CN117018195B - Application of small molecular compound or combination in preparation of medicine for starting liver in-situ regeneration - Google Patents

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

Application of small molecule compounds or combinations in the preparation of drugs for initiating liver regeneration in situ

Technical Field

The present invention belongs to the field of biomedicine, and specifically relates to the application of a combination of a TLR4 agonist or a prostaglandin E2 derivative and a GSK3 inhibitor in effectively initiating in situ liver regeneration.

Background technique

Liver disease is a major health and economic burden worldwide. With its increasing incidence, liver disease has become one of the leading causes of disease and death worldwide. In various chronic liver diseases, as liver disease progresses, hepatocytes gradually lose their strong regenerative ability and die or age, leading to liver fibrosis, which develops into cirrhosis and may eventually develop into liver cancer. When liver disease progresses to the terminal stage, the only effective treatment is liver transplantation, but lifelong immunosuppressants are required after transplantation, and the shortage of donor organs means that patients may die before waiting for a suitable transplant organ. In clinical practice, liver regeneration is a prerequisite for ensuring the survival and good prognosis of patients undergoing partial liver resection and partial liver transplantation. When the liver cannot regenerate effectively after partial liver resection or partial liver transplantation, it will cause small liver syndrome, which seriously threatens the life safety of patients. In patients with acute and chronic liver damage caused by factors such as drugs and hepatotoxic substances, the effective proliferation of hepatocytes is also an important guarantee for the good prognosis of patients. Therefore, it is of great clinical significance to establish effective means to initiate liver regeneration or repair the damaged regenerative ability of hepatocytes.

It is well known that the liver has excellent regenerative capacity. The rodent two-thirds liver resection model demonstrated this powerful regenerative ability for the first time. Most of the hepatocytes in the normal adult liver are in the G0 phase, and cell division rarely occurs. However, after partial hepatectomy, about 95% of the hepatocytes quickly re-enter the cell cycle, and the remaining liver quickly recovers to its original size in about 1 week. Liver regeneration is a multi-step, multi-factor, precise and orderly regulatory process involving multiple signal interactions. Due to the complexity of the liver regeneration process, the regulatory means and mechanisms for initiating liver regeneration still need to be further explored.

In summary, it can be seen that in the field of liver regeneration research, there is an urgent need to establish precise means to intervene in liver homeostasis in order to achieve precise and effective regulation of liver in situ regeneration, and then achieve the treatment of diseases related to liver regenerative disorders.

Summary of the invention

The present invention is directed to the above-mentioned problems, and in view of the limited means of liver regeneration and the yet to be elucidated mechanism in the prior art, provides an application of a small molecule compound or a combination in the preparation of a drug for initiating liver regeneration in situ.

This study selected a total of 28 small molecule compounds and combinations that can regulate biological behaviors such as the initiation of liver regeneration and the inhibition of liver regeneration termination. Through screening, it was found that the combined treatment of TLR4 agonists or prostaglandin E2 derivatives and GSK3 inhibitors can significantly promote the in situ proliferation of liver cells.

Wild-type C57BL/6 mice were intraperitoneally treated with TLR4 agonists or a combination of prostaglandin E2 derivatives and GSK3 inhibitors once every 24 hours for 120 hours (i.e., 5 days). 48 hours after stopping the drug (i.e., 2 days), effective initiation of liver in situ regeneration was detected.

In vitro cell experiments showed that the small molecule compound CRX-527 or the combination of dmPGE2 and CHIR99021 can effectively promote the in situ proliferation of mouse liver cells. The above method can effectively initiate the proliferation of in situ liver cells, and the proportions of Ki67-positive proliferating cells were 11.05%±1.65% and 17.41%±2.53%, respectively; the results of animal model experiments showed that in non-alcoholic fatty liver model mice, the treatment of the small molecule compound combination dmC2 and the small molecule compound CR promoted the proliferation of in situ cells in the liver of the non-alcoholic fatty liver mouse model, significantly increased the liver weight ratio of the model mice, reduced the proportion of fatty degeneration and activated hepatic stellate cells, and significantly improved the tissue structure of the liver.

