Use of a rioK3 inhibitor in the manufacture of a medicament for the treatment of sepsis-associated encephalopathy

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to the use of RIOK3 inhibitors in the preparation of a drug for treating sepsis-related encephalopathy. The application research finds that the expression of RIOK3 gene is significantly up-regulated in the pathological process of sepsis-related encephalopathy, and the inhibition of the expression or protein activity of RIOK3 gene can significantly reduce neuroinflammation, neuron and synapse damage, and improve cognitive dysfunction and learning and memory ability. The application discloses the use of RIOK3 as a new target for treating sepsis-related encephalopathy, and provides a new idea for the research and development of related drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to the use of RIOK3 inhibitors in the preparation of drugs for treating sepsis-associated encephalopathy. Background Technology

[0002] Sepsis is a disordered immune response in the host to infection, leading to life-threatening organ dysfunction and even death. Sepsis-associated encephalopathy (SAE), also known as sepsis-related brain injury, is an acute, diffuse brain dysfunction caused by infection outside the central nervous system (CNS). It primarily manifests as altered consciousness or delirium, seizures, and even coma in sepsis patients. Globally, there are over 48.9 million sepsis patients annually, and 50%–70% of these patients develop SAE. 60% of these patients face death within 30 days, making it one of the most common encephalopathy and causes of death in modern intensive care units (ICUs). Even those who survive SAE often suffer from long-term cognitive impairment or emotional disturbances, severely impacting their quality of life and consuming significant public health resources. Currently, clinical diagnosis of sepsis relies heavily on exclusionary diagnostic methods such as clinical manifestations, Glasgow Coma Scale (GCS), Reaction Level Scale (RLS), Mini-Mental State Examination (MMSE), electroencephalography (EEG), and neuroimaging. Specific biomarkers and effective prevention and treatment methods are lacking. Therefore, further research into the pathogenesis and pathophysiological mechanisms of SAE, and the identification of key markers and molecular targets, has significant theoretical and clinical implications.

[0003] RIO kinase 3 (RIOK3) is a widely expressed eukaryotic atypical protein kinase, first discovered in yeast. Structurally lacking a typical kinase domain, it retains kinase activity and belongs to the serine / threonine protein kinase (STPK) family, located on the long arm of human chromosome 18 at 18q11.2. Currently, the RIO kinase family includes RIOK1, RIOK2, and RIOK3. RIOKs play important roles in growth and development, cell proliferation and differentiation, cell cycle and senescence, and innate immunity. Studies have shown that RIOK3 is highly expressed in immune cells and the nervous system, negatively regulating the activation of retinoic acid-inducible gene I (RIG-I) / melanoma differentiation-associated gene 5 (MDA5), as well as type I interferon and inflammatory cytokine pathways, and positively regulating TLR3 / 4-mediated innate immune responses. Furthermore, RIOK3 can also interact with tumor necrosis factor receptor-associated factor 3 (TRAF3), catalyzing the ubiquitination of K48 lysine residues, degrading TRAF3, and thereby inhibiting NF-κκB-mediated inflammatory responses. Zhang et al. found that RIOK3 promotes the interaction between heat shock protein 90 (HSP90) and isocitrate dehydrogenase 1 (IDH1), enhancing the production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and improving the survival of colon cancer cells in a low-glucose environment. Yong et al. found that conditional knockout of RIOK3 upregulates the expression of TNF-α and IL-6 in RNA virus-induced bone marrow-derived macrophages (BMDMs). However, to date, there are no reported studies on RIOK3 in sepsis, especially SAE. Summary of the Invention

[0004] This invention discovered RIOK3 fl / fl After the RIOK3 gene was knocked out in mice, compared with RIOK3 mice without RIOK3 gene knockout... fl / flCompared to mice, RIOK3 gene conditional knockout mice showed significantly reduced cognitive impairment, neuronal and synaptic damage, and neuroinflammation, as well as markedly improved learning and memory. These results indicate that RIOK3 gene knockout can alleviate the disease progression of sepsis-associated encephalopathy (SEE). Based on these findings, the RIOK3 gene can be used as a drug target for screening drugs to treat SEE, and its inhibitors can be used to develop drugs for treating SEE.

[0005] The technical solution provided by this invention is as follows: In the first aspect, the use of RIOK3 inhibitors in the preparation of medicaments for treating sepsis-associated encephalopathy, wherein the RIOK3 inhibitor is an shRNA with a nucleotide sequence as shown in SEQ ID NO.10.

[0006] In some embodiments of the present invention, the sepsis-associated encephalopathy is accompanied by one or more of the following: (i) neuronal and synaptic damage; (ii) neuroinflammation; (iii) impairment of learning, memory and cognitive functions.

[0007] In some embodiments of the present invention, the treatment is used for one or more of the following: (i) reducing neuronal and synaptic damage; (ii) reducing neuroinflammation; and (iii) improving learning, memory, and cognitive functions.

[0008] In some embodiments of the present invention, the medicament comprises a RIOK3 inhibitor and a pharmaceutically acceptable carrier thereof.

[0009] In some embodiments of the present invention, the vector is a viral vector or a non-viral vector. Preferably, the viral vector is a recombinant adeno-associated virus.

[0010] Compared with the prior art, the present invention has the following advantages and effects: (1) This invention discovers that inhibiting the RIOK3 gene can improve sepsis-associated encephalopathy, and provides a new approach to treating sepsis-associated encephalopathy by using the RIOK3 gene as an inhibitory target.

