Application of the combination of lyophilized powder of LEECH and bear bile powder in the preparation of drugs for treating liver injury

Abstract

The application discloses application of a combination of lyophilized powder of Haemadipsa integrifascialis and bear gall powder in preparation of a medicine for treating liver injury, and belongs to the technical field of medicines. The application proves through in-vitro HepG2 and HSC-T6 cell experiments and in-vivo CCl4-induced mouse liver injury models that the combination of the lyophilized powder of Haemadipsa integrifascialis and the bear gall powder can significantly improve the survival rate of injured liver cells, inhibit the activation and fibrosis of hepatic stellate cells, reduce the level of inflammatory factors, improve the antioxidant capacity, reduce the pathological injury of liver tissues, and the synergistic effect is significantly better than that of single medicine, and the safety is good. The application provides a new medicine combination and experimental basis for developing a new liver-protecting and anti-liver fibrosis medicine, and has important clinical application and medicine development values.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of the combined use of lyophilized leech powder and bear bile powder in the preparation of drugs for treating liver damage. Background Technology

[0002] Hirudin, a natural antithrombin substance secreted by the salivary glands of the leech, is highly effective in treating cardiovascular and cerebrovascular diseases, especially stroke. Clinical and pharmacological studies have shown that hirudin has significant therapeutic effects on various diseases, including anti-inflammation, anti-oxidation, prevention of thrombosis, anti-organ fibrosis, protection of vascular endothelial cells, and improvement of hypoxia. Related studies have been conducted on the cardiovascular, urinary, and respiratory systems. Freeze-dried leech powder (LP) can improve liver blood circulation and promote hepatocyte regeneration, thus playing a certain role in protecting the liver.

[0003] Bear bile powder (BBP) is a traditional Chinese medicine. It is bitter and cold in nature, and enters the liver, gallbladder, and heart meridians. It has the effects of clearing heat, calming the liver, and improving eyesight. It can stabilize cells and has a certain repairing effect on liver cell damage. Natural bear bile powder is a traditional Chinese medicine used to treat liver dysfunction. Studies have shown that bear bile powder can improve liver damage by reducing the expression of inflammatory cytokines and hepatocyte apoptosis.

[0004] Currently, bear bile powder is used clinically, either alone or as a main ingredient in compound prescriptions. It is widely recorded in traditional Chinese medicine literature as being used to treat hepatobiliary diseases. Bear bile powder exhibits broad pharmacological effects and relatively few adverse reactions in treating hepatobiliary diseases. Particularly for liver tumors, it exerts anti-cancer activity by inhibiting tumor cell proliferation and angiogenesis, suggesting its significant potential for drug development and clinical application. It can play a role in all stages of liver disease development, including chronic hepatitis, liver fibrosis, cirrhosis, and liver cancer, possessing multiple effects such as hepatoprotection, choleretic activity, anti-liver fibrosis, improvement of hepatic steatosis, and anti-tumor activity. Clinically, it can treat various diseases such as hepatitis, cirrhosis, cholecystitis, gallstones, and cholestatic liver disease.

[0005] Combination therapy can improve drug efficacy, reduce drug dosage, and overcome drug resistance. While there are numerous reports on the use of lyophilized leech powder and bear bile powder alone, there are few reports on the combined use of these two drugs in liver disease research, and their effects on liver injury and fibrosis remain unexplored. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the combined use of lyophilized leech powder and bear bile powder in the preparation of drugs for treating liver injury, and to verify the efficacy of the combined use of lyophilized leech powder and bear bile powder in treating liver injury, thus providing a reference for the combined application of lyophilized leech powder and bear bile powder.

[0007] To achieve the above objectives, the present invention provides the application of lyophilized leech powder and bear bile powder in the preparation of drugs for treating liver damage.

[0008] Preferably, the liver injury is drug-induced liver injury, oxidative liver injury, liver fibrosis, carbon tetrachloride-induced acute liver injury, or subacute liver injury.

[0009] Preferably, the drug exerts its hepatoprotective effect by improving hepatocyte survival rate, inhibiting hepatocyte apoptosis, reducing the expression of inflammatory factors, enhancing antioxidant capacity, inhibiting hepatic stellate cell activation, and reducing collagen deposition.

[0010] Preferably, the mass ratio of lyophilized leech powder to bear bile powder in the drug is 3:1 to 3:10.

