Use of licochalcone B

Abstract

The present invention belongs to the technical field of biomedicine, and particularly relates to the use of licochalcone B. Licochalcone B promotes the proliferation of hepatocytes and significantly increases the liver / body weight ratio by inhibiting the activity of 15-PGDH protein, reducing the degradation of PGE2, increasing the level of endogenous PGE2, thereby promoting the regeneration and repair of the liver. The present invention discloses for the first time that licochalcone B is an effective small molecule inhibitor of 15-PGDH with the effect of promoting liver regeneration, and can be used to prepare drugs for promoting liver regeneration.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to the application of a small molecule inhibitor of 15-PGDH in the preparation of a drug for promoting liver regeneration. The small molecule inhibitor of 15-PGDH specifically refers to licochalcone B. Background Art

[0002] The liver is one of the important organs of the human body and plays an important role in maintaining metabolic homeostasis. The liver has strong regenerative ability. When damaged, various types of cells in the liver proliferate and differentiate to restore the relative stability of liver function and body homeostasis. Partial hepatectomy is the most effective way to treat end-stage liver disease and liver cancer clinically at present. However, liver dysfunction caused by hepatectomy is an important reason affecting the perioperative recovery and postoperative survival of patients, especially those who have undergone extensive hepatectomy such as semi-hepatectomy. Therefore, exploring drugs that can effectively promote the regeneration and repair of residual liver tissue has important clinical value.

[0003] Prostaglandin E2 (PGE2) is widely present in various tissue cells of the human body and plays a key role in mediating a series of cellular activities such as cell proliferation, differentiation, and apoptosis. At the same time, it is also involved in various pathological processes such as inflammation and cancer. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme for PGE2 degradation, which can catalyze the oxidation of active 15-hydroxyprostaglandin to inactive 15-ketoprostaglandin. Research reports show that 15-PGDH is a potential pharmacological target for promoting tissue regeneration. By inhibiting the 15-PGDH target, the degradation of PGE2 can be reduced, the accumulation of endogenous PGE2 can be increased, and thus the regeneration of liver tissue can be promoted.

[0004] Licochalcone B, with the CAS number 58749-23-8, molecular weight 286.28 g / mol, chemical name (E)-3-(3,4-dihydroxy-2-methoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one, and molecular formula C 16 H 14 O5, and its chemical structure is as follows:

[0005]

[0006] Licochalcone B is a natural flavonoid compound extracted from traditional Chinese medicines such as licorice and bitter almonds, and has various pharmacological effects such as anti-inflammatory, antibacterial, antioxidant, anti-cancer, and liver protection effects. However, so far, there has been no relevant report on the use of licochalcone B for promoting liver regeneration.

[0007] Therefore, the present invention provides a new use of licochalcone B. By inhibiting the activity of the 15-PGDH target, reducing the degradation of PGE2, promoting the accumulation of endogenous PGE2, and further promoting the regeneration and repair of the liver, it provides a new strategy for the drug treatment of liver diseases. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the deficiencies of the liver regeneration technology after extended hepatectomy. The purpose is to provide the application of licochalcone B in the preparation of drugs for promoting liver regeneration and its potential mechanism of action.

[0009] In order to achieve the above object, the technical solutions adopted by the present invention include the following aspects:

[0010] First, the present invention uses surface plasmon resonance (SPR) experiments to investigate the binding affinity between licochalcone B and the target protein 15-PGDH. SPR technology is a new technology centered on biosensor chips developed in the 1980s of the last century and is currently widely used to measure the affinity of molecular interactions.

[0011] Next, the present invention examines the effect of licochalcone B on the level of PGE2 in the serum of normal mice. PGE2 is an important liver regeneration regulatory factor that can effectively promote liver regeneration, but it is easily degraded by 15-PGDH in vivo. By measuring the change in the content of PGE2 in the serum of mice, the present invention further examines the ability of licochalcone B to inhibit 15-PGDH and increase the content of PGE2, thereby promoting liver repair.

