New application of GFT505 derivative

Abstract

The invention provides a new application of a GFT505 derivative, and relates to the technical field of drugs. According to DIA quantitative proteome data analysis differential proteins, 3d can target key proteins in a PPAR signal channel and a fatty acid degradation signal channel, and 3d can improve PPAR protein expression and activation, so that PPAR downstream protein expression is increased, the lipid metabolism process is enhanced, and lipid accumulation is reduced; the Alpha diversity and the Beta diversity of intestinal microbiota are obviously migrated to a normal group after administration treatment for 3 days, and a corresponding application direction is opened up for 3 days.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, in particular to new applications of GFT505 derivatives. Background Art

[0002] Non-alcoholic steatohepatitis (NASH) has become one of the most common chronic liver diseases in the world. With the increasing prevalence of metabolic syndromes such as obesity and diabetes, the number of NASH patients is expected to continue to grow rapidly in the next few years. NASH is a progressive liver disease that, if not treated in time, can gradually lead to serious complications such as liver fibrosis, cirrhosis, liver failure and liver cancer. This rapidly growing number of patients and the serious health hazards they bring have exacerbated the market's urgent need for NASH therapeutics. Clinical research on NASH has never stopped, and currently only one thyroid hormone receptor (THR-β) selective agonist, Resmetirom, has been approved by the FDA for the treatment of NASH. However, the treatment of NASH still faces huge difficulties, and current treatments mainly focus on lifestyle interventions, such as dietary changes and weight control.

[0003] Some small molecule targeted drugs have shown certain therapeutic prospects in early clinical trials, such as peroxisome proliferator-activated receptor (PPAR) agonists, farnesoid X receptor (FXR) agonists, and glucagon-like peptide-1 (GLP-1) receptor agonists. However, there is still a lack of fast, effective and widely accepted standard treatment options. GFT505 was once considered the first drug that was very likely to be approved by the FDA for the treatment of NASH. Although the drug has a good safety profile, it failed in a Phase III clinical study because its efficacy did not meet expectations.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide new applications of GFT505 derivatives, thereby opening up new application directions for GFT505 derivatives.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] The present invention provides the use of GFT505 derivatives in any of the following:

[0008] A1. Application in regulating PPAR signaling pathway or preparing products for regulating PPAR signaling pathway;

[0009] A2. Application in regulating fatty acid degradation signal pathway or preparing products for regulating fatty acid degradation signal pathway;

[0010] A3. Application in regulating intestinal microbial flora or in preparing products for regulating intestinal microbial flora;

[0011] The GFT505 derivative is GFT505 derivative 3d, and the structural formula of 3d is as follows:

[0012]

[0013] Furthermore, the regulation of the PPAR signaling pathway includes promoting the expression of PPAR protein and / or promoting the expression of PPAR downstream genes;

[0014] Furthermore, the PPAR downstream gene includes at least one of Acox1, Ehhadh or Acaa1.

[0015] Furthermore, the regulation of fatty acid degradation signal pathway includes at least one of promoting fatty acid oxidation, reducing mitochondrial dysfunction, increasing antioxidant enzyme activity or inhibiting inflammatory factor expression.

[0016] Furthermore, the oxidase includes SOD and / or GSH.

[0017] Furthermore, the inflammatory factor includes at least one of NO, IL-6 or TNF-α.

[0018] Furthermore, the regulating of the intestinal microbial flora includes at least one of increasing the diversity of the intestinal microbial flora, regulating flora abundance, activating beneficial flora, or inhibiting harmful flora.

[0019] Furthermore, the intestinal microbial flora diversity includes Alpha diversity and Beta diversity.

[0020] Furthermore, the regulating the abundance of the bacterial flora includes at least one of increasing the abundance of Firmicutes, increasing the abundance of Actinobacteria, decreasing the abundance of Deferribacteria, or decreasing the abundance of Proteobacteria;

[0021] The beneficial bacteria include Lactobacillus and / or Bifidobacterium;

[0022] The harmful bacteria include Helicobacter pylori.

[0023] Furthermore, the product includes a medicine.

[0024] The present invention provides a new application of the GFT505 derivative. The inventors analyzed the differential proteins based on DIA quantitative proteomic data and found that 3d can target key proteins in the PPAR signaling pathway and the fatty acid degradation signaling pathway, and 3d can increase the expression and activation of PPAR protein, increase the expression of PPAR downstream proteins, enhance the lipid metabolism process, and reduce lipid accumulation. After 3d drug treatment, the Alpha diversity and Beta diversity of the intestinal microbiota significantly migrated towards the normal group, opening up a corresponding application direction for 3d. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The results of the expression and quantitative analysis of proteins related to lipid metabolism in the in vivo HFD model for 3 days provided in Example 1 of the present invention;

[0027] Figure 2 The results of the expression and quantitative analysis of lipid metabolism-related proteins in HepG2 cells induced by FFAs at 3d provided in Example 1 of the present invention;

[0028] Figure 3 This is a statistical graph of the survival rates of RAW264.7 cells acted upon by different drugs provided in Example 1 of the present invention;

[0029] Figure 4 The effect of 3d provided in Example 1 of the present invention on inflammatory factors in RAW264.7 cells induced by LPS for 24 hours;

