Application of p97 / VCP inhibitor in preparation of pharmaceutical preparation for treating hepatic fibrosis

Abstract

The invention discloses application of a p97 / VCP inhibitor in preparation of a pharmaceutical preparation for treating hepatic fibrosis, and belongs to the field of biological medicine. The p97 / VCP inhibitor CB5081 disclosed by the invention shows an anti-fibrosis function by promoting the aging of hepatic stellate cells. When CB-5083 is used for treating fibrosis mice, activation of hepatic stellate cells is inhibited, and hepatic fibrosis is relieved. In addition, in-vivo experiments show that CB-5083 promotes HSC aging and reduces YAP expression. The invention emphasizes that the medicine taking p97 / VCP as a target spot has the effect of relieving hepatic fibrosis.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of a p97 / VCP inhibitor in the preparation of a pharmaceutical preparation for treating liver fibrosis. Background Art

[0002] Liver fibrosis is a pathological wound healing response that is associated with the progression of various chronic liver diseases, including nonalcoholic steatohepatitis (NASH) and hepatitis B / C virus infection, and often leads to cirrhosis and liver failure. The formation of fibrotic tissue is characterized by excessive accumulation of extracellular matrix (ECM) proteins, such as collagen and fibronectin, leading to distortion of liver architecture and subsequent functional loss. Hepatic stellate cells (HSCs), which account for approximately 15% of the total number of hepatocytes, are precursors of myofibroblasts and the main source of ECM. In the setting of chronic liver injury, continuous exposure to external immunogens can trigger the activation of quiescent HSCs. During HSC activation, it promotes them to enter a proliferative and fibrogenic state. Activated HSCs eventually transform into myofibroblasts, which are characterized by elevated expression of the intracellular protein α-SMA. Subsequently, they synthesize large amounts of ECM, mainly composed of collagen, and eventually form scar tissue. The core of liver fibrosis treatment is to inhibit and reverse the activation of hepatic stellate cells, including promoting apoptosis, senescence, or returning to an inactive state.

[0003] p97, also known as valosin-containing protein (VCP), is a member of the AAA+ ATPase family and is involved in protein degradation by extracting ubiquitinated proteins from protein complexes and organelles. p97 is an evolutionarily conserved component of the ubiquitin-proteasome system (UPS) and is overexpressed in various tumor types. In hepatocellular carcinoma (HCC), p97 expression is significantly elevated and correlated with advanced tumor lymph node metastasis stage and poor prognosis in patients. p97 inhibitors synergistically inhibit the IRE1a-XBP1 pathway with M1 virus, thereby inducing insoluble endoplasmic reticulum stress and promoting apoptosis of liver cancer cells. No relevant studies have been conducted on whether targeting p97 can regulate the activation state of hepatic stellate cells. Summary of the invention

[0004] The present invention provides an application of a p97 / VCP inhibitor in the preparation of a pharmaceutical preparation for treating liver fibrosis. The p97 / VCP inhibitor is CB-5083 (CAS No: 1542705-92-9), and its structure is shown below:

[0005] .

[0006] The liver fibrosis includes one or more of non-alcoholic fatty hepatitis, cholestatic liver disease, and autoimmune hepatitis.

[0007] The pharmaceutical preparation reduces the fibrotic activity of hepatic stellate cells by reducing the protein expression of α-SMA and collagen-Ⅰ in hepatic stellate cells, inhibiting the expression of Yes-related protein YAP in hepatic stellate cells, and inducing senescence of hepatic stellate cells.

[0008] The p97 / VCP inhibitor CB5081 of the present invention exhibits anti-fibrotic function by promoting the senescence of hepatic stellate cells. The present invention found that p97 expression was upregulated in diet- and chemical-induced non-alcoholic steatohepatitis and fibrosis mouse models. Intervention or knockout of p97 by the p97 antagonist CB-5083 can reduce the expression of α-SMA and collagen-I in mouse or human hepatic stellate cells. CB-5083 can induce HSC senescence, leading to upregulation of senescence markers such as p21, p53, GPX4 and SA-β-Gal. In addition, CB-5083 can also inhibit the expression of Yes-associated protein (YAP), which is an aging-related regulatory protein with pro-fibrotic function. We treated fibrotic mice with CB-5083 and found that the activation of hepatic stellate cells was inhibited and liver fibrosis was alleviated. In addition, in vivo experiments showed that CB-5083 promoted HSC senescence and reduced YAP expression. The present invention emphasizes that drugs targeting p97 / VCP have the effect of alleviating liver fibrosis. In vivo, we show that antagonizing p97 promotes senescence and inhibits proliferation of hepatic stellate cells in mice, highlighting the potential of targeting p97 / VCP for anti-fibrotic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a schematic diagram of the action principle of the p97 / VCP inhibitor of the present invention.

