Therapeutic application of si-PRMT6-based sepsis-ARDS

Abstract

The invention belongs to the technical field of biological medicine, provides novel application of PRMT6, and particularly relates to application of a PRMT6 inhibitor in preparation of a medicine for preventing or treating sepsis-ARDS. The invention discloses the application value of the PRMT6 as the sepsis-ARDS treatment target for the first time. According to the application disclosed by the invention, the expression of PRMT6 is inhibited in a targeted manner, ferroptosis in alveolar epithelial cells (AECs) can be effectively inhibited, and the histopathology related to ARDS is further improved, so that the disease progress is effectively improved, and a new path is opened up for targeted therapy of sepsis-ARDS.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to application of PRMT6 as a sepsis-ARDS treatment target, in particular to application of si-PRMT6 in sepsis-ARDS treatment. BACKGROUND

[0002] Sepsis is a devastating clinical disease that often leads to multiple organ failure in intensive care unit patients, causing a heavy burden on global health. Under the influence of sepsis, the lung becomes one of the most vulnerable organs, about 45% of cases develop from acute lung injury (ALI) to acute respiratory distress syndrome (ARDS), with high mortality and complex treatment, often requiring mechanical ventilation to maintain oxygenation, and there is still no effective drug treatment method.

[0003] The pathophysiological features of ALI include extensive damage to alveolar epithelial cells (AECs) and microvascular endothelial cells, pulmonary edema and excessive inflammatory responses induced by immune cells. Although apoptosis has been considered as the main form of AEC death, ferroptosis has become a key factor in various ALI models, including sepsis, oleic acid, radiation and ischemia-induced models.

[0004] As a form of iron-dependent programmed cell death marked by lipid peroxidation, ferroptosis plays a crucial role in various biological processes and pathological conditions, including infectious diseases: a study reported that glutathione peroxidase 4 (GPX4) inactivation led to lipid peroxidation accumulation, triggering cellular ferroptosis, resulting in sepsis multiple organ dysfunction; emerging evidence suggests that protein arginine methyltransferase (PRMT)-mediated epigenetic modification is a key regulator of ferroptosis. PRMTs catalyze the methylation of arginine residues in target proteins, significantly affecting the progression of pulmonary diseases. PRMT6, among them, is classified as type I, mainly producing H3R2me2a marks through asymmetric dimethylation, regulating the process of gene expression. PRMT6 plays a key role in the pathogenesis of inflammation, and PRMT6 is also related to ferroptosis in infectious diseases, it is speculated that PRMT6 may promote the occurrence and development of sepsis-ARDS by promoting ferroptosis, but no related work has been reported. SUMMARY

[0005] The present application is based on the above research, and aims to provide PRMT6 as a sepsis-ARDS treatment target, and to provide si-PRMT6 designed according to PRMT6 for treating sepsis-ARDS, and also provides a pharmaceutical composition with si-PRMT6 as an active component.

[0006] The application adopts a mouse cecal ligation puncture (CLP) model to induce sepsis-acute respiratory distress syndrome (ARDS), and divides the experimental mice into a sham operation group (sham group), a negative control group (CLP+DMSO group), a positive control group (CLP+EPZ020411 group), and an experimental group (CLP+si-PRMT6 LNP group). Histopathological analysis shows that PRMT6 is a key controllable node in the pathogenesis of sepsis-ARDS, and inhibition or knockout of PRMT6 mainly prevents ARDS through inhibition of ferroptosis pathway. Both PRMT6 inhibitor EPZ020411 and lung-targeting lipid nanoparticles (LNPs) encapsulating PRMT6-targeted siRNA (siPRMT6 LNPs) reproduce the protective effect of PRMT6 deletion, improve ARDS-related histopathology, and inhibit ferroptosis biomarkers.

