Use of enhancer 3C1 in the preparation of an enhancer of the interaction between FXR and Caspase 8 proteins

Abstract

The present invention discloses the use of enhancer 3C1 in the preparation of enhancer of interaction between FXR protein and Caspase8 protein.The enhancer of the present invention can promote the interaction between FXR protein and Caspase8 protein, inhibit the recruitment of Caspase8 to apoptotic complex, block the formation of apoptotic complex, inhibit the cleavage activation of Caspase8, block the process of apoptosis, and inhibit cell apoptosis.Therefore, the enhancer of the present invention is useful for the preparation of drugs for treating liver diseases with apoptotic phenotype, and has a promising market prospect.

Description

[Technical field]

[0001] The present invention relates to the use of an enhancer in the preparation of a protein interaction enhancer, in particular to the use of enhancer 3C1 in the preparation of an enhancer of the interaction between the FXR protein and the Caspase 8 protein. [Background technology]

[0002] Currently, various acute and chronic liver diseases (drug-induced liver injury, alcohol-induced liver injury, nonalcoholic fatty liver, cholestatic liver injury, viral hepatitis, liver fibrosis, liver cirrhosis, liver cancer, etc.) have a high incidence worldwide and are one of the important causes of death threatening human health. Currently, approximately 185 million people worldwide are infected with hepatitis C virus, the prevalence of nonalcoholic fatty liver disease (NAFLD) is over 25%, and more than 100 million people have progressed to nonalcoholic steatohepatitis (NASH). However, except for hepatitis C virus, there are currently few specific drugs available to treat other types of liver diseases.

[0003] Cell apoptosis is a fundamental biological phenomenon that plays an important role in the evolution / development and homeostasis of organisms. Cell apoptosis disorders are closely related to the onset and progression of many diseases. For example, hepatocyte apoptosis is a common feature and pathological mechanism of various liver diseases (drug-induced liver injury, alcohol-induced liver injury, NALFD / NASH, viral hepatitis, cholestatic liver injury, ischemia-reperfusion liver injury, hepatolenticular degeneration, liver fibrosis / cirrhosis, liver cancer, etc.). Inhibiting hepatocyte apoptosis is widely considered to be one of the key strategies in the treatment of liver diseases (Cellular and molecular mechanisms of liver injury. Gastroenterology, 2008, 134(6): 1641-54.).

[0004] Farnesoid X receptor (FXR, also known as NR1H4) is a member of the nuclear receptor superfamily and plays an important role in bile acid, sugar, and lipid metabolism, and is closely related to physiological and pathological processes such as energy balance, inflammation, cell fate, fibrosis, and liver regeneration. Therefore, FXR is widely considered as an important target for the development of hepatoprotective drugs (FXR modulators for enterohepatic and metabolic diseases. Expert OpinTher Pat. 2018 Nov;28(11):765-782.). Current drug development strategies targeting FXR mainly focus on developing FXR agonists or antagonists to control the transcriptional activity of FXR. Obeticholic acid is an FXR agonist approved by the US FDA in May 2016 for the treatment of anti-idiopathic biliary cirrhosis. This is the first successfully marketed drug targeting FXR, and a phase III clinical study on its effect on NASH has been completed, but its clinical efficacy has not been good (Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial. Lancet. 2019 Dec 14;394(10215):2184-2196.).

[0005] Research has shown that NASH and liver fibrosis are accompanied by significant hepatocyte apoptosis. FXR agonists have no effect in improving the significantly elevated hepatocyte apoptosis state in NASH and other diseases. This is one of the main reasons for the limited clinical efficacy of FXR agonists (Combined obeticholic acid and apoptosis inhibitor treatment alleviates liver fibrosis. Acta Pharm Sin B. 2019 May;9(3):526-536.). In addition, preliminary studies by the project team have shown that there is a stable protein-protein interaction between FXR and Caspase8 proteins in hepatocytes. When hepatocytes are stimulated by apoptosis, the interaction between FXR and Caspase8 proteins weakens, and Caspase8 binds to FADD and RIP1 to form an apoptosis complex. This complex promotes the cleavage activation of Caspase8 and promotes the process of apoptosis (Noncanonical farnesoid X receptor signaling inhibits apoptosis and impedes liver fibrosis.EBioMedicine. 2018 Nov;37:322-333.). Therefore, stabilizing and strengthening the interaction between FXR protein and Caspase8 protein is an important strategy to block apoptosis signaling and the occurrence and progression of apoptosis. However, it is still unclear how the interaction between FXR protein and Caspase8 protein is controlled, and there is currently no regulator for the interaction between FXR and Caspase8 protein. Summary of the Invention [Problem to be solved by the invention]

