Application of lncRNA-MALAT1 / miR-145 / BNIP3 regulation axis in preparation of medicine for treating hemorrhagic shock ischemia-reperfusion intestinal injury

Abstract

The invention discloses an application of an lncRNA-MALAT1 / miR-145 / BNIP3 regulation axis in preparation of a medicine for treating hemorrhagic shock ischemia reperfusion intestinal injury. In existing treatment, conventional liquid resuscitation easily causes serious inflammation and pathological apoptosis, the intestinal barrier repair effect is limited, and the molecular mechanism is not completely clarified. The invention discloses that a regulatory axis participates in an intestinal injury process through a ceRNA mechanism (MALAT1 is used as miR-145 sponge to inhibit regulation of BNIP3 and mediate controlled programmed cell death instead of pathological apoptosis), and provides a new molecular target for treatment. The medicine aiming at the regulation shaft can relieve inflammatory response, reduce pathological apoptosis and promote intestinal barrier repair, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the lncRNA-MALAT1 / miR-145 / BNIP3 regulatory axis in the preparation of drugs for treating ischemia-reperfusion intestinal injury in hemorrhagic shock. Background Technology

[0002] Hemorrhagic shock (HS) is a severe circulatory failure caused by trauma, surgery, or obstetric hemorrhage. Its core pathophysiological feature is a sharp reduction in effective circulating blood volume, leading to ischemia and hypoxia in tissues and organs such as the intestinal mucosa. The intestinal mucosa, being rich in blood vessels and sensitive to hypoxia, is prone to ischemia-reperfusion (I / R) injury: during ischemia, intestinal mucosal cells experience impaired energy metabolism and increased cell membrane permeability; during reperfusion, a large amount of oxygen free radicals are generated, triggering inflammatory responses and oxidative stress, leading to intestinal epithelial cell necrosis, disruption of tight junctions, and intestinal barrier dysfunction. After intestinal barrier disruption, bacteria and toxins translocate into the systemic circulation, triggering systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS), which are major causes of death in HS patients. Fluid resuscitation is a key measure in the treatment of HS, aiming to restore effective circulating blood volume and organ perfusion. Conventional fluid resuscitation (CFR) maintains a high mean arterial pressure (MAP) through rapid and large-volume fluid resuscitation. However, excessive fluid resuscitation can exacerbate pulmonary edema, abdominal compartment syndrome, and intensify intestinal mucosal inflammation and apoptosis due to reperfusion injury. In recent years, restrictive fluid resuscitation (RFR) has gradually gained attention. It maintains a lower MAP by controlling the amount of fluid administered, reducing reperfusion injury and improving patient outcomes. However, the molecular mechanisms by which RFR protects the intestinal mucosa are not yet fully elucidated.

[0003] With the deepening of molecular biology research, the role of non-coding RNA (ncRNA) in the regulation of intestinal barrier function has received increasing attention. Long non-coding RNA (lncRNA) and microRNA (miRNA) are two important classes of ncRNAs that participate in pathophysiological processes such as apoptosis and inflammatory responses through post-transcriptional regulation. Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is a highly conserved lncRNA, widely expressed in various tissues, and plays a pro-damage role in intestinal / respiratory injury (I / R). Studies have found that MALAT1 can act as a competitive endogenous RNA (ceRNA), inhibiting the regulation of target genes by sponging miRNAs such as miR-145. miR-145 is a tumor suppressor miRNA downregulated in various tumors and ischemic diseases. Its target genes include B-cell lymphoma / leukemia-2 interacting protein 3 (BNIP3). BNIP3 is a member of the Bcl-2 family, located in the mitochondrial membrane, and participates in mitophagy and apoptosis: moderately expressed BNIP3 can play a protective role by inducing mitophagy to clear damaged mitochondria; overexpression may lead to loss of mitochondrial membrane potential, release of cytochrome C, and trigger pathological apoptosis. Existing studies have shown that MALAT1, miR-145, and BNIP3 are all involved in infusion / reperfusion injury, but the specific regulatory relationship among the three in hemorrhage-induced intestinal injury (HS-I / R) and the differential regulation under different fluid resuscitation strategies remain unclear.