[0030] Figure 5 The effect of 3d on the MAPK signaling pathway in the in vivo HFD model provided in Example 1 of the present invention;

[0031] Figure 6 The effects of free fatty acids and 3d on L02 cell survival rate and lipid accumulation provided by Example 1 of the present invention;

[0032] Figure 7 The JC-1 probe provided in Example 1 of the present invention is used to detect the changes of MMP in L02 cells stimulated by FFAs for 24 hours;

[0033] Figure 8 The DCFH-DA probe provided in Example 1 of the present invention is used to detect the changes in ROS in L02 cells stimulated by FFAs for 24 hours;

[0034] Fig. 9 Annexin-V-FITC and PI staining were used to detect changes in apoptosis of L02 cells stimulated by FFAs for 24 hours, as provided in Example 1 of the present invention;

[0035] Fig.10 The effects of 3d on apoptosis-related proteins in the in vivo HFD model provided in Example 1 of the present invention;

[0036] Fig.11 A box plot showing the comparison of the Alpha diversity index groups provided in Example 2 of the present invention;

[0037] Fig.12 PCoA analysis of microbial composition provided in Example 2 of the present invention;

[0038] Fig.13 The species abundance of the intestinal flora at the phylum level under the 3d treatment provided in Example 2 of the present invention;

[0039] Fig.14 Comparison of species abundance of Firmicutes, Actinobacteria, Deferribacteria and Proteobacteria under the 3d treatment provided in Example 2 of the present invention;

[0040] Fig.15 The species abundance of intestinal flora at different levels under the 3d treatment provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0041] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0042] The present invention provides the use of GFT505 derivatives in any of the following:

[0043] A1. Application in regulating PPAR signaling pathway or preparing products for regulating PPAR signaling pathway;

[0044] A2. Application in regulating fatty acid degradation signal pathway or preparing products for regulating fatty acid degradation signal pathway;

[0045] A3. Application in regulating intestinal microbial flora or in preparing products for regulating intestinal microbial flora;

[0046] The GFT505 derivative is GFT505 derivative 3d, and the structural formula of 3d is as follows:

[0047]

[0048] The inventors analyzed the differential proteins based on DIA quantitative proteomic data and found that 3d can target key proteins in the PPAR signaling pathway and fatty acid degradation signaling pathway. 3d can increase PPAR protein expression and activation, increase PPAR downstream protein expression, enhance lipid metabolism, and reduce lipid accumulation. After 3d drug treatment, the Alpha diversity and Beta diversity of the intestinal microbiota significantly migrated toward the normal group, opening up corresponding application directions for 3d.

[0049] Further verification revealed that 3d can significantly activate the expression of PPARα, PPARγ and PPARδ proteins, and increase the activity of these receptors. In some specific embodiments, the regulation of the PPAR signaling pathway includes promoting the expression of PPAR proteins and / or promoting the expression of PPAR downstream genes.

[0050] In some specific embodiments, the PPAR downstream gene includes at least one of Acox1, Ehhadh or Acaa1.

[0051] By inhibiting p38 MAPK in the MAPK pathway, the generation of inflammatory factors is reduced, thereby reducing liver inflammatory response; it can alleviate mitochondrial dysfunction caused by excessive lipid accumulation, reduce ROS (reactive oxygen) generation in liver cells, alleviate oxidative stress response, and improve early cell apoptosis caused by mitochondrial dysfunction. In some specific embodiments, the regulation of fatty acid degradation signaling pathways includes promoting fatty acid oxidation, reducing mitochondrial dysfunction, increasing antioxidant enzyme activity, or inhibiting inflammatory factor expression.

[0052] In some specific embodiments, the oxidase comprises SOD and / or GSH.

[0053] 3d significantly reduced the production of inflammatory factors such as NO, IL-6, TNF-α, etc. in RAW264.7 macrophages induced by LPS. In some specific embodiments, the inflammatory factor includes at least one of NO, IL-6 or TNF-α.

[0054] In some specific embodiments, the regulating intestinal microbial flora includes at least one of increasing the diversity of intestinal microbial flora, regulating flora abundance, activating beneficial flora, or inhibiting harmful flora.

[0055] In some specific embodiments, the intestinal microbial flora diversity includes Alpha diversity and Beta diversity.

[0056] In some specific embodiments, the regulating bacterial abundance includes at least one of increasing the abundance of Firmicutes, increasing the abundance of Actinobacteria, decreasing the abundance of Deferribacteria, or decreasing the abundance of Proteobacteria.

[0057] Wherein, the beneficial bacteria include Lactobacillus and / or Bifidobacterium; the harmful bacteria include Helicobacter pylori.

[0058] In some specific embodiments, the product comprises a drug.

[0059] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0060] 3d:.

[0061] 1. Establishment of in vitro cellular lipid accumulation model

[0062] The cells were stimulated with 1.5mM FFAs for 24h. The experiment set up a blank group (control), a model group (FFAs) and a 3d drug group (3d). After the incubation was completed, the blank group was replaced with normal culture medium, and the model and drug groups were replaced with the configured FFAs culture medium. At the same time, drug treatment was given, the blank group and the model group were the drug solvent DMSO, and the drug group was 3d. The volume was consistent, that is, 30μL for small dishes and 5μL for 12-well plates, and they were incubated together for 24h.

