Application of Elov16 as drug target in preparation or screening of drugs for treating anxiety or depression related diseases

Abstract

The invention discloses an application of Elov16 as a drug target in preparation or screening of drugs for treating anxiety and / or depression related diseases. The invention finds that Elov16 affects anxiety behavior by regulating and controlling fatty acid composition of a prefrontal cortex. According to the present invention, the Elovl6 function deficiency results in the increase of palmitic acid and the reduction of oleic acid and linoleic acid, such that the PPARalpha-Allopregnanolone signal axis is inhibited, the neurosteroid Allopregnanolone synthesis is reduced, and the anxiety-like behavior is finally induced; the PPARalpha agonist GW7647 can be used for recovering the level of Allopregnanolone and remarkably improving the anxiety phenotype of an Elov16 knockout mouse. The invention provides a drug screening and diagnosis marker system taking Elov16 as a target spot and a precise intervention strategy for treating fatty acid metabolism disorder related anxiety diseases by activating a PPARalpha-Allopregnanolone pathway, and provides a theoretical basis and a conversion medical pathway for the development of anti-anxiety drugs.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of Elovl6 as a drug target in the preparation or screening of drugs for treating anxiety and / or depression-related diseases. Background Technology

[0002] Anxiety disorder is one of the most prevalent mental disorders worldwide, clinically characterized by excessive fear, worry, and avoidance, severely impairing patients' social functioning and quality of life. Currently, first-line drug treatment mainly relies on serotonin reuptake inhibitors (STIs), but these have drawbacks such as delayed onset of action, significant individual variability, and poor response in some patients. Therefore, elucidating new pathological mechanisms of anxiety disorder and identifying more effective intervention targets have become key research areas in this field. In recent years, research perspectives have gradually shifted from traditional neurotransmitter imbalances to the interaction between metabolic homeostasis and brain function. Among these, the role of lipid metabolism disorders in mental illness is becoming increasingly prominent, and how the composition of brain fatty acids and their metabolites participate in mood regulation is a cutting-edge direction worthy of in-depth exploration.

[0003] Elovl6, a long-chain fatty acid elongase 6 (ECL6), is a key enzyme catalyzing the elongation of C12–C18 saturated and monounsaturated fatty acids, and is widely expressed in the liver, adipose tissue, and central nervous system. Previous studies have largely focused on the role of Elovl6 in peripheral diseases such as metabolic syndrome and non-alcoholic fatty liver disease, while its significance in the regulation of higher brain functions remains unclear. Notably, several clues suggest that Elovl6 may be involved in the regulation of psychopathological states: for example, studies have found altered enzyme activity in serum samples from suicide victims; furthermore, Elovl6 has been reported as one of the genes with the most significant expression changes after treatment with the anti-anxiety drug fluoxetine. However, current evidence of these associations is insufficient to establish a causal role for Elovl6 in anxiety. Whether this enzyme affects mood-related neural circuits by regulating brain lipid composition, and what the specific molecular mechanisms are, remain important scientific questions that need to be elucidated. This invention aims to elucidate the role of Elovl6 in anxiety-like behaviors and to explore its underlying molecular mechanisms, providing new theoretical basis and potential targets for the prevention and treatment of anxiety disorders. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide an application of Elovl6 as a drug target in the preparation or screening of drugs for treating anxiety and / or depression-related diseases.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, this application provides the application of Elovl6 as a drug target in the preparation or screening of drugs for treating anxiety or depression-related diseases.

[0006] Secondly, this application provides the use of a reagent for detecting Elovl6 expression levels in the preparation of kits for diagnosing or prognostically assessing anxiety-related diseases.

[0007] Thirdly, this application provides the use of a reagent that regulates the PPARα-Allopregnanolone signaling axis in the preparation of a drug for treating anxiety disorders related to Elovl6 dysfunction.

[0008] Fourthly, this application provides a pharmaceutical composition for treating anxiety associated with Elovl6 dysfunction.