[0011] Preferably, the drug dosage form is a tablet, capsule, granule, lyophilized powder for injection, oral liquid, or powder.

[0012] Therefore, the combined use of lyophilized leech powder and bear bile powder in the preparation of drugs for treating liver injury has the following beneficial effects: (1) This invention induces in vitro hepatocyte damage by excessive APAP, and evaluates the protective activity of lyophilized leech powder and bear bile powder alone or in combination on damaged hepatocytes by measuring the survival rate of hepatocytes by the MTT assay. The experimental results show that the combined use of bear bile powder and lyophilized leech powder has a significant effect on improving the survival rate of hepatocytes.

[0013] (2) In the embodiments of the present invention, the combined action of lyophilized leech powder and bear bile powder on cells can significantly improve the cell survival rate of H2O2-induced hepatocyte damage, showing good protective and antioxidant activity.

[0014] (3) This invention uses hepatic stellate cells (HSC-T6) as target cells to establish an in vitro liver fibrosis model. The combined application of lyophilized leech powder and bear bile powder to HSC-T6 cells inhibits TGF-β-induced fibrotic growth in HSC-T6 cells, effectively reduces the expression levels of fibrosis markers α-SMA and Collagen I, and decreases ROS release, thereby improving oxidative stress. In the 0.2% CCl4-induced liver injury model group, serum HA levels were significantly increased, and the combined application of lyophilized leech powder and bear bile powder reduced HA levels. The combined application of lyophilized leech powder and bear bile powder showed better efficacy than either alone.

[0015] (4) This invention assesses oxidative liver injury by detecting serum ALT, AST, and HA levels, and liver tissue GSH and TNF-α levels in mice with liver injury induced by 0.2% and 5% CCl4. The combined administration of lyophilized leech powder and bear bile powder significantly increased serum ALT and AST levels, increased liver tissue GSH levels, and decreased TNF-α levels, indicating that the combined administration of lyophilized leech powder and bear bile powder can improve the oxidative stress level of CCl4-induced liver injury.

[0016] (5) The present invention found that the combined use of lyophilized leech powder and bear bile powder has a good effect on hepatocellular immune damage, oxidative damage and inhibition of cell fibrosis. The combined use of lyophilized leech powder and bear bile powder has good hepatoprotective activity.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention 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.

[0019] Figure 1 The effects of lyophilized leech powder (LP) and bear bile powder (BBP) on HepG2 cells; where A represents the toxicity of LP and BBP to HepG2 cells, B represents the protective effect of LP and BBP against APAP-induced acute liver injury, and C represents the protective effect of LP and BBP against H2O2-induced acute liver injury;* P <0.05,** P <0.01, *** P <0.001, compared with the control group; # P <0.05, ## P <0.01, compared to the model group; Figure 2 The effects of lyophilized leech powder (LP) and bear bile powder (BBP) on HSC-T6 cells; where A represents the cytotoxicity of LP and BBP on HSC-T6 cells, and B represents the protective effect of LP and BBP against TGF-β-induced acute liver injury; P <0.01, compared with the control group; # P <0.05, compared to the model group; Figure 3Laser confocal microscopy images and quantitative analysis of relative fluorescence intensity of AF594-labeled α-SMA cells co-incubated with HSC-T6 cells; where A is a laser confocal microscopy image, scale bar = 50µm; B is the quantitative analysis of relative fluorescence intensity;** P <0.01, compared with the control group; ## P <0.01 compared to the model group; Figure 4 The values ​​represent the amount of reactive oxygen species (ROS) generated in HSC-T6 cells (expressed as DFC fluorescence values) and the DFC fluorescence microfluidic index (MFI); where A represents the amount of ROS generated in HSC-T6 cells, and B represents the DFC fluorescence MFI. P <0.01, compared with the control group; # P <0.05, compared to the model group; Figure 5 Western blot (WB) bands for detecting α-SMA and Collagen I protein expression, and the relative gray values ​​of α-SMA or type I collagen relative to GAPDH; where A is the WB band for detecting α-SMA and Collagen I protein expression, B is the relative gray value of α-SMA relative to GAPDH, and C is the relative gray value of Collagen I protein relative to GAPDH. Figure 6 The effects of LP and BBP on the amelioration of 0.2% CCl4-induced subacute oxidative liver injury in mice were shown. Wherein, A represents body weight, B represents liver index, C represents spleen index, D represents serum aminotransferase level, E represents AST level, F represents hyaluronic acid level, G represents GSH expression level in liver homogenate, and H represents TNF-α expression level. P <0.05,** P <0.01, *** P <0.001, compared with the control group; # P <0.05, ## P <0.01, ### P <0.001, compared to the model group; Figure 7 The effects of LP and BBP on the amelioration of 0.5% CCl4-induced subacute oxidative liver injury in mice are shown. Wherein, A is the liver index, B is the spleen index, C is the aminotransferase content, D is the AST content, E is the LDH content, F is the SOD1 mRNA expression level in liver tissue, G is the CAT mRNA expression level in liver tissue, H is the GPX1 mRNA expression level in liver tissue, I is the percentage of area affected by inflammation / necrosis, J is the H&E staining result of pathological sections, K is the TNF-α mRNA expression level in mouse liver tissue, L is the IL-16 mRNA expression level in mouse liver tissue, and M is the IL-1β mRNA expression level in mouse liver tissue.P <0.05, ** P <0.01, *** P <0.001, compared with the control group; # P <0.05, ## P <0.01, P <0.001, compared with the model group. Specific implementation manners

