Medicaments and methods for treating radiation-induced liver damage

Abstract

The present application discloses a medicine and method for treating radiation-induced liver injury. Specifically, the research results of the present application show that the expression of Vcam1 gene in the liver of mice after whole-body irradiation by X-rays is significantly up-regulated in the case of Trim58 gene knockout, indicating that Trim58 may hinder the participation of Vcam1 in the radiation-induced liver injury protection mechanism, thereby aggravating the progress of inflammatory injury. On this basis, the present application provides a medicine and method for treating radiation-induced liver injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically, it relates to drugs and methods for treating radiation-induced liver injury. Background Technology

[0002] Radiation therapy is a crucial treatment for cancer, affecting approximately 50% of cancer patients, making it a valuable tool in the field. This treatment relies on ionizing radiation to induce tumor cell death. Ionizing radiation can directly or indirectly ionize matter, thereby disrupting the DNA structure of tumor cells and ultimately inhibiting tumor growth. The damage to biomolecules caused by radiation involves two main pathways: direct and indirect effects. On one hand, radiation can directly act on DNA molecules, causing DNA strand breaks and base repair damage. On the other hand, radiation indirectly generates reactive oxygen species (ROS) through the radiolytic decomposition of water. These ROS attack cell membranes, proteins, and DNA, triggering oxidative stress and leading to cell death pathways such as apoptosis, necrosis, or autophagy. According to research by Dr. Ravi A. Chandra's team, radiation therapy damages the DNA of cancer cells by releasing high-energy radiation, thereby inhibiting their proliferation. Because radiation has penetrating power, it allows for precise targeting of tumor cells while minimizing damage to surrounding healthy tissues.

[0003] The damage mechanism of ionizing radiation to cells is mainly achieved by disrupting intracellular biomolecules (especially DNA molecules). When radiation passes through a cell, its energy can induce the cell to produce free radicals. These free radicals interact with DNA, leading to DNA strand breaks or other forms of structural damage. After being exposed to radiation, cells activate endogenous repair mechanisms to cope with the DNA molecule damage. However, due to the biological characteristics of cancer cells and the limitations of their repair capabilities, their radiation sensitivity is usually higher than that of normal cells, ultimately resulting in a higher mortality rate.

[0004] Radiation therapy is widely used in clinical practice to treat various malignant tumors, including lung cancer, prostate cancer, breast cancer, and brain tumors. In cancer treatment strategies, radiation therapy can serve as a primary radical treatment for early-stage cancers, while for advanced-stage patients it can effectively alleviate symptoms and prolong survival. In medical practice, radiation therapy is often combined with other treatments such as surgical resection, chemotherapy, and immunotherapy to improve treatment outcomes. For patients with non-small cell lung cancer, stereotactic body radiotherapy (SBRT) has achieved efficacy similar to surgery in the early stages of the disease, while in advanced cases it alleviates clinical symptoms and improves the quality of life.

[0005] With the continuous development of modern radiotherapy technology, intensity-modulated radiotherapy (IMRT) and side-scaling radiotherapy (SBRT), with their precise dose distribution control and highly concentrated high-dose characteristics, have effectively improved tumor control while reducing the possibility of damage to surrounding normal tissues. However, the toxic reactions caused by radiation, such as acute mucositis, dermatitis, late-stage fibrosis, and vascular damage, limit further optimization of treatment doses. Therefore, the clinical application of radiotherapy still faces many challenges. In the future, with continuous technological advancements, radiotherapy will achieve breakthroughs in areas such as the development of personalized treatment plans, the optimization of combination therapy strategies, and the validation of novel radiotherapy technologies, thereby promoting higher levels of effectiveness and safety in cancer treatment.

[0006] Liver cancer, a globally prevalent malignant tumor, poses a significant threat to human health, with its incidence and mortality rates steadily increasing in recent years. Hepatocellular carcinoma (HCC), particularly common in Asia and Africa, is a major subtype of liver cancer, and studies have demonstrated a significant association between pathogenic factors such as chronic hepatitis B virus infection and the disease. Currently, clinical treatments for liver cancer include surgical resection, liver transplantation, local ablation, transarterial chemoembolization (TACE), and systemic therapy, among many other options. However, most patients are diagnosed at an advanced stage and often have underlying conditions such as cirrhosis, making surgery or other radical treatments unsuitable for many. The traditional chemotherapy drug cisplatin can induce tumor cell apoptosis, but its use is limited by drug resistance and hepatotoxicity. Against this clinical backdrop, radiotherapy (RT) has gradually become a core treatment for liver cancer, and its importance in comprehensive treatment plans will continue to rise for patients with liver cancer who are not candidates for surgical resection or are in a locally advanced stage.

