Application of indole-3-formaldehyde in preparation of product for treating type-2 diabetes mellitus complicated with metabolic dysfunction related steatohepatitis

Abstract

The invention discloses application of indole-3-formaldehyde in preparation of a product for treating type-2 diabetes mellitus complicated with metabolic dysfunction related steatohepatitis, and belongs to the technical field of biological medicine. According to the application disclosed by the invention, a T2DM-MASH mouse model is successfully established, not only is the effect of the type 2 diabetes mellitus in acceleration of MASLD fibrosis progress proved, but also a dual treatment mechanism of indole-3-formaldehyde (3-IAld) on the type 2 diabetes mellitus complicated with metabolic dysfunction related steatohepatitis is clarified for the first time; meanwhile, SIRT1 is activated to inhibit a TGF-beta / SMAD signal channel, so that the effect of treating type-2 diabetes mellitus complicated with metabolic dysfunction related steatohepatitis is achieved. These findings provide new therapeutic targets and strategies for the treatment of type-2 diabetes mellitus complicated with metabolic dysfunction related steatohepatitis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of indole-3-carboxaldehyde in the preparation of a product for treating type 2 diabetes mellitus combined with metabolic dysfunction associated steatohepatitis. BACKGROUND

[0002] The progression of liver fibrosis is a key turning point in the pathological mechanism of metabolic dysfunction associated steatohepatitis (MASLD), significantly increasing the risk of decompensated cirrhosis, liver failure and hepatocellular carcinoma, ultimately leading to increased liver-related mortality, cardiovascular mortality and all-cause mortality. Notably, MASLD has a significant epidemiological association with type 2 diabetes mellitus (T2DM). In the metabolic disorder associated steatohepatitis (MASH) cohort, the prevalence of T2DM in the population was 43.63%. A large amount of evidence confirms that type 2 diabetes mellitus (T2DM) is an independent risk factor for the progression of liver fibrosis to end-stage liver disease. This metabolic comorbidity has a bidirectional pathological interaction, synergistically exacerbating liver and extrahepatic disease outcomes, leading to a still gloomy prospect for the treatment of MASLD. Although the FDA approved Rezdiffra as the first targeted therapy for MASH combined with liver fibrosis in March 2024, its clinical application faces many limitations, including high cost and poor efficacy.

[0003] Increasing evidence suggests that the gut microbiota and its metabolites play a key role in the pathogenesis of MASLD. The mechanisms include: (1) regulation of intestinal nutrient absorption and barrier integrity; (2) liver transport of microbial metabolites and endotoxins, promoting steatosis and inflammatory cascades. Notably, the potential therapeutic role of 3-IAld in type 2 diabetes mellitus combined with metabolic dysfunction associated steatohepatitis has not been explored. SUMMARY

[0004] The purpose of the present application is to provide the application of indole-3-carboxaldehyde in the preparation of a product for treating type 2 diabetes mellitus combined with metabolic dysfunction associated steatohepatitis, in order to solve the problems existing in the prior art. The present application successfully established a type 2 diabetes mellitus combined with metabolic dysfunction associated steatohepatitis (T2DM-MASH) mouse model by combined induction of high-fat diet (HFD), carbon tetrachloride (CCl4) and streptozotocin (STZ). Fecal metabolomics analysis first revealed that the level of indole-3-carboxaldehyde (3-IAld) progressively decreased with disease progression, with the most significant decrease in T2DM-MASH mice. This study first systematically explores the therapeutic potential of 3-IAld in T2DM-MASH, aiming to elucidate the research progress of 3-IAld in T2DM-MASH and propose new treatment strategies.

[0005] To achieve the above object, the present application provides the following scheme:

[0006] In a first aspect, the present application provides the use of indole-3-carboxaldehyde in the preparation of a product for treating type 2 diabetes mellitus associated with metabolic dysfunction related fatty liver hepatitis.

[0007] Preferably, the product is a medicament for treating type 2 diabetes mellitus associated with metabolic dysfunction related fatty liver hepatitis.

[0008] Preferably, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

[0009] Preferably, the dosage form of the medicament comprises one or more of emulsion, capsule, pill, tablet, suspension, syrup, ointment, oral liquid, injection, granule.

