Application of mononuclear macrophage PKM2 as target in preparation of product for treating and / or preventing primary sclerosing cholangitis

Abstract

The invention discloses application of a mononuclear macrophage PKM2 in preparation of a product for treating and / or preventing primary sclerosing cholangitis (PSC), and belongs to the technical field of biological medicine. The expression or activity of the mononuclear macrophage PKM2 is regulated in a targeted mode from multiple angles, reactive bile duct hyperplasia can be remarkably reduced in the PSC treatment process, liver injury can be relieved, fibration around the bile duct can be inhibited, and the purpose of specifically and effectively inhibiting PSC development can be achieved. Therefore, the targeted mononuclear macrophage PKM2 provided by the invention can be used as a new strategy for PSC treatment, and is used for targeted treatment of PSC.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the treatment and / or reversal of primary sclerosing cholangitis, as well as the application of targeting mononuclear macrophage pyruvate kinase M2 (PKM2) in the preparation of products for the treatment and / or prevention of primary sclerosing cholangitis. Background Technology

[0002] Primary sclerosing cholangitis (PSC) is a cholestatic disease characterized by chronic, progressive inflammation of the bile ducts. It primarily manifests as inflammatory destruction of the intrahepatic and extrahepatic bile ducts, followed by cholestasis, liver fibrosis, and ultimately cirrhosis. In recent years, the incidence of PSC has been increasing annually, with a poor prognosis, and currently there are no effective drugs for treating PSC. Therefore, clarifying its molecular mechanisms of development and identifying potential therapeutic targets is of great significance for effectively preventing PSC from progressing to end-stage liver disease and improving patients' quality of life.

[0003] Bile duct reaction is a key pathological feature of post-hepatic cholangiocarcinoma (PSC), manifested as bile duct hyperplasia, inflammation, and fibrosis. Abnormal activation of immune cells is the core mechanism driving the bile duct reaction in PSC. Macrophages are significantly enriched around the bile ducts in PSC, participating in the regulation of bile duct inflammation and fibrosis, and playing a crucial role in PSC progression. On the one hand, mononuclear-derived macrophages are significantly enriched around the bile ducts in the liver tissue of PSC, exhibiting a lipid-associated macrophage (LAM)-like phenotype, and participating in the regulation of bile duct inflammation and fibrosis. Among them, SPP1... + Macrophage infiltration was significantly increased in fibrotic areas of the bile ducts, which was closely associated with fibrosis progression. Serum SPP1 expression was significantly elevated in patients with advanced PSC fibrosis, suggesting it may serve as a potential biomarker for PSC liver transplantation prognosis. Furthermore, bone marrow-derived macrophages were significantly enriched around the bile ducts in patients with PSC and metabolic liver disease, which may be a key factor driving the progression of PSC and metabolic liver disease.

[0004] Pyruvate kinase M2 (PKM2) is a key rate-limiting enzyme in the glycolysis pathway, playing a crucial role in the regulation of macrophage metabolism and function. In chronic liver disease, changes in hepatic PKM2 expression and activity are closely related to disease progression. Studies have shown that glycolysis levels in ihTh17 cells of mice with metabolic liver disease are significantly upregulated; and specific knockout of ihTh17 PKM2 can effectively improve liver inflammation. Furthermore, PKM2 expression and phosphorylation levels in macrophages of fibrotic liver tissue in humans and mice are significantly increased, which can induce a pro-inflammatory phenotype by enhancing cellular glycolysis, thus exacerbating tissue fibrosis. In hepatocellular carcinoma, high expression of PKM2 in tumor cells can induce M2 macrophage polarization and angiogenesis, promoting malignant progression of hepatocellular carcinoma. Overexpression of PKM2 in macrophages of mice with metabolic dysfunction-associated steatohepatitis (MASH) can drive their transformation to a pro-inflammatory phenotype, thereby activating hepatic stellate cells and promoting fibrosis progression; targeted inhibition of macrophage PKM2 can effectively improve MASH fibrosis. These findings suggest that PKM2 plays a crucial role in the progression of chronic liver disease, and that selectively targeting PKM2 in diseased cells may offer a new direction for the treatment of chronic liver disease.

