Metabolites for mafld based on multi-omics joint analysis and applications thereof

Abstract

The application belongs to the technical field of biological pharmacy, and provides metabolites for MAFLD based on multi-omics joint analysis and application thereof, wherein the metabolites include metabolites of Bacteroides uniformis bacteria for preparing a drug for treating MAFLD, the metabolites of Bacteroides uniformis bacteria are hexadecanedioic acid, and the application is an application of HDA in preparation of a drug for treating and / or relieving MAFLD. The application verifies a regulation effect of XBP1 as a key target molecule of HDA intervention on lipid metabolism disorder and ferroptosis in the MAFLD course, and verifies a relief effect of HDA on MAFLD in mice, thereby providing a theoretical basis and practical guidance for development of a new type of MAFLD treatment drug, and having important clinical significance.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to metabolites and their applications for MAFLD based on multi-omics joint analysis. Background Technology

[0002] A diagnosis of metabolic associated fatty liver disease (MAFLD) requires two core criteria: first, the patient's liver fat accumulation must reach or exceed the 5% threshold; second, it must be accompanied by at least one metabolic abnormality, which may manifest as obesity, type 2 diabetes, or other metabolic disorders. Studies show that the pathological changes in liver tissue of MAFLD patients are generally more severe than those of patients with non-alcoholic fatty liver disease (NAFLD), and the clinical prognosis is poorer. MAFLD presents with diverse and complex clinical manifestations, and as the disease progresses, patients may develop serious complications such as hepatitis, liver fibrosis, and even cirrhosis. It is often accompanied by other metabolic diseases, such as diabetes and hypertension. It is also associated with a variety of additional clinical manifestations, including obesity, insulin resistance (IR), and chronic kidney disease. In clinical practice, the diagnosis of MAFLD often relies on a combination of imaging examinations and biochemical indicators. Ultrasound examination is the most commonly used initial screening tool, while liver biopsy is considered the gold standard for diagnosis, although non-invasive testing methods are gradually gaining acceptance. Furthermore, there is currently a lack of effective drug treatments for MAFLD, as well as effective biomarkers to monitor treatment efficacy and disease progression. Therefore, early identification and classification of MAFLD are crucial for developing effective treatment plans.

[0003] As research into the interaction between gut microbiota composition and the pathogenesis of MAFLD deepens, researchers are gradually discovering potential novel therapeutic targets. These findings are expected to lead to breakthroughs in improving the long-term management and prognosis of MAFLD. In recent years, significant breakthroughs in omics methods have provided opportunities to discover MAFLD biomarkers in various biological specimens using technological advancements. These advances help identify and stratify the risk of MAFLD patients. Studies have shown that metabolomics can identify MAFLD-related biomarkers by analyzing changes in plasma and liver metabolites, thus providing a basis for early diagnosis and treatment. Therefore, through metabolomics analysis, researchers can better understand the sex differences in MAFLD and their role in disease progression, providing a new perspective for personalized treatment.

[0004] This invention aims to explore the gut microbiota characteristics of MAFLD patients and their relationship with metabolic disorders through multi-omics analysis. Fecal and plasma samples were collected from MAFLD patients and healthy controls (HC), and fecal metagenomic sequencing and plasma metabolomics analysis were performed, respectively. Furthermore, using a high-fat diet-induced MAFLD mouse model, the effects of key metabolites of *Bacteroides uniformis* on liver damage, steatosis, and inflammatory responses were evaluated. This provides a theoretical basis and practical guidance for the development of novel MAFLD therapeutics. Summary of the Invention

[0005] The purpose of this invention is to provide metabolites for MAFLD based on multi-omics joint analysis and their applications; to identify biomarkers for MAFLD diagnosis and / or risk prediction through fecal metagenomic sequencing and plasma metabolomics analysis; to identify key metabolites of Bacteroides uniformis that alleviate MAFLD through a high-fat diet-induced MAFLD mouse model, and to verify the pathways of key metabolites in alleviating MAFLD using an in vitro MAFLD model constructed by induction and in vivo mouse experiments; and to provide a theoretical basis and practical guidance for the development of novel diagnostic methods and therapeutic drugs for MAFLD.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides metabolites for MAFLD based on multi-omics joint analysis, the metabolites including metabolites of Bacteroides uniformis used to prepare drugs for treating MAFLD, wherein the metabolite of Bacteroides uniformis is hexadecanoic acid (HDA).