[0010] In a second aspect, the present application also provides a product for treating type 2 diabetes mellitus associated with metabolic dysfunction related fatty liver hepatitis, which comprises the indole-3-carboxaldehyde.

[0011] Preferably, the dosage form of the product comprises one or more of emulsion, capsule, pill, tablet, suspension, syrup, ointment, oral liquid, injection, granule.

[0012] Preferably, the product further comprises a pharmaceutically acceptable carrier or excipient.

[0013] In a third aspect, the present application also provides the use of the product in the preparation of a medicament for treating type 2 diabetes mellitus associated with metabolic dysfunction related fatty liver hepatitis.

[0014] The present application discloses the following technical effects:

[0015] The present application successfully establishes a T2DM-MASH liver fibrosis mouse model, not only confirms the role of type 2 diabetes mellitus in accelerating the progression of MASLD fibrosis, but more importantly, first elucidates the dual therapeutic mechanism of 3-IAld on type 2 diabetes mellitus associated with metabolic dysfunction related fatty liver hepatitis: by improving the intestinal barrier function to reduce endotoxemia, and simultaneously activating SIRT1 to inhibit the TGF-β / SMAD signaling pathway, so as to achieve the effect of treating type 2 diabetes mellitus associated with metabolic dysfunction related fatty liver hepatitis. These findings provide new therapeutic targets and strategies for the treatment of type 2 diabetes mellitus associated with metabolic dysfunction related fatty liver hepatitis. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description only need to be some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained based on these drawings without any creative work.

[0017] Figure 1 Figure 2 is a diagram of the research results of accelerating the progression of liver fibrosis for type 2 diabetes; wherein A is a diagram of the process of constructing a mouse model in different groups, B-D are the changes of body weight (B), liver weight (C) and the ratio of liver weight to body weight (D) of mice in different groups, E is a diagram of the liver appearance, H&E staining, reticular fiber staining and Masson staining of liver tissue of mice in different groups (scale bar = 100 pm), F is the non-alcoholic fatty liver disease activity score of mice in different groups, G is the liver fibrosis stage score of mice in different groups, H is the fasting blood glucose and serum insulin level of mice in different groups, I-N are the serum AST, ALT, TG, CHOL, GGT and TBIL levels of mice in different groups (data are expressed as mean ± standard error; significance is indicated as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0018] Figure 2 Figure 3 is a principal component analysis diagram of the changes of intestinal-derived metabolites of mice in different groups;

[0019] Figure 3 Figure 4 is a volcano plot identifying differential metabolites diagram (A-C) of the changes of intestinal-derived metabolites of mice in different groups;

[0020] Figure 4 Figure 5 is a K-means clustering analysis of significantly changed metabolites in the intestines of mice in different groups;

[0021] Figure 5 Figure 6 is a heat map of metabolites showing a downward trend in the intestines of mice in different groups;

[0022] Figure 6 Figure 7 is a correlation analysis of 3-IAld and liver inflammation score (A), hepatocyte steatosis score (B), non-alcoholic fatty liver disease activity score (C), fibrosis stage score (D) and blood glucose level (E) of mice in different groups;

[0023] Figure 7Figures for the results of feeding model mice with different doses of 3-IAld; wherein A is the experimental feeding timeline, B-D are the changes in body weight (B), liver weight (C), and liver to body weight ratio (D) of mice in different groups, E are the H&E staining images (top, scale bar = 500 pm) and transmission electron microscopy images (bottom, scale bar = 500 nm) of colon tissue sections of mice in different groups, F is the mRNA level analysis of ZO-1 and Occludin of mice in different groups, G is the gross image of colon (left) and quantitative analysis of colon length (right) of mice in different groups, H is the LPS and TNF-a levels in serum of mice in different groups (data are expressed as mean ± standard error; *p < 0.05, **p < 0.01, ***p < 0.001 compared with the CON group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the T2DM-MASH group);