[0063] 2. Establishment of in vitro cell inflammation model

[0064] RAW264.7 cells were cultured in DMEM high-glucose medium (4.5 g / L D-Glucose). RAW264.7 cells with good growth status were cultured at 1×10 5 Cells were inoculated in a 24-well plate at a density of 1 μg / mL, with 0.5 mL of cell solution per well. After 24 hours of stable cell adhesion and growth, the culture medium was replaced with serum-free culture medium. 1 μg / mL LPS was added 30 minutes later to induce a cell inflammation model (LPS).

[0065] 3. Establishment of HFD Diet Mouse Model

[0066] SPF male C57BL / 6 mice (18-22 g) were purchased from Vital River Co., Ltd. They were adaptively raised for 7 days, with free access to food and water, and controlled by a controllable light source cycle, i.e., 12-hour light-dark alternation, with a temperature controlled at 22±2°C and a relative humidity controlled at 55±5%.

[0067] After the adaptive feeding, the mice were randomly divided into three groups: a normal diet control group (control), a high-fat diet-induced model group (Model), and a treatment group (3d) that received oral administration of 30 mg / kg daily for 3 days. The feeding was continued for 24 weeks.

[0068] Example 13d Proteomic analysis and verification after treatment

[0069] 1. Lipid metabolism regulation

[0070] 1.1 Validation in HFD-induced mice.

[0071] Western Blot gel electrophoresis was performed on liver tissue homogenates of NASH mice induced by a high-fat diet and treated with 30 mg / kg for 3 days to verify the differential proteins. The results are as follows Figure 1 As shown in A; at the same time, the grayscale quantitative analysis of protein bands was performed using ImageJ software, and the results are shown in Figure 1 As shown in B. Among them, Model and Control group#p<0.05,###p<0.001, Model and 3d*p<0.05,**p<0.01,***p<0.001.

[0072] It can be seen that in the high-fat-induced model group, the activities of PPARα and γ were significantly inhibited (p<0.05). Compared with the blank group, the expression of PPARγ in the model group decreased by 60.21%; compared with the blank group, the expression of PPARα in the model group decreased by 59.63%. After 3 days of treatment, the decrease in the expression of PPARα and γ was significantly reversed (p<0.05). Compared with the model group, the expression of PPARγ in the 3-day treatment group increased by 63.76%; compared with the model group, the expression of PPARα in the 3-day treatment group increased by 47.72%, and 3 days could significantly activate the expression of PPAR protein. From the results, it can be seen that the expression of Acox1 in the high-fat-induced model group decreased (p<0.05). Compared with the blank group, the expression of Acox1 in the model group decreased by 23.58%. After 3 days of treatment, the decrease in Acox1 expression was significantly reversed (p<0.05), and the expression of Acox1 in the 3-day treatment group increased by 89.13% compared with the model group, and 3 days could significantly increase the expression of Acox1 protein.

[0073] Compared with the blank group, the expression of Ehhadh in the model group decreased by 53.22%. After 3 days of treatment, the decrease of Ehhadh expression was significantly reversed (p < 0.05). Compared with the model group, the expression of Ehhadh in the 3 days of treatment group increased by 98.75%. 3 days can significantly increase the expression of Ehhadh protein.

[0074] Compared with the blank group, the expression of Acaa1 in the model group decreased by 22.03%. After 3 days of treatment, the decrease in Acaa1 expression was significantly reversed (p < 0.05). Compared with the model group, the expression of Acaa1 in the 3 days of treatment group increased by 47.83%. 3 days can significantly increase the expression of Acaa1 protein.

[0075] 1.2 Detection of lipid metabolism-related proteins in HepG2 cells stimulated by exogenous free fatty acids

[0076] The cells were stimulated with 1.5 mM FFAs for 24 h and then collected for Western Blot electrophoresis. Figure 2 As shown, among them, FFAs and Control group###p<0.001; FFAs and 3d*p<0.05, **p<0.01, ***p<0.001. It can be seen that the experimental results are consistent with the in vivo results. 3d also has a significant activation effect on PPARα, PPARγ and PPARδ at the in vitro cell level. The activities of PPARα, PPARγ and PPARδ in the model group after exogenous free fatty acid stimulation are significantly inhibited (p<0.05). Compared with the blank group, the expression of PPARγ in the model group decreased by 38.40%; compared with the blank group, the expression of PPARα in the model group decreased by 54.69%; compared with the blank group, the expression of PPARδ in the model group decreased by 44.37%. After 3d administration, the decrease in the expression of PPARα, γ and δ was significantly reversed (p<0.05). Compared with the model group, the expression of PPARγ in the 3d administration group increased by 92.37%; compared with the model group, the expression of PPARα in the 3d administration group increased by 27.05%; compared with the model group, the expression of PPARδ in the 3d administration group increased by 76.38%. 3d can significantly activate the expression of PPAR protein.

[0077] After exogenous free fatty acid stimulation, Acox1 expression in the model group decreased. After 3 days of treatment, the decrease in Acox1 expression was significantly reversed (p < 0.05). Compared with the model group, Acox1 expression in the 3-day treatment group increased by 92.37%, and Acox1 protein expression was significantly increased in 3 days. After exogenous free fatty acid stimulation, Ehhadh expression in the model group decreased (p < 0.05). Compared with the blank group, Ehhadh expression in the model group decreased by 56.54%. After 3 days of treatment, the decrease in Ehhadh expression was significantly reversed (p < 0.05). Compared with the model group, Ehhadh expression in the 3-day treatment group increased by 52.58%, and Ehhadh protein expression was significantly increased in 3 days.

