Use of ion channel blockers in the treatment and / or prevention of liver fibrosis

Abstract

To provide drugs for more effective treatment and / or prevention of liver fibrosis.SOLUTION: There is provided, an ion channel blocker in production of drugs for treating and / or preventing liver fibrosis. The ion channel blocker is a potassium ion channel blocker. Specifically, use of gliclazide is provided.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to the technical field of ion channels and liver fibrosis, and in particular to the use of ion channel blockers in the treatment and / or prevention of liver fibrosis. [Background technology]

[0002] More than 800 million people worldwide suffer from chronic liver disease, accounting for 2 million deaths each year from various liver diseases. Hepatic fibrosis (HF) is not only the common pathological basis of all chronic liver diseases, but also an important part of the progression to cirrhosis and liver cancer. Research has shown that liver fibrosis, even early cirrhosis, is reversible. However, current anti-fibrosis strategies primarily focus on eliminating the etiology of liver fibrosis through antiviral therapy, alcohol abstinence, and dietary management. These strategies generally have drawbacks, including long treatment durations, limited efficacy, numerous side effects, and poor patient compliance. Therefore, researchers have sought methods to significantly reverse liver fibrosis.

[0003] Currently, hepatic stellate cell (HSC) activation and excessive hepatic extracellular matrix (ECM) deposition are considered to be important components of the pathophysiological changes of liver fibrosis. First discovered by von Kupffer in 1876, HSCs reside in the subendothelial space of Disse, located between hepatic sinusoidal endothelial cells (LSECs) and hepatocytes. They account for approximately 10% of all resident hepatocytes. In a normal liver, HSCs maintain a nonproliferative, quiescent phenotype. After liver injury or in vitro culture, HSCs are activated and transform from vitamin A-storing cells into myofibroblasts, characterized by proliferation, contraction, inflammation, chemotaxis, and increased extracellular matrix (ECM) production. HSC activation involves various regulatory mechanisms, including autophagy, endoplasmic reticulum stress, oxidative stress, retinol and cholesterol metabolism, epigenetics, and receptor-mediated signaling, demonstrating the complexity of HSC activation. Currently, targeting mechanisms of several important molecular channel disorders that lead to HSC activation, such as transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF), has been identified, and some of the mechanisms involved in HSC activation have already been elucidated. However, unfortunately, to date, there is still a lack of clinical anti-fibrotic therapeutic methods that target HSCs. Therefore, exploring other pathways that trigger HSC activation may lead to the discovery of more effective anti-fibrotic therapeutic targets.

[0004] Ion channels have been a hot topic of research in recent years, and some ion channels are thought to play important roles in the development, migration, invasion, and metastasis of gastrointestinal cancers, such as gastric cancer and liver cancer. Summary of the Invention

[0005] The object of the present invention is to solve the above-mentioned problems present in the prior art by providing the use of ion channel blockers in the treatment and / or prevention of liver fibrosis.

[0006] In order to achieve the above object, the present invention provides the following means. The present invention provides the use of an ion channel blocker in the manufacture of a medicament for treating and / or preventing liver fibrosis.

[0007] Preferably, the ion channel blocker is a potassium ion channel blocker.

[0008] Preferably, the potassium ion channel blocker comprises gliclazide.

[0009] Preferably, said liver fibrosis comprises CCl4-induced liver fibrosis.

[0010] The present invention further provides the use of gliclazide in the manufacture of a medicament for inhibiting autophagy in liver tissue.

[0011] The present invention further provides the use of gliclazide in the manufacture of a medicament for inhibiting the proliferation of hepatic stellate cells.

[0012] Preferably, the hepatic stellate cells comprise HSC-T6 cells.

[0013] The present invention further provides the use of gliclazide in the manufacture of a medicament for downregulating hepatocyte α-SMA.

[0014] The present invention further provides the use of gliclazide in the manufacture of a medicament for downregulating hepatocyte LC3.

[0015] The present invention provides the use of gliclazide in the manufacture of a medicament for upregulating hepatocyte P62.

