Application of poliumoside in preparation of FMO3 inhibitor

Abstract

The invention discloses an application of poliumoside in preparation of an FMO3 inhibitor. The invention reveals that poliumoside can be used as an FMO3 inhibitor to reduce the TMAO level for the first time, is used for treating renal interstitial fibrosis related to metabolic diseases, provides a brand new candidate compound and a solution for clinical anti-renal fibrosis treatment, and fills the technical blank in the specific field.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to the application of ginsenoside in the preparation of FMO3 inhibitors. Background Technology

[0002] Tubulointerstitial fibrosis (TIF) is the common pathway and core pathological step in the progression of various chronic kidney diseases (CKD) to end-stage renal disease. Its pathological features mainly include progressive damage and atrophy of renal tubular epithelial cells, persistent inflammatory cell infiltration, abnormal activation and proliferation of myofibroblasts, and the resulting excessive synthesis and abnormal deposition of extracellular matrix. This process is accompanied by the progressive loss of the peritubular capillary network, ultimately leading to the destruction of normal renal parenchymal structure and progressive loss of function. In recent years, increasing evidence suggests that the gut microbiota metabolite trimethylamine N-oxide (TMAO) and its key synthase flavin containing monooxygenase 3 (FMO3) play important roles in the development and progression of kidney disease. Elevated TMAO levels can exacerbate renal tubular epithelial damage, promote inflammatory responses and fibrosis, while inhibiting FMO3 activity and reducing TMAO levels have been shown to delay the onset of renal tubulointerstitial fibrosis. Therefore, the FMO3-TMAO axis has become a potential intervention target.

[0003] Despite extensive research on the mechanisms and treatment strategies of renal fibrosis both domestically and internationally in recent years, effective interventions for tubulointerstitial fibrosis remain lacking. Current treatments primarily focus on controlling the primary disease and slowing its progression, offering limited efficacy for existing fibrotic lesions. Particularly concerning is the emerging FMO3-TMAO axis, for which there are currently no clinically available, highly effective, and low-toxicity inhibitors. Therefore, developing drugs that target FMO3 activity, reduce TMAO levels, and effectively block the progression of renal fibrosis has become a crucial and urgent area of ​​research in nephrology. Summary of the Invention

[0004] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide the application of ginsenoside in the preparation of FMO3 inhibitors.

[0005] Another object of the present invention is to provide the use of sclerotin in the preparation of a medicament for treating kidney injury.

[0006] The objective of this invention is achieved through the following technical solution: the application of ginsenoside in the preparation of FMO3 inhibitors is demonstrated by experimental verification that ginsenoside can bind to FMO3, and the experimental results of reduced TMAO levels indicate that ginsenoside is an FMO3 inhibitor.

[0007] The application of sclerotin in the preparation of drugs for the prevention and / or treatment of kidney injury is that sclerotin, as the sole active ingredient, has the effect of improving renal fibrosis, reducing serum creatinine, and reducing renal inflammation.

[0008] The renal fibrosis is preferably renal tubular interstitial fibrosis.

[0009] The renal fibrosis is preferably renal fibrosis associated with metabolic diseases.

[0010] The metabolic disease mentioned is preferably diabetes.

[0011] The dosage form of the drug is oral or injectable; preferably injectable.

[0012] The present invention has the following advantages and effects compared with the prior art: (1) This invention reveals for the first time that ginsenoside can reduce TMAO levels as an FMO3 inhibitor and be used to treat metabolic disease-related renal interstitial fibrosis, providing a novel candidate compound and solution for clinical anti-renal fibrosis treatment and filling the technological gap in this specific field.

[0013] (2) The ginsenoside provided by the present invention has shown significant anti-renal tubulointerstitial fibrosis effects in various animal models of metabolic diseases.

[0014] (3) The active ingredient in this invention, ginsenoside, can be extracted and isolated from a variety of plants, and its sources are relatively wide, which provides feasibility for its subsequent development and application. Attached Figure Description

[0015] Figure 1 This is a flowchart of the screening of the FMO3 inhibitor ginsenoside by the "Tianhe-2" supercomputer in Embodiment 1 of the present invention.

[0016] Figure 2 This is a diagram showing the results of a surface plasmon resonance experiment in Example 1 of the present invention, which demonstrates that ginsenoside can bind to human FMO3 protein.

