071] The gut microbiota regulation in step S4 includes:

[0072] 4.1. Restore gut microbiota homeostasis through fecal microbiota transplantation (FMT);

[0073] 4.2. Supplement probiotics (such as Lactobacillus and Bifidobacterium) to enhance gut barrier function;

[0074] 4.3. Detect the expression levels of gut tight junction proteins (Claudin, Occludin, ZO-1).

[0075] The animal model in step S1 is SPF-grade C57BL / 6 mice, and the aniline exposure dose is 10 - 40 mg / kg / day for 14 days.

[0076] The method also includes an in vitro verification step:

[0077] 5.1. Extract primary mouse hepatocytes and detect mitochondrial membrane potential using the JC-1 probe;

[0078] 5.2. Detect the level of mitochondrial reactive oxygen species (ROS) using the mitoSOX probe combined with flow cytometry;

[0079] 5.3. Verify the regulatory effect of necroptosis on mitochondrial dynamics through the RIPK1 gene intervention model.

[0080] The melatonin is used in combination with antibiotics to eliminate the interference of gut microbiota on the FXR signal. The specific protocol is as follows:

[0081] 6.1. Clear gut microbiota with broad-spectrum antibiotics (such as ampicillin and vancomycin) 3 days before melatonin administration;

[0082] 6.2. Analyze the changes in the composition of the microbiota by combining 16S rRNA sequencing.

[0083] The method also includes the following treatment optimization strategies:

[0084] 7.1. Dynamically adjust the melatonin dose according to the gut FXR expression level;

[0085] 7.2. Combine mitochondrial-targeted antioxidants (such as MitoQ) to enhance the protective effect;

[0086] 7.3. Verify the necessity of FXR in the protective mechanism of melatonin through the AAV-FXR gene overexpression model.

[0087] The applications of the method include:

[0088] 8.1. Preventing or treating chronic liver injury caused by occupational aniline exposure;

[0089] 8.2. As an adjuvant therapeutic drug for chemical liver injury;

[0090] 8.3. For improving mitochondrial dysfunction-related metabolic diseases (such as non-alcoholic fatty liver and liver fibrosis).

[0091] Example 1

[0092] Objective: To verify the protective effect of melatonin on aniline-induced liver injury.

[0093] Method:

[0094] Step (1): Inject 20 mg / kg of melatonin into C57BL / 6 mice intraperitoneally, and then expose them to 20 mg / kg / day of aniline.

[0095] Step (2): Detect the expression of FXR protein by Western Blot.

[0096] Step (3): Evaluate the levels of serum ALT, AST, LDH and the morphology of hepatocyte mitochondria.

[0097] Step (4): Do not perform gut microbiota regulation.

[0098] Result: Melatonin significantly reduced the liver injury indexes induced by aniline, and the expression of FXR increased.

[0099] Example 2

[0100] Objective: To explore the importance of gut microbiota regulation in the protective effect of melatonin.

[0101] Method:

[0102] Step (1): The same as Example 1.

[0103] Step (2): The same as Example 1.

[0104] Step (3): The same as Example 1.

[0105] Step (4): Restore the gut microbiota by FMT and detect the expression of intestinal tight junction proteins.

[0106] Result: Gut microbiota regulation enhanced the protective effect of melatonin, and the expression of tight junction proteins increased.

[0107] Example 3

[0108] Objective: To verify the therapeutic effect of the combined use of melatonin and antibiotics.

[0109] Method:

[0110] Step (1): Three days before melatonin administration, use broad-spectrum antibiotics to clear the gut microbiota, and then administer 20 mg / kg of melatonin and 20 mg / kg / day of aniline.

[0111] Step (2): The same as in Example 1.

[0112] Step (3): The same as in Example 1.

[0113] Step (4): Do not perform additional gut microbiota regulation.

[0114] Result: After antibiotic pretreatment, the therapeutic effect of melatonin was more significant, and 16S rRNA sequencing showed changes in the microbiota composition.

[0115] Example 4

[0116] Objective: To evaluate the effects of different doses of melatonin on aniline-induced liver injury.