[0009] The first aspect of this application provides the use of Elovl6 as a drug target in the preparation or screening of drugs for treating anxiety or depression-related diseases.

[0010] Furthermore, Elovl6 includes the Elovl6 gene or the Elovl6 protein, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of which is shown in SEQ ID NO.2.

[0011] Furthermore, the drug exerts its effect through at least one of the following mechanisms:

[0012] (1) Upregulate the transcriptional level of the Elovl6 gene, the expression level of the Elovl6 protein, or the biological activity of the Elovl6 protein.

[0013] (2) Regulate the fatty acid composition in the prefrontal cortex of the subjects, specifically by reducing the relative content of palmitic acid and / or increasing the relative content of oleic acid and linoleic acid in the prefrontal cortex.

[0014] (3) Activate the pathway activity of the PPARα-Allopregnanolone signaling axis in the subject, including promoting nuclear transcriptional activation of PPARα and upregulating the expression of enzymes related to Allopregnanolone synthesis.

[0015] (4) Increase the amount of Allopregnanolone biosynthesis in the prefrontal cortex of the subjects, and restore it to the normal physiological range.

[0016] A second aspect of this application provides the use of a reagent for detecting Elovl6 expression levels in the preparation of a kit for diagnosing or prognostically assessing anxiety-related diseases. The definition of Elovl6 is the same as in the first aspect, the nucleotide sequence of the Elovl6 gene is shown in SEQ ID NO.1, and the amino acid composition of the protein encoded by the Elovl6 gene is shown in SEQ ID NO.2.

[0017] Furthermore, the reagents for detecting Elovl6 expression levels include: reagents for specifically detecting Elovl6 mRNA levels, selected from specific primers, specific probes, or gene chips related to the nucleotide sequence shown in SEQ ID NO.1; and / or reagents for specifically detecting Elovl6 protein levels, selected from specific monoclonal antibodies, polyclonal antibodies, or antigen-binding fragments designed for the amino acid sequence shown in SEQ ID NO.2.

[0018] The third aspect of this application provides the use of a drug that regulates the PPARα-Allopregnanolone signaling axis in the preparation of a drug for treating anxiety disorders related to Elovl6 dysfunction.

[0019] The Elovl6 dysfunction refers to a significant decrease in the expression level of the Elovl6 gene (based on the transcript of the sequence shown in SEQ ID NO.1) or the expression level / activity of the Elovl6 protein (based on the protein of the sequence shown in SEQ ID NO.2) compared to normal physiological levels, resulting in fatty acid metabolism disorders and inhibition of the PPARα-Allopregnanolone signaling axis mediated by the gene.

[0020] Furthermore, the drug that regulates the PPARα-Allopregnanolone signaling axis is a PPARα agonist; the drug restores the level of Allopregnanolone in the prefrontal cortex of the subject to the normal range by activating the PPARα-Allopregnanolone signaling axis, thereby improving anxiety-like behavior caused by Elovl6 dysfunction.

[0021] This application provides a fourth aspect of a pharmaceutical composition for treating anxiety related to Elovl6 dysfunction. The definition of Elovl6 dysfunction is the same as in the third aspect, namely, an abnormally reduced transcriptional level of the Elovl6 gene (nucleotide sequence SEQ ID NO.1) or an abnormally reduced expression level / activity of the Elovl6 protein (amino acid sequence SEQ ID NO.2).

[0022] The pharmaceutical composition contains an effective amount of a PPARα agonist and a pharmaceutically acceptable carrier.