[0008] Preferably, the metabolites further include any one or more of the following as diagnostic biomarkers for MAFLD: indolepyruvate, pyrrolidine, β-alanine, β-hydroxylated derivatives of phenylalanine, anecholine, γ-glutamyl isoleucine, lysine protease (P-18:1(9Z)), phenobarbital, soy isoflavones, and lysine protease (20:0 / 0:0).

[0009] The present invention also provides the use of the above metabolites in the preparation of medicaments for treating and / or alleviating MAFLD.

[0010] Preferably, the metabolite is hexadecanoic acid, which alleviates MAFLD symptoms by downregulating the XBP1-Hrd1 pathway and simultaneously activating the Nrf2 / SLC7A11 / GPX4 signaling axis to inhibit ferroptosis.

[0011] Preferably, the amount of hexadecanoic acid used is 40–80 μg / mL.

[0012] The present invention also provides the use of the above-mentioned metabolites as biomarkers in the preparation of detection kits for MAFLD diagnosis and / or risk prediction.

[0013] The present invention also provides the use of the above-mentioned metabolites as biomarkers in the preparation of MAFLD diagnostic and / or risk prediction products.

[0014] The present invention also provides the use of the above-mentioned metabolites as biomarkers in screening drugs for the treatment and / or relief of MAFLD.

[0015] Preferably, the metabolite used as a biomarker is derived from plasma.

[0016] The present invention also provides the application of a biomarker in the preparation of a diagnostic kit for MAFLD and / or risk prediction, wherein the biomarker is any one or more of the metabolites mentioned above as biomarkers.

[0017] Preferably, the biomarker is a combination of all the metabolites mentioned above that serve as biomarkers.

[0018] Preferably, the biomarkers also include any one or more of the following bacteria: s__Bacteroides_uniformis, s__Dialister_invisus, s__Phocaeicola_dorei, s__Collinsella_aerofaciens, s__Dorea_longicatena, and s__Clostridium_sp_AM22_11AC.

[0019] Preferably, the biomarker further includes a combination of bacteria s__Bacteroides_uniformis, s__Dialister_invisus, s__Phocaeicola_dorei, s__Collinsella_aerofaciens, s__Dorea_longicatena, and s__Clostridium_sp_AM22_11AC.

[0020] The beneficial effects of this invention are:

[0021] 1. Currently, MAFLD treatment mainly relies on lifestyle interventions and dietary control, and effective drug therapy is still lacking. This invention identifies HDA as a key metabolite that can alleviate MAFLD symptoms by downregulating the XBP1-Hrd1 pathway and simultaneously activating the Nrf2 / SLC7A11 / GPX4 signaling axis to inhibit ferroptosis. Using a palmitic acid / oleic acid (PA / OA) mixture-induced MAFLD model in the human hepatocellular carcinoma cell line HepG2 and a MAFLD mouse model as research models, this study further verifies the regulatory role of HDA on lipid metabolism disorders and ferroptosis in the in vitro and in vivo course of MAFLD. This provides a theoretical basis and practical guidance for the development of novel MAFLD therapeutics and has significant clinical implications.