The specific technical solutions of the present invention are as follows:

The first aspect of the present invention provides an application of a small molecule compound or a combination in the preparation of a drug for initiating in situ liver regeneration, wherein the small molecule compound is a TLR4 agonist; the combination is a first combination formed by a prostaglandin E2 derivative and a GSK3 inhibitor, or a second combination formed by a TLR4 agonist and the first combination.

In a second aspect of the present invention, a small molecule compound or a combination is provided for use in preparing a drug for a disease related to liver regeneration disorder, wherein the small molecule 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 a TLR4 agonist and the first combination. The disease related to liver regeneration disorder includes diseases such as insufficient liver regeneration or loss of liver regeneration ability.

In the above two aspects, the TLR4 agonist is selected from small molecule agonists of TLR4, such as any one or more of lipopolysaccharide analogs such as CRX-527, CRX-601, CRX-547, CRX-675, RS09, and GSK1795091;

The derivative of prostaglandin E2 can be a small molecule derivative of prostaglandin E2, such as 16,16-dimethylprostaglandinE2 (dmPGE2), KAG-308, Butaprost, Aganepag, CAY10580, Enprostil, etc.; the GSK3 inhibitor can 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 a drug that reduces fatty degeneration of liver tissue, promotes in situ cell proliferation in the liver, increases liver weight ratio or reduces the proportion of activated hepatic stellate cells. The drug has a combination of a TLR4 agonist or a prostaglandin E2 derivative and a GSK3 inhibitor as active ingredients.

In a third aspect, the present invention provides a form of a pharmaceutical composition for initiating in situ liver regeneration, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises a TLR4 agonist; another form of a pharmaceutical composition for initiating in situ liver regeneration, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises a prostaglandin E2 derivative and a GSK3 inhibitor.

Preferably, the liver is a mammalian liver.

Functions and Effects of the Invention

The present invention provides a new target for initiating liver regeneration program. Studies have shown that the in situ liver regeneration program can be initiated by activating TLR4 or administering a combination of a prostaglandin E2 derivative and a GSK3 inhibitor. Therefore, intervention in TLR4 and downstream signaling pathways or intervention in the related signaling pathways of a combination of a prostaglandin E2 derivative and a GSK3 inhibitor can promote the in situ proliferation of cells in the liver. These intervention methods include but are not limited to the use of a TLR4 agonist or a combination of a prostaglandin E2 derivative and a GSK3 inhibitor, and regulating the related signaling pathways by genetic means. Therefore, the present invention provides a new idea for initiating in situ liver regeneration and provides new inspiration for the treatment of diseases related to liver regeneration disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of Ki67 immunofluorescence staining of mouse liver tissue in the experimental group (CR and dmC2) and the control group. (A), (C), (E) Immunofluorescence staining of Ki67, a cell proliferation indicator; (B), (D), (F) are merged images of Ki67 immunofluorescence staining and DAPI immunofluorescence staining of the cell nucleus in the experimental group and the control group; (G) and (H) are statistical graphs of the proportion of Ki67 immunofluorescence staining positive cells in CR and dmC2 treatment and the corresponding control group; Vehicle is the control group treated with equal volume solvent; CR is the experimental group treated with small molecule compound CR; dmC2 is the experimental group treated with small molecule compound combined with dmC2. The results show that CR treatment or dmC2 treatment can initiate the in situ cell proliferation program in mouse liver.