[0016] The present invention discloses the following technical effects. The present invention is based on the idea that activation of hepatic stellate cells is an important part of anti-liver fibrosis, and in vivo and in vitro experiments have shown that gliclazide has the effect of inhibiting the activation of hepatic stellate cells. Furthermore, it has been found that gliclazide inhibits the activation of hepatic stellate cells mainly by inhibiting the autophagy pathway, and therefore gliclazide can be used to manufacture medicines for treating and / or preventing liver fibrosis. [Brief explanation of the drawings]

[0017] In order to more clearly describe the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without creative efforts. [Figure 1] FIG. 1 shows the degree of fibrosis in liver tissue observed by HE staining and picrosirius red staining in Example 1. [Figure 2] FIG. 1 shows the changes in serum AST (A) and ALT (B) levels detected in Example 1 and statistical charts. [Figure 3] 1 shows (A) changes in the expression of α-SMA in liver tissue detected by immunohistochemistry in Example 2, and a statistical diagram (B). [Figure 4] 1 shows (A) changes in the expression of α-SMA in liver tissue detected by Western blotting in Example 3, and a statistical diagram (B). [Figure 5] 1 shows (A) changes in LC3 expression in mouse liver tissue detected by Western blotting in Example 3, and a statistical diagram (B). [Figure 6] 1 shows (A) changes in expression of P62 in liver tissue detected by Western blotting in Example 3, and a statistical diagram (B). [Figure 7] FIG. 1 shows the effect of co-culture of gliclazide and TGF-β1 on HSC proliferation as detected by CCK8 in Example 4, and a statistical diagram. [Figure 8]1 shows (A) changes in the expression level of α-SMA protein in HSCs cells detected by Western blotting in Example 5, and a statistical diagram (B). [Figure 9] 1 shows (A) changes in LC3 expression in HSCs detected by Western blotting in Example 5, and a statistical diagram (B). [Figure 10] 1 shows (A) changes in the expression of p62 in HSCs cells detected by Western blotting in Example 5, and a statistical diagram (B). [Figure 11] 1 shows (A) changes in α-SMA expression in HSCs cells after the addition of rapamycin, as detected by Western blotting in Example 6, and a statistical diagram (B). DETAILED DESCRIPTION OF THE INVENTION

[0018] While various exemplary embodiments of the present invention are described in detail, it should be understood that the following detailed description is not intended to limit the invention, but rather to provide a more detailed description of certain aspects, features and embodiments of the present invention.

[0019] The technical solutions of the present invention are all common solutions in the art unless otherwise specified, and the reagents or raw materials used are all commercially available or existing unless otherwise specified.

[0020] Example 1: Carbon tetrachloride (CCl4)-induced liver fibrosis in mice Experimental materials Sixty healthy male C57 mice (20-25 g each, purchased from Chongqing Tengxin Biotechnology Co., Ltd.), a gavage needle, a 1 mL syringe, CCl4 solution, olive oil, and gliclazide solution were prepared.

[0021] Experimental Method CCl4 solution: CCl4 was dissolved in olive oil solution (CCl4:olive oil = 1:4) and prepared immediately before use. Gliclazide solution: Gliclazide solution was dissolved in saline and prepared immediately before use. The concentration was 500 μm.

[0022] The mice were randomly divided into four groups: a.Normal control group; b. Model group: Mice were orally administered CCl4 solution at 0.1 mL / 20 g (twice a week for a total of 8 weeks). c. CCl4 + glybcyst group: Mice were given CCl4 solution alone by oral gavage at 0.1 mL / 20 g twice a week for a total of 2 weeks, followed by co-administration of glybcyst and CCl4 solutions for 6 weeks. Glybcyst solution was administered intraperitoneally at 0.2 mL / 20 g once a day. d. Gliclazide group: Gliclazide was administered intraperitoneally for 8 weeks at a dose of 0.2 mL / 20 g once a day.

[0023] Mice were euthanized at 2, 4, 6, and 8 weeks after model establishment, with one mouse per group euthanized at each time. Liver samples were removed and flash-frozen in liquid nitrogen at -80°C. The remaining livers were fixed in 4% paraformaldehyde for 24 hours and then embedded in paraffin. The severity of liver fibrosis was assessed by appearance, picrosirius red staining, and HE staining. If the staining results indicated liver fibrosis, the model was considered successful. All mice were then euthanized, and eye blood was collected to measure liver function indices (ALT, AST). Liver tissue was fixed in formaldehyde and embedded in paraffin for further use.

[0024] Experimental results As shown in Figures 1-2, ocular blood samples were collected from the model group at week 8 and analyzed. Significant increases in ALT and AST were observed. HE staining revealed clear liver tissue structure, normal hepatic lobule structure, intact hepatocytes, and distinct patterns in the normal group. In the model group, liver tissue damage and significant fibrous tissue proliferation were observed. In the carbon tetrachloride + gliclazide group, liver tissue damage and fibrous tissue proliferation were significantly reduced. Picrosirius red staining revealed that in the model group, red collagen fibers became thicker and longer, with some forming pseudolobules, indicating successful establishment of a mouse liver fibrosis model. In the gliclazide group, no significant collagen fibers were observed, indicating that gliclazide at a concentration of 500 μM was unable to induce liver fibrosis in mice.