[0006] Object of the invention: The object of the present invention is to provide a use of enhancer 3C1 in the preparation of an enhancer of the interaction between FXR protein and Caspase 8 protein. Another object of the present invention is to provide a use of enhancer 3C1 in the preparation of a drug for treating liver fibrosis and liver injury as an enhancer of the interaction between FXR protein and Caspase 8 protein. [Means for solving the problem]

[0007] Technical solution: the use of enhancer 3C1 shown in formula (1) of the present invention in preparing an enhancer of the interaction between FXR protein and Caspase 8 protein.

[0008] [ka]

[0009] The enhancer 3C1 promotes the interaction between the FXR protein and the Caspase 8 protein under physiological or pathological conditions.

[0010] The enhancer blocks the formation of the apoptotic complex by blocking the recruitment of Caspase 8 protein to the apoptotic complex.

[0011] The enhancer subblocks the cleavage activation of Caspase 8 protein.

[0012] The enhancer prepares the formulation by adding pharma- ceutically acceptable auxiliary materials.

[0013] Said pharma- ceutically acceptable auxiliary materials include diluents, binders, disintegrants, glidants, lubricants, flavoring agents, encapsulating materials, and adsorbents.

[0014] The formulations are granules, powders, tablets, capsules, pills, and oral liquids.

[0015] The use of said enhancer 3C1 as an enhancer of the interaction between FXR protein and Caspase 8 protein in the preparation of a drug against hepatocyte apoptosis.

[0016] Use of said enhancer 3C1 as an enhancer of the interaction between FXR protein and Caspase 8 protein in the preparation of a hepatoprotective drug.

[0017] Use of said enhancer 3C1 as an enhancer of the interaction between FXR protein and Caspase 8 protein in the preparation of a therapeutic agent for liver fibrosis and liver injury. Effect of the Invention

[0018] Compared with the prior art, the present invention has the following significant advantages: (1) The enhancer can strengthen the interaction between FXR protein and Caspase8 protein in physiological state, with significant difference from the control group, optimally reaching ***P<0.001; (2) The enhancer can restore and strengthen the interaction between FXR protein and Caspase8 protein weakened in apoptotic state; (3) The enhancer can resist hepatocyte apoptosis caused by various causes, with significant difference from the control group, optimally reaching ***P<0.001; (4) The enhancer has precise targeting, high specificity, high accuracy, and many indications, and can be used in the preparation of drugs for treating various liver diseases with hepatocyte apoptosis phenotype. [Brief description of the drawings]

