Application of primrosein in the preparation of drugs for the treatment of liver fibrosis

Abstract

This invention discloses the application of primrosein in the preparation of drugs for the treatment of liver fibrosis. The chemical name of primrosein is 2-methoxy-6-pentyl-1,4-benzoquinone, and its molecular formula is C2. 12 O3H 16 Cellular experiments showed that primrosein reduced the proliferative activity of hepatic stellate cells but did not increase the release of lactate dehydrogenase from hepatic stellate cells, and had no significant inhibitory effect on hepatocyte proliferation. Animal experiments showed that primrosein reduced serum levels of markers in carbon tetrachloride-induced liver fibrosis model mice, decreased collagen fiber content in their liver tissue, and inhibited the expression of hepatic stellate cell activation markers, demonstrating excellent anti-liver fibrosis effects. This indicates that primrosein can be used to prepare effective drugs for the treatment of liver fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of primrosein in the preparation of drugs for the treatment of liver fibrosis. Background Technology

[0002] Liver fibrosis is a disease caused by long-term pathological damage and repair within the liver tissue due to various pathogenic factors such as viruses, alcohol, high-fat diets, or drugs. It is a common pathological process in the development of many chronic liver diseases into cirrhosis and / or hepatocellular carcinoma. Currently, liver fibrosis ranks eleventh in global disease mortality, seriously endangering human life and health, making the search for effective treatments for liver fibrosis an urgent priority.

[0003] The typical pathological feature of liver fibrosis lies in the abnormal deposition of extracellular matrix components within the liver tissue, leading to structural changes and functional disorders. Hepatic stellate cells are the primary source of extracellular matrix production in the liver. In a normal liver, hepatic stellate cells remain in a quiescent state with low proliferative activity and a weak ability to synthesize extracellular matrix components such as collagen. When the liver is injured, hepatic stellate cells are activated, their phenotype changing from a quiescent to an activated state. Activated hepatic stellate cells proliferate extensively and secrete extracellular matrix, participating in the formation of liver fibrosis and the remodeling of liver tissue structures. Therefore, hepatic stellate cell activation is a core link in the development and progression of liver fibrosis, and inhibiting hepatic stellate cell activation has become an important direction for exploring potential therapeutic strategies for liver fibrosis.

[0004] The basic treatment strategy for liver fibrosis is a combination of etiological treatment and anti-fibrotic therapy. For patients with liver fibrosis who lack specific etiological treatment or cannot undergo such treatment, active anti-fibrotic therapy should be implemented. Currently, there are no clearly defined chemical or biological drugs for the treatment of liver fibrosis in clinical practice. Colchicine, a monomeric compound extracted from the autumn crocus (Colchella asiatica), was used earlier in experimental liver fibrosis treatment, but its clinical application is limited due to its significant toxic side effects. Several traditional Chinese medicines with liver fibrosis as a registered indication are currently available in the Chinese pharmaceutical market, but they suffer from drawbacks such as large dosages, long treatment cycles, and unclear mechanisms of action. This underscores the urgent need for the development of safe and effective drugs for the treatment of liver fibrosis.

[0005] Primrosein is a natural benzoquinone compound extracted from the leaves and stem trichomes of Primula obconica Hance, a plant in the Primulaceae family. Current research shows that it has antibacterial and antitumor activities, but there are no reports on its anti-liver fibrosis activity. Summary of the Invention

[0006] The purpose of this invention is to provide the application of primrosein in the preparation of drugs for the treatment of liver fibrosis.

[0007] This invention first uses the normal mouse hepatocyte line AML12 and the mouse hepatic stellate cell line JS-1 to study the effects of different concentrations of primrosein (2, 4, 8 μM) on the proliferation and activity of hepatocytes and hepatic stellate cells. Using the mouse hepatic stellate cell line JS-1, the effect of primrosein on the release of lactate dehydrogenase within hepatic stellate cells was studied. Using the mouse hepatic stellate cell line JS-1, the effects of primrosein on the activation level and collagen synthesis level of hepatic stellate cells were studied at the transcriptional and translational levels. Using a carbon tetrachloride-induced mouse liver fibrosis model, the effects of different doses of primrosein (1.5, 3, 6 mg / kg) on ​​serum liver fibrosis markers, liver tissue hydroxyproline content, hepatic stellate cell activation level, collagen synthesis level, and liver tissue collagen content were studied at the animal level.