[0022] 2. This invention identifies potential biomarkers in the microbiome of MAFLD patients through metagenomic sequencing of human feces. By performing non-target metabolomics on plasma from MAFLD patients and controls, it clarifies potential biomarkers and related metabolic pathways in the plasma of MAFLD patients, providing a scientific basis for the pathogenesis and early screening of MAFLD. Attached Figure Description

[0023] Figure 1 The graph shows the LEfSe analysis of fecal differential flora between MAFLD patients and HC ((a) is a bar chart of LDA value distribution, (b) is a phylogenetic branching diagram);

[0024] Figure 2 The volcano plots show the HMDB classification annotations and differential expression of plasma metabolites in MAFLD patients and the HC group ((a) shows the HMDB classification annotations of plasma metabolites, and (b) shows the differential expression volcano plots of plasma metabolites).

[0025] Figure 3 The diagram shows the diagnostic models for MAFLD ((a) is a MAFLD diagnostic model based on combined plasma metabolites, (b) is a MAFLD diagnostic model based on microbiota, and (c) is a MAFLD diagnostic model based on microbiota + combined plasma metabolites).

[0026] Figure 4 Bu bacteria alleviated liver lipid deposition in a MAFLD mouse model (Oil Red O staining and white light microscopy).

[0027] Figure 5 These are common differential metabolites of Bu bacteria that exert a remission effect in MAFLD mice;

[0028] Figure 6 HDA alleviates lipid deposition in HepG2 cells, a MAFLD model in vitro (results of cell staining with Oil Red O followed by white light microscopy).

[0029] Figure 7 The effect of HDA on mitochondrial ferroptosis in an in vitro HepG2 cell MAFLD model was observed by transmission electron microscopy (electron microscope model: JEM1400, acceleration voltage: 80kV, 5000k and 10000k indicate the magnification of the electron microscope image).

[0030] Figure 8 The effect of HDA on the expression levels of XBP1, Nrf2, Hrd1, SLC7A11, and GPX4 mRNA in an in vitro HepG2 cell MAFLD model (compared with the Con group, ***P<0.001, ****P<0.0001; compared with the Mod group, ##P<0.01, ###P<0.001, ####P<0.0001; compared with the Mod+Erastin group, &P<0.05, &&P<0.01, &&&&P<0.0001).

[0031] Figure 9 HDA is used for Western blotting analysis of XBP1, Nrf2, Hrd1, SLC7A11, and GPX4 proteins in an in vitro HepG2 cell MAFLD model and quantitative analysis of protein expression levels using grayscale values.

[0032] Figure 10 The study found that live Bu bacteria, Bu bacteria supernatant, and exogenous HDA alleviated liver lipid deposition in a MAFLD mouse model (Oil Red O staining and white light microscopy).

[0033] Figure 11 The effect of HDA on mitochondrial ferroptosis in hepatocytes of MAFLD mice was observed by transmission electron microscopy (electron microscope model: JEM1400, accelerating voltage: 80kV, 5000k and 10000k indicate the magnification of the electron microscope image).

[0034] Figure 12 The effect of HDA on the expression levels of XBP1, Nrf2, Hrd1, SLC7A11, and GPX4 mRNA in MAFLD mice in vitro (compared with the NC group, ***P<0.001, ****P<0.0001; compared with the PBS group, ##P<0.01, ###P<0.001).

[0035] Figure 13 This is a quantitative analysis of the Western Blot images and gray values ​​of XBP1, Nrf2, Hrd1, SLC7A11, and GPX4 proteins in MAFLD mice using HDA. Detailed Implementation

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] Example 1: Metagenomic Analysis of Gut Bacteria in MAFLD Patients

[0040] 1. Objects and Methods

[0041] 1.1 Object

[0042] A total of 120 healthy controls and 120 patients with metabolic-associated fatty liver disease (MAFLD) were recruited.

[0043] 1.2 Shotgun metagenomic sequencing of gut bacteria

[0044] Sequencing and bioinformatics analysis were completed using Shanghai Baiqu Biomedical Technology Co., Ltd. Specifically, this included the following:

[0045] Fecal samples were collected, and fecal DNA was extracted by Novogene Bioinformatics Co., Ltd. (Beijing, China) using the SDS method. Finally, all samples were sequenced in pairs using the Illumina platform.