[0078] Therefore, 3d increased the expression of PPARα, γ and δ proteins both in vivo and in vitro, and reversed the inhibition of PPAR activity caused by lipid accumulation. At the same time, after 3d treatment, the expression of PPAR downstream target proteins was also affected, reversing the downregulation of downstream proteins Acox1, EHHADH and Acaa1. The above results show that the therapeutic effect of 3d on NASH is related to PPAR targets and can improve lipid accumulation.

[0079] 1.33d molecular docking simulation calculation of the target was performed to determine whether it is a PPAR agonist.

[0080] Through simulation calculation, the detailed binding position and hydrogen bond formation of 3d and the target protein were determined. Detailed docking mode analysis found that 3d formed a strong hydrogen bond interaction with the key amino acid residue ALA333 of PPARα, and formed Pi-sulfur bonds and multiple Pi-alkyl bonds with amino acid residues CYS275 and CYS276. 3d formed a Pi-sulfur bond and multiple Pi-alkyl bonds with the key amino acid residue MET364 of PPARγ. 3d formed a strong hydrogen bond interaction and multiple Pi-alkyl bonds with the key amino acid residue GLU295 of PPARδ. 3d binds to and matches well with the PPAR active pocket, and the formation of hydrogen bonds makes the conformation more stable. Therefore, 3d may be an agonist of PPAR.

[0081] Effect of 2.3d on LPS-induced inflammatory response in RAW264.7 macrophages

[0082] 2.1 Impact on survival rate

[0083] The MTT method was used to detect the effects of camptothecin (CPT), fenofibric acid and 3d on the proliferation of mouse RAW264.7 mononuclear macrophages. 5 The cells / mL were seeded in 96-well plates and incubated for 24 h. Compounds were added and cultured for another 24 h. The cell survival rate was detected using the MTT method.

[0084] The results are as follows Figure 3 As shown. It can be seen that fenofibric acid has no obvious inhibitory and killing effects on cells, while compound 3d has no obvious cytotoxicity at concentrations of 5, 10 and 20 μM, and the cell survival rate can reach 80% at a concentration of 40 μM. The cell survival rate is low at a concentration of 80 μM, and there is obvious cytotoxicity. Therefore, subsequent experimental studies selected 3d at concentrations of 10, 20 and 40 μM to continue the study on RAW264.7 cells.

[0085] 2.2 Effects of drugs on intracellular verification factors

[0086] Curcumin (Cur), a common natural compound with good anti-inflammatory effects in clinical practice, and fenofibric acid (FA), a fibrate-type lipid-lowering drug, were selected as positive drugs for anti-inflammatory activity test and acted on the RAW264.7 cell inflammation model induced by LPS for 24 hours. At the same time, a control group (Con) without drug use was set up.

[0087] like Figure 4 As shown, (A) nitric oxide (B) interleukin-6 (C) tumor necrosis factor-α. LPS vs. Control, ###p<0.001; LPS vs. 3d, *p<0.05, **p<0.01, ***p<0.001. Figure 4 As shown in Figure A, after administration of 1 μg / mL LPS, the NO content in the cell culture supernatant increased significantly (p < 0.05) to 29.87 μmol / L, while the addition of 10 μM curcumin 30 minutes after administration of LPS significantly reduced the NO content (p < 0.05) to 11.04 μmol / L. After the addition of 10 μM, 20 μM and 40 μM for 3 days, the NO in the cell culture supernatant showed a significant dose-dependent decrease trend, and the effect of 10 μM for 3 days was equivalent to that of the same dose of curcumin, which was 11.69 μmol / L.

[0088] Depend on Figure 4 As shown in Figure B, after administration of 1 μg / mL LPS, the IL-6 content in the cell culture supernatant increased significantly (p < 0.05) to 286.26 pg / mL, while the addition of 10 μM curcumin 30 minutes after administration of LPS significantly reduced the IL-6 content (p < 0.05) to 193.01 pg / mL. After the addition of 10 μM, 20 μM and 40 μM for 3 days, the IL-6 content in the cell culture supernatant was significantly reduced (p < 0.05), of which 20 μM for 3 days reduced the IL-6 content to 166.80 pg / mL, which was better than curcumin.

[0089] Depend on Figure 4 As shown in Figure C, after administration of 1μg / mL LPS, the TNF-α content in the cell culture supernatant increased significantly (p<0.05) to 629.25pg / mL, while the addition of 10μM curcumin 30min after administration of LPS reduced the TNF-α content to a certain extent, reaching 591.96pg / mL. After adding 10μM, 20μM and 40μM for 3d, the TNF-α content in the cell culture supernatant decreased significantly in a dose-dependent manner (p<0.05). At 10μM, the TNF-α content was reduced to 370.94pg / mL, which was significantly better than the therapeutic effect of curcumin at the same dose.

[0090] This result indicates that 3d may exert its anti-inflammatory effect by inhibiting the secretion of NO, TNF-α and IL-6 by cells, and this effect is consistent with the results of previous experiments.

