A pharmaceutical composition and use thereof in the preparation of a medicament for treating or preventing liver fibrosis

Abstract

The present application belongs to the technical field of medicine, and relates to a kind of pharmaceutical composition and its application in preparation for treating or preventing liver fibrosis drug. A kind of pharmaceutical composition for treating or preventing liver fibrosis, the pharmaceutical composition includes as active ingredient the protocatechuic acid (PCA) and gimalin (GER). The present application first confirms that protocatechuic acid and gimalin combined application has significant synergistic effect in the treatment of liver fibrosis, and its curative effect is better than any single compound single administration, can more effectively reduce liver injury index, improve liver pathological morphology, reduce intrahepatic collagen deposition. Based on its clear mechanism of action, the composition of the present application is especially suitable for treating liver fibrosis related to abnormal expression of RIPK3.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a pharmaceutical composition and its use in the preparation of drugs for the treatment or prevention of liver fibrosis. Background Technology

[0002] Hepatic fibrosis (HF) is a common pathological process in the progression of various chronic liver diseases (such as viral hepatitis, alcoholic liver disease, and non-alcoholic fatty liver disease) to cirrhosis. Its characteristic feature is that under the stimulation of persistent liver injury, hepatic stellate cells (HSCs) are activated, proliferate extensively, differentiate into myofibroblasts, and excessively secrete and deposit extracellular matrix (ECM), ultimately destroying the normal liver lobule structure and leading to decreased liver function or even liver failure. Therefore, effectively inhibiting or reversing liver fibrosis is a key aspect of treating chronic liver diseases.

[0003] Currently, there are no specific drugs for treating liver fibrosis in clinical practice. While some drugs, such as colchicine, have some efficacy, they often have adverse reactions and limited effectiveness. Traditional Chinese medicine (TCM) has a long history and unique advantages in treating liver fibrosis, often employing compound formulas or single herbs based on principles such as promoting blood circulation, removing blood stasis, soothing the liver, and strengthening the spleen. However, the complex composition of traditional Chinese medicine compound formulas, along with the difficulty in clearly defining their target mechanisms and quality control, limits their modernization.

[0004] Protocatechuic acid (PCA) is a phenolic acid active ingredient found in traditional Chinese medicine plants such as *Sparganium stoloniferum*. Modern research indicates that it possesses various pharmacological activities, including anti-inflammatory and antioxidant effects. Germacrone (GER) is a major sesquiterpene compound found in the traditional Chinese medicine *Curcuma zedoaria*, and has also been shown to have antitumor and anti-inflammatory effects. Both compounds have been reported to have certain hepatoprotective effects.

[0005] Although the pharmacological activities of protocatechuic acid and gemmaconazole have been partially studied, the combination of these two specific small molecule compounds, particularly for the treatment of liver fibrosis, has never been disclosed in the existing technology. Whether their combined use will produce a synergistic effect, their specific pharmacodynamic performance, and their underlying molecular mechanisms of action, especially their relationship with programmed cell death pathways (such as programmed necrosis), remain unknown. Therefore, developing a novel anti-liver fibrosis drug combination with clearly defined active ingredients, a well-defined mechanism of action, and significant therapeutic efficacy is a pressing technical problem to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a pharmaceutical composition for treating or preventing liver fibrosis.

[0007] The technical solution adopted by this invention to solve its technical problem is:

[0008] A pharmaceutical composition for treating or preventing liver fibrosis, the pharmaceutical composition comprising protocatechuic acid (PCA) and gemcitabine (GER) as active ingredients.

[0009] Preferably, the weight ratio of protocatechuic acid to gemmaconazole is 1:1. This specific ratio of composition has been shown in subsequent experiments to produce unexpected synergistic effects.

[0010] Preferably, the pharmaceutical composition also includes one or more pharmaceutically acceptable carriers or excipients to facilitate the formulation of various dosage forms suitable for clinical use, such as tablets, capsules, injections, etc.

[0011] The use of a pharmaceutical composition according to the present invention in the preparation of a drug for treating or preventing liver fibrosis.

