Use of K+ channel blockers in the manufacture of drugs for treating liver fibrosis

Abstract

To provide a new application of an existing K+ channel blocker.SOLUTION: Use of an existing K+ channel blocker (sotalol) in production of drugs for treating liver fibrosis is provided. It is demonstrated that sotalol can effectively inhibit liver fibrosis and reduce expression of a fibrosis-related protein α-SMA. A specific route is regulating expression and / or function of autophagy and EMT-based molecules so as to achieve inhibition of hepatic stellate cell fibrosis. The invention provides a new dosing choice for clinical treatment of liver fibrosis.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of medicine, specifically to the use of K in the manufacture of a therapeutic agent for liver fibrosis. + Regarding the use of channel blockers. [Background technology]

[0002] Liver fibrosis is a chronic injury to the liver caused by the long-term effects of reactions, which has many pathogenic factors and may further progress to liver cirrhosis or liver cancer, seriously threatening human health and life. The stage of liver fibrosis is not only a common pathological basis for chronic liver diseases, but also an important part of the progression to liver cirrhosis and HCC. In recent years, related research has found that liver fibrosis, and even early liver cirrhosis, can be reversed. At present, how to effectively prevent liver fibrosis has become the research focus of researchers at home and abroad.

[0003] Clinically, the treatment of fibrosis is mainly to remove the cause of liver fibrosis and perform treatment, mainly including, for example, antiviral treatment, abstinence from alcohol, and dietary control. However, these methods have disadvantages such as long treatment period, low therapeutic effect, many side effects, poor patient compliance, etc., and poor prognosis of patients. Therefore, finding new potential action targets for liver fibrosis and guiding clinical treatment of liver fibrosis more accurately and effectively is of positive significance to improving patient prognosis. Since the development cost of new drugs is high, it is desirable to find existing non-antifibrotic drugs that can be applied to the treatment of liver fibrosis in the clinical stage.

[0004] Ion channels have been attracting attention in recent research. They are crucial proteins that regulate cell signaling by tightly controlling the influx or outflow of ions into cells or organelles, thus affecting a wide range of physiological and pathological processes. In the study of ion channels and the digestive system, the influence of ion channels on liver fibrosis has attracted increasing attention. It has already been proven that potassium ion channels can promote the function of fibroblasts in multiple fibrotic diseases. In the liver, potassium ion channels promote fibrotic tissue and the expression of TGF-β, and promote liver fibrosis by activating HSCs. Due to the above association between potassium ion channels and liver fibrosis, the inventors hope to find potassium ion blockers that can be applied to the treatment of clinical liver fibrosis with the increasing repurposing of pharmaceuticals. Sotalol is a competitive β-adrenergic receptor antagonist, and in addition to having the action of class II (β-blocking), sotalol also has antiarrhythmic activity of class III, which can increase the action potential duration (APD) and prolong the repolarization of the atria and ventricles. Sotalol is also a potassium ion blocker and can block the expression of potassium channels. Currently, research on sotalol is limited to its effect on arrhythmia, and there are no reports on its effect and mechanism of action on liver fibrosis. Summary of the Invention

[0005] In response to the shortcomings of the prior art, the present invention provides a method for the preparation of a medicament for treating liver fibrosis. + The use of a channel blocker is provided.

[0006] To achieve the above objectives, the present invention provides the following technical solutions: The present invention relates to a method for the preparation of a medicament for treating liver fibrosis. + The use of channel blockers is disclosed.

[0007] Preferably, the K + The channel blocker is sotalol.

[0008] The present invention has the following beneficial effects: 1. Sotalol is a clinically used antiarrhythmic drug with + It has the property of inhibiting the channel. Experiments have demonstrated that sotalol can effectively suppress liver fibrosis and reduce the expression of fibrosis-related protein α-SMA. The specific pathway is to achieve inhibition of hepatic stellate cell fibrosis by controlling autophagy and the expression and / or function of EMT system molecules, providing a new drug option for clinically treating liver fibrosis.