[0091] 3. Effects of MAPK signaling pathway

[0092] Protein gel electrophoresis was used to verify the effect of compound 3d on the phosphorylation of p38 protein in the MAPK signaling pathway in the liver tissue of mice fed a high-fat diet. Figure 5 As shown, (A) protein expression (B) protein expression quantitative analysis results. Model and Control group#p<0.05; Model and 3d***p<0.001. It can be seen that the high-fat diet significantly activated the phosphorylation of p38 protein in the liver, and the degree of p38 phosphorylation was significantly reduced after 30mg / kg 3d treatment, reducing the stimulation of various inflammatory factors, thereby playing a therapeutic role in the inflammatory response during the disease process of NASH mice. Therefore, 3d alleviates the inflammatory response in vivo through the p38-MAPK pathway, which may be one of the mechanisms for the treatment of NASH by the compounds studied in this project.

[0093] 4.3d alleviates mitochondrial apoptosis caused by excessive lipid accumulation

[0094] 4.1 Effects of free fatty acids and 3d on L02 cell survival and lipid accumulation

[0095] To study the effect of lipid accumulation in hepatocytes on cell status, cells incubated with different concentrations of free fatty acids for 24 hours in 12-well plates were fixed with paraformaldehyde and stained with Oil Red O working solution to dye the lipid droplets in the cells red. At the same time, hematoxylin staining solution was used to dye the cell outline morphology purple to facilitate direct observation of cells under a microscope. After all staining treatments were completed, 50% glycerol was added to the 12-well plate, 0.5 mL per well, which was conducive to enhancing the refraction of light, making the results of microscopic photography clearer and long-term preservation.

[0096] like Figure 6 As shown, (A) Effect of 0-1.5 mM FFAs on lipid accumulation in L02 cells (B) Cell survival rate of L02 cells induced by 0.9 mM FFAs after 3 days of administration (C) Lipid accumulation of L02 cells induced by 0.9 mM FFAs after 3 days of administration, scale bar is 50 μm. The accumulation of lipid droplets in cells was observed using a 400x inverted biological microscope. Figure 6 As shown in Figure A, free fatty acid treatment promotes lipid accumulation in L02 cells, and the lipid droplets stained red by Oil Red O increase significantly in a concentration-dependent manner with the concentration of free fatty acids. High concentrations of free fatty acids cause intracellular lipid accumulation, triggering lipotoxicity and inducing cell death.

[0097] L02 cells were stimulated with 0.9 mM free fatty acids for 24 hours, and protected by 5 μM, 10 μM, 20 μM, 40 μM or 80 μM for 3 days. Figure 6 As shown in B, by using the MTT method to detect cell survival rate, 3 days significantly increased cell survival rate, reduced the stimulation of free fatty acids on normal human liver cells, and protected cells from lipotoxicity. At the same time, the cells after incubation were stained, such as Figure 6 As shown in C, after incubation with the same concentration of free fatty acids, the 3-day drug treatment group significantly reduced intracellular lipid accumulation and significantly improved the effects of lipotoxicity on cell proliferation and growth.

[0098] 4.2 Mitochondrial membrane potential

[0099] The JC-1 probe was used to detect mitochondrial membrane potential. In normal cells, JC-1 enters the mitochondria in a polarized manner and emits red fluorescence in the form of polymers. In damaged or apoptotic cells, due to the depolarization of the mitochondrial transmembrane potential, it is released from the mitochondria and converted into green fluorescent monomers. The normal human liver cell lipid accumulation model was used as an experimental object to detect the effect of excessive lipid accumulation on mitochondrial function. The cells after modeling and drug administration were collected and analyzed using flow cytometry after being treated with the JC-1 probe. Figure 7 As shown, (A) flow cytometry analysis (B) laser confocal microscopy photography (C) ImageJ quantitative analysis, scale bar, 50 μm. Control vs. FFAs, ###p<0.001; FFAs vs. 3d, **p<0.01. Figure 7 In the figure A, the green fluorescent monomer is detected by the B525 channel as the horizontal axis, and the red fluorescent polymer is detected by the B585 channel to draw a scatter plot. In the control group, the JC-1 probe of normal cells mostly exists in the form of polymers. In the carbonyl cyanide m-chlorophenylhydrazone (CCCP) group, which is a negative control group with complete collapse of mitochondrial function, the JC-1 probe exists in the form of monomers. Based on this principle, the coordinate axis is drawn, and the fluorescence intensity of the first quadrant is used as a measurement indicator. The first quadrant intensity of the control group was 76.09%. After induction by 0.9mM FFAs, the fluorescence intensity of the first quadrant decreased to 61.93%, the mitochondrial membrane potential was significantly reduced (p < 0.05), and the mitochondrial function was lost. After 20μM 3d administration, its fluorescence intensity was restored to 88.18%, the mitochondrial membrane potential was significantly restored, and the mitochondrial function remained stable.

