Application of a compound in the preparation of a drug for treating liver fibrosis and a NAMPT inhibitor

Abstract

The present invention relates to the technical field of drugs for treating liver fibrosis, and specifically to the use of a compound in the preparation of drugs for treating liver fibrosis and NAMPT inhibitors. Through single-cell transcriptomics research, it was found that the DNA repair pathway was significantly activated in hepatocytes with high HBV levels, and then the NAMPT-INSR pathway was found to play an important role in regulating the activation of hepatic stellate cells, thereby affecting the process of liver fibrosis. NAMPT can be used as a drug target for screening drugs for treating liver fibrosis, and then AZD6738 can be screened out as a NAMPT inhibitor to achieve the effect of inhibiting the activation of hepatic stellate cells and inhibiting liver fibrosis and cirrhosis. This technical solution can solve the technical problem of the lack of drugs for treating liver fibrosis in the prior art, which is of great significance for the development of effective anti-liver fibrosis drugs and has ideal promotion and application value.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs for treating liver fibrosis, and in particular to application of a compound in preparing drugs for treating liver fibrosis and NAMPT inhibitors. Background Art

[0002] Liver fibrosis is a pathological process, which refers to the abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Any liver damage will have a certain liver fibrosis process in the process of liver repair and healing. If the damaging factors cannot be removed for a long time, the fibrosis process will develop into cirrhosis. Liver fibrosis is an important pathological process for the development of cirrhosis. Liver fibrosis and cirrhosis are mainly caused by long-term damage to liver cells, which causes diffuse fibrosis of liver tissue, hardening of liver texture and impaired liver function. A variety of chronic liver diseases (such as hepatitis B and C, alcoholic hepatitis, non-alcoholic fatty hepatitis, schistosomiasis, autoimmune liver disease, drug-induced liver injury, etc.) can lead to the formation of liver fibrosis. Over the years, relevant research has been vigorously carried out, and the complex formation mechanism of liver fibrosis has been revealed from many aspects. Among them, hepatic stellate cells (HSCs) are still the hot spot and focus of liver fibrosis research. This cell is located in the Disse gap between the liver sinusoidal endothelial cells and hepatocytes, scattered throughout the liver, and accounts for about 10% of all liver intrinsic cells. Under physiological conditions, HSCs exhibit a non-proliferative quiescent phenotype, with retinyl ester (vitamin A or retinoic acid) lipid droplets rich in the cytoplasm around the cell nucleus. Under the influence of external stimuli, HSCs change from a quiescent state to an activated state, the lipid droplets in the cytoplasm gradually disappear, and collagen and other extracellular matrices (ECM) are formed in large quantities. This transformation is crucial for the formation of liver fibrosis. HSCs are the main cell source of liver myofibroblasts. Blocking the activation of HSCs or inactivating activated HSCs and promoting the enhanced activity of fibrinolytic substances are potential targets for anti-liver fibrosis. Research on new targets and new pathways for HSCs activation is of great significance for the development of effective anti-liver fibrosis drugs.

[0003] Hepatitis B virus (HBV) infection is one of the causes of liver fibrosis and liver cancer. However, the occurrence and development of liver cancer and liver fibrosis are two relatively independent pathological processes. Liver cancer is mainly caused by the malignant transformation of liver cells, forming solid tumors. Liver fibrosis is mainly caused by abnormal activation of hepatic stellate cells. Liver fibrosis and cirrhosis are mostly benign lesions with slow progression. Generally, early treatment has a good prognosis. However, liver cancer is a malignant tumor lesion. The liver undergoes necrosis, and most of its functions cannot be restored. It also develops rapidly, making it difficult to control the disease and the prognosis relatively poor. Only some patients with cirrhosis may develop liver cancer, and the liver tissues of some patients with liver cancer may show certain symptoms of liver fibrosis. The therapeutic targets for liver cancer and liver fibrosis are also completely different. The former needs to inhibit the proliferation and metastasis of liver cancer cells, while the latter needs to inhibit the activation of hepatic stellate cells. At present, researchers have developed a large number of drugs for the treatment of liver cancer, such as PD-1, lenfatinib, ramucirumab, etc. However, these drugs for the treatment of liver cancer have no significant therapeutic effect on liver fibrosis and cirrhosis, and some may even trigger liver inflammation and promote the occurrence of liver fibrosis. Summary of the invention

[0004] The present invention is intended to provide the use of AZD6738 in the preparation of a drug for treating liver fibrosis, so as to solve the technical problem of the lack of drugs for treating liver fibrosis in the prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] Application of AZD6738 in the preparation of medicines for treating liver fibrosis.

[0007] Furthermore, the structural formula of AZD6738 is shown in formula (1):

[0008]

[0009] Furthermore, the liver fibrosis is caused by infection of hepatitis B virus.

[0010] Furthermore, AZD6738 is used to inhibit the expression of NAMPT protein in normal hepatocytes or hepatoma cells.

[0011] The principle and beneficial effects of adopting the above technical solution are:

[0012] AZD6738 (Ceralasertib) can inhibit the expression of NAMPT protein in normal hepatocytes or hepatoma cells. Since the NAMPT-INSR signaling pathway plays an important role in HSCs cell activation, AZD6738 can inhibit the activation of hepatic stellate cells and thus inhibit the progression of liver fibrosis and cirrhosis. In normal hepatocytes or hepatoma cells infected with hepatitis B virus, the expression of NAMPT protein is significantly upregulated. Therefore, AZD6738 can be used for hepatic stellate cell activation and liver fibrosis caused by hepatitis B virus infection.

[0013] Example 2 Figure 2 C shows that HBV infection in normal liver cells L02 or HepG2 liver cancer cells can lead to upregulation of NAMPT protein expression. Figure 3 F and Figure 3 G shows the phenomenon that normal hepatocytes and hepatoma cells infected with HBV overexpressing NAMPT are co-cultured with hepatic stellate cells, which leads to the upregulation of activation markers of hepatic stellate cells HSCs. HSCs activation means the progress of liver fibrosis, and NAMPT is positively correlated with liver fibrosis. After applying AZD6738 to normal hepatocytes and hepatoma cells overexpressing NAMPT, the expression of NAMPT can be significantly reduced (Example 2 Figure 2 DF), thereby resisting the process of liver fibrosis. In addition, in vivo experiments also verified the inhibitory effect of AZD6738 on liver fibrosis (Example 6 Figure 8 ).

