Application of 18 beta-glycyrrhetinic acid in TMAO inhibitor

Abstract

The invention relates to the technical field of medicines, discloses an application of 18 beta-glycyrrhetinic acid in a TMAO inhibitor, and further discloses an application of 18 beta-glycyrrhetinic acid in preparation of medicines for treating chronic diseases such as chronic kidney disease (CKD) and the like. The invention provides 18 beta-glycyrrhetinic acid (18 beta-GA) which is a substance capable of effectively inhibiting choline in intestinal tracts from being metabolized into TMAO for the first time, and the 18 beta-GA is efficiently combined with CutC / D and FMO to inhibit the generation of TMAO, so that the aggravating effect of TMAO on chronic kidney diseases is relieved; the invention further provides a method for improving kidney diseases through a CXCL12 / CXCR4 signal axis after the TMAO is reduced by the 18 beta-glycyrrhetinic acid, a new thought and a new method are provided for developing medicines for treating the TMAO related diseases, and a new way is expected to be developed for treating the chronic kidney diseases.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and more specifically, to the use of 18β-glycyrrhetinic acid (18β-GA) in TMAO inhibitors, and the use of 18β-glycyrrhetinic acid in the preparation of drugs for treating chronic diseases such as chronic kidney disease (CKD). Background Art

[0002] Chronic diseases, such as chronic kidney disease (CKD), are global health problems with high morbidity and mortality. In recent years, studies have revealed that the enteric toxin, trimethylamine hyperoxide (TMAO), plays an important role in the progression of these chronic diseases. TMAO is mainly derived from the metabolism of choline in the intestine and can aggravate the deterioration of chronic kidney disease and other related diseases. Although existing treatments mostly intervene in the direct symptoms of the disease, there are few reports of drugs that can safely and effectively regulate the production of TMAO. Therefore, the development of a drug that can effectively inhibit the metabolism of choline to TMAO is of great significance for the treatment or relief of TMAO-mediated kidney and other organ damage. Summary of the invention

[0003] In view of the problems existing in the prior art, the present invention aims to provide a drug that can effectively inhibit the metabolism of choline into TMAO in the intestine, thereby reducing the production of TMAO and alleviating its aggravating effect on chronic diseases.

[0004] In order to achieve the above invention objectives, this application adopts the following technical solutions:

[0005] Drug screening: Through virtual drug screening technology, candidate drugs that can efficiently bind to CutC / D and FMO, the key enzymes that metabolize choline to TMAO, were screened from a large number of compounds. Among them, 18β-glycyrrhetinic acid (18β-GA) showed high binding affinity with CutC / D and FMO enzymes and was identified as a potential therapeutic drug.

[0006] In vitro experimental verification: The inhibitory effect of 18β-GA on CutC / D enzyme and FMO enzyme was verified by using experimental models such as bacterial culture medium, Caenorhabditis elegans, and BL21 bacteria carrying relevant plasmids. The experimental results showed that 18β-GA can significantly reduce the concentration of TMA and TMAO, and has no significant effect on other bacterial species containing cholinesterase.

[0007] Mechanism of action analysis: The mechanism of action of 18β-GA on the TMAO / CXCL12 / CXCR4 signaling axis was analyzed by single-cell sequencing technology and cell interaction network diagrams. The experimental results showed that 18β-GA can regulate this signaling axis, reduce the infiltration and proliferation of immune cells, and thus reduce the inflammatory response and damage of the kidneys.

[0008] Based on the above research, in a first aspect, the present application provides the use of 18β-glycyrrhetinic acid in TMAO inhibitors.

[0009] Furthermore, the 18β-glycyrrhetinic acid inhibits TMAO generation by efficiently binding to CutC / D and FMO.

[0010] In a second aspect, the present application provides a TMAO inhibitor comprising 18β-glycyrrhetinic acid as an active substance.

[0011] In a third aspect, the present application provides the use of 18β-glycyrrhetinic acid in the preparation of drugs for treating chronic diseases.