[0004] Although current technologies provide some understanding of the molecular mechanisms of HS-I / R intestinal injury, significant limitations remain: First, the role of the MALAT1 / miR-145 / BNIP3 regulatory axis in HS-I / R intestinal injury has not been systematically studied, particularly how this axis mediates the intestinal protective effects of different fluid resuscitation strategies; second, existing therapeutic drugs mostly target downstream effects such as inflammation or apoptosis, lacking targeted drugs against this regulatory axis, making it difficult to fundamentally block the molecular pathways of intestinal injury; third, the dual role of BNIP3 in HS-I / R intestinal injury (protective autophagy vs. pathological apoptosis) remains unclear, limiting its application as a therapeutic target. Therefore, elucidating the mechanism of action of the MALAT1 / miR-145 / BNIP3 regulatory axis in HS-I / R intestinal injury and developing drugs targeting this axis are of great significance for improving the prognosis of HS patients.

[0005] This invention reveals the crucial role of the MALAT1 / miR-145 / BNIP3 regulatory axis in HS-I / R intestinal injury, clarifying that this axis regulates the survival and death of intestinal mucosal cells through the pathway of "MALAT1 sponging miR-145 → miR-145 inhibiting BNIP3 → BNIP3-mediated mitophagy / apoptosis." Simultaneously, it elucidates that restrictive fluid resuscitation exerts an intestinal protective effect by downregulating MALAT1, upregulating miR-145, and moderately activating BNIP3-mediated "protective autophagy," thereby reducing pathological apoptosis and inflammatory responses. This discovery fills a gap in the study of the molecular mechanisms of HS-I / R intestinal injury, provides a novel theoretical basis for drug development targeting this regulatory axis, and holds promise as a new strategy for the treatment of HS-I / R intestinal injury. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the existing technology and provide the application of the lncRNA-MALAT1 / miR-145 / BNIP3 regulatory axis in the preparation of drugs for treating ischemic reperfusion intestinal injury in hemorrhagic shock, thus providing a new approach for the treatment of ischemic reperfusion intestinal injury in hemorrhagic shock.

[0007] The technical solution to achieve the purpose of this invention is: the application of the lncRNA-MALAT1 / miR-145 / BNIP3 regulatory axis in the preparation of drugs for treating ischemia-reperfusion intestinal injury in hemorrhagic shock. The mechanism of action of the regulatory axis is: lncRNA-MALAT1 acts as a competitive endogenous RNA (ceRNA) sponge to adsorb miR-145, inhibiting the targeted inhibition of BNIP3 by miR-145, thereby regulating the expression of BNIP3; the drug improves intestinal mucosal barrier function, reduces inflammatory response and pathological cell apoptosis by regulating this regulatory axis.

[0008] As a further improvement, the drug comprises an lncRNA-MALAT1 inhibitor, which relieves the inhibition of miR-145 by reducing the expression or activity of lncRNA-MALAT1.

[0009] As a further improvement, the drug comprises a miR-145 mimic or agonist, which inhibits BNIP3 expression by enhancing the expression or activity of miR-145.

[0010] As a further improvement, the drug contains a BNIP3 activator, which mediates protective programmed cell death by promoting the expression or activity of BNIP3.

[0011] As a further improvement, the lncRNA-MALAT1 inhibitor is selected from antisense oligonucleotides (ASO), small interfering RNA (siRNA), short hairpin RNA (shRNA), or gene editing vectors.

[0012] As a further improvement, the miR-145 mimic is selected from miR-145-5p mimic, miR-145-5pagomir, or modified miRNA oligonucleotides.

[0013] As a further improvement, the BNIP3 activator is selected from BNIP3 expression vectors, BNIP3 recombinant proteins, or BNIP3 promoter activators.

[0014] As a further improvement, the drug is used in combination with a restrictive fluid resuscitation strategy, which maintains the mean arterial pressure (MAP) at 60-70 mmHg.

[0015] As a further improvement, the pathological features of the hemorrhagic shock ischemia-reperfusion intestinal injury include destruction of intestinal mucosal villi, infiltration of inflammatory cells, loss of tight junction proteins, and increased cell apoptosis.