[0014] This protocol also provides the application of NAMPT protein in screening drugs for treating liver fibrosis.

[0015] Furthermore, the drug is used to inhibit the expression of NAMPT protein.

[0016] Furthermore, the liver fibrosis is caused by hepatitis B virus infecting liver cells and activating hepatic stellate cells.

[0017] The principle and beneficial effects of adopting the above technical solution are:

[0018] Through single-cell transcriptomics research, the inventors found that DNA repair pathways and glycolysis are significantly activated in hepatocytes with high levels of HBV, which are characteristics of hepatocytes with high levels of HBV transcription. It was found that 50 genes related to ligand-receptor interactions in hepatic stellate cells (HSCs) were enriched in hepatocytes with high levels of HBV, including NAMPT (nicotinamide phosphoribosyltransferase, a ligand expressed in hepatocytes) and INSR (receptor on hepatic stellate cells HSCs). The NAMPT-INSR signaling pathway plays a significant role in the activation of HSCs cells caused by HBV transcription, compared with other signaling pathways between hepatocytes and hepatic stellate cells formed by receptors and ligands, and is a key pathway affecting HBV-induced liver fibrosis. Using the above findings, we can have a deeper understanding of the pathological mechanisms of liver fibrosis and cirrhosis caused by HBV, and use the NAMPT-INSR signaling pathway, especially NAMPT, as a target for the treatment of related diseases to screen drugs for the treatment of liver fibrosis and cirrhosis caused by HBV. If the drug can inhibit the transcription level or protein level of NAMPT, it has the potential to be used as a drug for liver fibrosis and cirrhosis. As an example, this program screened out a small molecule drug AZD6738 that can treat liver fibrosis by using NAMPT as the drug target.

[0019] This scheme also provides the use of AZD6738 in the preparation of NAMPT inhibitors.

[0020] Furthermore, the NAMPT inhibitor is used to reduce the expression level of NAMPT protein in normal hepatocytes or hepatoma cells.

[0021] Furthermore, normal liver cells or liver cancer cells are infected with hepatitis B virus.

[0022] The principle and beneficial effects of adopting the above technical solution are:

[0023] In normal liver cells or liver cancer cells infected by HBV virus, the expression of NAMPT increases (which in turn causes HSCs cell activation and promotes the progression of liver fibrosis). However, under the action of AZD6738, the expression of NAMPT is significantly downregulated, avoiding the activation of HSCs cells and the progression of liver fibrosis caused by HBV. These research results show that AZD6738 can be used as an inhibitor of NAMPT, which can be used for the treatment of liver fibrosis or basic research purposes (non-therapeutic purposes). In basic research, the expression of NAMPT can be regulated to study its mechanism of action, upstream and downstream signaling molecules, etc. The most intuitive means of regulating the expression of NAMPT is to directly control the expression of the gene, such as overexpression through transgenic and reducing the expression of the target gene through RNA interference. However, the above process is very complicated and requires the design of related vectors and transgenic operations, which increases the cost of experimental research. If small molecules can be used to affect the expression of NAMPT by direct administration and incubation, the research process will be greatly shortened. There is not necessarily a suitable small molecule inhibitor for every gene or protein. In this study, the inventors found that AZD6738 can effectively inhibit the expression of NAMPT. It is a new type of small molecule compound that can be used as a potential NAMPT inhibitor. It has achieved unexpected technical effects and created conditions for basic research related to NAMPT. In fact, commercial small molecule inhibitors and agonists have very wide applications as basic scientific research tools. In addition to medical uses, the discovery of new NAMPT inhibitors also has other non-medical uses and can be used as basic tool reagents in the scientific research process. In this technical solution, the inventors regulated the expression of NAMPT protein by administering or not administering AZD6738, and then studied the effect of NAMPT expression on poly (ADP ribose) polymerase 1 (PARP1) (for example: Example 6) Figure 8 ). Therefore, AZD6738 not only has medical uses, but can also be used as a basic scientific research reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The single cell analysis and HBV transcription detection experimental results in HCCs of Example 1.

[0025] Figure 2 This is the research result of Example 2 that HBV transcriptionally activates the NAMPT-INSR pathway to promote the interaction between hepatocytes and HSCs.

[0026] Figure 3 These are the results of the inhibitor screening, NAMPT overexpression, and the interaction between hepatocytes and HSCs in Example 3.

[0027] Figure 4 These are the research results of Example 4 on the effect of NAMPT on liver cancer stem cells.

[0028] Figure 5 This is the Masson staining image of Example 4.

[0029] Figure 6 This is the research result of Example 5 on the crosstalk between hepatocytes and HSCs mediated by the NAMPT ligand-INSR receptor signaling pathway.

[0030] Figure 7 These are the research results of Example 5 on the overactivation of PARP1 lactylation and telomere maintenance induced by the NAMPT-INSR signaling pathway.

[0031] Figure 8 The liver tissues of Example 6, H&E and Masson staining pictures, and immunoblot pictures of ATR phosphorylation, NAMPT expression, and PARP1 lactylation (AZD6738 treatment, AG14361 treatment). DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial channels. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art.

