CN111481537A - Application of small molecular compound in preparation of medicine for promoting liver regeneration - Google Patents
Application of small molecular compound in preparation of medicine for promoting liver regeneration Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, and particularly relates to application of genistein in preparation of a medicine for promoting liver regeneration. Genistein can increase the in vivo accumulation of PGE2, improve the level of endogenous PGE2, and promote the proliferation of liver cells, thereby effectively promoting the regeneration and repair of liver tissues and increasing the specific gravity of regenerated liver. The invention discloses the genistein as an effective small molecular compound with the effect of promoting liver regeneration for the first time, can be used for preparing a medicament for promoting liver regeneration, provides new insights for solving the current situation of lack of clinically effective liver regeneration medicaments, and has potential and good application prospects in the field of liver tissue repair and regeneration medicaments.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and relates to application of a small molecular compound in preparation of a medicine for promoting liver regeneration, in particular to application of genistein in preparation of a medicine for promoting liver regeneration.
Background
The treatment of liver diseases is still a great test faced by the medical community, especially for liver cancer, acute liver failure and other serious liver function impairment, and the current treatment methods mainly include liver transplantation, artificial liver technology and liver resection. Liver transplantation is a more effective treatment way, but the serious shortage of donors limits the wide development of liver transplantation; the existing bioartificial liver technology can not completely replace the liver function, and the proliferation of the liver cells after being transplanted into the body is limited, thereby limiting the treatment effect. Hepatectomy is currently the most common method of treating a variety of benign and malignant diseases of the liver. Although the liver has huge potential regeneration capacity, after the liver is damaged, parenchymal cells, non-parenchymal cells, various in-vivo cell factors and growth factors in the liver and various liver stem cells and the like can respond quickly to restore the volume of the liver to the original size, and the liver function is restored to be normal, the liver is mechanically damaged by liver resection and is subjected to ischemia reperfusion injury, the regeneration process is a complex pathophysiological process, and the liver failure is easy to occur after the enlarged liver resection. Therefore, the effective method for promoting liver regeneration is artificially applied, so that lost liver histiocytes can be promoted to be rapidly supplemented, the liver function can be compensated, the liver failure can be prevented, and the disease and the wound can be cured as soon as possible. Currently, drugs are still the main means for treating liver diseases, but clinically effective liver regeneration drugs are limited, and the action mechanism of some drugs is unclear, so that research and development of liver regeneration promoting drugs are urgent.
Liver regeneration is a complex, precise and ordered regulation process with multiple stages and multiple factors, which mainly comprises three major parts, namely a cytokine regulation network, a growth factor regulation network and a metabolism regulation network, including parenchymal hepatocyte regeneration and liver tissue structure reconstruction, and various in vivo cytokines and growth factors participate in regulation through different mechanisms.
Prostaglandin E2(PGE2) is an important regulator of liver regeneration, is an eicosanoid unsaturated fatty acid, is a metabolite of arachidonic acid cyclooxygenase, is produced by 3 consecutive enzymatic reactions, and is degraded in vivo by 15-hydroxy prostaglandin dehydrogenase (15-PGDH). In the study of a partial hepatectomy model, rats are found to have increased cyclooxygenase-2 (COX-2) synthesis and increased prostaglandin synthesis after hepatectomy, and when the liver volume is gradually recovered, the secretion is gradually reduced to the same level as that of a sham operation group, while if the COX-2 action is selectively inhibited, the liver regeneration is obviously inhibited, and the liver apoptosis is increased, which indicates that the generation of endogenous PGE2 plays an important role in the initiation and control of liver regeneration. Researchers at the southwestern medical center, university of Kaes West reservoir and university of Texas, found that the degradation of PGE2 could be reduced by inhibiting 15-PGDH, promoting liver regeneration. Thus, endogenous PGE 2-mediated tissue repair can be increased by increasing synthesis of PGE2, or inhibiting rapid degradation of PGE2 in vivo.
Genistein (Genistein), also known as Genistein, with CAS number of 446-72-0, molecular weight of 270.24g/mol, chemical name of 4',5,7-Trihydroxyisoflavone, and molecular formula of C15H10O5The chemical structure is as follows:
genistein is a polyphenol compound which can be found in plants such as soybean, red clover and the like, has a molecular structure similar to 17 β -estradiol, has oxidation resistance and high affinity to estrogen receptors, can inhibit the activities of tyrosine protein kinase (PTK) and topo-I (isozyme II), has the effects of inducing programmed cell death, improving anticancer efficacy, inhibiting angiogenesis and the like, is a main phytoestrogen researched at present, has been widely researched for the benefit of the genistein on human health, is a very potential cancer preventive, and has no related report on the aspect of promoting liver regeneration after hepatectomy.
