Use of SBFI-26 in the preparation of drugs for the prevention of liver fibrosis

Abstract

This invention relates to the field of biomedical technology, and particularly to the use of SBFI-26 in the preparation of drugs for the prevention of liver fibrosis. This invention provides the use of SBFI-26 in the preparation of drugs for the prevention of liver fibrosis. A mouse model of liver fibrosis was established by intraperitoneal injection of CCL4. SBFI-26 injection significantly protected against the occurrence of liver fibrosis. The livers of the mice in the prevention group were generally in good condition, with smooth surfaces and no fatty liver-like deformities. Prophylactic administration of SBFI-26 significantly reduced the expression of α-SMA and phosphorylated Smad2 / 3 in mice with liver fibrosis, suggesting that SBFI-26 inhibits the occurrence and progression of liver fibrosis. Therefore, SBFI-26 provides a new therapeutic approach for the prevention of liver fibrosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of SBFI-26 in the preparation of drugs for the prevention of liver fibrosis. Background Technology

[0002] Hepatic fibrosis (HF) refers to the process in which hepatocytes are repeatedly damaged and regenerated during the development of chronic liver disease. It involves the diffuse excessive deposition and abnormal distribution of extracellular matrix such as collagen, glycoproteins, and proteoglycans in the liver. It is a pathological repair response of the liver to chronic damage and a key step in the progression of various chronic liver diseases to cirrhosis, as well as an important factor affecting the prognosis of chronic liver diseases.

[0003] When the liver is repeatedly or continuously damaged, fibrosis develops. After a single, even severe injury (such as acute hepatitis), the liver can usually repair itself by regenerating hepatocytes and combining them with the connective tissue network (internal structure) left behind when older hepatocytes die. However, if the damage is repeated or persistent (such as in chronic hepatitis), hepatocytes attempt to repair the damage, leading to scar tissue (fibrosis). Liver fibrosis caused by bile duct obstruction develops more rapidly. Scar tissue replaces hepatocytes, but unlike the latter, it does not perform any function. Scar tissue deforms the internal structure of the liver, blocking blood flow into and within the liver, restricting blood supply to hepatocytes. When blood supply is insufficient, hepatocytes die, leading to the formation of even more scar tissue. After months or even years of repeated or continuous damage, permanent, widespread fibrosis can develop. Further progression of liver fibrosis can cause structural disorder of the liver, nodular regeneration of hepatocytes, and the formation of pseudolobular structures, i.e., cirrhosis. Liver fibrosis is histologically reversible and can be reversed with aggressive treatment during its development. Otherwise, if liver fibrosis progresses to cirrhosis, reversal becomes very difficult, and the prognosis is relatively poor.

[0004] There are currently no marketed drugs that can effectively and safely prevent the formation of scar tissue, meaning that there is a lack of drugs to prevent liver fibrosis in clinical practice. Therefore, there is an urgent need to develop new drugs to prevent liver fibrosis. Summary of the Invention

[0005] The purpose of this invention is to provide the use of SBFI-26 in the preparation of drugs for the prevention and treatment of liver fibrosis. Through research, this invention has found that SBFI-26 has a good inhibitory effect on liver fibrosis, indicating that SBFI-26 can provide a new treatment option for liver fibrosis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides the use of SBFI-26 in the preparation of medicaments for the prevention and treatment of liver fibrosis.

[0008] The mechanism of action of this application: Measurements of α-SMA expression and collagen production showed that inhibition of FABP5 reduced TGF-β-stimulated HSC activation. Furthermore, HSC activation mirrors the energy requirements observed in cancer cells; therefore, a better understanding of HSC metabolic regulation is becoming a new priority in the field of liver fibrosis, significantly impacting pericellular metabolism in other tissues. Lipid droplet metabolism during HSC activation provides fatty acids for β-oxidation. In this regard, inhibition of mitochondrial fatty acid catabolism prevents HSC activation, highlighting the relevance of lipid metabolism in HSC biology. Moreover, HSC activation is also controlled by major transcriptional regulators of fatty acid content. These regulators include peroxisome proliferator-activated receptor γ (PPARγ) and sterol regulatory element-binding protein-1 (SREBP-1c). In our study, we demonstrated that inhibiting FABP5 can prevent the occurrence of liver fibrosis. The mechanism is to inhibit the activation of HSC cells. The possible mechanisms for inhibiting HSC cell activation are as follows: ① inhibiting fatty acid uptake, which leads to weakened fatty acid β oxidation and insufficient energy supply; ② inhibiting the activation of the PPARγ pathway, which in turn inhibits the activation pathway of HSCs; ③ inhibiting the activation of the SREBP-1c pathway, which in turn inhibits the activation pathway of HSCs.