[0007] With the rapid development of radiotherapy technology, emerging techniques such as stereotactic body radiotherapy (SBRT) and proton beam therapy have become increasingly widespread. These techniques not only allow for precise targeting of high-dose radiation to the tumor area but also effectively protect surrounding normal tissues from damage. Their application has significantly improved the local control and long-term survival of liver cancer. Stereotactic body radiotherapy (SBRT) can deliver high-dose radiation to tumor tissue in a relatively short time, and studies have shown that its treatment effect on early-stage liver cancer patients is similar to that of surgical resection. Radiation segmentectomy, as an emerging technique, employs highly precise radioembolization technology combined with a targeted dose distribution scheme. This effectively controls the extent of tumor growth while maximizing the protection of normal liver tissue function, thus providing favorable conditions for patients to undergo subsequent surgical intervention or systemic treatment.

[0008] Currently, radiotherapy plays an important role in the clinical treatment of liver cancer, especially for patients with liver cancer who cannot undergo surgical resection. However, radiation-induced liver injury is the most prominent dose-limiting toxicity during radiotherapy, which directly affects the promotion and application of radiotherapy technology in this field. Therefore, in-depth understanding of the pathological mechanism of radiation-induced liver injury and the development of targeted prevention and treatment programs have become core issues that need to be addressed in the field of radiotherapy research for liver cancer.

[0009] The pathogenesis of liver-derived disease (RILD) involves a complex regulatory process involving multiple factors and stages. Ionizing radiation can directly cause DNA double-strand breaks and promote the production of large amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS), thereby triggering oxidative stress responses. These changes ultimately lead to damage and even apoptosis of hepatocytes and non-parenchymal cells. Radiation activates inflammatory signaling pathways, leading to the release of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), thus exacerbating liver tissue damage and the progression of fibrosis. Studies have shown that the occurrence of RILD is significantly related to factors such as radiation dose, irradiation range, and the patient's liver function status. Currently, the clinical diagnosis of RILD mainly relies on clinical manifestations and imaging results. However, due to the lack of early and sensitive biomarkers, some patients are diagnosed at an advanced stage of the disease, thus missing the optimal treatment opportunity. In the treatment of liver cancer, the combined use of radiotherapy with other treatments such as TACE and immunotherapy can enhance the treatment effect of liver cancer, but it may also increase the risk of RILD.

[0010] Therefore, how to improve the tumor control rate while minimizing the probability of RILD is a major challenge in the comprehensive treatment of liver cancer. In-depth analysis of the pathogenesis of radiation-induced liver injury has become an important research direction in the field of cancer radiotherapy. Summary of the Invention

[0011] The purpose of this invention is to provide a drug and method for treating radiation-induced liver injury.

[0012] In a first aspect, the present invention provides a medicament for treating radiation-induced liver injury, the medicament comprising a Trim58 inhibitor and / or a Vcam1 activator.

[0013] In another preferred embodiment, the Trim58 inhibitor is an inhibitor of the Trim58 gene.

[0014] Preferably, it is: Trim58 gene-specific siRNA or its precursor, Trim58 gene-specific microRNA or its precursor, inhibitor of the Trim58 gene promoter, or a combination thereof.

[0015] In another preferred embodiment, the inhibitor of the Trim58 gene is a Trim58 gene-specific siRNA or its precursor, or a microRNA or its precursor.

[0016] In another preferred embodiment, the siRNA specifically targets the Trim58 gene sequence or its complementary sequence.

[0017] In another preferred embodiment, the Trim58 inhibitor is an inhibitor of the protein encoded by the Trim58 gene.

[0018] In another preferred embodiment, the inhibitor of the Trim58 gene-encoded protein is selected from the group consisting of: antibodies against the Trim58 gene-encoded protein and binding proteins of the Trim58 gene-encoded protein.

[0019] In another preferred embodiment, the Vcam1 activator is a substance (such as a compound or protein) that enhances the expression of the Vcam1 gene.