[0012] Furthermore, the chronic disease includes chronic kidney disease.

[0013] Furthermore, the 18β-glycyrrhetinic acid improves renal injury via the CXCL12 / CXCR4 signaling axis.

[0014] In a fourth aspect, the present application provides a drug for treating chronic kidney disease, comprising 18β-glycyrrhetinic acid as an active ingredient.

[0015] Furthermore, the drug also includes a pharmaceutically acceptable carrier or excipient.

[0016] Furthermore, the pharmaceutical dosage forms include tablets, powders, granules, capsules, oral solutions, injections or sustained-release preparations.

[0017] In summary, compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention proposes for the first time to use 18β-glycyrrhetinic acid (18β-GA) as a drug for the treatment of chronic kidney disease, by inhibiting the key enzyme that metabolizes choline to TMAO, thereby reducing the aggravating effect of TMAO on chronic kidney disease. The results of in vivo and in vitro experiments show that 18β-GA can significantly reduce the concentration of TMAO, reduce pathological damage to the kidneys, improve renal function, and regulate the structure of intestinal flora. Therefore, the present invention not only provides a new drug option for the treatment of chronic kidney disease, but also provides new ideas and methods for the development of therapeutic drugs for TMAO-related diseases, which is expected to open up new avenues for the treatment of chronic kidney disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :Drug screening. (A: In vitro and in vivo experimental process; B: Results of molecular docking technology; C: Top five compounds and scores of molecular docking)

[0020] Figure 2:Inhibitory effect of 18β-glycyrrhetinic acid on CutC / D enzyme and FMO enzyme (in vitro experiment). (A: Flow chart; B: Changes in the concentrations of trimethylamine (TMA) and trimethylamine oxide (TMAO) in bacterial culture medium; C: Expression of the Fmo3 gene in Caenorhabditis elegans (C.elegans); D: Dose-concentration curve of the inhibitory effect of 18β-GA on the metabolism of choline to TMA and TMAO; E: The inhibitory effect of 18β-GA on the production of TMA by BL21 bacteria carrying the CutD-pET28a plasmid; F: The effect of 18β-GA on the growth of BL21 bacteria carrying the CutD-pET28a plasmid in a culture medium with choline as the sole carbon source; G: The inhibitory effect of 18β-GA on the production of TMAO by BL21 bacteria carrying the FMO-pET28a plasmid; H: Verification of the inhibitory effect of 18β-GA on the oxidation of TMA to TMAO in the purified FMO enzyme system; I: The effect of 18β-GA on the ability of other strains containing choline metabolizing enzymes to produce TMA)

[0021] Figure 3 :Inhibitory effect of 18β-glycyrrhetinic acid on CutC / D enzyme and FMO enzyme (in vitro experiment). (A: serum TMAO level; B: kidney pathological staining; C: mouse blood creatinine (Scr) and urea nitrogen (BUN) values; D: mouse intestinal flora β diversity analysis; E: distribution of intestinal flora at the taxonomic level; F: abundance ratio heat map of different bacterial species; G: abundance ratio of four bacteria containing CutC / D genes)

[0022] Figure 4 :18β-Glycyrrhetinic acid alleviates renal injury aggravated by CXCL12 / CXCR4 signaling axis by reducing TMAO. (A: Distribution of renal cell types in mice after 18β-GA treatment; B: Cell interaction network diagram; C: Differences in CXCL signaling pathways between I / R+Cho group and I / R+Cho+GA group; D: Dot plot; E: Flow cytometry analysis; F: Western blotting analysis; G: Immunofluorescence staining results; H: Changes in plasma TMAO concentration after 18β-GA intervention and mean ± standard deviation; I: Plasma TMAO value and its changes over time) DETAILED DESCRIPTION

[0023] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0024] It is worth noting that the materials used in the examples are commercially available unless otherwise specified; the methods used in the examples are existing methods unless otherwise specified.