[0012] Finally, the present invention constructs a 70% hepatectomy mouse model as a liver regeneration model, and designs 6 groups respectively: blank control group, positive drug group (SW033291), sham operation group, high-dose licochalcone B group (100 mg / kg), medium-dose licochalcone B group (50 mg / kg), and low-dose licochalcone B group (25 mg / kg). The drugs are administered by intraperitoneal injection. The mice are sacrificed 1 day, 2 days, 3 days, and 5 days after administration respectively. Before sacrifice, BrdU is intraperitoneally injected into the mice, the liver tissues are removed, weighed, and immunohistochemical staining experiments are carried out. The positive rate of 5-bromo-2-deoxyuridine (BrdU) is used as an index to characterize the proliferation of hepatocytes in mice after hepatectomy. At the same time, the recovery of the liver weight of mice is measured according to the generally recognized liver / body weight ratio in the academic community. The ability of licochalcone B to promote liver regeneration and repair is investigated.

[0013] The present invention has the following beneficial effects:

[0014] The present invention shows that liquiritigenin B has good binding affinity with 15-PGDH protein; in addition, liquiritigenin B can significantly increase the content of PGE2 in the serum of mice. By using the classical 70% hepatectomy model and administering liquiritigenin B, it can effectively promote the proliferation of mouse hepatocytes, increase the proportion of the regenerated liver, and promote the recovery of liver function, and the effect is better than that of the reported compound SW033291; at the same time, the above results indicate that liquiritigenin B has a significant promoting effect on the regenerative capacity of the liver after hepatectomy, and the mechanism of action involves inhibiting the activity of 15-PGDH protein and increasing the level of endogenous PGE2. The present invention provides a new method and means for the development of drugs for clinically regulating liver regeneration after hepatectomy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a diagram of the result of BrdU immunohistochemical staining.

[0016] Figure 2 It is a schematic diagram of the statistical analysis result of the BrdU positive cell rate.

[0017] Figure 3 It is the statistical analysis result of the BrdU positive cell rate of each experimental group after 3 days and 5 days of drug administration.

[0018] Figure 4 It is a schematic diagram of the effect of liquiritigenin B on the liver / body weight ratio of mice after partial hepatectomy after 3 days and 5 days of drug administration.

[0019] Figure 5 It is a schematic diagram of the binding affinity of the interaction between liquiritigenin B and 15-PGDH protein.

[0020] Figure 6 It is a schematic diagram of the effect of liquiritigenin B on the content of PGE2 in the serum of mice. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention will be further described below in conjunction with the drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, equipment and methods used in the present invention are the reagents, equipment and methods commonly purchased in the art.

[0022] Example 1: Study on the binding affinity between liquiritigenin B and 15-PGDH target protein

[0023] To investigate the binding ability of licochalcone B to 15-PGDH, the present invention used SPR experiments to evaluate the binding affinity between licochalcone B and the 15-PGDH target protein. In this experiment, a CM5 sensor chip was selected, and the measurement was carried out using a Biacore 8k (GE Healthcare, USA) instrument at 25°C. The specific experimental steps are as follows:

[0024] (1) Preparation of the instrument: Turn on the power switches of the Biacore 8K system and the computer, and place the buffer solution, chip, and sample tray into the instrument.

[0025] (2) Ligand coupling: Dilute the 15-PGDH protein to 10 μg / mL with sodium acetate at different pH values (pH 5.5, 5.0, 4.5, 4.0) and inject samples sequentially for 180 s, so that licochalcone B begins to accumulate on the chip surface through electrostatic interaction. Finally, select the sodium acetate with the mildest conditions that can reach the target coupling amount for subsequent coupling. In this experiment, pH 5.0 was determined as the optimal coupling condition, and its response value was much higher than the target coupling amount. Dilute the ligand solution to 2 μg / mL with sodium acetate at pH 5.0 for ligand coupling. Then select the Immobilization template in the Surface preparation menu, and immobilize the 15-PGDH protein into the Flow Cell 2 sample channel of the CM5 chip by the amino coupling method. The coupling time is 420 s, and the speed is 10 μL / min.