[0020] In a second aspect, the present invention provides a method for treating radiation-induced liver injury, the method comprising the steps of inhibiting the expression of the Trim58 gene or inhibiting the activity of the Trim58 protein in a subject, and / or increasing the expression of the Vcam1 gene or increasing the activity of the Vcam1 protein in a subject.

[0021] In another preferred embodiment, the method includes the step of administering the drug described in the first aspect of the invention to a subject.

[0022] In another preferred embodiment, the Trim58 inhibitor is an inhibitor of the Trim58 gene.

[0023] Preferably, it is: Trim58 gene-specific siRNA or its precursor, Trim58 gene-specific microRNA or its precursor, inhibitor of the Trim58 gene promoter, or a combination thereof.

[0024] In another preferred embodiment, the inhibitor of the Trim58 gene is a Trim58 gene-specific siRNA or its precursor, or a microRNA or its precursor.

[0025] In another preferred embodiment, the siRNA specifically targets the Trim58 gene sequence or its complementary sequence.

[0026] In another preferred embodiment, the Trim58 inhibitor is an inhibitor of the protein encoded by the Trim58 gene.

[0027] In another preferred embodiment, the inhibitor of the Trim58 gene-encoded protein is selected from the group consisting of: antibodies against the Trim58 gene-encoded protein and binding proteins of the Trim58 gene-encoded protein.

[0028] In another preferred embodiment, the Vcam1 activator is a substance (such as a compound or protein) that enhances the expression of the Vcam1 gene.

[0029] A third aspect of the invention provides the use of an inhibitor of the Trim58 gene or its encoded protein for the preparation of a medicament for the prevention or treatment of radiation-induced liver injury.

[0030] In another preferred embodiment, the inhibitor of the Trim58 gene is: a Trim58 gene-specific siRNA or its precursor, a Trim58 gene-specific microRNA or its precursor, an inhibitor that inhibits the Trim58 gene promoter, or a combination thereof.

[0031] In another preferred embodiment, the inhibitor of the Trim58 gene is a Trim58 gene-specific siRNA or its precursor, or a microRNA or its precursor.

[0032] In another preferred embodiment, the siRNA specifically targets the Trim58 gene sequence or its complementary sequence.

[0033] In another preferred embodiment, the Trim58 inhibitor is an inhibitor of the protein encoded by the Trim58 gene.

[0034] In another preferred embodiment, the inhibitor of the Trim58 gene-encoded protein is selected from the group consisting of: antibodies against the Trim58 gene-encoded protein and binding proteins of the Trim58 gene-encoded protein.

[0035] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0036] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0037] Figure 1 Principal component analysis (PCA) results of RNA-seq data.

[0038] Figure 2 Venn diagram of common differentially expressed genes in different sample groups.

[0039] Figure 3 Vcam1 RNAseq level expression.

[0040] Figure 4 qPCR was used to verify Vcam1 expression. Unirradiated Trim58+ / + mice were used as the control group for analysis. (A) Analysis of variance between the control group and each experimental group showed a significant upregulation of Vcam1 gene expression. (B) Without X-ray irradiation, there was little difference in gene expression levels between Trim58- / - and Trim58+ / + mice. (C) On the third day after whole-body irradiation, the Vcam1 gene expression level in the liver tissue of Trim58- / - mice was significantly higher than that of Trim58+ / + mice. (D) On the third day, the Vcam1 gene expression level in the liver tissue of Trim58+ / + mice was significantly higher than that of unirradiated mice. (E) On the third day, the Vcam1 gene expression level in the liver tissue of Trim58- / - mice was significantly higher than that of unirradiated mice. n = 3. Trim58+ / + vs. Trim58- / -: P < 0.05, P<0.01, P<0.001. This indicates whether the difference between the compared objects is significant in a significance test. ns indicates no significant difference. This indicates that P < 0.05. This indicates that P < 0.01. This means P < 0.0001. Detailed Implementation

[0041] TRIM58 The TRIM (Tripartite Motif) family of proteins is a class of multifunctional molecules widely involved in ubiquitination, signal transduction, and cellular homeostasis regulation. They share a common characteristic: a conserved N-terminal triple domain consisting of a RING (Really Interesting New Gene) domain, a B-box zinc finger domain, and a coiled-coil domain. The RING domain endows TRIM proteins with E3 ubiquitin ligase activity, regulating protein degradation, localization, or function by mediating substrate ubiquitination. For example, in cell cycle regulation, TRIM proteins can influence mitosis by ubiquitinizing key kinases or phosphatases. Furthermore, the RING domain can significantly affect its catalytic activity through self-polymerization.