[0019] [Figure 1] The interaction between FXR and Caspase 8 proteins in cell apoptotic states. [Diagram 2] The present invention relates to the regulation of the interaction between FXR and Caspase 8 proteins in healthy cells by enhancer 3C1. [Diagram 3]The present invention relates to the regulation of the interaction between FXR protein and Caspase 8 protein in apoptotic cells by enhancer 3C1. [Figure 4] Protective effect of enhancer 3C1 of the present application against cell apoptosis: (A) hepatocyte apoptosis induced by ActD / TNFα, (B) hepatocyte apoptosis induced by CHX / FasL, (C) hepatocyte apoptosis induced by TRAIL. *P<0.05, **P<0.01, ***P<0.001. [Figure 5A] Protective effect of the present enhancer 3C1 against liver injury induced by CCl4. (A) Serum ALT level, *P<0.05, **P<0.01, ***P<0.001. [Figure 5B] Protective effect of the present enhancer 3C1 against liver injury induced by CCl4. (B) Serum AST levels, *P<0.05, **P<0.01, ***P<0.001. [Figure 5C] Protective effect of enhancer 3C1 of the present application against liver injury induced by CCl4. (C) Liver H&E staining, *P<0.05, **P<0.01, ***P<0.001. [Figure 5D] Protective effect of enhancer 3C1 of the present application against liver injury induced by CCl4. (D) Sirius red staining of liver, *P<0.05, **P<0.01, ***P<0.001. [Figure 5E] Protective effect of enhancer 3C1 of the present application against liver injury induced by CCl4. (E) Hepatic Timp1 mRNA level, *P<0.05, **P<0.01, ***P<0.001. [Figure 5F] Protective effect of enhancer 3C1 of the present application against liver injury caused by CCl4. (F) Hepatic Timp2 mRNA level, *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Example 1 Changes in the interaction between FXR and Caspase 8 proteins during cell apoptosis 1. Experimental Materials DMEM medium was purchased from GIBCO (Grand Island, New York, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, Utah, USA). Trypsin was purchased from Amersco (Solon, Ohio, USA). Cell culture consumables were purchased from Costar (USA). Actinomycin D (ActD) and cycloheximide (CHX) were purchased from MedChem Express (NJ, USA). Recombinant human tumor necrosis factor alpha (TNFα) was purchased from Peprotech (Rocky Hill, USA), and recombinant human FasL protein and recombinant human TRAIL protein were purchased from Sino Biological (Wayne, PA, USA).

[0021] 2. Experimental Method 2.1. Cell Culture and Treatment Human hepatocyte cell line L02 was cultured in DMEM complete medium containing 10% fetal bovine serum and placed in a cell incubator at 37°C and 5% CO2. 2 × 10 cells in the logarithmic growth phase were cultured. 5 Cells / well were seeded in 12-well plates. After 24 h, apoptosis models were created using TRAIL, ActD / TNFα, and CHX / FasL methods. The administered concentration of TRAIL was 50 ng / ml, and the treatment time with TRAIL was 0, 2, 4, 8, 12, and 24 h. The administered concentrations of ActD / TNFα were 0.2 μM and 20 ng / ml, ActD was pre-administered for 30 min, and the treatment time with ActD was 0, 1, 2, 4, 8, and 12 h. The administered concentrations of CHX / FasL were 50 μM and 50 ng / ml, CHX was pre-administered for 30 min, and the treatment time with CHX was 0, 2, 4, and 8 h.

[0022] 2.2 Co-immunoprecipitation (Co-IP) 1) The cells were washed twice with pre-chilled PBS, and finally, an appropriate amount of pre-chilled PBS was added, and the cells were scraped off from the culture dish or culture bottle using a pre-chilled cell scraper, and the suspension was transferred to a 1.5 ml EP tube. Centrifuged at 4°C and 4000 rpm for 5 min, the supernatant was discarded, an appropriate amount of NP40 dissolving solution was added, and the cells were shaken uniformly, and the cells were disrupted by ultrasonication for 5 seconds each time, a total of three times, to completely dissolve the cells. Next, centrifuged at 4°C and 14,000 g for 10 min, and the supernatant was collected in a new EP tube to obtain a protein dissolution solution. Protein concentration was measured by the BCA method.

[0023] 2) 25μl of Protein A Beads were taken, blocked with 5% BSA at room temperature for 1h, washed three times with PBS, centrifuged at 14,000g for 1min each time, and the supernatant was discarded. A homogenous concentration of protein lysate was added to the Beads, shaken at 4℃ for 1h, and the sample was pre-cleared. Centrifuged at 14,000g for 1min, and the supernatant was transferred to a new EP tube. Caspase8 antibody and IgG were added to the protein lysate, shaken overnight on a shaker at 4℃, and the antigen-antibody complex was prepared. The next day, the Beads were washed twice, and then centrifuged at 14,000g for 1min at 4℃. Next, the Ag-Ab mixture was added to the Beads, shaken on a shaker at 4℃ for 3h, and the Beads-antigen-antibody complex was prepared. Next, centrifuged at 14,000g for 1min at 4℃. The supernatant was discarded, and the bottom ones (Beads) were left. The beads were washed three times and centrifuged instantaneously. 2× Loading Buffer (30 μl) was added to the beads, which were then boiled in boiling water for 5 min and centrifuged at 12,000 rpm for 5 min. Western blot and gel imaging analysis were performed.