Therefore, the invention provides a new application of genistein, and can well promote the proliferation of liver cells and the regeneration and repair of liver by increasing the level of endogenous PGE2, thereby providing a new insight for solving the current situation of lack of clinically effective liver regeneration drugs.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects of the existing liver regeneration technology after hepatectomy and aims to provide the application of genistein in preparing liver regeneration medicines and an action mechanism thereof. The genistein has obvious promotion effect on the regeneration capacity of the liver after hepatectomy, can increase the level of PGE2 in vivo, and provides a new method and means for clinically regulating and controlling liver regeneration after hepatectomy or liver transplantation.
In order to solve the above-mentioned purpose, the invention is realized by the following technical scheme:
firstly, establishing a mouse liver regeneration model by 70% hepatectomy, respectively designing a blank group (containing 1% DMSO normal saline), a model drug (genistein) and a positive drug group (SW033291), respectively administering the drugs in an intraperitoneal injection mode, representing the proliferation of liver cells after the mouse hepatectomy by using the positive rate of BRDU (5-Bromo-2-deoxyUridine, 5-bromodeoxyuridine) as an index through an immunohistochemical method, simultaneously evaluating the liver regeneration effect through a liver/body weight ratio, and respectively comparing the recovery difference of the residual liver of the model mouse in the administration group with the blank group and the positive drug group after 1 day, 2 days, 3 days and 5 days of administration to prove the promotion effect of the genistein on liver repair and regeneration.
After the genistein is used for treating model mice, the liver regeneration and proliferation trend is consistent with the treatment trend of a positive medicament SW033291, and the proliferation amount of liver cells of mice of each test group reaches a peak value two days after administration. Genistein is an effective active compound with the effect of promoting liver regeneration, can remarkably promote the proliferation of liver cells and promote the regeneration and repair of mouse liver after hepatectomy, and the promotion effect of genistein on liver regeneration is better than that of a positive control medicament SW 033291. The regulation and control of the liver regeneration process by the medicament not only requires that the proliferation amount of the liver cells can be effectively increased, but also can quickly enter a termination stage when the liver regeneration amount is enough, and the hyperproliferation of the liver cells cannot be caused by continuous administration. When the administration dosage of genistein is 50mg/kg, sufficient hepatocyte proliferation can be effectively promoted after two or three days of administration, the termination stage can be approached after five days of administration, the liver regeneration process can be regulated and controlled within a controllable range, and liver cancer and the like possibly caused by excessive liver regeneration can be prevented.
The present inventors then examined the effect of genistein on the levels of PGE2 in the serum of normal mice. Many studies have shown that PGE2 is an important regulator of liver regeneration and can effectively promote liver regeneration. PGE2 exists in various types of cells, is rapidly metabolized by 15-PGDH in vivo due to its unstable chemical structure, and acts only in the local environment of its synthesis by autocrine or paracrine, and thus, PGE2 content is stable in normal physiological environment. Under the stimulation of no pathological condition, whether the drug can regulate the metabolic process of PGE2 can be accurately judged by observing the influence of genistein on the content of PGE2 in a normal mouse. According to the invention, by comparing the content of PGE2 in mouse serum after the genistein and the blank solution are treated, the genistein can obviously increase the accumulation of PGE2 in a normal mouse body, and the content of endogenous PGE2 is increased. Therefore, the mechanism of promoting liver regeneration by genistein may be to increase the in vivo content of PGE2 by increasing the synthesis of PGE2 or decreasing its degradation, thereby promoting the proliferation of hepatocytes and the repair and regeneration of liver tissue.
The genistein can effectively promote the proliferation of liver cells, promote the regeneration of liver tissues, increase the specific gravity of regenerated livers, promote the recovery of liver functions and quality, reduce the death rate when most of the liver tissues are lost, has better action effect than that of a reported compound SW033291, and improves the content of endogenous PGE2 in a mechanism for promoting the liver regeneration after hepatectomy. The invention discloses genistein which can be used for preparing a medicine for treating diseases related to liver regeneration deficiency and an action mechanism of genistein for promoting liver regeneration.