[0033] In order to clarify the mechanism of drug action, the cell models used in this technical solution include:

[0034] HBV replication cell models: L02-pHBV4.1 (normal liver cells infected with HBV), HepG2.2.15 (liver cancer cells infected with HBV), MIG-MYC-pHBV4.1 (liver cancer stem cells infected with HBV); MIG-MYC (liver cancer stem cells, formed by overexpression of MYC in liver stem cells); MIG-MYC-NAMPT (liver cancer stem cells overexpress NAMPT); L02-NAMPT (normal liver cells overexpress NAMPT); HepG2-NAMPT (liver cancer cells overexpress NAMPT); MIG-MYC-pHBV4.1-shNampt (liver cancer stem cells infected with HBV and expressing shRNA that inhibits NAMPT). The above cell lines and models can be obtained by commercial means or prepared by conventional means of the prior art. The above cell models are prepared for evaluating and testing the function of the drugs or reagents to be tested, and can be obtained by conventional transfection of relevant plasmids, and the technical means used are conventional means well known to those skilled in the art; the experimental methods used are conventional methods and can be carried out according to the described recombinant technology (see Molecular Cloning, Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). See also the relevant literature and patents previously published by the inventor:

[0035] Tang H,McLachlan AA pregenomic RNA sequence adjacent to DR1 and complementary to epsilon influences hepatitis B virus replication efficiency. Virology.2002Nov10;303(1):199-210.doi:10.1006 / viro.2002.1645.PMID:12482672.

[0036] Zhou LY,Chen EQ,Wang ML,Chen LL,Liu CP,Zeng F,Tang H.Biologicalcharacteristics comparison of HBV rtA181T mutants with truncated orsubstituted HBsAg expression in vitro and in vivo model systems.SciRep.2016Dec 15;6:39260.doi:10.1038 / srep39260.PMID:27976732; PMCID:PMC5157016.

[0037] CN114917348B The invention discloses the application of neural adhesion factor in preparing reagent for inhibiting and marking the metastasis of liver cancer cells.

[0038] The abbreviations and their meanings involved in this technical solution are as follows:

[0039] HCCs: hepatocellular carcinomas; HSCs: hepatic stellate cells; HBV: hepatitis B virus; NAMPT: nicotinamide phosphoribosyltransferase; INSR: insulin receptor; ATM: ataxia telangiectasia mutated gene; ATR: ATM-related protein; aHSCs: activated HSCs, HSCs with high expression of COL1A1; sHSCs: stable HSCs, HSCs with low transcription level of COL1A1; myHSCs: myofibroblastic stellate cells; LCSCs: liver cancer stem cells; PARP1: poly (ADP-ribose) polymerase 1; TMMs: telomere maintenance mechanisms.

[0040] Example 1: Single cell transcriptomics study

[0041] Single-cell transcriptome sequencing (scRNA-seq) was performed on tumor tissues from 10 patients with hepatocellular carcinoma (HCCs) to investigate the biological characteristics and interactions between hepatocytes and hepatic stellate cells (HSCs) during the progression of HCCs. Differential gene expression analysis was performed using transcriptome data from 71,083 single cells to identify cluster-specific markers. Pseudo-time analysis revealed the impact of HBV transcription on the developmental trajectory of hepatocytes. Nine different cell states were identified in the potential hepatocyte trajectory (colored by state 1-9), and the red arrows show the potential evolutionary direction. Experimental results can be found in Figure 1 (Single-cell analysis and detection of HBV transcription in HCCs), where Figure 1 A shows ten single-cell RNA sequencing datasets generated from HCCs tumor tissues, providing transcriptome information for 71,083 individual cells; the violin plot shows the normalized expression of cell type-specific marker genes. Figure 1 B shows the result of assigning cell types to clusters. Figure 1 C is the hepatocyte trajectory analysis (arrows indicate potential evolutionary directions): the potential trajectory of all hepatocytes, nine different cell states were found (colored by states 1-9) along the potential hepatocyte trajectory; HBV transcription in hepatocytes was also shown, and hepatocytes with high HBV transcription levels were mainly located in state 1 of the trajectory. Figure 1 D shows the classification of hepatocytes into HBV low and HBV high clusters based on HBV transcription levels. Figure 1E is a heat map showing the expression changes of 20 highly variable pathways associated with low or high HBV transcription, among which DNA repair and glycolysis pathways were activated in hepatocytes with high HBV expression.

[0042] Analysis of experimental data revealed that hepatocytes with high levels of HBV transcription (HBV high-level hepatocytes) were mainly located in state 1 of the trajectory, while hepatocytes with low levels of HBV transcription (HBV low-level hepatocytes) appeared in states 2-9 ( Figure 1 C). These results suggest that HBV transcriptional activity plays a role in the developmental trajectory of hepatocytes during the progression of HCCs. Differential expression analysis of samples stratified based on HBV transcription (high or low) revealed differential expression of 20 highly variable pathways ( Figure 1 D, E). Notably, genes associated with DNA repair pathways (P = 0.014) and glycolysis (P = < 0.001) were significantly activated in hepatocytes with high HBV levels. These results suggest that activation of DNA repair pathways and glycolysis may be characteristics of hepatocytes with high levels of HBV transcription. The experimental results show that HBV transcription affects the developmental trajectory of hepatocytes.

[0043] Example 2: Study on the effect of HBV transcriptional activation on the interaction between hepatocytes and hepatic stellate cells

[0044] Further analysis of the test results of Example 1 revealed that 50 genes related to ligand-receptor interactions in hepatic stellate cells (HSC) were enriched in hepatocytes with high levels of HBV, including NAMPT (nicotinamide phosphoribosyltransferase, a ligand expressed in hepatocytes) and INSR (a receptor on HSC). Figure 2 (HBV transcriptional activation of the NAMPT-INSR pathway promotes the interaction between hepatocytes and HSCs). Figure 2 A is a possible ligand-receptor interaction between hepatocytes with high HBV transcription and HSC. Figure 2 B is the expression of NAMPT in hepatocytes with high or low HBV expression: the expression of HSC marker COL1A1 and receptor INSR in aHSCs and sHSCs. HSCs with high expression of COL1A1 are defined as activated HSCs (aHSCs), and HSCs with low COL1A1 transcription levels are defined as stable HSCs (sHSCs). From the statistical data, it can be seen that INSR is highly expressed in aHSC clusters.