[0042] Members of the TRIM protein family exert dual pro- and anti-tumor effects in tumors by regulating the ubiquitin-proteasome system, inflammatory responses, and cell signaling pathways. Recent studies have confirmed a significant association between TRIM family proteins and the self-renewal capacity of cancer stem cells. Specifically, TRIM25 can promote the proliferation and drug resistance of breast cancer stem cells by maintaining the expression of the oncogene c-Myc. Abnormal expression of TRIM proteins is closely related to the development and progression of various cancers. TRIM31, for instance, shows significantly elevated expression levels in colorectal cancer tissues and enhances the invasive and metastatic capabilities of tumor cells by activating the Wnt / β-catenin signaling pathway. These studies suggest that TRIM family members may influence important oncogenic pathways or epigenetic modification mechanisms, thus possessing potential value as targets for cancer therapy.

[0043] TRIM58, a relatively unexplored member of the TRIM family, has been the subject of numerous studies in recent years, revealing its close association with the progression of various cancers. TRIM proteins exhibit different functional properties in liver cancer (HCC), with TRIM58 effectively inhibiting HCC metastasis by influencing the expression of cyclins (such as Cyclin D1) and epithelial-mesenchymal transition (EMT)-related molecules. Further research has shown that TRIM58 has a protective effect in radiation-induced liver injury (RILD), potentially mitigating radiation-induced hepatocyte apoptosis by repairing DNA double-strand breaks (DSBs). Hypermethylation of the Trim58 gene and downregulation of its mRNA expression are associated with HCC tumorigenesis and poor prognosis in patients after hepatectomy. The expression level of TRIM58 is closely related to the prognosis of HCC patients, with patients expressing low levels of TRIM58 being more prone to recurrence after surgery and having a poorer response to radiotherapy, suggesting that TRIM58 could be a potential target for combination therapy.

[0044] Vcam1 gene Vascular cell adhesion molecule-1 (VCAM-1) is an important member of the immunoglobulin superfamily and a key cell adhesion molecule. Its molecular structure consists of three parts: an extracellular region, a transmembrane region, and an intracellular region. The extracellular region contains 6-7 immunoglobulin-like domains. Among these domains, the first and fourth Ig-like domains can bind to integrin α4β1 (VLA-4), thereby mediating the adhesion and migration of leukocytes to endothelial cells. In liver tissue, VCAM-1 expression is mainly concentrated in hepatic sinusoidal endothelial cells (LSECs), and its expression level is influenced by various factors, including inflammatory factors (TNF-α, IL-1β), oxidative stress, and mechanical stress.

[0045] VCAM-1 plays a crucial role in the progression of liver inflammation. In 1996, Italian medical expert Mario Pirisi and his research group discovered that the serum levels of soluble VCAM-1 (sVCAM-1) were significantly elevated in patients with acute and chronic liver disease, and that changes in its concentration were positively correlated with disease severity. Guo Qianqian et al. conducted research on a non-alcoholic steatohepatitis (NASH) model, and their results indicated that elevated VCAM-1 expression levels on liver stromal cells (LSECs) may promote monocyte adhesion and infiltration, thereby exacerbating liver inflammation and fibrosis.

[0046] In tumor research, Zhang Dan's team confirmed through clinical sample detection combined with cell model experiments that the expression level of VCAM-1 in colorectal cancer tissue was significantly increased. This phenomenon is related to tumor metastasis ability and poor patient prognosis. Related mechanism studies have shown that VCAM-1 can promote tumor cell invasion by inducing EMT and transendothelial migration (TEM)

[30] . In addition, Professor Martin Schlesinger pointed out in his review that VCAM-1 has a dual regulatory role in tumor angiogenesis: the expression level of VCAM-1 inside newly formed tumor blood vessels is reduced, which helps the tumor evade the monitoring of the body's immune system; on the other hand, the abnormal expression of VCAM-1 on the surface of tumor cells can attract the aggregation of macrophages and osteoclasts, thereby creating suitable conditions for tumor metastasis. In 2018, Deok-Hoon Kong's team clarified in a review that the abnormal expression of VCAM-1 in liver cancer cells mediates the interaction with tumor-associated macrophages and activates the PI3K / Akt related signaling pathway, thereby promoting the migration and invasion of tumor cells.

