CN117018195B - Application of small molecular compound or combination in preparation of medicine for starting liver in-situ regeneration - Google Patents
Application of small molecular compound or combination in preparation of medicine for starting liver in-situ regeneration Download PDFInfo
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- CN117018195B CN117018195B CN202310975771.1A CN202310975771A CN117018195B CN 117018195 B CN117018195 B CN 117018195B CN 202310975771 A CN202310975771 A CN 202310975771A CN 117018195 B CN117018195 B CN 117018195B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
- A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
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- Engineering & Computer Science (AREA)
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- General Chemical & Material Sciences (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to the technical field of medicines, and provides an application of a small molecular compound or a combination in preparing a medicine for starting liver in-situ regeneration, wherein the small molecular compound is a TLR4 agonist, and the combination is a first combination formed by a prostaglandin E2 derivative and a GSK3 inhibitor or a second combination formed by the TLR4 agonist and the first combination. The method can effectively start the proliferation of normal liver in-situ cells, and the proportion of Ki67 positive proliferation cells is 11.05+/-1.65% and 17.41+/-2.53% respectively; animal disease model experimental results show that in a non-alcoholic fatty liver model mouse, the small molecular compound combination and the treatment of the TLR4 agonist promote the proliferation of in-situ cells in the liver of the non-alcoholic fatty liver model mouse, remarkably improve the liver weight ratio of the model mouse, reduce the proportion of hepatic stellate cells with steatosis and activation, and remarkably improve the tissue structure of the liver.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application of a TLR4 agonist or a prostaglandin E2 derivative and a GSK3 inhibitor combination in effectively starting liver in-situ regeneration.
Background
Liver disease is a significant health and economic burden worldwide, and as its incidence increases, liver disease has become one of the leading causes of disease and death worldwide. In various chronic liver diseases, as the liver disease progresses, liver cells gradually lose their strong regenerative ability and progress to death or aging, thereby causing liver fibrosis, which progresses to cirrhosis, and eventually may progress to liver cancer. When liver disease progresses to the end stage, the only effective treatment is liver transplantation, but immunosuppressants need to be taken throughout the life after transplantation, and donor organ starvation means that the patient may die before waiting for the appropriate transplanted organ. In clinic, a prerequisite for ensuring survival and a good prognosis for patients with partial hepatectomy and partial liver transplantation is liver regeneration. When the liver is not regenerated effectively after partial hepatectomy or partial liver transplantation, a small liver syndrome which seriously threatens the life safety of the patient is caused. In patients with acute and chronic liver injury caused by factors such as medicines, hepatotoxic substances and the like, effective proliferation of liver cells is also an important guarantee of good prognosis of the patients. Therefore, establishing an effective means to initiate liver regeneration or repair the damaged regeneration capacity of hepatocytes is of great clinical significance.
It is well known that liver has excellent regeneration capacity, and rodent two-thirds hepatectomy models demonstrate this powerful regeneration capacity for the first time. The majority of hepatocytes in normal adult liver are in G0 phase and cell division rarely occurs, however, after partial hepatectomy, about 95% of hepatocytes rapidly reenter the cell cycle and the remaining liver rapidly returns to its original size around 1 week. Liver regeneration is a multi-step, multi-factor, precise and orderly regulatory process involving a variety of signal interactions. Due to the complexity of liver regeneration processes, the means and mechanisms of regulation to initiate liver regeneration remain to be explored further.
In summary, in the field of liver regeneration research, it is urgently required to establish an accurate means to intervene in liver homeostasis so as to realize accurate and effective regulation and control of liver in-situ regeneration, and further realize treatment of liver regeneration disorder related diseases.
Disclosure of Invention
The invention aims at the problems that the liver regeneration means are limited and the mechanism is to be elucidated in the prior art, and provides an application of a small molecular compound or a combination in preparing a drug for starting liver in-situ regeneration.
The study selects 28 small molecular compounds and combinations capable of regulating and controlling biological behaviors such as liver regeneration start and liver regeneration stop, and the combination treatment of the TLR4 agonist or the prostaglandin E2 derivative and the GSK3 inhibitor can obviously promote the in-situ proliferation of liver cells through screening.
Wild type C57BL/6 mice were given either a TLR4 agonist treatment or a prostaglandin E2 derivative in combination with a GSK3 inhibitor, once every 24 hours, for 120 hours (i.e., 5 days) and after stopping the administration for 48 hours (i.e., 2 days), an effective onset of liver regeneration in situ was detected.