[0024] Figure 8 Figures for the results of 3-IAld improving liver fibrosis in T2DM-MASH mice; wherein A are the liver appearance, H&E staining, reticular fiber staining, and Masson staining images (scale bar = 100 pm) of liver tissue of mice in different groups, B-G are the serum AST, ALT, TG, CHOL, GGT, and TBIL levels of mice in different groups, H are the fasting blood glucose and serum insulin levels of mice in different groups (data are expressed as mean ± standard error; *p < 0.05, **p < 0.01, ***p < 0.001 compared with the CON group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the T2DM-MASH group);

[0025] Figure 9 Figures for 3-IAld reducing liver fibrosis in DM-MASH mice through the SIRT1 / TGF-β / SMAD pathway; wherein A are the staining images of a-SMA and TGF-β1 in liver tissue sections of mice in different groups (scale bar = 100 pm), B is the molecular docking analysis image, C is the Western blot analysis result, D is the mRNA level analysis results of TIMP1, SIRT1, TGF-β1, and a-SMA (data are expressed as mean ± standard error; *p < 0.05, **p < 0.01, ***p < 0.001). DETAILED DESCRIPTION

[0026] The various illustrative embodiments of the present application will now be described in detail below. This description should be considered in connection with the accompanying drawings, which are included to provide a description of the various illustrative embodiments of the present application, and are not intended to limit the present application in any way.

[0027] Example 1

[0028] 3-IAld solution was prepared according to a previously reported method (Swann, A., Gifford, C. R., DeFilippis, Z., Langara, S., Sharma, A., Urevska Antonova, A., et al. Intestinal microbiota-produced indole limits graft-versus-host disease through the type I interferon signaling pathway. Blood. 2018; 132(23): 2506-2519. doi: 10.1182 / blood-2018-03-838193.).

[0029] Standard chow (carbohydrate 70% kcal, protein 20% kcal, fat 10% kcal), high-fat chow (HFD; fat 60% of total calories, carbohydrate 20% of total calories, protein 20% of total calories) were purchased from Sino-Biotech (Tianjin) Co., Ltd.; streptozotocin (STZ) was purchased from Sigma-Aldrich; anti-α-SMA antibody was purchased from Affinity Biosciences (Cat. No. BF9212); anti-TGF-β1 antibody was purchased from Wuhan Say Imag Biotech Co., Ltd. (proteintech, Cat. No. 81746-2-RR); AMCA-AffiniPure goat anti-rabbit IgG was purchased from Jackson ImmunoResearch (Cat. No. 111-155-003); TNF-α (Cat. No. HA722022), MMP1 (Cat. No. HA723118), and TIMP1 (Cat. No. HA721741) were purchased from Hangzhou Huaan Biotechnology Co., Ltd., P-SMAD2 (Cat. No. 110472-RO010), α-SMA (Cat. No. A5835), and SMAD (Cat. No. 100424-T36) were purchased from Beijing Yiqioshengzhou Biotechnology Co., Ltd., SIRT1 (Cat. No. S0B1328) and SMAD2 (Cat. No. SOB0947) were purchased from Hangzhou Sutai Biotechnology Co., Ltd., P-SMAD3 was purchased from Suzhou Sizuobai Biotechnology Co., Ltd. (Cat. No. R380775), and TGFβ1 was purchased from Hangzhou Zhenyoupin Biotechnology Co., Ltd. (Cat. No. YP-mAb-15967).

[0030] 1. Experimental methods

[0031] 1.1 Mouse source and model construction method

[0032] Male C57BL / 6L mice (6-8 weeks old) were purchased from Beijing Verh Laboratory Animal Technology Co., Ltd. All animals were raised under specific conditions, with a 12-hour light-dark cycle, temperature 22-25℃, free access to water and food. Before the experiment, the mice were adapted to the environment for one week. All experimental operations were in accordance with the “China Experimental Animal Management Technical Regulations” and the experimental protocol approved by the Animal Ethics Committee of Tianjin Medical University (Approval No. KY2025K162).

