A micropeptide, nucleic acids encoding the same and use in modulating microglial inflammatory activation

Abstract

This invention discloses a micropeptide, its encoding nucleic acid, and its application in regulating inflammatory activation of microglia. The amino acid sequence of the micropeptide is shown in SEQ ID NO:3. Simultaneously, this invention discloses the nucleic acid encoding the micropeptide, which contains either the nucleotide sequence shown in SEQ ID NO:1 or the nucleotide sequence shown in SEQ ID NO:2, wherein SEQ ID NO:2 is a small open reading frame located within the sequence of SEQ ID NO:1. This invention validated the translational expression of the micropeptide in microglia by constructing an EGFP reporter gene fusion vector and a SUMO tag fusion expression vector. Functional experiments showed that under oxygen-glucose deprivation / reperfusion injury conditions, the micropeptide significantly inhibited inflammatory activation of microglia. This invention provides a new potential target and candidate drug for the treatment of neuroinflammatory diseases such as stroke.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a micropeptide and its encoded nucleic acid and its application in regulating the inflammatory activation of microglia. Background Technology

[0002] Microglia are important innate immune cells in the central nervous system, playing a crucial role in maintaining neural microenvironment homeostasis and responding to pathological stimuli. During the development of various central nervous system diseases, microglia can be abnormally activated by pathological stimuli and transform into a pro-inflammatory phenotype, thereby releasing various pro-inflammatory cytokines and inflammatory mediators, amplifying neuroinflammatory responses, and leading to secondary nerve damage.

[0003] In existing research, stroke, especially ischemic stroke leading to ischemia-reperfusion injury, is considered one of the important pathological conditions inducing microglial inflammatory activation. Ischemia and subsequent reperfusion can trigger energy metabolism disorders, oxidative stress, and abnormal activation of inflammatory signals, thereby promoting the transformation of microglia into a pro-inflammatory state, exacerbating neuroinflammatory responses, and leading to brain tissue damage. Therefore, the regulation of microglial inflammatory activation under ischemia-reperfusion-related conditions has always been an important technical direction in stroke-related neuroinflammatory research.

[0004] To address the aforementioned pathological processes, existing technologies have proposed various intervention strategies to regulate the inflammatory activation of microglia. These mainly include small-molecule anti-inflammatory drugs, biologics, or interventions targeting inflammatory signaling pathways, such as inhibiting inflammatory signaling pathways like NF-κB and MAPK to reduce the inflammatory response of microglia. However, these interventions typically suffer from limitations such as limited target specificity, high risk of systemic side effects, and insufficient target specificity, making it difficult to achieve precise regulation of stroke-related neuroinflammatory responses.

[0005] With the development of high-throughput sequencing and proteomics technologies, researchers have discovered open reading frames (ORFs) in some endogenous transcripts that can be translated into functional micropeptides. Compared to traditional drug molecules, micropeptides originate from endogenous biological processes and have advantages such as small molecular weight and good biocompatibility, showing potential research value in the field of inflammation regulation. However, current research on the role of micropeptides in the regulation of microglial inflammatory activation remains limited, especially under stroke-related pathological conditions such as ischemia-reperfusion, where systematic research and validation of functional micropeptides targeting microglial inflammatory responses are still lacking.

[0006] Given that the role of endogenous micropeptides in regulating microglial inflammatory activation under ischemia-reperfusion-related conditions is not yet clearly defined in existing technologies, and there is a lack of corresponding application technologies, it is necessary to conduct further research on the functions and applications of related micropeptides. Summary of the Invention

[0007] The present invention aims to address the problem in the prior art of lacking safe, effective and biocompatible precise control methods for microglial inflammatory activation under stroke-related pathological conditions (especially ischemia-reperfusion conditions).

[0008] Existing intervention strategies for microglial inflammatory activation mainly rely on small molecule anti-inflammatory drugs or biologics that target inflammatory signaling pathways. While these strategies can suppress inflammatory responses to some extent, they generally suffer from problems such as insufficient target specificity, significant potential toxic side effects, limited safety with long-term use, and difficulty in achieving precise regulation. Consequently, they fail to meet the needs for precise and biocompatible regulation of neuroinflammation in central nervous system diseases such as stroke.