[0010] Figure 2p97 levels are elevated in fibrotic livers and TGF-β-stimulated HSCs. (A) p97 mRNA levels in hepatocellular carcinoma (LIHC) were analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA) database. (B) H&E staining and immunohistochemical analysis of α-SMA and collagen I in liver sections of mice in the sham and fibrotic groups. (C) Immunohistochemical analysis of p97 in liver sections of mice in the sham and fibrotic groups. (D) Immunofluorescence staining of p97 (red) and Desmin (green, a marker of hepatic stellate cells) in liver sections of mice in the sham and fibrotic groups. (E) Expression of p97 in hepatic stellate cells of mice treated with the profibrotic factor TGF-β. Mouse hepatic stellate cells were stimulated with TGF-β (20 ng / ml) for 12 hours. (F, G) Expression of p97 in human hepatic stellate cells LX-2 treated with TGF-β (20 ng / ml) or 10% fetal bovine serum for 12 hours after starvation (western blot or qRT-qPCR). Scale bar: 100 μm (B, D), 200 μm (C), all results are expressed as mean ± SD. *p<0.05; **p<0.01.

[0011] Figure 3 The p97 antagonist CB-5083 or shRNA interference can reduce the fibrotic activity of HSCs. Wherein: (A) Chemical structure of p97 antagonist CB-5083. (B) To monitor the cytotoxic effect, the viability of mouse hepatic stellate cells or human LX-2 cells was detected by MTT assay after 12 h of CB-5083 treatment. (C) Effects of different concentrations of CB-5083 treatment for 12 h on the mRNA expression of α-SMA and type I collagen in mouse hepatic stellate cells. qRT-PCR was used to detect mRNA levels. (D) Effects of CB-5083 treatment on the mRNA expression of α-SMA and type I collagen in human LX-2 cells. (E) Western blotting analysis of p97 knockout in mouse hepatic stellate cells by shRNA recombinant lentivirus (sh-p97-1, sh-p97-2, sh-p97-3 and random shRNA control). p97 knockout efficiency, vimentin, type I collagen, and GAPDH were detected in total cell lysates. (F) qRT-PCR was used to detect the mRNA expression of α-SMA and type I collagen in mouse hepatic stellate cells after p97 knockout. (G) Western blotting was used to analyze the knockout efficiency of p97 in human LX-2 cells and the expression of type I collagen after knockout. (H) qRT-PCR was used to detect the mRNA expression of α-SMA and type I collagen in human LX-2 cells after p97 knockout. All results are expressed as mean ± SD. *p < 0.05; **p < 0.01.

[0012] Figure 4 p97 antagonist CB-5083 or shRNA interference can induce HSC senescence. Wherein: (A) Western blotting analysis of p21, p53 and GPX4 expression in mouse hepatic stellate cells. Mouse hepatic stellate cells were cultured in 0.3 or 0.1 μM CB-5083 for 12 hours. (B) Senescence-associated β-galactosidase (SA-β-gal) assay was performed in mouse hepatic stellate cells treated with 0.3 μM CB-5083 for 12 hours. (C) Human LX-2 cells were treated with 0.3 μM CB-5083 for 12 h and SA-β-gal senescence assay was performed. (D) Western blotting analysis after knockdown of p97, p21 and GPX4 in mouse hepatic stellate cells by recombinant shRNA lentivirus. Scale bar: 100 μm (B, C). All results are expressed as mean ± SD. *p<0.05; **p<0.01.