[0011] (2) This invention provides a RIOK3 inhibitor and its use in the preparation of drugs for treating sepsis-associated encephalopathy, enriching the drug library for sepsis-associated encephalopathy. Attached Figure Description

[0012] Figure 1The study compared RIOK3 mRNA levels in PBMCs from healthy individuals and patients with sepsis-associated encephalopathy (SAE). Correlation analysis between RIOK3 mRNA levels and SAE showed consistent upregulation of neuron-specific enolase (NSE) and S100 calcium-binding protein B (S100B), key indicators of brain injury assessment. RIOK3 mRNA expression was significantly upregulated in PBMCs with SAE and positively correlated with SAE. (p < 0.001 vs. normal group) A: Compared with healthy volunteers, SAE patients had significantly higher serum NSE levels; B: Compared with healthy volunteers, SAE patients had significantly higher serum S100B levels; C: Compared with healthy volunteers, SAE patients had significantly higher RIOK3 mRNA levels in PBMCs; D: The area under the ROC curve (AUC) is 0.913, the sensitivity is 90%, and the specificity is 85%, indicating strong diagnostic efficacy.

[0013] Figure 2 This is a graph showing the expression and statistical distribution of RIOK3 in the hippocampus of C57BL / 6 mice 24 hours after sham surgery and cecal ligation and perforation (CLP) modeling. β-actin was used as an internal control. RIOK3 expression in the hippocampus was upregulated when sepsis-related encephalopathy occurred. (p < 0.01 vs sham surgery group) A: Western blot analysis of RIOK3 expression in hippocampal tissue before and after CLP modeling; B: Statistical analysis of RIOK3 protein levels in hippocampal tissues of Sham and CLP mice; C: Immunofluorescence double label co-localization staining pattern.

[0014] Figure 3 This is a graph showing the protein expression and statistical distribution of RIOK3 in primary microglia of neonatal rats after LPS stimulation in vitro, with β-actin as an internal control. RIOK3 expression was upregulated after LPS stimulation. (p<0.001 vs. control group) A: Western blot analysis of RIOK3 expression in primary microglia before and after LPS stimulation; B: Statistical analysis of RIOK3 protein levels in primary microglia of the non-stimulated control group and the LPS-stimulated group.

[0015] Figure 4 This is a structural map of the Lv-shRIOK3 plasmid and a statistical graph showing the expression of RIOK3 protein after RIOK3 gene knockdown. β-actin was used as an internal control, and RIOK3 expression was found to be significantly reduced. (p<0.001 vs Lv-shRNA group); A: Structure diagram of Lv-shRIOK3 plasmid; B: Western blot analysis of RIOK3 expression in primary microglia after Lv-shRNA or shRIOK3 infection; Statistical analysis of RIOK3 protein levels in primary microglia of the C:Lv-shRNA and Lv-shRIOK3 groups.

[0016] Figure 5 The levels of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in the supernatant of cultured primary rat microglia infected with lentiviral Lv-shRNA and Lv-shRIOK3 and stimulated with LPS for 12 hours were observed. The lentiviral interference with RIOK3 inhibited the production of pro-inflammatory cytokines in microglia. #1: p < 0.001 vs Lv-shRNA control group, #2: p < 0.01 vs Lv-shRNA LPS stimulation group. A: ELISA was used to detect the TNF-α content in the supernatant of primary microglia in each group; B: ELISA was used to detect the IL-6 content in the supernatant of primary microglia in each group; C: ELISA was used to detect the IL-1β content in the supernatant of primary microglia in each group.

[0017] Figure 6 It's RIOK3 fl / fl Cx3CR1 Cre Mouse construction strategy, genotype identification, and RIOK3 protein expression level detection diagram. (RIOK3...) fl / fl Mice and CX3CR1 Cre RIOK3 was obtained by crossbreeding mice. fl / fl CX3CR1 Cre In mice, Cre recombinase mediates a specific deletion of the E3–E5 exon segment of the RIOK3 gene between loxP sites; RIOK3 fl / flCX3CR1 Cre Genotyped mouse DNA can simultaneously amplify two target bands, 523 bp and 368 bp, and simultaneously interact with RIOK3. fl / fl Compared to control mice, RIOK3 fl / fl Cx3CR1 Cre The expression level of RIOK3 protein in mouse microglia was significantly reduced. p < 0.001 vs RIOK3 fl / fl The control group indicates that the RIOK3 conditional knockout mouse for microglia was successfully constructed.

[0018] A: RIOK3 fl / fl Cx3CR1 Cre Schematic diagram of mouse construction; B: Genotyping of mouse tails; C: RIOK3 protein expression in primary microglia; D: Statistical analysis of RIOK3 protein levels in primary microglia.

[0019] Figure 7 It's RIOK3 fl / fl CX3CR1 Cre Mice and littermate wild-type RIOK3 fl / fl Statistical graphs of learning and cognitive function assessments (Y maze, new object recognition, and Morris water maze tests) in mice 7 days after sham surgery or CLP surgery modeling. p < 0.05 p < 0.01 vs RIOK3 fl / fl Sham surgery group, #: p < 0.05, ##: p < 0.01 vs RIOK3 fl / fl Model group); A: Representative trajectory heatmaps of mice in each group during the Y-maze experiment; B:Y Maze Test New Different Arm Dwell Time Percentage Statistical Analysis Chart; C: Representative trajectory diagrams of mice in each group during the new object recognition experiment; D: Statistical analysis chart of new object recognition test results and new object preference index; E: Representative trajectory diagrams of mice in each group during the orientation and navigation phase of the Morris water maze experiment; F: Quantitative statistical analysis of escape latency during the Morris water maze experiment training phase; G: Statistical analysis chart of the target quadrant dwell time during the Morris water maze test phase; H: Statistical analysis chart of the number of times the platform was traversed during the Morris water maze experiment testing phase.