[0100] At the same time, the JC-1 probe was used to detect the mitochondrial membrane potential, and a laser confocal microscope was used to take pictures for qualitative observation, such as Figure 7As shown in B. Under the microscope field of view at 400× magnification, the control group had fewer green fluorescent JC-1 monomers. In the free fatty acid-induced group, the green fluorescent monomers increased significantly and were evenly distributed around the cells. JC-1 monomers were released from the mitochondria, and the mitochondrial membrane potential was severely depolarized. After 3 days of treatment with 20 μM, the fluorescence intensity decreased significantly, the mitochondrial membrane potential partially returned to normal, and mitochondrial function was maintained. The fluorescence intensity was quantitatively analyzed using ImageJ software for the results of microscopic photography, with the ratio of green fluorescence to red fluorescence intensity as the vertical coordinate, as shown in Figure 2. Figure 7 As shown in C. There was a significant difference between the control and free fatty acid induced groups (p < 0.05). After free fatty acid treatment, the mitochondrial membrane potential of the cells collapsed. After 20 μM 3d was given, the cells were protected from the stimulation of excessive lipid accumulation caused by FFAs, and mitochondrial function remained steady. Therefore, this experiment tested the mitochondrial membrane potential from both qualitative and quantitative perspectives, confirming the protective effect of 3d on mitochondrial membrane potential in an environment of excessive lipid accumulation, and further revealing the positive role of 3d in maintaining mitochondrial functional homeostasis.

[0101] Therefore, 3d rescued the mitochondrial function in hepatocytes and maintained its functional homeostasis, which could curb the formation of fibrosis and protect liver tissue from irreversible damage. ROS is a key mediator of the oxidative stress process, including superoxide free radicals, hydrogen peroxide and its downstream products. The increase in ROS content will affect many physiological and pathological processes such as cell growth, proliferation, aging and apoptosis. Uncontrolled oxidative stress can interrupt mitochondrial ATP production and induce mitochondrial dysfunction. Flow cytometry was used to analyze the cells induced by the lipid accumulation model with DCFH-DA dye, which was oxidized by intracellular ROS to produce the fluorescent substance DCF, and the fluorescence intensity was proportional to the level of reactive oxygen. Figure 8 As shown, A is the analysis by flow cytometry, and B is the quantitative analysis by ImageJ. Control vs. FFAs, ###p<0.001; FFAs vs. 3d, ***p<0.001. After stimulation with 0.9mM FFAs, the intracellular reactive oxygen content was significantly increased by 44.62% (p<0.05) compared with the blank group, while after treatment with 20μM 3d, the intracellular reactive oxygen was significantly reduced by 11.13% (p<0.05). Therefore, the above experiments can prove that 3d can effectively reduce the generation of intracellular ROS, reduce oxidative stress, and protect mitochondrial function.

[0102] 5. Effects of high-fat injury on cell viability in vitro

[0103] In order to further understand the mechanism by which excessive lipid accumulation reduces cell viability, a cell apoptosis experiment was performed to study the effect of 3d on the apoptosis index of hepatocytes treated with FFAs. Fig. 9As shown, A is the analysis by flow cytometry, and B is the quantitative analysis by ImageJ. Control vs. FFAs, ###p<0.001; FFAs vs. 3d, ***p<0.001. Fig. 9 As shown in Figure A, 0.9 mM FFAs induced a significant increase in the number of apoptotic cells (p < 0.05), from 6.24% in the blank group to 47.35%, while 20 μM 3-day administration significantly reduced the number of apoptotic cells (p < 0.05) to 10.08%, close to the normal level. In the presence of excessive FFAs, the viability of hepatocytes was significantly reduced, and 3 days reversed this effect.

[0104] Further, it was proved that mitochondrial membrane potential would decrease with lipid accumulation and reverse to normal level after 3 days of treatment. To prove that it reversed the trend of cell apoptosis at the molecular level, Western Blot gel electrophoresis was performed on the liver tissue of NASH mice induced by HFD diet. Fig.10 As shown, (A) protein expression (B) quantitative analysis results, Model and Control group###p<0.001; Model and 3d*p<0.05, **p<0.01, ***p<0.001. Fig.10 As shown in Figure A, HFD induction led to the activation of Caspase8 (cysteine-aspartate specific protease) and downregulation of anti-apoptotic protein Bcl-2 compared with the blank group (p < 0.05). The addition of 3d reversed the activation of Caspase8 and significantly upregulated the expression of anti-apoptotic protein Bcl-2, indicating that 3d restored the cell apoptosis induced by HFD diet.

[0105] Example 23d Effects on the intestinal flora of HFD-induced NASH mice

[0106] 1. Sample Processing

[0107] The NASH model mice constructed in Example 1 were taken, and the feces of the mice were collected in EP tubes in the last week of continuous feeding for 24 weeks, and quickly frozen in liquid nitrogen in time, and stored at -80°C for a long time. Anshan (Tianjin) Biotechnology Co., Ltd., Tianjin Chemicos Technology Co., Ltd. and Hebei Chemicos Technology Co., Ltd. were commissioned to conduct the test.

[0108] 2. Experimental results

[0109] 2.1 Alpha Diversity Analysis

[0110] The results are as follows Fig.11 As shown by Fig.11As shown in A and B, the community richness in the experimental group did not change significantly, and there was no statistical difference. The Simpson index and Shannon index can be used to evaluate the species diversity and uniformity of the sample. The lower the species diversity in the sample, the closer the Simpson index value is to 1. The Shannon index is more sensitive to abundance, and the higher the value, the higher the species diversity. Fig.11 As shown in C and D, species diversity increased significantly after feeding on a high-fat diet, and the species diversity of the normal diet control group was significantly less than that of the model group. After 3 days of drug treatment, its species diversity shifted towards the normal diet group.

