Application of mTOR inhibitor Torin1 in the preparation of drugs for cholestatic bile duct injury

Abstract

The present invention relates to the field of biotechnology, and specifically to the use of the mTOR inhibitor Torin1 in the preparation of drugs for cholestatic bile duct injury. mTOR is an evolutionarily conserved serine / threonine kinase that plays a key role in cell growth and metabolism. Torin 1 is an inhibitor of mTOR. mTOR regulates cell growth, movement and metabolism by forming two multiprotein complexes, mTORC1 and mTORC2. mTORC1 is sensitive to nutrients, and mTORC2 is regulated by PI3K and growth factor signaling pathways. The present invention found that Torin 1 has a significant therapeutic effect on cholestatic bile duct injury. After treatment, it can significantly improve liver function indicators and pathological changes such as inflammatory cell infiltration, bile duct hyperplasia, and periductal liver fibrosis in patients with cholestatic bile duct injury; this inhibitor can significantly reduce the level of inflammatory factors in animal models of cholestatic bile duct injury.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to application of an mTOR inhibitor, Torin1, in preparing a drug for cholestatic bile duct injury. Background Art

[0002] Primary sclerosing cholangitis (PSC) is a chronic, progressive bile duct disease caused by cholestatic bile duct damage. It is characterized by progressive inflammation and fibrosis of the intrahepatic and extrahepatic bile ducts, leading to multifocal bile duct strictures. Most patients eventually develop cirrhosis, portal hypertension, and hepatic decompensation. Currently, there is no effective treatment. Bile duct hyperplasia and bile duct activation are early manifestations of the disease. Biliary epithelial cells secrete various cytokines and related proteins, which in turn induce the proliferation of periductal fibrosis, leading to cholestatic liver fibrosis. This fibrous proliferation, in turn, stimulates morphological changes in bile duct cells, including hypertrophy, metaplasia, atrophy, and necrosis. Many foci of hyperplasia and fibrosis in the portal tract gradually connect, forming cirrhosis.

[0003] The establishment of an animal model of cholestatic bile duct injury provides a pathway for basic research, therapeutic studies, and drug screening for the prevention and treatment of cholestatic bile duct injury. Chronic DDC (3,5-Diethoxycarbonyl-1,4-dihydrocollidine) feeding is a xenobiotic-induced cholestatic bile duct injury model. This model involves feeding mice a diet containing 0.1% DDC. DDC feeding increases the secretion of porphyrins, forming porphyrin emboli, leading to abnormal proliferation, degeneration, and necrosis of bile duct epithelial cells and hepatocytes. It also increases the expression of vascular adhesion molecules, osteopontin (OPN), and tumor necrosis factor α (TNF-α) in bile duct epithelial cells, ultimately leading to an increase in peribiliary inflammatory cells and bile duct proliferation. This model mimics the pathogenesis of PSC and is currently recognized as an ideal model for studying the pathogenesis of PSC. The establishment of this animal model provides a way for basic research, treatment research and screening of drugs for the prevention and treatment of cholestatic bile duct injury. Summary of the Invention

[0004] The present invention aims to address the technical problem that Torin 1 inhibits the mTOR signaling pathway, alleviating its role in DDC-induced bile duct injury, specifically cholestatic bile duct injury. By inhibiting the mTOR signaling pathway, Torin 1 aims to clarify the therapeutic effect of Torin 1 in DDC-induced bile duct injury.

[0005] Specifically, the use of the mTOR inhibitor Torin1 in the preparation of drugs for cholestatic bile duct injury.

[0006] As a preferred embodiment of the use of the mTOR inhibitor Torin1 in the preparation of a drug for cholestatic bile duct injury according to the present invention: the mTOR regulates cell growth, movement and metabolism by forming two multiprotein complexes, mTORC1 and mTORC2.

[0007] As a preferred embodiment of the use of the mTOR inhibitor Torin1 in the preparation of a drug for cholestatic bile duct injury according to the present invention: Torin1 is an inhibitor of the mTOR complexes mTORC1 and mTORC2.

[0008] As a preferred embodiment of the use of the mTOR inhibitor Torin1 in the preparation of a medicament for cholestatic bile duct injury according to the present invention: Torin1 inhibits the phosphorylation of mTORC1 and mTORC2 substrates at concentrations of 2 nM and 10 nM, respectively.

[0009] A drug for treating cholestatic bile duct injury, comprising the mTOR inhibitor Torin1.

[0010] The drug for treating cholestatic bile duct injury also contains a pharmaceutically acceptable carrier.