[0045] Immunofluorescence analysis was further performed to investigate the co-localization of NAMPT and INSR in HCCs cells and adjacent normal cells. Figure 1C, Immunofluorescence staining to identify the expression of NAMPT (green) and INSR (red) in tumors and adjacent normal tissues. Figure 1 D is the results of immunoblotting analysis of NAMPT in HBV replication cell models (including L02-pHBV4.1, HepG2.215 and MIG-MYC-pHBV4.1) (P<0.01: **, P<0.001; ***, P<0.0001: ***). The above HBV replication cell models are HBV infection models constructed for human normal hepatocytes, hepatoma cells and liver cancer stem cells, respectively.

[0046] The above experimental results show that NAMPT (ligand expressed in hepatocytes) and INSR (receptor on HSC) are enriched in hepatocytes and hepatic stellate cells (HSC) with high HBV levels ( Figure 2 A). NAMPT is an important factor in the DNA repair pathway and is highly expressed in hepatocytes with high HBV levels. Based on the expression of COL1A1, HSCs are classified into activated HSCs (aHSCs) and stable HSCs (sHSCs). INSR receptors are only expressed in aHSCs ( Figure 2 B). These findings suggest that high levels of HBV transcription in hepatocytes may activate DNA damage and repair pathways and promote interaction with HSCs through NAMPT. Although there are many types of ligands involved in hepatocytes with high HBV transcription levels, and there are many types of receptors on HSC cells that respond to the above ligand signals, through the above transcriptomic studies and data analysis, it can be found that the NAMPT-INSR signaling pathway plays a significant role in the activation of HSC cells caused by HBV transcription, compared with other receptor-ligand-formed signaling pathways between hepatocytes and hepatic stellate cells, and is a key pathway affecting HBV-induced liver fibrosis. Among them, HSC cell activation participates in the process of liver fibrosis and cirrhosis through proliferation and secretion of extracellular matrix. Using the above findings, we can have a deeper understanding of the pathological mechanism of liver fibrosis and cirrhosis caused by HBV, and use the NAMPT-INSR signaling pathway, especially NAMPT, as a therapeutic target for related diseases to screen drugs for the treatment of liver fibrosis and cirrhosis caused by HBV. If the drug can inhibit the transcription level or protein level of NAMPT, it has the potential to be used as a drug for liver fibrosis and cirrhosis.

[0047] Further experiments investigated the expression of NAMPT and INSR. NAMPT was activated in all HBV replication cell models (including L02-pHBV4.1, HepG2.2.15, and MIG-MYC-pHBV4.1 organoids), further demonstrating the positive correlation between HBV transcriptionally activated NAMPT and the promotion of HSC activation through the INSR receptor ( Figure 2 C, where L02 cells are normal human liver cells and HepG2 are human liver cancer cells). After the normal liver cells L02 were transfected with HBV to form a HBV replication cell model, the expression of NAMPT protein increased, indicating that the expression of NAMPT protein was positively correlated with the HBV transcription level. And the above positive correlation pattern also exists in liver cancer cells HepG2. There is no necessary connection between HBV infection and hepatocellular carcinoma (whether liver cells are transformed into cancer cells). After normal liver cells are infected with HBV, certain symptoms of liver cell damage will appear, but liver cells will not necessarily be transformed into liver cancer cells. The expression level of NAMPT protein is also mainly related to whether the cells are infected with HBV, and has no necessary relationship with whether the cells are cancerous.

[0048] The above research results show that HBV transcription can activate the NAMPT-INSR pathway to promote the interaction between hepatocytes and hepatic stellate cells. Hepatic stellate cells are a group of resident fibroblasts located in the gap between hepatic sinusoidal endothelial cells and hepatocytes, and are the main effector cells in the process of liver fibrosis. Once the liver is damaged, quiescent hepatic stellate cells will be activated and transdifferentiated into proliferative, profibrotic, and contractile myofibroblasts, secreting a large amount of extracellular matrix components, especially collagen, which in turn promotes the process of liver fibrosis. The NAMPT-INSR pathway plays a key role in liver fibrosis or cirrhosis caused by HBV transcription. This is the first time that the inventors have discovered that NAMPT can be used as a potential target for drugs to treat liver fibrosis caused by HBV transcription, and a series of drug screening has been carried out.

[0049] Embodiment 3:

[0050] (1) Study on hepatocytes overexpressing NAMPT

[0051] Since Example 2 found that NAMPT is a potential therapeutic target, in order to study the significance of the NAMPT-INSR pathway in the interaction between hepatocytes and HSCs, this example further studied hepatocytes overexpressing NAMPT. More specifically, this example established cell lines L02-NAMPT and HepG2-NAMPT (NCBI ID of NAMPT is 10135) that overexpress NAMPT. In normal human hepatocytes L02 and human hepatoma cells HepG2, conventional transgenic means were used to overexpress the NAMPT gene in L02 cells and HepG2. The above process can be obtained by entrusting relevant institutions to conventional means of the prior art, and will not be repeated here. The L02 cells transferred into an empty vector were named L02-Vec cells, and the L02 cells transferred into a vector integrated with NAMPT were named L02-NAMPT cells. The HepG2 cells transferred into an empty vector were named HepG2-Vec cells, and the HepG2 cells transferred into a vector integrated with NAMPT were named HepG2-NAMPT cells. For details on the transcription of NAMPT (mRNA) in L02-Vec cells, L02-NAMPT cells, HepG2-Vec cells, and HepG2-NAMPT cells, see Figure 3 A. NAMPT protein expression is detailed in Figure 3 B.

[0052] In the experimental results, it was observed that overexpression of NAMPT did not directly affect the colony formation ability of L02 and HepG2 cells (L02 65.67±6.01 vs. L02-NAMPT 66.67±4.10, P=0.897; HepG2 32.33±5.93 vs. HepG2-NAMPT 43.67±5.46, P=0.232) or the apoptosis rate (L02 14.27±0.56 vs. L02-NAMPT 13.77±0.71, P=0.611; HepG2 9.23±0.24 vs. HepG2-NAMPT 9.11±0.95, P=0.906) ( Figure 3 C, D), nor does it promote the growth of subcutaneous tumors in nude mice ( Figure 3 E).