[0023] Furthermore, PPARα agonists include, but are not limited to, the following categories and representatives (including natural agonists): Clinical fibrates: fenofibrate, bezafibrate, gemfibrozil, etc. (with well-established safety profiles and suitability for clinical translation); Classic utility drugs: GW7647, WY14643, etc. (high specificity, with GW7647 having a preferred dose of 1-10 mg / kg, administered via intraperitoneal injection or oral administration; the doses of other agonists can be routinely optimized based on the subject's weight, disease severity, and drug potency); Novel agonists: selective PPARα modulators (such as K-877, LY518674, etc., with higher specificity; the dose can be optimized based on in vitro activity assay data and in vivo pharmacokinetic results); Natural agonists: including unsaturated fatty acids (such as arachidonic acid, EPA, DHA), plant extracts known to have PPARα agonistic activity (such as curcumin, resveratrol, etc.) (naturally derived, with good biocompatibility; the dose needs to be adjusted based on the purity of the extract and in vitro / in vivo activity assay results). Furthermore, the composition also includes an auxiliary active ingredient that modulates the fatty acid composition, the auxiliary active ingredient being selected from one or more of oleic acid, linoleic acid, or pharmaceutically acceptable derivatives thereof, which can synergistically modulate the fatty acid composition of the prefrontal cortex with PPARα agonists, thereby enhancing the anti-anxiety effect.

[0024] Furthermore, pharmaceutically acceptable carriers can be selected according to the route of administration, including pharmaceutically acceptable fillers, binders, disintegrants, lubricants, solubilizers, etc.; among them, oral formulations are suitable for carriers such as starch and lactose, while intraperitoneal injection formulations are suitable for injectable carriers such as physiological saline and phosphate buffer.

[0025] Beneficial Effects: This invention reveals for the first time the core mechanism of action of the Elovl6-fatty acid-PPARα-Allopregnanolone signaling axis in the regulation of anxiety-like behavior, providing innovative theoretical basis and technical means for the precision diagnosis and treatment of anxiety disorders. This invention achieves key breakthroughs in theoretical innovation, technological practicality, and clinical translation, addressing the industry pain points of unclear pathogenesis, lack of diagnostic biomarkers, and limited therapeutic targets for anxiety disorders, possessing significant scientific value and broad application prospects. This discovery provides a molecular bridge for understanding the comorbidity mechanism of metabolic disorders and mental disorders, filling the gap in the mechanism of action of Elovl6 in the regulation of central nervous system function.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] (1) Innovative Breakthrough in Scientific Mechanism of the Invention: This invention elucidates a novel "metabolism-brain-behavior" signaling pathway, directly linking the function of Elovl6, a key enzyme in lipid metabolism, to emotion regulation, thus overcoming the limitations of traditional neurotransmitter imbalance theories. Studies have confirmed that Elovl6 regulates the fatty acid composition of the prefrontal cortex, reducing palmitic acid and increasing the oleic acid / linoleic acid ratio, thereby activating the PPARα-Allopregnanolone signaling axis and ultimately affecting GABAergic inhibitory neurotransmission.

[0028] (2) Accuracy and practicality of diagnostic biomarkers: This invention provides a multi-level, quantifiable diagnostic biomarker system: At the gene level, Elovl6 mRNA and protein expression levels can serve as predictive indicators of anxiety susceptibility and disease severity; at the metabolic level, the ratio of palmitic acid / oleic acid / linoleic acid in the prefrontal cortex constitutes a fatty acid profile diagnostic biomarker, which is particularly suitable for identifying metabolically abnormal anxiety subgroups; at the molecular level, changes in PPARα activity and Allopregnanolone content can reflect the functional status of pathways in real time, providing objective evidence for efficacy monitoring. Compared with the subjectivity of traditional behavioral assessments, the biomarker system of this invention has the outstanding advantages of high specificity, early warning capability, and ease of dynamic monitoring.

[0029] (3) High efficiency and targeting of the treatment strategy: This invention verifies the feasibility of PPARα as a key drug target. Its beneficial effects are reflected in: rapid onset of action: overcoming the limitation of SSRI drugs having an onset of action over multiple weeks, it achieves significant improvement in behavioral phenotypes in animal models with just two days of intervention; precise mechanism: it specifically supplements Allopregnanolone deficiency caused by Elovl6 dysfunction, restoring GABAA receptor function, rather than generally regulating monoamine neurotransmitters; broad applicability: it is not only applicable to primary anxiety disorder, but also provides a "one-target, two-effect" treatment possibility for patients with comorbid anxiety in metabolic syndrome (such as obesity and diabetes), solving the clinical problem of poor response to conventional anti-anxiety drugs in such patients.