In vitro cell experiments show that the combination of the small molecular compound CRX-527 or dmPGE2 and CHIR99021 can effectively promote the in-situ proliferation of mouse liver cells, the method can effectively start the in-situ proliferation of liver cells, and the Ki67 positive proliferation cell proportion is 11.05% +/-1.65% and 17.41% +/-2.53% respectively; animal model experiment results show that in a non-alcoholic fatty liver model mouse, the small molecular compound composition dmC and the small molecular compound CR promote proliferation of in-situ cells in the liver of the non-alcoholic fatty liver model mouse, remarkably improve the liver weight ratio of the model mouse, reduce the proportion of hepatic stellate cells with steatosis and activation, and remarkably improve the tissue structure of the liver.
The specific technical scheme of the invention is as follows:
In a first aspect of the invention, there is provided the use of a small molecule compound or combination in the manufacture of a medicament for initiating liver regeneration in situ, wherein the small molecule compound is a TLR4 agonist; the combination is a first combination of a derivative of prostaglandin E2 and a GSK3 inhibitor or a second combination of a TLR4 agonist and the first combination.
In a second aspect of the invention, there is provided the use of a small molecule compound or combination which is a TLR4 agonist in the manufacture of a medicament for use in the treatment of a disease associated with liver dysgenesis, the combination being a first combination of a derivative of prostaglandin E2 and a GSK3 inhibitor or a second combination of a TLR4 agonist and the first combination. The liver regeneration disorder related diseases comprise liver regeneration deficiency or liver regeneration capacity loss and other diseases.
In both aspects, the TLR4 agonist is selected from small molecule agonists of TLR4, such as any one or more of lipopolysaccharide analogues of CRX-527, CRX-601, CRX-547, CRX-675, RS09, GSK1795091, etc.;
The derivative of prostaglandin E2 may be a small molecule derivative of prostaglandin E2, such as 16, 16-dimethylprostadine E2 (16, 16-DimethylProstaglandinE, dmPGE 2), KAG-308, butaprost, aganepag, CAY10580, enprostil, etc.; the GSK3 inhibitor may be a small molecule inhibitor of GSK3, such as CHIR99021, SB216763, LY2090314, tideglusib, BIO, TWS119, AZD2858, CHIR-98014, CP21R7, SB415286, etc.
Preferably, the drug is one that reduces steatosis in liver tissue, promotes in situ cell proliferation in the liver, increases liver weight ratio, or reduces the proportion of activated hepatic stellate cells. The medicine takes a combination of a TLR4 agonist or a prostaglandin E2 derivative and a GSK3 inhibitor as active components.
In a third aspect of the invention, there is provided a form of a pharmaceutical composition for initiating regeneration of the liver in situ comprising an active ingredient and a pharmaceutically acceptable adjuvant, wherein the active ingredient comprises a TLR4 agonist; another form of a pharmaceutical composition for initiating regeneration of the liver in situ comprises an active ingredient comprising a derivative of prostaglandin E2 and a GSK3 inhibitor, and pharmaceutically acceptable excipients.
Preferably, the liver is mammalian.
Effects and effects of the invention
The invention provides a new target for starting liver regeneration procedures, and researches show that the liver in-situ regeneration procedures can be started by combined administration of the derivative of activated TLR4 or prostaglandin E2 and the GSK3 inhibitor, so that the in-situ proliferation of cells in the liver can be promoted by interfering TLR4 and downstream signaling paths or interfering related signaling paths of the derivative of prostaglandin E2 and the GSK3 inhibitor. Such interventions include, but are not limited to, the use of TLR4 agonists or the use of prostaglandin E2 derivatives in combination with GSK3 inhibitors to modulate related signaling pathways by genetic means. Therefore, the invention provides a new idea for the start of in-situ liver regeneration and a new hint for the treatment of diseases related to liver regeneration disorder.
Drawings
FIG. 1 shows the Ki67 immunofluorescence staining results of mouse liver tissue from the experimental group (CR and dmC 2) and the control group. (A) Immunofluorescent staining of (C), (E) cell proliferation index Ki 67; (B) (D) and (F) are combined images of immunofluorescent staining of Ki67 and DAPI immunofluorescent staining of nuclei of the experimental and control groups respectively (Merged); (G) And (H) a statistical plot of the proportion of Ki67 immunofluorescent-stained positive cells for CR and dmC2 treatments versus the corresponding control group; vehicle is an equivalent volume solvent treated control group; CR is the experimental group treated by small molecule compound CR; dmC2 is the experimental group treated with the small molecule compound combination dmC. The results show that either CR treatment or dmC treatment can initiate an in situ cell proliferation procedure in the mouse liver.