Drawings
FIG. 1 is a graph showing the results of BrdU immunohistochemical staining.
FIG. 2 is a graph showing statistical analysis of BrdU positive cell rate of each administration group with reference to a blank control, and investigating whether the model drug and the positive drug have promoting effect on the proliferation of mouse hepatocytes after hepatectomy.
FIG. 3 is a graph showing the results of statistical analysis of the BrdU positive cell rate among experimental groups, and the differences in the effects of model drugs, positive drugs, and different doses of model drugs on the proliferation of mouse hepatocytes after hepatectomy were examined two and three days after administration.
FIG. 4 is a graph showing the effect of genistein on the liver/body weight ratio of mice after hepatectomy.
FIG. 5 is a PGE2 content measurement standard curve.
FIG. 6 is a graph showing the effect of genistein on the content of PGE2 in mouse serum.
Detailed Description
The invention will be further described by the following specific examples in conjunction with the drawings, which are provided for illustration only and are not intended to limit the scope of the invention.
The clean male Kunming mice used in the invention are purchased from a clean animal house of the center of experimental animals of Jiangsu university, the mice are 7-8 weeks old during the experiment, the mice are raised in the clean animal house, the room temperature is stabilized at about 25 ℃, all operations comply with the experimental animal use regulations prepared by the ethical Committee of the country and Jiangsu university in the whole in-vivo experiment process, BRDU is purchased from Dalian American biotechnology limited company, genistein is purchased from Ensa chemical technology (Shanghai) limited company, BRDU antibody, goat anti-rat IgGH & L (HRP) are purchased from Abcam company, PGE2 kit is purchased from Nanjing Beiga biotechnology limited company, all experimental results are obtained by independent 3-6 repeated experiments, unpaired double-sample t-test analysis data are adopted, and SEM is expressed by Shi, and P value <0.05 is a significant difference standard (< 0.05, < 0.01).
The first embodiment is as follows: genistein can remarkably promote liver regeneration.
Carrying out 70% hepatectomy on mice, randomly dividing model mice which are successfully modeled into five groups, starting intraperitoneal injection administration on the first day after surgery, and carrying out blank group: glucose solution containing 1% DMSO, 50mg/kg, once a day; positive drug group: SW033291, 5mg/kg twice a day; model drug group: genistein 25mg/kg, 50mg/kg, 100mg/kg once a day. Mice were sacrificed 1 day, 2 days, 3 days, 5 days after dosing, two hours prior to sacrifice, BRDU 50mg/kg was injected into the mice, liver tissue was harvested, weighed, immunohistochemically stained, and the rate of BRDU uptake by hepatocytes was examined. The number of BrdU positive cells and the number of negative cells were counted in 5 fields randomly selected under a 20-fold mirror, and the rate of BrdU positive cells per mouse was characterized as an average value.
The results show that the liver regeneration and proliferation trend of genistein after being used for the treatment of model mice is consistent with the treatment trend of the positive drug SW 033291. After the administration for one day, the proliferation amount of the liver cells of each experimental group of mice does not change obviously, only a few liver cells are in a cell division state, and the liver cells are not different from those of blank groups of mice and are in the initial stage of liver regeneration; after two days of administration, the liver regeneration reaction is completely activated, the proliferation amount of the liver cells of mice in each test group reaches the peak value, compared with a blank control group, the BrdU positive cell rate of the positive drug group, the 25mg/kg genistein group and the 50mg/kg genistein group is obviously higher, the proliferation amount of the liver cells is more, and the liver regeneration degree of the mice in the 100mg/kg genistein group is not obviously different from that of the blank control group; after three days of administration, the liver regeneration reaction starts to be inhibited, and compared with the second day, the liver cells in a cell division state are reduced, the proliferation amount of the liver cells is obviously reduced, the BrdU positive cell rate of a genistein group of only 50mg/kg is obviously higher than that of a blank control group, and other experimental groups have no obvious difference with the blank control group; five days after administration, the liver regeneration reaction of mice of the blank control group, the positive drug control group and the 50mg/kg genistein group tends to be in a termination stage, the liver regeneration reaction of the mice of the 25mg/kg genistein group and the 100mg/kg genistein group is still continued, the BrdU positive cell rate is obviously higher than that of the blank control group, probably because the early regeneration is insufficient, the regenerated liver cells are not enough to maintain the physiological function of the liver, and the liver cells continuously divide after five days of administration so as to maintain the survival requirements of the liver and the organism (figure 1 and figure 2).