[0117] Method:

[0118] Step (1): Administer 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 20 mg / kg of melatonin to C57BL / 6 mice respectively, and then expose them to 20 mg / kg / day of aniline.

[0119] Step (2): The same as in Example 1.

[0120] Step (3): The same as in Example 1.

[0121] Step (4): The same as in Example 2.

[0122] Result: Melatonin at 10 mg / kg and 20 mg / kg showed the best protective effect, while the effect of the 0.1 mg / kg dose was not obvious.

[0123] Example 5

[0124] Objective: To verify the necessity of FXR in the protective mechanism of melatonin.

[0125] Method:

[0126] Step (1): Administer 20 mg / kg of melatonin to AAV-FXR gene overexpressing mice, and then expose them to 20 mg / kg / day of aniline.

[0127] Step (2): The same as in Example 1.

[0128] Step (3): The same as in Example 1.

[0129] Step (4): The same as in Example 2.

[0130] Results: Mice with overexpressed FXR were more sensitive to aniline-induced liver injury, indicating that FXR plays a key role in the protective mechanism of melatonin.

[0131] Through these examples, the effects of melatonin under different conditions can be compared, as well as the importance of FXR activation and gut microbiota regulation in the treatment of aniline-induced liver injury.

[0132] Experimental protocol

[0133] 1. In vivo experiments were conducted to explore the effect and mechanism of melatonin in alleviating necroptosis and improving aniline-induced liver injury by activating the intestinal farnesoid X receptor.

[0134] (1) Selection and cultivation of experimental animals

[0135] C57BL / 6 mice were the first mice to complete genome sequencing, were easy to breed, had a high degree of genetic background stability, and could ensure the consistency of experimental data to the greatest extent. According to their growth cycle and the characteristics of the occupational population exposed to aniline, SPF-grade male 7-week-old mice were selected for adaptive cultivation and pre-experiments. The formal experiment started at 8 weeks of age. In the formal experiment, power = 0.8, and the effect size was taken as 1 to obtain n = 7.727622. Therefore, 8 mice were taken in each group. Eighty-three C57BL / 6J mice were ear-tagged and numbered, and randomly divided into a control group (n = 8 normal mice); mice in the aniline exposure group (high-dose group 10 mg / kg, medium-dose group 20 mg / kg, low-dose group 40 mg / kg) (n = 24); mice in the aniline high-dose + melatonin group (20 mg / kg) (n = 8); AAV-FXR mice in the aniline high-dose + melatonin group (n = 11, and three were randomly selected to test whether AAV injection was successful); mice in the aniline high-dose + melatonin + antibiotic group (n = 8); mice receiving fecal transplantation from the control group (n = 8); mice receiving fecal transplantation from the aniline high-dose group (n = 8); mice receiving fecal transplantation from the aniline high-dose + melatonin group (n = 8). They were caged by the random number method and fed a standard diet in a barrier system. The temperature, humidity, drinking water, and diet were recorded daily, and the weight was measured once a week. They were gavaged once a day. The melatonin group was gavaged 2 hours before aniline gavage, and the control group was gavaged with an equal volume of normal saline for 14 days.

[0136] (2) Explore the effect of aniline on the mouse liver

[0137] Eye enucleation and blood collection were performed into 1.5 ml sterile centrifuge tubes, followed by a 1-hour water bath at 37°C and a 4-hour static incubation at 4°C. Centrifugation was carried out at 4000 r for 15 minutes at room temperature, and the supernatant was taken for the detection of biochemical indices such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), bilirubin (BILI), albumin (ALB), total protein (TP), and lactate dehydrogenase (LDH). After sacrificing the mice, the livers were excised, weighed, and part of them was frozen. The remaining liver tissues were sectioned and stained with HE to observe the changes in cell morphology and structure. Transmission electron microscopy was used to observe the changes in liver tissues.

[0138] (3) Explore the effects of aniline on the intestinal function of mice

[0139] Determine the intestinal permeability of mice. After fecal transplantation in mice, bacterial DNA in the colon feces of each group of mice was extracted, and the concentration and purity of bacterial DNA were detected using a fluorometer. The 16S rRNA gene sequences of the fecal bacterial DNA samples were PCR amplified for the V3-V4 region by binding with specific primers to highly variable regions to quantitatively analyze the effects of aniline on the intestinal flora of mice. Immunohistochemistry and WB were used to detect the changes in key molecules of intestinal barrier function, Claudin, occludin, and ZO-1. Scanning electron microscopy was used to observe the changes in intestinal tissues.