[0030] (4) Standardization and high-throughput of drug screening models: This invention establishes a multi-dimensional drug screening platform based on SH-SY5Y cells and gene-edited animals, which can simultaneously detect indicators such as Elovl6 expression, fatty acid composition, PPARα activity, and Allopregnanolone synthesis efficiency. This platform has the characteristics of clear mechanism, clear endpoint, and short cycle, which can significantly improve the efficiency and success rate of anti-anxiety drug development and reduce development costs.

[0031] (5) Breakthrough value in translational medicine: This invention transforms basic metabolic research into clinically applicable diagnostic and treatment solutions: The "drug repurposing" strategy of PPARα agonists (such as fenofibrate and other marketed drugs) can significantly shorten the clinical translation cycle; Precise subtyping based on fatty acid profiles helps to achieve individualized treatment of anxiety disorders and promotes the advancement of psychiatry towards precision medicine. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Explanation of abbreviations in the attached diagram:

[0034] Figure 1 This is a diagram illustrating the anxiety-like behavior induced in mice by Elovl6 gene knockout in this invention. Figure 1 A represents the wild-type (WT) and Elovl6 knockout (Elovl6) of this invention. - / - A representative movement trajectory diagram of mice in an open field experiment. Figure 1 B- Figure 1 C represents the quantitative analysis of the open field experiment in this invention: compared with WT mice, Elovl6 - / - The distance the mouse traveled in the central region ( Figure 1 B) and length of stay ( Figure 1 C) All were significantly reduced (n=6, *p<0.05, **p<0.01). Figure 1 D represents the WT and Elovl6 of this invention. - / - A representative movement trajectory diagram of mice in the elevated cross maze experiment. Figure 1 E- Figure 1 F represents the quantitative analysis of the elevated cross-maze experiment of this invention: Elovl6 - / - The percentage of mice entering the open arm ( Figure 1 E) and length of stay ( Figure 1 F) were significantly lower than those in WT mice (n=6, p<0.01); Figure 1 G represents the invention WT and Elovl6. - / - Representative images of the mouse bead embedding experiment. Figure 1 H represents the quantitative analysis of the embedded bead experiment in this invention: Elovl6 - / - The number of marbles buried in mice was significantly greater than that in WT mice (n=6, *p<0.001). Figure 1 I- Figure 1 J represents the invention WT and Elovl6 - / -Quantitative analysis of mouse self-grooming experiment: Elovl6 - / - Number of self-grooming times in mice ( Figure 1 I) and cumulative time ( Figure 1 The values ​​of J) were significantly higher than those of WT mice (n=6, p<0.01, ***p<0.001) (Figure). Figure 1 K represents the WT and Elovl6 of this invention. - / - A representative image from the mouse nesting experiment. Figure 1 L is the nesting experiment scoring chart of this invention: Elovl6 - / - The nesting score of the mice was significantly lower than that of the WT mice (n=6, *p<0.05).

[0035] Figure 2 The Elovl6 knockout of this invention leads to disordered fatty acid composition in the prefrontal cortex and inhibits the PPARα-Allo pathway. Figure 2 A is a statistical diagram of fatty acid composition in the mouse prefrontal cortex tissue according to the present invention. Figure 2 B is a graph showing the expression level of PPARα in mouse prefrontal cortex tissue detected by RT-qPCR according to the present invention. Figure 2 C is a statistical graph showing the expression levels of the Allo biosynthetic genes StAR, Cyp11a1, and 5α-RI in the mouse prefrontal cortex tissue of this invention. Figure 2 D is a statistical graph of Allo content in the mouse prefrontal cortex tissue according to the present invention.