[0024] 2.3 Western blot 1) Cells were collected and placed on ice, and 100 μl of RIPA cell lysis solution, 1% Protease Inhibitor Cocktail (Sigma), and 1% phosphatase inhibitor were added per 20 μl packed cell volume. The aggregates were thoroughly pipetted and lysed on ice for 30 min, followed by ultrasonic disruption 5 times for 2 s each, with a pause for a few seconds to allow the lysis solution to cool. The cells were centrifuged at 13,000 g for 10 min, and the resulting supernatant was transferred to a new centrifuge tube as the extracted total protein sample, and the protein content was measured using the BCA method. 4× loading buffer was added, mixed uniformly, and boiled for 10 min in preparation for loading.

[0025] 2) SDS-PAGE of 3%-5% stacking gel and 6%-12% resolving gel were prepared, and protein samples were separated by electrophoresis. Samples were thoroughly shaken, and equal amounts of 50-100 μg total protein were loaded from each sample. Electrophoresis was performed with a gradient of 75 V for 40 min for stacking gel and 115 V for 60 min for resolving gel. Protein transfer was performed under semi-dry transfer conditions, and filter paper, cut gel, and PVDF membrane were equilibrated with semi-dry transfer buffer. The filter paper was placed flat in a semi-dry transfer tank, and the PVDF membrane, gel, and another piece of filter paper were placed on top of it in order, carefully removing air bubbles between all layers. The tank was covered with a cover for semi-dry transfer, and the constant voltage was set to 25 V, and the transfer was performed for 30 min.

[0026] 3) After the transfer, the PVDF membrane was blocked in a 5% skim milk blocking solution prepared in TBST solution and blocked at 37°C for 1 h. An appropriate amount of primary antibody prepared in the blocking solution was added at 100 μl / cm 2 The relative amounts of PVDF were added and incubated overnight at 4° C. The PVDF membrane was washed four times with TBST solution for 5 min each. The membrane was incubated in HRP-conjugated secondary antibody dilution at 37° C. for 60 min, and the PVDF membrane was washed four times with TBST solution for 5 min each.

[0027] 4) Exposure was performed using a ChemiDocTM XRS+ gel imaging system (Bio-Rad), and densitometric analysis and semi-quantitative comparison were performed using Image LabTM software.

[0028] 3. Experimental Results As shown in Figure 1, under physiological conditions, there is an interaction between FXR and Caspase 8 proteins, but the interaction between FXR and Caspase 8 was weakened in the ActD / TNFα-induced apoptosis model.

[0029] Example 2 Enhancer 3C1 regulates the interaction between FXR and Caspase 8 proteins 1. Experimental Materials Compound 3C1 was purchased from TargetMol and has the following structural formula: [ka] The remaining reagents were the same as in Example 1.

[0030] 2. Experimental Method 2.1 Cell culture and treatment Human hepatocyte cell line L02 was cultured in DMEM complete medium containing 10% fetal bovine serum and placed in a cell incubator at 37°C and 5% CO2.

[0031] 2.2 Regulatory effect of compound 3C1 on FXR-Caspase8 protein interaction under physiological conditions 2 x 10 cells in logarithmic growth phase 5 Cells / well were seeded in a 6-well plate. After 24 h, different concentrations of compound 3C1 (1 μM, 10 μM, and 100 μM) were administered, and the cells were harvested 2 h later.

[0032] 2.3 Regulatory effect of compound 3C1 on FXR-Caspase8 protein interaction under apoptotic conditions 2 x 10 cells in logarithmic growth phase 5Cells / well were seeded in a 6-well plate. The apoptosis induction method was the same as in Example 1. While administering the apoptosis inducer, different concentrations of compound 3C1 (1 μM, 10 μM, and 100 μM) were administered, and the cells were collected 2 h later.

[0033] 2.4 Co-IP This is the same as in Example 1.