[0013] Figure 5 The p97 antagonist CB-5083 inhibits the expression of YAP in mouse HSCs, while YAP exhibits profibrotic function. Among them: (A) Effect of CB-5083 on YAP mRNA expression in mouse hepatic stellate cells. mRNA levels were detected by qRT-PCR. (B) Immunofluorescence staining and quantification of YAP in mouse HSCs after CB-5083 treatment. Mouse HSCs were cultured with 0.3 μM CB-5083 for 12 hours. (C) Western blotting analysis of YAP protein after p97 knockout in mouse HSCs by shRNA recombinant lentivirus. (D) qRT-PCR detection of α-SMA and collagen I mRNA expression in mouse hepatic stellate cells after YAP downregulation. (E) Chemical structure of YAP antagonist verteporfin. (F) Effect of 12-hour treatment of YAP inhibitor verteporfin on α-SMA and collagen I mRNA expression in mouse hepatic stellate cells. mRNA levels were detected by qRT-PCR. Scale bar: 200 μm (B). All results are expressed as mean ± SD. *p < 0.05; **p < 0.01.

[0014] Figure 6CB-5083 promotes hepatic stellate cell senescence and inhibits YAP expression in mice. (A) Sirius red staining and immunohistochemistry of collagen I in liver sections of mice with liver fibrosis in the sham, 10% DMSO control and CB-5083 groups. (B) Immunofluorescence staining and quantification of Desmin in liver sections of mice in the sham, 10% DMSO control and CB-5083 groups. (C) Immunofluorescence staining and quantification of p21, a senescence marker, in liver sections of mice treated with sham, 10% DMSO and CB-5083. (D) Immunofluorescence co-staining of p21 (red) and Desmin (green) in liver sections of mice treated with sham, 10% DMSO and CB-5083. (E) Western blotting and quantification of p21 and GPX4 proteins in liver sections of mice treated with sham, 10% DMSO and CB-5083. (F) Immunofluorescence staining and quantitative analysis of YAP in liver sections of sham-operated, 10% DMSO- and CB-5083-treated mice. (G) Western blotting and quantitative analysis of YAP protein in liver tissues of sham-operated, 10% DMSO- and CB-5083-treated mice. Scale bars: 200 μm (A, B, C, F) and 100 μm (D). All results are expressed as mean ± SD. *p < 0.05; **p < 0.01. DETAILED DESCRIPTION

[0015] 1. Mouse model

[0016] A diet- and chemical-induced NASH mouse model with rapid progression of fibrosis was established by using a Western diet and weekly low-dose intraperitoneal injection of carbon tetrachloride (CCl4) as an accelerator. SPF male C57BL / 6 mice were purchased from Jicui Yaokang and were 8 weeks old. Mice were fed with a Western diet containing 41% sucrose, 21.1% fat, and 1.25% cholesterol (w / w) purchased from Beijing HFK Biotechnology Co., Ltd. for 8 weeks to establish the NASH and fibrosis model. At the same time, mice were given a high-glucose solution containing 23.1 g / L d-fructose and 18.9 g / L d-glucose. In addition, mice were intraperitoneally injected with 0.32 g / kg carbon tetrachloride (CCl4) once a week. CCl4 was dissolved in olive oil at a concentration of 20% (v / v), and olive oil alone was used as a control. Mice were intraperitoneally injected with CB-5083 (sourced from targetmol, dose of 30 mg / kg) at a dose of 100 μL every 3 days. The CB-5083 solution was dissolved in saline containing 10% dimethyl sulfoxide (DMSO). The saline plus 10% DMSO served as the control group.

[0017] 2. Tissue collection and histological examination of mouse samples

[0018] Paraformaldehyde (PFA) was used for tissue analysis and immunohistochemistry; the second segment was embedded in optimal cutting temperature compound (OCT) for cryosectioning; the remaining tissue was stored at -80°C for mRNA or western blotting analysis. For histological analysis, tissues were fixed in 10% PFA for at least 24 h, embedded in paraffin, sectioned into 4-µm-thick slices, and then dewaxed and rehydrated. Fibrotic features were observed by staining with Sirius red or hematoxylin-eosin (H&E). For immunohistochemistry, antigen retrieval was performed by boiling the slides in 10 mM sodium citrate buffer, pH 6.0, for 10 min. The slides were then washed with phosphate-buffered saline (PBS) and incubated with 3% hydrogen peroxide for 10 min. The slides were blocked with 5% goat serum, followed by the addition of primary antibodies and incubation overnight at 4°C. The slides were visualized using an anti-rabbit / mouse immunohistochemistry kit (from ZSGB-BIO). For immunofluorescence, liver tissues in OCT were cut into 6-µm thick sections and then fixed in 10% PFA for 10 minutes. After fixation, they were permeabilized with 0.1% Triton X-100 solution for 10 minutes. After blocking with 5% goat serum for 30 minutes, primary antibodies were added and incubated overnight at 4°C. Secondary antibodies were incubated for 1 hour. Slides were stained with DAPI and then mounted. Antibody related information is as follows.