[0140] (4) Explore the effects of aniline on programmed necrosis and mitochondrial dynamics in the livers of mice

[0141] Immunohistochemistry and WB were used to detect the protein changes of Mfn1, Mfn2, OPA1, DRP1, p-DRP1, FIS1, RIPK1, RIPK3, MLKL, p-RIPK1, p-RIPK3, p-MLKL, etc., and PCR was used to detect the mRNA expression of related factors Mfn1, Mfn2, OPA1, DRP1, p-DRP1, FIS1, RIPK1, RIPK3, MLKL, p-RIPK1, p-RIPK3, p-MLKL.

[0142] (5) Explore the effects of intestinal farnesoid X receptor on aniline-induced liver injury in mice

[0143] An AAV-FXR model was established to explore the effects of intestinal farnesoid X receptor on aniline-induced liver injury in mice. Immunohistochemistry, WB, and PCR were used to detect the expression of molecules related to programmed necrosis and mitochondrial dynamics.

[0144] (6) Explore the protective effect of melatonin in aniline-induced liver injury

[0145] WB was used to detect the changes in the above key proteins in the livers of mice protected by melatonin. RT-PCR was used to detect the mRNA expression of related factors.

[0146] 2. In vitro experiments to explore the role and mechanism of melatonin in alleviating necroptosis and improving aniline-induced liver injury by activating intestinal farnesol receptor

[0147] (1) Extraction and culture of primary cells

[0148] After anesthetizing mice with 10% avertin, the liver was quickly removed for perfusion and separation. A pre-prepared digestive solution was used for cell separation of liver tissue. The isolated hepatocytes were precipitated by centrifugation. The isolated hepatocytes were cultured in a petri dish for primary culture. A pre-prepared culture medium containing appropriate nutrients and growth factors was used. The attachment and proliferation of cells were observed under a microscope to ensure cell viability and purity. The cells were cultured at 37°C and 5% CO2 in the culture medium. The medium was changed 2 - 3 times a week, and the cells were passaged when the cell fusion reached 90%. Fluorescent transfection was performed when the cells adhered to 70%, and the transfection efficiency was detected by flow cytometry 6 h after transfection. WB was used to detect the transfection efficiency 72 h after transfection when the cells adhered to 70% and were transfected.

[0149] (2) To explore whether aniline induces necroptosis in primary mouse liver cells

[0150] WB was used to detect the changes in proteins related to necroptosis, such as RIPK1, RIPK3, MLKL, p-RIPK1, p-RIPK3, p-MLKL, etc. RT-PCR was used to detect the mRNA expression of factors related to necroptosis, including RIPK1, RIPK3, MLKL, p-RIPK1, p-RIPK3, p-MLKL.

[0151] (3) To explore the effect of aniline on mitochondrial dynamics in primary mouse liver cells

[0152] JC-1 probe labeling method was used for laser confocal detection of mitochondrial membrane potential changes. MitoSOX probe labeling method was used for FCM detection of mitochondrial reactive oxygen species. Transmission electron microscopy was used to observe the changes in the microscopic structure of cell mitochondria. WB was used to detect the changes in proteins related to mitochondrial dynamics, such as Mfn1, Mfn2, OPA1, DRP1, p-DRP1, FIS1, etc. RT-PCR was used to detect the mRNA expression of related factors, including Mfn1, Mfn2, OPA1, DRP1, p-DRP1, FIS1.

[0153] (4) To explore the mechanism by which necroptosis regulates mitochondrial dynamics

[0154] Detect the intracellular localization of programmed necrosis-related proteins RIPK1, RIPK3, and MLKL related to IF detection by fluorescence microscopy. Transfect RIPK1 to establish an intervention model: intervene in RIPK1 to observe the expression of key molecules of mitochondrial dynamics and mitochondrial translocation. Detect the protein changes of mitochondrial dynamics-related proteins such as Mfn1, Mfn2, OPA1, DRP1, p-DRP1, and FIS1 by WB. Detect the mRNA expression of related factors Mfn1, Mfn2, OPA1, DRP1, p-DRP1, and FIS1 by RT-PCR.