[0012] Preferably, when the drug is administered to mammals (such as humans or mice), the dosage of protocatechuic acid and gemcitabine is 25-50 mg / kg body weight / day, respectively. This dosage range has been proven to be safe and effective.

[0013] Preferably, the drug treats or prevents liver fibrosis by promoting the RIPK3-mediated programmed necrosis pathway in hepatic stellate cells.

[0014] Preferably, the PCA and GER regulate the programmed necrosis pathway by binding to the RIPK3 protein, inhibiting the expression of hepatic stellate cell (HSC) activation markers α-SMA, Collagen I, and Fibronectin, and downregulating programmed necrosis markers pRIPK3, pRIPK1, and pMLKL.

[0015] Through in-depth research, the inventors have surprisingly discovered that the anti-hepatic fibrosis effect of protocatechuic acid and gemmaconazole has a unique molecular mechanism. In one specific embodiment, the drug treats or prevents liver fibrosis by promoting the RIPK3-mediated programmed necrosis pathway in hepatic stellate cells. More specifically, protocatechuic acid and gemmaconazole directly bind to the RIPK3 protein, altering its protein thermostability, thereby regulating the programmed necrosis pathway, effectively inhibiting the expression of hepatic stellate cell (HSC) activation markers α-SMA, collagen I, and fibronectin, and significantly downregulating the phosphorylation levels of key proteins in the programmed necrosis pathway, namely p-RIPK3, p-RIPK1, and p-MLKL.

[0016] The application of the pharmaceutical composition described in this invention in the preparation of drugs for treating liver fibrosis, including inflammatory infiltration of liver tissue, cell necrosis, abnormal proliferation and deposition of extracellular matrix.

[0017] As a preferred option, the serum ALT and AST levels in the combined drug administration group decreased by ≥40% compared with the model group, and the collagen area ratio in liver tissue decreased by ≥60% (Masson staining).

[0018] The compositions of the present invention exhibit excellent in vivo efficacy. In one specific embodiment, the effects of the drug are manifested as follows: compared with the model group, combined administration can reduce the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) by not less than 40%, and reduce the collagen area ratio of liver tissue after Masson staining by not less than 60%.

[0019] A method for promoting programmed necrosis of activated hepatic stellate cells, the method comprising contacting the cells with an effective amount of a composition of protocatechuic acid and gemmaconazole, wherein the composition is capable of binding to the intracellular RIPK3 protein and altering its thermal stability.

[0020] The beneficial effects of this invention are:

[0021] This invention is the first to demonstrate that the combined use of protocatechuic acid and gemcitabine has a significant synergistic effect in the treatment of liver fibrosis. Its efficacy is superior to either single compound administered alone, and also superior to positive control drugs. It can more effectively reduce liver damage indicators, improve liver pathological morphology, and reduce intrahepatic collagen deposition.

[0022] This invention uses two high-purity single small molecule compounds as active ingredients, overcoming the shortcomings of traditional Chinese medicine compound ingredients being complex and difficult to control in terms of quality, and laying the foundation for stable drug production and standardized application.

[0023] This invention reveals for the first time a novel mechanism by which the combination of protocatechuic acid and gemcitabine exerts its anti-liver fibrosis effect by directly targeting the RIPK3 protein and regulating the programmed necrosis pathway, providing a new target and theoretical basis for the treatment of liver fibrosis.

[0024] Based on its well-defined mechanism of action, the composition of the present invention is particularly suitable for treating liver fibrosis associated with abnormal RIPK3 expression, providing new possibilities for achieving precision treatment. Attached Figure Description

[0025] Figure 1 MTT assay results for gemcitabine (left) and protocatechuic acid (right);

[0026] Figure 2 The left image shows the results of Western blot analysis of rat HSCs and the statistical results of grayscale values.