[0005] In cell biology and in vivo animal studies related to liver diseases, various PKM2 inhibitors and allosteric agents have been developed. PKM2 inhibitors, by modulating the activity of the PKM2 protein, can inhibit its role in glycolysis, thereby affecting the energy metabolism and growth of tumor cells. For example, Shikonin is a potent and specific PKM2 inhibitor with multiple biological activities, including anti-tumor, anti-inflammatory, and wound-healing effects. Furthermore, the development of PKM2 allosteric inhibitors or activators has opened new avenues for cancer treatment. For example, DASA-58 is a potent PKM2 allosteric activator and has been used in metabolic and various cancer studies. To date, no experimental results have shown that monocyte / macrophage PKM2 is related to the development and progression of PSC, and no experimental data have confirmed that monocyte / macrophage PKM2 can serve as a therapeutic target for PSC. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide an application of mononuclear macrophage PKM2 as a target in the preparation of products for the treatment of primary sclerosing cholangitis.

[0007] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides the use of PKM2 mononuclear macrophages as a target in the preparation of products for the treatment and / or prevention of primary sclerosing cholangitis. Herein, PKM2 mononuclear macrophages refer to PKM2 in mononuclear macrophages.

[0008] Furthermore, the application is achieved by inhibiting the expression level of PKM2 in monocytes and macrophages.

[0009] Furthermore, the application of substances that inhibit PKM2 expression levels in mononuclear macrophages in the preparation of products for the treatment and / or prevention of primary sclerosing cholangitis.

[0010] Furthermore, the product may be a medicine.

[0011] Furthermore, the drug includes a substance that effectively inhibits the expression level of PKM2 in monocytes and macrophages for treatment and / or prevention.

[0012] Furthermore, the substances that inhibit the expression level of PKM2 in monocytes and macrophages include antisense RNA, siRNA, shRNA or sgRNA that target PKM2 in monocytes and macrophages, or inhibitors and / or allosteric agents that bind to PKM2 in monocytes and macrophages, etc.

[0013] Preferably, the shRNA is shPKM2, and its target sequence is as follows: 5'-CCGTGGGTGCCGTGGAGGC-3'.

[0014] The target sequence in this invention is highly specific to the target gene mRNA sequence and is used to guide shRNA to specifically bind to and silence the expression of the target gene. Moreover, the target sequence has been verified to be a sequence with high target gene knockdown efficiency.

[0015] Furthermore, the drug also contains pharmaceutically acceptable excipients or carriers. The excipients or carriers may be adjusted according to the drug's dosage form, application, etc., and this invention does not limit the specific types and amounts of pharmaceutical excipients or carriers included.

[0016] Furthermore, the excipients or carriers may include at least one of the following: adhesives, diluents, surfactants, lubricants, humectants, preservatives, disintegrants, adsorbents, fillers, etc.

[0017] Furthermore, the aforementioned drugs can be prepared into various pharmaceutical dosage forms using conventional methods. These dosage forms include: capsules, hard capsules, soft capsules, lozenges, powders, granules, tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, drops, tinctures, pills, suspensions, tinctures, oral liquids, and other oral administration dosage forms, as well as injections and other non-oral administration dosage forms, such as injections.

[0018] This invention is based on a novel PSC treatment strategy regulated by PKM2 in mononuclear macrophages. By targeting and inhibiting PKM2 expression or nuclear translocation, it regulates macrophage phenotype and function, thereby reducing bile duct hyperplasia, inflammation and fibrosis, and improving disease progression.

[0019] Secondly, the present invention provides a method for treating, preventing, or alleviating primary sclerosing cholangitis by targeting PKM2 in mononuclear macrophages, comprising administering to a subject in need a pharmaceutical composition containing an effective amount of a PKM2 inhibitor / allosteric agent. The PKM2 inhibitor / allosteric agent can promote macrophage phenotypic and functional transformation, inhibit the release of inflammatory mediators and pro-fibrotic factors, thereby alleviating bile duct hyperplasia, inflammation, and fibrosis.