[0036] Figure 3 This invention provides a cellular-level validation diagram of the regulatory role of fatty acids in the PPARα-Allo pathway. Figure 3 A is a cell viability graph of sh-sy5y cells after treatment with different fatty acids, as detected by CCK-8 assay according to the present invention. Figure 3 B is a graph showing the expression levels of PPARα protein in cells after intervention with fatty acids and GW7647 according to the present invention. Figure 3 C represents the mRNA expression levels of the Allo biosynthesis genes StAR, Cyp11a1, and 5α-RI in cells after intervention with fatty acids and GW7647 according to the present invention. Figure 3 D is a statistical graph of the fatty acid and GW7647 intervention in cells after the present invention.

[0037] Figure 4 This is a diagram showing the anxiety phenotype of Elovl6 knockout mice that can be reversed by activating PPARα according to the present invention. Figure 4 A represents the intervention of GW7647 in this invention to improve Elovl6. - / - A graph showing the Allo content in the prefrontal cortex of mice. Figure 4 Figure B shows the increase in the number of times mice entered the open arm after intervention with GW7647 according to this invention. Figure 4Figure C shows the increase in time for mice to enter the open arm after intervention with GW7647 according to this invention. Figure 4 Figure D shows the reduction in the number of self-grooming sessions in mice after intervention with GW7647 according to this invention. Figure 4 E is a graph showing the reduction in self-grooming time in mice after intervention with GW7647 according to the present invention. Figure 4 F shows the reduction in the number of embedded beads in mice after GW7647 intervention according to the present invention. Detailed Implementation

[0038] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed.

[0039] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0040] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. Here, A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0041] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c are single or multiple.

[0042] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0043] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0044] The first aspect of this application provides the use of Elovl6 as a drug target in the preparation or screening of drugs for treating anxiety or depression-related diseases.

[0045] In some embodiments, Elovl6 includes the Elovl6 gene or the Elovl6 protein; wherein: the nucleotide sequence of the Elovl6 gene is as shown in SEQ ID NO.1 (SEQ ID NO.1 is a human sequence; the present invention also covers other mammalian (e.g., mouse, rat) functional homologous sequences that have more than 85% homology with SEQ ID NO.1 and encode the Elovl6 protein with fatty acid elongation enzyme activity, specifically the full-length coding region sequence of the corresponding species encoding fatty acid elongation enzyme 6); the Elovl6 protein is the expression product of the Elovl6 gene, and its amino acid sequence is as shown in SEQ ID NO.2.

[0046] In some embodiments, the drug exerts its therapeutic effect on anxiety or depression-related disorders through at least one of the following mechanisms, and said mechanisms can be verified by in vitro cell experiments or in vivo animal experiments:

[0047] (1) Upregulate the expression level or activity of Elovl6: Specifically, this can be achieved by upregulating the transcription efficiency of the Elovl6 gene, promoting the stable expression of Elovl6 mRNA, enhancing the post-translational modification of Elovl6 protein (such as phosphorylation modification), or inhibiting the degradation of Elovl6 protein. The upregulation effect can be verified by detecting the Elovl6 mRNA level by real-time quantitative PCR (qPCR), detecting the Elovl6 protein level by Western blotting, and detecting the Elovl6 fatty acid elongation enzyme activity by in vitro enzyme activity assay.

[0048] (2) Regulate the fatty acid composition of the prefrontal cortex: Specifically, reduce the relative content of palmitic acid (C16:0) in the prefrontal cortex tissue of the subjects, and / or increase the relative content of oleic acid (C18:1) and linoleic acid (C18:2) in the prefrontal cortex; the changes in the content of the above fatty acids in the prefrontal cortex can be quantitatively detected by gas chromatography-mass spectrometry (GC-MS); (3) Activate the PPARα-Allopregnanolone signal axis: Specifically, promote the nuclear localization of PPARα protein, enhance the binding ability of PPARα to the promoter region of downstream target genes, and upregulate the transcription and expression of target genes; the nuclear localization of PPARα can be detected by immunofluorescence, the binding of PPARα to target genes can be verified by chromatin immunoprecipitation (ChIP), and the activation effect of the signal axis can be verified by qPCR detection of target gene expression level;

[0049] (4) Increase the biosynthesis of Allopregnanolone: ​​Specifically, increase the content of Allopregnanolone in the prefrontal cortex to the normal physiological range; the changes in the content of Allopregnanolone in the prefrontal cortex can be quantitatively detected by liquid chromatography-mass spectrometry (LC-MS).