[0007] Based on the above research, the application provides the following technical solutions:

[0008] In a first aspect of the application, the application of PRMT6 as a sepsis-ARDS treatment target is provided, and inhibition or knockout of PRMT6 mainly prevents sepsis-ARDS through inhibition of the ferroptosis pathway.

[0009] In a second aspect of the application, the application of a substance that inhibits or silences PRMT6 is provided, i.e., the application in the preparation of a drug for preventing or treating sepsis-ARDS.

[0010] Preferably, the substance that inhibits or silences PRMT6 is selected from any of the following cases:

[0011] (1) a small molecule inhibitor of PRMT6, such as EPZ020411;

[0012] (2) shRNA or siRNA that inhibits the expression of PRMT6;

[0013] (3) a recombinant expression vector or transgenic cell line of the above shRNA or siRNA;

[0014] (4) a liposome (LNP) or nanoparticle coated with the above shRNA or siRNA.

[0015] The sequence of the siRNA is as follows:

[0016] Sense strand: 5'-GAUAUGAGCUGCAUGGAGATT-3' (SEQ ID NO. 1);

[0017] Antisense strand: R: 5'-UCUCCAUGCAGCUCAUAUCTT-3' (SEQ ID NO. 2).

[0018] The PRMT6 siRNA-coated liposome and PRMT6 small molecule inhibitor EPZ020411 were used as therapeutic drugs in the present application, and the results showed that LNP characterization showed good biocompatibility and no detectable effect on physiological organ function. LNP-siPRMT6 mediated dose-dependent and sustained PRMT6 inhibition at mRNA and protein levels in MLE-12 cells and mouse lung tissues. Both strategies reproduced the protective effect of PRMT6 deletion, improved ARDS-related histopathology, and inhibited ferroptosis biomarkers.

[0019] In a fourth aspect of the present application, a pharmaceutical composition for treating sepsis-ARDS is provided, comprising an active ingredient, preferably a PRMT6 inhibitor, and a pharmaceutically acceptable carrier.

[0020] The PRMT6 inhibitor is selected from any of the following:

[0021] (1) a small molecule inhibitor of PRMT6, such as EPZ020411;

[0022] (2) shRNA or siRNA that inhibits the expression of PRMT6;

[0023] (3) a recombinant expression vector or transgenic cell line of the above shRNA or siRNA;

[0024] (4) a liposome (LNP) or nanoparticle coated with the above shRNA or siRNA.

[0025] Preferably, the sequence of the siRNA is as shown in SEQ ID NO. 1 and 2 above.

[0026] The pharmaceutical composition can be used to treat sepsis-ARDS, specifically by treating the inflammatory response in the lung tissue of sepsis-ARDS, and further by inhibiting the ferroptosis response in the lung tissue of sepsis-ARDS to achieve disease treatment.

[0027] Preferably, the pharmaceutical composition is used in combination with other therapeutic drugs.

[0028] In the preferred embodiment of the present application, si-PRMT6 LNPs coated with PRMT6 siRNA are used as therapeutic drugs, and the preparation method is as follows:

[0029] (1) LNP encapsulating siRNA is prepared using 5A2SC8, DOPE, cholesterol ester, DMG-PEG2000, and DOTAP as raw materials, and the molar ratio of the above components in the final raw materials is 11.90 : 11.90 : 23.81 : 2.38 : 50;

[0030] (2) The raw materials in step (1) are dissolved in an ethanol solution to prepare a lipid storage solution, siRNA is dissolved in a 10 mM citric acid buffer with a pH of 4.0, and the two solutions are mixed at a volume ratio of 30:1. Among them, in the lipid storage solution, the concentrations of 5A2SC8, DOPE, cholesteryl ester, DMG-PEG2000 and DOTAP used to encapsulate 1 μg of siRNA are 0.442 mM, 0.442 mM, 0.883 mM, 0.088 mM and 1.854 mM, respectively.