[0046] Metagenome samples were identified and analyzed using the MetaPhlAn analysis platform. The LEfSe software was used to determine the characteristics most likely to explain the differences between groups, and the results were displayed visually in the form of a phylogenetic chart.

[0047] Gut microbiota analysis results of the 2MAFLD group and HC group

[0048] Differential microbiota analysis was performed using the LEfSe algorithm (linear discriminant analysis of effect size), with an LDA value of 2 as the screening threshold, to identify key microbiota that could distinguish different groups at each taxonomic level (see [link to analysis]). Figure 1(a)-(b)). Analysis revealed that Bacteroides was significantly enriched in the healthy control group (LDA = 4.479), making it the most significant distinguishing phylum from the MAFLD group. Compared to the MAFLD group, the genera *g__Bacteroides* (LDA = 4.094) was significantly enriched in the HC group and played a crucial role in taxonomy. Compared to the HC group, the prominent genera in the MAFLD group were *g__Lachnospiraceae_unclassified* (LDA = 3.903), *g__Collinsella* (LDA = 3.566), *g__Dorea* (LDA = 3.274), and *g__Veillonella* (LDA = 2.806). Compared to the MAFLD group, the prominent species in the HC group were *s__Bacteroides_uniformis* (LDA = 3.818), *s__Dialister_invisus* (LDA = 3.263), and *s__Phocaeicola_dorei* (LDA = 3.023). Compared to HC, the prominent species in the MAFLD group were s__Collinsella_aerofaciens (LDA = 3.536), s__Dorea_longicatena (LDA = 3.115), and s__Clostridium_sp_AM22_11AC (LDA = 2.954).

[0049] Example 2: Plasma Non-target Metabolomics in MAFLD Patients

[0050] 1 Experimental Methods

[0051] The detection and bioinformatics analysis of plasma non-target metabolism in the subjects studied in Example 1 were completed using Shanghai Baiqu Biomedical Technology Co., Ltd., as detailed below:

[0052] 100 μL of plasma sample was transferred to an EP tube, then methanol extract was added, mixed, and allowed to stand. The mixture was then centrifuged for 15 minutes. The supernatant was collected and placed in a sample vial for analysis. Chromatographic separation was performed using a Thermo Fisher Scientific Vanquish ultra-high performance liquid chromatography system with a Waters ACQUITYUPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm). Chromatographic and mass spectrometric analyses were then performed.

[0053] The raw data was converted to mzXML format using the open-source software ProteoWizard. Then, an R language package (based on the XCMS core) was used to perform data processing including peak identification, extraction, alignment, and integration. Matching was performed with a self-built secondary mass spectrometry library in BiotreeDB (V2.1), with the matching algorithm's cutoff value set to 0.3. Metabolite identification was then completed.

[0054] Metabolomics data analysis: After preprocessing the raw data, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to assess differences between groups and similarities within groups. Student's t-test was applied to calculate the significance of metabolite differences between the two groups, and changes in fold expression were analyzed for double screening.