FIG. 2 shows the Ki67 immunofluorescent staining results of liver tissue of mice in the experimental (dmC and CR) and control groups after an optimized dosing time of 5 days (120 hours). (A) Immunofluorescent staining of (C), (E) cell proliferation index Ki 67; (B) (D) and (F) are combined images of immunofluorescent staining of Ki67 and DAPI immunofluorescent staining of nuclei of the experimental and control groups respectively (Merged); (G) A statistical chart of the proportion of the small molecule compound combination dmC days treated with the corresponding control group Ki67 immunofluorescence staining positive cells; (H) A statistical plot of the proportion of Ki67 immunofluorescent staining positive cells in both treatment groups administered for 2 days and 5 days for small molecule compound combination dmC; (I) A statistical chart of the proportion of the small molecule compound CR treated for 5 days and the corresponding control group Ki67 immunofluorescence staining positive cells; j is a statistical graph of the proportion of Ki67 immunofluorescence staining positive cells under the treatment of the small molecule compound CR treatment group after 2 days and 5 days; vehicle is an equivalent volume solvent treated control group; dmC2 is the experimental group treated with the small molecule compound combination dmC; CR is the experimental group of small molecule compound CR treatment. The results showed that the proportion of proliferating cells in both dmC day and CR treated groups was significantly higher than that in the control group, there was no statistical difference in the proportion of proliferating cells in CR treated groups 5 days relative to the proportion of proliferating cells in CR treated groups 2 days, while the proportion of proliferating cells in dmC treated groups 5 days relative to the proportion of proliferating cells in CR treated groups 2 days was increased.
FIG. 3 is a graph showing the appearance of the liver of mice in the experimental group (dmC 2) and the control group and a statistical graph of the weight ratio of the liver. (A), (B), (C) and (D) are external views of mouse liver, and (E) is a statistical graph of the weight ratio of liver weight corresponding to the treatment. Vehicle is an equivalent volume solvent treated control group; dmC2 is an experiment of the small molecule compound combination dmC2 treatment; 2day represents a group of 2 days of small molecule compound combination treatment; 5day represents the group treated with the small molecule compound combination for 5 days. The results showed that there was no statistical difference between the liver weights of the dmC day group treated with 2 and the control group, and that the liver weight ratio of the dmC2 combination treated with 5 days was significantly higher than that of the control group.
FIG. 4 is a graph showing the appearance of liver of mice in experimental (CR) and control groups and a statistical graph of the weight ratio of liver. (A), (B), (C) and (D) are external views of mouse liver, and (E) is a statistical graph of the weight ratio of liver weight corresponding to the treatment. Vehicle is an equivalent volume solvent treated control group; CR is the experimental group treated by small molecule compound CR; 2day represents the group of 2 days of treatment with small molecule compounds; 5day represents the group treated with small molecule compounds for 5 days. The results showed that the liver weight ratio of the group treated for 2 days was significantly higher than that of the control group, but there was no statistical difference, and the liver weight ratio of the group treated for 5 days with the small molecule compound CR was significantly higher than that of the control group.
FIG. 5 shows immunofluorescent staining results of small molecule compounds and liver tissue of mice in the combined experimental and control groups. (A) Immunofluorescent staining of the (D), (G) cell mitotic marker PH 3; (B) Immunofluorescent staining of (E) and (H) cell proliferation indexes Ki 67; (C) (F), (I) are combined images of immunofluorescent staining of PH3 and Ki67 immunofluorescent staining of each of the experimental and control groups (Merged); vehicle is an equal volume solvent treated control group (5 days); dmC2 is the experimental group treated with the small molecule compound combination dmC2 (5 days); CR is the group of small molecule compound CR treatment (5 days). Experimental results show that dmC treatment with CR initiates the procedure for in situ cell proliferation of the liver.