[0020] Thirdly, this invention also covers intervening in macrophage metabolism and functional reprogramming by inhibiting PKM2 expression and activity, thereby blocking its pro-inflammatory and pro-fibrotic functions. For example, targeted delivery systems of small molecule compounds or traditional Chinese medicines and their natural products (such as nanomedicine delivery systems), siRNA, or gene editing technologies (such as CRISPR-Cas9) can be used to downregulate PKM2 expression or nuclear translocation in monocytes and macrophages to inhibit the activation of functions related to biliary response, thereby improving bile duct injury.

[0021] Fourthly, this invention proposes a combined treatment regimen that combines macrophage PKM2 regulators (allosteric agents, inhibitors, traditional Chinese medicine and their natural products) with existing PSC treatment drugs (such as obeticholic acid and UDCA) to synergistically enhance efficacy. Experimental data show that targeted macrophage PKM2 intervention can significantly downregulate serum alkaline phosphatase (ALP) levels, inhibit bile duct hyperplasia (KRT19) and fibrosis (Masson staining), reduce periductal inflammatory cell infiltration, and delay disease progression.

[0022] Among them, the gene number of human PKM2 is Gene ID: 5315 (PKM pyruvate kinase M1 / 2 [ Homosapiens (human) ]).

[0023] The present invention has the following advantages and effects compared with the prior art: This invention significantly reduces reactive bile duct hyperplasia, alleviates liver damage, and inhibits perichondral fibrosis during PSC treatment by targeting and regulating the expression or activity of PKM2 on monocytes and macrophages. In other words, the targeted regulation of PKM2 on monocytes and macrophages provided by this invention treats PSC from multiple angles, achieving a specific and effective inhibition of PSC progression. Furthermore, in vivo experimental results show that intervention targeting PKM2 on monocytes and macrophages not only effectively improves bile duct hyperplasia and reduces serum ALP levels, but also significantly inhibits perichondral fibrosis. In vitro experimental results show that the absence of PKM2 on monocytes and macrophages downregulates SPP1 expression and secretion, thereby blocking downstream integrin signaling, inhibiting the differentiation of hepatic progenitor cells into bile duct cells, and alleviating bile duct reactions. Therefore, the targeted regulation of PKM2 on monocytes and macrophages provided by this invention can serve as a novel strategy for the targeted treatment of PSC. Attached Figure Description

[0024] Figure 1 The expression of PKM2 in mononuclear macrophages was significantly elevated in the peribiliary bile ducts of human and mouse PSC livers. Specifically, A: flow cytometry analysis was used to determine the proportions of mononuclear cells (Mono), mononuclear macrophages (MoKC), Kupffer cells (KC), neutrophils (NEUT), T cells, and B cells in mouse PSC liver tissue; B: multiplex immunofluorescence assay was used to detect the expression of F4 / 80, CLEC4F, and PKM2 in mouse PSC liver tissue; C: multiplex immunofluorescence assay was used to detect the expression of CD68, VSIG4, and PKM2 in human PSC liver tissue.

[0025] Figure 2 The study showed that PKM2-specific deletion of mononuclear macrophages effectively alleviated PSC induced by DDC diet and BDL in mice. In the first two sections, A and B showed changes in serum ALT, AST, and ALP levels in DDC model mice. In the second section, H&E, Masson staining, and immunofluorescence staining were used to detect liver lesions, fibrosis, and KRT19 expression in mice (left), and the Masson-positive staining area and KRT19-positive region were statistically compared (right). In the third section, D and E showed changes in serum ALT, AST, and ALP levels in BDL model mice. In the fourth section, H&E, Masson staining, and immunofluorescence staining were used to detect liver lesions, fibrosis, and KRT19 expression in mice (left), and the Masson-positive staining area and KRT19-positive region were statistically compared (right).