[0008] This invention is the first to discover that primoriside has a preventive or therapeutic effect on liver fibrosis. Cellular experiments showed that primoriside did not affect the proliferative activity of mouse hepatocytes, but significantly reduced the proliferative activity of mouse hepatic stellate cells; primoriside did not alter the release of lactate dehydrogenase from mouse hepatic stellate cells and had no significant cytotoxicity; primoriside significantly inhibited the transcription and translation levels of the hepatic stellate cell activation marker α-SMA and the collagen gene COL1A1, exhibiting a concentration-dependent effect. Animal experiments showed that primoriside reduced the levels of serum diagnostic markers laminin, hyaluronic acid, type III procollagen, and type IV collagen in mice with liver fibrosis; primoriside reduced the content of hydroxyproline in the liver of mice with liver fibrosis and decreased the mRNA and protein expression of α-SMA and COL1A1; primoriside significantly reduced the collagen content in the liver of mice with liver fibrosis.

[0009] This invention reveals the therapeutic effect and molecular mechanism of primrosein on liver fibrosis, providing an important basis for its preparation as a drug for treating liver fibrosis. Attached Figure Description

[0010] Figure 1 The effect of primrosein on the proliferative activity of normal mouse hepatocytes AML12.

[0011] Figure 2 The effect of primrosein on the proliferative activity of mouse hepatic stellate cells JS-1. *P<0.05, **P<0.01 and ***P<0.001 vs. primrosein 0 μM group.

[0012] Figure 3 The effect of primrosein on the lactate dehydrogenase content in the supernatant of JS-1 hepatic stellate cells culture medium.

[0013] Figure 4The effect of primrose extract on the mRNA expression of activation-related genes α-SMA and collagen gene COL1A1 in mouse hepatic stellate cells JS-1. *P<0.05, **P<0.01 and ***P<0.001 vs. primrose extract 0 μM group.

[0014] Figure 5 The effect of primrosein on the protein expression of activation-related genes α-SMA and COL1A1 in mouse hepatic stellate cells JS-1.

[0015] Figure 6 The effects of primrose extract on serum diagnostic markers of liver fibrosis, including laminin, hyaluronic acid, type III procollagen, and type IV collagen. ### P<0.001 vs. solvent group; *P<0.05, **P<0.01 and ***P<0.001 vs. primoriside 0 mg / kg + carbon tetrachloride group.

[0016] Figure 7 The effects of primrosein on the content of hydroxyproline, and the mRNA and protein expression levels of α-SMA and COL1A1 in mouse liver tissue were investigated. ### P<0.001 vs. solvent group; *P<0.05, **P<0.01 and ***P<0.001 vs. primoridin 0 mg / kg + carbon tetrachloride group.

[0017] Figure 8 The effect of primrosein on collagen content in mouse liver tissue. Detailed Implementation

[0018] 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.

[0019] This invention proposes a promising anti-liver fibrosis drug, and the specific embodiments of this invention are described in further detail below.

[0020] Example 1

[0021] Effects of primrosein on the proliferative activity of mouse hepatocytes AML12 and hepatic stellate cells JS-1 (in vitro experiment)

[0022] Collect cells in the logarithmic growth phase, adjust the cell suspension concentration, and seed them in 96-well plates at 180 μl per well (approximately 1 × 10⁶ cells per well). 4Cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin for 24 h at 37°C and 5% CO2 until cell adhesion was achieved. Cells were divided into four groups: a normal control group treated with an equal volume of solvent, and the other three groups treated with primrosein (2, 4, and 8 μM) for 24 h. Each group had six replicates. After drug treatment, 10 μl of CCK-8 solution was added to each well, and the cells were cultured for another 4 h. The absorbance at 450 nm was measured using an ELISA reader.

[0023] like Figure 1 and Figure 2 As shown, 2, 4, and 8 μM primoridin had no significant effect on the proliferation of hepatocytes cultured in vitro, but it significantly inhibited the proliferation of hepatic stellate cells, and this effect increased with increasing concentration, showing a clear concentration-dependent effect. This indicates that primoridin effectively inhibits the proliferation of hepatic stellate cells without affecting the proliferation of hepatocytes.

[0024] Example 2

[0025] Cytotoxic effects of primrosein on mouse hepatic stellate cells (in vitro experiment)

[0026] Collect cells in the logarithmic growth phase, adjust the cell suspension concentration, and seed them in 96-well plates at 180 μl per well (approximately 1 × 10⁶ cells per well). 4 Cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin for 24 h at 37°C and 5% CO2 until cell adhesion was achieved. Cells were divided into four groups: a normal control group treated with an equal volume of solvent, and the other three groups treated with primrosein (2, 4, and 8 μM) for 24 h. Each group had six replicates. After drug treatment, the lactate dehydrogenase activity in the cell culture supernatant was detected using a micro-enzyme labeling method according to the manufacturer's instructions.