[0016] After adopting the above technical solution, the present invention has the following positive effects: (1) Significantly improves the histopathological changes of ischemia-reperfusion intestinal injury and protects the integrity of the intestinal mucosal barrier. This invention assessed intestinal mucosal morphology using H&E staining. The results showed that the villous structure of the ileum tissue in rats in the restricted fluid resuscitation (RFR) group, which targeted and regulated the lncRNA-MALAT1 / miR-145 / BNIP3 axis, was relatively intact, and the degree of inflammatory infiltration, tissue edema, and vascular congestion was significantly reduced; the intestinal mucosal injury score (6.8±1.2) was significantly lower than that in the conventional fluid resuscitation (CFR) group (9.4±1.6) (P<0.05). This indicates that regulation of this axis can effectively inhibit the destruction of the intestinal mucosal structure caused by hemorrhagic shock and ischemia-reperfusion (I / R), maintain intestinal barrier function, and prevent bacterial translocation and subsequent infection risk.

[0017] (2) It effectively inhibits the inflammatory response and reduces serum inflammatory factor levels. ELISA detection of serum inflammatory markers showed that the serum levels of tumor necrosis factor-α (TNF-α: 126.83 pg / ml) and interleukin-6 (IL-6: 89.66 pg / ml) in the RFR group (regulatory axis activation group) were significantly lower than those in the CFR group (TNF-α: 152.44 pg / ml; IL-6: 115.41 pg / ml) (both P < 0.001). This indicates that regulation of the lncRNA-MALAT1 / miR-145 / BNIP3 axis can effectively inhibit I / R-induced inflammatory cascade, reduce the release of inflammatory mediators, and lower the risk of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS).

[0018] (3) Regulating apoptosis patterns, reducing pathological apoptosis, and promoting protective programmed cell death. This invention detected apoptosis using TUNEL staining. The results showed that the apoptotic cell rate in the RFR group (12.8±2.1%) was significantly lower than that in the CFR group (19.6±3.4%) (P<0.05). Furthermore, the apoptotic cells in the RFR group exhibited a dispersed distribution and weak fluorescence intensity, suggesting BNIP3-mediated protective programmed cell death (such as controlled autophagy or apoptosis); while the apoptotic cells in the CFR group showed aggregation and high-intensity fluorescence, indicating pathological apoptosis (the main form leading to tissue damage). This indicates that this regulatory axis can guide cell death towards a "protective" mode, clearing damaged cells while preventing further damage to the intestinal mucosa from excessive apoptosis.

[0019] (4) The molecular mechanism is clear, providing precise target sites for drug development. This invention constructed a lncRNA-MALAT1 / miR-145 / BNIP3 ceRNA regulatory network (MALAT1 acts as a "sponge" for miR-145, inhibiting its regulation of the target gene BNIP3) through bioinformatics analysis (cross-species, multi-dataset validation). The correlation between changes in this axis expression and its protective effect against intestinal injury was verified by qRT-PCR (MALAT1: RFR group 2.58 vs CFR group 3.93, P=0.027; miR-145-5P: RFR group 0.60 vs CFR group 0.38, P=0.009; BNIP3: RFR group 4.48 vs CFR group 3.63, P=0.025) and Western Blot (BNIP3 protein: RFR group 1.144±0.044 vs CFR group 0.863±0.020, P<0.05). The well-defined molecular mechanism provides a precise target for the development of drugs targeting this axis (such as MALAT1 antisense oligonucleotides, miR-145 mimics, BNIP3 agonists, etc.), avoiding the drawbacks of traditional drugs such as "broad targets and multiple side effects".