[0053] L02-NAMPT / HepG-NAMPT and human hepatic stellate cells LX2 were co-cultured using conventional co-culture methods to study the proliferation and apoptosis of L02-NAMPT / HepG-NAMPT cells and the surface markers of human hepatic stellate cells LX2. After co-culture with LX2, the colony-forming ability of L02-NAMPT (57.67±4.81 vs. 81.33±2.19, P=0.011) and HepG-NAMPT (43.67±11.62 vs. 81.33±4.98, P=0.041) was enhanced ( Figure 3 H); the apoptosis rate of L02-NAMPT (14.17±1.02 vs.4.28±0.45, P<0.001) and HepG-NAMPT (9.25±1.07 vs.3.13±0.50, P=0.007) cells was significantly decreased ( Figure 3 I). It was observed that the expression of ACTA2 (α-SMA) and COL1A1 in LX2 increased, and TGFB1 (TGF-β) was upregulated, showing the characteristics of myHSC ( Figure 3 F, G). Hepatic stellate cells have two subpopulations, cyHSCs and myHSCs. In myHSCs (myofibroblastic HSCs), related molecules of the extracellular matrix are aggregated, and HSCs are highly activated. The cells show the characteristics of myHSC, indicating that HSC is activated, and the cells participate in the formation of liver fibrosis and the reconstruction of intrahepatic structure through proliferation and secretion of extracellular matrix. By increasing the expression level of NAMPT in normal hepatocytes and hepatoma cells, the activation level of stellate cell HSC can be increased, thereby promoting the process of liver fibrosis. It further illustrates that NAMPT is a target for the treatment of liver fibrosis. Increasing the expression of NAMPT can promote the progression of liver fibrosis, while reducing the expression of NAMPT can reduce the activation level of stellate cell HSC, prevent its proliferation and secretion of extracellular matrix, and inhibit liver fibrosis.

[0054] (2) Screening of NAMPT inhibitors

[0055] In order to inhibit the expression of NAMPT, the commonly used means in the prior art are to use RNA interference to inhibit gene transcription and expression, or to use some inhibitors to inhibit the transcription and expression of NAMPT. The former is more complicated to use, requiring the construction of shRNA and cell transfection, while the use of some small molecule inhibitors can directly apply drugs and quickly produce effects. The inventors have studied small molecule inhibitors that inhibit NAMPT.

[0056] The inventors used Western blot to analyze and detect the activation of various proteins related to DNA damage in L02 (normal human liver cells) and L02-pHBV4.1 cells (HBV replication cell model), and screened potential small molecule compounds with NAMPT inhibitory efficacy.

[0057] The results showed that compared with L02 cells, the phosphorylation level of ATR in L02-pHBV4.1 cells was significantly increased, and the expression of NAMPT was increased. At the same time, there was no significant difference in the phosphorylation level of ATM between the two cell lines ( Figure 2 D). HBV transcription in L02-pHBV4.1 cells is at an activated level. Compared with normal hepatocytes not infected with HBV, the expression or activation level of proteins related to DNA repair, including NAMPT, has changed to a certain extent. Next, KU60019 and AZD6738 were used to act on L02-pHBV4.1 cells to observe the expression of NAMPT. When KU60019 was used, ATM-mediated CHEK2 phosphorylation was inhibited, but there was no significant effect on the expression of NAMPT. AZD6738 significantly reduced ATR autophosphorylation and NAMPT expression, but did not inhibit ATM-mediated CHEK2 phosphorylation ( Figure 2 E). In addition, AZD6738 also effectively reduced the increase in NAMPT expression caused by HBV replication in HepG2.2.15 cells ( Figure 2F). In normal liver cells or liver cancer cells infected by HBV virus, the expression of NAMPT increases (which in turn causes HSC cell activation and promotes the progression of liver fibrosis). However, under the action of AZD6738, the activation of NAMPT by HBV is blocked, and the expression of NAMPT is significantly downregulated, avoiding the activation of HSC cells and the progression of liver fibrosis caused by HBV. These research results show that AZD6738 can be used as an inhibitor of NAMPT for the treatment of liver fibrosis or for basic research purposes. In basic research, regulating the expression of NAMPT can further study its mechanism of action and upstream and downstream signaling molecules. The most intuitive means of regulating the expression of NAMPT is to directly control the expression of the gene, such as overexpression through transgenics and reducing the expression of the target gene through RNA interference. However, the above process is very complicated and increases the cost of experimental research. If small molecules can be used to affect the expression of NAMPT by direct administration and incubation, the research process will be greatly shortened. However, there may not be a suitable small molecule inhibitor for each gene or protein. In this study, the inventors found that AZD6738 can effectively inhibit the expression of NAMPT. It is a new type of small molecule compound that can be used as a potential NAMPT inhibitor. It has achieved unexpected technical effects and created conditions for basic research related to NAMPT. In fact, commercial small molecule inhibitors and agonists have very wide applications as basic scientific research tools. The discovery of new NAMPT inhibitors has other non-medical uses in addition to medical uses. In the prior art, shRNA is usually used to downregulate the expression of the target gene. The inventors have also tried to use conventional shRNA methods to inhibit the transcription of the NAMPT gene. The effect produced by using shRNA is similar to that of using the NAMPT inhibitor AZD6738. In the WB experiment, it shows the same Figure 2 E and F show similar experimental results.