Figure 2 shows the results of Ki67 immunofluorescence staining of liver tissues of mice in the experimental group (dmC2 and CR) and the control group after the optimized administration time was 5 days (120 hours). (A), (C), (E) Immunofluorescence staining of cell proliferation index Ki67; (B), (D), (F) are merged images of Ki67 immunofluorescence staining and DAPI immunofluorescence staining of cell nucleus in the experimental group and the control group respectively; (G) is a statistical graph of the proportion of Ki67 immunofluorescence positive cells in the small molecule compound combination dmC2 treated for 5 days and the corresponding control group; (H) is a statistical graph of the proportion of Ki67 immunofluorescence positive cells in the small molecule compound combination dmC2 treated group under the treatment of 2 days and 5 days; (I) is a statistical graph of the proportion of Ki67 immunofluorescence positive cells in the small molecule compound CR treated group under the treatment of 2 days and 5 days; J is a statistical graph of the proportion of Ki67 immunofluorescence positive cells in the small molecule compound CR treated group under the treatment of 2 days and 5 days; Vehicle is the control group treated with equal volume solvent; dmC2 is the experimental group treated with the small molecule compound combination dmC2; CR is the experimental group treated with the small molecule compound CR. The results showed that the proportion of proliferative cells in the 5-day treatment groups of dmC2 and CR was significantly higher than that in the control group. There was no statistical difference in the positive proportion of proliferative cells in the 5-day treatment group of CR compared with that in the 2-day treatment group, while the positive proportion of proliferative cells in the 5-day treatment group of dmC2 increased to a certain extent compared with that in the 2-day treatment group.

Figure 3 shows the appearance of the liver of mice in the experimental group (dmC2) and the control group, and the statistical graph of the liver weight to body weight ratio. (A), (B), (C), and (D) are the appearance of the mouse liver, and (E) is the statistical graph of the liver weight to body weight ratio of the corresponding treatment. Vehicle is the control group treated with an equal volume of solvent; dmC2 is the experiment treated with a small molecule compound combined with dmC2; 2day represents the group treated with a small molecule compound combination for 2 days; 5day represents the group treated with a small molecule compound combination for 5 days. The results showed that there was no statistical difference in liver weight between the dmC2 treatment group for 2 days and the control group, and the liver weight ratio of the dmC2 combination treatment group for 5 days was significantly higher than that of the control group.

Figure 4 shows the appearance of the liver of mice in the experimental group (CR) and the control group, and the statistical graph of the liver weight to body weight ratio. (A), (B), (C), and (D) are the appearance of the mouse liver, and (E) is the statistical graph of the liver weight to body weight ratio of the corresponding treatment. Vehicle is the control group treated with an equal volume of solvent; CR is the experimental group treated with small molecule compounds CR; 2day represents the group treated with small molecule compounds for 2 days; 5day represents the group treated with small molecule compounds for 5 days. The results showed that although the liver weight ratio of the group treated for 2 days was higher than that of the control group, there was no statistical difference, and the liver weight ratio of the group treated with small molecule compounds CR for 5 days was significantly higher than that of the control group.

Figure 5 shows the results of immunofluorescence staining of liver tissue of mice in the experimental group and the control group of small molecule compounds and combinations. (A), (D), (G) Immunofluorescence staining of PH3, a marker of cell mitosis; (B), (E), (H) Immunofluorescence staining of Ki67, an indicator of cell proliferation; (C), (F), (I) are merged images of immunofluorescence staining of PH3 and Ki67 in the experimental group and the control group; Vehicle is the control group treated with an equal volume of solvent (5 days); dmC2 is the experimental group treated with the small molecule compound combination dmC2 (5 days); CR is the experimental group treated with the small molecule compound CR (5 days). The experimental results show that the treatment of dmC2 and CR initiated the process of in situ cell proliferation in the liver.

Figure 6 shows the results of H&E staining, Oil Red O staining and Masson trichrome staining of liver tissues of model mice in the experimental group and control group of the small molecule compound dmC2 combination. (A) and (B) are H&E staining of liver tissues of model mice in the experimental group and control group; (C) and (D) are Oil Red O staining of lipid detection indicators of liver tissues of model mice in the experimental group and control group; (E) and (F) are Masson trichrome staining of liver tissues of model mice in the experimental group and control group; Vehicle is the control group treated with equal volume solvent; dmC2 is the experimental group treated with the small molecule compound combination dmC2. The results show that the treatment of the small molecule compound combination dmC2 significantly alleviated the fatty degeneration of liver tissue and improved the tissue structure of the liver.