[0020] Figure 8 It's RIOK3 fl / fl CX3CR1 Cre Mice and littermate wild-type RIOK3 fl / fl HE and Nissl staining and neuronal damage statistics of hippocampal tissue 24 hours after mouse sham surgery or CLP surgery modeling. p < 0.001 vs RIOK3 fl / fl Sham surgery group, ###: p < 0.001 vs RIOK3 fl / fl Model group); A: HE and Nissl staining were used to assess hippocampal neuronal damage; B: Statistical analysis of neuronal damage assessed by HE staining; Statistical analysis of C:Nissl staining for assessing neuronal damage.

[0021] Figure 9 It's RIOK3 fl / fl CX3CR1 Cre Mice and littermate wild-type RIOK3 fl / fl Ultrastructural analysis of hippocampal neuronal synapses 24 hours after sham surgery or CLP surgery in mice. p < 0.001 vs RIOK3 fl / fl Sham surgery group, ##: p < 0.01 vs RIOK3 fl / fl Model group); A: Transmission electron microscopy was used to observe the changes in the ultrastructure of hippocampal synapses, postsynaptic membrane density, and synaptic cleft width. The red arrows point to the synaptic structure. SV: synaptic vesicles; SC: synaptic cleft; PSD: postsynaptic compact zone; scale bar is 500 nm. B: Statistical graph of quantitative analysis of postsynaptic compact band thickness; C: Statistical graph of quantitative analysis of synaptic cleft width.

[0022] Figure 10 It's RIOK3 fl / fl CX3CR1 Cre Mice and littermate wild-type RIOK3 fl / fl Fluorescence staining and statistical graph of Iba1 marker in mouse hippocampal microglia 24 h after sham surgery or CLP surgery modeling; statistical graph of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α. p < 0.001 vs RIOK3 fl / fl Sham surgery group, ##: p < 0.01 vs RIOK3 fl / fl Model group); A: Iba1 immunofluorescence staining was used to detect the activation of microglia in the hippocampus of mice in each group. The scale bar is 50 μm. B: The number of Iba1-positive cells in the hippocampus was quantitatively analyzed using ImageJ software; C: ELISA was used to detect the IL-6 content in the hippocampus of mice in each group; D: ELISA was used to detect the TNF-α content in the hippocampus of mice in each group; E: ELISA was used to detect the IL-1β content in the hippocampus of mice in each group.

[0023] Figure 11 This is a technical roadmap for evaluating cognitive function in mice. Figure 12 This is a structural map of the AAV-shRIOK3 plasmid, along with validation and statistical diagrams of RIOK3 mRNA and protein expression after RIOK3 gene knockdown. β-actin was used as an internal control, and RIOK3 expression was found to be significantly reduced. (p<0.001 vsAAV-shNC group); A: Structure diagram of AAV-shRIOK3 plasmid; B: qRT-PCR was used to detect the expression of RIOK3 mRNA in hippocampal tissues after bilateral stereotactic injection of AAV-shNC or AAV-shRIOK3 into the hippocampus. C: Western blot analysis of RIOK3 protein expression in hippocampal tissue of mice infected with AAV-shNC or AAV-shRIOK3 via intraventricular injection. Statistical analysis of RIOK3 protein levels in hippocampal tissue of the AAV-shNC or AAV-shRIOK3 group (D: AAV-shNC or AAV-shRIOK3 group).

[0024] Figure 13 This is a statistical graph showing the learning and cognitive function assessment (new object recognition and Morris water maze test) of mice 7 days after sham surgery or CLP surgery modeling following bilateral stereotactic injection of AAV-shNC or AAV-shRIOK3 into the hippocampus 3 weeks later. p < 0.05 #: p < 0.01 vs AAV-shNC sham surgery group, #: p < 0.05, ##: p < 0.01 vs AAV-shNC model group). A: Representative trajectory diagrams of mice in each group during the new object recognition experiment; B: Statistical analysis chart of new object recognition test results and new object preference index; C: Representative trajectory diagrams of mice in each group during the orientation and navigation phase of the Morris water maze experiment; D: Quantitative statistical analysis of escape latency during the Morris water maze experiment training phase; E: Statistical analysis chart of the target quadrant dwell time during the Morris water maze test phase; F: Statistical analysis chart of the number of times the platform traversed during the Morris water maze experiment testing phase.

[0025] Figure 14 Three weeks after bilateral stereotactic injection of AAV-shNC or AAV-shRIOK3 into the hippocampus, and 24 hours after sham surgery or CLP surgery, the levels of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in the hippocampus tissue of mice were observed; RIOK3 interfered with adeno-associated virus inhibition of pro-inflammatory cytokine production in microglia. : p < 0.001 vs AAV-shNC sham surgery group, ##: p < 0.01 vs AAV-shNC model group). A: ELISA was used to detect the IL-6 content in the hippocampus of mice in each group; B: ELISA was used to detect the TNF-α content in the hippocampus of mice in each group; C: ELISA was used to detect the IL-1β content in the hippocampus of mice in each group. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0027] This invention provides the use of RIOK3 inhibitors in the preparation of drugs for treating sepsis-associated encephalopathy (SAE). This invention is the first to discover that inhibiting RIOK3 expression or activity can effectively reverse cognitive impairment in SAE model animals. Example 3 of this invention uses RIOK3... fl / fl CX3CR1 Cre Mice and littermate wild-type RIOK3 fl / flUsing mice as experimental subjects, a sepsis-related brain injury model was induced by cecal ligation and puncture (CLP) to investigate the function of the RIOK3 gene. The results showed that, compared with the sham-operated group, RIOK3 expression was significantly upregulated in the wild-type C57BL / 6 mouse CLP model group. Figure 2 ); and wild-type RIOK3 from the same litter as the model group. fl / fl Compared to mice, the model group RIOK3 fl / fl CX3CR1 Cre Cognitive impairment, neuronal and synaptic damage, and neuroinflammation were significantly reduced in mice, and learning and memory were significantly improved. Figure 7-10 These results indicate that microglia-specific RIOK3 gene knockout can alleviate the disease progression of sepsis-associated encephalopathy (SAE). Therefore, the RIOK3 gene can serve as a drug target. In vitro cell or animal models with RIOK3 gene knockout can be constructed to screen for drugs and / or biological agents for the treatment of SAE, achieving therapeutic goals through genetic engineering. Furthermore, small molecule inhibitors can be designed targeting the RIOK3-Mus CDS sequence (SEQ ID NO.1). Using in vitro cell or animal models with RIOK3 gene overexpression, molecules that specifically inhibit RIOK3 can be screened to provide new therapeutic molecules for the treatment of SAE.