[0020] This invention provides a novel strategy for the clinical treatment of hemorrhagic shock and supports the molecular theoretical basis of restrictive fluid resuscitation. It reveals the molecular mechanism by which restrictive fluid resuscitation exerts its intestinal protective effect by regulating the lncRNA-MALAT1 / miR-145 / BNIP3 axis, providing strong molecular theoretical support for the clinical use of restrictive fluid resuscitation as an alternative to conventional fluid resuscitation in the treatment of hemorrhagic shock. Compared to conventional resuscitation, restrictive resuscitation can reduce the inflammatory response, protect the intestinal barrier, and regulate apoptosis patterns through this axis, thereby reducing the incidence of complications (such as infection and MODS) and improving patient prognosis. This discovery will drive the transformation of hemorrhagic shock treatment from "empirical fluid resuscitation" to "precise molecular regulation," and has significant clinical translational value.

[0021] In summary, this invention achieves multidimensional protection (tissue, inflammation, apoptosis, molecular mechanisms) against ischemia-reperfusion intestinal injury in hemorrhagic shock by targeting the lncRNA-MALAT1 / miR-145 / BNIP3 regulatory axis, providing a novel approach and precise target for drug development to treat this disease, and has significant clinical application potential. Attached Figure Description

[0022] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 Trend and functional enrichment analysis of the MALAT1 / miR-145 / BNIP3 regulatory axis in ischemia-reperfusion injury of this invention: (AD), MALAT1 expression analysis of four datasets; (E) BNIP3 expression profile; (FG), miR-145 expression changes and time series analysis; (HI), functional enrichment analysis (GO biological processes and KEGG pathway).

[0023] Figure 2 The morphological effects of different treatments on the intestinal mucosa of this invention (H&E staining of ileal tissue from each group of rats, visually demonstrating the pathological differences in intestinal mucosal tissue among the groups: left column: 100x magnification; right column: 200x magnification. Scale bar: left column 200μm, right column 100μm): (A1-A2), NC group; (B1-B2), HS group; (C1-C2), CFR group; (D1-D2), RFR group; (E1-E2), SEC Group.

[0024] Figure 3 To illustrate the differences in apoptotic cell levels in rat ileum tissue among different groups using fluorescent TUNEL staining (each row from left to right: DAPI staining (blue, cell nuclei), TUNEL staining (green, apoptotic cells), and a merged image; all images were taken at 400x magnification. Scale bar: 50μm): (A1-A3), NC group; (B1-B3), HS group; (C1-C3), CFR group; (D1-D3), RFR group; (E1-E3), SEC Group.

[0025] Figure 4 The effects of different fluid resuscitation strategies of this invention on the MALAT1 / miR-145 / BNIP3 axis and inflammatory cytokines: (AB) qPCR analysis: Quantitative analysis of the relative expression levels of bnip3, MALAT1, and miR-145-5P in the ileum tissue of rats in each group (data were analyzed using 2-...). ΔΔCt The method was calculated and normalized to the NC group as a control). (CD) ELISA analysis: Measurement of serum inflammatory factors IL-6 and TNF-α concentrations; (E) Western Blot analysis: Using β-actin (42kDa) as a loading control, the expression level of BNIP3 protein (22kDa) in the ileum tissue of each group was detected. Detailed Implementation

[0026] The embodiments of the present invention are described in detail below. It should be noted that these embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0027] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; primer sequences, vector construction details, etc., can be supplemented according to the actual situation, and those skilled in the art can repeat them through conventional experimental methods; unless otherwise specified, the methods used in this invention are all conventional methods in the art.

[0028] Specifically, the relevant verification embodiments in this invention are as follows: Example 1

[0029] 1. Experimental Materials 1.1 Laboratory Animals One hundred male Sprague-Dawley (SD) rats, weighing 220-250g, were purchased from an experimental animal center (SPF grade). After one week of acclimatization, they were randomly divided into 5 groups: Normal control group (NC, n=10): No treatment; Hemorrhagic shock group (HS, n=20): No resuscitation after modeling; Routine fluid resuscitation group (CFR, n=20): Routine resuscitation with 0.9% normal saline; Restrictive fluid resuscitation group (RFR, n=20): 7.5% hypertonic saline restrictive resuscitation; Ischemia-reperfusion control group (SEC, n=30): superior mesenteric artery clamping reperfusion.