[0050] The second aspect of this application provides the use of a reagent for detecting Elovl6 expression levels in the preparation of a kit for diagnosing or prognostically assessing anxiety-related diseases.

[0051] In some embodiments, the definition of Elovl6 is the same as in the first aspect, the nucleotide sequence of the Elovl6 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Elovl6 gene is shown in SEQ ID NO.2.

[0052] In some embodiments, the reagent for detecting Elovl6 expression levels includes: primers, probes, or chips that specifically detect Elovl6 mRNA levels, and / or antibodies that specifically detect Elovl6 protein levels.

[0053] The reagents for specifically detecting Elovl6 mRNA levels are designed based on the nucleotide sequence shown in SEQ ID NO.1, including primers, probes, or gene chips. The primers must meet amplification specificity requirements, with a fragment length of 100-300 bp, and can be used for amplification techniques such as qPCR and RT-PCR. The probes can be labeled with fluorescent groups (such as FAM or HEX) or quencher groups (such as BHQ1) for in situ hybridization or quantitative fluorescence detection. The gene chip is a nucleic acid chip immobilized with Elovl6-specific probes, which can be used for high-throughput detection of the relative expression level of Elovl6 mRNA in samples.

[0054] Reagents for specifically detecting Elovl6 protein levels: designed based on the amino acid sequence shown in SEQ ID NO.2, including monoclonal antibodies, polyclonal antibodies, or their antigen-binding fragments (such as Fab, F(ab')2 fragments); the antibodies must have high specificity and high affinity, and the expression level of Elovl6 protein in samples can be detected by methods such as immunohistochemistry (IHC), immunofluorescence (IF), Western blotting, and enzyme-linked immunosorbent assay (ELISA).

[0055] In some embodiments, the kit may further include pharmaceutically acceptable auxiliary reagents, specifically: buffers (such as PBS buffer, Tris-HCl buffer), enzymes (such as Taq enzyme, reverse transcriptase), substrates (such as chromogenic substrate TMB, fluorescent substrate), positive controls (standards containing Elovl6 mRNA or standards containing Elovl6 protein), negative controls (blank samples without Elovl6), and washing solutions, etc.

[0056] The third aspect of this application provides the use of a reagent that regulates the PPARα-Allopregnanolone signaling axis in the preparation of a medicament for treating anxiety disorders related to Elovl6 dysfunction.

[0057] In some embodiments, the Elovl6 dysfunction refers to a significantly lower transcriptional level of the Elovl6 gene (SEQ ID NO.1) or expression level / activity of the Elovl6 protein (SEQ ID NO.2) than the normal reference value of healthy individuals, which in turn leads to disordered fatty acid composition of the prefrontal cortex, inhibition of the PPARα-Allopregnanolone signaling axis, and reduced Allopregnanolone content.

[0058] In some embodiments, the reagent that regulates the PPARα-Allopregnanolone signaling axis is a PPARα agonist. The PPARα agonist includes, but is not limited to, the following categories: clinical fibrates: fenofibrate, bezafibrate, gemfibrozil, etc. (with established safety profiles and suitability for clinical translation); classic utility drugs: GW7647, WY14643, etc. (high specificity, suitable for mechanism validation); novel agonists: selective PPARα modulators (such as K-877, LY518674, etc., with higher specificity); natural agonists: unsaturated fatty acids (such as arachidonic acid, EPA, DHA), plant extracts known to have PPARα agonist activity (such as curcumin, resveratrol, etc.) (good biocompatibility).

[0059] In some embodiments, the drug improves anxiety-like behavior by restoring allopregnanolone levels in the prefrontal cortex to a normal range.