[0009] 2. In the course of research, the present invention found that sotalol can improve the degree of liver fibrosis by suppressing activated hepatic stellate cells, specifically by inhibiting epithelial-mesenchymal transition (EMT) and autophagy pathways.

[0010] 3. In this invention, K + A channel blocker (sotalol) was used to study its effect on liver fibrosis, which may provide more options for inhibiting hepatic stellate cell activation, improving and reversing liver fibrosis. [Brief description of the drawings]

[0011] [Figure 1] 1 shows the degree of fibrosis of liver tissue observed by HE staining and picrosirius red staining in Example 1. [Diagram 2] FIG. 2 shows changes and statistical diagrams of serum ALT and AST levels detected in Example 1. [Diagram 3] FIG. 13 shows the expression changes and statistical diagrams of α-SMA in liver tissue detected by immunohistochemistry in Example 2. [Figure 4] FIG. 13 shows the expression changes and statistical diagrams of α-SMA in liver tissue detected by Western blotting in Example 3. [Diagram 5]FIG. 13 shows changes in expression of autophagy-related indicators LC3 and P62 in liver tissue detected by Western blotting and statistical diagrams in Example 3. [Figure 6] FIG. 13 shows statistical diagrams and changes in expression of epithelial-mesenchymal transition mesenchymal-like cell markers N-cadherin and Vimentin in liver tissue detected by Western blotting in Example 3. [Figure 7] FIG. 13 shows the expression changes and statistics of ZO-1, an epithelial-mesenchymal transition mesenchymal-like cell marker, in liver tissue detected by Western blotting in Example 3. [Figure 8] FIG. 13 is a statistical diagram showing the effect of co-culture of sotalol and TGF-β1 on HSCs cell proliferation detected by CCK8 in Example 4. [Figure 9] FIG. 13 shows changes in the expression level of α-SMA protein in HSCs cells detected by Western blotting and statistical charts in Example 5. [Figure 10] FIG. 13 shows changes in the expression level of LC3 protein in HSCs cells detected by Western blotting and statistical charts in Example 5. [Figure 11] FIG. 13 shows changes in the expression level of P62 protein in HSCs cells detected by Western blotting and statistical charts in Example 5. [Figure 12] FIG. 13 shows changes in the expression level of Z0-1 protein in HSCs cells detected by Western blotting and statistical diagrams in Example 5. [Figure 13] FIG. 13 shows changes in the expression level of N-cadherin protein in HSCs cells detected by Western blotting and statistical charts in Example 5. [Figure 14] FIG. 13 shows changes in the expression level of Vimentin protein in HSCs cells detected by Western blotting in Example 5, and a statistical diagram. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, the technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the drawings in the embodiments of the present invention, but obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts are all included in the protection scope of the present invention.

[0013] Unless otherwise specified, the technical means used in the examples are common means well known to those skilled in the art.

[0014] The present invention relates to a method for the preparation of a therapeutic agent for liver fibrosis. + The use of a channel blocker, wherein K + Channel blockers improve liver fibrosis, specifically, + The channel blocker is sotalol.

[0015] The present invention will now be further described with reference to specific examples.

[0016] Example 1: Carbon tetrachloride (CCl4)-induced mouse liver fibrosis Experimental materials 60 healthy male C57 mice (20-25 g / mouse), gavage needles, 1 mL syringes, CCl4 solution, olive oil, sotalol solution.

[0017] Experimental method: CCl4 was dissolved in olive oil solution (CCl4: olive oil = 1:4, v / v) (prepared at the time of use; 0.1mL / 20g). Gavage was performed twice a week for a total of 8 weeks. Sotalol solution was mixed with saline (prepared at the time of use; concentration 1mm; 0.2mL / 20g). Intraperitoneal injection was performed once a day for a total of 6-8 weeks. Mice were randomly divided into a. normal control group, b. model group (mice were gavage-administered CCl4 for 8 weeks), c. CCl4 + sotalol group (mice were administered CCl4 alone for 2 weeks, followed by co-administration of sotalol and CCl4 for 6 weeks), and d. sotalol group (sotalol was intraperitoneally injected for 8 weeks). Mice were euthanized in turn at 2, 4, 6, and 8 weeks after model establishment, with one mouse from each group being euthanized each time. The liver samples were removed and stored in a -80℃ refrigerator for quick freezing, and the other liver was fixed in 4% paraformaldehyde for 24 hours and then embedded in paraffin. The severity of liver fibrosis was observed by appearance, picrosirius red staining, and HE staining, and if the staining results showed liver fibrosis, the model was considered to be successfully established. Then, all mice were euthanized, eyeball blood was collected to measure liver function indexes (ALT, AST), and liver tissue was fixed in formaldehyde and embedded in paraffin for use.