[0026] Figure 3Activated monocytes and macrophages highly express PKM2; Western blot was used to detect changes in PKM2 expression in THP-1 cells (human monocyte cell line) after in vitro induction for 0, 12, 24 and 48 h.

[0027] Figure 4 The study showed that PKM2-specific loss in monocytes / macrophages alleviates biliary reactivity by inhibiting SPP1-mediated differentiation of hepatic progenitor cells. A: A schematic diagram of co-culture of THP-1 cells and HepaRG cells (human hepatic progenitor cells); B: Immunofluorescence assay of KRT19 expression in HepaRG cells; C: qPCR assay of changes in the expression of biliary tract markers (KRT19, KRT7, EPCAM, SOX9) in HepaRG cells; D: qPCR assay of changes in the expression of integrin signaling genes (ITGA2, ITGA5, ITGA6, ITGB1, ITGB4) in HepaRG cells; E: ELISA assay of SPP1 content in THP-1 cell culture supernatant; F: Immunofluorescence assay of changes in KRT19 expression in HepaRG cells after treatment with SPP1 inhibitor (Compound 11) and integrin inhibitor (CWHM-12).

[0028] Figure 5 Targeted PKM2 therapy for mononuclear macrophages (AAV8-shPKM2) effectively inhibited the progression of PSC. Among them, A and B: partial detection of changes in serum ALT, AST and ALP levels in mice in each treatment group after AAV8-shPKM2 treatment; C: partial detection of liver lesions and fibrosis in mice in each treatment group using H&E and Masson staining (left), and statistical comparison of Masson positive staining area (right); D: partial detection of KRT19 expression in liver tissue of mice in each treatment group using immunofluorescence (left), and statistical comparison of KRT19 positive area (right). Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents used in the following embodiments are all commercially available conventional reagents, and the experimental methods are all conventional methods in the art unless otherwise specified.

[0030] Example 1: Expression of PKM2 in human and mouse PSC liver mononuclear macrophages Step 1: Constructing a DDC-induced mouse PSC model: Ten male C57BL / 6J mice, aged 7 weeks, were purchased and randomly divided into two groups according to body weight, named the control group (ND) and the PSC model group (DDC). The DDC group was fed a diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) to induce the PSC mouse model for 3 weeks. The control group was fed a normal diet. After 3 weeks, the mice were anesthetized for the corresponding experiments.

[0031] Step 2: Analyze the proportion of major immune cells in the liver of PSC mice using flow cytometry and antibody combination: Mice were anesthetized with isoflurane, and non-parenchymal liver cells were isolated by in situ perfusion and in vitro digestion. After routine flow cytometry antibody labeling and staining, T cells (CD45+) were analyzed using spectroscopic flow cytometry. + CD3 + CD19 - ), B cells (CD45) + CD3 - CD19 + ), monocytes (CD45) + CD11b + ), mononuclear macrophages (CD45) + CD11b + F4 / 80 + Kupffer cells (CD45) + CD11b - F4 / 80 + ) and neutrophils (CD45) + Ly6G + The percentage of ). The results are as follows Figure 1 As shown in Figure A, this indicates that the liver mononuclear macrophages (CD45) in the PSC model mouse are... + CD11b + F4 / 80 + Infiltration increased significantly.

[0032] Step 3: Multiplex immunofluorescence detection of PKM2 expression and distribution in mouse and human PSC liver tissue mononuclear macrophages: Mouse and human PSC liver tissues were routinely paraffin-embedded and sectioned. After dewaxing and hydration, multiplex immunofluorescence staining of mouse liver tissues was performed using a TSA kit according to the manufacturer's instructions to detect the expression of F4 / 80, CLEC4F, and PKM2 in mouse PSC liver tissues, and the expression of CD68, VSIG4, and PKM2 in human PSC liver tissues. The fluorescently stained liver tissue sections were observed and photographed under a fast laser scanning confocal microscope. Results are as follows: Figure 1As shown in B and C, this indicates that compared to KC cells (mouse: CLEC4F) + F4 / 80 + Person: VSIG4 + CD68 + MoKC cells (mouse: CLEC4F) surrounding bile ducts in the liver of mice and humans (PSCs) - F4 / 80 + Person: VSIG4 - CD68 + The infiltration was significantly increased, accompanied by significant PKM2 expression.