[0020] The technical solution of the present invention will be further described below through the drawings and embodiments.

[0021] In order to make the purpose, technical solution and advantages of the present application clearer, more thorough and complete, the technical solution of the present invention will be clearly and completely described below through the drawings and embodiments. The following detailed descriptions are all descriptions of embodiments, aiming to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms adopted by the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which the present application belongs.

[0022] The instrument equipment and reagent materials used in the embodiments are all obtained through commercial channels.

[0023] The model animals used in the embodiments are 6 - 8 week - old SPF - grade male ICR mice (weighing about 22 g), provided by Beijing Huafukang Biotechnology Co., Ltd., with the animal license number: SCKX (Beijing) 2019 - 0008. The animals are housed in the GLP animal experiment center of the Institute of Materia Medica, Chinese Academy of Medical Sciences. The room temperature is 22 - 24°C, and the relative humidity is 40% - 60%. The minimum air change rate is 20 times per hour, the lights are turned on every 12 h, and there is light. The animals are housed in IVC cages, with 4 - 5 animals in each cage. All animals are raised and managed by trained personnel, and the animals are kept free in diet and activities throughout the feeding process. The animals and related disposals used in the embodiments meet the requirements of animal welfare, and the experiments have been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Materia Medica, Chinese Academy of Medical Sciences (approval numbers: 00004033, 00004034).

[0024] The human hepatocellular carcinoma HepG2 cells and rat HSC - T6 cells used in the embodiments are both grown in DMEM culture medium containing 10% fetal bovine serum (containing penicillin 100 U·mL -1 , streptomycin 100 μg·mL -1 ), and the culture conditions are 37°C, 5% CO2, and saturated humidity. The cells are digested and passaged about every 2 - 3 days with a solution containing 0.25% trypsin and 0.02% EDTA. The cells in the logarithmic growth phase are used for the experiments.

[0025] Example 1 (1) HepG2 cells were fed with 8×10 3 Cells / well were seeded into 96-well cell culture plates and cultured for 24 h. Then, LP 0.1 μg / mL was added to each well. -1 BBP 0.3 μg·mL -1 LP 0.1μg·mL -1 +BBP 0.3μg·mL -1 The drug was tested, and a positive control of bicyclol (10 μmol·L⁻¹) was also provided. -1 The study included three groups: a solvent control group, a solvent control group, and an acetaminophen (APAP) model group. Except for the solvent control group, all other groups were treated with an acetaminophen (APAP) model at a final concentration of 8 mmol / L. -1 APAP was continued to act on the cells for 24 hours. The culture medium was then discarded, and 100 µL of 0.5 mg / mL solution was added to each well. -1 MTT solution was added to each well, and the cells were cultured for another 4 hours. The MTT solution was then discarded, and 150 µL of dimethyl sulfoxide (DMSO) was added to each well. The absorbance was measured at 570 nm using a microplate reader.

[0026] Cell viability (%) = [(average OD value of drug-treated cells - OD value of blank control group) / (average OD value of solvent control cells - OD value of blank control group)] × 100%.