[0018] Experimental results: As shown in Figure 1-2, the results of collecting and detecting eyeball blood from the model group at the 8th week showed that both ALT and AST were significantly elevated. The staining results are shown in Figure 1. As a result of HE staining, in the normal group, the structure of the liver tissue was clear, the structure of the liver lobule was normal, the liver cells were intact, and the pattern was clear. In the model group, the liver tissue was damaged and fibrous tissue was significantly proliferated. In the carbon tetrachloride + sotalol group, the degree of liver tissue damage and fibrous tissue proliferation were both significantly reduced. As can be seen from the Sirius red staining results, in the model group, the red collagen fibers became thicker and longer, and some formed pseudolobules, indicating that the mouse liver fibrosis model was successfully established. In the sotalol group, no obvious collagen fibers were found, indicating that at a concentration of 1mm, sotalol cannot cause liver fibrosis in mice.

[0019] Example 2: Immunohistochemical detection of protein expression levels of α-SMA, an indicator of liver fibrosis among different groups Experimental materials: C57 mice, α-SMA primary antibody, immunohistochemistry kit, PBS solution, sodium citrate solution, etc.

[0020] Experimental method: The paraffin block of Example 1 was taken, and after sectioning, immunohistochemistry was performed 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, repaired antigens with sodium citrate repair solution, cooled and washed with PBS, blocked at room temperature for 30 minutes, added primary antibody, incubated at 4°C overnight, and incubated with secondary antibody at room temperature for 1 hour the next day, stained with DBA, re-stained with hematoxylin, differentiated with hydrochloric acid alcohol, dehydrated, sealed, and finally observed and photographed under a microscope.

[0021] Experimental Results: As shown in Figure 3, sotalol can downregulate the expression of α-SMA in carbon tetrachloride-induced liver fibrosis mice.

[0022] Example 3: Detection of the expression status of liver fibrosis indicator α-SMA and autophagy-related indicators, epithelial-mesenchymal transition (EMT), etc. in different experimental groups by Western blot technique Experimental materials: Mouse liver tissue, tweezers, scissors, 2 mm steel ball, magnet, cell lysis solution, PBS solution, BCA protein quantification kit, skim milk powder, α-SMA, autophagy and EMT-related primary antibodies and corresponding secondary antibodies, etc.

[0023] Experimental method: Take the liver tissue of Example 1, cut out pieces the size of a grain of rice with tweezers and scissors, and add them into a marked 1.5mL EP tube. Put two 2mm steel balls into each EP tube, add 200-250μL of cell lysis solution, set the parameters, put into a grinder and grind thoroughly, then use a magnet to suck out the steel balls, work on ice for 30min, then centrifuge at 12000rpm, 4℃ for 30min. A standard curve was made, and 1μL of protein sample, 19μL of PBS solution, and 200μL of BCA working solution (50:1) were added to each well of a 96-well plate, and two additional wells were added to each well. Placed in a drying oven at 37℃ for 30min. ELISA was used to measure the OD value at 560nm, and the protein concentration was calculated. The protein was boiled in boiling water for 5min, and then the sample was loaded, followed by gel electrophoresis, transfer, and sealing, and then incubated with primary and secondary antibodies and exposed to light.

[0024] Experimental Results: As shown in Figures 4-7, after treatment with sotalol, the expression of α-SMA and LC3 in liver tissue induced by CCl4 was significantly reduced, the expression of N-cadherin and Vimentin was suppressed, and the accumulation of P62 and ZO-1 was promoted.