Figure 7 shows the α-SMA immunohistochemical staining, Ki67 immunohistochemical staining, liver appearance and liver weight ratio of the model mice in the experimental group and control group of the small molecule compound dmC2 combination. (A) and (B) are α-SMA immunohistochemical staining of the liver tissue of the experimental group and the control group of mice; (C) and (D) are Ki67 immunohistochemical staining of the liver tissue of the experimental group and the control group of mice; (E) and (F) are the appearance of the liver of the experimental group and the control group of mice; (G) is a statistical chart of the ratio of mouse liver weight to body weight; Vehicle is the control group treated with an equal volume of solvent; dmC2 is the experimental group treated with the small molecule compound combination dmC2. The results showed that the treatment of the small molecule compound combination dmC2 promoted the proliferation of in situ cells in the liver of the non-alcoholic fatty liver mouse model, significantly increased the liver weight ratio of the model mice, and reduced the proportion of activated hepatic stellate cells.

Figure 8 Results of H&E staining, Oil Red O staining and Masson staining of liver tissues of model mice in the experimental group and control group of small molecule compound CR. (A) and (B) are H&E staining of liver tissues of model mice in the experimental group and control group; (C) and (D) are Oil Red O staining of lipid detection indicators of liver tissues of model mice in the experimental group and control group; (E) and (F) are Masson staining of liver tissues of model mice in the experimental group and control group; Vehicle is the control group treated with equal volume of solvent; CR is the experimental group treated with small molecule compound CR. The results show that the treatment of small molecule compound CR significantly improved the tissue structure and fatty degeneration of non-alcoholic fatty liver model mice.

Figure 9 shows the α-SMA immunohistochemical staining, Ki67 immunohistochemical staining, liver appearance and liver weight ratio of model mice in the experimental group and control group of small molecule compound CR. (A) and (B) are α-SMA immunohistochemical staining of liver tissue of mice in the experimental group and control group; (C) and (D) are Ki67 immunohistochemical staining of liver tissue of mice in the experimental group and control group; (E) and (F) are appearance of the liver of mice in the experimental group and control group; (G) is a statistical chart of the ratio of mouse liver weight to body weight; Vehicle is the control group treated with equal volume solvent; CR is the experimental group treated with small molecule compound CR. The results showed that the treatment of small molecule compound CR significantly reduced the proportion of activated hepatic stellate cells in the liver of model mice, increased the proportion of in situ proliferating cells in the liver of model mice and the liver weight ratio of model mice.

Detailed ways

The present invention is described in detail below in conjunction with the embodiments and drawings. However, the following embodiments should not be considered as limiting the scope of the present invention.

The reagents and raw materials used in the present invention are all commercially available or can be prepared according to literature methods. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

Example 1: TLR4 agonists or prostaglandin E2 derivatives combined with GSK3 inhibitors can initiate liver regeneration in situ

1. Small molecule compounds and combinations (CR and dmC2) promote in situ proliferation of liver cells

This study selected a total of 28 small molecule compounds and combinations that can regulate biological behaviors such as the initiation of liver regeneration and the inhibition of liver regeneration termination. Through screening, it was found that the combination of TLR4 agonists or prostaglandin E2 derivatives and GSK3 inhibitors can significantly promote the proliferation of liver cells in situ. C57BL/6 wild-type mice were intraperitoneally injected with TLR4 agonist small molecule compound CRX-527 (this treatment was recorded as CR group) or the combination of prostaglandin E2 derivative dmPGE2 and GSK3 inhibitor CHIR99021 (this treatment was recorded as dmC2 group), and the control group (Vehicle) was intraperitoneally injected with an equal volume of solvent (all compounds were diluted in 10% 2-hydroxypropyl-β-cyclodextrin). The drug was administered once every 24 hours for 48 hours. After stopping the drug administration for 48 hours, the mice were killed by dislocation, and the mouse livers were collected for Ki67 immunofluorescence staining. By immunofluorescence staining of the proliferation index Ki67, it was found that the proportion of Ki67-positive proliferating hepatocytes in the CR-treated experimental group and the dmC2-treated experimental group was 12.66%±2.94% and 9.62%±2.36%, respectively, which was significantly higher than that in the control group (**p<0.01), and Ki67-positive cell staining was localized in the cell nucleus (Figure 1A-1H). In summary, the small molecule compound CRX-527 or the combination of dmPGE2 and CHIR99021 can effectively promote the proliferation of mouse liver cells in situ.