[0033] Mice were randomly divided into three groups: control group (CON), MASH group and T2DM-MASH group. CON group mice were fed with standard chow for 14 weeks; MASH model group mice were fed with high-fat diet (HFD) for 8 weeks, then intraperitoneal injection of 5% CCl4 (2 mL / kg) twice a week for a total of 6 weeks (injection at 1 pm and 4 pm every week), while continuing to feed HFD for 6 weeks; T2DM-MASH model group mice were first fed with HFD for 8 weeks, then fasted for 12 hours, then intraperitoneal injection of STZ (40 mg / kg, dissolved in 0.1 M citric acid buffer, pH = 4.5) for 3 consecutive days, at this time the CON group and the MASH group received the same volume of citric acid buffer. One week after STZ injection, mice with blood glucose levels > 16.7 mmol / L (300 mg / dL) and accompanied by polydipsia, polyuria, and polyphagia were diagnosed as diabetic, and were fed with CCl4 (same as above) combined with high-fat diet for 6 weeks.

[0034] To explore the protective effect of 3-IAld on T2DM-MASH model mice, diabetic mice were randomly divided into three groups one week after STZ injection: low-dose 3-IAld group (25 mg / kg / day, L 3-IAld), high-dose 3-IAld group (50 mg / kg / day, H 3-IAld) and control group (T2DM-MASH group without 3-IAld). Treatment was administered once daily by oral gavage for 6 weeks until the completion of T2DM-MASH induction.

[0035] The body weight and blood glucose level of the experimental mice were monitored every week. At week 16, the mice were euthanized, the whole liver and colon were excised, weighed and photographed. One cm sections of colon and liver tissue were taken for histological analysis, and the remaining portions were rapidly frozen in liquid nitrogen and stored at -80°C for subsequent testing. Faecal samples were collected for intestinal metabolite analysis, and serum samples were collected for subsequent studies.

[0036] 1.2 Histological staining and immunohistochemical analysis

[0037] The obtained liver and colon tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 5 pm. The colon sections were stained with H&E to evaluate histological changes. The liver sections were also stained with H&E to evaluate steatosis, ballooning, and lobular inflammation. In addition, the liver sections were also subjected to reticulin staining (for reticulin deposition) and Masson's trichrome staining (for collagen deposition) to quantitatively evaluate the degree of liver fibrosis. All stained sections were observed under a light microscope (ECLIPSE Ni-U, Nikon). Liver biopsy specimens were independently evaluated by two experienced pathologists who were blinded to the experimental groups to ensure objectivity of the evaluation. The histopathological changes in the liver tissue were scored using the nonalcoholic steatohepatitis activity score (NAS) system, which evaluates steatosis (0-3), lobular inflammation (0-3), and hepatocellular ballooning (0-2). The stage of liver fibrosis was determined according to the METAVIR scoring system (F0-F4).

[0038] In the immunohistochemistry (IHC) experiment, the paraffin-embedded liver tissue sections were subjected to deparaffinization, rehydration, and antigen retrieval. The sections were incubated with the following primary antibodies overnight at 4°C: anti-a-SMA antibody (dilution ratio 1 :200) and anti-TGF-β1 antibody (dilution ratio 1 :300). After washing, the sections were incubated with HRP-labeled secondary antibody AMCA-AffiniPure goat anti-rabbit IgG (dilution ratio 1 : 100). The immunoreaction was developed by 3,3'-diaminobenzidine (DAB), followed by hematoxylin counterstaining. The stained sections were imaged under an ECLIPSE Ni-U light microscope (Nikon).

[0039] 1.3 Broadly targeted metabolomics analysis

[0040] Metabolomics extraction and analysis were performed by Wuhan Meiwu Biological Technology Co., Ltd. The brief steps are as follows:

[0041] Take 50 mg of fecal sample, homogenize with 500 pL of ice-cold 70% (by volume) methanol, vortex for 3 minutes, and centrifuge at 12,000 x g for 10 minutes at 4°C. Take 300 pL of supernatant, perform protein precipitation at -20°C, then centrifuge at 12,000 x g for 10 minutes, filter through a protein precipitation plate, and the obtained filtrate is the final injection solution. For derivatization, mix 30 pL of injection solution with 5 pL of 200 mM 3-nitrophenylhydrazine (3-NPH) and 5 pL of 150 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), incubate at 30°C in the dark for 60 minutes, terminate the reaction with 1,460 pL of ice-cold acetonitrile after the reaction is complete, and collect the supernatant by centrifugation for subsequent analysis.