[0028] In some embodiments of the present invention, the RIOK3 inhibitor is an shRNA with a nucleotide sequence as shown in SEQ ID NO. 10. This sequence is optimized to specifically target the key functional region of the RIOK3 gene, exhibiting extremely high gene silencing efficiency and low off-target risk, ensuring the precision and safety of the inhibitory effect from the source. Simultaneously, the RIOK3 inhibitor is combined with a viral or non-viral vector to construct a recombinant vector, which not only effectively protects the shRNA from degradation by nucleases in vivo but also significantly improves its transmembrane delivery efficiency and stability in target cells, overcoming the technical bottleneck of easy degradation and difficult delivery of nucleic acid drugs in vivo. Example 9 shows that, after administration of the shRNA shown in SEQ ID NO. 10, the treatment group showed significant improvement compared to the model group in terms of neuroinflammatory factor levels or neuronal pathological damage.

[0029] In some embodiments of the present invention, the sepsis-associated encephalopathy is accompanied by one or more of the following: (i) neuronal and synaptic damage; (ii) neuroinflammation; (iii) impairment of learning, memory and cognitive functions.

[0030] In some embodiments of the present invention, the treatment of sepsis-associated encephalopathy manifests as an improvement in one or more of the following effects: (i) reduction of neuronal and synaptic damage; (ii) reduction of neuroinflammation; (iii) improvement of learning, memory, and cognitive function. Example 4 uses the Morris water maze test (e.g.) Figure 7 As shown in the figure, the escape latency and number of platform crossings in mice were recorded, directly demonstrating the ameliorative effect of RIOK3 gene knockout on cognitive dysfunction. Example 5 observed neuronal morphology (e.g., HE staining and Nissl staining) using HE and Nissl staining. Figure 8 As shown in the figure), the protective effect on neuronal structure was confirmed. Example 6: Electron microscopy was used to observe the ultrastructure of hippocampal synapses (e.g., Figure 9 As shown in the figure, the protective effect on synaptic structures was confirmed. Furthermore, Iba1 immunofluorescence staining was used to detect microglial activation and inflammatory factor levels (such as...). Figure 10 As shown in the figure, the inhibitory effect of RIOK3 gene knockout on neuroinflammation was confirmed. Example 9 used ELISA to detect the levels of inflammatory factors (such as IL-1β, IL-6, TNF-α) in brain tissue. Figure 14 As shown in the figure, the data indicate that inflammatory factors are significantly reduced after treatment with RIOK3 inhibitors.

[0031] The technical solution of the present invention will be described in detail below through specific embodiments: Laboratory animals and their care: Experimental animals: Microglia-specific RIOK3 gene knocked out as constructed in Example 1 fl / fl CX3CR1 Cre Mice and their littermates wild-type RIOK3 fl / fl Mice: Male, 6-8 weeks old, weighing 20-25g.

[0032] Housing environment: All laboratory animals were housed in the Specific Pathogen Free (SPF) Laboratory Animal Center of Wuhan University Institute of Medical Sciences.

[0033] Feeding conditions: The room temperature should be between 22 and 24 degrees Celsius, the humidity between 50% and 70%, the light should be alternating between light and dark for 12 hours, and the animals should have free access to water and food.

[0034] Example 1: RIOK3 fl / fl CX3CR1 Cre Mouse construction RIOK3 for 8-10 week old babies fl / fl Mice (purchased from Jicui (Nanjing) Biotechnology Co., Ltd.) and CX3CR1 Cre Mice (purchased from Jicui (Nanjing) Biotechnology Co., Ltd.) were mated, RIOK3fl / fl Mice carry the Riok3 gene targeting region enclosed by the loxP site, CX3CR1 Cre Mice carry a Cre recombinase expression element driven by the Cx3cr1 promoter. Using the Cre-loxP recombination system, a conditional knockout model of RIOK3 gene deletion was constructed in CX3CR1-expressing cells by achieving specific deletion of the RIOK3 gene. After mating, mice were tail-dissected for enzymatic gene identification, and RIOK3 was successfully screened. fl / fl CX3CR1 Cre Mice (e.g.) Figure 6 The primer pairs used in the enzymatic digestion method are: F1 (SEQ ID NO.2), R1 (SEQ ID NO.3), F2 (SEQ ID NO.4), and R2 (SEQ ID NO.5).

[0035] Example 2: Expression of RIOK3 in peripheral blood mononuclear cells (PBMCs) of SAE patients Healthy volunteers and SAE patients were selected. Blood samples were collected, and serum levels of neuron-specific enolase (NSE) and S100B, key indicators for assessing brain injury, were measured. Total RNA was extracted from peripheral blood PBMCs and subjected to qRT-PCR to detect RIOK3 mRNA levels, followed by correlation analysis. Results are as follows: Figure 1 As shown, consistent with the upregulation of NSE and S100B, the expression of RIOK3 mRNA in sepsis-associated encephalopathy PBMCs was significantly upregulated. The primer pair used for qRT-PCR was F3 (SEQ ID NO.6) and R3 (SEQ ID NO.7).