[0025] Example 2: Immunohistochemical detection of the protein expression level of α-SMA, an indicator of liver fibrosis, between different groups Experimental materials: C57 mice, α-SMA primary antibody, immunohistochemistry kit, PBS solution, sodium citrate solution, etc.

[0026] Experimental method: The paraffin blocks from Example 1 were taken, sections were prepared, and then immunohistochemistry procedures were carried out according to the procedure. The sections were immersed in 100%, 95%, 80%, and 75% ethanol for 5 minutes, washed with PBS, removed catalase with hydrogen peroxide, and repaired the antigens with sodium citrate repair solution. After cooling, the sections were washed with PBS, blocked at room temperature for 30 minutes, and then added with primary antibody. The sections were incubated at 4°C overnight, and the next day, incubated with secondary antibody at room temperature for 1 hour. The sections were then stained with DBA, re-stained with hematoxylin, differentiated with hydrochloric acid alcohol, dehydrated, sealed, and finally observed and photographed under a microscope.

[0027] Experimental Results: As shown in Figure 3, gliclazide can downregulate the expression of α-SMA in mice with carbon tetrachloride-induced liver fibrosis.

[0028] Example 3: Detection of the expression status of liver fibrosis indicator α-SMA and autophagy-related indicators (LC3, P62) in different experimental groups using Western blot technique Experimental materials: mouse liver tissue, tweezers, scissors, 2 mm steel ball, magnet, cell lysis solution, PBS solution, BCA protein quantification kit, nonfat powdered milk, αSMA, LC3, and P62 primary antibodies and corresponding secondary antibodies, etc.

[0029] Experimental Method: The liver tissue from Example 1 was taken and cut into pieces the size of rice grains using tweezers and scissors. These were then placed in marked 1.5 mL EP tubes. Two 2 mm steel balls were placed in each EP tube, and 200-250 μL of cell lysis solution was added. After setting the parameters, the tubes were placed in a grinder and thoroughly ground. The steel balls were then removed using a magnet. The tubes were then incubated on ice for 30 minutes and centrifuged at 12,000 rpm for 30 minutes at 4°C. A standard curve was prepared, and 1 μL of protein sample, 19 μL of PBS, and 200 μL of BCA working solution (50:1) were added to each well of a 96-well plate. Two additional wells were added to each well. The wells were then placed in a drying oven at 37°C for 30 minutes. The OD at 560 nm was measured using ELISA to calculate the protein concentration. The proteins were boiled in boiling water for 5 minutes before loading. The samples were then subjected to gel electrophoresis, transfer, and sealing, followed by incubation with primary and secondary antibodies and subsequent exposure.

[0030] Experimental results: As shown in Figures 4-6, gliclazide significantly reduced the expression of αSMA and LC3 in liver tissue and significantly increased the expression level of the autophagy substrate P62 in liver tissue.

[0031] Example 4: Detection of the effect of co-culture of gliclazide and TGF-β1 on HSC proliferation by CCK8 Experimental materials: HSC-T6 cells, DMEM complete medium, 96-well plate, counting plate, CCK-8 working solution, etc.

[0032] Experimental method: First, HSC-T6 hepatic stellate cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% double antibody (penicillin-streptomycin solution), and 1% non-essential amino acids (NEAA) at 37°C and 5% CO2 until the cells reached 80%-90% confluence. After that, they were digested with trypsin and passaged. Then, they were seeded onto plates at a density of 7 x 10 6 A cell suspension containing cells / L was added at 100 μL per well to a 96-well culture plate, with six wells per group. Experiments were conducted using control, TGF-β1, and TGF-β1 + gliclazide at different concentrations (50 μM, 100 μM, 150 μM, and 200 μM). After cell adhesion, the corresponding concentrations of gliclazide were added to the control and TGF-β1 groups, and after 3 h, TGFβ1 (10 ng / mL) was added to the corresponding groups to stimulate mouse hepatic stellate cells. After 48 h of incubation, 100 μL of a 10% CCK8 + 90% medium mixture was added to each well. The cells were incubated at 37°C for 1–4 h. The wavelength was set to 450 nm, and the absorbance values were read and recorded using an automated microplate reader. CCK8 experiments were used to detect the effect of gliclazide on cell proliferation and to screen the optimal time point and drug concentration.