[0034] 2.5 Western blot This is the same as in Example 1.

[0035] 3. Experimental Results 3.1 Regulatory effect of compound 3C1 on FXR-Caspase8 protein interaction under physiological conditions As shown in FIG. 2, in normal cells, administration of compound 3C1 significantly enhanced the interaction between FXR protein and Caspase 8 protein, and this effect was dose-dependent.

[0036] 3.2 Regulatory effect of compound 3C1 on FXR-Caspase8 protein interaction under apoptotic conditions As described in Example 1, FXR-Caspase8 protein interaction was downregulated under apoptotic conditions. As shown in Figure 3, under apoptotic conditions of ActD / TNFα-induced L02, the interaction between FXR protein and Caspase8 protein was weakened, and compound 3C1 had a significant reversal effect on this. The above results confirmed that compound 3C1 is an enhancer of the interaction between FXR and Caspase 8 protein.

[0037] Example 3 Pharmacological activity of enhancer 3C1 against cell apoptosis 1. Experimental Materials Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was purchased from MedChem Express (NJ, USA). The remaining reagents were the same as in Example 1.

[0038] 2. Experimental Method 2.1 Cell culture, transfection and treatment Human hepatic cell line L02 was cultured in DMEM complete medium containing 10% fetal bovine serum, and then placed in a cell incubator at 37°C and 5% CO2. 5 × 10 cells were cultured in the logarithmic growth phase. 3 Cells / well were seeded in a 96-well plate. After 24 h, the cells were transfected with 50 nM, and apoptosis modeling was performed 48 h after transfection. The specific methods are as follows: TRAIL was administered at a concentration of 50 ng / ml, and the treatment time with TRAIL was 24 h. ActD / TNFα was administered at a concentration of 0.2 μM and 20 ng / ml, ActD was pre-administered for 30 min, and the treatment time with ActD was 8 h. CHX / FasL was administered at a concentration of 50 μM and 50 ng / ml, CHX was pre-administered for 30 min, and the treatment time with CHX was 8 h. While the cells were being treated with the apoptotic agent, they were also treated with different concentrations of compound 3C1: 1 μM, 10 μM, and 100 μM.

[0039] 2.2 Detection of cell viability After the administration period was completed, 20 μl of MTT (5 mg / ml) was added to each well and incubated at 37°C for 4 h. The medium was then aspirated and discarded, and 150 μl of DMSO was added to each well and incubated at 37°C for 10 min. The absorbance was measured at a wavelength of 490 nm.

[0040] 3. Experimental Results As shown in Figure 4, the FXR-Caspase8 protein interaction enhancer 3C1 exhibited a significant anti-apoptotic effect in three classical apoptosis models, TRAIL (Figure 4A), ActD / TNFα (Figure 4B), and CHX / FasL (Figure 4C), and this anti-apoptotic effect was dose-dependent.

[0041] Example 4 Protective effect of enhancer 3C1 against liver injury induced by CCl4 1. Experimental Materials Experimental mice (C57BL / 6) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. CCl4 was purchased from Shanghai Lingfeng Chemical Reagents Co., Ltd., mineral oil was purchased from Sigma-Aldrich Co., Ltd., Trizol RNAisoplus was purchased from Takara Co., Ltd., reverse transcription kit and SYBR Green were purchased from Applied Biosystems Co., Ltd., and the primers used were designed and synthesized by Invitrogen Co., Ltd. (Shanghai, China). DEPC water was purchased from Beyotime Institute of Biotechnology (Shanghai, China). The remaining biological materials / reagents are commercially available. The remaining experimental materials were the same as in Example 1.

[0042] 2. Experimental Method 2.1 Effect of enhancer 3C1 on CCl4-induced mouse liver injury Before the experiment, C57BL / 6 mice were housed for one week at room temperature of 22-26°C, relative humidity of 40%-60%, and alternating light and dark periods of 12 h. All mice were housed in separate cages, allowed free access to water and food, and maintained at a body weight of 20-22 g. The mice were divided into a blank control group, a model group, a 3C1 low-dose administration group (5 mg / kg), and a 3C1 high-dose administration group (10 mg / kg).