[0111] 2.2 Beta diversity analysis

[0112] Principal coordinate analysis (PCoA) can be used to analyze the similarities and differences between samples. In the study of intestinal flora, it can be used to analyze the differences in microbial composition between different samples. The PCoA results are visualized. The closer the distance between samples, the closer their microbial composition is; the farther the distance, the greater the difference in their microbial composition. Fig.12 As shown, the blank group (control) is purple, the model group (Model) is gray, and the 3d drug administration group (3d) is blue. The blank group and the model group show a large separation trend, indicating that their microbial compositions are quite different; and the 3d drug administration group shifts toward the blank group and shows a partial aggregation trend, indicating that the microbial composition after drug treatment is similar to that of the blank group, and there is a certain degree of similarity.

[0113] 2.3 Species composition analysis

[0114] The species composition of intestinal flora at the phylum (PHYLUM) level showed obvious differences, such as Fig.13 The top ten species in terms of abundance are Patescibacteria, Firmicutes, Epsilonbacteraeota, Deferribacteres, Actinobacteria, Tenericutes, Verrucomicrobia, Bacteroidetes, and Proteobacteria.

[0115] At the phylum level, Firmicutes and Actinobacteria in each group of samples accounted for more than 90% of the total and were the main dominant phyla.

[0116] Firmicutes are involved in bile acid metabolism, and the uncoupling enzyme bile salt hydrolase (BSH) is synthesized by Firmicutes during bile acid metabolism. Bile acids are metabolized into beneficial bile salts, such as ursodeoxycholic acid, through oxidation and epimerization. The bile acid metabolism process plays a key role in many physiological processes. It not only contributes to fat digestion and absorption, but also affects the composition and energy balance of the intestinal microbiota. In the enterohepatic circulation, bile acids are co-regulated by the hepatic biosynthesis and metabolism of the intestinal microbiota, and the interaction between bile acids and the intestinal microbiota is bidirectional. The intestinal flora can convert primary bile acids into secondary bile acids, and the abundance of such bacteria is significantly reduced in the HFD-induced NASH mouse model, such as Fig.14 The figure in A shows Firmicutes. The abundance of Firmicutes in the model group mice decreased from 80.5% to 76.3%, which not only reduced the stimulation of bile acid receptors by secondary bile acids, but also led to further disturbance of intestinal flora. After 3 days of treatment, the abundance of Firmicutes increased significantly to normal levels, which was 9.5% higher than that in the model group, which was beneficial to regulate the bile acid cycle, maintain the diversity of symbiotic bacterial communities, and maintain their balanced growth.

[0117] A decrease in the number of Actinobacteria is closely related to obesity, diabetes, and immune response. Actinomycetes are one of the four major gates of the intestinal microbiota. Although they only account for a small proportion, they are crucial in maintaining intestinal homeostasis and can promote tight junction function and maintain intestinal integrity. Short-chain fatty acids, the metabolites of symbiotic bacteria Actinobacteria, have been shown to regulate host microbiota and inflammatory responses. Among them, butyrate, as one of the most effective anti-inflammatory mediators in the intestine, plays an important role in reducing local intestinal inflammation. At the same time, it can regulate intestinal permeability, reduce the risk of bacteria and LPS transferring to the systemic circulation, and inhibit the impact on systemic inflammatory responses. Fig.14 B in the middle shows Actinomycetes. The abundance of Actinomycetes in the blank group was 11.4%, and the abundance of Actinomycetes in the HFD-induced model group was significantly reduced to 5.6%. However, after 3 days of treatment, the abundance of Actinomycetes increased significantly to 23.6, which significantly increased the abundance of Actinomycetes, which is beneficial to reduce intestinal inflammation, enhance the intestinal mucosal barrier, and protect the stability of intestinal microecology.

[0118] Deferribacteres are associated with diabetes and are enriched during gastrointestinal inflammation, triggering IgG-mediated immune responses. Fig.14 D in the middle shows Deferribacter. The abundance of Deferribacter in the model group induced by HFD diet increased to 5.386%, which was 5.37% higher than 0.016% in the blank group. 3d could restore the abundance of Deferribacter to 0.056%, close to the level of the normal group.

[0119] Most Proteobacteria are harmful bacteria. Salmonella, plague bacteria, Vibrio cholerae, Pseudomonas aeruginosa and Escherichia coli all belong to Proteobacteria, which can cause serious diseases in humans. In addition, studies have shown that NASH causes a significant increase in the number of Proteobacteria that produce alcohol in the intestine compared to the normal group. Excessive ethanol is metabolized by alcohol dehydrogenase in the liver, causing fluctuations in the intracellular redox potential and aggravating the inflammatory response. Fig.14 The figure C shows Proteobacteria. The proportion of Proteobacteria in the blank group was very low, only 0.8%. However, the number of Proteobacteria in the HFD diet-induced model group increased significantly, reaching 8.5%. After 3 days of drug treatment, it dropped to 2.5%, significantly reducing the abundance of Proteobacteria in mice, reducing the colonization of harmful bacteria, and improving abnormal ethanol metabolism.

