Application of erythrinine in preparation of medicine for promoting liver regeneration

Abstract

The invention discloses an application of erythrinine in preparation of a medicine for promoting liver regeneration, in particular to promotion of liver regeneration after partial liver resection. The invention discovers that the erythrinine has certain effects of repairing the liver injury and promoting the liver proliferation for the first time, and further researches the related mechanism of the erythrinine for repairing the liver injury and promoting the liver proliferation. The erythrinine can be used as an active ingredient to be applied to the medicine for treating liver regeneration after partial liver resection, a new application of the erythrinine is developed, and a new choice is provided for treating complications such as acute liver injury and liver failure after partial liver resection.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the application of erythrina alkaloids in the preparation of drugs for promoting liver regeneration. Background Art

[0002] Statistics as of January 2024 show that the incidence of malignant liver diseases is still increasing year by year worldwide. Complications of cirrhosis and hepatocellular carcinoma are the main causes of liver disease-related deaths. At present, the treatment principle of malignant liver diseases such as hepatocellular carcinoma or cirrhosis is still comprehensive treatment based on surgery, among which partial hepatectomy (PHx) is the main surgical treatment. However, liver failure after partial hepatectomy is the most serious complication and one of the most common causes of death in the perioperative period of liver resection, which seriously reduces the overall survival rate of patients with liver tumors.

[0003] Modern pharmacological studies have shown that the extract of Vaccaria segetalis contains a large number of biologically active ingredients with important pharmacological properties. Active ingredients with functions such as promoting wound healing and reducing inflammatory responses have been discovered one after another. Hypaphorine (HYP) is a small molecule indole alkaloid compound isolated from Vaccaria segetalis, a traditional Chinese medicine. Its structural formula is shown below:

[0004]

[0005] Although fenugreek has a long history of use as a medicine, and its pharmacologically active ingredient erythrinain has made progress in repairing skin wounds and promoting wound healing, there are currently few studies on whether erythrinain can promote liver regeneration after partial hepatectomy and whether it can repair acute liver damage. Summary of the invention

[0006] The purpose of the present invention is to provide application of erythrine in preparing a drug for promoting liver regeneration.

[0007] Furthermore, the drug for promoting liver regeneration promotes liver regeneration and / or liver proliferation after partial liver resection.

[0008] Furthermore, the liver regeneration promoting drug repairs liver cell proliferation after partial liver resection.

[0009] Furthermore, the liver regeneration promoting drug increases the secretion of repair cytokines in the liver after partial hepatectomy.

[0010] The present invention first discovered that erythrina alkaloids have certain effects of repairing liver damage and promoting liver proliferation, and further studied the related mechanisms of erythrina alkaloids in repairing liver damage and promoting liver proliferation. Erythrina alkaloids can be used as an active ingredient in drugs for treating liver regeneration after partial hepatectomy, opening up new uses for erythrina alkaloids and providing new options for treating complications such as acute liver damage and liver failure after partial hepatectomy. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 These are the results of the effect of erythrine on the proliferation activity of AML12 cells.

[0012] Figure 2 These are the results of the effect of erythrine on the proliferation level of AML12 cells.

[0013] Figure 3 The results show the effect of erythrine on the apparent state of liver after 70% hepatectomy in C57 mice.

[0014] Figure 4 The results show the effect of erythrine on the liver weight to body weight ratio in C57 mice after 70% hepatectomy.

[0015] Figure 5 The results show the effect of erythrine on the mRNA levels of proliferation-related cyclins in the remaining liver tissue of C57 mice after 70% hepatectomy.

[0016] Figure 6 The results show the effect of erythrine on the mRNA levels of proliferation-related cytokines in the remaining liver tissue of C57 mice after 70% hepatectomy. DETAILED DESCRIPTION

[0017] The present invention provides the use of erythrina alkaloids in the preparation of drugs for promoting liver regeneration, in particular promoting liver regeneration after partial liver resection. The present invention is the first to discover that erythrina alkaloids have certain effects of repairing liver damage and promoting liver proliferation, and further studies the related mechanisms of erythrina alkaloids in repairing liver damage and promoting liver proliferation.

[0018] First, the CCK8 method was used to evaluate the effect of erythrina alkaloids on the proliferation of AML12 cells. The cells were digested with trypsin in the logarithmic growth phase and inoculated in a 96-well plate. The cells were divided into a control group and groups with different concentrations of erythrina alkaloids. The results showed that erythrina alkaloids could significantly promote cell proliferation in the range of 6.25 to 100 μmol / L, and the concentration of 25 μmol / L was the best. Cell proliferation was evaluated by EdU staining experiment. AML12 cells in the logarithmic growth phase were inoculated and divided into a control group and a drug-treated group, the latter of which used a culture medium containing 25 μmol / L erythrina alkaloids; the results showed that the proportion of EdU-positive cells in the drug-treated group was significantly higher than that in the control group, indicating that erythrina alkaloids increased the level of cell proliferation.