[0047] In recent years, VCAM-1 has received widespread attention as a therapeutic target for liver diseases. Martin Schlesinger and Gerd Bendas pointed out that targeting VCAM-1 with antibodies or small molecule inhibitors can significantly inhibit leukocyte adhesion and hinder tumor metastasis. This therapeutic strategy has been demonstrated to have potential efficacy in various disease models. Sara A. El-Mokhtar and her team found through experimental studies that the mRNA expression level of VCAM-1 in peripheral blood mononuclear cells of patients with cirrhosis was significantly increased. This trend was positively correlated with the levels of inflammatory factors such as TNF-α and IL-17A, suggesting that VCAM-1 may be involved in the chronic inflammatory process of cirrhosis. These findings lay the theoretical foundation for developing treatments for liver diseases targeting VCAM-1.

[0048] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, parts and percentages are by weight.

[0049] Materials and methods laboratory animals Trim58 wild-type mice (represented as "Trim58+ / +" and "WT") and Trim58 knockout mice (represented as "Trim58- / -" and "KO") were genotyped and individually numbered at 4 weeks of age by harvesting toe tissue. They were then separated into different cages and kept under normal housing conditions. At 6 weeks of age, male mice weighing between 19-21 g were selected for model construction. The mice were placed in a fixed box with an X-ray irradiator and subjected to whole-body X-ray irradiation with a dose of 6 Gy, controlled at a dose rate of 120 cGy / min. The irradiated Trim58+ / + and Trim58- / - mice were subsequently housed in an SPF-grade laboratory animal facility, fed sterile feed and sterile drinking water, with free access to food and water.

[0050] Experimental methods Total RNA extraction from tissues 1. Autoclaved scissors and forceps were used for dissection to remove liver tissue from the mouse.

[0051] 2. After euthanizing the mice, the liver tissue was completely dissected and rinsed three times with PBS to remove any remaining blood.

[0052] 3. After cleaning, divide the liver tissue into 10-20 mg tissue blocks, weigh them and record the weights, and then place the tissue blocks into the corresponding EP tubes.

[0053] 4. Samples not used in this experiment were placed in liquid nitrogen for rapid freezing, and then transferred to a freezer at -80°C for long-term storage.

[0054] 5. Add lysis buffer RL containing 1% β-mercaptoethanol to an EP tube containing liver tissue, and grind it thoroughly using a mechanical grinding device (Tiangen grinding pestle) to break down the liver tissue until it is homogeneous, thus obtaining a tissue homogenate.

[0055] 6. Add a mixture containing Proteinase K prepared with RNase-Free dd H2O to the homogenate, mix with a pipette, and then place it in a 56℃ water bath for 15 minutes to achieve protein degradation.

[0056] 7. Place the EP tube into a centrifuge, set the parameters of the high-speed refrigerated centrifuge to 4℃, 12000×g, and centrifuge for 3.5 minutes. Collect the supernatant containing tissue RNA and transfer it to a new EP tube.

[0057] 8. Add anhydrous ethanol for RNA precipitation to a new EP tube, mix well, and transfer the mixture to an RNA adsorption column. Perform high-speed centrifugation and discard the waste liquid to separate the precipitate.

[0058] 9. The protein removal solution RW1, DNAse I working solution, protein solution RW1, and ethanol-containing rinsing solution RW were added sequentially for washing, multiple centrifugation, and waste liquid disposal to finally remove impurities from the waste liquid.

[0059] 10. RNase-free dd H2O was used to elute and purify the RNA adsorbed on the column membrane, thereby obtaining a high-purity RNA solution.

[0060] 11. Take 1.5 μL of the extract and use a NanoVue micro spectrophotometer to measure the A260 / A280 ratio and concentration, and record the experimental results.

[0061] 12. Seal the RNase-Free centrifuge tubes containing RNA solution with paraffin film and then store them in a freezer at -80°C for later use.

[0062] RNA is reverse transcribed into cDNA During RNA extraction, the concentration of the extracted RNA solution varies due to the different sizes of various tissues. To ensure the reliability of subsequent experimental results, the extracted RNA solutions need to be diluted to a standard concentration of 0.8 μg / μL beforehand. Subsequently, the reverse transcription kit from Acrel Biotech Co., Ltd. was used to reverse transcribe the tissue RNA into cDNA according to the provided procedure. The specific steps are as follows: (1) Removal of genomic DNA To obtain high-purity template RNA, subsequent purification steps are required: ① Add 9 μL of premixed solution and 1 μL of diluted RNA to a 0.2 mL PCR tube, and slowly mix using a pipette to complete the preparation of the system. At the same time, a blank control sample needs to be prepared simultaneously. Replace the RNA sample with Milli-Q ultrapure water used to dissolve the RNA, and add an equal volume of 9 μL of premixed solution to mix.