FIG. 6 is a graph showing the results of H & E staining, oil red O staining and Marsonian staining (Massontrichromestaining) of liver tissue of mice model in a combination of small molecule compound dmC and control. (A) (B) H & E staining of liver tissue of mice model of experimental and control groups; (C) (D) oil red O staining which is an index for detecting lipid in liver tissue of mice in experimental group and control group models; (E) (F) masson staining of liver tissue of mice in experimental and control groups; vehicle is an equivalent volume solvent treated control group; dmC2 is the experimental group treated with the small molecule compound combination dmC. The results show that the treatment of the small molecule compound combination dmC significantly relieves the steatosis of liver tissue and improves the tissue structure of the liver.
FIG. 7 shows the results of the combined experimental and control group models of small molecule compound dmC, mouse liver tissue alpha-SMA immunohistochemical staining, ki67 immunohistochemical staining, and liver profile and liver weight ratio statistics. (A) (B) immunohistochemical staining of mouse liver tissue α -SMA in experimental and control groups; (C) (D) Ki67 immunohistochemical staining of mouse liver tissue for experimental and control groups; (E) (F) is the appearance diagram of the liver of the mice in the experimental group and the control group; (G) is a statistical plot of the ratio of liver weight to body weight of mice; vehicle is an equivalent volume solvent treated control group; dmC2 is the experimental group treated with the small molecule compound combination dmC. The results show that the treatment of the small molecular compound composition dmC promotes the proliferation of in-situ cells in the liver of a non-alcoholic fatty liver mouse model, remarkably improves the liver weight ratio of the model mouse and reduces the proportion of activated hepatic stellate cells.
FIG. 8 shows the results of H & E staining, oil red O staining and Pinus massoniana staining of mouse liver tissue in the small molecule compound CR experimental group and the control group model. (A) (B) H & E staining of liver tissue of mice model of experimental and control groups; (C) (D) oil red O staining which is an index for detecting lipid in liver tissue of mice in experimental group and control group models; (E) (F) masson staining of liver tissue of mice in experimental and control groups; vehicle is an equivalent volume solvent treated control group; CR is the experimental group of small molecule compound CR treatment. The results show that the treatment of small molecule compound CR significantly improves the tissue structure and steatosis of non-alcoholic fatty liver model mice.
FIG. 9 shows the immunohistochemical staining of small molecule compound CR experimental group and control group model mouse liver tissue alpha-SMA, the immunohistochemical staining of Ki67, the appearance of liver and the statistical graph of liver weight ratio. (A) (B) immunohistochemical staining of mouse liver tissue α -SMA in experimental and control groups; (C) (D) Ki67 immunohistochemical staining of mouse liver tissue for experimental and control groups; (E) (F) is the appearance diagram of the liver of the mice in the experimental group and the control group; (G) is a statistical plot of the ratio of liver weight to body weight of mice; vehicle is an equivalent volume solvent treated control group; CR is the experimental group of small molecule compound CR treatment. The results show that the treatment of the small molecular compound CR obviously reduces the proportion of activated hepatic stellate cells in the liver of the model mouse, and improves the proportion of in-situ proliferation cells in the liver of the model mouse and the liver weight ratio of the model mouse.
Detailed Description
The present invention will be described in detail with reference to the following examples and the accompanying drawings. The following examples should not be construed as limiting the scope of the invention.
The reagents and starting materials used in the present invention are commercially available or may be prepared by literature procedures. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
Example 1: the combined administration of a TLR4 agonist or a prostaglandin E2 derivative and a GSK3 inhibitor can initiate liver in situ regeneration
1. Small molecule compounds and combinations (CR and dmC 2) for promoting in situ proliferation of liver cells
The study selects 28 small molecular compounds and combinations capable of regulating and controlling biological behaviors such as liver regeneration start and liver regeneration stop, and the combination treatment of the TLR4 agonist or the prostaglandin E2 derivative and the GSK3 inhibitor can obviously promote the in-situ proliferation of liver cells through screening. C57BL/6 wild-type mice were given intraperitoneal injections of the TLR4 agonist small molecule compound CRX-527 (treatment indicated as CR group) or a combination of the prostaglandin E2 derivative dmPGE2 and the GSK3 inhibitor CHIR99021 (treatment indicated as dmC group), control groups (Vehicle) were given intraperitoneal injections of an equal volume of solvent (all compounds diluted in 10% 2-hydroxypropyl-beta-cyclodextrin), once every 24 hours, for 48 hours, after stopping the administration for 48 hours, the mice were euthanized, the livers of the mice were harvested, and Ki67 immunofluorescence staining was performed. The ratio of Ki67 positive proliferating hepatocytes was found to be 12.66% ± 2.94% and 9.62% ± 2.36% by proliferating index Ki67 immunofluorescence staining for CR-treated and dmC 2-treated groups, respectively, significantly higher than control group (p < 0.01) and Ki67 positive cell staining was localized to the nuclei (fig. 1A-1H). In conclusion, the combination of small molecule compound CRX-527 or dmPGE2 and CHIR99021 can effectively promote in situ proliferation of mouse liver cells.