[0019]

[0020] 3. Cell culture and drug treatment

[0021] Mouse HSCs were purchased from iCell Bioscience and cultured in primary stellate cell complete medium (iCellBioscience, PriMed-icell-009). Human HSCs (LX-2 cells) were cultured in RPMI-1640 complete medium (containing 10% [v / v] fetal bovine serum [FBS]) at 37°C in a humidified incubator with 5% CO2. Initially, HSCs were seeded in 24-well plates and cultured overnight at 37°C. Subsequently, cells were starved in serum-free basal medium for 12 h. Cells were stimulated with TGF-β or different concentrations of CB-5083. After 12 h, cells were collected for further examination. The cytotoxicity of CB5083 was detected using the MTT cell proliferation and cytotoxicity assay kit (Solarbio, M1020) according to the manufacturer's protocol.

[0022] (IV) Senescence-associated β-galactosidase staining (SA-β-gal) analysis

[0023] The Cell Senescence β-Galactosidase Staining Kit (YEASEN, 40754ES60) was used according to the protocol. In brief, after treatment, HSCs were gently washed once with PBS. Cells were fixed with fixative solution for 15 min and then washed again with PBS. Then, the precisely prepared staining solution was applied to the cells and then incubated at 37 °C for 36 h to ensure optimal staining. Finally, the cells were washed twice with PBS to make the staining pattern clearly visible and imaged on an automated microscope.

[0024] 5. Immunoblotting analysis

[0025] Proteins were extracted from HSC cells or liver tissues using lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.1% SDS, 2 mM EDTA, and protease inhibitor cocktail (Roche). Liver tissue sections were placed in lysis buffer for sonication and centrifugation. The protein concentration in the lysate was determined using Coomassie Brilliant Blue G-250. The proteins were bound to the loading buffer by SDS-PAGE and then transferred to a 0.45 µm PVDF membrane. After blocking with 5% skim milk solution in tris-buffered saline for 2 h, the membrane was incubated with primary antibodies overnight at 4°C. Proteins were identified using an ultrasensitive enhanced chemiluminescence kit (from Oriscience Biotechnology) and quantified using ImageJ software. Antibody information is shown in Table 1.

[0026] 6. Real-time quantitative polymerase chain reaction (qRT-PCR)

[0027] Total RNA was extracted from mouse liver tissue or HSC cells using TRIzol reagent according to standard protocols. The mRNA level was quantified using the OneStep qRT-PCR SYBR Green Kit (from Vazyme). The target gene and GAPDH mRNA levels were detected using the ΔΔCT method. The qRT-PCR primer sequences are shown in Table 2.

[0028]

[0029] 7. Construction of stable knockdown cell lines

[0030] Using LV-2 plasmid (pGLVU6 / Puro vector) as a vector, shRNA recombinant lentivirus targeting mouse or human p97 or YAP was constructed. LV-2 plasmid containing shRNA sequence and auxiliary plasmid (pMD2). G and pSPAX2 were co-transfected into HEK293T cells using Lipofectamine 3000 transfection reagent (from Invitrogen). After 48h, the supernatant was collected, filtered, and frozen at -80℃. HSCs were infected with lentivirus and cultured at 37℃ for 24h. For stable knockdown in cell lines, cells were incubated in selected medium containing 10 μg / ml puromycin (from InvivoGen). All knockdown cell lines were confirmed by western blotting or qRT-PCR. The shRNA targeting sequences used in this study are shown in Table 3.

[0031]

[0032] 8. Statistical methods

[0033] GraphPad Prism 6 software was used for statistical analysis of the data. The results are expressed as mean ± standard deviation (SD). Two-tailed Student's t test was used for comparison between two groups, and one-way analysis of variance and Dunnett's post hoc test were used for comparison between multiple groups. A p value less than 0.05 was considered statistically significant, and a p value less than 0.01 was considered extremely significant (*p < 0.05; **p < 0.01).

[0034] The present study showed that the level of p97 / VCP was elevated in mouse liver fibrosis tissue and mainly concentrated in the fibrotic area. In both mouse and human HSCs, downregulation of p97 or antagonism with CB-5083 could reduce the protein expression of α-SMA and collagen-Ⅰ. In addition, inhibition of p97 expression in HSCs induced cell senescence and reduced the expression of the pro-proliferation and anti-aging protein YAP.