[0017] Figure 3 The image shows the LC-MS results of serum TMAO content in ob / ob mice 4 weeks after administration in Example 2 of this invention.

[0018] Figure 4 This is a graph showing the change in serum creatinine levels in ob / ob mice after 4 weeks of drug administration in Example 2 of the present invention.

[0019] Figure 5 This is a PAS staining result of kidney tissue in ob / ob mice 4 weeks after administration of the drug in Example 2 of the present invention.

[0020] Figure 6 This is a Sirius Red staining result of kidney tissue in ob / ob mice 4 weeks after administration of the drug in Example 2 of the present invention.

[0021] Figure 7 This is a graph showing the detection results of mRNA levels of genes related to inflammation, fibrosis, and kidney injury in the kidneys of ob / ob mice 4 weeks after administration in Example 2 of the present invention.

[0022] Figure 8 The image shows the LC-MS results of serum TMAO content in diet-induced obese (DIO) mice after 4 weeks of drug administration in Example 3 of this invention.

[0023] Figure 9 This is a graph showing the change in serum creatinine levels in DIO mice after 4 weeks of administration in Example 3 of the present invention.

[0024] Figure 10 This is a PAS staining result of kidney tissue in DIO mice 4 weeks after administration in Example 3 of the present invention.

[0025] Figure 11 This is a Sirius Red staining result of kidney tissue in DIO mice 4 weeks after administration in Example 3 of the present invention.

[0026] Figure 12 This is a graph showing the detection levels of mRNA related to inflammation, fibrosis, and kidney injury in the kidneys of DIO mice 4 weeks after administration in Example 3 of this invention. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0028] Example 1: Screening of FMO3 inhibitor ginsenoside by the "Tianhe-2" supercomputer Screening process as follows Figure 1 As shown, using the binding sites of FMO3 and its substrate NAD (nicotinamide adenine dinucleotide) as virtual binding pockets, small molecule compounds that can competitively bind to FMO3 with NAD, which is essential for its enzyme activity, were screened. Using the Tianhe-2 supercomputer and based on the Schrödinger virtual docking algorithm, the binding pockets of the FMO3 protein crystal structure were subjected to high-throughput virtual docking screening with the Shanghai Institute of Materia Medica's small molecule compound database (n=1499). The compounds were sorted according to the strength of their virtual binding energies to screen for small molecule inhibitors that may directly bind to FMO3. Figure 2 As shown, surface plasmon resonance experiments have verified that ginsenoside can bind to human FMO3 protein.

[0029] Example 2: The effect of ginsenoside on renal interstitial fibrosis in ob / ob mice Eight-week-old male ob / ob mice (Guangdong Yaokang Biotechnology Co., Ltd.) and age-matched C57BL / 6J control mice (Guangdong Yaokang Biotechnology Co., Ltd.) were selected and housed in an SPF-grade animal experimental environment with a 12-hour / 12-hour light / dark cycle and free access to food and water. After one week of acclimatization, the mice were randomly divided into a control group (Lean), a model group (ob / ob+Veh), and a treatment group (ob / ob+POL), with eight mice in each group. The control and model groups were continuously fed standard diets. The treatment group, under the same feeding conditions, received intraperitoneal injections of ginsenoside solution (20 mg / kg) daily starting from week 5 for four consecutive weeks. The control and model groups received an equal volume of physiological saline injection.

[0030] During the experiment, mouse body weight, water intake, and food intake were recorded. After 4 weeks of treatment, mice were fasted for 6 hours and then anesthetized with isoflurane. Blood was collected via cardiac sampling to measure serum TMAO and creatinine (CREA) levels. Both kidneys were dissected and separated. Part of the kidney tissue was fixed in 4% paraformaldehyde for 48 hours, routinely embedded in paraffin, and sectioned for PAS and SiriusRed staining to evaluate the degree of tubulointerstitial injury and fibrosis. Part of the kidney tissue was used for RNA extraction to detect the expression levels of inflammation and fibrosis-related genes.

[0031] Serum TMAO level test results as follows Figure 3 As shown, the serum TMAO level in the model group was significantly higher than that in the control group. After administration, the serum TMAO level in ob / ob mice could be reduced to the level of normal mice.

[0032] Serum creatinine test results are as follows Figure 4 As shown, the serum creatinine level in the model group was significantly higher than that in the control group, and the serum creatinine level in ob / ob mice was significantly reduced after administration.