[0027] Figure 3 Liver index (left) and liver weight (right) results for each group of mice. Note: * P<0.03, ** P<0.002, *** P<0.001;

[0028] Figure 4 The results of serum biochemical AST (left) and ALT (right) measurements in each group of mice are shown below. Note: * P<0.03, ** P<0.002, *** P<0.001;

[0029] Figure 5 These are actual photographs of the livers of mice in each group;

[0030] Figure 6 HE staining images for each group (200X);

[0031] Figure 7 Masson staining diagrams (200X) for each group;

[0032] Figure 8 The results of liver fibrosis-related protein detection in each group of mice are shown below. * P<0.03, ** P<0.002, *** P<0.001;

[0033] Figure 9 Docking diagram of protocatechuic acid (top) and gemmaconazole (bottom) with RIPK3 protein molecules;

[0034] Figure 10 The binding curves of protocatechuic acid and gemcitabine to RIPK3 protein were verified for the Cetsa assay (left) and Western blotting assay (right).

[0035] Figure 11 The image shows the Western blot results of rat HSCs (top left) and the statistical results of grayscale values.

[0036] Figure 12 The image shows the results of Western blot detection of programmed necrosis protein in rat HSCs (top left) and the statistical results of grayscale values. Detailed Implementation

[0037] The technical solution of the present invention will be further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0038] In this invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0039] Unless otherwise specified, the reagents used in the following examples can be purchased from a regular biochemical reagent store.

[0040] The animal model protocol is as follows: (1) Male BALB / c mice of SPF grade 8-9 weeks were randomly divided into the following 6 groups (n=6 mice / group): Control group, Model group, Positive drug group (colchicine) (0.2 mg / kg), Protocatechuic acid alone group (50 mg / kg), Gemmazone alone group (50 mg / kg), Low-dose group of protocatechuic acid and Gemmazone combined administration (25 mg / kg + 25 mg / kg), High-dose group of protocatechuic acid and Gemmazone combined administration (50 mg / kg + 50 mg / kg), prepared with 0.25% sodium carboxymethyl cellulose. (2) After BALB / c mice were acclimatized for one week, except for the normal control group, the other groups were subcutaneously injected with a mixture of 40% carbon tetrachloride and olive oil (3 mL / kg body weight), (CCl4: olive oil = 2:3), twice a week, once in the first week, with the dose doubled. The treatment groups were administered according to the above protocol for eight weeks.

[0041] Example 1: Effect of combined administration of protocatechuic acid and gemcitabine on activated hepatic stellate cells

[0042] 1. Sample source: Protocatechuic acid and gemmaconazole (LOT: 23022442) were purchased from Beijing Bettercare Biomedical Technology Co., Ltd., with a purity of ≥98%.

[0043] 2. Dosage Design: Cells were divided into a control group, a model group, and a protocatechuic acid-gemardone combined treatment group. After cell adhesion, HSCs were treated with the drug and cultured in an incubator for 24 h. Cells were then collected for subsequent experiments. Specific drug treatments for each group are as follows:

[0044] Control group (Normal): free DMEM.

[0045] Model group: free DMEM + 10 ng / mL TNF-α.

[0046] Protocatechuic acid and gemcitabine combined administration group: 50 μmol / L protocatechuic acid and 50 μmol / L gemcitabine + free DMEM + 10 ng / mL TNF-α.

[0047] 3. Experimental methods and results:

[0048] (1) MTT assay to determine drug concentration: HSCs cultured to the logarithmic growth phase were digested with trypsin and then the cell density was adjusted to 2.5 × 10⁻⁶ cells / year using complete culture medium. 4 Cells were seeded at a density of 1 / mL in 96-well plates, with 200 μL of cell suspension added to each well. Cells were incubated overnight in a cell culture incubator to allow adherence. Concentrations of protocatechuic acid and gimazone were prepared at 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 μmol / L. The solvent was 10% PBS. Different concentrations of drug-containing culture medium were added to the adhered cells in 96-well plates, with six replicates per concentration. After 24 h of incubation in the dark, 10 μL of MTT solution was added to each well. After incubation for 4 h, the supernatant was carefully aspirated, and 150 μL of DMSO solution was added to each well. The plates were shaken for 10 min, and the absorbance at 492 nm was measured using a microplate reader. The absorbance values ​​are shown in the attached table. Figure 1 .