[0055] 2 Experimental Results

[0056] The identified differentially metabolites were categorized using the HMDB compound classification system (Superclass level). Analysis showed that these differentially metabolites mainly belong to two major categories: lipids and lipid-like compounds, and organic acids and their derivatives (see [link to HMDB classification system]). Figure 2 (a)). Based on VIP value (see Figure 2 (b) Showing the top 10 differentially expressed metabolites (all with VIP values ​​greater than 3.46), six of which were upregulated: indolepyruvate (VIP = 4.3683, P = 4.97E-13), pyrrolidine (VIP = 4.1661, P = 2.27E-09), β-Alanine (VIP = 3.7988, P = 0.00000000187), β-hydroxylated derivative of DL-Dopa phenylalanine (VIP = 6.89E-10, P = 3.22E-07), reticuline (VIP = 3.5883, P = 0.00002807), gamma- Glutamylisoleucine γ-glutamyl isoleucine (VIP = 3.4643, P = 0.00000005559); the four differentially regulated metabolites were LysoPC (P-18:1(9Z)) lysine protease (P-18:1(9Z)) (VIP = 3.8085, P = 1.62E-10), Phenobarbital (VIP = 3.6712, P = 1.63E-07), Daidzein (VIP = 3.5585, P = 1.29E-09), and LysoPC (20:0 / 0:0) lysine protease (20:0 / 0:0) (VIP = 3.4780, P = 1.07E-09).

[0057] Example 3: Validation of the diagnostic efficacy of microbial community and metabolomics analysis for MAFLD

[0058] The receiver operating characteristics (ROC) of the differentially expressed microorganisms screened in Example 1 and the differentially expressed plasma metabolites screened in Example 2 were analyzed using the R package "pROC". ROC curves were plotted to evaluate the diagnostic efficacy of microbiome and metabolomics for MAFLD.

[0059] The metabolites with the highest AUC values ​​for individual metabolites were Indolepyruvate (0.78), Daidzein (0.74), and LysoPC (P-181(9Z)) (0.74) (see Table 1). The combined metabolite MAFLD diagnostic model had a higher AUC value (0.91) than the microbial AUC value (0.8). Multi-omics data (microbial community + metabolite) had better predictive value than single-omics data, with an AUC value of 0.93 (see Table 1). Figure 3 ).

[0060] The results showed that individual metabolites such as Indolepyruvate, Daidzein, and LysoPC (P-181(9Z)) can be used to diagnose MAFLD. Combined predictive models of metabolites and the combination of metabolites and microorganisms showed high accuracy in diagnosing MAFLD. The accuracy of combined metabolite predictions was superior to that of microbial community, and multi-omics data (combined metabolite + microbial community) had higher predictive value than single-omics data. In other words, plasma metabolomics and microbial community can both serve as biomarkers for the diagnosis of MAFLD.

[0061] Table 1 Results of the single metabolite diagnostic model

[0062] Metabolite name AUC Specificity Sensitivity Indolepyruvate 0.7751 0.7417 0.7250 pyrrolidine 0.7300 0.6291 0.7042 β-alanine 0.7208 0.5500 0.8167 β-hydroxylated derivatives of phenylalanine 0.7328 0.7167 0.6833 γ-Glutamyl isoleucine 0.7113 0.5917 0.7583 LysoPC(P-18:1(9Z)) 0.7384 0.5896 0.7854 Phenobarbital 0.6827 0.7583 0.5500 Soy isoflavones 0.7383 0.8542 0.5875 LysoPC(20:0 / 0:0) 0.7151 0.7458 0.6458

[0063] Example 4: Constructing an in vivo model to analyze the metabolites of Bu bacteria that exert their effects in MAFLD mice.

[0064] 1. Experimental Materials

[0065] Experimental strain: Bacteroides uniformis (Bu) BNCC139204 was purchased from Beina Innovation Biotechnology Co., Ltd.

[0066] Laboratory animals: Healthy male C57BL / 6J mice were purchased from Spiford (Beijing) Biotechnology Co., Ltd. Mice maintenance diet and Western diet D12492 (60% high fat) were purchased from Beijing Botai Hongda Biotechnology Co., Ltd.