[0025] Example 1: Drug Screening

[0026] This example conducts virtual drug screening for the intestinal metabolite trimethylamine oxide (TMAO). The screening experimental process is as follows: Figure 1 As shown in A, Figure 1 A depicts the in vitro and in vivo experimental process in detail in the form of a schematic diagram. First, 150 metabolism-related and clinically safe compounds were screened from a comprehensive library of 3,000 compounds through computer simulation and molecular docking technology. Then, the binding affinity of these compounds to CutC / D and FMO enzymes was calculated using AutoDock Vina software to screen out candidate drugs with the highest binding energy. Finally, the efficacy and safety of these candidate drugs were verified in in vitro and in vivo experiments.

[0027] Figure 1 B shows the docking of 18β-glycyrrhetinic acid (18β-GA), the compound with the highest binding energy for CutC / D enzyme, and Hesperidin, the compound with the highest binding energy for FMO enzyme, with their respective target enzymes. Through detailed docking analysis, the interaction patterns and key binding sites between these compounds and target enzymes were revealed.

[0028] Figure 1 C lists the top five compounds with the highest molecular docking scores and displays their total scores (TotalScore). These scores are calculated based on comprehensive factors such as the binding affinity and hydrogen bond formation ability of the compound with the target enzyme. By comparing these scores, the most promising candidate drugs can be screened out.

[0029] Through virtual drug screening technology, this experiment successfully screened out several candidate drugs that can efficiently bind to CutC / D and FMO enzymes from a large number of compounds. Among them, 18β-glycyrrhetinic acid and hesperidin showed high binding affinity to CutC / D and FMO enzymes, respectively. This discovery provides important clues and basis for the development of new therapeutic drugs for TMAO-mediated kidney damage. In the future, these candidate drugs will be further verified by in vitro and in vivo experiments to evaluate their efficacy and safety, and are expected to open up new avenues for the treatment of kidney diseases.

[0030] Example 2: 18β-Glycyrrhetinic acid has an inhibitory effect on CutC / D enzyme and FMO enzyme (in vitro experiment)

[0031] In order to further explore the inhibitory effect of 18β-glycyrrhetinic acid on CutC / D enzyme and FMO enzyme, the following experiments were conducted in this example:

[0032] (1) Changes in the concentrations of trimethylamine (TMA) and trimethylamine oxide (TMAO) in bacterial culture medium

[0033] Proteus mirabilis (P.mir) was used as the experimental strain to measure the concentrations of TMA and TMAO in a culture medium containing different concentrations of the above 10 Chinese medicine monomers. The results are as follows: Figure 2 As shown in B. The results showed that 18β-GA could significantly reduce the concentrations of TMA and TMAO (***p<0.001, n=3).

[0034] (2) Expression of Fmo3 gene in Caenorhabditis elegans (C. elegans).

[0035] By feeding the above-mentioned Chinese medicine monomers with different concentrations, ox-LDL was used as a positive control to detect the expression level of the Fmo3 gene in nematodes. The results are as follows Figure 2 C. The results showed that 18β-GA had no significant effect on the expression of Fmo3 gene (n=3).

[0036] (3) Dose-concentration curve of the inhibitory effect of 18β-GA on choline metabolism to TMA and TMAO.

[0037] In P.mir, different concentrations of 18β-GA were used to treat and its inhibitory effect on choline metabolism to TMA and TMAO was determined. The results are as follows: Figure 2 D. The results showed that the inhibitory effect of 18β-GA on TMA and TMAO was dose-dependent, with EC50 values ​​of 56nM and 33nM, respectively.

[0038] (4) 18β-GA inhibits the production of TMA by BL21 bacteria carrying CutD-pET28a plasmid.

[0039] BL21 bacteria were transformed into strains carrying the CutD-pET28a plasmid, and the production of TMA was measured under treatment with different concentrations of 18β-GA. BL21 bacteria carrying an empty plasmid were used as negative controls. Figure 2 As shown in E. The results showed that 18β-GA could significantly inhibit the process of choline decomposition into TMA catalyzed by CutD enzyme (***p<0.001, n=3).