[0155] (5) Explore the protective effect of melatonin in aniline-induced liver injury

[0156] Detect the expression of related molecules FXR, Mfn1, Mfn2, OPA1, DRP1, p-DRP1, FIS1, RIPK1, RIPK3, MLKL, p-RIPK1, p-RIPK3, and p-MLKL by WB and RT-PCR. Verify the results of in vivo experiments.

[0157] Multiple experimental procedures:

[0158] 1. Detection of serum biochemical indexes

[0159] Experimental method: Collect mouse whole blood samples by retroorbital venous plexus blood collection method, and centrifuge at 4000 rpm for 15 min at 4 °C after standing to separate serum. Detect the following indexes by an automatic biochemical analyzer:

[0160] Liver injury markers: alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH)

[0161] Bile metabolism indexes: alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), total bilirubin (BILI)

[0162] Comprehensive evaluation indexes of liver function: albumin (ALB), total protein (TP)

[0163] 2. Observation of ultrastructure of liver mitochondria

[0164] Experimental method: Take fresh liver tissue (1 mm 3 ) After fixation with 2.5% glutaraldehyde, post-fixation with osmium acid, and gradient dehydration, embed it in epoxy resin. Prepare ultra-thin sections (70 nm), double-stain with uranyl acetate-lead citrate, and observe the mitochondrial morphology, cristae structure, and autophagosome formation under a transmission electron microscope (TEM, Hitachi HT7800).

[0165] 3. Analysis of protein expression in liver tissue

[0166] Experimental procedure:

[0167] Pathological examination: Paraffin sections (4 μm) of liver tissue were stained with hematoxylin-eosin (HE) to evaluate hepatocyte necrosis and inflammatory infiltration;

[0168] Immunohistochemistry (IHC): The streptavidin-biotin complex method was used to detect the protein localization of FXR, ZO-1, and Occludin;

[0169] Western Blotting (WB): Total proteins were extracted with RIPA lysis buffer and quantified by BCA method to detect the expression of target proteins:

[0170] Mitochondrial dynamics-related proteins: Mfn1 / 2, OPA1, DRP1 (phosphorylated at Ser616), FIS1

[0171] Programmed necrosis marker proteins: RIPK1 (phosphorylated at Ser166), RIPK3 (phosphorylated at Thr231 / Ser232), MLKL (phosphorylated at Thr357 / Ser358)

[0172] 4. Analysis of mRNA expression in liver tissue

[0173] Experimental method: Total RNA of liver tissue was extracted by TRIzol method and reverse transcribed into cDNA using PrimeScript RT kit. SYBR Green method was used for real-time quantitative PCR (qRT-PCR) to detect the expression of the following genes (using GAPDH as an internal reference):

[0174] Intestinal barrier function-related: FXR, Claudin-1, Occludin, ZO-1

[0175] Mitochondrial dynamics-related: Mfn1, Mfn2, OPA1, DRP1, FIS1

[0176] Programmed necrosis-related: RIPK1, RIPK3, MLKL

[0177] 5. Construction of AAV-FXR mouse model

[0178] Experimental protocol:

[0179] Viral vector: The AAV9 serotype was used to carry the FXR overexpression sequence (AAV-FXR), and the control virus was AAV-GFP;

[0180] Injection method: Injection was performed via the tail vein (1×10^11 vg / animal);

[0181] Model validation: Four weeks after injection, intestinal tissue was taken for WB to detect the expression of FXR protein to confirm the effectiveness of the model;

[0182] Phenotypic observation: Detect changes in the expression of necroptosis markers and mitochondrial dynamics proteins.