[0009] Furthermore, current technologies have not clearly defined the role of endogenous micropeptides in regulating microglial inflammatory activation under stroke-related pathological conditions, and there is a lack of application technologies based on endogenous micropeptides to inhibit microglial inflammatory activation. Therefore, it is necessary to explore new biological regulatory pathways to compensate for the shortcomings of existing technologies.

[0010] To address the aforementioned technical problems, this invention provides an application of endogenous micropeptides in inhibiting inflammatory activation of microglia.

[0011] This invention provides a micropeptide, the sequence of which is shown in SEQ ID NO.3.

[0012] The present invention provides a nucleic acid encoding a micropeptide as described above.

[0013] According to a specific implementation, the encoded nucleic acid comprises a sequence as shown in SEQ ID NO.1.

[0014] According to a specific embodiment, the encoding nucleic acid comprises the sequence shown in SEQ ID NO: 2. SEQ ID NO: 2 is a small open reading frame located within the sequence of SEQ ID NO: 1.

[0015] The micropeptides are functional peptide molecules produced by the translation of endogenous transcripts. They have the characteristics of short amino acid sequence length, small molecular weight, and easy expression in cells, and can play a biological regulatory role at the cellular level.

[0016] The present invention provides a recombinant expression vector comprising the aforementioned encoded nucleic acid.

[0017] The present invention provides a host cell comprising the recombinant expression vector described above, or having the encoded nucleic acid integrated into its genome.

[0018] The technical solutions of this invention include, but are not limited to: introducing, expressing, or enhancing the expression level of the micropeptides in microglia, enabling them to participate in the regulation of cellular inflammatory responses under stroke-related pathological stimuli, thereby inhibiting the inflammatory activation process of microglia. This invention provides new potential targets and candidate drugs for the treatment of stroke and other neuroinflammatory diseases.

[0019] The present invention provides the use of the aforementioned micropeptide or the aforementioned encoded nucleic acid in the preparation of a medicament for inhibiting inflammatory activation of microglia.

[0020] The inflammatory activation of microglia mentioned above is induced by ischemia-reperfusion injury.

[0021] The drug is used to prevent or treat stroke.

[0022] The inhibition of microglial inflammatory activation includes: reducing the level of one or more of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 produced by the cells; downregulating the expression of iNOS; and / or upregulating the expression of Arg1.

[0023] In one specific embodiment, an expression vector encoding the micropeptide is introduced into microglia cultured in vitro, enabling the microglia to express the micropeptide; under ischemia-reperfusion-related simulated stimulation conditions, the inflammatory activation state of the microglia is detected to evaluate the inhibitory effect of the micropeptide on the inflammatory response of microglia.

[0024] In other embodiments, the micropeptides can also be applied to microglial inflammatory activation models under different induction conditions to study or regulate the inflammatory response process of microglia. The technical solutions of this invention are not limited to the specific embodiments described above; those skilled in the art can make corresponding modifications or substitutions without departing from the concept of this invention.

[0025] Compared with the prior art, the present invention has the following beneficial effects: (1) The micropeptides described in this invention are derived from the translation of endogenous transcripts. They have small molecular weight and good biocompatibility. Compared with exogenous protein drugs or chemically synthesized small molecule drugs, they can effectively reduce the risk of potential immunogenicity and toxic side effects, and are more suitable for the regulation of inflammatory responses related to the central nervous system.

[0026] (2) The results of cell-level experiments show that under ischemia-reperfusion-related stimulation conditions, the introduction and expression of the micropeptide described in this invention can significantly inhibit the inflammatory activation level of microglia and reduce inflammatory response-related indicators, indicating that the micropeptide has a clear technical effect in the regulation of stroke-related neuroinflammation.