[0019] Afterwards, an injection containing erythrina alkaloids was prepared, and C57BL / 6 mice were randomly divided into a sham operation group (sham), a model group (70% PH), and a drug-treated group (70% PH+HYP). After 70% hepatectomy, mice in the drug-treated group were treated with erythrina alkaloids, and the results showed that the color and volume of the remaining liver tissue were relatively healthy, indicating that the level of liver proliferation was high. The livers of mice in each group were sampled 36 hours after surgery and the liver weight to body weight ratio was calculated. The results showed that the liver weight to body weight ratio of the drug-treated group was significantly higher than that of the model group, indicating that erythrina alkaloids promoted liver regeneration. The expression of proliferation-related genes in mouse liver tissue was detected, and the results showed that the mRNA expression of CyclinA2, CyclinB1, CyclinD1, and CyclinE1 in the liver was significantly increased after erythrina alkaloid treatment; in addition, the expression level of hepatocyte growth factor HGF was also significantly increased, further supporting that erythrina alkaloids help maintain liver homeostasis and promote regeneration.

[0020] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0023] Example 1

[0024] Cell experiments

[0025] 1. CCK8 Experiment

[0026] When the mouse hepatocytes AML12 grew to the logarithmic phase, the cells were digested with trypsin, washed and blown away, and inoculated in a 96-well plate with about 5000 cells per well, with a volume of 100 μl per well. The cells were inoculated and cultured 24 hours in advance for CCK8 experiments. Erythrina alkali was dissolved in DMSO solution to prepare a mother solution with a concentration of 50 mmol / L. After the cells adhered to the wall, they were randomly divided into a control group and different concentrations of erythrina alkali (final concentrations of 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μmol / L), and a blank group containing no cells but only AML12 complete culture medium was set up, and 3 replicates were set up for each group. The control group was added with 100 μL of AML12 complete medium, and each concentration group of erythrine was added with 100 μL of complete medium containing the corresponding concentration of drug. After culturing for 36 h, 10 μL of CCK8 solution was added to the cells of each group. After incubation at 37°C for 2 h, the absorbance (OD) of each well was measured at a wavelength of 450 nm using a microplate reader, and the cell proliferation rate was calculated:

[0027]

[0028] from Figure 1 It can be seen that erythrine has the effect of promoting the proliferation activity of mouse hepatocyte AML12 in the range of 6.25-100 μmol / L, and the effect is most significant at the concentration of 25 μmol / L.

[0029] 2. EdU staining of cells

[0030] Mouse hepatocytes AML12 in the logarithmic growth phase were inoculated in 3.5 cm culture dishes and randomly divided into two groups: control group and drug administration group. The next day, when the cells were attached to the wall and the density reached about 50%, the drug administration group was replaced with DMEM medium containing 10% fetal bovine serum with 25 μmol / L erythrine, and the control group was replaced with DMEM medium without erythrine and 10% fetal bovine serum, and the cells were placed in the incubator for further culture for 36 hours.

[0031] When the cells were cultured for 36 hours, the cells were washed three times with sterile PBS, replaced with fresh medium containing 20 μmol / L EdU, and placed in the incubator for further culture for 2 hours. The cells were then digested with trypsin, centrifuged at 200×g for 5 minutes, and the supernatant was discarded to collect the cells. After permeabilization and fixation of the cells, 488Fluorophore-FC antibody was added and incubated in the dark for 30 minutes. After centrifugation, the cells were washed three times with PBS, and finally resuspended with 1 mL PBS to detect the proportion of EdU-positive cells by flow cytometry.

[0032] like Figure 2 As shown, compared with the control group, the proportion of EdU-positive cells in mouse hepatocyte AML12 cultured in the medium containing 25 μmol / L erythrine increased significantly.

[0033] The above results indicate that erythrine can enhance the proliferation level of mouse hepatocyte AML12.

[0034] Example 2

[0035] Animal experiments

[0036] 1. In vivo administration in mice

[0037] Prepare an injection containing erythrine in advance, the solvent is a mixture of DMSO, PEG300, Tween 80 and saline (volume ratio 2:8:1:9). Under a sterile environment, first add 6 mg of erythrine to 200 μL DMSO, then add 800 μL PEG300, 100 μL Tween 80, 900 μL saline in sequence and vortex mix to prepare a 3 mg / mL stock solution. According to the weight of the mouse, each mouse is administered 25 mg / kg.