[0063] ② The instrument needs to be set to remove gDNA reaction, with the reaction temperature maintained at 42℃ and the duration set to 120 seconds.

[0064] (2) Add an equal volume of reverse transcription premix (10 μL) to the gDNA-cleansed RNA (10 μL), and gently mix with a pipette to prepare a 20 μL reverse transcription reaction system.

[0065] (3) Perform the reverse transcription reaction according to the reaction procedure.

[0066] Example 1. RNA-Seq This study used 8 male Trim58+ / + (n=8) and Trim58- / - (n=8) mice aged 6 weeks and weighing approximately 19-21g, all of which were C57BL / 6J mice and their genotypes were verified by PCR. The mice were housed in an SPF environment and acclimatized for 7 days before the experiment.

[0067] Sixteen mice were randomly divided into four groups (n=4 in each group) according to the following conditions: WT_D0 group: Trim58+ / + mice, which were not subjected to radiation treatment and had their liver samples collected on day 0; KO_D0 group: Trim58- / - mice, which were not subjected to radiation treatment, and liver samples were collected on day 0; WT_D3 group: Trim58+ / + mice were irradiated with 6 Gy X-rays all over their bodies, and liver samples were collected on the 3rd day after irradiation; KO_D3 group: Trim58- / - mice were irradiated with 6 Gy X-rays all over their bodies, and liver samples were collected on the 3rd day after irradiation.

[0068] All samples were flash-frozen in liquid nitrogen and then stored at -80°C.

[0069] PCA analysis Principal component analysis (PCA), a multivariate statistical method, is widely used to examine population variability and intra-group consistency in biological samples. Based on the principles of linear algebra, this method performs dimensionality reduction on massive gene expression variables and extracts key principal components. This study uses FPKM-normalized expression levels for PCA modeling. Theoretically, the distribution of samples in the dimensionality-reduced space should exhibit characteristics of discrete between groups and clustered within groups, reflecting the rationality of the experimental design and the controllability of data quality.

[0070] Analysis of the principal component analysis (PCA) results revealed that the first two principal components jointly explained 43.74% of the heterogeneity among samples (PC1: 33.54%; PC2: 10.20%). On the PC1 axis, the control group (WT_D0) and KO_D0 showed negative dispersion with high clustering within each group; while the WT_D3 and KO_D3 groups showed positive dispersion, with relatively high dispersion between groups but relatively tight clustering within each group. On the PC2 axis, the WT_D0 and KO_D0 groups were concentrated near the origin with low dispersion; the KO_D3 group showed a positive shift, while the WT_D3 group showed a negative shift, indicating a clear separation trend on the PC2 axis.

[0071] Statistical results show that there is no significant difference between the WT_D0 group and the KO_D0 group, but there is a significant difference between the KO_D3 group and the KO_D0 group; there is a significant difference between the WT_D3 group and the WT_D0 group, and there is a significant difference between the WT_D3 group and the KO_D3 group. Figure 1 The results of principal component analysis (PCA) of the RNA-seq data are shown.

[0072] Statistics on differentially expressed genes Without X-ray irradiation, there was little difference in gene expression levels between Trim58- / - and Trim58+ / + mice, with 326 genes showing significant differences in expression levels. However, on day 3 post-irradiation, liver gene expression changed in both Trim58- / - and Trim58+ / + mice. Trim58+ / + mice showed significant changes in the expression of 1278 genes on day 3 compared to unirradiated mice (Day 0). Trim58- / - mice showed significant changes in the expression of 1388 genes on day 3 compared to unirradiated mice (Day 0). Furthermore, 599 genes showed differences between Trim58+ / + and Trim58- / - mice on day 3 after irradiation.