[0035] The present study used the Gene Expression Profiling Interactive Analysis (GEPIA) database to analyze transcriptome data from 369 human hepatocellular carcinoma (LIHC) samples and 50 healthy controls, indicating that p97 expression was upregulated in the human HCC group. In a mouse liver fibrosis model, immunohistochemical analysis showed that p97 was significantly increased in fibrotic mouse liver tissue. Immunofluorescence analysis showed that the induced expression of p97 was mainly located in areas where fibrosis occurred, and Desmin staining was positive. p97 was highly expressed in TGF-β-treated mouse HSCs. In human HSC LX-2 cells, TGF-β or FBS treatment promoted high expression of P97 protein ( Figure 2 ).

[0036] The present study showed that p97 / VCP antagonist CB-5083 or p97 knockdown can reduce the fibrotic activity of HSCs. The p97 antagonist CB-5083 was used in mouse HSCs and human LX2 cells ( Figure 3 A). Mouse HSCs were treated with 0.3, 0.1 and 0.03 μM CB-5083 for 12 hours, and CB-5083 reduced the mRNA expression of α-SMA and collagen I. After the p97 antagonist CB-5083 acted on human LX2 cells, CB-5083 also reduced the mRNA expression of α-SMA and collagen I. The present invention uses shRNA to knock down the expression of endogenous p97 in cells. Stable knockout cell lines were established in mouse HSCs and human LX2 cells. After shRNA knocked down the expression of p97 protein in mouse HSCs, the expression of type I collagen and vimentin was reduced ( Figure 3 ).

[0037] The present invention shows that treatment with the p97 antagonist CB5083 can induce HSC senescence. The present invention treated mouse HSCs with 0.3 and 0.1 μM CB-5083. CB-5083 treatment increased the expression of p21, p53 and GPX4 proteins, inducing mouse HSC cell senescence. CB-5083 treatment of mouse HSCs promoted SA-β-Gal activity. The present invention promoted SA-β-Gal activity by treating human LX-2 cells with CB-5083. Knockdown of endogenous p97 by shRNA also increased the expression of p21, p53 and GPX4 proteins in mouse HSCs, indicating that inhibition of p97 can induce HSC senescence ( Figure 4 ).

[0038] The present study showed that treatment with the p97 antagonist CB5083 inhibited YAP expression in HSCs. qRT-PCR and immunofluorescence analysis showed that 0.3 and 0.1 μM CB-5083 reduced the expression of YAP in mouse HSCs. p97 knockdown can also reduce the expression of YAP in mouse HSCs. The present invention uses shRNA to knock down the expression of endogenous YAP in cells, and YAP knockdown reduces the mRNA expression levels of α-SMA and collagen I in mouse HSCs. The YAP inhibitor verteporfin can reduce the expression of α-SMA and collagen I mRNA in mouse HSCs. The present invention shows that p97 inhibition reduces YAP protein expression in HSCs, and YAP has the function of activating HSCs ( Figure 5 ).

[0039] The present study showed that the p97 antagonist CB5083 induced HSCs senescence and improved liver fibrosis in mice. In the diet and CCl4-induced mouse liver fibrosis model, the CB-5083-treated group showed reduced HSCs activation. CB-5083-treated mice had reduced liver fibrosis, including reduced collagen I deposition and weakened activation of mouse HSCs. CB-5083 treatment promoted HSCs senescence in mouse livers, including promoting the expression of p21 and GPX4 in hepatic stellate cells, while inhibiting the expression of YAP ( Figure 6 ).

Claims

1. Application of p97 / VCP inhibitors in the preparation of pharmaceutical preparations for the treatment of liver fibrosis.

2. The use according to claim 1, characterized in that: The p97 / VCP inhibitor is CB-5083, and its structure is shown below: 。 3. The use according to claim 1, characterized in that: The liver fibrosis includes one or more of non-alcoholic fatty hepatitis, cholestatic liver disease, and autoimmune hepatitis.

4. The use according to claim 1, characterized in that: The pharmaceutical preparation reduces the fibrotic activity of hepatic stellate cells by reducing the protein expression of α-SMA and collagen-Ⅰ in hepatic stellate cells, inhibiting the expression of Yes-related protein YAP in hepatic stellate cells, and inducing senescence of hepatic stellate cells.

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