[0031] Compared with the prior art, the beneficial effects of the present application are as follows:

[0032] The application value of PRMT6 as a sepsis-ARDS treatment target is disclosed for the first time. By targeting and inhibiting the expression of PRMT6, the ferroptosis in alveolar epithelial cells (AECs) can be effectively inhibited, thereby improving the ARDS-related histopathology, and effectively improving the disease progression, thereby opening up a new path for the targeted treatment of sepsis-ARDS. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 It is shown that PRMT6 aggravates ARDS through ferroptosis: a lung section hematoxylin-eosin (H&E) and lung injury score of each group of mice, scale, 50 μm; b TUNEL staining and TUNEL staining quantification of lung sections of mice in different groups, scale, 50 μm; c GPX4 immunohistochemical staining and GPX4 expression quantification of lung sections of mice in different groups, scale, 50 μm; d 4-HNE immunohistochemical staining and 4-HNE expression quantification of lung sections of mice in each group, scale, 50 μm; e MDA and GSH levels in lung tissues of mice in different groups.

[0034] Figure 2 It is shown that PRMT6 aggravates ARDS inflammation: a Western blot analysis of PRMT6 expression in lung tissues of mice in different groups; b total protein content in bronchoalveolar lavage fluid of mice in each group; c lung wet / dry ratio of mice in different groups; d changes in IL-1β, IL-6, TNF-α levels in bronchoalveolar lavage fluid of mice in each group; e total cell count and neutrophils in bronchoalveolar lavage fluid of mice in each group.

[0035] Figure 3The siPRMT6 LNPs effectively target lung tissue and reduce the expression of PRMT6: a, size distribution, polydispersity index (PDI), zeta potential and encapsulation efficiency of siPRMT6 LNPs; b, cryogenic transmission electron microscopy images of siPRMT6 LNPs, scale bar, 50 nm; c, H&E staining of lung, liver, spleen, kidney, scale bar, 50 μm; d, serum ALT, AST, BUN, CK-MB levels; e, PRMT6 protein and mRNA levels in MLE-12 cells after 24 h of dose-dependent LNP treatment; f, PRMT6 protein and mRNA levels in mouse lungs after 24 h of dose-dependent LNP treatment; g, PRMT6 protein and mRNA levels in mouse lungs after 0-7 days of 0.5 mg / kg LNP treatment.

[0036] Figure 4 Targeted inhibition of PRMT6 can reduce ARDS: a, H&E, TUNEL staining, GPX4, 4-HNE immunohistochemical staining of lung sections of mice in different groups, scale bar, 50 μm; b, lung injury score, TUNEL staining quantification, GPX4 expression, 4-HNE expression quantification; c, lung BALF and wet / dry total protein content of mice in different groups; d, changes in IL-1β, IL-6, TNF-α levels in the alveolar lavage fluid of mice in each group; e, total cell count and neutrophils in the alveolar lavage fluid of mice in each group. DETAILED DESCRIPTION

[0037] The present application will be described in detail below with reference to the embodiments and drawings, but the implementation of the present application is not limited to this.

[0038] The reagents and raw materials used in the present application are commercially available or can be prepared according to the literature method. The experimental methods in the following examples are generally carried out according to the conventional conditions or according to the conditions recommended by the manufacturer, unless otherwise specified.

[0039] 1. Experimental materials

[0040] 48 male clean C57BL / 6 mice (divided into 4 groups, 12 mice in each group, 22-25 g), MLE-12 cells, si-PRMT6 LNPs (synthesized by self), sevoflurane, hematoxylin-eosin staining agent, TUNEL kit, GPX4 antibody, 4-HNE antibody, BCA kit, Ly6G flow antibody, EPZ020411 inhibitor (PRMT6 activity inhibitor).