[0030] 1.2 Main Reagents and Kits Molecular biology reagents: TRIzol reagent (Invitrogen), cDNA synthesis kit (TaKaRa), qRT-PCR kit (Applied Biosystems), RIPA lysis buffer (Beyotime), PVDF membrane (Millipore). Protein detection reagents: BNIP3 primary antibody (Abcam), miR-145 primary antibody (Cell Signaling Technology), MALAT1 primary antibody (Santa Cruz), β-actin primary antibody (Sigma), HRP-labeled secondary antibody (Jackson Immuno Research). Functional assay reagents: ELISA kit (TNF-α, IL-6, R&D Systems), H&E staining kit (Solarbio), TUNEL apoptosis assay kit (Roche). Other: 1% sodium pentobarbital, 0.9% normal saline, 7.5% hypertonic saline.

[0031] 1.3 Main Instruments Real-time quantitative PCR instrument (ABI 7500), microplate reader (BioTek Synergy H1), inverted microscope (Olympus BX53), Western blot system (Bio-Rad), flow cytometer (BD FACSCanto II), and constant temperature heating pad (Harvard Apparatus).

[0032] 2 Experimental Methods 2.1 Establishment of a hemorrhagic shock ischemia-reperfusion model 2.1.1 Anesthesia and Cannulation: Rats were fasted for 8 hours (with free access to water) and anesthetized with 1% sodium pentobarbital (1 ml / 100 g, intraperitoneal injection). They were then fixed in a supine position on a constant-temperature heating pad (25°C). Under aseptic conditions, the left carotid artery was cannulated (for hemodynamic monitoring) and the tail vein was cannulated (for fluid administration).

[0033] 2.1.2 Hemorrhagic shock modeling: In the HS, CFR, and RFR groups, arterial bloodletting was performed to maintain a mean arterial pressure (MAP) of 35-40 mmHg for 60 minutes to simulate hemorrhagic shock.

[0034] 2.1.3 Recovery Strategy: HS group: No intervention after modeling; CFR group: 0.9% normal saline was infused within 30 minutes, with a target MAP of approximately 80 mmHg; RFR group: 7.5% hypertonic saline infusion, maintaining MAP≈60mmHg (restrictive resuscitation); SEC group: superior mesenteric artery clamped for 15 / 30 / 45 minutes, followed by 5 minutes of reperfusion (simulating ischemia-reperfusion injury).

[0035] 2.2 Sample Collection 2.2.1 Blood samples: Blood was collected from the carotid artery at baseline and 30 minutes after recovery. Serum was separated for ELISA testing. 2.2.2 Tissue samples: After euthanasia, ileal tissue was taken from 5 cm proximal to the cecum. Part of the tissue was fixed in 4% paraformaldehyde (for H&E and TUNEL staining), and the other part was flash-frozen in liquid nitrogen (for qRT-PCR and Western blot).

[0036] 2.3 Bioinformatics Analysis Four ischemia-reperfusion datasets were retrieved from the GEO database: GSE37013 (human gut I / R, bulk RNA-seq); GSE190581 (mouse intestinal regeneration, scRNA-seq); GSE163638 (mouse intestinal healing, scRNA-seq); GSE21405 (rat muscle I / R, miRNA microarray).

[0037] Data preprocessing (GEOquery package), single-cell analysis (Seurat v5.0), differential expression analysis (FindMarker, |log2FC|>0.5, adj P<0.05), and functional enrichment (clusterProfiler, GO / KEGG pathway) were performed using R software (v4.3.0) to construct the MALAT1 / miR-145 / BNIP3 ceRNA network.

[0038] 2.4 Molecular expression detection 2.4.1 qRT-PCR: Total RNA was extracted from the intestinal mucosa using TRIzol and reverse transcribed into cDNA. Using β-actin as an internal control, the MALAT1, miR-145, and BNIP3 genes were amplified, and their relative expression levels (2-) were calculated. ΔΔCt Law).

[0039] 2.4.2 Western Blot: Total protein was extracted using RIPA lysis buffer, SDS-PAGE electrophoresis (20-40 μg protein / well), transferred to PVDF membrane, BNIP3 detection was performed using a β-actin-normalized specific primary antibody, and density analysis (ImageJ) was used to quantify relative protein expression.