[0009] This application proposes that prophylactic intraperitoneal injection of 2 mg / kg SBFI-26 into mice can effectively prevent the occurrence of liver fibrosis, thus suggesting that SBFI-26 may be used as a preventive drug for liver fibrosis. Considering the lipid-soluble nature of SBFI-26, oral, intramuscular, and intravenous formulations can be developed. Furthermore, considering its liver-targeting properties, liver-targeting drugs such as GalNAc-conjugated SBFI-26 can be developed.

[0010] The beneficial technical effects of this invention are:

[0011] This invention provides the use of SBFI-26 in the preparation of drugs for the prevention of liver fibrosis. This application establishes a mouse model of liver fibrosis through intraperitoneal injection of CCL4 and sets up a prophylactic SBFI-26 administration group, observing indicators such as gross liver morphology, pathophysiological changes, α-SMA expression, Smad2 / 3 expression, phosphorylated Smad2 / 3 expression, and collagen fiber distribution. This invention, through research, has found that:

[0012] (1) After continuous intraperitoneal injection of SBFI-26 at a concentration of 2 mg / kg / d for 6 weeks, the activity of FABP5 in vivo was significantly inhibited. SBFI-26 injection significantly protected against liver fibrosis. The livers of mice in the prevention group were in good condition, with smooth surfaces and no fatty liver-like deformities.

[0013] (2) Masson’s trichrome staining showed that SBFI-26 effectively prevented the occurrence of liver fibrosis.

[0014] (3) Prophylactic administration of SBFI-26 significantly reduced the expression of α-sma and phosphorylated smad2 / 3 in mice with liver fibrosis, suggesting that SBFI-26 inhibited the occurrence and progression of liver fibrosis.

[0015] (4) In vivo and in vitro tests on compound SBFI-26 revealed that SBFI-26 has a strong inhibitory effect on FABP5. The anti-liver fibrosis effect of SBFI-26 is achieved by inhibiting FABP5, which in turn inhibits TGF-β1-induced α-SMA synthesis. Therefore, the preventive effect of SBFI-26 on liver fibrosis may be achieved by inhibiting FABP5 in liver tissue.

[0016] In conclusion, SBFI-26 provides a new treatment option for the prevention of liver fibrosis. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Gross photographs of liver tissue from different groups;

[0019] Figure 2 HE staining of liver tissue sections from different groups;

[0020] Figure 3 Masson staining of liver tissue sections from different groups;

[0021] Figure 4 Immunohistochemical staining of α-SMA in liver tissues from different groups;

[0022] Figure 5 The FABP5 inhibitor SBFI-26 can reduce the expression levels of α-SMA and phosphorylated smad2 / 3 in vitro;

[0023] Figure 6 SBFI-26 treatment can reduce the migration ability of HepG2 and HUH7 cell lines;

[0024] Figure 7SBFI-26 treatment can reduce the invasive ability of HepG2 and HUH7 cell lines;

[0025] Figure 8 SBFI-26 treatment can reduce the long-chain fatty acid uptake capacity of HepG2 and HUH7 cell lines; Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1

[0028] I. Experimental Methods

[0029] 1. Establishment of a CCL4-induced liver fibrosis model

[0030] Thirty male C57BL / 6 mice, aged six to eight weeks and weighing approximately 18–20 g, were selected and examined for signs of discomfort (e.g., changes in respiration, rough fur, abnormal behavior, kyphosis). Animals exhibiting these abnormalities were excluded from the study. These mice were randomly assigned to a control group (n=10), a model group (n=10), and a liver fibrosis prevention group (n=10). The control group received an intraperitoneal injection of 1 mL / kg of sterile corn oil; the model group received an intraperitoneal injection of 1 mL / kg of sterile corn oil with a 20% CCL4 concentration; and the liver fibrosis prevention group received an intraperitoneal injection of 1 mL / kg of sterile corn oil with a 20% CCL4 concentration, along with an injection of 2 mg / kg of SBIF-26. All injections were administered twice weekly for six weeks.

[0031] 2. Anesthesia, euthanasia, and sampling of laboratory animals

[0032] (1) After weighing the mice, place them in a small animal gas anesthesia machine (RWD R500) using isoflurane as the anesthetic. Isoflurane is evaporated by airflow. Induction anesthesia is performed at a concentration of 2-3%, and maintenance anesthesia is performed at a concentration of 1.5-2%. After anesthesia, blood is collected from the heart using a 1 mL disposable sterile syringe. After centrifugation with 1.5 mL of the syringe, the blood is allowed to stand for 30 min, then centrifuged at 3000 r / min for 15 min. The supernatant serum is collected and stored at -80℃ for later use.

[0033] (2) Fix the mouse’s limbs to the dissection table, expose the entire chest and abdomen of the mouse, trim the hair off the abdomen, and disinfect with 75% alcohol.