[0049] Depend on Figure 1 It is known that gemmaconazole and protocatechuic acid at a concentration of 50 μmol / L have no cytotoxicity to HSCs cultured in conventional culture medium, while they can promote the death of TNF-α-stimulated activated HSCs.

[0050] (2) Western blot detection of proteins:

[0051] Cells from each group after drug administration were washed three times with pre-cooled PBS. Cells from each group were collected using a cell scraper, and 200 μL of cell lysis buffer was added to lyse the collected cells. Cells were incubated on ice for 30 min, then centrifuged at 10,000 rpm for 30 min at 4°C. The supernatant was collected to obtain total cell protein, which was diluted to an appropriate concentration with PBS. 5× protein loading buffer (SDS-PAGE Protein Loading Buffer, [Beyotime, batch number: P0015]) was added, and the mixture was quantified. The mixture was vortexed, and proteins from each group were separated using SDS-PAGE. The proteins were then transferred to a PVDF membrane. Next, the PVDF membrane was blocked at room temperature with a 5% skim milk powder-PBST mixture for 1 hour. Subsequently, the PVDF membrane was incubated overnight at 4°C with primary antibodies containing α-SMA (1:1000), Col I (1:1000), and GAPDH (1:15000). After repeated washing with PBST, the PVDF membranes were incubated with rabbit / mouse secondary antibodies at room temperature for 2 hours. The target protein bands were visualized using a ChemiScope 6100 multichemiluminescence imaging system. The final results of changes in liver fibrosis marker proteins in different groups of mice are shown below. Figure 2 As shown.

[0052] Depend on Figure 2 It was found that, compared with the control group, the expression of α-SMA and Collagen I proteins in the model group was significantly upregulated. After administration of gemcitabine and protocatechuic acid, the expression of α-SMA and Collagen I proteins decreased. Among them, the combined administration of protocatechuic acid and gemcitabine significantly downregulated the levels of α-SMA and Collagen I proteins, and the effect was stronger than that of the compounds alone (P<0.05). This result indicates that the combined administration of protocatechuic acid and gemcitabine can significantly regulate protein levels and thus improve liver fibrosis.

[0053] Example 2: Effects of protocatechuic acid-gemmazone combined administration on a mouse model of liver fibrosis.

[0054] 1. Sample source: Protocatechuic acid and gemmaconazole (LOT: 23022442) were purchased from Beijing Bettercare Biomedical Technology Co., Ltd., with a purity of ≥98%.

[0055] 2. Dosage Design: The control group, model group, positive control group (colchicine) (0.2 mg / kg), protocatechuic acid alone (50 mg / kg), gemimadone alone (50 mg / kg), low-dose combination of protocatechuic acid and gemimadone (25 mg / kg + 25 mg / kg), and high-dose combination of protocatechuic acid and gemimadone (50 mg / kg + 50 mg / kg) were prepared using 0.25% sodium carboxymethyl cellulose. All groups were administered the drugs according to the above regimen for eight weeks.

[0056] 3. Animal model of liver fibrosis: After one week of acclimatization, BALB / c mice, except for the normal control group, were subcutaneously injected with a mixture of 40% carbon tetrachloride and olive oil (3 mL / kg) twice a week, with the dose doubled in the first week.

[0057] 4. Experimental methods and results:

[0058] (1) Body weight to liver weight ratio: Thirty-six male BALB / c mice were weighed and randomly divided into 6 groups (n=6 mice / group) according to their body weight. After one week of acclimatization, they were fed continuously for 8 weeks according to the dosage design. They were weighed twice a week during the experimental period. On the last day of the eighth week, they were fasted and weighed the following day before being sacrificed. Liver weight was measured, and changes in body weight and liver weight ratio were recorded. Figure 3 .

[0059] Depend on Figure 3It was found that, compared with the control group, the body weight of mice after modeling was significantly reduced (P<0.01), and the body weight trend of each treatment group was similar to that of the control group, indicating that the physical condition of the mice gradually improved after drug intervention. In addition, compared with the control group, the liver index of the model group was significantly increased, and after drug treatment, the liver index decreased significantly (P<0.01).