[0043] C57BL / 6 mice in the 3C1 low-dose and 3C1 high-dose groups were pretreated with 5 mg / kg / day or 10 mg / kg / day 3C1 by intraperitoneal injection. The remaining groups received the same dose of vehicle control. CCl4 modeling was performed 3 days later, and the blank control group received the same dose of mineral oil. Mice were euthanized 48 h later. Blood and liver of the mice were collected.

[0044] 2.2 Detection of serum transaminases After standing at room temperature for 0.5 h, the whole blood was centrifuged at 8,000 rpm for 10 min, and the supernatant was transferred to a new EP tube to obtain serum. Serum ALT and AST were detected according to the instructions of the ALT and AST kits.

[0045] 2.3 RT-PCR 2.3.1 Extraction of total RNA from animal tissue samples 1) 20 mg of liver tissue sample was weighed from each mouse, 1.0 ml of RNAiso Plus was added, and the tissue was homogenized using a homogenizer. The homogenate was transferred to a centrifuge tube and left at room temperature for 5 minutes, after which it was centrifuged at 12,000 g and 4°C for 5 minutes.

[0046] 2) 800 μl of the supernatant was transferred, 160 μl of chloroform was added, and the mixture was shaken vigorously for 15 s, left at room temperature for 5 min, and centrifuged at 12,000 g for 15 min. The sample was separated into three layers: a yellow organic lower layer, a colorless aqueous upper layer, and an intermediate layer.

[0047] 3) 300 μl of the upper aqueous phase was carefully transferred to a new tube, 300 μl of isopropyl alcohol was added, the mixture was mixed evenly by inversion, the mixture was allowed to stand at room temperature for 10 minutes, and the mixture was centrifuged at 12,000 g for 10 minutes, and the supernatant was discarded.

[0048] 4) The RNA precipitate was washed with 1.0 ml of pre-cooled 75% ethanol and centrifuged at 12,000 g for 5 min. The supernatant was discarded to obtain total RNA, which was redissolved in 20 μl of DEPC water, quantified, and then diluted to 0.5 μg / μl.

[0049] 2.3.2 Reverse transcription Following the system ratios listed in the instructions, RNA solution and kit components were mixed to a total of 20 μl of the system, and the temperature was programmed for reverse transcription. The specific ratio requirements are as follows:

[0050] [Table A]

[0051] 2.3.3PCR The PCR system is as follows. [Table B]

[0052] The PCR conditions used were as follows: Stage 1: Pre-denaturation, 95℃, 1min Stage 2: PCR reaction, 95°C, 15s, e.g., 60°C, 30s, 40 cycles; 72°C, 30s Stage 3: Melting curve analysis 65~95℃, 0.5℃ / 5sec

[0053] 2.3.4 The primers used in the fluorescent quantitative PCR are shown in Table 1 below. [Table 1]

[0054] 2.4 Pathological analysis of the liver H&E staining and Sirius Red staining of this section were commissioned to Wuhan Google Biotechnology Co., Ltd.

[0055] 3. Experimental results 3.1 Effects of Enhancer 3C1 on serum biochemical indices As shown in Figures 5A and 5B, CCl4 causes a significant increase in serum ALT and AST levels in mice, but enhancer 3C1 can significantly reduce the increase in serum ALT and AST levels caused by CCl4 injection.

[0056] 3.2 Ameliorative effect of enhancer 3C1 on liver pathology According to the liver pathology analysis and H&E staining results (Figure 5C), in the mice of the control group, the structure of the hepatic lobule was intact, the hepatocytes were arranged radially around the central vein, and there was no fibrosis and inflammatory cells. The hepatocytes were large in size, with round nuclei in the center. After the injection of CCl4, the hepatic sinusoids were obviously congested and expanded, the hepatocytes underwent granular degeneration, there were many partial hepatocyte necrotic foci, some hepatocytes showed edema and degeneration and inflammatory cell infiltration, some of the hepatic lobule structure in the liver was destroyed, there was abnormal proliferation of fibrous tissue in the portal vein area, and collagen fibers were significantly proliferated and showed band-like proliferation. In the 3C1-treated group, the structural disorder of the hepatic lobule was significantly improved, the infiltration of inflammatory cells was reduced, and collagen fibers in the portal vein area were reduced. This indicates that 3C1 has obvious hepatoprotective effect. According to the Sirius Red staining results of liver pathology analysis (Figure 5D), there was less red collagen fiber staining in the liver of the control group. After CCl4 injection, there were many red collagen fibers in the portal vein area. After 3C1 administration, the proliferation of red collagen fibers in the liver was significantly improved.