[0034] (3) Cut the abdominal skin along the midline of the abdomen from the upper part of the penis upwards to the xiphoid process, and bluntly peel away the skin and subcutaneous tissue to expose the superficial muscle layer of the abdominal wall. Bluntly separate the abdominal wall muscles along the linea alba, cut the peritoneum to expose the abdominal cavity, turn the liver upwards to expose the porta hepatis, and use ophthalmic scissors to remove the connective tissue and blood vessels around the liver.

[0035] (4) The hepatic hilum system was ligated and cut off. The rat liver was removed completely by blunt and sharp means, placed in a sterile culture dish, rinsed with physiological saline, weighed and recorded.

[0036] ① Three tissue samples, each approximately 1mm × 1mm × 1mm in size, were taken from the left lobe of the liver and fixed with 4-glutaraldehyde;

[0037] ② The left lobe of liver tissue was fixed by immersion in 4% paraformaldehyde solution;

[0038] ③ After weighing and recording the right lobe liver tissue, the samples were aliquoted and placed in sterile cryovials for liquid nitrogen freezing and storage for future use.

[0039] 3. Immunohistochemical detection of α-SMA expression in liver tissue

[0040] Immunohistochemistry was used. Tissues from the blank control group, model group, and liver fibrosis prevention group were selected. After routine fixation with 4% paraformaldehyde, dehydration, paraffin embedding, and 4μm thick serial sections, the sections were prepared. The sections were baked at 60℃ for 30 min, dewaxed with xylene, hydrated with graded ethanol, then endogenous peroxidase was inactivated with 3% H2O2, and antigen was repaired with sodium citrate buffer. After cooling to room temperature, 5% goat serum was added for blocking at room temperature for 30 min. Primary antibody against α-SMA protein (ab124964) was added, and the sections were incubated overnight at 4℃. The next day, secondary antibody was added, and the sections were incubated at 37℃ for 30 min, washed three times with PBS, developed with DAB, counterstained with hematoxylin, dehydrated with graded ethanol, and mounted with neutral resin for microscopic observation. The immunohistochemical procedures were strictly performed according to the kit instructions.

[0041] 4. HE staining and Masson staining of liver tissues from different groups

[0042] HE staining: After euthanizing mice, a portion of fresh liver tissue was placed in fixative, routinely fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned into 4μm thick serial sections. After development in 40℃ warm water, the sections were lifted out onto a glass slide with the rough side facing up, air-dried, dewaxed and hydrated, and then stained with hematoxylin and eosin respectively. After rinsing and dehydration, the sections were mounted with resin and observed and photographed under a light microscope.

[0043] Masson staining: After hydration, sections were stained with Weigert iron hematoxylin solution for 5 min, acidified with hydrochloric acid for 2 s until blue return, rinsed with double-distilled water, stained with Ponceau S and fuchsin solution for 15 min, washed with phosphomolybdic acid solution for 3 min, stained with aniline blue solution for 3 min, washed with glacial acetic acid for 1 min, dehydrated, cleared, and mounted with neutral resin. Microscopic observation was performed, and images at the same magnification were exported and measured using ImageJ (V1.8.0.112) software. Three Masson-stained images were randomly selected from each group to calculate CVF and the average value was taken.

[0044] 5. Immunoblotting detection of α-SMA, Smad2 / 3 and phosphorylated Smad2 / 3 in liver tissue

[0045] Weigh 50 mg of mouse liver tissue from each group, add RIPA lysis buffer, homogenize rapidly on ice, incubate at 4°C for 3 h, centrifuge at high speed, collect supernatant for protein quantification, add sample buffer, denature at 100°C for 10 min, and store at -20°C for later use; after electrophoresis, transfer the sample to a membrane, block with 5% skim milk powder for 1 h after transfer, wash with PBST, add α-sma (ab124964), smad2 / 3 (ab202445) and phosphorylated smad2 / 3 (ab254407) primary antibodies, incubate at 4°C overnight, wash with PBST the next day, add secondary antibody and incubate for 1 h, wash again, prepare developing solution, and expose using a chemiluminescence imaging system.

[0046] 6. Cell line culture

[0047] Human HCC cell lines (HepG2, HuH7) were obtained from the American Center for Type Culture Collection (Manassas, Virginia, USA). Cells were cultured in Dulbecco modified Eagle medium (DMEM, Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin under a humid atmosphere of 5% CO2.

[0048] 7. Cell proliferation, migration, invasion, apoptosis, and cell cycle analysis

[0049] Cell migration was assessed using a Transwell assay. Cell invasion was assessed using a BioCoat Matrigel Invasion Chamber (BDBiosciences). The number of migrating and invading cells was counted in three randomized fields of view. All experiments were repeated three times.