[0120] like Fig.15 As shown in A, the species with the highest abundance at the class level are: Bacillus, α-Proteobacteria, Actinobacteria, Saccharomyces, Clostridia, Deferribacter, δ-Proteobacteria, Red Bugs, Erysipelotrichia, γ-Proteobacteria, Bacteroidetes, Campylobacter, and Soft Film. Among them, Erysipelotrichia and Clostridia are the main dominant bacteria. Clostridia can play a positive ecological role in the intestine, but some are pathogens. The overgrowth of some Clostridia bacteria in the intestine can damage the intestinal barrier, allowing endotoxins to enter the blood circulation through the intestinal mucosa, thereby triggering inflammatory responses and liver damage, and promoting the development of NASH. In this experiment, the number of species of Clostridia increased significantly in the model group, which was reversed after 3d treatment. Some bacteria in the Actinobacteria are considered to have the potential of probiotics and can help maintain the balance of intestinal microecology and metabolic health. As can be seen from the figure, the number of Actinomycetes species increased significantly in the blank group (control) and the 3d medication group (Mpl-3d), and 3d effectively increased the abundance of probiotics.

[0121] like Fig.15 As shown in B, the species with the highest abundance at the order level are: Rhodotorulales, Campylobacteriales, Fusobacteriales, β-Proteobacteriales, Erysipelothrales, Deferribacillales, Bacillusales, Desulfovibrioales, Bifidobacteriales, Bacteroideales, and Lactobacillales. Among them, Fusobacteriales and Erysipelothrales are the main dominant bacteria.

[0122] like Fig.15 As shown in C, the species with the highest abundance at the FAMILY level are: Erysipelothrixaceae, Lactobacillaceae, Mirabiliaceae, Desulfovibrioaceae, Enterococcaceae, Peptostreptococcaceae, and Lactobacillaceae.

[0123] like Fig.15 As shown in D, the top species in abundance at the genus level are: Odoribacter, Lactobacillus, Dubosiella, Bacteroides, Enterorhabdus, Bifidobacterium, Erysipelatoclostridium, Blautia, Helicobacter, Ileibacterium, Desulfovibrio, Faecalibaculum. 3d can increase the number of beneficial bacteria at the genus level, such as Lactobacillus. At the same time, 3d can also reduce the number of harmful bacteria, such as Helicobacter.

[0124] After 3 days of treatment, the Alpha diversity and Beta diversity of the intestinal microbiota significantly migrated toward the normal group. Analysis of species composition found that the main microbiota involved in the changes in this study were Firmicutes, Actinobacteria, Deferribacteria and Proteobacteria. The species abundance of Proteobacteria and Deferribacteria was positively correlated with the disease severity of NASH, while the reduction of Firmicutes and Actinobacteria would induce NASH deterioration. 3d has an improvement effect on the above-mentioned NASH-related flora. At the same time, 3d significantly increased the species abundance of probiotics, such as Lactobacillus and Bifidobacterium; and inhibited the species abundance of harmful bacteria, such as Helicobacter pylori. It shows that 3d has the potential to treat NASH, and can target the intestinal microbiota through the liver-intestinal axis to normalize the flora structure, showing a certain flora recovery effect.

[0125] Example 33d Pharmacokinetics

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of GFT505 derivatives, characterized in that: The application includes any of the following: A1. Application in regulating PPAR signaling pathway or preparing products for regulating PPAR signaling pathway; A2. Application in regulating fatty acid degradation signal pathway or preparing products for regulating fatty acid degradation signal pathway; A3. Application in regulating intestinal microbial flora or in preparing products for regulating intestinal microbial flora; The GFT505 derivative is GFT505 derivative 3d, and the structural formula of 3d is as follows:

2. The use according to claim 1, characterized in that: The regulation of the PPAR signaling pathway includes promoting the expression of PPAR protein and / or promoting the expression of PPAR downstream genes.

3. The use according to claim 2, characterized in that: The PPAR downstream gene includes at least one of Acox1, Ehhadh or Acaa1.

4. The use according to claim 1, characterized in that: The regulation of fatty acid degradation signal pathway includes at least one of promoting fatty acid oxidation, reducing mitochondrial dysfunction, increasing antioxidant enzyme activity or inhibiting inflammatory factor expression.

5. The use according to claim 4, characterized in that: The antioxidant enzymes include SOD and / or GSH.

6. The use according to claim 4, characterized in that: The inflammatory factors include at least one of NO, IL-6 or TNF-α.

7. The use according to claim 1, characterized in that: The regulating of the intestinal microbial flora includes at least one of increasing the diversity of the intestinal microbial flora, regulating flora abundance, activating beneficial flora, or inhibiting harmful flora.

8. The use according to claim 7, characterized in that: The intestinal microbial flora diversity includes Alpha diversity and Beta diversity.

9. The use according to claim 7, characterized in that: The regulating the abundance of the bacterial flora includes at least one of increasing the abundance of Firmicutes, increasing the abundance of Actinobacteria, decreasing the abundance of Deferribacteria, or decreasing the abundance of Proteobacteria; The beneficial bacteria include Lactobacillus and / or Bifidobacterium; The harmful bacteria include Helicobacter pylori.

10. The use according to any one of claims 1 to 9, characterized in that: The products include pharmaceuticals.

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