[0073] In addition, through the Figure 2 Preliminary analysis of the Venn diagrams revealed 1388 differentially expressed genes (58.9%) in the KO_D3 vs. KO_D0 group, 1278 differentially expressed genes (54.2%) in the WT_D3 vs. WT_D0 group, and 599 differentially expressed genes (24.5%) in the KO_D3 vs. WT_D3 group. Further analysis of the intersection of differentially expressed genes among the groups was conducted. ①The KO_D3 vs. KO_D0 group and the WT_D3 vs. WT_D0 group shared 23.8% of the differentially expressed genes; ②The KO_D3 vs. KO_D0 group and the KO_D3 vs. WT_D3 group shared 9.9% of the differentially expressed genes; ③The WT_D3 vs. WT_D0 group and the KO_D3 vs. WT_D3 group shared 6.9% of the differentially expressed genes; In summary, the KO_D3 vs. KO_D0 group showed the highest number of differentially expressed genes, which may suggest that X-ray-induced changes in liver gene expression are more significant in the context of Trim58 knockout, indicating that Trim58 knockout may exacerbate radiation-induced gene expression changes. Compared to the WT_D3 vs. WT_D0 group, the KO_D3 vs. KO_D0 group may suggest that regardless of Trim58 knockout, X-ray irradiation can induce common expression changes in approximately one-quarter of the genes in the liver, and these genes may be involved in the basic biological processes of radiation damage.

[0074] The number of differentially expressed genes in the KO_D3 vs. WT_D3 group (599) was significantly greater than that in the KO_D0 vs. WT_D0 group (326). It is speculated that the deletion of the Trim58 gene may enhance the sensitivity and specificity of the liver to radiation-induced gene expression changes, revealing that knocking out the Trim58 gene interferes with the regulatory network of specific genes under radiation stress, thereby triggering significant changes in differentially expressed genes.

[0075] Effect of irradiation on Vcam1 expression levels in the liver of Trim58- / - mice Through the Figure 3 Analysis revealed no significant difference (ns) in Vcam1 expression levels in the livers of Trim58+ / + and Trim58- / - mice in the initial state before radiation treatment. This clearly demonstrates that, under normal physiological conditions and without radiation stress, the presence or absence of the Trim58 gene does not significantly affect the basal expression level of Vcam1 in the liver.

[0076] Changes in the WT group: There was no significant difference in Vcam1 expression levels (ns) between the WT_D3 group and the WT_D0 group, indicating that without Trim58 knockout, Vcam1 expression showed no significant change on day 3 after X-ray irradiation. Changes in the KO group: Vcam1 expression levels were also significantly increased in the KO_D3 group compared to the KO_D0 group. This indicates that in Trim58- / - mice, whole-body X-ray irradiation significantly increased Vcam1 expression in the liver on day 3 post-irradiation. On day 3 after whole-body X-ray irradiation, there was a highly significant difference in Vcam1 expression levels between the WT_D3 and KO_D3 groups. Furthermore, the expression level of the WT_D3 group was lower than that of the KO_D3 group.

[0077] This clearly demonstrates that, on the third day after radiation stress, the knockout of the Trim58 gene promoted the expression of Vcam1 in the liver. In other words, the presence of the Trim58 gene can inhibit the expression of Vcam1 in the liver after radiation stimulation, thereby hindering Vcam1's participation in the radiation-induced liver injury protective mechanism.

[0078] Figure 4 The results show the expression of Vcam1 as verified by qPCR. Unirradiated Trim58+ / + mice were used as the control group for analysis. (A) Analysis of variance between the control group and each experimental group showed a significant upregulation of Vcam1 gene expression. (B) Without X-ray irradiation, there was little difference in gene expression levels between Trim58- / - and Trim58+ / + mice. (C) On the third day after whole-body irradiation, the expression level of Vcam1 gene in the liver tissue of Trim58- / - mice was significantly higher than that of Trim58+ / + mice. (D) On the third day, the expression level of Vcam1 gene in the liver tissue of Trim58+ / + mice was significantly higher than that of unirradiated mice. (E) On the third day, the expression level of Vcam1 gene in the liver tissue of Trim58- / - mice was significantly higher than that of unirradiated mice. n = 3. Trim58+ / + vs. Trim58- / -: P < 0.05, P<0.01, P<0.001. This indicates whether the difference between the compared objects is significant in a significance test. ns indicates no significant difference. This indicates that P < 0.05. This indicates that P < 0.01. This means P < 0.0001.