[0040] 2.5 Functional Indicator Testing 2.5.1 Detection of inflammatory factors: Serum TNF-α and IL-6 concentrations were detected by ELISA. The procedure was strictly followed according to the kit instructions, and the absorbance was read at 450 nm using an ELISA reader.

[0041] 2.5.2 Histopathological assessment: Ileal tissue was paraffin-embedded, sectioned (4μm), stained with H&E, and observed under a microscope (100×, 200×). Scoring was performed according to "villi integrity, inflammatory infiltration, edema, and congestion" (0-4 points for each item, 0-16 points in total).

[0042] 2.5.3 Apoptosis detection: TUNEL staining (fluorescence method: using a commercial kit (Roche, Germany). After dewaxing and antigen recovery (sodium citrate, pH 6.0, 15 min), sections were subjected to peroxidase inhibition (3% h2O2, 10 min) and TUNEL reaction (37°C, 60 min)). Cell nuclei were counterstained with DAPI, observed under a microscope (400×), and the apoptosis index (TUNEL-positive cells / total cell count × 100%) was calculated.

[0043] 2.6 Statistical Analysis Statistical analysis was performed using R software (v4.3.0) and SPSS 26.0: (1) Data are expressed as mean ± SD; (2) Intergroup comparison: Student's t test was used for two groups, and one-way ANOVA (Bonferroni correction) was used for multiple groups. (3) The difference was statistically significant: P<0.05.

[0044] 3. Results Analysis 3.1 Bioinformatics Verification of the Existence of the Regulatory Axis Cross-analysis of four GEO datasets revealed (e.g.) Figure 1 (as shown) 3.1.1 MALAT1 upregulation: In human intestinal I / R (GSE37013), mouse intestinal regeneration (GSE190581), and mouse intestinal healing (GSE163638), MALAT1 was significantly increased after injury (|log2FC|>0.5, P<0.05). 3.1.2 miR-145 downregulation: In rat muscle I / R (GSE21405), miR-145 decreased to 25% of the control group 4 hours after reperfusion (P<0.01). 3.1.3 BNIP3 upregulation: Multiple datasets showed that BNIP3 (miR-145 target) was significantly increased after injury (P<0.05). 3.1.4 Functional enrichment: The regulatory axis involved the pathways of "apoptosis", "inflammatory response" and "tissue repair" (GO / KEGG, P<0.05), supporting the MALAT1 / miR-145 / BNIP3ceRNA regulation hypothesis.

[0045] 3.2 Differential expression of regulatory axis molecules (qRT-PCR / Western blot) Compared with the CFR group, the RFR group (e.g. Figure 4 (AB) and Figure 4 (E) shows: MALAT1 expression was significantly reduced: 2.58 vs 3.93 (P=0.027); miR-145 expression was significantly increased: 0.60 vs 0.38 (P=0.009); BNIP3 expression was significantly increased: mRNA (4.48 vs 3.63, P=0.025) and protein (1.144±0.044 vs 0.863±0.020, P<0.05).

[0046] This indicates differences in the activation of regulatory pathways among resuscitation strategies; RFR promotes pro-apoptotic signaling, while CFR enhances cell protection mechanisms. 3.3 Reduction of inflammatory response (ELISA) (e.g.) Figure 4 (CD) Serum inflammatory factor levels in the RFR group were significantly lower than those in the CFR group: TNF-α: 126.83 pg / ml vs 152.44 pg / ml (P<0.001); IL-6: 89.66 pg / ml vs 115.41 pg / ml (P<0.001).

[0047] The difference (25.6–25.8 pg / ml) indicates that CFR induced more significant inflammatory activation compared to the restrictive approach. 3.4 Improvement of intestinal injury repair (H&E staining) (e.g.) Figure 2 (As shown) The intestinal mucosal injury score in the RFR group was significantly lower than that in the CFR group (6.8±1.2 vs 9.4±1.6, P<0.05), as shown in the following: The fluff structure is more complete; Reduced inflammatory infiltration; Edema and congestion are reduced.