[0033] PAS staining results of kidney tissue as follows Figure 5 As shown, ginsenoside A can improve renal tubular basement membrane thickening and interstitial damage.

[0034] Sirius Red staining results of kidney tissue as follows Figure 6 As shown, after administration of ginsenoside A, collagen fibers decreased and the area of ​​positive regions shrank.

[0035] The expression levels of inflammation-related gene IL-6 and fibrosis-related gene Timp-1 are as follows: Figure 7 As shown, ginsenoside A can downregulate the expression of inflammatory cytokines IL-6 and fibrosis-related gene Timp-1 in the kidneys of ob / ob mice.

[0036] Example 3: The effect of ginsenoside on renal interstitial fibrosis in DIO mice Male C57BL / 6J mice aged 6-8 weeks were selected and, after one week of acclimatization, randomly divided into four groups: a control group (WT), a WT group receiving L-carnitine (WT+LC+Veh), a model group (DIO+LC+Veh), and a treatment group (DIO+LC+POL), with 8 mice in each group. Mice in the model and treatment groups were fed a 60% high-fat diet (HFD) for 12 weeks for model establishment and experimentation, while the control and WT groups receiving L-carnitine were fed a normal diet. Eight weeks after model establishment, the treatment group received daily intraperitoneal injections of sclerotinib (20 mg / kg) for 4 consecutive weeks; the control and WT groups received the same volume of physiological saline as the model group. During weeks 11-12 of the experiment, mice in the model and treatment groups were simultaneously administered L-carnitine (50 mg / kg / day) orally via gavage to increase plasma TMAO levels. Body weight, diet, and water intake were continuously recorded throughout the treatment period. After the experiment, mice were fasted for 6 hours, and blood was collected from their hearts after anesthesia to measure serum TMAO and creatinine levels. At the same time, kidney tissue was separated, part of which was used for paraffin sectioning and histological analysis, and part of which was used for RNA extraction to detect the expression level of fibrosis-related genes.

[0037] Serum TMAO level test results as follows Figure 8 As shown, the serum TMAO levels in the other three groups were elevated compared to the control group. After administration, the serum TMAO levels in the DIO+LC+POL administration group were significantly lower than those in the DIO+LC+Veh model group.

[0038] Serum creatinine test results are as follows Figure 9 As shown, there was no difference in serum creatinine between the control group and the WT+LC+Veh group. The serum creatinine level in the DIO+LC+Veh model group was significantly higher than that in the control group, while the serum creatinine level in the DIO+LC+POL administration group was lower than that in the DIO+LC+Veh model group.

[0039] PAS staining results of kidney tissue as follows Figure 10 As shown, it can be seen that ginsenoside can improve the thickening of the renal tubular basement membrane and interstitial damage in mice in the DIO+LC+POL administration group.

[0040] Sirius Red staining results of kidney tissue as follows Figure 11 As shown, after administration of ginsenoside, the collagen fibers of mice in the DIO+LC+POL administration group decreased, and the area of ​​positive regions was significantly reduced.

[0041] The expression levels of inflammation-related gene IL-6 and fibrosis-related genes Timp-1, Col1a1, and Col3a1 are as follows: Figure 12 As shown, ginsenoside can downregulate the expression of inflammatory cytokines IL-6, fibrosis-related genes Timp-1, Col1a1, and Col3a1 in the kidneys of mice in the DIO+LC+POL treatment group.

[0042] In summary, the ginsenoside provided by this invention has significant application value in the preparation of drugs for the prevention and / or treatment of renal interstitial fibrosis associated with FMO3 / TMAO-mediated metabolic diseases.

[0043] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of aesculin in the preparation of an inhibitor of FMO3.

2. Use of aesculin in the preparation of a medicament for preventing and / or treating renal injury.

3. Use according to claim 2, characterized in that: The renal injury is at least one of renal fibrosis, elevated serum creatinine, and renal inflammation.

4. Use according to claim 3, characterized in that: The renal fibrosis is tubulointerstitial fibrosis.

5. Use according to claim 3, characterized in that: The renal fibrosis is metabolic disease-related renal fibrosis.

6. Use according to claim 3, characterized in that: The metabolic disease is diabetes.

7. Use according to any one of claims 2 to 6, characterized in that: The medicament is in the form of an oral agent or an injection.

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