[0067] 2 Experimental Methods

[0068] Twenty-four C57BL / 6J mice were housed under standard laboratory conditions: 25±1℃, relative humidity 55±5%, alternating 12h light and 12h dark cycles, and free access to drinking water. After one week of acclimatization, a mixture of antibiotics (vancomycin, 100 mg / kg; neomycin sulfate, metronidazole, and ampicillin, 200 mg / kg) was administered to the mice via gavage in 200 μL every morning for 7 consecutive days. Fecal smear assays and 16S rRNA testing further confirmed the formation of germ-free mice (ABX mice). The 24 mice were randomly divided into three groups: NC, high-fat diet (HFD), and Bu. The NC group was fed a standard diet for 12 weeks, 3 times a week. The other two groups were fed a high-fat diet (D12492, 60% high-fat) for 12 weeks, 3 times a week. The Bu group was gavaged every other day, concurrently with the establishment of the fatty liver model, for a period of 12 weeks. The gavage substance contained approximately 2 × 10⁻⁶ bacteria. 8 Mice were given PBS containing cfu Bu bacteria, while other groups were given approximately 200 μL of PBS by gavage.

[0069] Hepatic and Escherichia coli (H&E) staining and Oil Red staining were used to analyze pathological changes in liver tissue. Real-time quantitative PCR and Western blotting were used to detect the expression levels of liver-related genes (XBP1, Hrd1, Nrf2, SLC7A11, and GPX4). Metabolomics studies were performed on mouse feces, plasma, liver tissue, and Bubacillus supernatant using GC-MS, following methods partially identical to those in Example 2.

[0070] 3 Results

[0071] The liver oil red and hematoxylin staining of mice in the Bu intervention group were improved compared with those in the HFD group (see...). Figure 4 Intersection of metabolites from in vitro Bu culture supernatant, reversible plasma metabolites after Bu intervention, fecal metabolites, liver metabolites, and differentially expressed plasma metabolites from MAFLD patients revealed that hexadecanoic acid (HDA) was the only trend-compliant beneficial metabolite intersection (see [link to article]). Figure 5 In other words, the common differential metabolite that Bacteroides uniformis uses to alleviate symptoms in MAFLD mice is HDA. A search of the KEGG database for Bacteroides uniformis annotated genes revealed the presence of the fabZ gene, which catalyzes the dehydration of 3-hydroxyacyl-ACP (HDA precursor) to 2,3-enoyl-ACP, a necessary step in HDA synthesis.

[0072] Example 5: Application of DHA in in vitro models and in mice

[0073] 1. Materials and Methods

[0074] 1.1 Materials

[0075] Experimental cell line: Human HepG2 was purchased from Wuhan Saibaikang (Shanghai) Biotechnology Co., Ltd. (Catalog No.: iCell-h092 (STR identification)).

[0076] HDA working solution: Weigh 1 mg of HDA and dissolve it in 100 μL of ethanol to prepare a 1 mg / ml solution, then dilute with water / culture medium.

[0077] OA / PA fatty liver modeling working solution:

[0078] OA: Stock solution concentration 12mM → Effective concentration 0.5mM, diluted 24 times; PA: Stock solution concentration 6mM → Effective concentration 0.25mM, diluted 24 times; Total volume of 24 portions = 1 portion OA + 1 portion PA + 22 portions complete culture medium. Taking 1ml as an example, OA plus 1 / 24 = 0.0417ml, PA plus 1.2 / 24 = 0.0417ml, the remainder is culture medium.

[0079] 1.2 Methods

[0080] Using the human hepatocellular carcinoma cell line HepG2 as a research model, an in vitro MAFLD model was constructed by inducing with a mixture of palmitic acid / oleic acid (PA / OA). Lipid deposition was observed by Oil Red O staining, and the improvement effect of HDA on the in vitro MAFLD model was confirmed by combining PCR, immunoblotting analysis and electron microscopy.