[0048] 3.5 Altered apoptosis patterns (TUNEL staining) TUNEL testing showed (as follows) Figure 3 (as shown) The apoptotic cell rate in the RFR group (12.8±2.1%) was significantly lower than that in the CFR group (19.6±3.4%) (P<0.05). The apoptotic cells in the RFR group were scattered and had weak fluorescence intensity, suggesting that it was BNIP3-mediated protective programmed cell death. The apoptotic cells in the CFR group showed aggregation and high-intensity fluorescence, indicating pathological apoptosis.

[0049] in conclusion This experiment confirms that the lncRNA-MALAT1 / miR-145 / BNIP3 regulatory axis is a key molecular mechanism of ischemia-reperfusion intestinal injury in hemorrhagic shock. Restrictive fluid resuscitation reduces intestinal mucosal damage, inflammatory response, and pathological apoptosis by decreasing MALAT1 expression, increasing miR-145 activity, and promoting BNIP3-mediated protective apoptosis.

[0050] The working principle of this invention is as follows: This invention intervenes in the molecular pathological process of intestinal injury in hemorrhagic shock and ischemia-reperfusion (I / R) by targeting and regulating the lncRNA-MALAT1 / miR-145 / BNIP3 axis. Specifically, inhibiting lncRNA-MALAT1 expression can relieve its negative regulation of miR-145, promote the increase of miR-145 activity, and thus relieve the inhibitory effect of miR-145 on the pro-apoptotic protein BNIP3; moderately upregulated BNIP3 mediates controllable programmed cell death (protective apoptosis), rather than pathological apoptosis, thereby reducing intestinal epithelial damage, inhibiting the release of inflammatory factors (TNF-α, IL-6), maintaining the integrity of villous structure, and ultimately repairing the intestinal mucosal barrier function. Regulators of this axis (such as miR-145 mimics, MALAT1 inhibitors, and BNIP3 activators) can be used to prepare drugs for treating intestinal injury in hemorrhagic shock and ischemia-reperfusion by coordinating the balance between cell death and tissue homeostasis.

[0051] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of the lncRNA-MALAT1 / miR-145 / BNIP3 regulatory axis in the preparation of drugs for treating ischemia-reperfusion intestinal injury in hemorrhagic shock, characterized in that, The mechanism of action of the regulatory axis is as follows: lncRNA-MALAT1 acts as a competitive endogenous RNA (ceRNA) sponge to adsorb miR-145, inhibiting the targeted inhibition of BNIP3 by miR-145, thereby regulating the expression of BNIP3; the drug improves intestinal mucosal barrier function, reduces inflammatory response and pathological cell apoptosis by regulating this regulatory axis.

2. The application according to claim 1, characterized in that, The drug contains a lncRNA-MALAT1 inhibitor, which relieves the inhibition of miR-145 by reducing the expression or activity of lncRNA-MALAT1.

3. The application according to claim 1, characterized in that, The drug comprises a miR-145 mimic or agonist, which inhibits BNIP3 expression by enhancing the expression or activity of miR-145.

4. The application according to claim 1, characterized in that, The drug contains a BNIP3 activator, which mediates protective programmed cell death by promoting the expression or activity of BNIP3.

5. The application according to claim 2, characterized in that, The lncRNA-MALAT1 inhibitor is selected from antisense oligonucleotides (ASO), small interfering RNA (siRNA), short hairpin RNA (shRNA), or gene editing vectors.

6. The application according to claim 3, characterized in that, The miR-145 mimic is selected from miR-145-5p mimic, miR-145-5p agomir, or modified miRNA oligonucleotides.

7. The application according to claim 4, characterized in that, The BNIP3 activator is selected from BNIP3 expression vectors, BNIP3 recombinant proteins, or BNIP3 promoter activators.

8. The application according to any one of claims 1-7, characterized in that, The medication is used in conjunction with a restrictive fluid resuscitation strategy, which maintains mean arterial pressure (MAP) at 60-70 mmHg.

9. The application according to any one of claims 1-7, characterized in that, The pathological features of hemorrhagic shock-ischemia-reperfusion intestinal injury include destruction of intestinal mucosal villi, infiltration of inflammatory cells, loss of tight junction proteins, and increased cell apoptosis.

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