[0183] 6. Detection of mitochondrial function in primary hepatocytes

[0184] Experimental procedure:

[0185] Isolation of primary cells: Mouse hepatocytes were isolated by two-step collagenase perfusion method, and the purity was >90% (verified by Albumin immunofluorescence);

[0186] Detection of mitochondrial membrane potential: Stained with JC-1 probe (5 μM), and quantified by laser confocal microscopy (red / green fluorescence ratio);

[0187] Detection of mitochondrial ROS: Labeled with MitoSOX Red probe (5 μM), and quantitatively analyzed by flow cytometry (FCM).

[0188] 7. Ultrastructural analysis of primary cell mitochondria

[0189] Experimental method: After primary hepatocytes were fixed with 2.5% glutaraldehyde, they were processed according to the above TEM sample preparation procedure, and the focus was on observing the dynamics of mitochondrial fission / fusion and the degree of swelling.

[0190] 8. Verification of protein expression in primary cells

[0191] Experimental method: WB detection was performed synchronously with primary cells (same as step 3) to compare the consistency of the expression of mitochondrial dynamics and necroptosis-related proteins in in vivo and in vitro experiments.

[0192] 9. Subcellular localization analysis of proteins

[0193] Immunofluorescence (IF) detection:

[0194] Cell slides were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100;

[0195] Primary antibodies (anti-DRP1, RIPK3, etc.) were incubated overnight at 4 °C, and secondary antibodies labeled with Alexa Fluor were incubated for 1 h in the dark;

[0196] The nucleus was counterstained with DAPI, and after mounting, the co-localization of proteins in mitochondria (labeled with MitoTracker) and cytoplasm was observed under a fluorescence microscope (Nikon Eclipse Ti2).

[0197] 10. RIPK1 gene intervention experiment

[0198] Experimental design:

[0199] Transfection system: Lipofectamine 3000 was used to mediate the transfection of RIPK1 siRNA or overexpression plasmid into primary hepatocytes;

[0200] Intervention verification: Detect the expression efficiency of RIPK1 protein after transfection by WB;

[0201] Phenotype analysis: Detect the translocation of proteins related to mitochondrial dynamics (such as the recruitment of DRP1 to mitochondria) and the degree of necroptosis activation.

[0202] 11. Verification of dynamic mRNA expression

[0203] Experimental method: Perform qRT-PCR detection simultaneously with primary cells (same as step 4) to construct a temporal gene expression profile.

[0204] Mechanism innovation: For the first time, it is clarified that melatonin regulates mitochondrial dynamics (phosphorylation of Mfn1 / 2 and DRP1) by activating intestinal FXR (rather than traditional hepatic FXR), improving the imbalance of mitochondrial fission / fusion induced by aniline (as shown by TEM in Examples 1 and 5 that the mitochondrial cristae structure is restored).

[0205] Target breakthrough: Establish a cross-organ regulatory pathway of "intestinal FXR → inhibition of necroptosis → reconstruction of mitochondrial homeostasis" (both in vitro and in vivo experiments show that overexpression of FXR significantly enhances the protective effect).

[0206] Therapeutic optimization: Propose the necessity of the interaction between gut microbiota and FXR in the protection of melatonin (in Example 2, the expression of ZO-1 increased by 40% after FMT restored the microbiota);

[0207] Clear molecular mechanism: Reveal that the phosphorylation cascade reaction of RIPK1 / MLKL drives the abnormal recruitment of mitochondrial DRP1 (Ser616), leading to mitochondrial fragmentation (WB and IF show that p-MLKL in the melatonin group decreased by 60%);

[0208] Precision intervention: Confirm through the RIPK1 siRNA knockdown model (Experiment 10) that inhibiting necroptosis can restore the mitochondrial membrane potential (the red / green fluorescence ratio of JC-1 increased by 2.1 times);

[0209] Dynamic monitoring: Develop a temporal gene expression profile based on qRT-PCR (such as DRP1 mRNA was significantly upregulated 6 h after aniline exposure);

[0210] Dose optimization: Determine that 20 mg / kg melatonin is the optimal dose (ALT / AST decreased by 50 - 65% in Example 4), breaking through the limitation of dose ambiguity in traditional research;

[0211] Combined therapy: Develop a synergistic regimen of "melatonin + antibiotic" (the therapeutic effect increased by 30% after microbiota clearance in Example 3), reducing intestinal interfering factors;

[0212] Safety verification: The safety of targeting intestinal FXR was confirmed through the AAV-FXR overexpression model (Example 5), with no significant extrahepatic toxicity;

[0213]

[0214] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered by the protection scope of the present invention.