[0057] 3.3 Ameliorative effect of enhancer 3C1 on liver fibrosis genes Real-time RT-PCR results (Figures 5E and 5F) showed that in the CCl4-induced mouse acute liver injury model, the levels of liver fibrosis genes Timp1 and Timp2 mRNA were significantly increased (P<0.001), and enhancer 3C1 could significantly reduce the mRNA levels of Timp1 and Timp2 (P<0.001), which was dose-dependent. Based on the above results, it was suggested that enhancer 3C1 had a clear hepatoprotective effect.

[0058] Example 5 50 mg of Enhancer 3C1 was mixed with 50 mg of microcrystalline cellulose and 60 mg of starch, and made into a soft material using an appropriate amount of 20% ethanol as a wetting agent, granulated in a conventional manner, mixed with talc and magnesium stearate, and compressed into tablets.

[0059] 50 mg of Enhancer 3C1 was mixed with 50 mg of starch, 10 mg of dextrin, and 10 mg of sucrose, softened with 30% ethanol as a wetting agent, and wet granulated to produce granules.

[0060] 50 mg of Enhancer 3C1 was mixed with 50 mg of starch, 10 mg of dextrin, and 10 mg of sucrose, and made into a soft mass using 30% ethanol as a wetting agent, wet granulated, and filled into capsules to prepare capsules.

[0061] 50 mg of Enhancer 3C1 was mixed with 40 mg of starch and 10 mg of talc and a powder was prepared using conventional techniques.

[0062] 50 mg of Enhancer 3C1 was mixed with 15 mg of powdered sugar and 8 mL of water to prepare a syrup using conventional techniques, then 70 mg of starch was added and pills were prepared using conventional techniques.

[0063] An oral solution was prepared using conventional techniques using 50 mg of Enhancer 3C1, povidone, sucrose, and water.

Claims

1. Use of enhancer 3C1 of formula (1) in the preparation of an enhancer of the interaction between FXR protein and Caspase 8 protein. 【Chemistry 3】 Formula (1)

2. The use according to claim 1, characterized in that the enhancer 3C1 promotes the interaction between the FXR protein and the Caspase 8 protein in physiological or pathological conditions.

3. The use according to claim 1, characterized in that the enhancer blocks the formation of an apoptotic complex by blocking the recruitment of Caspase 8 protein to the apoptotic complex.

4. The use according to claim 1, characterized in that the enhancer subblocks the cleavage activation of Caspase 8 protein.

5. The use according to claim 1, characterized in that the enhancer is prepared by adding a pharma- ceutically acceptable auxiliary material to the formulation.

6. The use according to claim 5, characterized in that the pharma- ceutically acceptable auxiliary materials include diluents, binders, disintegrants, glidants, lubricants, flavoring agents, encapsulating materials, and adsorbents.

7. The use according to claim 5, characterized in that the formulation is a granule, a powder, a tablet, a capsule, a pill, or an oral liquid.

8. Use of enhancer 3C1 shown in formula (1) in the preparation of a drug for resisting hepatocyte apoptosis as an enhancer of the interaction between FXR protein and Caspase 8 protein. 【Chemistry 4】 Formula (1)

9. Use of enhancer 3C1 shown in formula (1) in the preparation of a hepatoprotective drug as an enhancer of the interaction between FXR protein and Caspase 8 protein. 【Chemistry 5】 Formula (1)

10. Use of enhancer 3C1 represented by formula (1) in the preparation of a therapeutic agent for liver fibrosis / liver injury as an enhancer of the interaction between FXR protein and Caspase 8 protein. 【Chemistry 6】 Formula (1)

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