[0079] The experimental results presented the differences in the relative mRNA expression levels of the Vcam1 gene in the liver tissues of mice in each group. In the unirradiated group, there was no significant difference (ns) in Vcam1 gene expression in the liver tissues of Trim58+ / + mice and Trim58- / - mice, indicating that under normal physiological conditions, Trim58 gene knockout has no significant effect on Vcam1 expression; on day 3 after irradiation, the Vcam1 expression in Trim58+ / + mice was higher than that in their unirradiated group ( This indicates that X-ray irradiation may induce upregulation of Vcam1 expression in the liver of Trim58+ / + mice. Notably, Vcam1 expression in Trim58- / - mice was significantly higher than in the unirradiated group (…). ), and was also significantly higher than Trim58+ / + mice at the same time point ( This indicates that after X-ray irradiation, Trim58 gene knockout can significantly enhance Vcam1 gene expression.

[0080] In summary, the experimental results indicate that X-ray irradiation may induce increased expression of the Vcam1 gene in the liver of Trim58- / - mice, suggesting that Trim58 deficiency may enhance the liver's response to radiation through some mechanism, making the upregulation of Vcam1 expression more significant. Therefore, it is speculated that Trim58 may hinder Vcam1 from participating in the protective mechanism against radiation-induced liver injury.

[0081] X-ray-induced radiation-induced liver injury models (Trim58+ / + and Trim58- / -) were established by subjecting mice to whole-body X-ray radiation at a dose of 6 Gy. The mRNA expression profiles of liver tissues from Trim58+ / + and Trim58- / - mice after radiation irradiation were analyzed using RNA-seq technology, and qPCR experiments were conducted to verify Vcam1 gene expression. The results showed that after X-ray irradiation, the Vcam1 expression level in the liver tissue of Trim58- / - mice was significantly higher than that in Trim58+ / + mice.

[0082] Meanwhile, analysis of different experimental groups and control groups revealed that: without X-ray irradiation, the gene expression levels of Trim58- / - mice and Trim58+ / + mice were similar; however, on the third day after whole-body irradiation, the expression level of the Vcam1 gene in the liver tissue of Trim58- / - mice was significantly higher than that of Trim58+ / + mice; on the other hand, the expression level of the Vcam1 gene in the liver tissue of Trim58+ / + mice on the third day was significantly higher than that of unirradiated mice; and the expression level of the Vcam1 gene in the liver tissue of Trim58- / - mice on the third day was also significantly higher than that of unirradiated mice.

[0083] The results showed that in the case of Trim58 gene knockout, Vcam1 gene expression in mouse liver was significantly upregulated after whole-body X-ray irradiation, indicating that Trim58 may have hindered Vcam1's participation in radiation-induced liver injury protection mechanisms, thereby aggravating the progression of inflammatory damage.

[0084] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A drug for treating radiation-induced liver injury, characterized in that, The drug includes a Trim58 inhibitor and / or a Vcam1 activator.

2. The drug as described in claim 1, characterized in that, The Trim58 inhibitor is an inhibitor of the Trim58 gene.

3. The drug as described in claim 2, characterized in that, The inhibitor of the Trim58 gene is a Trim58 gene-specific siRNA or its precursor, a Trim58 gene-specific microRNA or its precursor, an inhibitor that inhibits the Trim58 gene promoter, or a combination thereof.

4. The drug as described in claim 3, characterized in that, The inhibitor of the Trim58 gene is a Trim58 gene-specific siRNA or its precursor, or a microRNA or its precursor.

5. The drug as described in claim 5, characterized in that, The siRNA specifically targets the Trim58 gene sequence or its complementary sequence.

6. The drug as described in claim 1, characterized in that, The Trim58 inhibitor is an inhibitor of the protein encoded by the Trim58 gene.

7. The drug as described in claim 6, characterized in that, The inhibitor of the Trim58 gene-encoded protein is selected from the following group: antibodies against the Trim58 gene-encoded protein and binding proteins of the Trim58 gene-encoded protein.

8. A method for treating radiation-induced liver injury, characterized in that, The method includes the steps of inhibiting the expression of the Trim58 gene or inhibiting the activity of the Trim58 protein in the subject, and / or increasing the expression of the Vcam1 gene or increasing the activity of the Vcam1 protein in the subject.

9. Use of inhibitors of the Trim58 gene or its encoded protein for the preparation of drugs to prevent or treat radiation-induced liver injury.

10. The use as described in claim 9, characterized in that, Inhibitors of the Trim58 gene are Trim58 gene-specific siRNA or its precursor, or microRNA or its precursor.

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