[0060] (2) Serum biochemical indicators: The administration method for each group was the same as in section 2 above. In week 9 of the experimental period, blood was collected from the orbital venous plexus of mice in each group. After standing at room temperature for 30 min, the blood was centrifuged at 3000 rpm for 10 min, and the supernatant serum was collected. The levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) were measured using an automated biochemical analyzer. The final serum biochemical indicators of different groups of mice are as follows: Figure 4 As shown.

[0061] Depend on Figure 4 It was found that, compared with the control group, AST and ALT were significantly elevated in the model group, and significantly downregulated by protocatechuic acid, gemcitabine, and the combined administration of the two drugs (P<0.05), with the high dose of the combined drugs showing a more significant effect (P<0.001). Gemcitabine has a stronger regulatory effect on ALT than protocatechuic acid.

[0062] (3) Liver morphology observation: The administration method for each group was the same as in item 2 above. Liver tissues of mice in each group were taken and observed. The results are as follows. Figure 5 As shown.

[0063] Depend on Figure 5 The control group mice exhibited smooth, reddish-brown, soft, and elastic livers with clear edges. The model group mice had brownish-yellow, hard, and blunt livers with thickened edges and numerous grayish-white nodules. After drug administration, the livers became reddish, soft, and clearly textured, with a significant reduction in nodules. The most significant reduction was observed with a high dose of protocatechuic acid and gemcitabine combined.

[0064] (4) H&E and Masson pathological tests: The administration methods for each group were the same as in section 2 above. Liver tissues of mice in each group were taken and fixed with formalin. The liver tissue samples were removed, rinsed with water, and liver tissue of appropriate thickness was cut and rinsed with running water for more than 2 hours. The samples of each group were dehydrated using a fully automatic dehydrator. The tissues were embedded in paraffin, pre-cooled, and then serially sectioned to a thickness of 4 μm. The sections were baked in a 60℃ oven for more than 2 hours. The samples were then dewaxed, hydrated, stained with eosin-hematoxylin (HE), stained with Masson, mounted with neutral resin, and observed under an optical microscope. The final pathological changes of mice in different groups were as follows: Figure 6 , Figure 7 As shown.

[0065] Depend on Figure 6 , Figure 7 It was observed that in the normal control group, mouse hepatocytes were arranged in an orderly manner, centered on the central vein, with intact lobular structure, orderly arranged hepatic cords, and only a small amount of blue-stained collagen fiber connective tissue in the vessel wall. In the model group, hepatocytes showed edema, fatty degeneration, disordered cell arrangement, disordered lobular structure, enlarged portal vein area, formation of numerous pseudolobules, extensive collagen fiber deposition in the central vein area, significantly widened blue-stained areas of collagen fiber bands, and the formation of fibrous septa. Significant improvements in liver histology were observed after HE and Masson staining in all treatment groups. The high-dose group treated with a combination of protocatechuic acid and gemcitabine showed more intact hepatocyte structure and less collagen fiber deposition.

[0066] (5) Western blot detection: The administration method for each group was the same as in section 2 above. Liver tissue from each group of mice was taken, and 20 mg of liver tissue was weighed. 100 μL of a mixture (protein lysis buffer: PMSF = 100:1) was added in proportion. Magnetic beads were added and the tissue was homogenized using a tissue cell disruptor. After homogenization, the tissue was centrifuged for 10 min at 15000 rpm and the supernatant was collected. The supernatant was diluted with PBS solution to an appropriate concentration, and 5× protein loading buffer (SDS-PAGE Protein Loading Buffer, [Beyotime, batch number: P0015]) was added and mixed to quantify. The mixture was vortexed and the proteins of each group were separated by SDS-PAGE and then transferred to a PVDF membrane. Next, the PVDF membrane was blocked in a 5% skim milk powder-PBST mixture at room temperature for 1 hour. Subsequently, the PVDF membrane was incubated overnight at 4°C with primary antibodies containing α-SMA (1:1000), Col I (1:1000), FN (1:1000), and GAPDH (1:15000). After multiple washes with PBST, the PVDF membrane was incubated with rabbit / mouse secondary antibodies at room temperature for 2 hours. The target protein bands were visualized using a ChemiScope 6100 multichemiluminescence imaging system. The final results of changes in liver fibrosis marker proteins in different groups of mice are shown below. Figure 8 As shown.