[0042] Metabolic profiling was performed using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) system (Exion LC TM ADUPLC and 6500+ mass spectrometer, SCIEX Analysis Instruments company). Chromatographic separation and mass spectrometry detection parameters were set according to previous literature reports (Guo H, Zhang L, Tang Z, Yu X, Wu J, et al. Correlation analysis of metabolomics and transcriptomics reveals dynamic regulation of purine metabolism and flavonoid synthesis during cotton somatic embryogenesis [J]. International Journal of Molecular Sciences. 2019;20:2070. doi: 10.3390 / ijms20092070.).

[0043] Metabolite identification was performed using the Metware database (MWDB, Metware Biotechnology Co., Ltd. company). Differential metabolites were defined as metabolites with a variable importance projection (VIP) score > 1 and |log2(fold change)|≥1.

[0044] 1.4 Biochemical analysis

[0045] Serum biochemical parameters including glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (CHOL), gamma glutamyl transpeptidase (GGT), and total bilirubin (TBIL) were quantitatively determined using a fully automatic biochemical analyzer (Shandong Boke Biological Industry Co., Ltd.). In addition, the levels of lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), and insulin in serum were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits.

[0046] 1.5 Transmission electron microscopy observation

[0047] Mouse colon tissues were fixed with 2.5% (volume concentration) glutaraldehyde (dissolved in 0.1 M PBS, pH = 7.4) at 4°C for 2-4 hours, then washed with PBS, and then fixed with 1% osmium tetroxide at 4°C for 1.5-2 hours. The samples were dehydrated with a gradient ethanol series (30%→100%, 15 minutes per step), then embedded with Epon 812 epoxy resin, and polymerized at 60°C for 48 hours. After the reaction was completed, ultrathin sections (50-70 nm) were cut using an ultramicrotome, stained with 2% uranyl acetate solution for 20 minutes, and then stained with a lead citrate solution for 5-10 minutes (avoiding carbon dioxide), and observed under a JEM1230 transmission electron microscope (80 kV) produced by Hitachi, Ltd.

[0048] 1.6 Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis

[0049] RNA extraction was performed using TRIzol reagent (Invitrogen, USA). First-strand cDNA synthesis was performed using HiScript II qRT Super Mix (Vazyme, China). Quantitative real-time PCR was performed using qPCR SYBR Green PCR Master Mix (Yease, China) and gene-specific primers listed in detail in Table 1. The expression level of all target genes was normalized to the reference gene GAPDH or β-actin, and the relative expression fold change was calculated using 2 -ΔΔCT Method calculation.

[0050] Table 1 Primer sequences

[0051]

[0052] 1.7 Western blot analysis

[0053] Liver tissues were washed twice with cold phosphate-buffered saline (PBS) and then lysed in cold RIPA buffer containing protease inhibitors for 30 min. The buffer was composed of 50 mM Tris-HCl (pH = 7.4), 150 mM NaCl, 1% (volume concentration) Triton X-100, 1% (volume concentration) sodium deoxycholate, and 0.1% (volume concentration) SDS. The supernatant was obtained by centrifugation (at a speed of 12000 rpm for 5 min). The proteins were separated by 10% or 15% SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The membrane was blocked with a TBST solution containing 5% skim milk, followed by overnight incubation with the primary antibody, and then incubation with a HRP-labeled secondary antibody. The signal was detected using a Tanon ECL system. The primary antibody can be selected as follows: TNF-a, a-SMA, MMP1, TIMP1, SIRT1, SMAD2, SMAD, P-SMAD2, P-SMAD3, TGFp1.

[0054] 1.8 Molecular docking

[0055] Analysis was performed using CB-Dock2 (https: / / cadd.labshare.cn / cb-dock2 / php / index.php). The 2D structure of 3-IAld (PubChem: https: / / pubchem.ncbi.nlm.nih.gov[PubChem CID: 10256]) was energy-minimized after adding hydrogen atoms, while the SIRT1 crystal structure (UniProt: https: / / www.uniprot.org / [UniProt ID: Q923E4]) was prepared by hydrogenation and solvent removal. CB-Dock2 generates the optimal binding conformation based on the minimum free energy, resulting in a binding affinity of 3-IAld interacting with SIRT1 of -6.8 kcal / mol.