[0011] The pharmaceutically acceptable carrier refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance with a reasonable benefit / risk ratio.

[0012] This inhibitor, Torin 1, is a potent mTOR inhibitor with an IC50 of 3 nM when formulated as a drug for cholestatic bile duct injury. Torin 1 inhibits the mTORC1 / 2 complex.

[0013] Compared with the existing technology, it has the following beneficial effects:

[0014] After extensive and in-depth research, it was discovered for the first time that the mTOR inhibitor Torin1 has a significant therapeutic effect on cholestatic bile duct injury, and it was also discovered for the first time that the mTOR inhibitor Torin1 has a new use in the treatment of cholestatic bile duct injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1, showing that Torin1 improves DDC-induced cholestatic bile duct injury. A: Liver gross appearance; B: Liver weight / body weight ratio; C: ALT; D: ALP; E: TBIL, compared with the corresponding groups, * p <0.05,*** p <0.001.

[0016] Figure 2 , shows that Torin1 ameliorates liver pathological changes in DDC-induced cholestatic bile duct injury (H&E staining). Black arrows indicate bile duct epithelial cell proliferation and inflammatory cell infiltration.

[0017] Figure 3 , showing that Torin1 improves collagen fiber deposition in DDC-induced cholestatic bile duct injury (Masson staining) A: Collagen fiber deposition in the liver of mice in each group B: Masson staining image analysis results, compared with the corresponding groups, * p <0.05,** p <0.01,*** p <0.001.

[0018] Figure 4 , showing that Torin1 reduces the proliferation of bile duct epithelial cells (CK19) in DDC-induced cholestatic bile duct injury. A: Distribution of CK19 in liver tissues of mice in each DDC model group. B: Semi-quantitative analysis of CK19 expression, compared with the corresponding groups, ** p <0.01,*** p <0.001.

[0019] Figure 5 , showing that Torin1 reduces the proliferation of hepatocytes in DDC-induced cholestatic bile duct injury. A: Distribution of Ki67 in liver tissues of mice in each DDC model group B: Semi-quantitative analysis of Ki67 expression, compared with the corresponding groups, * p <0.05,*** p <0.001.

[0020] Figure 6 , indicating that Torin1 reduces the expression of proinflammatory cytokines in DDC-induced cholestatic bile duct injury. qRT-PCR results showed that compared with the DDC group, the liver tissue of mice in the DDC+Torin1 group Il6 (A) Mcp1 (B) The expression levels of pro-inflammatory cytokines such as Il10 (C) Arg1 (D) The expression levels of anti-inflammatory cytokines such as IL-6 and IL-7 increased. Compared with the corresponding groups, * p <0.05,** p <0.01,***p <0.001.

[0021] Figure 7 , showing that Torin1 reduces the expression levels of related proteins in the DDC-induced cholestatic bile duct injury model. A: Western blot analysis of mTOR, P-mTOR, Akt, P-Akt (Ser473), p65, and P-p65 protein expression. BD: Semi-quantitative analysis of related proteins compared with the corresponding groups. * p <0.05,** p <0.01. DETAILED DESCRIPTION

[0022] Example 1: Torin1 improves DDC-induced cholestatic bile duct injury

[0023] 1. Establishment and grouping of animal models of cholestatic bile duct injury

[0024] Experimental animals: Experimental animal grouping and model establishment: 6-8 week old SPF female C57BL / 6J mice with an average weight of 20-30 g, good activity, were housed in a specific pathogen-free environment.

[0025] Experimental mouse groups

[0026] Before establishing the model mice, the experimental mice were divided into groups. Twenty-four mice were randomly assigned to a solvent group (NMP, N-methylpyrrolidone, served as the solvent control group), a Torin1-treated group, a DDC+NMP group, and a DDC+Torin1 group, with six mice in each group. (Note: Torin1, as used throughout this article, refers to the mTOR inhibitor).

[0027] Establishment of an animal model of induced bile duct injury

[0028] The treatment group received an intraperitoneal injection of Torin1 every week, and both groups received injections every other day for 3 consecutive weeks. During this period, the control group received an intraperitoneal injection of 0.1% (NMP) every day. Torin1 was dissolved in NMP according to the instructions, with a storage concentration of 30 mg / ml. It was intraperitoneally injected every two days at a dose of 10 mg / kg body weight. Samples were collected and related tests were performed 24 hours after the last injection of Torin1. After 3 weeks, the mice were killed, the livers were separated, and the gross pathological changes of the mouse livers were observed with the naked eye. It was found that: in the NMP solvent group and the Torin1 treatment group, the mouse livers were bright red, with a smooth surface, a clear interface, and a soft texture. The livers of the mice in the DDC group were obviously congested and swollen, with a dark brown color, a granular surface, and a tough texture. In the (DDC+Torin1) group, the liver congestion and swelling were significantly reduced, the color was dark red, the surface was not granular, and the texture was slightly tough (see Figure 1 A), the liver weight / body weight was also significantly improved compared with the DDC group (see Figure 1 B, p <0.05).