[0033] The above detection results in this embodiment ( Figure 1 The results indicate that there is significant infiltration of mononuclear macrophages around the bile ducts in human and mouse PSCs, and that PKM2 is highly expressed.

[0034] Example 2: Effects of PKM2-specific deletion in monocytes and macrophages on PSC progression in mice Step 1, constructing a DDC diet-induced PKM2 knockout mouse PSC model of mononuclear macrophages: Pkm2 F / F (Strain#: 024048, Jackson lab, USA) and LysM Cre mice (Strain#: 004781, Jacksonlab, USA) were crossbred and bred in cages for at least 10 generations to construct homozygous mice with PKM2-specific knockout of mononuclear macrophages. Pkm2 △MAC ),give Pkm2 △MAC and its control mice Pkm2 F / F Mice were fed a 0.1% DDC diet in two groups of six male mice (6-8 weeks old). Mouse weight was observed and recorded weekly. Mice were sacrificed and samples were collected after three weeks. All animal experiments followed the National Institutes of Health (NIH) guidelines for mouse care and use and were approved by the Laboratory Animal Management Center of Jinan University. Pkm2 F / F and LysM -Cre mice are also disclosed in the literature "Zhang Di, Wang Lijuan, Li Chong, et al. PKM2 deficiency promotes mucosal repair in ulcerative colitis through macrophage polarization [J]. Chinese Journal of Pathophysiology, 2024, 40(7):1163-1172."

[0035] Step 2, constructing a bile duct ligation (BDL)-induced PKM2 knockout mononuclear macrophage (PSC) mouse model: 6-8 week old male mice were selected. Pkm2 △MAC and its control mice Pkm2 F / F Two groups of eight mice each underwent common bile duct ligation surgery. The procedure was briefly described as follows: Mice were anesthetized with 8% tribromoethanol via intraperitoneal injection. Hair was removed and the skin was prepared. Ophthalmic scissors were used to incise the skin and muscle tissue layer by layer, making an incision 0.5 cm above the xiphoid process to expose the bile duct. A double ligation was performed at the common bile duct using 6-0 surgical sutures. The abdomen was closed. After ligation, the mice were placed in a dry, warm environment for resuscitation. The mice's growth was observed daily, and their weight was recorded every three days. After two weeks, the mice were sacrificed and their tissues collected. All animal experiments followed the guidelines for mouse care and use from the National Institutes of Health (NIH) and were approved by the Laboratory Animal Management Center of Jinan University.

[0036] Step 3: Collect serum from each group of mice for routine serum biochemical tests, including liver function (ALT and AST) and cholestasis (ALP) levels. Results are as follows: Figure 2 As shown in AB and DE, the absence of PKM2 in monocytes and macrophages can significantly downregulate serum ALT, AST and ALP levels in DDC and BDL-induced PSC mice, suggesting that targeting PKM2 in monocytes and macrophages can help alleviate cholestasis and liver damage.

[0037] Step 4, histopathological staining of mouse liver tissue: Routine H&E staining was used to observe changes in liver tissue structure, hepatocyte damage, bile duct hyperplasia, and inflammatory infiltration; Masson staining was used to assess the regression of liver fibrosis; and KRT19 fluorescence staining was used to assess bile duct cell hyperplasia. Results are as follows: Figure 2 As shown in C and F, the absence of PKM2 in mononuclear macrophages can significantly inhibit bile duct hyperplasia, inflammation, and fibrosis in PSC mice.

[0038] The above detection results in this embodiment ( Figure 2 The study suggests that targeting PKM2 in monocytes and macrophages can effectively alleviate PSC.