2. Optimization of administration time of small molecule compounds and combinations (CR and dmC 2)
Based on the prior study, the optimal administration time length is 120 hours, the administration is carried out once every 24 hours, the mice are killed after 48 hours of stopping the administration, and the livers of the mice are harvested for Ki67 immunofluorescence staining. The proportion of Ki67 positive cells in the liver of mice in the group 5 days from treatment with the small molecule compound combination dmC was significantly increased (fig. 2H) (< 0.05) compared to the liver of mice in the group 2 days from treatment with the small molecule compound combination dmC, and the proportion of Ki67 positive cells in the liver of mice in the group 5 days from treatment with the small molecule compound combination dmC was 17.41% ± 2.53% (fig. 2C, 2D), which was also significantly higher (0.63% ± 0.15%) than the proportion of Ki67 positive cells in the liver of mice in the control group (fig. 2G) (< 0.01); while the ratio of Ki67 positive cells in the liver of mice in the 5-day group to which the small molecule compound CR was administered was somewhat reduced compared to the 2-day group to which the small molecule compound CR was administered, but there was no statistical difference (fig. 2J) (ns, p > 0.05), the ratio of Ki67 positive cells in the liver of mice in the 5-day group to which the small molecule compound CR was administered was 11.05% ± 1.65% (fig. 2E, 2F), significantly higher than the ratio of Ki67 positive cells in the liver of mice in the control group (0.50% ± 0.15%) (fig. 2I) (. P < 0.01), similar to the other three groups of small molecule compounds.
The results show that the positive proportion of proliferation cells of the group treated by the small molecular compound CR is reduced by a certain proportion relative to the group treated by 2 days, but the small molecular compound CR has no statistical difference, that is, continuous administration does not lead to the in-situ cell proliferation of the liver of the mice, but is controlled to fluctuate within a certain range; the small molecule compound composition dmC days of treatment has a certain proportion of increase of the positive proportion of the proliferation cells relative to the group treated for 2 days, namely, the positive proportion of the proliferation cells in the liver of the mice can be continuously increased after dmC groups are continuously dosed for 5 days.
3. Liver regeneration index detection
To further confirm the proliferation of liver cells, the present study counted the ratio of liver weights of mice in the experimental and control groups and examined the expression of mitotic markers in the livers of mice.
Statistical analysis of liver weight ratios for 2 days and 5 days for small molecule compounds and combinations (dmC and CR) of the experimental and control groups found: small molecule compound dmC2 combined treatment for 2 days had a liver weight ratio of 4.61% ± 0.31% for 2 days, although higher than the control (fig. 3A, 3B), but no statistical difference (fig. 3E) (ns, p > 0.05); the dmC day combined treatment had a liver weight ratio of 5.79% ± 0.33% significantly higher than the control (fig. 3C, 3D, 3E) (×p < 0.01). Small molecule compound CR was similar to small molecule compound dmC in combination with a2 day group liver weight ratio of 4.27% ± 0.04% for 2 days treatment, although higher than control (fig. 4A, 4B), but without statistical differences (fig. 4E) (ns, p > 0.05); the liver weight ratio of the small molecule compound CR treated for 5 days was 5.72% ± 0.64% significantly higher than that of the control group (fig. 4C, 4D, 4E) (×p < 0.01).
Proliferation index Ki67 is detectable in all active phases (G1, S, G2, M) of the cell cycle, and is undetectable only in stationary phase (G0), whereas phosphorylation modification of histone H3 occurs only in mitosis, and therefore phosphorylated histone H3 (Phospho-HistoneH, PH 3) is a marker of cell mitosis. By immunofluorescent staining of PH3 and Ki67, it can be seen that the control group had only sporadic PH3 and Ki67 positive cells, and PH3 and Ki67 positive cells were co-localized to the same nucleus (FIGS. 5A, 5B, 5C); consistent with immunofluorescence staining results of proliferation index Ki67, the proportion of cells mitotic marker PH3 positive cells in the liver of dmC and CR treated mice was significantly higher than control group (p < 0.01), and most of PH3 positive cells could co-localize to the same nucleus as Ki67 positive cells, and a small fraction did not localize to the same nucleus, possibly due to cells in different cell cycles. It was further confirmed by co-staining with PH3 and Ki67 that dmC2 and CR initiated the procedure for liver in situ cell proliferation (FIGS. 5D-5I).