[0058] The detailed experimental results of the above experiments can be found in Figure 2 and Figure 3 , Figure 2 and Figure 3 The specific test settings are as follows:

[0059] Figure 2 D is the immunoblot results of ATM, P-ATM, ATR, P-ATR and NAMPT levels in L02 and L02-pHBV4.1 cell lines, with ACTB (β-Actin) as the internal reference control. Figure 2E: L02-pHBV4.1 cells were treated with KU60019 (10 μM) or AZD6738 (2 μM) for 24 h, and the protein levels of ATM, P-ATM, ATR, P-ATR, CHEK2, P-CHEK2, and NAMPT were analyzed by immunoblotting, with ACTB (β-Actin) as the internal control. Figure 2 F: HepG2 and HepG2.215 cells were treated with AZD6738 (2 μM) for 24 h, and the protein levels of ATR, P-ATR, and NAMPT were analyzed by immunoblotting, with ACTB (β-Actin) as the internal control. Figure 3 A and B are the results of immunoblot analysis of NAMPT in L02 cells and HepG2 cells overexpressing NAMPT or transformed with empty vector, with ACTB (β-Actin) as the internal reference control. Figure 3 C and Figure 3 D is the cell colony formation and apoptosis analysis of L02-Vec cells, L02-NAMPT cells, HepG2-Vec cells and HepG2-NAMPT cells. Figure 3 E is the construction of L02-Vec / L02-NAMPT or HepG2-Vec / HepG2-NAMPT nude mouse subcutaneous tumor models to detect the growth of subcutaneous tumors after cell injection. The experimental results showed that overexpression of NAMPT had no significant effect on the tumor growth caused by L02 cells and HepG2 cells (mainly the latter). Figure 3 F is the immunoblot results of ACTA2 (α-SMA), COL1A1, and TGFB (TGF-β) levels in LX2 cells co-cultured with or without L02-NAMPT / HepG2-NAMPT. Figure 3 G is the immunofluorescence detection results of relevant markers in LX2 cells co-cultured with or without L02-NAMPT. This experiment was used to identify the immunofluorescence staining of INSR (red) and COL1A1 (green). Figure 3 H is colony formation of L02-NAMPT / HepG2-NAMPT cells with or without co-culture of LX2. Figure 3 I is apoptosis analysis of L02-NAMPT / HepG2-NAMPT cells with or without LX2 co-culture. The data in the bar graphs in the above figures are represented as mean±SD (n=3), P>0.05: NS, P<0.05: *, P<0.01: **, P<0.001: ***.

[0060] Example 4: Effect of NAMPT on liver cancer stem cells

[0061] In order to study the effect of NAMPT on liver cancer stem cells (LCSCs) and the effect on liver fibrosis, the inventors constructed LCSCs (MIG-MYC-NAMPT cells) overexpressing NAMPT and carried out three-dimensional (3D) culture. The construction process of MIG-MYC-NAMPT cells can be found in the inventor's prior patent (CN114917348B Application of neural adhesion factors in the preparation of reagents for inhibiting and marking liver cancer cell metastasis, "(2) Construction of LCSCs cells with NRCAM inhibition expression and overexpression" in the patent), except that the overexpressed target gene is changed from NRCAM to NAMPT, which is a conventional operation method in the prior art and will not be described here. MIG-MYC-NAMPT cells overexpressing NAMPT and MIG-MYC cells not overexpressing NAMPT are obtained by the above method. Liver stem cells overexpressing MYC can be used as liver cancer stem cells.

[0062] Immunofluorescence confocal microscopy was used to detect the expression of stem cell markers, such as Epcam and Afp, in MIG-MYC-NAMPT and MIG-MYC organoids (cells formed into organoids through 3D culture). It was found that the expression of Epcam and Afp in MIG-MYC-NAMPT organoids was similar to that in MIG-MYC organoids ( Figure 4 A). After co-culturing MIG-MYC-NAMPT organoids with LX2, it was found that MIG-MYC-NAMPT organoids could activate the expression of ACTA2, COL1A1, and TGFB1 in HSCs ( Figure 4 B). Epcam is also upregulated in MIG-MYC-NAMPT organoids ( Figure 4 C). In addition, NAMPT did not directly affect the colony-forming ability of LCSCs (MIG-MYC 77.67±2.52 vs. MIG-MYC-NAMPT 78.33±4.04, P=0.820) or the apoptosis rate (MIG-MYC 5.69±0.23 vs. MIG-MYC-NAMPT 5.56±0.33, P=0.684) ( Figure 4 D) and does not promote the growth of subcutaneous tumors in nude mice ( Figure 4 E). After co-culture with LX2, the colony formation ability of MIG-MYC-NAMPT organoids was enhanced (80.67±3.51 vs. 93.00±2.00, P=0.006), and the apoptosis rate of MIG-MYC-NAMPT organoids was significantly reduced (5.24±0.36 vs. 3.34±0.35, P=<0.001) ( Figure 4F). These results suggest that overexpression of NAMPT does not directly affect the stemness of MIG-MYC. MIG-MYC-NAMPT organoids interact with HSCs, promoting the differentiation of mouse HSCs into myHSC phenotypes and promoting the progression of liver fibrosis.

[0063] MIG-MYC and MIG-MYC-NAMPT organoids were transplanted into radiation-induced transient immunodeficient C57BL / 6 mice by intrahepatic injection to form a mouse tumor gene-driven HCCs model. The mice were killed 28 days after allogeneic transplantation, and their livers were fixed and subjected to H&E and Masson staining. The adjacent liver tissues of the MIG-MYC-NAMPT group and the MIG-MYC group were examined by Masson staining. We also found pseudolobule formation and severe fibrosis in the tissues of the MIG-MYC-NAMPT group ( Figure 5 ). These results indicate that HBV-induced increase in NAMPT expression does not directly promote the malignant transformation of hepatocytes, but may promote the progression of liver fibrosis through interaction with myHSCs.

[0064] For detailed experimental results of this example, see Figure 4 and Figure 5 . Figure 4 We demonstrated the impact of the NAMPT-INSR pathway on the stemness properties of LCSCs in a mouse oncogene-driven allogeneic HCCs model. Figure 4 A: Afp and Epcam were detected by immunofluorescence staining in cell colonies of MIG-MYC-Vec and MIG-MYC-NAMPT organoids. Figure 4 B is the result of immunoblot analysis of ACTA2, COL1A1, and TGFB levels in LX2 cells co-cultured with or without MIG-MYC-NAMPT. Figure 4 C is an experimental image of detecting Afp and Epcam by immunofluorescence staining in MIG-MYC-NAMPT cell colonies with or without LX2 co-culture. Figure 4 D shows the results of colony formation and apoptosis analysis of MIG-MYC-Vec and MIG-MYC-NAMPT cell colonies. Figure 4 E is the formation curve of cell-derived subcutaneous tumors in nude mice. Figure 4 F shows the results of colony formation and apoptosis analysis of MIG-MYC-NAMPT cell colonies co-cultured with or without LX2. Figure 5 Masson staining of MIG-MYC-Vec (Mouse: 1-1) and MIG-MYC-NAMPT cell colony (Mouse: 2-1) groups.