[0040] (5) Effect of 18β-GA on the growth of BL21 bacteria carrying CutD-pET28a plasmid in culture medium with choline as the sole carbon source.

[0041] BL21 bacteria were transformed into strains carrying the CutD-pET28a plasmid, and bacterial growth under treatment with different concentrations of 18β-GA was measured in a minimal medium containing 30 mM choline. Figure 2 F. The results showed that 18β-GA could significantly inhibit the growth of BL21 bacteria carrying CutD-pET28a plasmid in a medium with choline as the sole carbon source (***p<0.001, n=3), further confirming the inhibitory effect of 18β-GA on CutC / D enzyme.

[0042] (6) 18β-GA inhibits the production of TMAO by BL21 bacteria carrying FMO-pET28a plasmid.

[0043] BL21 bacteria were transformed into strains carrying the FMO-pET28a plasmid, and the production of TMAO was measured under treatment with different concentrations of 18β-GA. BL21 bacteria carrying an empty plasmid were used as negative controls. Figure 2 G. The results showed that 18β-GA could significantly inhibit the process of TMA oxidation to TMAO catalyzed by FMO enzyme (***p<0.001, n=3).

[0044] (7) Verification of the inhibitory effect of 18β-GA on the oxidation of TMA to TMAO in the purified FMO enzyme system.

[0045] Using the purified FMO enzyme system, the production of TMAO was measured under treatment with different concentrations of 18β-GA. The results are shown in Figure 2 H. The results showed that 18β-GA could directly inhibit the activity of FMO enzyme (***p<0.001, n=3).

[0046] (8) The effect of 18β-GA on the ability of other bacteria containing cholinesterase to produce TMA.

[0047] Using bacteria containing different cholinesterases, such as Acinetobacter calcoaceticus (ATCC 19606), the production of TMA was measured in a medium containing 18β-GA. The results are shown in Figure 2 I. The results showed that 18β-GA had no significant effect on the ability of other strains containing cholinesterase to produce TMA (ns>0.05), further confirming the specific inhibitory effect of 18β-GA on CutC / D enzyme and FMO enzyme.

[0048] In summary, the results of in vitro experiments show that 18β-glycyrrhetinic acid (18β-GA) has a significant inhibitory effect on the CutC / D enzyme that decomposes choline in the intestine and the FMO enzyme that oxidizes trimethylamine (TMAO). By using experimental models such as Proteus mirabilis (P. mirabilis) and Caenorhabditis elegans (C. elegans), this embodiment verifies that 18β-GA can significantly reduce the concentration of TMA and TMAO, and has no significant effect on the expression of the Fmo3 gene. Further, using BL21 bacteria carrying CutD-pET28a and FMO-pET28a plasmids, and a purified FMO enzyme system, the inhibitory mechanism of 18β-GA on CutC / D enzyme and FMO enzyme is elaborated in detail. The results show that 18β-GA can directly inhibit the process of choline decomposition into TMA and TMA oxidation into TMAO, and has no significant effect on other strains containing metabolic choline enzymes. These findings provide new ideas for the development of therapeutic drugs for TMAO-related diseases.

[0049] Example 3: 18β-Glycyrrhetinic acid has an inhibitory effect on CutC / D enzyme and FMO enzyme (in vivo experiment)

[0050] In order to further explore the inhibitory effect of 18β-glycyrrhetinic acid on CutC / D enzyme and FMO enzyme, the following experiments were conducted in this example:

[0051] (1) Serum TMAO levels Figure 3 As shown in A, the figure shows the changes in serum TMAO levels in mice after administration of 18β-GA (50 mg / kg / d). The experimental results showed that the concentration of TMAO in the serum of mice administered with 18β-GA was significantly reduced, with a statistical difference compared with the control group. This indicates that 18β-GA has the effect of inhibiting the production of TMAO in vivo.

[0052] (2) Kidney pathological staining.