[0027] like Figure 3 As shown, treatment with primrosein at concentrations of 8 μM and below did not significantly alter the activity of lactate dehydrogenase in the supernatant of hepatic stellate cell culture medium, suggesting that primrosein did not affect the release of lactate dehydrogenase from hepatic stellate cells and that primrosein had no significant cytotoxic effect on hepatic stellate cells.

[0028] This indicates that the inhibitory effect of primordin on the proliferation of hepatic stellate cells within a certain concentration range does not originate from direct cytotoxicity.

[0029] Example 3

[0030] Effects of primrosein on mRNA expression of hepatic stellate cell activation markers and collagen genes (in vitro experiments)

[0031] Cells in the logarithmic growth phase were collected, and the cell suspension concentration was adjusted. Cells were seeded in 6-well plates in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, and cultured at 37°C and 5% CO2 for 24 h until cell adhesion was achieved. When the cell density reached 50%, the cells were divided into four groups. Except for the normal control group, which was treated with an equal volume of solvent, the other three groups were treated with primrosein (2, 4, and 8 μM) for 24 h. The culture medium was discarded, and the cells were washed three times with pre-cooled phosphate-buffered saline (PBS) to completely remove the culture medium. 300 μl of Trizol was added to each well, and the cells were gently pipetted repeatedly for 1 min to transfer the solution into 1.5 ml EP tubes. 60 μl of chloroform was added to each tube, the EP tube caps were tightened, and the tubes were vortexed for 15 s and then allowed to stand at room temperature for 3 min. The tubes were then centrifuged at 4°C and 12000 rpm for 15 min. Transfer the upper aqueous layer to a new 1.5 ml EP tube, add 150 μl of isopropanol and mix well. Incubate at room temperature for 10 min, then centrifuge at 4°C and 12000 rpm for 10 min. The resulting precipitate at the bottom of the tube is the RNA. Discard the supernatant, add 300 μl of 75% ethanol, vortex for a few seconds, and centrifuge at 4°C and 7500 rpm for 10 min. Discard the supernatant, place the EP tube in a fume hood for 30 min to allow the ethanol to evaporate completely, then add 20 μl of DEPC water. Perform reverse transcription and amplification experiments according to the kit instructions.

[0032] The primer sequences are as follows:

[0033] α-SMA(F):5′-CATCCACGAAACCACCTA-3′(Seq_1)

[0034] α-SMA(R):5′-GGGCAGGAATGATTTGGA-3′(Seq_2)

[0035] COL1A1(F):5′-GAGAGAGCATGACCGATGGATT-3′(Seq_3)

[0036] COL1A1(R):5′-TGTAGGCTACGCTGTTCTTGCA-3′(Seq_4)

[0037] GAPDH(F):5′-CTATGACCACAGTCCATGC-3′(Seq_5)

[0038] GAPDH(R): 5′-CACATTGGGGGTAGGAACAC-3′ (Seq_6).

[0039] like Figure 4As shown, primrosein inhibits the mRNA expression of hepatic stellate cell activation marker α-SMA and collagen gene COL1A1 in a concentration-dependent manner.

[0040] This indicates that primrosein can inhibit the transcription of genes related to the activation of hepatic stellate cells.

[0041] Example 4

[0042] Effects of primrosein on activation markers and collagen expression in hepatic stellate cells (in vitro experiment)

[0043] Logarithmic growth phase cells were collected, and the cell suspension concentration was adjusted. Cells were seeded in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics for 24 hours at 37°C and 5% CO2 until cell adhesion was achieved. When the cell density reached 50%, the cells were divided into four groups. Except for the normal control group, which was treated with an equal volume of solvent, the other three groups were treated with primrosein (2, 4, and 8 μM) for 24 hours. The culture medium was discarded, and the cells were washed three times with pre-chilled PBS to completely remove the medium. Cells were lysed with pre-chilled RIPA lysis buffer, and the cell lysates were collected and stored overnight at -80°C. The next day, after the cell lysates thawed, they were centrifuged at 12000 rpm for 20 minutes at 4°C. The supernatant was collected, and protein concentration was determined using a BCA assay kit. Protein was loaded at a rate of 25 μg and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by PVDF transfer. Bovine serum albumin (BSA) was used to block non-specific binding sites on PVDF. The corresponding primary antibody was added and the mixture was incubated overnight at 4°C. Horseradish peroxidase-labeled secondary antibody was then added for hybridization. ECL chemiluminescence buffer was added, and imaging and analysis were performed using a gel imaging system (Bio-Rad).