[0050] 8. Fatty acid uptake capacity test

[0051] HepG2 and HuH7 cells in the logarithmic growth phase were selected and seeded in laser confocal microplates. A blank control group, a WT group, and an SBFI-26 intervention group (50 μM) were set up. After 24 h, the cells were washed twice with PBS, starved for 10 min with sterile Hank's equilibration buffer, and then incubated for 5 min with 200 nM BODIPY FLFAs in Hank's equilibration buffer. Flow cytometry was then used to detect the cells, with average fluorescence intensity as the statistical index. All experiments were repeated three times.

[0052] II. Results

[0053] 1. CCL4 can effectively induce liver fibrosis, while SBFI-26 administration can prevent the occurrence of liver fibrosis.

[0054] Exposing the livers of mice in different groups revealed that, to the naked eye, the liver tissue of the CCL4 model group was dark red, shrunken, lighter, and tougher, exhibiting a "turtle-back" texture. Figure 1 B), while no abnormalities were observed in the corn oil control group. Figure 1 A). After HE staining, the CCL4 model group showed degenerated, necrotic, and regenerated hepatocytes, as well as regenerated hepatocyte nodules. The regenerated hepatocytes were large, with large and deeply stained nuclei, or binucleated; the pseudolobules were surrounded by fibrous septa, and inflammatory cell infiltration and small bile duct proliferation were observed within the fibrous septa. Figure 2 B), while the corn oil control group showed little or no of the above-mentioned denaturation. Figure 2 A). After Masson staining, the CCL4 model group showed a large amount of blue-stained collagen fiber tissue (A). Figure 2 B), while the corn oil control group and the SBFI-26 prevention group showed little or no of the above-mentioned denaturation. Figure 3 A). Furthermore, immunohistochemical staining of α-Sma expression levels in the liver tissues of the three mouse groups revealed that the CCL4 model group showed significantly elevated α-Sma expression levels and a wider distribution. Figure 4 In summary, the results indicate that the CCL4-induced liver fibrosis model was successful.

[0055] 2. The FABP5 inhibitor SBFI-26 can effectively prevent the occurrence of liver fibrosis in vivo.

[0056] To determine whether the FABP5 inhibitor SBFI-26 could prevent liver fibrosis, researchers administered SBFI-26 concurrently with intraperitoneal injection of CCL4 solution. After 6 weeks of treatment, liver tissue samples were collected and found that the livers of the SBFI-26 prevention group were grossly normal, with no signs of fibrosis. Figure 1 C), HE staining and Masson staining showed no abnormalities or collagen aggregation. Figure 2 C. Figure 3C) suggests that SBFI-26 can effectively prevent liver fibrosis caused by CCL4.

[0057] 3. The FABP5 inhibitor SBFI-26 can reduce the expression levels of α-SMA and phosphorylated Smad2 / 3 in vitro.

[0058] Western blot analysis showed that TGFβ (10 ng / mL) treatment significantly increased the expression levels of α-SMA and phosphorylated smad2 / 3 in JS-1 cells, while the total smad2 / 3 expression level showed no difference. Furthermore, simultaneous treatment with SBFI-26 (50 μM) and TGFβ significantly reduced the expression levels of α-SMA and phosphorylated smad2 / 3 compared to the TGFβ-only group, while the total smad2 / 3 expression level showed no difference. This indicates that SBFI-26 treatment can effectively prevent the TGFβ-induced increase in α-SMA and phosphorylated smad2 / 3 expression. Figure 5 .

[0059] 4. The FABP5 inhibitor SBFI-26 can inhibit the migration and invasion of liver cancer cell lines in vitro.

[0060] Considering that liver fibrosis may ultimately lead to hepatocellular carcinoma associated with cirrhosis, further evaluation was conducted to determine whether SBFI-26 had an impact on the malignant behavior of tumor cells. Results showed that SBFI-26 intervention effectively inhibited the migration and invasion abilities of HepG2 and HuH7 cells. Figure 6 , 7 ).

[0061] 5. The FABP5 inhibitor SBFI-26 may exert its effect by inhibiting the uptake of long-chain fatty acids in cells.

[0062] Based on previous results, this study preliminarily demonstrated that prophylactic administration of SBFI-26 can completely prevent CCL4-induced liver fibrosis and also inhibit the malignant phenotype of hepatocellular carcinoma cell lines. To further clarify its possible pharmacological mechanism, researchers examined the long-chain fatty acid uptake function of cells and found that SBFI-26 intervention significantly reduced the cells' ability to uptake long-chain fatty acids. This suggests that SBFI-26 may exert its preventive and anti-tumor effects by influencing fatty acid uptake, thereby leading to lipid metabolism reprogramming. Figure 8 ).

[0063] Finally, it should be noted that the above embodiments are only used to illustrate and not limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Use of SBFI-26 for the preparation of a medicament for the prevention of liver fibrosis induced by carbon tetrachloride.

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