2. Optimization of administration time of small molecule compounds and combinations (CR and dmC2)

Based on the previous study, this study chose to optimize the administration time of 120 hours, with administration once every 24 hours. After stopping administration for 48 hours, the mice were killed by dislocation, and the mouse livers were collected for Ki67 immunofluorescence staining. The proportion of Ki67-positive cells in the livers of mice treated with the small molecule compound combination dmC2 for 5 days was significantly higher than that of the small molecule compound combination dmC2 group for 2 days (Figure 2H) (*p < 0.05), and the proportion of Ki67-positive cells in the livers of mice treated with the small molecule compound combination dmC2 for 5 days was 17.41% ± 2.53% (Figure 2C, 2D), which was also significantly higher than the proportion of Ki67-positive cells in the livers of mice in the control group (0.63% ± 0.15%) (Figure 2G) (**p < 0.01); while the small molecule compound The proportion of Ki67-positive cells in the liver of mice in the 5-day CR treatment group was lower than that in the 2-day CR treatment group of small molecule compounds, but there was no statistical difference (Figure 2J) (ns, p>0.05). Similar to the other three groups of small molecule compounds, the proportion of Ki67-positive cells in the liver of mice in the 5-day CR treatment group of small molecule compounds was 11.05%±1.65% (Figures 2E and 2F), which was significantly higher than the proportion of Ki67-positive cells in the liver of mice in the control group (0.50%±0.15%) (Figure 2I) (**p<0.01).

The above results show that the positive proportion of proliferating cells in the group treated with the small molecule compound CR for 5 days was reduced by a certain proportion compared with the group treated for 2 days, but there was no statistical difference, that is, continuous administration did not lead to unlimited proliferation of in situ cells in the mouse liver, but was controlled to fluctuate within a certain range; and the positive proportion of proliferating cells in the group treated with the small molecule compound combination dmC2 for 5 days was increased by a certain proportion compared with the group treated for 2 days, that is, continuous administration of the dmC2 group for 5 days can continue to increase the positive proportion of proliferating cells in the mouse liver.

3. Detection of liver regeneration indicators

In order to further confirm the proliferation of liver cells, this study counted the liver weight ratio of mice in the experimental group and the control group and detected the expression of mitotic markers in the mouse liver.

Statistical analysis of the liver weight ratios of the experimental group and the combination (dmC2 and CR) of small molecule compounds and the control group after 2 days and 5 days of treatment revealed that although the liver weight ratio of the group treated with the small molecule compound dmC2 combination for 2 days was higher than that of the control group (Figures 3A and 3B), there was no statistical difference (Figure 3E) (ns, p>0.05), and the liver weight ratio of the group treated for 2 days was 4.61%±0.31%; the liver weight ratio of the group treated with the dmC2 combination for 5 days was 5.79%±0.33%, which was significantly higher than that of the control group (Figures 3C, 3D, 3E) (**p<0.01). The combination of small molecule compound CR and small molecule compound dmC2 is similar. Although the liver weight ratio of the 2-day treatment group is higher than that of the control group (Figures 4A and 4B), there is no statistical difference (Figure 4E) (ns, p>0.05). The liver weight ratio of the 2-day treatment group is 4.27%±0.04%; the liver weight ratio of the 5-day treatment group of small molecule compound CR is 5.72%±0.64%, which is significantly higher than that of the control group (Figures 4C, 4D, 4E) (**p<0.01).

The proliferation index Ki67 can be detected in the active phases of the cell cycle (G1, S, G2, M), but not in the stationary phase (G0). The phosphorylation modification of histone H3 only occurs in the mitotic phase, so phosphorylated histone H3 (PH3) is a marker of the cell mitotic phase. Through immunofluorescence staining of PH3 and Ki67, it can be seen that there are only sporadic PH3 and Ki67 positive cells in the control group, and PH3 and Ki67 positive cells are co-localized in the same cell nucleus (Figure 5A, 5B, 5C); consistent with the results of immunofluorescence staining of the proliferation index Ki67, the proportion of PH3 positive cells, a marker of the cell mitotic phase, in the liver of mice treated with dmC2 and CR was significantly higher than that in the control group (**p < 0.01), and most of the PH3 positive cells can be co-localized with Ki67 positive cells in the same cell nucleus, and a small number of them are not localized in the same cell nucleus, which may be due to the cells being in different cell cycles. Co-staining of PH3 and Ki67 further confirmed that dmC2 and CR initiated the program of in situ cell proliferation in the liver ( Figures 5D-5I ).