[0081] (1) Cell experiments

[0082] 1) HepG2 cells were induced with 1 mM PA / OA fatty acid modeling solution for 24 h. HDA concentrations of 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 320 μg / mL, 160 μg / mL, 80 μg / mL, 40 μg / mL, 20 μg / mL, and 10 μg / mL were administered to the cells. Cell viability was assessed using CCK8 assay. Experimental groups: ① Control group (Con): HepG2 cells; ② Model group (Mod): HepG2 cells + 1 mM PA / OA fatty acid modeling solution for 24 h; ③ Experimental groups: Mod + 20 μg / mL HDA, Mod + 40 μg / mL HDA, Mod + 60 μg / mL HDA, and Mod + 80 μg / mL HDA.

[0083] 2) Based on the results of step 1), the final experimental groups were obtained: ① Blank group (Con): HepG2 cells; ② Model group (Mod): HepG2 cells + PA / OA1 mM fatty acid modeling solution for 24 h; ③ Low-dose HDA group (Mod+HDA-L): HepG2 cells + HDA 40 μg / mL + pre-administered PA / OA1 mM fatty acid modeling solution for 24 h; ④ High-dose HDA group (Mod+HDA-H): HepG2 cells + HDA 80 μg / mL + pre-administered PA / OA1 mM fatty acid modeling solution for 24 h; ⑤ Ferraphobia induction group (Mod+Erastin): HepG2 cells + PA / OA1 mM fatty acid modeling solution for 24 h + Erastin 10 μmol / L; ⑥ Ferraphobia induction group + Low-dose HDA group (Mod+Erastin+HDA-L): HepG2 cells + PA / OA1 mM fatty acid modeling solution for 24 h + Erastin 10 μmol / L. mM fatty acid modeling solution damage for 24h + Erastin 10μmol / L + HDA 40μg / mL; ⑦ Ferrocyte induction group + high-dose HDA group (Mod + Erastin + HDA-H): HepG2 cells + PPA / OA1 mM fatty acid modeling solution damage for 24h + Erastin 10μmol / L + HDA 80μg / mL.

[0084] (2) Animal experiments

[0085] Fifty-three C57BL / 6J mice were housed under the same conditions as before. After one week of acclimatization, the mice were administered an antibiotic mixture to form germ-free mice (ABX mice) according to the same protocol. The 53 mice were divided into 9 groups: NC control group (n=9), HFD high-fat diet group (n=8), PBS buffer group (n=5), Bu live bacteria group (n=6), inact Bu inactivated bacteria group (n=5), Bu_SN bacterial supernatant group (n=5), CBA sterile culture medium group (n=5), HDA-L low-dose exogenous HDA group (n=5), and HDA-H high-dose exogenous HDA group (n=5). The NC group mice were fed a normal diet for 12 weeks, while the other groups were fed a high-fat diet D12492 (60% high fat) for 12 weeks. Mice were administered PBS via gavage every other day, concurrently with the establishment of the fatty liver model, for a period of 12 weeks. Groups 1 and 3 were given 200 μL of PBS buffer by gavage, while the Bu, inact Bu, Bu_SN, CBA, HDA-L, and HDA-H groups were given 0.2 ml of PBS containing 2×10⁻⁶ mg / L of PBS by gavage. 8 CFU contains Bacillus subtilis, with a concentration of 2×10⁻⁶. 8 CFU-inactivated Bacillus subtilis, Bacillus subtilis supernatant, Bacillus subtilis-free Columbia Blood Agar (CBA), 8 mg low-dose HDA, 16 mg high-dose HDA.

[0086] Electron microscopy was used to observe signs of mitochondrial ferroptosis, H&E staining was used to analyze pathological changes in colon tissue, and real-time quantitative PCR and Western blotting were used to detect the expression levels of liver-related genes (XBP1, Hrd1, Nrf2, SLC7A11 and GPX4).

[0087] 2 Results

[0088] 2.1 HDA can alleviate lipid deposition and hepatocyte damage in HepG2 cells, a MAFLD model, in vitro.

[0089] like Figure 6 It can be seen that, compared with the model group, the lipid deposition and hepatocyte damage in HepG2 cells were lowest when the HDA concentration was 80ug / mL and 40ug / mL. Therefore, 80ug / mL and 40ug / mL HDA were selected for subsequent mechanism studies.