[0036] Example 3: Construction of a CLP surgical model With RIOK3 fl / fl CX3CR1 Cre Mice and littermate wild-type RIOK3 fl / fl In mice, a mouse model of sepsis-related encephalopathy was established using cecal ligation and perforation (CLP). The procedure is as follows: 1. Preoperative preparation (1) Anesthesia: First, weigh the mice and calculate the required amount of anesthetic (2% sodium pentobarbital) based on 45 mg / kg body weight. Administer the anesthetic via intraperitoneal injection and record the injection time. Successful anesthesia is defined as no obvious reaction to tail or toe clamping and the mouse being in good condition (generally, there is no obvious reaction about 10 minutes after injection; the optimal time for surgery is about 30 minutes after anesthesia, with the mouse showing a toe clamping reaction approximately 50 minutes after anesthesia).

[0037] (2) Preparation of the surgical area: Remove hair from the lower quadrant of the abdomen and disinfect with iodine solution three times.

[0038] (3) Fully expose the cecum: Make a longitudinal incision of about 1.0 cm along the midline of the abdomen, bluntly separate the abdominal wall tissue, expose the abdominal cavity, gently explore the cecum and place it outward intact.

[0039] 2. CLP surgery Model group: A non-invasive silk suture was used to ligate the cecum at approximately the distal half of the ileocecal valve. Then, using a 21G needle, a single perforation was made into the distal cecum, avoiding the mesenteric vessels. A small amount of contents was squeezed out by gently pressing the cecum to confirm successful perforation. After perforation, the cecum was repositioned in the abdominal cavity, and the incision was sutured along the abdominal muscles and skin layers. The surgical area was then disinfected again with povidone-iodine.

[0040] Sham surgery group: After freeing the cecum, it was not ligated or perforated, and the remaining steps were the same as those in the sepsis-associated encephalopathy group.

[0041] 3. Postoperative care Immediately after CLP surgery, mice were given a subcutaneous injection of 1 mL of preheated 0.9% saline (for prophylactic fluid resuscitation) and buprenorphine 0.05 mg / kg for analgesia. The animals were kept warm on a temperature-controlled mat until fully awake. They were allowed free access to food and water post-surgery, and their condition and recovery were closely monitored. RIOK3 fl / fl CX3CR1 Cre Mice and littermate wild-type RIOK3 fl / fl The mice were tested for various indicators 24 hours after surgery.

[0042] 4. Validation of RIOK3 expression in hippocampal tissue of CLP model mice: Hippocampal tissues from wild-type C57BL / 6 mice (sham-operated group and model group) 24 h after CLP surgery were selected. Proteins were extracted from the hippocampus and subjected to SDS-PAGE-Western blot assay. The expression of RIOK3 protein was measured using an antibody specifically recognizing RIOK3 protein, with β-actin as an internal control. Results are as follows: Figure 2 As shown, the expression of RIOK3 protein was significantly upregulated after CLP surgery.

[0043] Example 4: Evaluation of cognitive function in mice For RIOK3 fl / fl CX3CR1 Cre Mice and littermate wild-type RIOK3 fl / fl Mice (6-8 weeks old, 20-25 g) underwent Sham and CLP surgeries simultaneously, and were treated according to the following regimen 7 days post-surgery. Figure 11 The experimental technique roadmap shown is used to conduct related behavioral experiments: 4.1 Y-maze experiment The Y-maze consists of three arms of equal length, intersecting at a 120° angle in the center to form a typical Y-shape. Before the experiment, three arms were randomly assigned as the start arm, novel arm, and other arms, with the central triangular area serving as a common transition zone. During the training phase, the novel arm was first closed with a barrier. The mouse was gently placed at the entrance of the start arm, with its head facing the distal end of the arm and away from the central area, allowing it to move freely in the two open arms for 10 minutes. After exploration, the mouse was returned to its original cage, and the maze was thoroughly wiped with 75% ethanol to remove odor traces before the next mouse was tested. One hour after the training phase, the test phase began. The novel arm barrier was removed, and the mouse was placed back at the entrance of the start arm, starting the test in the same manner, allowing it to explore freely in the three arms for 5 minutes. Throughout the process, a top-mounted camera system tracked and recorded the mouse's movement trajectory, the number of times it entered each arm, and the duration of its stay in each arm in real time.

[0044] 4.2 New Object Recognition Experiment In a quiet and dimly lit environment, mice were placed in an opaque box (40 cm × 40 cm × 40 cm) for 3 minutes before the experiment to acclimatize. The experiment then entered the exploration phase, with mice placed in an area containing two identical objects, A1 and A2, positioned diagonally. Mice were allowed to explore freely for 5 minutes before being removed. 24 hours later, the identification experiment began. The familiar object (A1) was replaced by a new object (A3) with a different shape but identical properties, allowing the mouse to explore for 5 minutes. The mice's movement parameters were monitored and analyzed using a camera connected to the Any-Maze animal tracking system software. The exploration time for the familiar and novel objects was recorded, and a preference index was calculated. The preference index was defined as: [Time spent exploring the new object (s) (A3) / Total time spent exploring both objects (s) (A2 + A3)] × 100%. After each experiment, the floor and sidewalls of the square testing area were wiped with 75% ethanol to remove odors and secretions that could influence the olfactory cues of the next mouse.