In summary, the proportion of Ki67-positive cells, a cell proliferation index, in the liver of mice treated with dmC2 and CR for 2 days and 5 days was significantly higher than that in the control group. The proportion of Ki67-positive cells in the liver of mice treated with the small molecule compound combination dmC2 for 5 days was significantly higher than that in the 2-day treatment group. At the same time, the liver weight ratio of mice treated with dmC2 and CR for 5 days was significantly higher than that in the control group. Consistent with the immunofluorescence staining results of the proliferation index Ki67, the proportion of PH3-positive cells, a cell mitosis marker, in the liver of mice treated with dmC2 and CR was significantly higher than that in the control group. Based on the immunofluorescence results of the proliferation index Ki67, the statistical results of the liver weight ratio, and the immunofluorescence results of the cell mitosis marker PH3, it can be seen that dmC2 and CR can effectively promote the proliferation of in situ cells in the mouse liver and initiate the liver regeneration program.

Example 2: Efficacy of small molecule compounds and combinations in aplastic non-alcoholic fatty liver disease model

1. Efficacy of the small molecule compound combination dmC2 in the non-alcoholic fatty liver disease model

Wild-type C57BL/6 mice were fed a low-methionine and choline-deficient high-fat diet (Research Diet, A0607130) for 6 weeks to establish a mouse model of non-alcoholic fatty liver disease. After 6 weeks, they were randomly divided into two groups, with 6 mice in each group. The experimental group was treated with the small molecule compound combination dmC2 for 5 days, and the control group was treated with the small molecule compound solvent (Vehicle) for 5 days. After 2 days of stopping the treatment, the mice were killed by dislocation, and the mouse liver and serum were collected for subsequent analysis. The results of H&E staining showed that there were a large number of inflammatory cell infiltration and vacuolar degeneration in the liver tissue of the non-alcoholic fatty liver model mice in the control group (Figure 6A), while the vacuoles in the liver tissue of the non-alcoholic fatty liver model mice in the experimental group treated with the small molecule compound combination dmC2 became smaller, and the number of vacuolar degeneration was significantly reduced (Figure 6B); the results of lipid detection indicator Oil Red O staining showed that compared with the control group, the small molecule compound combination dmC2 treatment group significantly reduced the size and number of lipid droplets in the liver, and the fatty degeneration and tissue structure of the liver were improved (Figures 6A, 6B, 6C, 6D); Masson staining results showed that no significant collagen fiber deposition was observed in the liver tissue of the non-alcoholic fatty liver model mice in both the experimental and control groups (Figures 6E, 6F). The extracellular matrix in liver fibrosis mainly comes from activated hepatic stellate cells. Therefore, this study detected the hepatic stellate cell activation marker α-SMA. The α-SMA immunohistochemistry results showed that the α-SMA positive area in the small molecule compound combination dmC2 treatment group was 2.08% ± 0.46%, which was significantly lower than the α-SMA positive area in the control group (5.06% ± 0.52%). That is, the treatment of the small molecule compound combination dmC2 significantly reduced the expression of α-SMA (Figure 7A, 7B) (**p < 0.01); Next, this study detected the expression of Ki67, a cell proliferation indicator, in the small molecule compound combination dmC2 experimental group and the control group. By Ki67 immunohistochemical staining It can be seen that the proportion of Ki67-positive cells in the experimental group treated with the small molecule compound combination dmC2 (9.98% ± 1.55%) was significantly higher than that in the control group (2.92% ± 0.67%) (Figure 7C, 7D) (**p < 0.01), that is, in the non-alcoholic fatty liver model mice, the treatment with the small molecule compound combination dmC2 initiated the proliferation program of in situ cells in the liver; at the same time, this study detected the liver weight ratio of the experimental group and the control group of non-alcoholic fatty liver model mice treated with the small molecule compound combination dmC2. The liver weight ratio results showed that compared with the control group, the liver weight ratio of the experimental group treated with the small molecule compound combination dmC2 was significantly increased (Figure 7E, 7F, 7G) (*p < 0.05).