[0041] 2. Experimental methods

[0042] Animal model preparation: After the mice were anesthetized with sevoflurane, CLP operation was used to induce sepsis-ARDS. The experimental mice were divided into sham group, CLP+DMSO group, CLP+EPZ020411 group, CLP+LNP group. After 18 hours of observation, the mice were sacrificed, and the lung tissue or lung lavage fluid was taken. Hematoxylin-eosin and TUNEL staining was performed on lung tissue sections, GPX4 and 4-HNE immunohistochemistry was performed, lung lavage fluid was measured for protein concentration by BCA method, and flow cytometry was used to detect cell count in lavage fluid.

[0043] si-PRMT6 LNP preparation: DOTAP-50 LNPs (si-PRMT6 LNPs) targeting the lungs were prepared by ethanol dilution method. (1) Lipid stock solution was prepared in ethanol, with 4.42 mM 5A2SC8, 4.42 mM DOPE, 8.83 mM cholesterol, 0.88 mM DMG-PEG2000, 18.54 mM DOTAP as raw materials to prepare DOTAP-50 LNPs encapsulating 10 μg siRNA, with a final molar ratio of 11.90 / 11.90 / 23.81 / 2.38 / 50; (2) siRNA was dissolved in 10 mM citric acid buffer (pH 4.0); (3) the lipid phase 10 μL was mixed with the siRNA-water phase 30 μL, so that the total lipid:siRNA ratio was 30:1, to maximize the safety and efficacy of DOTAP-50 LNPs.

[0044] DOTAP-50 LNPs were diluted to 0.5 μg / mL siRNA for dynamic light scattering (DLS) and RiboGreen experiments, and to 4 μg / mL for in vitro silencing experiments. All DOTAP-50 LNPs were dialyzed against PBS (Pur-A-Lyzer Midi Dialysis Kit, WMCO 3.5 kDa) for 2 hours, and were intravenously injected after appropriate dilution in PBS. DOTAP-50 LNPs were appropriately diluted in PBS, and the hydrodynamic diameter, polydispersity index (PDI), and zeta potential were measured on a Zetasizer Nano ZS (Malvern Instruments). The encapsulation efficiency of siRNA was determined using a modified Quant-iT RiboGreen RNA assay (Invitrogen). Cryo-TEM (ThermoFisher) was used to image DOTAP-50 LNPs.

[0045] 3. Experimental results

[0046] Histopathological analysis using hematoxylin and eosin (H&E) staining showed that PRMT6 knockout significantly alleviated CLP-induced ARDS, manifested as reduced pulmonary congestion, reduced alveolar wall thickening, and decreased inflammatory infiltration. Figure 1 a). PRMT6 deficiency further reduced pulmonary edema, and significantly decreased total protein levels and lung wet / dry weight ratio in BALF (a). Figure 2 As expected, CLP significantly increased the BALF concentrations of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. Figure 2 d), PRMT6 deficiency significantly reduced this concentration. PRMT6 deficiency also inhibited CLP-induced accumulation of total cells and neutrophils in BALF (d). Figure 2 e). Furthermore, PRMT6 deficiency reduced markers of ferroptosis in the lungs during CLP challenges, including cell death, GPX4, lipid peroxidation markers 4-hydroxynonenal (4-HNE), MDA, and GSH ( Figure 1 be).

[0047] To validate PRMT6 as a controllable therapeutic target, we employed pharmacological and targeted genetic interventions. We designed and characterized the PRMT6 inhibitor EPZ020411 and lung-targeting lipid nanoparticles (LNPs) encapsulating PRMT6-targeting siRNA (siPRMT6 LNPs). LNP characterization showed good biocompatibility and no detectable effect on physiological organ function. Figure 3 ad. LNP-siPRMT6 mediates dose-dependent and sustained PRMT6 inhibition of mRNA and protein levels in MLE-12 cells and mouse lung tissue. Figure 3 e.g., both strategies reproduced the protective effect of PRMT6 deficiency and improved ARDS-related histopathology. Figure 4 a~b), improved inflammation levels ( Figure 4 c~e) inhibited ferroptosis biomarkers. Overall, these findings suggest that PRMT6 is a key node in the pathogenesis of sepsis-ARDS, and that siPRMT6 LNPs can effectively improve disease progression.