In summary, the ratio of the cell proliferation index Ki67 positive cells in the livers of mice treated with dmC days and mice treated with dmC days by adopting the CR is significantly higher than that of the control group, wherein the ratio of the cell proliferation index Ki67 positive cells in the livers of mice treated with 5 days by adopting the small molecule compound combination dmC2 is significantly higher than that of the mice treated with 2 days; at the same time, the liver weight ratio of mice in dmC days of CR treatment is obviously higher than that of mice in a control group. Consistent with immunofluorescence staining results of proliferation index Ki67, the proportion of cells positive for the mitotic marker PH3 in the liver of dmC and CR-treated mice was significantly higher than that of the control group. The result of the comprehensive proliferation index Ki67 immunofluorescence, the result of the liver weight ratio statistics and the result of the cell mitosis marker PH3 immunofluorescence can be seen that dmC and CR can effectively promote the proliferation of the mouse liver in-situ cells, and the liver regeneration procedure is started.
Example 2: small molecule compounds and their therapeutic effects in model of aplastic non-alcoholic fatty liver disease
1. Efficacy of small molecule compound combination dmC2 in non-alcoholic fatty liver disease model
Wild type C57BL/6 mice were given a diet low in methionine and choline deficient in high fat (RESEARCHDIET, a 0607130) for 6 weeks for modeling of non-alcoholic steatohepatitis mice, and after 6 weeks were randomized into two groups of 6 mice each, the experimental group was given a small molecule compound combination dmC2 treatment for 5 days, the control group was given a small molecule compound solvent (Vehicle) treatment for 5 days, after stopping the treatment for 2 days, the mice were sacrificed from dislocation and the liver and serum of the mice were harvested for subsequent analysis. H & E staining results show that the liver tissue of the non-alcoholic fatty liver model mouse in the control group has massive inflammatory cell infiltration and cavitation degeneration (figure 6A), while the liver tissue of the non-alcoholic fatty liver model mouse in the experimental group treated by the small molecular compound composition dmC2 has small cavitation and obviously reduced cavitation degeneration quantity (figure 6B); lipid detection index oil red O staining results showed that the treatment group of small molecule compound combination dmC2 significantly reduced the size and number of lipid droplets in the liver and the steatosis and tissue structure of the liver were improved relative to the control group (fig. 6A, 6B, 6C, 6D); the results of the masson staining showed that no significant collagen fiber deposition was seen in liver tissue of the non-alcoholic fatty liver model mice of both the experimental and control groups (fig. 6E, 6F). The extracellular matrix in liver fibrosis is mainly derived from activated hepatic stellate cells, so that the research detects hepatic stellate cell activation markers alpha-SMA, and an alpha-SMA immunohistochemical result shows that the area of an alpha-SMA positive area of a small molecular compound combination dmC2 treatment group is 2.08% +/-0.46%, which is significantly lower than that of an alpha-SMA positive area of a control group (5.06% +/-0.52%), namely that the expression of alpha-SMA is significantly reduced by the treatment of the small molecular compound combination dmC2 (shown in fig. 7A and 7B) (. P < 0.01); next, the expression of the cell proliferation index Ki67 of the small molecule compound combination dmC and the control group was detected in the present study, and it can be seen by Ki67 immunohistochemical staining that the Ki67 positive cell proportion (9.98% ± 1.55%) of the small molecule compound combination dmC2 is significantly higher than that of the control group (2.92% ± 0.67%) (fig. 7C, 7D) (. P < 0.01), i.e. the treatment of the small molecule compound combination dmC2 initiated the proliferation procedure of in situ cells in the liver in the non-alcoholic fatty liver model mice; meanwhile, the liver weight ratio of a non-alcoholic fatty liver model mouse experimental group treated by the small molecular compound composition dmC and a control group is detected, and the liver weight ratio result shows that compared with the control group, the liver weight ratio of the mouse experimental group treated by the small molecular compound composition dmC is obviously improved (figures 7E, 7F and 7G) (. P is less than 0.05).