In summary, in non-alcoholic fatty liver model mice, treatment with the small molecule compound combination dmC2 promoted the proliferation of in situ cells in the liver of non-alcoholic fatty liver mouse model mice, significantly increased the liver weight ratio of model mice, reduced the proportion of fatty degeneration and activated hepatic stellate cells, and significantly improved the tissue structure of the liver.

2. Efficacy of the small molecule compound CR in the non-alcoholic fatty liver disease model

The non-alcoholic fatty liver model mice were established by the above method (low methionine and choline deficient high-fat diet). After the model was successfully established, the mice were randomly divided into two groups, with 6 mice in each group. The experimental group and the control group were treated with small molecule compound CR and control solvent, respectively. After the treatment, the mice were killed by dislocation, and the mouse serum and liver tissue were collected for subsequent research. Similar to the results of the treatment with the small molecule compound combination dmC2, the H&E staining results of the liver tissue of the experimental group of mice treated with the small molecule compound CR showed that the large number of inflammatory cell infiltration and vacuolar degeneration in the control group were improved (Figures 8A, 8B), and the results of lipid detection index Oil Red O staining showed that the treatment group of the small molecule compound CR significantly reduced the size and number of lipid droplets in the liver, and the fatty degeneration of the liver was improved (Figures 8A, 8B, 8C, 8D); the same Masson staining results showed that no significant collagen fiber deposition was found in the liver tissue of the non-alcoholic fatty liver model mice in the experimental group and the control group (Figures 8E, 8F). The detection results of α-SMA, a marker of hepatic stellate cell activation, showed that the positive area of α-SMA in the small molecule compound CR treatment group was 2.01% ± 0.47%, which was significantly lower than that in the control group (4.94% ± 0.47%), that is, the treatment of small molecule compound CR significantly reduced its positive area (Figure 9A, 9B) (**p < 0.01); Next, this study detected the effect of small molecule compound CR on the expression of Ki67, a cell proliferation indicator in the liver of mice with non-alcoholic fatty liver disease model. Ki67 immunohistochemical staining showed that the proportion of Ki67 positive cells in the control group was significantly lower than that in the control group.

(2.70% ± 0.44%), the proportion of Ki67-positive cells in the liver tissue of the experimental group of mice treated with small molecule compound CR (13.75% ± 0.95%) was significantly increased (Figure 9C, 9D) (**p < 0.01), that is, the treatment with small molecule compound CR promoted the proliferation of in situ cells in the liver of non-alcoholic fatty liver model mice; at the same time, this study explored the effect of treatment with small molecule compound CR on the liver weight ratio of non-alcoholic fatty liver model mice, and the treatment with small molecule compound CR significantly increased the liver weight ratio of model mice (Figure 9E, 9F, 9G) (**p < 0.01).

In summary, treatment with the small molecule compound CR significantly increased the proportion of in situ proliferating cells in the liver of model mice and the liver-to-weight ratio of model mice, reduced the proportion of activated hepatic stellate cells in the liver of model mice, and improved the tissue structure and fatty degeneration of non-alcoholic fatty liver model mice.

The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims (5)

1. Use of a small molecule compound in the preparation of a drug for initiating in situ liver regeneration, characterized in that the small molecule 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. Use of a small molecule compound in the preparation of a drug for liver regenerative disorder-related diseases, characterized in that the small molecule 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, Wherein, the disease related to liver regeneration disorder is non-alcoholic fatty liver disease. 3. The use according to claim 1 or 2, characterized in that: Wherein, the drug has the TLR4 agonist as an active ingredient. 4. The use according to claim 1, characterized in that: Wherein, the drug is a drug that promotes in situ cell proliferation in the liver or increases the liver weight ratio. 5. The use according to claim 2, characterized in that: The drug is a drug that reduces fatty degeneration of liver tissue, promotes in situ cell proliferation in the liver, increases liver weight ratio or reduces the proportion of activated hepatic stellate cells.