[0053] The tissue morphology and inflammatory cell infiltration of mouse kidneys were observed by PAS (periodic acid Schiff staining), MASSON (Masson staining) and anti-CD45 immunofluorescence staining. Figure 3 As shown in Figure B. The results showed that although the renal tubular vacuolar degeneration and tubular expansion still existed in the kidneys of mice in the 18β-GA administration group, the atrophy and collapse of the renal tubules, the infiltration of inflammatory cells in the renal interstitium and the degree of fibrosis were significantly reduced compared with those in the control group, and the protein casts were also significantly reduced. This indicates that 18β-GA can reduce the pathological damage of the kidneys.

[0054] (3) Changes in serum creatinine (Scr) and blood urea nitrogen (BUN) values ​​in mice after administration of 18β-GA. Figure 3C. The experimental results showed that the Scr and BUN values ​​of mice in the 18β-GA administration group were lower than those in the control group, indicating that 18β-GA can improve the renal function of mice.

[0055] (4) Analysis of β-diversity of mouse intestinal flora.

[0056] The β diversity of the mouse intestinal flora was analyzed by 16S rRNA gene sequencing (V3-V4 region). Figure 3 D. The results showed that the beta diversity of the intestinal flora in the I / R+Cho group (i.e., high choline + ischemia-reperfusion group) was significantly different from that in other groups. This indicates that high choline and ischemia-reperfusion treatment had a significant effect on the intestinal flora of mice.

[0057] (5) Distribution of intestinal flora at the taxonomic level. Figure 3 As shown in Figure E, NMDS (non-metric multidimensional scaling) showed that the intestinal flora of the I / R+Cho group was significantly different from that of the other groups, mainly manifested in the increased abundance of Bacteroidia and Clostricia.

[0058] (6) Heat map of abundance of different bacterial species Figure 3 As shown in F, the abundance ratios of different bacterial species in the intestines of mice in different groups are shown. By comparison, it can be seen that the abundance of certain bacteria (such as Bacteroides, Blautia, Peptococcus, and Lachnoclostridium) in the I / R+Cho group increased significantly. These bacteria are rich in CutC / D enzymes and are important players in choline decomposition and TMA production.

[0059] (7) The abundance of four bacteria containing CutC / D genes, such as Figure 3 G shows the abundance ratio of four bacteria (Bacteroides, Blautia, Peptococcus and Lachnoclostridium) containing CutC / D genes in the intestines of different groups of mice. The experimental results showed that the abundance of these four bacteria in the intestines of mice given 18β-GA was significantly reduced compared with the I / R+Cho group. This indicates that 18β-GA can inhibit the growth of these bacteria rich in CutC / D enzymes, thereby reducing the production of TMA and TMAO.

[0060] In summary, the in vivo experimental results verified the effect of 18β-glycyrrhetinic acid (18β-GA) in inhibiting the production of trimethylamine oxide (TMAO) in vivo. The experimental results showed that 18β-GA could significantly reduce the concentration of TMAO in mouse serum, alleviate kidney pathological damage, improve renal function, regulate the structure of intestinal flora, and reduce the growth of bacteria rich in CutC / D enzymes. These results further support the feasibility of 18β-GA as a potential drug for the treatment of kidney disease.

[0061] Example 4: Mechanism of action of 18β-glycyrrhetinic acid on the TMAO / CXCL12 / CXCR4 signaling axis

[0062] In order to further explore the mechanism of action of 18β-glycyrrhetinic acid in improving kidney disease through the CXCL12 / CXCR4 signaling axis after reducing TMAO, as well as its potential impact on the progression of chronic kidney disease (CKD) in a clinical setting, the following experiments were conducted in this example:

[0063] Figure 4 The mechanism of action of 18β-GA against the TMAO / CXCL12 / CXCR4 axis and its effectiveness in the clinical setting are demonstrated.

[0064] Through single-cell sequencing technology, t-SNE visualization analysis of all subcell types was performed. Figure 4 A shows the distribution of renal cell types in mice after 18β-GA treatment.