[0038] Nine C57BL / 6 mice of similar age and weight were randomly divided into three groups: sham operation group, model group (70% hepatectomy) and drug administration group (drug administration after 70% hepatectomy). After anesthesia, the abdomen of the mice in the sham operation group was incised and sutured, and the 70% hepatectomy group and drug administration group underwent 70% hepatectomy, respectively, and the resected parts were the left lateral lobe (accounting for 30% of the total volume of the liver) and the middle lobe (accounting for 40% of the total volume of the liver). After surgery, all mice needed to receive appropriate postoperative care, keep warm and provide water to prevent dehydration and hypothermia. 12 hours after surgery, mice in the drug administration group were injected with 25 mg / kg of erythrine by intraperitoneal injection. 24 hours after drug administration, that is, 36 hours after surgery, blood was collected from the eyeballs and tissue samples were taken, and the in vivo status of the mouse liver was recorded by photographing.

[0039] like Figure 3 As shown, compared with the model group (70% PH), the remaining liver tissue of the mice in the drug-treated group (70% PH+HYP) was healthier in color and the tissue volume tended to increase slightly. This result indicated that the mice treated with erythrinain after surgery were in better health and had a higher level of liver proliferation.

[0040] Calculation of liver weight to body weight ratio

[0041] Tissue samples were collected from mice in different groups at 36 hours after 70% hepatectomy or sham operation. The weight of mice was weighed and recorded before sampling. When sampling, the remaining liver tissue (including the large right lobe, small right lobe, and caudal lobe) was weighed and the total weight of the remaining liver was recorded.

[0042]

[0043] like Figure 4As shown, compared with the model group (70% PH), the liver weight of mice in the drug-treated group (70% PH+HYP) was higher and there was a significant difference. This result indicated that the liver tissue of mice treated with erythrinain after surgery proliferated faster.

[0044] 3. Detection of the expression of proliferation-related genes in mouse liver tissue by real-time quantitative PCR

[0045] Take fresh tail lobe tissue from the remaining liver of the mouse after surgery, wash it twice with pre-cooled PBS, add 1mL Trizol, and let it stand for 10min. Take the cell lysate, transfer it to an enzyme-free 1.5mL centrifuge tube, add 0.2mL chloroform, vortex and shake to mix, let it stand for 5min, and centrifuge it at 12000rpm for 15min. Take the supernatant to a new enzyme-free 1.5mL centrifuge tube, add an equal volume of isopropanol, gently invert and let it stand for 10min, and centrifuge it at 12000rpm for 10min. Discard the supernatant, add 1mL of anhydrous ethanol, and centrifuge it at 12000rpm for 5min. Discard the anhydrous ethanol, leave it at room temperature until the ethanol evaporates, and add 30μL of enzyme-free water to dissolve it. Measure the RNA concentration and purity. Subsequently, reverse transcription was performed according to the instructions of the reverse transcription kit of Nanjing Novozyme Biological Company. After obtaining cDNA, the real-time fluorescence quantitative PCR detection kit of Nanjing Novozyme Biological Company was used for operation, and the results were relatively quantified using the ΔΔCT method, with GAPDH as the internal reference gene.

[0046] Real-time quantitative PCR was used to detect the mRNA expression levels of proliferation-related genes in fresh liver tissues of mice after surgery. Figure 5 As shown in the figure, 24 hours after administration, the mRNA expression levels of cyclins CyclinA2, CyclinB1, CyclinD1, and CyclinE1 in the liver tissue of mice treated with erythrine were higher than those in the model group. Figure 6 As shown, 24 hours after administration, the mRNA expression level of HGF, a classical hepatocyte growth factor that regulates hepatocyte proliferation, was higher in the liver tissue of mice treated with erythrine.

[0047] In summary, erythrine can increase the secretion of repair cytokines in the liver, maintain liver homeostasis in postoperative mice, and promote the regeneration of the remaining liver.

Claims

1. Application of erythrine in the preparation of drugs for promoting liver regeneration.

2. The use according to claim 1, characterized in that: The liver regeneration promoting drug promotes liver regeneration after partial liver resection.

3. The use according to claim 1, characterized in that: The liver regeneration promoting drug repairs liver cell proliferation after partial liver resection.

4. The use according to claim 1, characterized in that: The liver regeneration promoting drug increases the secretion of repair cytokines in the liver after partial hepatectomy.

5. A drug for promoting liver regeneration, characterized in that: Erythrine is used as the sole active ingredient or the main active ingredient.

6. The drug according to claim 5, characterized in that The medicament further includes a pharmaceutically acceptable carrier.

7. Use of erythrine in the preparation of a drug for treating acute liver injury or liver failure caused by partial liver resection.

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