[0065] Example 5: Study on Overactivation of PARP1 Lactylation and Telomere Maintenance Induced by Hepatocyte (NAMPT)-HSCs (INSR) Communication

[0066] The inventors studied the knockout of INSR, a receptor for NAMPT in HSCs, and found that the activation effect of hepatocytes on HSCs disappeared ( Figure 6 AB), in mice, after knocking out INSR in liver HSCs, the activation effect of mouse tumor stem cells on HSCs also disappeared. Mouse tumor stem cells can continue to form liver cancer, but will no longer cause liver fibrosis (6C-H).

[0067] in, Figure 6 We demonstrated that NAMPT ligand-INSR receptor signaling mediates crosstalk between hepatocytes and HSCs. Figure 6 A is INSR in LX2, LX2 - INSR - / - (INSR knockout in LX2 cells) and LX2 - INSR - / - Expression in INSR cells (rescue experiment). Figure 6 B shows the results of co-culture of LX2, LX2 and HepG2-NAMPT - INSR - / - and LX2 - INSR - / - -Western blot results of INSR, ACTA2, COL1A1, and TGFB levels in cells. Figure 6 C shows Insr fl / fl ; Lrat-Cre mouse model construction workflow. Figure 6 D shows Lrat-P2A-iCre mice and ins fl / fl ; IF staining results of Cre (green) and Insr (red) in HSCs of Lrat-Cre mice. Figure 6 E shows Lrat-P2A-iCre mice and ins fl / fl ; Statistical results of INSR expression in HSCs of Lrat-Cre mice. Figure 6 F shows the expression of mouse HSCs and HSCs-Insr after co-culture with MIG-MYC-NAMPT organoids. - / - Western blot results of Insr, Acta2, Col1a1, and Tgfb levels in cells. Figure 6 G shows the relationship between HSCs and HSCs-Insr - / - IF staining results of Afp and Epcam in MIG-MYC-NAMPT organoids after cell co-culture. Figure 6 H shows Lrat-P2A-iCre mice and ins fl / fl ; The allogeneic liver cancer model established by Lrat-Cre mice using MIG-MYC-NAMPT organoids (n=6 in each group) was evaluated by H&E and Masson staining for tumor formation and liver fibrosis. The statistical results are expressed as: P≤0.001(***), P≤0.0001(****).

[0068] Telomeres are repetitive nucleotide sequences located at the ends of chromosomes and are essential for maintaining genome stability and cell lifespan. In certain cell types, including stem cells and cancer cells, telomere length is maintained by specific telomere maintenance mechanisms (TMMs). To investigate the impact of communication between hepatocytes (NAMPT) and hepatic stellate cells (INSR) on telomere length, L02-NAMPT cells were co-cultured with LX2 cells and telomere length was measured using FISH. A significant increase in telomere length was observed in L02-NAMPT cells ( Figure 7 A), showing the activation of TMMs in L02-NAMPT cells.

[0069] Maintenance of T-loop structure is a key aspect of TMMs. Cohesion complex plays an important role in resolving T-loop structure to prevent telomere dysfunction, ultimately promoting telomere maintenance and cell lifespan regulation. To confirm the activation of T-loop structure maintenance, L02-NAMPT cells were co-cultured with LX2 cells and subjected to Western blotting analysis. The activation of major Cohesion complex components SMC1, SMC3, and RAD21 was significantly increased, CTCF levels were increased, and TRF2 levels were decreased ( Figure 7 B), demonstrating activation of TMMs and inhibition of telomere shortening.

[0070] NAMPT is an enzyme that limits the rate of NAD+ synthesis and increases the tolerance of cells to extracellular glucose depletion. INSR can induce a metabolic shift to glycolysis, suggesting that the NAMPT-INSR pathway may affect the metabolic pattern of hepatocytes and HSCs. The Seahorse Cell Energy Metabolism Analyzer was used to observe the changes in L02-NAMPT metabolism after co-culture with LX2 cells, and it was found that the glycolysis of L02-NAMPT was significantly enhanced ( Figure 7 C) and produces lactate dehydrogenase A (LDHA), a key enzyme in glycolysis and lactate production ( Figure 7 D).

[0071] In addition to its role in DNA repair, PARP1 is also involved in telomere length and T-loop formation. However, the study found that co-culturing L02-NAMPT cells with LX2 cells had no effect on PARP1 expression in L02-NAMPT cells (e.g. Figure 7 B). PARP1 can be lactylated at specific sites, and studies have shown that after co-culture with LX2, the total lactylation level in L02-NAMPT cells was significantly increased ( Figure 7 E). To investigate the effect of co-culture on PARP1 lactylation, we used PARP1 antibody and Protein A / G magnetic beads to perform PARP1 immunoprecipitation (IP) on L02-NAMPT protein solution ( Figure 7 F). Then, the lactylation level of PARP1 was measured using a full lactylation antibody, and it was found that co-culture with LX2 promoted the lactylation of PARP1 ( Figure 7 G).