[0065] Figure 4 B reveals the changes in the quantity and intensity of intercellular communication before and after 18β-GA intervention.

[0066] Figure 4 C compared the differences in CXCL signaling pathway between the I / R+Cho (ischemia-reperfusion + high choline diet) group and the I / R+Cho+GA (ischemia-reperfusion + high choline diet + 18β-GA) group.

[0067] Figure 4 D shows the communication possibilities of all signaling pathways upregulated in the I / R+Cho group.

[0068] Figure 4 E shows flow cytometry analysis of the proportion of CD11B+_CXCR4+ and CD4+_CXCR4+ cells in the kidney, evaluating the effect of 18β-GA on immune cell infiltration.

[0069] Figure 4 F shows Western blotting analysis of the protein expression levels of COL1A1, ITGAV, and CXCL12 after 18β-GA treatment.

[0070] Figure 4 G shows the immunofluorescence staining results of CXCL12, visually demonstrating the effect of 18β-GA on CXCL12 expression.

[0071] Figure 4 H shows the changes in plasma TMAO concentrations and mean ± standard deviation of 20 patients with end-stage renal disease (ESRD) after 0, 14, and 28 days of 18β-GA intervention. Figure 4 I shows the individual plasma TMAO values ​​of 10 patients and their changes over time.

[0072] In summary, the following conclusions can be drawn:

[0073] (1) Effects of 18β-GA on the TMAO / CXCL12 / CXCR4 axis:

[0074] Single-cell sequencing results showed that after 18β-GA treatment, the number of renal tubular epithelial cells in I / R+Cho+GA mice was restored, and the infiltration of Mono_Mac (monocytes / macrophages) and T_Lymphocytes (T lymphocytes) in the kidneys was significantly decreased, indicating that 18β-GA may alleviate renal inflammation by regulating immune cell infiltration.

[0075] Cell interaction analysis further confirmed that after 18β-GA treatment, the amount of intercellular communications was significantly reduced, especially the PTS3a cell-mediated CXCL pathway was significantly reduced, and the LR pair interaction of CXCL12-CXCR4 also decreased, suggesting that 18β-GA may alleviate the progression of CKD by inhibiting this key signaling axis.

[0076] (2) Effectiveness in clinical settings:

[0077] In a clinical study involving 20 ESRD patients, this example evaluated the effect of compound glycyrrhizic acid tablets (containing glycyrrhizic acid, which can be hydrolyzed into the active form of 18β-GA) on plasma TMAO concentration. The results showed that compared with the placebo group, the plasma TMAO concentration in the glycyrrhizic acid group was significantly reduced on the 28th day, and the individual plasma TMAO values ​​of most patients showed a downward trend. This preliminary result shows that compound glycyrrhizic acid tablets may help reduce TMAO levels in hemodialysis patients, providing a strong basis for further large-scale research.

[0078] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. 18 Application of β-glycyrrhetinic acid in TMAO inhibitors.

2. The use according to claim 1, characterized in that: The 18β-glycyrrhetinic acid inhibits TMAO production by efficiently binding to CutC / D and FMO.

3. A TMAO inhibitor, characterized in that Includes 18β-glycyrrhetinic acid as the active ingredient. 4.18 Application of β-glycyrrhetinic acid in the preparation of drugs for the treatment of chronic diseases.

5. The use according to claim 4, characterized in that: The chronic disease includes chronic kidney disease.

6. The use according to claim 5, characterized in that: The 18β-glycyrrhetinic acid improves renal injury via the CXCL12 / CXCR4 signaling axis.

7. A drug for treating chronic kidney disease, characterized in that: Includes 18β-glycyrrhetinic acid as the active ingredient.

8. The drug according to claim 7, characterized in that The medicine also includes a pharmaceutically acceptable carrier or excipient.

9. The drug according to claim 7, characterized in that The pharmaceutical dosage forms include tablets, powders, granules, capsules, oral liquids, injections or sustained-release preparations.

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