[0067] Depend on Figure 8 It was found that, compared with the control group, the expression of α-SMA, Collagen I, and Fibronectin proteins in the model group was significantly upregulated (P<0.05); compared with the model group, the expression of α-SMA, Collagen I, and Fibronectin in the compound intervention group was decreased (P<0.05), with the most significant decrease in the high-dose group of protocatechuic acid and gemcitabine combined administration.

[0068] Example 3: In vitro experiment on the regulation of programmed necrosis by protocatechuic acid and gemcitabine targeting RIPK3.

[0069] 1. Sample source: Protocatechuic acid and gemmaconazole (LOT: 23022442) were purchased from Beijing Bettercare Biomedical Technology Co., Ltd., with a purity of ≥98%.

[0070] 2. Dosage Design: Cells were divided into a control group, a model group, and a protocatechuic acid-gemardone combined treatment group. After cell adhesion, HSCs were treated with the drug and cultured in an incubator for 24 h. Cells were then collected for subsequent experiments. Specific drug treatments for each group are as follows:

[0071] Control group (Normal): free DMEM.

[0072] Model group: free DMEM + 10 ng / mL TNF-α.

[0073] Protocatechuic acid and gemcitabine combined administration group: 50 μmol / L protocatechuic acid + 50 μmol / L gemcitabine + free DMEM + 10 ng / mL TNF-α.

[0074] 3. Experimental methods and results:

[0075] (1) Molecular docking: First, the structural information of the RIPK3 protein was obtained from the PDB database, ensuring that the resolution of the crystal structure was <3 Å and that the crystal structure was obtained using X-ray diffraction. The pdb format file of the target protein crystal structure was downloaded from the PDB database. It was processed in AutoDock for conformational clustering, interaction analysis, etc. Then, the molecular docking results were visualized using PyMOL.

[0076] Depend on Figure 9 It is known that the binding energies of protocatechuic acid, gemcitabine, and RIPK3 are -4.03 and -3.38 kcal / mol, respectively. The binding ability is strong and multiple hydrogen bonds are formed, indicating that protocatechuic acid, gemcitabine, and RIPK3 can dock and establish a binding relationship with the receptor protein.

[0077] (2) CETSA experiment to verify drug-protein binding: Protein extraction refers to the method described in Example 2 (5) Western blot detection. The experimental system was prepared as 90 μL protein + 10 μL drug solution, which was serially diluted with cell lysis buffer from the stock solution. Protocatechuic acid and gemcitabine were prepared as 20 mM stock solution with DMSO. The effect of a single drug concentration (20 μM) on the expression of proteins at 8 temperature gradients was investigated, with DMSO as the control group (concentration same as drug). The operation was kept on ice, the cell lysis buffer was thawed in advance, and the caps of the eight tubes were prepared. After lysis, the experimental grouping was performed: DMSO (control group) was added to one tube, and the small molecule of the required concentration was added to the other tube. After mixing evenly, the tubes were placed at room temperature for 10-30 min (1-3 min is sufficient) (10 μL drug + 90 μL lysis buffer). Different temperature gradients were set up, and the above treatment groups were then aliquoted into eight-tube strips, 100 μL / tube, with the number of aliquots matching the number of temperature gradients, i.e., 40, 42, 44, 48, 53, 57, 61, 67, 72, and 75 ℃. Each sample was heated for 3 min at each gradient temperature (which can be set using a PCR instrument), cooled to room temperature for 3 min, and then centrifuged (12000 rpm, 20 min, 4 ℃). 80 μL of supernatant protein was aspirated into a 600 μL centrifuge tube, 5X loading buffer was added, and the mixture was denatured in a metal bath at 100 ℃ for 10 min. The proteins from each group were separated by SDS-PAGE and then transferred to a PVDF membrane. Next, the PVDF membrane was blocked at room temperature with a 5% skim milk powder-PBST mixture for 1 hour. Subsequently, the PVDF membrane was incubated overnight at 4 ℃ with RIPK3 (1:1000) primary antibody. After multiple washes with PBST, the PVDF membrane was incubated with rabbit / mouse secondary antibody at room temperature for 2 hours. The target protein band was visualized using a ChemiScope 6100 multichemiluminescence imaging system. The final results of RIPK3 changes in samples treated at different temperatures are shown below. Figure 10 As shown.