[0027] The toxicity of lyophilized leech powder (LP) and bear bile powder (BBP) to HepG2 cells is as follows: Figure 1 As shown in Figure A, the results indicate that LP is 0.1 μg·mL. -1 BBP 0.3 μg·mL -1 LP 0.1μg·mL -1 +BBP 0.3μg·mL -1 After 24 hours of treatment, the cell survival rate of HepG2 cells was above 90%. LP and BBP, alone or in combination, did not have significant cytotoxicity on HepG2 cells.

[0028] The protective effects of lyophilized leech powder (LP) and bear bile powder (BBP) against APAP-induced acute liver injury are as follows: Figure 1 As shown in Figure B, after 24 hours of APAP treatment, the cell viability of the model group was significantly reduced compared with the control group; LP 0.1 μg·mL -1 +BBP 0.3μg·mL -1 The combined drug group significantly improved the cell survival rate of APAP-induced HepG2 cell damage, with effects comparable to those of the positive control drug bicyclol.

[0029] (2) HepG2 cells were fed with 8×10 3Cells were seeded per well in a 96-well cell culture plate and cultured for 24 hours. Then, LP was added to each well to a final concentration of 0.1 μg / mL. -1 BBP 0.3 μg·mL -1 LP 0.1μg·mL -1 +BBP 0.3μg·mL -1 The drug was tested, and a positive control of bicyclol (10 μmol·L⁻¹) was also provided. -1 Cells were incubated for 24 hours in three groups: a solvent control group, a solvent control group, and an H2O2 model group. Except for the solvent control group, which received an equal volume of culture medium, all other groups received H2O2 solution (final concentration 400 μg·mL⁻¹). -1 The cells were further treated for 3 hours. The culture medium was then discarded, and cell viability was assessed using the MTT assay.

[0030] The protective effects of lyophilized leech powder (LP) and bear bile powder (BBP) against H2O2-induced acute liver injury are as follows: Figure 1 As shown in Figure C, after 24 hours of H2O2 treatment, the cell viability of the model group was significantly reduced compared with the control group; LP 0.1 μg·mL -1 +BBP 0.3μg·mL -1 The combined drug group significantly improved the cell survival rate of HepG2 cells damaged by H2O2, and its effect was comparable to that of the positive control drug bicyclol.

[0031] Example 2 HSC-T6 cells were used at 8×10 3 Cells / well were seeded into 96-well cell culture plates and cultured for 24 h. Then, LP 0.1 μg / mL was added to each well. -1 BBP 0.3 μg·mL -1 LP 0.1μg·mL -1 +BBP 0.3μg·mL -1 At the same time, a positive control drug, colchicine (10 mmol·L⁻¹), was set up. -1 The study included three groups: a control group, a solvent blank control group, and a TGF-β model group. Except for the solvent control group, all other groups were treated with a final concentration of 10 ng / mL. -1 TGF-β was applied to the cells for another 24 hours. After another 24 hours of treatment, the culture medium was discarded, and the OD value was measured using the MTT assay.

[0032] Hepatic stellate cell activation inhibition rate (%) = (mean OD of model group - mean OD of treatment group) / (mean OD of model group) × 100%.

[0033] The toxicity of lyophilized leech powder (LP) and bear bile powder (BBP) to HSC-T6 cells is as follows: Figure 2 As shown in Figure A, the results indicate that LP is 0.1 μg·mL. -1 BBP 0.3 μg·mL -1 LP 0.1μg·mL -1 +BBP 0.3μg·mL -1 After 24 hours of treatment, the cell survival rate of HSC-T6 cells was greater than 90%. LP and BBP, alone or in combination, did not have significant cytotoxicity on HSC-T6 cells.

[0034] The protective effects of LP and BBP against TGF-β-induced acute liver injury, such as Figure 2 As shown in Figure B, the results indicate that LP is 0.1 μg·mL. -1 +BBP 0.3μg·mL -1 The group can significantly inhibit the fibrosis and growth of HSC-T6 cells, with an effect comparable to that of the positive control drug colchicine.