[0029] 2. Sample Collection and Processing

[0030] Collect liver tissue samples

[0031] Three weeks after modeling, blood was collected from the eyeballs and the mice were sacrificed by cervical scissors. The chest cavity was opened at the linea alba with ophthalmic scissors, the liver tissue was exposed, and the gallbladder was removed from the liver with scissors. Serum was separated, and the eyeball blood was centrifuged at 3500 rpm and 4°C for 15 minutes in a low-temperature centrifuge. The blood was aliquoted and stored frozen at -80°C until further use.

[0032] The removed liver tissue was placed on a glass block on ice. A piece of liver tissue (1 cm × 1 cm × 1 cm) was taken 4 mm from the edge of the liver tissue and fixed in 4% paraformaldehyde solution for HE staining. The remaining liver tissue was cut into several pieces (1 cm × 1 cm × 1 cm) and frozen in a -80°C refrigerator for later use.

[0033] Detection of ALT, ALP, and TBIL in serum

[0034] After blood was allowed to clot, it was centrifuged at 3500 rpm for 15 minutes, and serum was separated and immediately tested. Serum ALT, ALP, and TBIL levels were measured using an automatic biochemical analyzer provided by the Department of Laboratory Medicine of the Affiliated Hospital of Xuzhou Medical University.

[0035] The results are as follows Figure 1 The results showed that liver damage was obvious after DDC administration, and the serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin (TBIL) were significantly increased ( p <0.001), while after Torin1 treatment, ALT content was significantly increased compared with that in the DDC group (see Figure 1 C), ALP content (see Figure 1 As shown in D, p <0.001) and TBIL levels were significantly reduced (see Figure 1 As shown in E, p <0.001). The results showed that Torin1 could significantly reduce the liver and gallbladder injury indicators induced by DDC.

[0036] dyeing

[0037] In order to study the liver pathological damage induced by DDC in C57BL / 6 mice, we used conventional HE staining to observe the liver pathology. Figure 2As shown in A, the liver lobules in the NMP solvent group and the Torin1 treatment group were intact with relatively clear boundaries, and the liver cell structure was complete and full, and no bile duct hyperplasia was found; while in the DDC model group, more inflammatory cells such as neutrophils and monocytes infiltrated around the central vein, the bile duct became thicker and larger, and hyperplasia was obvious. There was protoporphyrin embolism in the bile duct, indicating that the bile duct was obstructed and accompanied by hyperplasia. The number of collagen fibers increased, and more obvious fibrous septa were seen. Fibroblasts gathered around the bile duct, indicating that liver fibrosis occurred. Compared with the DDC group, the DDC+Torin1 group had less inflammatory cell infiltration in the liver, less bile duct hyperplasia, less porphyrin embolism in the bile duct, and less fibroblasts gathered around the bile duct (see Figure 2 A, B, p <0.05). The results showed that Torin1 treatment could significantly reduce DDC-induced inflammatory cell infiltration and bile duct epithelial cell proliferation.

[0038] dyeing

[0039] In order to observe the collagen fiber deposition in the liver of each group of mice, we performed Masson staining on the liver tissue of each group of mice (Masson staining kit was purchased from Nanjing Jiancheng Bioengineering Institute) and observed the Masson staining of the mouse liver tissue under an ordinary optical microscope. The results are as follows Figure 3 As shown in A: The liver lobule structure of mice in the normal group (NMP solvent group and Torin1 treatment group) was clear and complete, and the hepatocytes were arranged radially with the central vein as the center. There was no collagen fiber proliferation. A small amount of blue collagen fibers were distributed on the vascular walls of the central vein and the portal area of ​​the hepatic sinusoids. The vascular walls of the portal area and the central vein area were blue. The liver lobule structure of mice in the model group (DDC group) was disordered, with obvious deposition of collagen fibers. There was also obvious blue deposition around the dilated bile duct and along the course of the bile duct. Image J software analysis showed that compared with the control group, the blue area was significantly increased, indicating that the increased deposition of collagen fibers occurred around the bile duct. After Torin1 treatment, there was a significant improvement, the deposition area of ​​collagen fibers around the bile duct was reduced, and the degree of fibrosis was alleviated (see Figure 3 B, p <0.05).