[0078] Depend on Figure 10 It can be seen that the degradation rate of RIPK3 protein increases with increasing temperature, and decreases significantly at 53 °C. However, the degradation rate of RIPK3 protein with added gimazone and protocatechuic acid is significantly slowed down, and binding still occurs at 61 °C. After grayscale analysis of the bands, it was found that 20 μM gimazone and protocatechuic acid have a significant effect on the thermal stability of RIPK3, and both can directly bind to RIPK3.

[0079] (3) Western blot detection: The detection method is the same as described in (5) Western blot detection above.

[0080] Depend on Figure 11 It can be seen that, under RIPK3 knockdown, the protein expression of α-SMA, CollagenI, and Fibronectin was upregulated in the TNF-induced activated HSCs model, and was significantly downregulated after drug administration.

[0081] (4) Western blot detection of programmed necrosis-related proteins: The detection method is the same as described in (5) Western blot detection of proteins above.

[0082] Depend on Figure 12 It was found that a TNF-α-induced HSCs model was established on shRIPK3 HSCs cells, and protocatechuic acid and gemmaconazole were administered for intervention. Compared with the model group, the protein expression of pRIPK3, pRIPK1, and pMLKL was significantly increased after protocatechuic acid and gemmaconazole intervention.

[0083] (5) Summary: The interaction between protocatechuic acid and gemimazone and RIPK3 protein was verified by molecular docking and CETSA experiments. Then, TNF-α induction was performed on shRIPK3-inducing HSCs, followed by administration of protocatechuic acid and gemimazone. It was found that protocatechuic acid and gemimazone could promote programmed necrosis of activated HSCs and improve liver fibrosis caused by decreased RIPK3 protein expression, suggesting that the drug may have other pathways of action.

[0084] In summary, compared with the protocatechuic acid monotherapy group and the gemcitabine monotherapy group, the high-dose combination therapy group (50+50 mg / kg) showed superior efficacy in several key indicators, such as a more significant downregulation of AST and ALT. Figure 4 The improvement in liver morphology (reduction of nodules) is more pronounced. Figure 5 It showed greater improvement in liver histopathology (H&E and Masson staining) and less collagen fiber deposition. Figure 6 , 7 ), with the strongest inhibitory effect on liver fibrosis marker proteins (α-SMA, Collagen I, Fibronectin). Figure 8 This synergistic effect cannot be directly and easily predicted from the known pharmacological activities of the two compounds.

Claims

1. A pharmaceutical composition for treating or preventing liver fibrosis, characterized in that, The pharmaceutical composition contains protocatechuic acid (PCA) and gemcitabine (GER) as active ingredients.

2. The pharmaceutical composition according to claim 1, characterized in that, The weight ratio of protocatechuic acid to gemmaconazole is 1:0.5-1.

5.

3. The pharmaceutical composition according to claim 1, characterized in that: The pharmaceutical composition also contains one or more pharmaceutically acceptable carriers or excipients.

4. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for treating or preventing liver fibrosis.

5. The application according to claim 4, characterized in that, The drug treats or prevents liver fibrosis by promoting the RIPK3-mediated programmed necrosis pathway in hepatic stellate cells.

6. The application according to claim 4, characterized in that, The PCA and GER regulate the programmed necrosis pathway by binding to the RIPK3 protein, inhibiting the expression of hepatic stellate cell (HSC) activation markers α-SMA, Collagen I, and Fibronectin, and upregulating programmed necrosis markers pRIPK3, pRIPK1, and pMLKL.

7. A method for promoting programmed necrosis of activated hepatic stellate cells in vitro, comprising contacting the cells with an effective amount of a PCA and GER composition, wherein the composition alters the thermal stability of the RIPK3 protein.

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