[0035] Example 3 HSC-T6 cells were used at 5×10 4 Cells were seeded in laser confocal culture dishes and cultured for 24 hours. Then, LP was added to a final concentration of 0.1 μg / mL. -1 BBP 0.3 μg·mL -1 LP 0.1μg·mL -1 +BBP 0.3μg·mL -1 The drug and TGF-β (final concentration 10 ng / mL) -1 Simultaneously, a solvent blank control group and a model group were set up. Cells were treated for another 24 hours. Cells were fixed with 2 mL of 4% paraformaldehyde for 20 min, the fixative was discarded, and the cells were washed twice with PBS. Then, 2 mL of 0.1% Triton X-100 was added for 10 min of infiltration, followed by blocking with 2 mL of 3% goat serum for 2 h. α-SMA antibody was diluted 1:500 (using PBS as solvent) and incubated overnight at 4°C. AF594-labeled secondary antibody was diluted 1:200 (using PBS as solvent), incubated at room temperature for 1 h, washed twice with pre-cooled PBS, and 200 µL of anti-fluorescence quencher was added. Intracellular α-SMA expression was observed using a laser confocal microscope (Ex=588 nm, Em=604 nm).

[0036] Laser confocal microscopy images and quantitative analysis of relative fluorescence intensity of AF594-labeled α-SMA cells co-incubated with HSC-T6 cells are shown below. Figure 3 As shown, the results indicate that LP 0.1 μg·mL -1 +BBP 0.3μg·mL -1After combined drug treatment, intracellular α-SMA expression was significantly reduced in HSC-T6 cells (LP 0.1 μg / mL). -1 +BBP 0.3μg·mL -1 The combined drug therapy was significantly superior to LP 0.1 μg·mL. -1 Used alone.

[0037] Example 4 HSC-T6 cells were used at 5×10 4 Cells were seeded in 6 cm diameter culture dishes and cultured for 24 h. Then, LP was added to a final concentration of 0.1 μg / mL. -1 BBP 0.3 μg·mL -1 LP 0.1μg·mL -1 +BBP 0.3μg·mL -1 The drug and TGF-β (final concentration 10 ng / mL) -1 Simultaneously, a solvent blank control group and a model group were set up. Cells were treated for another 24 hours. The culture medium for HSC-T6 cells was removed, the cells were washed twice with pre-cooled PBS, digested with trypsin, resuspended in culture medium, and incubated at 1000 rpm. -1 Centrifuge for 5 min to obtain HSC-T6 cells. Dilute with serum-free medium at a ratio of 1:1000 and load the probe DCFH-DA in situ to a final concentration of 10 μmol·L⁻¹. -1 Add 500 µL of prepared DCFH-DA solution to each group of cells. Incubate at 37°C for 30 min, then wash the cells three times with serum-free cell culture medium to thoroughly remove any uninfiltrated DCFH-DA. Detect DCF green fluorescence expression using the FITC channel of a flow cytometer.

[0038] The amount of reactive oxygen species generated in HSC-T6 cells (expressed as DFC fluorescence value) is as follows: Figure 4 As shown in Figure A, the DFC fluorescence MFI is as follows: Figure 4 As shown in Figure B, the results indicate that LP is 0.1 μg·mL. -1 +BBP 0.3μg·mL -1 When combined with other drugs, the intracellular ROS content of HSC-T6 cells decreased, and the combined effect was better than that of LP or BBP alone.

[0039] Example 5 HSC-T6 cells were used at 5×10 4 Cells were seeded in 6 cm diameter culture dishes and cultured for 24 h. Then, LP was added to a final concentration of 0.1 μg / mL. -1 BBP 0.3 μg·mL -1 LP 0.1μg·mL -1+BBP 0.3μg·mL -1 The drug and TGF-β (final concentration 10 ng / mL) -1 Simultaneously, a solvent blank control group and a model group were set up. Cells were treated for another 24 hours. The culture medium for HSC-T6 cells was removed, and the cells were washed twice with pre-chilled PBS. Cells were scraped off using a cell scraper, resuspended in pre-chilled PBS, and incubated at 1000 rpm. -1 Centrifuge for 5 minutes, then centrifuge twice to obtain HSC-T6 cells.

[0040] Add 100 µL of high-efficiency RIPA lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor, and lyse cells thoroughly using a cell sonicator at 40 W power for 30 min on ice. -1 After centrifugation at 4℃ for 10 min, the cell lysate was collected, and proteins were quantified and electrophoresed using a BCA assay kit. After transfer, the PVDF membrane was incubated with primary antibody overnight. The membrane was then washed with PBST, and secondary antibody was added. Incubation was continued at room temperature for 1 h, followed by three PBST washes for 10 min each. ECL chemiluminescence solutions A and B were added to the PVDF membrane, and the reaction was allowed to proceed for 1-2 min. The membrane was then detected using a chemiluminescence analyzer to assess the expression of α-SMA and Collagen I proteins.