[0025] Example 4: Detection of the effect of co-culture of sotalol and TGF-β1 on HSCs cell proliferation by CCK8 Experimental materials: HSC-T6 cells, DMEM complete medium, 96-well plate, counting plate, CCK-8 working solution, etc.

[0026] Experimental method: First, HSC-T6 hepatic stellate cells were cultured in DMEM culture 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 cell confluence reached 80%-90%. After that, they were digested with trypsin and passaged. Then, they were seeded on a plate at a density of 7×10 6The cell suspension of 100μL / well was taken and added to a 96-well culture plate at 100μL / well. There were 6 wells in each group. The experiment was divided into a control group, a TGF-β1 group, a TGF-β1 + sotalol drug group with different concentrations (200μM, 400μM, 800μM, 1000μM), and a sotalol drug group (200μM, 400μM, 800μM, 1000μM). After the cells attached to the wall, sotalol was added to the remaining groups at the corresponding concentrations in addition to the control group and the TGF-β1 group, and after 3h, TGFβ1 (10ng / mL) was added to the corresponding groups to stimulate the mouse hepatic stellate cells. After 48h incubation, 100μL of "10% CCK8 + 90% medium" mixed solution was added to each well, incubated at 37℃ for 1-4h, the wavelength was set to 450nm, and the absorbance value was read and recorded at the corresponding wavelength by a fully automated microplate reader. The CCK8 experiment was used to detect the effect of sotalol on cell proliferation and screen the optimal action time point and drug concentration.

[0027] Experimental results: As shown in FIG. 8, compared with the normal value, the proliferation of HSC-T6 cells could be significantly induced after stimulation with TGF-β1, and the TGF-β1-induced proliferation tendency was suppressed by co-culture with sotalol and TGF-β1.

[0028] Example 5: Detection of expression status of α-SMA, etc. in HSCs cells by Western blot technique Experimental materials: HSC-T6 cells, DMEM complete medium, cell culture dish, cell lysis solution, PBS solution, BCA protein quantification kit, skim milk powder, αSMA, autophagy and EMT primary antibodies and corresponding secondary antibodies, etc.

[0029] Experimental method: First, the subjects were divided into a normal group, a TGF-β1 group, a sotalol + TGF-β1 group, and a sotalol group. Here, the TGF-β1 stimulation concentration was 10 ng / mL, and the sotalol concentration was 400 μm. HSC-T6 cells were cultured using cell culture dishes, cultured under the conditions described in Example 4, and drugs were added and TGF-β1 was added. After 48 h, the incubated cells were washed 3-4 times with pre-cooled PBS, and then the remaining PBS liquid was aspirated and dried with filter paper. 50 μL of RIPA and 0.5 μL of 100 mol / L PMSF (100:1) were added to a 50 mL culture flask, and the cells were allowed to act on ice for 30 minutes, after which the protein was scraped off with a cell scraper, and the protein was aspirated into an EP tube and centrifuged at 12000 rpm, 30 minutes, and 4 ° C. A standard curve was made, 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, and two additional wells were added to each well. The plates were placed in a dry oven at 37°C for 30 min. ELISA was used to measure the OD value at 560 nm, and the protein concentration was calculated. The proteins were boiled in boiling water for 5 minutes before loading the samples, and gel electrophoresis, transfer, and sealing were performed in sequence, and the primary and secondary antibodies were incubated and then exposed to light.

[0030] Experimental results: As shown in Figures 9-14, sotalol significantly reduced the expression of α-SMA in HSCs cells. In the autophagy process, sotalol significantly inhibited the expression of LC3 and promoted the accumulation of P62. In the epithelial-mesenchymal transition process, sotalol promoted the accumulation of ZO-1 and inhibited the expression of N-cadherin and Vimentin.

[0031] The above embodiments are merely illustrative of the 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 be included within the scope of protection defined by the claims of the present invention.

Claims

【Request 1】 Sotalol in the manufacture of hepatic fibrosis treatments ＋ Use of channel inhibitors.

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