[0045] 4.3 Morris Water Maze Experiment The Morris water maze test was used to assess the spatial learning and memory abilities of mice. The experimental setup consisted of a circular pool with a diameter of 100 cm and a depth of 50 cm, divided into four quadrants after being filled with clean water. A platform with a diameter of 10 cm was fixed in the target quadrant, with its surface approximately 1 cm underwater. The water temperature was maintained at 23±1℃ throughout the experiment. Swimming trajectories and related parameters were recorded using a camera device combined with the Any-Maze animal behavior analysis system. Orientation and navigation training was conducted continuously for 4 days, 4 times daily, with 20-minute intervals between each session. Each time, mice were placed in the water from a random entry point facing away from the platform and allowed to freely search for the hidden platform for 60 seconds. If a platform was found, the mouse stayed for 10 seconds; if not, it was guided to the platform and stayed for 30 seconds. The escape latency and swimming trajectory were recorded to evaluate spatial learning ability. On day 5, a spatial exploration experiment was conducted. After removing the platform, the mice were placed in the water from the opposite quadrant and allowed to swim freely for 60 seconds. The time spent in the target quadrant, the number of times they crossed the original platform position, and the swimming path were recorded to assess spatial memory retention.

[0046] RIOK3 fl / fl CX3CR1 Cre Mice and littermate wild-type RIOK3 fl / fl The results of behavioral experiments performed on mice after CLP surgery are shown in the figure below. Figure 7 In the Y-maze test, microglia RIOK3 gene knockout effectively prolonged the exploration time of novel arms; in the new object recognition experiment, microglia RIOK3 gene knockout improved the decline in the preference index; in the MWM test, microglia RIOK3 gene knockout effectively reduced the delay in reaching the platform and increased the time spent in the target quadrant and the number of platform crossings. In summary, CLP surgery can induce memory and cognitive dysfunction in mice, while RIOK3 deficiency can significantly alleviate the learning, memory, and cognitive function impairments in CLP mice, suggesting that RIOK3 may be involved in the development and progression of SAE-related neurological dysfunction.

[0047] Example 5: Hippocampal histopathological examination For RIOK3 fl / fl CX3CR1 Cre Mice and littermate wild-type RIOK3 fl / fl Mice (6-8 weeks old, 20-25 g) underwent Sham and CLP surgeries simultaneously. Hippocampal tissue was obtained 24 hours post-surgery for pathological examination according to the following steps: 1. Get materials (1) Preliminary work: Pre-cool the dissection instruments, culture dishes / filter paper and PBS on ice in advance; prepare labeled EP tubes (mouse number, group, type of surgery and date of collection) and 10 mL centrifuge tubes containing 4% paraformaldehyde fixative.

[0048] (2) Sample Collection: Mice were fully anesthetized and fixed. Once deep anesthesia was confirmed and the toe-clamping reflex was absent, the scalp was quickly cut open, and the skull was cut forward along the foramen magnum. The bone flap was gently lifted, and the whole brain was completely removed and immediately placed in a pre-chilled culture dish on ice. The sections were preserved intact and placed in 4% paraformaldehyde. For index determination, the hippocampus was isolated: the brain tissue was divided into two hemispheres along the midsagittal plane. One hemisphere was placed with its inner surface facing up, and the covering cortex was gently peeled off to expose the hippocampal structure. The intact hippocampus was carefully separated along the boundary. After removing the residual cortex, white matter, choroid plexus, and other attached tissues under ice-cold conditions, the removed hippocampus was quickly transferred to a pre-chilled EP tube, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0049] 2. Pathological examination 2.1 Preparation of paraffin-embedded specimen sections The main procedures include: complete and fixed brain → embedding frame processing → rinsing with running water → dehydration → clearing → wax infiltration → embedding → sectioning → spreading → air drying or baking for later use.

[0050] 2.2 Hematoxylin-eosin staining (HE) and Nissl staining Main steps: Paraffin-embedded specimens baked at 60℃ for 30 min were placed in xylene for 5 min × 3 times → 100% ethanol for 5 min × 2 times → 95% ethanol for 5 min → 70% ethanol for 5 min → rinsed with distilled water for 5 min × 2 times → rinsed with PBS for 5 min → rinsed with PBS for 10 min → discarded PBS, and then placed in pH 6.0 sodium citrate buffer for antigen retrieval, followed by rinsing with PBS. For HE staining, the sections were first stained with hematoxylin for nuclei, differentiated with acid water, and blued with ammonia water, then counterstained with eosin for cytoplasm, followed by graded ethanol dehydration, xylene clearing, and mounting. For Nissl staining, the sections were stained with 1% toluidine blue, and then subjected to the same dehydration, clearing, and mounting processes. Finally, the morphological and pathological changes of neurons in the hippocampus were observed and evaluated under a microscope.

[0051] RIOK3 fl / fl CX3CR1 Cre and its wild-type RIOK3 fl / fl Phenotypic results of mice after CLP surgery are shown in Figure 8 In the Sham group, hippocampal neurons were neatly arranged, with intact cell structures, uniform staining of cytoplasm and nucleus, and clear cell morphology. In contrast, RIOK3... fl / fl The +CLP group showed significant pathological changes in the hippocampus, particularly in the CA3 region: disordered neuronal arrangement, increased intercellular spaces, accompanied by deepened cytoplasmic staining and nuclear pyknosis, and a significantly increased number of damaged neurons. This was in contrast to the RIOK3 group. fl / fl+CLP group, RIOK3 fl / fl CX3CR1 Cre The CLP group showed a reduction in the degree of pathological damage, manifested as a decrease in neuronal degeneration, cytoplasmic hyperstaining, and nuclear pyknosis.

[0052] Example 6: Observation of the ultrastructure of hippocampal synapses For RIOK3 fl / fl CX3CR1 Cre Mice and littermate wild-type RIOK3 fl / fl Mice (6-8 weeks old, 20-25 g) underwent Sham and CLP surgeries simultaneously. Hippocampal tissue was obtained for electron microscopy 24 h post-surgery according to the following steps: 1. Preliminary preparations (1) Preparatory work: Pre-cool the dissection instruments, culture dishes / filter paper and PBS on ice in advance; prepare labeled EP tubes containing electron microscopy fixative (mouse number, group, type of surgery and date of collection).