[0072] To further investigate the effect of PARP1 lactylation on telomere maintenance in hepatocytes, PARP1 was knocked out in L02 using CRISPR / Cas9. In addition, a PARP1-7KR (K498 / 505 / 506 / 508 / 518 / 521 / 524R) mutant plasmid was constructed to completely abolish the lactylation of PARP1. L02-PARP1- / - cells were heterologously expressed with wild-type PARP1 and PARP1-7KR by transfection with PARP1 and PARP1-7KR plasmids. To induce lactylation, cells were treated with L-lactic acid (25 mM) ( Figure 7 H). We then measured the lactylation level of PARP1 and the results showed that PARP1-7KR completely abolished the lactylation of PARP1 ( Figure 7 I, J). IF and FISH analysis showed that the telomeres of cells expressing the PARP1-7KR mutant were significantly shorter than those of L02 cells expressing wild-type PARP1 ( Figure 7 K). In addition, L02 cells expressing the PARP1-7KR mutant had significantly lower Cohesion complexes and CTCF, and higher levels of TRF2, compared with cells expressing wild-type PARP1 ( Figure 7 L), indicating that PARP1 lactylation plays a key role in the activation of TMMs in hepatocytes.

[0073] Detailed test results can be found in Figure 7 ,in, Figure 7 A is a telomere FISH experiment to measure telomere length. Figure 7 B is the immunoblot analysis of key Cohesion complex factors (SMC1, SMC3 and RAD21), CTCF, TRF2 and PARP1. Figure 6C is Seahorse XF glycolysis assay. Figure 7 D is the expression of GLUT1, GLUT3, HK2, LDHA, and PFK. Figure 6 E is the lactic acid level detection result. Figure 7 F is the result of immunoprecipitation (IP) study in PARP1 protein lysate. Figure 6 G is the results of the detection of PARP1 lactylation level of L02-NAMPT with or without LX2 co-culture. Figure 7 H is the lactic acid level detection result. Figure 7 I is the immunoprecipitation (IP) result of PARP1 in protein lysate. Figure 7 J is the detection result of PARP1 lactylation level. Figure 7 K is telomere FISH experiment. Figure 7 L is the key factor of the Cohesion complex, immunoblot analysis of CTCF, TRF2 and PARP1 (for L02 cells expressing wild-type PARP1 or PARP1-7KR after treatment with L-lactic acid (25 mM)). Among them, the statistical results are P>0.05 (NS), P<0.05 (*), P<0.01 (**).

[0074] Example 6: In vivo experimental study of a method for treating hepatitis B-related liver fibrosis

[0075] To investigate the involvement of the ATR-NAMPT pathway and PARP1 lactylation in the progression of liver fibrosis in vivo, organoids obtained by cell culture of MIG-MYC cells, MIG-MYC-pHBV4.1 cells, and MIG-MYC-pHBV4.1-shNampt cells were transplanted into C57BL / 6 mice that were transiently irradiated to induce immunodeficiency. The role of the inhibitor AZD6738 in inhibiting liver fibrosis in vivo was investigated. Seven days after modeling, mice were intragastrically administered (ig) AZD6738 (50 mg / Kg) or DMSO (control). After 28 days, all mice were killed and their liver tissues were subjected to immunoblotting, H&E, and Masson staining. MIG-MYC cells are mouse liver stem cells expressing MYC (a model of liver cancer stem cells), MIG-MYC-pHBV4.1 cells are liver cancer stem cells with enhanced HBV transcription, and MIG-MYC-pHBV4.1-shNampt cells are liver cancer stem cells with enhanced HBV transcription that use shRNA to inhibit NAMPT expression.

[0076] Masson staining showed that regardless of whether AZD6738 was applied, the MIG-MYC-pHBV4.1-shNampt group had reduced inflammation and fibrosis compared with the MIG-MYC-pHBV4.1 group due to the inhibition of NAMPT expression; the MIG-MYC-pHBV4.1 group had increased inflammation and fibrosis compared with the MIG-MYC group due to HBV virus transcription. Comparing the cases with or without AZD6738, the MIG-MYC-pHBV4.1-shNampt group, the MIG-MYC-pHBV4.1 group, and the MIG-MYC group all showed reduced inflammation and fibrosis, and there were no pseudolobules or obvious fibrosis in the AZD6738-treated group ( Figure 8 A) In Figure 8 In B, tissue clearing and image analysis of representative MIG-MYC-pHBV4.1 mice and MIG-MYC-pHBV4.1-shnampt mice treated with DMSO or AZD6738, respectively. MYC-labeled tumor cells are stained green, while ACTA2-positive cells are stained red. shNampt and inhibition of Nampt with AZD6738 attenuated the activation of ACTA2 around liver cancer, where ACTA2 activation represents the activation of HSCs and the activation of liver fibrosis ( Figure 8 B). In addition, immunoblot analysis showed that ATR phosphorylation, NAMPT expression, and PARP1 lactylation were completely suppressed in HCC tissues from the MIG-MYC, MIG-MYC-pHBV4.1, and MIG-MYC-pHBV4.1-shNampt groups under AZD6738 treatment ( Figure 8 C).

[0077] PARP1 lactylation was further investigated in the MIG-MYC-pHBV4.1 group using the PARP1-specific inhibitor AG14361. In mouse livers, tumor xenografts were associated with significantly reduced tumorigenicity after AG14361 treatment (DMSO: 6 / 6 hepatocellular carcinoma vs. AG14361: 2 / 6 hepatocellular carcinoma). AG14361 could achieve a certain anti-tumor effect. However, there was no significant reduction in inflammation and fibrosis in tumor-adjacent normal liver tissue ( Figure 8 D). In addition, immunoblot analysis showed that the expression and lactation of PARP1 were completely inhibited in the liver tissue of mice in the MIG-MYC-pHBV4.1 group treated with AG14361. There was no significant change in NAMPT expression and total cellular lactation level after AG14361 treatment ( Figure 8E) The above experimental results further provide evidence that PARP1 lactylation plays a key role in the formation of hepatocellular carcinoma. However, although AG14361 can inhibit tumors to a certain extent, it has no inhibitory effect on liver fibrosis. This study shows that among the small molecule compounds tested by the inventors, only AZD6738 can inhibit NAMPT expression, thereby inhibiting the activation of hepatic stellate cells and thus inhibiting the process of liver fibrosis.

[0078] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. The use of AZD6738 in the preparation of a drug for treating liver fibrosis, characterized in that: The structural formula of AZD6738 is shown in formula (1): Formula (1) The liver fibrosis is caused by infection of hepatitis B virus.

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