[0041] Western blots for detecting α-SMA and Collagen I protein expression are shown below. Figure 5 As shown in Figure A, the relative gray values ​​of α-SMA with respect to GAPDH are as follows: Figure 5 As shown in Figure B, the relative gray values ​​of Collagen I protein relative to GAPDH are as follows: Figure 5 As shown in Figure C. The results show that LP 0.1 μg·mL -1 +BBP 0.3μg·mL -1 After combined drug administration to HSC-T6 cells, the expression of intracellular α-SMA and Collagen I decreased.

[0042] Example 6 After adapting to the environment, mice were randomly divided into 8 groups according to their body weight: blank control group, 0.2% CCl4 model group, LP (60 mg / kg) group, and LP (60 mg / kg) group. -1 Group ) and BBP (100 mg·kg -1 Group ) and BBP (200 mg·kg -1 Group ) LP (60mg·kg -1 ) + BBP (100mg·kg) -1 Group ) LP (60mg·kg -1 ) + BBP (200mg·kg) -1Group ) and positive control drug bicyclol (200 mg·kg) -1 The model group consisted of 10 mice per group. Mice in the model group were administered 10 mL / kg of solution. -1 Subacute liver injury in mice was induced by subcutaneous injection of 0.2% CCl4, administered every other day for 14 consecutive days. In the treatment group, the appropriate dose of the drug was administered by gavage 1-2 hours after the subcutaneous injection of 0.2% CCl4 at a dose of 10 mL / kg. -1 The mice were administered the drug once daily for 14 consecutive days. The control group was given an equal volume of saline solution once daily for 14 consecutive days. After fasting for 6 hours but with free access to water, the mice were anesthetized by inhalation of 2% isoflurane and then dislocated at the neck.

[0043] The body weight, liver index, spleen index, serum aminotransferase (ALT), AST, and hyaluronic acid (HA) levels of mice, as well as the expression levels of GSH and TNF-α in liver homogenate, were measured to evaluate the ameliorative effects of LP and BBP on 0.2% CCl4-induced subacute oxidative liver injury in mice.

[0044] Detection of serum ALT, AST, LDH and HA levels: Blood was drawn from the ocular venous plexus under 2% isoflurane inhalation anesthesia, allowed to stand at room temperature for 2 hours, and then infused at 3500 rpm. -1 Centrifuge for 20 min, collect the supernatant to obtain serum. AST, ALT, and LDH levels in the serum were detected using a fully automated biochemical analyzer. HA levels in the serum were determined according to the ELISA kit instructions.

[0045] Content of GSH and TNF-α in liver tissue homogenate: Liver tissue was weighed and added to pre-cooled physiological saline at a mass-to-volume ratio of 1:9. The tissue was cut into small pieces, steel balls were added, and the homogenate was ground using a low-temperature tissue homogenizer. 4℃, 3500 r·min -1 Centrifuge for 10 min, collect the supernatant, and prepare a 10% liver tissue homogenate. Measure the GSH and TNF-α levels according to the kit instructions.

[0046] Test results are as follows Figure 6 As shown, the data are expressed as mean ± standard deviation, n=10. P <0.05、** P <0.01、*** P <0.001, compared with the control group; # P <0.05、## P <0.01、### P<0.001, compared with the model group. Results showed that, compared with the model group, LP alone did not significantly change the body weight of mice, BBP alone showed a decreasing trend in body weight, and the combined administration of LP and BBP resulted in a decrease in body weight. Compared with the model group, LP (60 mg / kg) -1 ) + BBP (100mg·kg) -1 The combined medication group significantly reduced liver and spleen indices; compared with the model group, LP (60 mg / kg) -1 ) + BBP (100mg·kg) -1 The combined use of these drugs can significantly reduce ALT and AST levels, decrease serum HA levels, increase GSH levels in liver tissue, and reduce TNF-α levels.