[0048] To characterize the ferroptosis-dependent mechanism underlying PRMT6-mediated ARDS pathology, we evaluated the therapeutic overlap between the ferroptosis inhibitor lipresstatin-1 (Lip-1) and PRMT6 deficiency. Lip-1's protective effect against ALI was comparable to that of PRMT6 deficiency, but combination therapy failed to further improve damage or ferroptosis markers in PRMT6-deficient mice, suggesting that PRMT6 deficiency primarily prevents ARDS by inhibiting the ferroptosis pathway.

[0049] To sum up, the application designs the lung-targeted nano-liposome (LNP) for wrapping PRMT6 siRNA, and confirms the treatment effect of anti-si-PRMT6 LNP on ARDS, which not only can bring a new direction for the treatment of ARDS, but also has strong biocompatibility and important clinical significance.

[0050] The unexplained part in the present application is the same as the prior art or realized by using the prior art. The applicant declares that the present application is illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed methods, that is, it does not mean that the present application must rely on the above-mentioned detailed methods to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. Application of PRMT6 as a therapeutic target for sepsis-ARDS.

2. Application of PRMT6 inhibitors in the preparation of drugs for the prevention or treatment of sepsis-ARDS.

3. The application according to claim 2, characterized in that, The PRMT6 inhibitor is selected from any of the following: (1) Small molecule inhibitors of PRMT6; (2) shRNA or siRNA that inhibits PRMT6 expression; (3) Recombinant expression vectors or transgenic cell lines of the above-mentioned shRNA or siRNA; (4) Liposomes (LNPs) or nanoparticles coated with the above-mentioned shRNA or siRNA.

4. The application according to claim 3, characterized in that, The small molecule inhibitor of PRMT6 is selected from EPZ020411.

5. The application according to claim 3, characterized in that, The sequence of the siRNA is shown in SEQ ID NO.1 and 2.

6. A pharmaceutical composition for treating sepsis-ARDS, characterized in that, It includes an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is selected from PRMT6 inhibitors.

7. The pharmaceutical composition for treating sepsis-ARDS according to claim 6, characterized in that, The PRMT6 inhibitor is selected from any of the following: (1) Small molecule inhibitors of PRMT6; (2) shRNA or siRNA that inhibits PRMT6 expression; (3) Recombinant expression vectors or transgenic cell lines of the above-mentioned shRNA or siRNA; (4) Liposomes or nanoparticles coated with the above-mentioned shRNA or siRNA, The sequence of the siRNA is shown in SEQ ID NO.1 and 2.

8. The pharmaceutical composition for treating sepsis-ARDS according to claim 6, characterized in that, The pharmaceutical composition is used in combination with other therapeutic agents.

9. The pharmaceutical composition for treating sepsis-ARDS according to claim 6, characterized in that, The preparation method of liposomes si-PRMT6 LNPs coated with PRMT6 siRNA is as follows: (1) LNPs encapsulating siRNA were prepared using 5A2SC8, DOPE, cholesterol ester, DMG-PEG2000, and DOTAP as raw materials. The final molar ratio of the above components in the raw materials was 11.90 : 11.90 : 23.81 : 2.38 :

50. (2) Dissolve the raw materials in step (1) in an ethanol solution to prepare a lipid storage solution, dissolve the siRNA in a 10 mM citrate buffer at pH 4.0, and mix the two solutions at a volume ratio of 30:

1.

10. The pharmaceutical composition for treating sepsis-ARDS according to claim 9, characterized in that, In the lipid storage solution of step (2), the concentrations of 5A2SC8, DOPE, cholesterol ester, DMG-PEG2000 and DOTAP used to encapsulate 1 μg siRNA were 0.442 mM, 0.442 mM, 0.883 mM, 0.088 mM and 1.854 mM, respectively.

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