[0033] Experimental results: As shown in Figure 7, compared with the normal values, HSC-T6 cell proliferation could be significantly induced after stimulation with TGF-β1. Co-culture with gliclazide and TGF-β1 at concentrations of 100 μM, 150 μM, and 200 μM suppressed the TGF-β1-induced proliferation tendency, and the difference in the results was statistically significant (P<0.05).

[0034] Example 5: Detection of the expression status of α-SMA and autophagy-related markers (LC3, p62) in HSCs cells by Western blot technique Experimental materials: HSC-T6 cells, DMEM complete medium, cell culture dish, cell lysate, PBS solution, BCA protein quantification kit, nonfat powdered milk, αSMA, LC3, and p62 primary antibodies and corresponding secondary antibodies, etc.

[0035] Experimental Method: The cells were divided into a normal group, a TGF-β1 group, a gliclazide + TGF-β1 group, and a gliclazide group. The TGF-β1 stimulation concentration was 10 ng / mL, and the gliclazide concentration was 100 μM. HSC-T6 cells were cultured in cell culture dishes under the conditions described in Example 4, with the addition of drugs and TGF-β1. After 48 h, the cells were washed 3-4 times with pre-chilled PBS and then dried by aspirating the remaining PBS liquid with filter paper. A 50 mL culture flask was added with 50 μL of RIPA and 0.5 μL of 100 mol / L PMSF (100:1). The cells were incubated on ice for 30 minutes, after which the proteins were scraped off with a cell scraper. The proteins were aspirated into EP tubes and centrifuged at 12,000 rpm for 30 minutes at 4°C. To prepare a standard curve, 1 μL of protein sample, 19 μL of PBS, and 200 μL of BCA working solution (50:1) were added to each well of a 96-well plate. Two additional wells were added to each well. The wells were then placed in a 37°C dry oven for 30 min. The OD at 560 nm was measured using ELISA, and the protein concentration was calculated. The protein was boiled in boiling water for 5 minutes before loading. The gel was then electrophoresed, transferred, and sealed. After incubation with primary and secondary antibodies, the gel was exposed to light.

[0036] Experimental results: As shown in Figures 8-10, gliclazide significantly reduced the expression of αSMA and LC3 in HSCs, and significantly increased the expression level of the autophagy substrate P62 in HSCs.

[0037] Example 6: Detection of α-SMA expression levels in HSCs cells after addition of an autophagy agonist (rapamycin) by Western blot technique Experimental materials: HSC-T6 cells, Rapamycin, DMEM complete medium, cell culture dish, cell lysate, PBS solution, BCA protein quantification kit, nonfat powdered milk, αSMA, LC3, and P62 primary antibodies and corresponding secondary antibodies, etc.

[0038] Experimental Method: Similarly, the cells were divided into a normal group, a TGF-β1 group, a gliclazide + TGF-β1 group, and a gliclazide + TGF-β1 + rapamycin group. The TGF-β1 stimulation concentration was 10 ng / mL, the gliclazide concentration was 100 μM, and the rapamycin concentration was 100 nM. The TGF-β1 addition time was as described in Example 5, and rapamycin and gliclazide were added simultaneously. After 48 h of incubation, protein was quantified and detected by Western blot.

[0039] Experimental results: As shown in Figure 11, in the co-culture group (TGFβ1 + gliclazide), the expression level of α-SMA protein was significantly decreased compared with the TGFβ1 alone group, and compared with another co-culture group (TGFβ1 + gliclazide + rapamycin), the expression level of α-SMA protein was significantly increased, and the difference was statistically significant (P<0.05).

[0040] The above-mentioned embodiments are merely illustrative of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the design concept of the present invention should fall within the scope of protection defined by the claims of the present invention.

Claims

1. 1. Use of gliclazide in the manufacture of a medicament for treating and / or preventing liver fibrosis, comprising: Gliclazide inhibits autophagy in liver tissue, thereby inhibiting the proliferation of hepatic stellate cells induced by TGF-β1; Use of gliclazide in the manufacture of a medicament for the treatment and / or prevention of liver fibrosis, characterized in that the liver fibrosis is CCl4-induced liver fibrosis.

2. 2. The use of gliclazide according to claim 1 in the manufacture of a medicament for treating and / or preventing liver fibrosis, wherein the hepatic stellate cells comprise HSC-T6 cells.

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