In summary, in the non-alcoholic fatty liver model mouse, the treatment of the small molecular compound composition dmC promotes the proliferation of in-situ cells in the liver of the non-alcoholic fatty liver model mouse, remarkably improves the liver weight ratio of the model mouse, reduces the proportion of hepatic stellate cells with steatosis and activation, and remarkably improves the tissue structure of the liver.
2. Therapeutic effect of small molecule compound CR in non-alcoholic fatty liver disease model
Non-alcoholic fatty liver model mice were prepared by the above method (low methionine and choline deficient high fat diet method), and after successful modeling, the mice were randomly divided into two groups of 6 mice each, and the mice in experimental group and control group were respectively given a small molecule compound CR treatment and a control solvent treatment, and after the treatment was completed, the mice were dislocation-killed, and the serum and liver tissues of the mice were collected for subsequent study. Similar to the treatment results of small molecule compound combination dmC2, the H & E staining results of small molecule compound CR treated mice liver tissue showed a significant improvement in inflammatory cell infiltration and vacuolation present in the control group (fig. 8A, 8B), the lipid detection index oil red O staining results showed that the small molecule compound CR treated group significantly reduced the size and number of lipid droplets in the liver, and the liver steatosis was improved (fig. 8A, 8B, 8C, 8D); no significant collagen fiber deposition was seen in liver tissue of non-alcoholic fatty liver model mice of the same masson staining results, experimental and control groups (fig. 8E, 8F). The detection result of the hepatic stellate cell activation marker alpha-SMA shows that the area of the small molecular compound CR treated group alpha-SMA positive area is 2.01% +/-0.47% which is obviously lower than that of the control group alpha-SMA positive area (4.94% +/-0.47%), namely the treatment of the small molecular compound CR obviously reduces the positive area (figures 9A and 9B) (. Times.p < 0.01); next, the present study examined the effect of small molecule CR on the expression of Ki67, a cell proliferation index in the liver of a non-alcoholic fatty liver model mouse, and the ratio of Ki67 positive cells relative to control group, as seen in the Ki67 immunohistochemical staining
(2.70% ± 0.44%), The proportion of Ki67 positive cells in liver tissue of mice of the experimental group treated with small molecule compound CR (13.75% ± 0.95%) was significantly increased (fig. 9C, 9D) (. P < 0.01), i.e. the treatment with small molecule compound CR promoted proliferation of cells in situ in liver of mice of the non-alcoholic fatty liver model; meanwhile, the study explores the influence of the treatment of the small molecular compound CR on the liver weight ratio of a non-alcoholic fatty liver model mouse, and the treatment of the small molecular compound CR obviously improves the liver weight ratio of the model mouse (figures 9E, 9F and 9G) (. Times.p < 0.01).
In conclusion, the treatment of the small molecular compound CR obviously improves the proportion of in-situ proliferation cells in the liver of the model mouse and the liver weight ratio of the model mouse, reduces the proportion of activated hepatic stellate cells in the liver of the model mouse, and improves the tissue structure and steatosis of the non-alcoholic fatty liver model mouse.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. The application of a small molecular compound in preparing a drug for activating liver in-situ regeneration is characterized in that the small molecular compound is a TLR4 agonist, and the TLR4 agonist is selected from any one or more of CRX-527, CRX-601, CRX-547 and CRX-675.
2. The application of a small molecular compound in preparing medicaments for liver regeneration disorder related diseases is characterized in that the small molecular compound is a TLR4 agonist, the TLR4 agonist is selected from any one or more of CRX-527, CRX-601, CRX-547 and CRX-675,
Wherein the liver regeneration disorder related disease is non-alcoholic fatty liver.
3. Use according to claim 1 or 2, characterized in that:
wherein the medicament takes the TLR4 agonist as an active component.
4. The use according to claim 1, characterized in that:
wherein the medicine is a medicine for promoting in-situ cell proliferation in liver or improving liver weight ratio.
5. The use according to claim 2, characterized in that:
Wherein the medicine is used for reducing the steatosis of liver tissues, promoting the proliferation of in-situ cells in the liver, improving the liver weight ratio or reducing the proportion of activated hepatic stellate cells.
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| CN116509898A (en) * | 2022-11-29 | 2023-08-01 | 中国人民解放军空军军医大学 | Application of liver c-kit positive cells in preparation of medicines for treating liver diseases |
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| CN118453877A (en) | 2024-08-09 |
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