[0090] 2.2 Effects of HDA on oxidative stress and ferroptosis in HepG2 cell MAFLD model

[0091] Compared with the Control group, the Mod group and the Mod+Erastin group showed upregulated mitochondrial ferroptosis under electron microscopy, which was reversed in the HDA-treated group. The effect was most significant with HDA 80 μg / mL, showing a dose-response relationship (see [link to original text]). Figure 7 ).

[0092] 2.3 HDA downregulates ferroptosis via XBP1-Hrd1-Nrf2 / SLC7A11 / GPX4, delaying MAFLD in an in vitro HepG2 cell model.

[0093] Compared with the Control group, the Mod group and the Mod+Erastin group showed significantly increased XBP1 / Hrd1 mRNA and protein expression levels, and decreased Nrf2 / SLC7A11 / GPX4 gene and protein expression levels. These effects were reversed after HDA administration, with HDA at 80 μg / mL showing the most significant effect and exhibiting a dose-response relationship (see [link to original text]). Figure 8-9 ).

[0094] 2.4 Effects of live bacteria Bu, inactivated bacteria inact Bu, bacterial supernatant Bu_SN, sterile culture medium CBA and exogenous HDA on liver function and blood lipids in MAFLD mice

[0095] Except for the Inact Bu and CBA groups, the Bu, Bu_SN, and HDA intervention groups showed lower lipid deposition in the livers of mice compared to the PBS group (see [link to relevant documentation]). Figure 10 ).

[0096] 2.5 The metabolite HDA downregulates signs of mitochondrial ferroptosis in MAFLD mouse hepatocytes

[0097] Compared with NC, liver cells in the PBS group showed mitochondrial abnormalities, such as increased membrane density, outer membrane rupture, and reduced or absent cristae, but did not exhibit apoptosis characteristics such as chromatin condensation, nor cytoplasmic and organelle swelling, plasma membrane rupture, or other signs of cell necrosis, nor the double-membrane vesicle characteristics of autophagy. These characteristics improved after HDA intervention (see [link to HDA ​​intervention]). Figure 11 ).

[0098] 2.6 Metabolite HDA delays the progression of MAFLD in mice by activating XBP1-Hrd1-Nrf2-SLC7A11-GPX4 and downregulating ferroptosis.

[0099] Compared with the NC group, PCR and WB showed that XBP1 and Hrd1 were upregulated and Nrf2-SLC7A11-GPX4 was downregulated in the HFD group. These indicators were reversed after HDA intervention (see [link to HDA ​​intervention]). Figure 12-13 ).

[0100] In summary, the in vitro MAFLD model induced by this invention demonstrates that HDA downregulates ferroptosis through a common mechanism XBP1 / Hrd1 / Nrf2 / SLC7A11 / GPX4, thus delaying MAFLD progression. In vivo mouse experiments further confirm that HDA downregulates ferroptosis through the same mechanism XBP1 / Hrd1 / Nrf2 / SLC7A11 / GPX4, thus delaying MAFLD progression.

[0101] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. The use of the metabolite hexadecanoic acid in the preparation of drugs for treating and / or alleviating MAFLD.

2. The application according to claim 1, characterized in that, The metabolite hexadecanoic acid alleviates MAFLD symptoms by downregulating the XBP1-Hrd1 pathway and simultaneously activating the Nrf2 / SLC7A11 / GPX4 signaling axis to inhibit ferroptosis.

3. The application according to claim 1, characterized in that, The amount of the metabolite hexadecanoic acid used is 40~80 μg / mL.

4. The use of the metabolite hexadecanoic acid as described in claim 1 in screening drugs for the treatment and / or relief of MAFLD.

5. The application according to claim 4, characterized in that, The metabolite hexadecanoic acid is derived from blood plasma.

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