[0044] like Figure 5 As shown, primrosein inhibited the protein expression of hepatic stellate cell activation markers α-SMA and collagen COL1A1 in a concentration-dependent manner.

[0045] This indicates that primrosein can inhibit the translation of proteins related to the activation of hepatic stellate cells.

[0046] Example 5

[0047] Effects of primrosein on liver fibrosis markers in mice (in vivo experiment)

[0048] Ninety healthy male ICR mice, weighing 20-25 grams, were purchased from the Experimental Animal Center of Nantong University. The animals had free access to food and water, were fed standard pelleted feed, and were acclimatized to a standard light-dark cycle (12 hours light, 12 hours darkness), room temperature of 21-25℃, and constant humidity environment. Water was changed daily, and bedding was changed every other day. A 50% olive oil solution was prepared by mixing carbon tetrachloride and olive oil at a 1:1 volume ratio. Primrosein was prepared as a solution using sodium carboxymethyl cellulose. The animals were randomly divided into six groups: normal group, model group, low-dose primrosein treatment group (1.5 mg / kg), medium-dose primrosein treatment group (3 mg / kg), high-dose primrosein treatment group (6 mg / kg), and colchicine positive control group (0.25 mg / kg), with 15 mice in each group. Five groups of mice, excluding the control group, were administered carbon tetrachloride-olive oil intraperitoneally (1 ml / kg) every other day for 8 weeks. Starting in week 5, mice in each treatment group were administered primrosein or colchicine in sodium carboxymethyl cellulose solution by gavage once daily for 4 weeks. The normal control group received an equal volume of the solvent in the same manner. After the experiment, blood was collected from the orbital cavity of each mouse, and the mice were euthanized and their livers were removed intact from the abdominal cavity. The mouse blood was allowed to stand for 2 hours, then centrifuged at 3000 rpm for 20 minutes. The supernatant was collected, stored at -80℃, and analyzed as soon as possible.

[0049] Laminin, hyaluronic acid, type III procollagen, and type IV collagen are four indicators for assessing liver fibrosis. Laminin is a non-collagenous structural protein unique to the basement membrane and is positively correlated with the degree of liver fibrosis. Hyaluronic acid, synthesized and secreted by hepatic stellate cells, can accurately and sensitively reflect the amount of fibrosis generated in the liver and the extent of liver function impairment. Type III procollagen reflects the synthesis of type III collagen in the liver, and its serum content is consistent with the degree of liver fibrosis. Type IV collagen is a major component of the basement membrane, and its serum content gradually increases with the severity of liver fibrosis, making it one of the early markers of liver fibrosis. The levels of fibrosis indicators laminin, hyaluronic acid, type III procollagen, and type IV collagen in mouse serum were detected using an enzyme-linked immunosorbent assay (ELISA) kit.

[0050] Hydroxyproline, a unique component of collagen, can be used as an indicator of collagen content. Following the kit instructions, the hydroxyproline kit was used to detect the hydroxyproline content in mouse liver tissue in order to analyze the deposition of collagen fibers in mouse liver tissue.

[0051] Real-time PCR was used to analyze the mRNA expression levels of key fibrosis genes α-SMA and COL1A1 in liver tissue. A suitable amount of liver tissue was placed in a 1.5 ml EP tube, and 300 μl of Trizol was added to each tube. The tubes were thoroughly ground, and 60 μl of chloroform was added. The tubes were tightly capped, vortexed for 15 seconds, and then allowed to stand at room temperature for 3 minutes. The tubes were then centrifuged at 12000 rpm for 15 minutes at 4°C. The supernatant was transferred to a new 1.5 ml EP tube, and 150 μl of isopropanol was added and mixed. The tubes were allowed to stand at room temperature for 10 minutes, and then centrifuged at 12000 rpm for 10 minutes at 4°C, allowing RNA to adhere to the bottom of the tube. The supernatant was discarded, and 300 μl of 75% ethanol was added. The tubes were vortexed for a few seconds and then centrifuged at 7500 rpm for 10 minutes at 4°C. Discard the supernatant, place in a fume hood for 30 minutes, and after the ethanol has evaporated, add 20 μl of DEPC water. Perform reverse transcription and amplification experiments according to the kit instructions.