[0039] Example 3: PKM2 expression during monocyte / macrophage activation THP-1 cells (human monocytic leukemia cell line THP-1, derived from human monocytic leukemia patients, purchased from Wuhan Pronosei Life Sciences Co., Ltd.) in the exponential growth phase were seeded in 6-well plates and co-cultured with 30 ng / mL CSF1 (colony-stimulating factor 1) and 50 ng / mL Ac-LDL (acetylated low-density lipoprotein). Cells were collected at 0, 12, 24, and 48 h of culture, and proteins were extracted. Western blot was used to detect the expression of PKM2 in THP-1 cells after 0, 12, 24, and 48 h of in vitro induction. Results are as follows: Figure 3As shown, the expression of PKM2 gradually increases during the differentiation of monocytes into mature macrophages.

[0040] Example 4: Effect of PKM2 deficiency in mononuclear macrophages on bile duct reactivity Differentiation of hepatic progenitor cells into bile duct cells is key to the biliary response in post-spinal cholangiocarcinoma (PSC). To observe the effect of PKM2 deletion in monocytes / macrophages on hepatic progenitor cell differentiation in vitro, a stable PKM2 knockout cell line of monocytes / macrophages was first constructed. The target sequence of shPKM2 was 5'-CCGTGGGTGCCGTGGAGGC-3', and the nonsense sequence was designated as the target sequence of shNC.

[0041] Step 1, PKM2 Specific short hairpin RNA (shPKM2) lentivirus and control short hairpin RNA (shNC) lentivirus were constructed by Shanghai Jikai Gene Technology Co., Ltd. Following the lentivirus operation manual provided by Jikai, PKM2 stably knocked-out THP-1 cells were constructed. Single-clonal cell lines were screened and expanded. Western blot was used to identify and select PKM2 stably knocked-out THP-1 cells (PKM2-KO) for subsequent expansion culture.

[0042] Step 2: The experiment was divided into the following 3 groups: control group, shNC group, and shPKM2 group. A schematic diagram of the co-culture is shown below. Figure 4 As shown in A in the diagram. In the shPKM2 and shNC groups, PKM2-KO and its control cell lines were seeded in the upper chamber of a 6-well plate. After induction for 48 h with 30 ng / mL CSF1 and 50 ng / mL Ac-LDL, HepaRG cells (purchased from Shanghai Qiansi Biotechnology Co., Ltd.) with hepatic progenitor cell characteristics were seeded in the lower chamber. In the Control group, control cell lines were seeded in the upper chamber of a 6-well plate without induction. After 48 h, HepaRG cells were seeded in the lower chamber. Cells from both the upper and lower chambers were collected after 7 days of culture. Immunofluorescence and qPCR were used to detect the mRNA expression of KRT19 protein, bile duct differentiation-related genes (KRT19, KRT7, EPCAM, SOX9), and integrin signaling genes (ITGA2, ITGA5, ITGA6, ITGB1, ITGB4) in HepaRG cells. ELISA was used to detect the SPP1 (secretory phosphoprotein 1) content in the culture supernatant of the upper chamber. The results are shown in the diagram. Figure 4 As shown in B and CE, PKM2 deficiency can reduce the expression and secretion of SPP1 in monocytes and macrophages, thereby inhibiting the differentiation of hepatic progenitor cells into bile duct cells by downregulating cytokinin signal transduction.

[0043] Step 3: Using the same cell co-culture method as in Step 2, THP-1 cells were seeded in the upper chamber and induced with a combination of 30 ng / mL CSF1 and 50 ng / mL Ac-LDL for 48 h. HepaRG cells, exhibiting hepatic progenitor cell characteristics, were then seeded in the lower chamber. Experimental groups were as follows: the model group (Vehicle) received no drug treatment; the SPP1 inhibitor group received 1 μM of Compound 11 (CAS: 842964-18-5, Selleck) in the upper chamber; and the integrin inhibitor group received 10 nM of CWHM-12 (CAS: 1564286-55-0, MCE) in the lower chamber. After culturing for another 24 h, the expression of KRT19 protein in HepaRG cells was detected by immunofluorescence. Results are as follows: Figure 4 As shown in F, blocking SPP1-Intergrin axis signal transduction can inhibit the differentiation of hepatic progenitor cells into bile duct cells.