The genistein with different doses has great influence on the liver regeneration of mice after hepatectomy, when the administration dose is 25mg/kg and 50mg/kg, the promotion effect on the liver regeneration is more obvious, and when the administration dose is 100mg/kg, the promotion effect on the liver regeneration is not obvious. When the dosage is 25mg/kg, the BrdU positive cell rate of the mouse in the model drug group is obviously higher than that of the positive control group when the mouse is administrated for two days, and the BrdU positive cell rate is not different from that of the genistein group at 50 mg/kg; when the dosage is 50mg/kg and the administration is carried out for two days, the BRDU positive cell rate of the model drug group mouse is obviously higher than that of the positive control group; when the dosage is 50mg/kg, the BrdU positive cell rate of the mice in the model drug group is obviously higher than that of the genistein group and the positive control group at the time of three days of administration (figure 3). And three days after administration, the liver/body weight ratio of the positive drug group and the model drug group mice was significantly higher than that of the blank control group (fig. 4).
The results show that genistein is an effective traditional Chinese medicine active ingredient with the effect of promoting liver regeneration, can remarkably promote the proliferation of liver cells and promote the regeneration and repair of mouse liver after hepatectomy. When the dosage of genistein is 50mg/kg, the promotion effect on liver regeneration is obviously better than that of a positive control medicament SW033291, and the genistein can effectively promote the proliferation of sufficient liver cells after two or three days of administration, can approach to a termination stage after five days of administration, and can regulate and control the liver regeneration process within a controllable range.
Example two: genistein promotes liver regeneration by increasing the level of endogenous PGE 2.
In order to investigate the mechanism of genistein regulation of liver regeneration process, the invention investigated the influence of genistein on the level of PGE2 in the serum of normal mice. Dividing normal mice into a blank control group and a drug group, injecting 50mg/kg of glucose solution containing DMSO% into the abdominal cavity of the blank control group, injecting 50mg/kg of genistein into the abdominal cavity of the drug group, killing the mice after administration for 4h, taking blood from eyeballs, preparing serum samples, and measuring the content of PGE2 in each sample by a PGE2 Elisa kit. The result shows that the content of PGE2 in the mouse serum after genistein treatment is obviously higher than that of a blank control group, and under the stimulation of no pathological condition, genistein can obviously increase the accumulation of PGE2 in a normal mouse and increase the content of endogenous PGE 2.
The above results suggest that the mechanism of genistein promoting liver regeneration may be to increase accumulation of PGE2 in vivo by increasing synthesis or decreasing degradation of PGE2, thereby promoting proliferation of hepatocytes and repair and regeneration of liver tissue (fig. 5 and 6).
The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above embodiments, and all embodiments are within the scope of the present invention as long as the requirements of the present invention are met.
Claims (3)
1. The application of the small molecular compound in preparing the medicine for promoting liver regeneration is characterized in that the small molecular compound is genistein.
2. The use of claim 1, wherein the small molecule compound genistein is a molecule with a known structure having a CAS number of 446-72-0, a molecular weight of 270.24g/mol, a chemical name of 4',5, 7-trihydroxysoflavone, and a molecular formula of C15H10O5The chemical structure is as follows:
3. the use of claim 1, wherein genistein is effective in increasing endogenous PGE2 levels, promoting hepatocyte proliferation, and promoting liver tissue regeneration and repair by increasing PGE2 accumulation in vivo.
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| WO2012136859A1 (en) * | 2011-04-07 | 2012-10-11 | Helmholtz-Zentrum für Infektionsforschung GmbH | Medicament for liver regeneration and for treatment of liver failure |
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| WO2012136859A1 (en) * | 2011-04-07 | 2012-10-11 | Helmholtz-Zentrum für Infektionsforschung GmbH | Medicament for liver regeneration and for treatment of liver failure |
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| Title |
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| 李彦: "以iTＲAQ 技术分析金雀异黄酮作用大鼠肝星状细胞差异表达的蛋白", 《中国新药杂志》 * |
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Application publication date: 20200804 |