[0027] (3) Starting from endogenous micropeptides, a novel functional molecule, this invention provides a new technical approach for the precise biological regulation of stroke-related neuroinflammation, which is different from traditional small molecule anti-inflammatory drugs and biological agents. Attached Figure Description

[0028] Figure 1 The sequence and characteristic map of PEP331 peptide; Figure 2 The following diagrams show the detection of PEP331 micropeptide expression and presence in microglia: (A) fluorescence detection based on the EGFP reporter system; (B) Western blot detection based on SUMO tag fusion. Figure 3 Schematic diagram of the inhibitory effect of PEP331 micropeptide on microglial inflammatory activation under ischemia-reperfusion-related stimulation: (A) Detection of pro-inflammatory factors TNF-α, IL-1β, and IL-6; (B) Detection of mRNA expression of Nos2 and Arg1; (C) Cell viability detection (CCK-8); (D) Cytotoxicity detection (LDH release). Detailed Implementation

[0029] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. Those skilled in the art should understand that the following embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Equivalent substitutions or modifications made to the embodiments without departing from the technical concept of the present invention should fall within the scope of protection of the present invention.

[0030] Example 1: Source and sequence characteristics of PEP331 micropeptide Transcriptome sequencing analysis identified a long non-coding RNA (lncRNA) whose expression changed in mouse microglia under ischemia-reperfusion-related pathological conditions, designated MSTRG.41731.1. The sequence of the MSTRG.41731.1 transcript is shown in SEQ ID NO.1, with a full length of 330 bases, and its genomic location is in the region 86802792-86803945 of mouse chromosome 5.

[0031] Bioinformatics analysis of the MSTRG.41731.1 transcript revealed the presence of a small open reading frame (smORF), denoted as MSTRG.41731.1-smORF. The sequence of MSTRG.41731.1-smORF, as shown in SEQ ID NO.2, is 123 bases in length. This MSTRG.41731.1-smORF encodes a 40-amino acid PEP331 peptide, the sequence of which is shown in SEQ ID NO.3.

[0032] Analysis of the physicochemical properties of the PEP331 peptide revealed that its molecular weight is approximately 4.84 kDa, its isoelectric point is approximately 10.59, and it exhibits weak positive charge under neutral conditions. Figure 1 As shown in the figure. The above analysis results indicate that the PEP331 micropeptide has the typical structural characteristics of a small molecule functional peptide, providing a structural basis for its biological function in cells.

[0033] Example 2: Detection of translational expression of PEP331 micropeptide in microglia To verify whether MSTRG.41731.1-smORF can translate and express a micropeptide in cells, the nucleotide sequence corresponding to MSTRG.41731.1-smORF was cloned into a eukaryotic expression vector to construct the fusion expression vector pEGFP-N1-PEP331. During the construction process, after removing the stop codon from MSTRG.41731.1-smORF, it was ligated to the N-terminus of the coding sequence of enhanced green fluorescent protein (EGFP) after removing the start codon, so that the fusion protein shares the start codon of MSTRG.41731.1-smORF and the stop codon of EGFP.

[0034] Simultaneously, a control vector pEGFP-N1-PEP331 with start codon mutation was constructed. ATG-MUT The start codon ATG of MSTRG.41731.1-smORF is mutated into the sequence ATA, which does not have the function of initiating translation.

[0035] The aforementioned vectors were transfected into the microglia cell line BV-2 using liposome transfection. After transfection, the cells were cultured for a predetermined time, and the expression of the fusion protein was analyzed by fluorescence signal detection. Figure 2As shown in Figure A, a significant fluorescent signal was detected in cells transfected with pEGFP-N1-PEP331, while no corresponding signal was detected in cells transfected with the start codon mutant vector, indicating that the MSTRG.41731.1-smORF can initiate translation in cells.

[0036] Given that the PEP331 peptide is a newly discovered peptide and there is a lack of specific antibodies to directly detect its endogenous expression, in order to further verify its translational expression, a SUMO (small ubiquitin-like modifier) ​​tag fusion expression vector pcDNA3.1-SUMO-PEP331 and its corresponding start codon mutation vector pcDNA3.1-SUMO-PEP331 were constructed. ATG-MUT .

[0037] The above vectors were transfected into BV-2 cells, and total cell protein was extracted after transfection. The expression of the fusion protein was detected using a protein immunoassay. Figure 2 As shown in -B, the corresponding molecular weight fusion protein signal was detected only in cells transfected with pcDNA3.1-SUMO-PEP331, while no corresponding signal was detected in the start codon mutation group and the untransfected control group.