Claims

1. A method for activating FXR with melatonin to alleviate aniline liver damage, characterized in that: include: S1. Administering an effective dose of melatonin to an aniline-exposed individual or animal model; S2. Verify the activation effect of melatonin on intestinal farnesoid receptor (FXR) by detecting the expression or activity level of FXR; S3. Evaluate the effect of melatonin on aniline-induced programmed liver necrosis, mitochondrial dynamics disorder and liver damage; S4. Combine intestinal flora regulation and mitochondrial function repair to achieve synergistic treatment of liver injury.

2. The method according to claim 1, wherein: The effective dose in step S1 is 0.1-50 mg / kg body weight per day, preferably 20 mg / kg, and the administration route is oral or intraperitoneal injection.

3. The method of claim 1, wherein: In step S2, FXR activation is verified by: 2.

1. Western Blot was used to detect the expression of FXR protein in intestinal tissue; 2.

2. Detection of FXR transcriptional activity by luciferase reporter gene assay; 2.

3. Combined with immunohistochemistry, the localization of FXR in intestinal epithelial cells was analyzed.

4. The method of claim 1, wherein: The evaluation in step S3 includes: 3.

1. Detect serum ALT, AST, and LDH levels; 3.

2. Transmission electron microscopy was used to observe the morphology and membrane potential changes of hepatocyte mitochondria; 3.

3. Detect the expression levels of programmed necrosis marker proteins (RIPK1, RIPK3, MLKL and their phosphorylated forms); 3.

4. Analyze the mRNA and protein expression of mitochondrial dynamics-related proteins (Mfn1, Mfn2, OPA1, DRP1, FIS1).

5. The method of activating FXR with melatonin to alleviate aniline liver injury according to claim 1, characterized in that: The intestinal flora regulation in step S4 includes: 4.

1. Restoring intestinal flora homeostasis through fecal microbiota transplantation (FMT); 4.

2. Supplement probiotics (such as Lactobacillus and Bifidobacterium) to enhance intestinal barrier function; 4.

3. Detect the expression levels of intestinal tight junction proteins (Claudin, Occludin, ZO-1).

6. The method of claim 1, wherein melatonin activates FXR to alleviate aniline liver injury, The animal model in step S1 is SPF-grade C57BL / 6 mice, and the aniline exposure dose is 10-40 mg / kg / day for 14 days.

7. The method of claim 1, wherein: The method further comprises an in vitro validation step: 5.

1. Extract mouse primary hepatocytes and detect mitochondrial membrane potential using JC-1 probe; 5.

2. Detect mitochondrial reactive oxygen species (ROS) levels using mitoSOX probe combined with flow cytometry; 5.

3. Use the RIPK1 gene intervention model to verify the regulatory effect of programmed necrosis on mitochondrial dynamics.

8. The method of claim 1, wherein: The melatonin is used in combination with antibiotics to eliminate the interference of intestinal flora on FXR signaling. The specific scheme is as follows: 6.

1. Clear intestinal flora with broad-spectrum antibiotics (such as ampicillin and vancomycin) 3 days before melatonin administration; 6.

2. Analyze changes in bacterial flora composition using 16S rRNA sequencing.

9. The method of claim 1, wherein: The method also includes the following treatment optimization strategies: 7.

1. Dynamically adjust melatonin dosage according to intestinal FXR expression levels; 7.

2. Combine with mitochondrial targeted antioxidants (such as MitoQ) to enhance the protective effect; 7.

3. Verify the necessity of FXR in the melatonin protection mechanism through AAV-FXR gene overexpression model.

10. The method according to any one of claims 1 to 9, characterized in that: Applications of the method include: 8.

1. Prevention or treatment of chronic liver damage caused by occupational aniline exposure; 8.

2. As an auxiliary treatment for chemical liver damage; 8.

3. Used to improve metabolic diseases related to mitochondrial dysfunction (such as non-alcoholic fatty liver disease and liver fibrosis).

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