[0026] (3) Determination of sample affinity: Prepare a concentration gradient of the compound to be tested (250 μM, 125 μM, 62.5 μM, 31.25 μM, 15.625 μM, 7.813 μM, 3.906 μM, 1.953 μM) using a PBST solution containing 5% DMSO for binding affinity determination. Use a DPBS solution containing 5% DMSO as the buffer, inject samples at a speed of 30 μL / min, set the binding time to 120 s, and the dissociation time to 180 s. Use the analysis software to automatically perform fitting and output the results.

[0027] As Figure 5 shown, licochalcone B can have a strong interaction with the 15-PGDH protein, and its dissociation constant (Kd) is 13.2 μM, and the maximum response value is about 80 RU, indicating that licochalcone B can bind to the 15-PGDH target protein at the molecular level and has an affinity at the micromolar level.

[0028] Example 2: Study on the inhibition of 15-PGDH by licochalcone B to increase the content of serum PGE2

[0029] To further investigate whether licochalcone B can inhibit 15-PGDH, increase the content of PGE2 in vivo, and thus regulate liver regeneration, the present invention examined the effect of licochalcone B on the content of PGE2 in the serum of normal mice. Male Balb / c mice aged 6-8 weeks were randomly divided into 3 groups: blank control group (normal saline, 50 mg / kg), licochalcone B group (50 mg / kg), and positive drug group (SW033291, 5 mg / kg), and the drugs were administered by intraperitoneal injection. Three mice were used at each time point, and the mice were sacrificed at 0, 2, 4, 8, and 12 h after administration. After taking blood from the eyeballs, the supernatant was obtained by centrifugation at 3700 rpm / min for 10 min, and the content of PGE2 in each serum sample was determined by a PGE2 ELISA kit.

[0030] As Figure 6 shown, at 4 h after administration, the content of PGE2 in the serum of the mice in the licochalcone B group was significantly higher than that in the blank control group and the positive drug group. In addition, the change trends of the content of PGE2 in the serum of the blank control group and the licochalcone B group were the same, reaching the peak at 4 h after administration, while the positive group reached the peak at 2 h, and all experimental groups basically returned to the normal level at 12 h. Therefore, licochalcone B can significantly increase the content of PGE2 in mice.

[0031] Example 3: Study on the effect of licochalcone B on liver regeneration after hepatectomy

[0032] 1. Experimental design

[0033] (1) Experimental animals: Kunming mice, male, 7-8 weeks old, purchased from the Experimental Animal Center of Jiangsu University, and the animal certificate number is SCXK (Jiangsu) 2018-0012. Before the operation, the mice drank water and ate normally without any special treatment, and all experimental operations complied with the "Regulations on the Use of Experimental Animals" formulated by the national and Jiangsu University Ethics Committees.

[0034] (2) Experimental grouping: The successfully modeled mice were randomly divided into 6 groups, namely the blank control group (saline), the positive drug group (SW033291), the sham operation group (saline), the low-dose liquiritigenin B group (25 mg / kg), the medium-dose liquiritigenin B group (50 mg / kg), and the high-dose liquiritigenin B group (100 mg / kg). Administration was by intraperitoneal injection. The administration dose of the positive drug group was 5 mg / kg, twice a day; the administration doses of the blank control group and the sham operation group were 50 mg / kg, once a day; the high-, medium-, and low-dose liquiritigenin B groups were all once a day. On the first day after surgery, the mice were administered drugs after waking up. The mice were sacrificed 1 day, 2 days, 3 days, and 5 days after administration, and BrdU (50 mg / kg) was intraperitoneally injected into the mice 4 times before sacrifice, with an interval of 2 hours each time. All liver tissues were taken, weighed, and subjected to immunohistochemical staining.

[0035] 2. Observation of hepatocyte proliferation by BrDU immunohistochemical staining of liver tissue

[0036] BrdU is an artificially synthesized thymidine analogue, which can also replace thymine (T) and infiltrate into the replicating DNA molecule (S phase) during the cell proliferation period. Therefore, the degree of BrdU uptake by hepatocytes represents the DNA synthesis situation, that is, the BrdU positive cell rate in liver sections characterizes the proliferation level of hepatocytes.