[0038] Example 3: Effects of PEP331 micropeptide on inflammatory activation of microglia under ischemia-reperfusion-related stimulation. To investigate the effect of the PEP331 micropeptide on microglial inflammatory activation under ischemia-reperfusion-related stimulation, the following eukaryotic expression vectors were constructed: the expression vector pcDNA3.1-PEP331 for the PEP331 micropeptide and its start codon mutation vector pcDNA3.1-PEP331. ATG-MUT pcDNA3.1-PEP331, a micropeptide expression vector with a coding frame shift FS The vectors overexpressing lncRNA MSTRG.41731.1, pcDNA3.1-MSTRG.41731.1, and pcDNA3.1-MSTRG.41731.1 with smORF removed, were also included. ΔPEP331 .

[0039] The aforementioned vectors were transfected into BV-2 cells using liposome transfection. After transfection, the cells were subjected to oxygen-glucose deprivation / reoxygenation (OGD / R) treatment to simulate ischemia-reperfusion-related pathological stimuli.

[0040] At predetermined time points after reoxygenation, cell culture supernatant was collected and inflammatory factor levels were detected, along with the expression of microglial inflammatory activation-related markers. Furthermore, the degree of cell damage and cell viability were assessed. The results are as follows: Figure 3 As shown.

[0041] The embodiments described in this invention provide a technical solution that can be implemented and reproduced by those skilled in the art without creative effort. Through the above embodiments, the inhibitory effect of the micropeptide PEP331 on the inflammatory activation of microglia under relevant stimuli can be verified, and overexpression of PEP331 does not cause significant toxic damage to cells.

[0042] sequence list SEQ ID NO.1: GTGAGACACCATCAGAAAGGGTGTTATGTCTTTCTTTGCCAGGGCTACACAGAGAAACCTTGACTTGGAAAACAGAGAGAGACAGACAGACAGACAGACAGTTGCTATAGTGATAAGACCCCTTTTTATGGAGAAAAGCTAGTACAGAGGCCCTCCTGT GGAGGTGAAGACTGATGGTAAAAGATAAGAGACAATAACAAATAAGCACTAATTGTCTTCGGAAAGCACATACAAAGACTTTATGAAGATCTGAGGGGCATTCCAGTAACCAGGAATGGAGGTGAATGAAGATCTGCGGGGCATTCCAGTAACCAGGAATGGAGG SEQ ID NO.2: ATGTCTTTCTTTGCCAGGGCTACACAGAGAAACCTTGACTTGGAAAACAGAGAGAGACAGACAGACAGACAGACAGTTGCTATAGTGATAAGACCCCTTTTTATGGAGAAAAGCTAG SEQ ID NO.3: MSFFARATQRNLDLENRERDRQTDRQTVAIVIRPLFMEKS.

Claims

1. A micropeptide, characterized in that, The sequence of the micropeptide is shown in SEQ ID NO.

3.

2. A method encoding a nucleic acid, characterized in that, It encodes the micropeptide of claim 1.

3. The encoded nucleic acid according to claim 2, characterized in that, The encoded nucleic acid comprises a sequence as shown in SEQ ID NO.

1.

4. The nucleic acid encoding method according to claim 2, characterized in that, The encoded nucleic acid comprises a sequence as shown in SEQ ID NO.

2.

5. A recombinant expression vector, characterized in that, It includes the encoded nucleic acid as described in any one of claims 2-4.

6. A host cell comprising the recombinant expression vector of claim 5, or having the encoded nucleic acid of any one of claims 2-4 integrated into its genome.

7. Use of the micropeptide of claim 1 or the encoded nucleic acid of any one of claims 2-4 in the preparation of a medicament for inhibiting inflammatory activation of microglia.

8. The use according to claim 7, wherein the inflammatory activation of the microglia is induced by ischemia-reperfusion injury.

9. The use according to claim 7 or 8, wherein the drug is used for the prevention or treatment of stroke.

10. The use according to claim 7 or 8, wherein the inhibition of microglial inflammatory activation comprises: Reduce the level of one or more of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 produced by the cells; Downregulate iNOS expression and / or upregulate Arg1 expression.

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