[0052] The primer sequences are as follows:

[0053] α-SMA(F):5′-CATCCACGAAACCACCTA-3′(Seq_1)

[0054] α-SMA(R):5′-GGGCAGGAATGATTTGGA-3′(Seq_2)

[0055] COL1A1(F):5′-GAGAGAGCATGACCGATGGATT-3′(Seq_3)

[0056] COL1A1(R):5′-TGTAGGCTACGCTGTTCTTGCA-3′(Seq_4)

[0057] GAPDH(F):5′-CTATGACCACAGTCCATGC-3′(Seq_5)

[0058] GAPDH(R): 5′-CACATTGGGGGTAGGAACAC-3′ (Seq_6).

[0059] An appropriate amount of liver tissue was collected and lysed using pre-cooled RIPA lysis buffer. The lysate was collected and stored overnight at -80°C. The next day, after the lysate thawed, it was centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was collected, and the protein concentration was determined using a BCA kit. 25 μg of protein was loaded and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). PVDF was transferred to a membrane. Bovine serum albumin (BSA) was used to block non-specific binding sites on PVDF. After adding the corresponding primary antibody, the membrane was incubated overnight at 4°C. Horseradish peroxidase-labeled secondary antibody was added for hybridization. ECL chemiluminescence buffer was added, and the membrane was imaged and analyzed using a gel imaging system (Bio-Rad).

[0060] Liver collagen staining was performed using Masson's trichrome staining and Sirius red staining. Masson's trichrome staining is a classic technique for staining collagen fibers; after staining, muscle fibers appear red, and collagen fibers appear blue, primarily used to distinguish between collagen and muscle fibers. Sirius red staining is a classic technique for distinguishing between type I and type III collagen fibers. Under polarized light microscopy, type I collagen fibers appear orange-yellow or red, while type III collagen fibers appear green. Mouse liver tissue was fixed overnight in 4% paraformaldehyde, embedded in paraffin, cut into 6 μm thick sections, dewaxed, and stained with Masson's trichrome and Sirius red, respectively. The collagen staining was observed under an optical microscope, and images were acquired by randomly selecting fields of view.

[0061] like Figure 6 As shown, compared with the normal control group, the serum levels of four liver fibrosis indicators—lamin, hyaluronic acid, type III procollagen, and type IV collagen—were significantly increased in mice with carbon tetrachloride-induced liver fibrosis, while primrosein dose-dependently reduced the levels of these indicators.

[0062] like Figure 7 As shown, compared with the normal control group, the content of hydroxyproline in the liver tissue of carbon tetrachloride-induced liver fibrosis mice was significantly increased, while primrosein reduced the hydroxyproline content in liver tissue in a dose-dependent manner. Primrosein significantly reduced the mRNA and protein content of α-SMA and COL1A1 in the liver tissue of liver fibrosis mice in a dose-dependent manner.

[0063] like Figure 8 As shown, Masson's trichrome staining revealed a large amount of blue collagen fiber deposition in the liver tissue of mice with carbon tetrachloride-induced liver fibrosis compared to the normal control group. The fibers extended outwards from the portal area, and the fibers were thicker and stained more deeply. Collagen deposition was significantly reduced in the liver tissue of mice treated with primrose oil. Sirius red staining revealed a large amount of red type I collagen fibers in the liver tissue of mice with carbon tetrachloride-induced liver fibrosis compared to the normal group. Type I collagen fiber deposition was significantly reduced in the liver tissue of mice treated with primrose oil.

[0064] The above results indicate that primrosein can significantly reduce the degree of liver fibrosis in mice.

Claims

1. The application of primrosein in the preparation of drugs for the treatment of liver fibrosis, wherein the structural formula of primrosein is shown below: 。 2. The application according to claim 1, characterized in that: The liver fibrosis mentioned is a disease caused by excessive activation of hepatic stellate cells.

3. The application according to claim 1, characterized in that: Primrose extract can reduce hepatic stellate cell activity, decrease the mRNA and protein expression of hepatic stellate cell activation marker α-SMA and fibrosis marker COL1A1, reduce the content of serum laminin, hyaluronic acid, type III procollagen and type IV collagen in liver fibrosis, reduce the content of hydroxyproline in liver tissue, decrease the mRNA and protein expression of α-SMA and COL1A1 in liver tissue, and improve collagen fiber deposition in liver tissue.

4. The application according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

5. The application according to claim 1, characterized in that: The dosage form of the drug is tablets, granules, pills, capsules, or injections.

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