[0037] After preparing the liver tissue into paraffin blocks, the sections were subjected to steps such as dewaxing and hydration, antigen repair, DNA denaturation, serum blocking, dropping the primary antibody and incubating overnight, dropping the secondary antibody, DAB color development, hematoxylin counterstaining, dehydration, and sealing. Five fields of view were randomly selected under a 20-fold microscope, and Image J software was used for image analysis of the immunohistochemical results to count the BrdU positive cell rate (%), and the average value was taken to characterize the regeneration degree of each mouse's liver.

[0038] As Figure 1 and 2As shown, licochalcone B can significantly promote the proliferation of hepatocytes in mice after hepatectomy. One day after administration, the change in the proliferation of hepatocytes in each experimental group of mice was not obvious, and only a very small number of hepatocytes were in the state of cell division. Two days after administration, the BrdU-positive cell rate in the positive drug group of mice was significantly higher than that in other groups. Three days after administration, the proliferation of hepatocytes in each experimental group of mice reached the peak. The BrdU-positive cell rates in the 25 mg / kg licochalcone B group and the 50 mg / kg licochalcone B group were significantly increased, showing significant differences compared with the blank control group and being higher than those in the positive drug group. Five days after administration, the proliferation of hepatocytes in each experimental group decreased significantly, but the BrdU-positive cell rate in the 100 mg / kg licochalcone B group was significantly higher than that in the blank control group, indicating that the liver regeneration reaction in this group of mice continued. This may be due to insufficient regeneration in the early stage, and the regenerated hepatocytes were not sufficient to maintain the physiological functions of the liver. Therefore, five days after administration, the hepatocytes continued to divide continuously to maintain the physiological needs of the liver and the body. The above results indicate that licochalcone B has a significant promoting effect on the proliferation of hepatocytes after hepatectomy.

[0039] As Figure 3 shown, licochalcone B at different administration doses has a great influence on the proliferation of hepatocytes in mice after hepatectomy. When the administration dose is 25 mg / kg and 50 mg / kg, the promoting effect on liver regeneration is more obvious. Three days after administration, the BrdU-positive cell rate in the 25 mg / kg licochalcone B group was significantly higher than that in the 50 mg / kg licochalcone B group, the 100 mg / kg licochalcone B group, and the positive drug group. Five days after administration, the liver regeneration reaction in the 25 mg / kg licochalcone B group and the 50 mg / kg licochalcone B group of mice had terminated according to the physiological needs, while the liver regeneration reaction in the 100 mg / kg licochalcone B group of mice continued.

[0040] 3. Determination of liver weight recovery index

[0041] According to the formula of liver / body weight ratio recognized in the academic community to measure the liver weight recovery of mice. As Figure 4 shown, three days after administration, the liver / body weight ratios of mice in the 25 mg / kg licochalcone B group and the 100 mg / kg licochalcone B group were significantly higher than those in the blank control group and higher than those in the positive drug group. Five days after administration, the liver / body weight of the 25 mg / kg licochalcone B group was significantly higher than that in the blank control group and slightly lower than that in the positive drug group. The above results indicate that licochalcone B can significantly promote the recovery of liver weight after hepatectomy.

[0042] The above results indicate that licochalcone B promotes the proliferation of hepatocytes and the repair and regeneration of the liver by inhibiting the activity of 15-PGDH protein, reducing the degradation of PGE2, and increasing the content of endogenous PGE2.

Claims

1. Use of licochalcone B, characterized in that, It is used as a small molecule inhibitor of 15-PGDH for the preparation of drugs to promote liver regeneration.

2. The application according to claim 1, characterized in that, Licochalcone B acts on 15-PGDH protein, reduces the degradation of PGE2, increases the endogenous PGE2 level, and thus promotes the proliferation of hepatocytes and the regeneration and repair of the liver.

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