[0040] Immunohistochemical detection of bile duct epithelial cell proliferation

[0041] Paraffin sections (4 μm thick) were microwave-treated (0.01 mmol / L citrate buffer, pH 6.0) and blocked with 5% BSA for 30 minutes at room temperature. Monoclonal mouse anti-CK19 and anti-Ki67 antibodies (1:100 dilution; Abcam) were then used. A control group was treated with 1% BSA and incubated in a humidified chamber at 4°C overnight. The next day, the slides were removed and a secondary antibody diluted according to the manufacturer's instructions was added. The slides were then placed in a humidified chamber and incubated at 37°C for 30 minutes. DAB color development solution was prepared and applied dropwise to the sections, and color development was observed under a microscope. Hematoxylin stain was then applied dropwise to the slides, incubated at room temperature for 5–10 seconds, rinsed with running water, dehydrated, and mounted. Positive distribution was observed under a light microscope and quantified using ImageJ.

[0042] The results are as follows Figure 4 、 Figure 5 The results showed that compared with the normal group, the expression of CK19 and Ki67 in the DDC diet group was significantly increased, and Torin1 could reduce the expression of CK19 in the liver of mice fed with DDC diet ( Figure 4 A, B, p <0.05), Ki67 expression (see Figure 5 A, B, p <0.05). Therefore, this example once again confirmed that Torin1 alleviates DDC-induced bile duct injury by inhibiting mTOR signaling.

[0043] Expression of inflammatory factors in mouse liver tissue

[0044] The relative mRNA expression levels of pro-inflammatory factors (IL-6 and MCP1) and anti-inflammatory factors (IL-10 and Arg1) were determined. Total RNA from liver tissues of each group was extracted using the Trizol method according to the manufacturer's instructions. RNA was reverse transcribed into cDNA and then amplified by PCR. β-actin was used as an internal reference, and the target gene mRNA expression was measured using 2 -∆∆ Ct calculation, primer sequences are shown in Table 1.

[0045] qRT-PCR results are as follows Figure 6 The results showed that compared with the DDC group, the liver tissue of mice in the DDC+Torin1 group Il6 ( p <0.001) Mcp1 ( p <0.01) and other pro-inflammatory cytokines, Il10 ( p <0.001) Arg1 ( p <0.001). Therefore, Torin1 can reduce DDC-induced liver inflammation in mice.

[0046] Table 1 Primer sequences used in this example

[0047]

[0048] Western blot was used to detect the levels of mTOR, P-mTOR, Akt, P-Akt (Ser473), p65, and P-p65 proteins and their phosphorylation levels.

[0049] Total protein from each group was extracted and the concentration was measured using the BCA method. 40 μg of protein sample was loaded into each well for SDS-PAGE electrophoresis and wet transfer was performed using a Bio-Rad standard transfer device. The membrane was blocked with 5% skim milk at room temperature for 2 h, incubated with the primary antibody on a shaker at 4°C overnight, and washed. The membrane was incubated with the secondary antibody at room temperature for 2 h, washed, and exposed to light with ECL developer. The results were analyzed using ImageLab software. The ratio of the gray value of the target protein to the gray value of the corresponding internal reference protein was used for statistical analysis. The experiment was repeated three times.

[0050] The results showed that the phosphorylation levels of P-mTOR, P-AKT and P-p65 in the DDC group were significantly increased, while those in the DDC+Torin1 group were significantly decreased compared with the DDC group (see Figure 7 , p <0.05). Therefore, Torin1 effectively inhibited DDC-induced activation of the Akt / mTOR / NF-κB pathway.

[0051] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all within the scope defined by the claims of this application.

Claims

1. Application of mTOR inhibitor Torin1 in the preparation of drugs for cholestatic bile duct injury.

2. Use of the mTOR inhibitor Torin1 according to claim 1 in the preparation of a drug for cholestatic bile duct injury, characterized in that: The mTOR regulates cell growth, motility and metabolism by forming two multiprotein complexes, mTORC1 and mTORC2.

3. Use of the mTOR inhibitor Torin1 according to claim 2 in the preparation of a drug for cholestatic bile duct injury, characterized in that: Torin1 is an inhibitor of the mTOR complexes mTORC1 and mTORC2.

4. Use of the mTOR inhibitor Torin1 according to claim 2 in the preparation of a drug for cholestatic bile duct injury, characterized in that: Torin1 inhibits the phosphorylation of mTORC1 and mTORC2 substrates at concentrations of 2 nM and 10 nM, respectively.

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