[0053] (2) Electron microscopy sampling: Mice were anesthetized and then perfused and fixed via the left ventricle. First, approximately 50 mL of pre-cooled physiological saline was injected until the right atrial appendage effluent became clear and blood was removed. Then, 50 mL of 4% paraformaldehyde solution was injected for initial fixation in vivo. After the mice were fully rigid and the tissues were completely fixed, the brain tissue was rapidly separated, and approximately 1 mm of the hippocampus was harvested. 3 The tissue block was placed into the aforementioned EP tube.

[0054] 2. Electron microscopy observation of synaptic ultrastructure Immediately after sampling, tissues were fixed in 2.5% glutaraldehyde at 4°C for 2–4 h or overnight, followed by rinsing three times with 0.1 M PBS for 10 min each time, and then post-fixed in 1% osmium tetroxide for 1–2 h. The fixed samples were then dehydrated sequentially with 50%–100% graded ethanol, replaced with propylene oxide, and then infiltrated with resin, finally embedded in Epon 812 epoxy resin and polymerized at 60°C for 24–48 h. After curing, sections approximately 70 nm thick were prepared using an ultramicrotome, placed on a 200-mesh copper grid, and stained sequentially with uranium acetate and lead citrate before air-drying. Finally, transmission electron microscopy was used to observe and image the ultrastructure of hippocampal synapses, and the thickness of postsynaptic dense patches and the width of synaptic gaps were measured and analyzed. Multiple fields of view and multiple synapses were randomly selected for each group for quantification to ensure the representativeness and reliability of the results. RIOK3 fl / fl CX3CR1 Cre Mice and littermate wild-type RIOK3 fl / fl Synaptic changes in mice after CLP surgery are shown in the figure. Figure 9In the CLP group, synaptic structure was blurred, presynaptic membrane terminal swelling was significant, postsynaptic membrane density was reduced, and synaptic cleft was significantly widened. RIOK3 fl / fl CX3CR1 Cre +CLP group compared to RIOK3 fl / fl The +CLP group showed a thicker postsynaptic density and a narrower synaptic gap, suggesting reduced damage to the synaptic structure.

[0055] Example 7: Expression analysis of RIOK3 in primary microglia stimulated with either control group (PBS) or lipopolysaccharide (LPS) Primary microglia from neonatal Sprague-Dawley (SD) mice (1-3 days old) were cultured. After 24 hours of inoculation and culture, the medium was changed (the specific procedure for culturing primary neonatal SD mouse microglia is described in Example 8 below). Serum-free high-glucose DMEM was added to starve the microglia for 12 hours to synchronize them. Then, the cells were stimulated with PBS and lipopolysaccharide (LPS, 1 μg / mL) for 12 hours respectively. Proteins were extracted from the microglia and subjected to SDS-PAGE-Western blot analysis. The expression of RIOK3 protein was measured using an antibody specifically recognizing RIOK3 protein, with β-actin as an internal control. The results are as follows: Figure 3 As shown, the expression of RIOK3 in microglia was significantly upregulated after stimulation with lipopolysaccharide (LPS).

[0056] Example 8: Effect of RIOK3 interference (Lv-shRIOK3) on the release of pro-inflammatory cytokines in LPS-stimulated primary microglia. 1. Primary microglia culture (1) Pretreatment of culture plates: 0.01% L-poly-L-lysine solution is evenly applied to the bottom of the desired culture dish or culture plate and incubated at 37°C for 4 h or left to stand at room temperature overnight to enhance cell adhesion. After incubation, the coating solution is discarded, and the plate is rinsed 1-2 times with sterile deionized water and then completely air-dried for later use.

[0057] (2) Tissue acquisition and digestion: Newborn mice aged 1-3 days were selected, and whole brain tissue was quickly isolated and placed in ice-cold calcium- and magnesium-free PBS. After the meninges were removed, the brain tissue was cut into small pieces, and 0.25% Trypsin-EDTA solution was added. The tissue was digested in a 37°C incubator for about 5-15 minutes. After the edges of the tissue pieces became loose, an equal volume of complete culture medium containing 10% fetal bovine serum (FBS) was added to stop the digestion.

[0058] (3) Cell dispersion, filtration and centrifugation: Gently pipette to mix the tissue digestion solution to ensure full cell dispersion. Filter the cell suspension through a 70 μm cell sieve to remove undigested tissue fragments. Then centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cell pellet in culture medium containing 10% FBS.

[0059] (4) Cell culture and isolation and purification of microglia: The resuspended cells were seeded into pre-coated 75 cm² plates. 2 In culture flasks, incubate statically at 37°C in a 5% CO2 incubator for 7–10 days without changing the culture medium to form a typical mixed glial cell layer. After confluence, gently tap the side of the culture flask or place it on a shaker at 200 rpm for about 10 minutes to detach microglia from the surface of the astrocyte layer. Collect the supernatant obtained from shaking and centrifuge at 1000 rpm for 5 minutes; the resulting cell pellet is the isolated microglia. Seed into suitable culture plates and continue culturing for subsequent experiments.

[0060] 2. RIOK3 expression interference To construct a lentivirus that targets and knocks down RIOK3, the plasmid pLKO.1-U6-RIOK3-shRNA-Puro (structure shown in [see SEQ ID NO.8]) containing the shRNA sequence targeting RIOK3 was used. Figure 4 The lentiviral packaging plasmid psPAX2 and envelope plasmid pMD2.G were co-transfected into HEK 293T cells to obtain the lentiviral Lv-RIOK3-shRNA-Puro, abbreviated as Lv-shRIOK3. After large-scale amplification and purification, the titer and expression level of the target gene RIOK3 were detected (see...). Figure 4 (For later use)

[0061] Simultaneously, a negative control virus was constructed: the empty vector plasmid pLKO.1-U6-shRNA-Puro containing the non-specific negative control shRNA sequence (SEQ ID NO.9: 5'-UUCUCCGAACGUGUCACGUTT-3') was co-transfected with the lentiviral packaging plasmid psPAX2 and the envelope plasmid pMD2.G into HEK 293T cells to obtain the lentiviral Lv-shRNA-Puro, abbreviated as Lv-shRNA. After large-scale amplification using the same process, it was purified, and the titer was detected for later use.