[0056] 1.9 Statistical analysis

[0057] Data are expressed as mean ± standard error (SEM). For comparisons involving two groups, Student’s t-test was used; for multiple group analysis, one-way analysis of variance (ANOVA) with post hoc test was used. Pearson correlation analysis was used to assess the association between two variables. The threshold for statistical significance was set at P < 0.05. All analyses were performed in GraphPad Prism.

[0058] 2. Experimental results

[0059] 2.12 Type 2 diabetes mellitus (T2DM) exacerbates liver fibrosis in MASH mice

[0060] Hyperglycemia is an independent risk factor for accelerated fibrosis progression in metabolic disorder-associated steatohepatitis (MASH), but the mechanism is not clear. To investigate this relationship, the present invention established a new T2DM-MASH mouse model by combining a high-fat diet with low-dose streptozotocin and CCl4 injection. Figure 1 In the A), the MASH group only received a high-fat diet plus CCl4 injection. It was found that Figure 1 From Fig. H, it can be seen that the blood glucose and insulin levels of the T2DM-MASH group were significantly higher than those of the MASH group.

[0061] After 16 weeks of dietary intervention, both experimental groups of animals showed an increase in liver weight and liver-to-body weight ratio, with the T2DM-MASH group showing more significant changes (Fig. B-D). Macroscopic observation showed that both the T2DM-MASH group and the MASH group showed obvious liver pallor compared with the CON group (Fig. E). Histopathological analysis showed that the steatosis of the T2DM-MASH group mice was reduced, but the hepatocyte ballooning and lobular inflammation were aggravated (Fig. F and G). Figure 1 Figure 1 After 16 weeks of dietary intervention, both experimental groups of animals showed an increase in liver weight and liver-to-body weight ratio, with the T2DM-MASH group showing more significant changes (Fig. B-D). Macroscopic observation showed that both the T2DM-MASH group and the MASH group showed obvious liver pallor compared with the CON group (Fig. E). Histopathological analysis showed that the steatosis of the T2DM-MASH group mice was reduced, but the hepatocyte ballooning and lobular inflammation were aggravated (Fig. F and G).​Figure 1 Compared with the MASH group, the T2DM-MASH group had a significantly higher non-alcoholic fatty liver disease activity score (NAS). Figure 1 F), significantly increased fibrotic deposition in the liver ( Figure 1 China E and Figure 1 In the middle G); serum biochemical indicators showed that the levels of total bilirubin (TBIL) were comparable, but the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG), total cholesterol (CHOL), and gamma-glutamyl transferase (GGT) were significantly elevated. Figure 1 The presence of inflammatory markers (IN) in the liver was consistent with increased hepatocellular damage and impaired liver function. In summary, these findings suggest that type 2 diabetes mellitus (T2DM) can enhance liver inflammation and fibrosis in MASH mice.

[0062] 2.2 Abnormal gut-derived metabolites in T2DM-MASH mice

[0063] To elucidate the metabolic disorders associated with the accelerated progression of MASH in mice with type 2 diabetes mellitus (T2DM), this invention performed extensive targeted metabolomics analysis on fecal samples from mice in the T2DM-MASH group, MASH group, and control group. Principal component analysis (PCA) revealed significant clustering patterns among the three groups, with clear separation between the T2DM-MASH group, MASH group, and CON group. Figure 2 Metabolites that satisfy VIP>1 and |log2(foldchange)|≥1 are identified as significantly altered metabolites. Comparative analysis showed that there were 68 upregulated metabolites and 47 downregulated metabolites between the T2DM-MASH group and the MASH group. Figure 3 (A); There were 24 upregulated metabolites and 136 downregulated metabolites between the MASH group and the CON group. Figure 3 (B); There were 37 upregulated metabolites and 126 downregulated metabolites between the T2DM-MASH group and the CON group. Figure 3 (C)