[0044] The above detection results in this embodiment ( Figure 4 The results suggest that PKM2 on monocytes and macrophages can promote the expression and secretion of SPP1 in vitro, thereby activating integrin signaling in hepatic progenitor cells and inducing their differentiation into bile duct cells. Therefore, targeting PKM2 on monocytes and macrophages can reduce SPP1 secretion, inhibit the differentiation of hepatic progenitor cells into bile duct cells, and thus alleviate bile duct reactions.

[0045] Example 5: The effect of PKM2-targeted mononuclear macrophage therapy on PSC progression Based on the shPKM2 and shNC sequences in Example 4, adeno-associated virus (AAV8-shPKM2) targeting PKM2 in monocytes and macrophages, and a control short hairpin RNA (shNC) adeno-associated virus (AAV8-shNC) were constructed by Shanghai Jikai Gene Technology Co., Ltd. Six- to eight-week-old male mice (purchased from Vital River) were randomly divided into two groups: a control group (AAV8-shNC) and a treatment group (AAV8-shPKM2), with eight mice in each group. All mice were fed a diet containing 0.1% DDC. One week later, the mice in the treatment group were treated with a tail vein injection of AAV8-shPKM2 virus at a dose of 1×10⁻⁶. 13 Vg / mouse; control mice were injected with an equal volume of AAV8-shNC via the tail vein and sacrificed 4 weeks later for tissue collection. Mouse serum was used for ALT, AST, and ALP detection; liver tissue was used for H&E and Masson staining and KRT19 fluorescence detection. All animal experiments followed the National Institutes of Health (NIH) guidelines for mouse care and use and were approved by the Laboratory Animal Management Center of Jinan University. Results are as follows: Figure 5As shown, targeting hepatic mononuclear macrophages PKM2 can significantly downregulate serum ALT, AST and ALP levels, and inhibit hepatic bile duct hyperplasia and fibrosis.

[0046] The above detection results in this embodiment ( Figure 5 The study suggests that targeting PKM2 in monocytes and macrophages can effectively inhibit the progression of PSC.

[0047] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of PKM2 mononuclear macrophages as a target in the preparation of products for the treatment and / or prevention of primary sclerosing cholangitis, characterized in that, The application is achieved by inhibiting the expression level of PKM2 in monocytes and macrophages.

2. Application of substances that inhibit PKM2 expression levels in mononuclear macrophages in the preparation of products for the treatment and / or prevention of primary sclerosing cholangitis.

3. The application of substances that inhibit the expression level of PKM2 in mononuclear macrophages in combination with drugs for the treatment of primary sclerosing cholangitis in the preparation of products for the treatment and / or prevention of primary sclerosing cholangitis.

4. The application according to claim 1, 2, or 3, characterized in that: The product is a medicine.

5. The application according to claim 4, characterized in that: The drug includes a substance that can treat and / or prevent the expression of PKM2 in monocytes and macrophages in an effective amount.

6. The application according to claim 5, characterized in that: The substances that inhibit the expression level of PKM2 in monocytes and macrophages include antisense RNA, siRNA, shRNA or sgRNA that target PKM2 in monocytes and macrophages, or inhibitors and / or allosteric agents that bind to PKM2 in monocytes and macrophages.

7. The application according to claim 6, characterized in that: The shRNA is shPKM2, and its target sequence is as follows: 5'-CCGTGGGTGCCGTGGAGGC-3'.

8. The application according to claim 4, characterized in that: The drug also contains pharmaceutically acceptable excipients or carriers.

9. The application according to claim 8, characterized in that: The excipients or carriers include at least one of the following: adhesives, diluents, surfactants, lubricants, humectants, preservatives, disintegrants, adsorbents, and fillers.

10. The application according to claim 4, characterized in that: The drugs described herein are prepared into various pharmaceutical dosage forms using conventional methods. These dosage forms include: capsules, hard capsules, soft capsules, lozenges, powders, granules, tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, drops, tinctures, pills, suspensions, tinctures, oral liquids, and other oral dosage forms such as injections.

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