[0062] In an LPS-induced microglial inflammation model, primary microglia re-inoculated with Lv-shRNA and Lv-shRIOK3 viral solutions (10 MOI) were used to infect cells that had been cultured for 2 days. After 48 hours, cells were stimulated for 12 hours with either 1 μM lipopolysaccharide (LPS) (Sigma, L4516) or PBS, resulting in four groups: Lv-shRNA+PBS, Lv-shRNA+LPS, Lv-shRIOK3+PBS, and Lv-shRIOK3+LPS. Cell culture supernatants from these four groups were then collected for ELISA. The results showed that the levels of pro-inflammatory factors IL-6, IL-1β, and TNF-α in the Lv-shRIOK3+LPS group were significantly lower than those in the Lv-shRNA+LPS group. Figure 5 ).

[0063] Example 9: Effect of pre-administration of recombinant adeno-associated virus containing shRNA as shown in SEQ ID NO. 10 on sepsis-associated encephalopathy 1. Pre-administration of AAV-shRIOK3 An AAV expression plasmid containing the RIOK3-specific shRNA sequence shown in SEQ ID NO. 10 (5′-GCCTACTATCAGACTCTTCATTTCAAGAGAATGAAGAGTCTGATAGTAGGCTTTTTT-3′) was co-transfected with an AAV helper packaging plasmid and a capsid packaging plasmid into HEK293T packaging cells. Recombinant adeno-associated virus (AAV) carrying the RIOK3-targeting shRNA sequence was obtained via adeno-associated virus packaging system. An AAV expression plasmid containing a non-specific negative control shRNA sequence was packaged using the same method to obtain the negative control virus AAV-shNC. After purification, concentration, and titer detection, the resulting virus was used to perform bilateral stereotactic injections into the hippocampus of mice, followed by hippocampal extraction to detect the expression level of the target gene RIOK3 (see...). Figure 12 (For later use)

[0064] Three weeks prior to CLP modeling, mice underwent bilateral hippocampal stereotactic injections of the aforementioned constructed AAV-shRIOK3 or AAV-shNC. After anesthesia, mice were fixed on a stereotactic brain imaging system, and the injection sites in the bilateral hippocampuses were determined using a stereotactic brain map. The corresponding viruses were then slowly injected via a microinjection system. The total viral load injected into the bilateral hippocampuses of each mouse was 2 × 10⁻⁶. 9 vg, viral titer approximately 2 × 10 12The injection rate was vg / mL, with a total injection volume of 1 μL (0.5 μL bilaterally). After injection, the needle was left in place for an appropriate time to reduce viral reflux, then the needle was slowly withdrawn and the incision sutured. Routine postoperative care was provided, and the patient continued to be fed for 3 weeks to ensure adequate AAV-mediated shRNA expression and achieve sustained RIOK3 knockdown.

[0065] 2. Evaluation of cognitive function in mice Mice that had received bilateral stereotactic injections of AAV-shNC or AAV-shRIOK3 into the hippocampus three weeks prior were used as experimental subjects. A mouse sepsis-related encephalopathy model was established using cecal ligation and perforation (CLP). Seven days post-surgery, the mice were treated according to the following... Figure 11 The experimental technique roadmap shown is used to conduct relevant behavioral experiments: see Example 4 for specific experimental methods.

[0066] Behavioral experiments were performed on mice that underwent CLP surgery 3 weeks after bilateral stereotactic injection of AAV-shNC or AAV-shRIOK3 into the hippocampus. (See attached table for details.) Figure 13 In the novel object recognition experiment, hippocampal RIOK3 gene knockdown improved the decline in the preference index; in the MWM test, hippocampal RIOK3 gene knockdown effectively reduced the delay in reaching the platform and increased the time spent in the target quadrant and the number of times the platform was crossed. In summary, CLP surgery can induce memory and cognitive dysfunction in mice, while RIOK3 deficiency can significantly alleviate the learning, memory, and cognitive function impairments in CLP mice, suggesting that RIOK3 may be involved in the development of SAE-related neurological dysfunction.

[0067] 3. Inflammatory factor detection 24 hours after CLP surgery in mice 3 weeks following bilateral stereotactic injection of AAV-shNC or AAV-shRIOK3 into the hippocampus, hippocampal homogenates were extracted for ELISA. Results showed that the levels of pro-inflammatory factors IL-6, IL-1β, and TNF-α in the AAV-shRIOK3+CLP group were significantly lower than those in the AAV-shNC+CLP group. Figure 14 ).

[0068] 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 use of RIOK3 inhibitors in the preparation of drugs for treating sepsis-associated encephalopathy, characterized in that: The RIOK3 inhibitor is an shRNA with a nucleotide sequence as shown in SEQ ID NO.

10.

2. The use according to claim 1, characterized in that: The sepsis-related encephalopathy is accompanied by one or more of the following: (i) Neuronal and synaptic damage; (ii) Nerve inflammation; (iii) Impairment of learning, memory and cognitive functions.

3. The use according to claim 1, characterized in that: The treatment is used for one or more of the following: (i) Reduce neuronal and synaptic damage; (ii) Reduce nerve inflammation; (iii) Improve learning, memory and cognitive functions.

4. The use according to claim 1, characterized in that: The drug comprises a RIOK3 inhibitor and its pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that: The vector can be a viral vector or a non-viral vector.

6. The use according to claim 5, characterized in that: The viral vector is a recombinant adeno-associated virus.

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