[0064] K-means clustering analysis identified ten different metabolic patterns. Figure 4 Among them, cluster 4 (29 metabolites) showed a trend of gradually decreasing expression levels in the CON group, MASH group, and T2DM-MASH group. Figure 5 Notably, 3-IAld was significantly depleted in the T2DM-MASH group mice. Correlation analysis showed that 3-IAld levels were correlated with hepatitis scores (r = -0.6728, p = 0.0043). Figure 6 (A) Hepatocellular emphysema score (r = -0.7158, p = 0.0018); Figure 6Medium B) and non-alcoholic fatty liver disease (NAFLD) activity score (r = -0.7343, p = 0.0012; Figure 6 Medium C), liver fibrosis stage (r = -0.7178, p = 0.0017; Figure 6 Medium D), fasting blood glucose concentration (r = -0.7254, p = 0.0015; Figure 6 Medium E) significantly correlated.

[0065] These findings suggest that the depletion of 3-IAld can contribute to the accelerated fibrosis observed in the T2DM-MASH group. The strong negative correlation with key disease parameters indicates that 3-IAld can be a potential mediator of gut-liver axis dysregulation in the progression of T2DM-MASH.

[0066] 2.33-IAld improves intestinal mucosal barrier function and integrity in T2DM-MASH mice

[0067] To explore the therapeutic effect of 3-IAld on intestinal mucosal barrier dysfunction in T2DM-MASH mice, in vivo experiments were conducted. T2DM-MASH mice were orally administered 3-IAld at a dose of 25 mg / kg (low dose, L 3-IAld) or 50 mg / kg (high dose, H 3-IAld), with specific protocols as shown in Figure 7 Medium A. 3-IAld treatment significantly reduced the increase in body weight, liver weight, and liver-to-body weight ratio induced by the T2DM-MASH model ( Figure 7 Medium B-D). Compared with the normal control group, the colon length of T2DM-MASH mice was significantly shortened, while this index was significantly restored after 3-IAld administration ( Figure 7 Medium G). H&E staining and transmission electron microscopy results showed that Figure 7 Medium E), the colonic microvilli of control mice were arranged in an orderly manner, and the intercellular connections were intact. In contrast, the colonic microvilli structure of T2DM-MASH mice was disordered, and the tight junction integrity was impaired. Notably, 3-IAld treatment alleviated the ultrastructural changes induced by T2DM-MASH.

[0068] In addition, compared with the untreated group, 3-IAld treatment significantly upregulated the expression of tight junction proteins (ZO-1 and Occludin) (p < 0.05, Figure 7 Medium F), the levels of pro-inflammatory cytokines (TNF-a) and endotoxins (LPS) in serum were also significantly reduced in 3-IAld-treated mice (p < 0.05, Figure 7 Medium H), the high-dose group showed better therapeutic effect.

[0069] These results collectively indicate that 3-IAld protects against the progression of T2DM-MASH by maintaining intestinal barrier integrity and alleviating endotoxemia, thereby reducing liver damage. The dose-dependent efficacy suggests that 3-IAld may be a potential therapeutic candidate for T2DM-MASH-related intestinal and liver pathologies.

[0070] 2,43-IAld alleviates liver inflammation and fibrosis progression in T2DM-MASH mice.

[0071] Experimental results showed that 3-IAld treatment significantly reduced liver pathological changes in T2DM-MASH mice. Histological analysis ( Figure 8 The results showed that, compared with the untreated group, mice given 3-IAld for 6 consecutive weeks had significantly reduced liver inflammatory activity and fibrotic deposition. This anti-fibrotic effect was accompanied by a significant decrease in circulating hepatocyte injury markers (AST, ALT), lipid metabolites (TG, CHOL), and cholestasis indicators (TBIL, GGT) (p<0.05). Figure 8 (BG). In addition, 3-IAld treatment improved systemic metabolic homeostasis, as evidenced by decreased blood glucose levels and improved insulin sensitivity (p<0.05); Figure 8 (H). These data indicate that 3-IAld has a protective effect against liver inflammation and fibrosis progression in T2DM-MASH mice by alleviating liver damage, improving metabolic disorders, and inhibiting fibrotic remodeling.