[0047] Example 7 After adapting to the environment, mice were randomly divided into 6 groups according to their body weight: blank control group, 5% CCl4 model group, LP (60 mg·kg) group, and LP (60 mg·kg) group. -1 Group ) and BBP (100 mg·kg -1 Group ) LP (60mg·kg -1 ) + BBP (200mg·kg) -1 Group ) and positive control drug bicyclol (200 mg·kg) -1 Group 1, with 8 animals in each group. The treatment group received the appropriate dose of drug via gavage, at a dose of 10 mL / kg. -1 The medication was administered twice daily, once in the morning and once in the afternoon, for two consecutive days. One to two hours after the last administration, all groups except the blank control group received 10 mL / kg of the medication. -1 Mice were injected intraperitoneally with 5% CCl4 once, while the control group was given an equal volume of physiological saline. After fasting for 6 hours but with free access to water, the mice were anesthetized by inhalation of 2% isoflurane and then dislocated at the neck.

[0048] The study measured the liver and spleen indices in mice; the levels of aminotransferase (ALT), AST, and lactate dehydrogenase (LDH); the mRNA expression levels of superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione peroxidase 1 (GPX1) in liver tissue; the H&E staining results of pathological sections (scale bar = 100µm or 200µm); the percentage of area affected by inflammation / necrosis; and the mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-16), and interleukin-1β (IL-1β) in mouse liver tissue.

[0049] RT-qPCR was used to detect the expression of relevant genes: 100 mg of liver tissue was weighed, homogenized, and total RNA was extracted using the TransZOLUP plus RNA Kit. The mRNA was then reverse transcribed into cDNA using the Reverse Transcription Kit. The expression of the target genes was detected using the TransStart Tip Green Qpcr superMix. Detailed primer sequences are shown in Table 1. β-actin was used as an internal control for calibration, and the relative expression levels of each gene were calculated.

[0050] Table 1 Primer Sequences

[0051] Pathological section H&E staining: Mouse liver tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. The sections were stained with hematoxylin and eosin (H&E). Pathological changes in the liver tissue were observed under a microscope.

[0052] Test results as follows Figure 7 As shown, the data are expressed as mean ± standard deviation, n=10. P <0.05,** P <0.01, *** P <0.001, compared with the control group; # P <0.05, ## P <0.01, ### P <0.001, compared to the model group.

[0053] The results showed that, compared with the model group, LP (60 mg·kg) -1 ) + BBP (200mg·kg) -1 The combined use of these drugs significantly increased liver indices, decreased spleen indices, significantly reduced serum ALT, AST, and LDH levels, promoted the expression of SOD1, CAT, and GPX1, and improved liver pathological morphology. Simultaneously, it reduced the expression of TNF-α, IL-6, and IL-1β in liver tissue. In the positive control group, bicyclol significantly increased liver indices, significantly decreased spleen indices, significantly reduced serum ALT, AST, and LDH levels, and improved liver pathological morphology. LP (60 mg / kg) -1 ) + BBP (200mg·kg) -1 The combined use of these drugs is superior to LP and BBP alone in reducing serum ALT, AST and LDH levels and regulating the expression of SOD1, CAT, GPX1, TNF-α, IL-6 and IL-1β.

[0054] In conclusion, the combined use of lyophilized leech powder and bear bile powder has a good effect on hepatocellular immune damage, oxidative damage and inhibition of cell fibrosis, and the combined use of lyophilized leech powder and bear bile powder has good hepatoprotective activity.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of the combined use of lyophilized leech powder and bear bile powder in the preparation of drugs for treating liver damage.

2. The application of the combined use of lyophilized leech powder and bear bile powder according to claim 1 in the preparation of drugs for treating liver injury, characterized in that: The liver injury referred to includes drug-induced liver injury, oxidative liver injury, liver fibrosis, carbon tetrachloride-induced acute liver injury, or subacute liver injury.

3. The application of the combined use of lyophilized leech powder and bear bile powder according to claim 1 in the preparation of drugs for treating liver injury, characterized in that: The drug exerts its hepatoprotective effect by improving hepatocyte survival rate, inhibiting hepatocyte apoptosis, reducing the expression of inflammatory factors, enhancing antioxidant capacity, inhibiting hepatic stellate cell activation, and reducing collagen deposition.

4. The application of the combined use of lyophilized leech powder and bear bile powder according to claim 1 in the preparation of drugs for treating liver injury, characterized in that: The mass ratio of lyophilized leech powder to bear bile powder in the drug is 3:1 to 3:

10.

5. The application of the combined use of lyophilized leech powder and bear bile powder according to claim 1 in the preparation of drugs for treating liver injury, characterized in that: The drug dosage form is tablet, capsule, granule, lyophilized powder for injection, oral liquid or powder.

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