[0072] 2.53-AId Ameliorate Liver Fibrosis and Inflammation in T2DM-MASH Mice by Regulating the SIRT1 / TGF-β / SMAD Pathway. SIRT1 plays a crucial role in regulating hepatic stellate cell (HSC) phenotypic transformation, as evidenced by significant downregulation in (i) mouse models of liver fibrosis, (ii) cirrhotic patient specimens, and (iii) activated HSCs compared to resting state. In diabetic nephropathy (DKD), indole-3-propionic acid (IPA) significantly ameliorate proteinuria by inhibiting SIRT1 ubiquitination-mediated degradation. However, the precise molecular mechanism by which 3-AId regulates metabolic disorder-associated steatohepatitis (MASH) via the SIRT1 signaling pathway remains unclear.

[0073] Immunohistochemical analysis showed that, compared with the untreated T2DM-MASH control group, 3-IAld treatment significantly reduced the expression of fibrosis markers (α-SMA and TGF-β1) in the liver and alleviated collagen deposition. Figure 9 (A). Molecular docking studies (Vina score: -6.8 kcal / mol) confirmed the high-affinity binding between 3-IAld and SIRT1. Figure 9Fig. 3B, suggesting its direct targeting effect. The experimental results showed that 3-IAld administration presented a dose-dependent effect: (i) restoring physiological level of SIRT1 expression, (ii) inhibiting TGF-β / SMAD signaling pathway activation, (iii) achieving extracellular matrix homeostasis by down-regulating TIMP-1 and up-regulating MMP-1 Figure 9 Fig. 3C and Figure 9 Fig. 3D). Meanwhile, the reduction of pro-fibrotic markers (a-SMA, collagen deposition) and inflammatory mediators (TNF-a) further confirmed the dual anti-fibrotic and anti-inflammatory effects of 3-IAld Figure 9 Fig. 3C and Figure 9 Fig. 3D). These results indicated that 3-IAld is a promising therapeutic candidate targeting agent for T2DM-MASH treatment, which is unique in that it can simultaneously address metabolic derangements, inflammatory responses, and fibrotic progression through SIRT1-mediated TGF-β / SMAD signaling pathway modulation.

[0074] The present application successfully established a T2DM-MASH liver fibrosis mouse model, not only confirming the role of type 2 diabetes in accelerating the progression of MASLD fibrosis, but more importantly, for the first time elucidating the dual therapeutic mechanism of 3-IAld for type 2 diabetes mellitus combined with metabolic dysfunction-related steatohepatitis: by improving intestinal barrier function to reduce endotoxemia, and simultaneously activating SIRT1 to inhibit the TGF-β / SMAD signaling pathway, thereby achieving the effect of treating type 2 diabetes mellitus combined with metabolic dysfunction-related steatohepatitis. These findings provide new therapeutic targets and strategies for the treatment of type 2 diabetes mellitus combined with metabolic dysfunction-related steatohepatitis.

[0075] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application, and various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of protection of the present application as defined by the claims.

Claims

1. Application of indole-3-carboxaldehyde in the preparation of products for the treatment of type 2 diabetes mellitus complicated with metabolic dysfunction-related steatohepatitis.

2. The application according to claim 1, characterized in that, The product is a medication for treating type 2 diabetes mellitus complicated with metabolic dysfunction-related steatohepatitis.

3. The application according to claim 2, characterized in that, The drug contains a pharmaceutically acceptable carrier or excipient.

4. The application according to claim 2, characterized in that, The dosage forms of the drug include one or more of the following: emulsion, capsule, pill, tablet, suspension, syrup, ointment, oral liquid, injection, and granule.

5. A product for treating type 2 diabetes mellitus complicated with metabolic dysfunction-related steatohepatitis, characterized in that, The product includes indole-3-carboxaldehyde as described in claim 1.

6. The product according to claim 5, characterized in that, The dosage forms of the product include one or more of the following: emulsion, capsule, pill, tablet, suspension, syrup, ointment, oral liquid, injection, and granule.

7. The product according to claim 6, characterized in that, The product also contains pharmaceutically acceptable carriers or excipients.

8. The use of the product according to any one of claims 5-7 in the preparation of a medicament for treating type 2 diabetes mellitus complicated with metabolic dysfunction-related steatohepatitis.

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