[0060] The fourth aspect of this application provides a pharmaceutical composition for treating anxiety related to Elovl6 dysfunction, characterized in that it comprises an effective amount of a PPARα agonist and a pharmaceutically acceptable carrier.

[0061] In some embodiments, the PPARα agonists include, but are not limited to, GW7647, fenofibrate, bezafibrate, WY14643, K-877, arachidonic acid, curcumin, etc.; among which, GW7647 is preferred, with a dosage of 1-10 mg / kg (based on the subject's weight), administered via intraperitoneal injection or oral administration; the dosage of other PPARα agonists can be routinely optimized and determined based on their potency, in vivo pharmacokinetic data, and the subject's weight and disease severity.

[0062] In some embodiments, the pharmaceutical composition further comprises an auxiliary active ingredient for regulating fatty acid composition, wherein the auxiliary active ingredient is selected from one or more of oleic acid, linoleic acid, or pharmaceutically acceptable derivatives thereof; the pharmaceutically acceptable derivatives include ethyl oleate, glyceryl oleate, ethyl linoleate, glyceryl linoleate, etc.; the effective dose of the auxiliary active ingredient is a dose that can synergistically regulate the fatty acid composition of the prefrontal cortex (reducing palmitic acid content and increasing oleic acid / linoleic acid content) with a PPARα agonist, and the specific dose can be optimized and determined by GC-MS detection of fatty acid content. It also comprises an auxiliary active ingredient for regulating fatty acid composition, wherein the auxiliary active ingredient is selected from one or more of oleic acid, linoleic acid, or derivatives thereof.

[0063] In some embodiments, the pharmaceutically acceptable carrier is selected according to the route of administration and includes pharmaceutically acceptable fillers, binders, disintegrants, lubricants, solubilizers, preservatives, stabilizers, etc.

[0064] Example 1

[0065] The present invention relates to the application of Elovl6 as a drug target in the preparation or screening of drugs for treating anxiety or depression-related diseases.

[0066] This embodiment aims to screen candidate drugs that can treat anxiety-related diseases by upregulating Elovl6 expression levels. The specific implementation process is as follows: Mouse prefrontal cortex neuronal cell lines with low Elovl6 expression, a library of 100 small molecule compounds for screening, a qPCR detection kit, and a Western blotting detection kit were used as experimental materials. First, Elovl6-low-expressing neuronal cells were seeded in 96-well plates and cultured until cell confluence reached 70%-80%. Then, different concentrations of the selected compounds (0.1-10 μM) were added to each well. A blank control group without the added compounds and a positive control group with a known Elovl6 upregulating reagent were also set up, with three replicates in each group. After 24 hours of culture, the expression level of Elovl6 mRNA in each well was detected by qPCR using primers designed based on SEQ ID NO.1, and the expression level of Elovl6 protein was detected by Western blotting using an antibody prepared based on SEQ ID NO.2. Drugs that significantly upregulate Elovl6 compared to the blank control group were screened. Compounds with mRNA and / or protein levels (upregulated ≥2-fold, p<0.05) were selected as candidate drugs; to verify the effectiveness of the candidate drugs, they were further applied to Elovl6. - / - In mice, the effects of the drug on improving anxiety-like behavior were detected through open field and elevated cross maze tests. At the same time, changes in fatty acid composition and allopregnanolone content in the prefrontal cortex of mice were detected to clarify the mechanism of action of the drug.

[0067] Elovl6 includes the Elovl6 gene or the Elovl6 protein.

[0068] Drugs exert their effects through at least one of the following mechanisms:

[0069] (1) Upregulate the expression level or activity of Elovl6;

[0070] (2) Adjust the fatty acid composition of the prefrontal cortex, reduce the relative content of palmitic acid and / or increase the relative content of oleic acid and linoleic acid;

[0071] (3) Activate the PPARα-Allopregnanolone signal axis;

[0072] (4) Increase the biosynthesis of Allopregnanolone.

[0073] Example 2

[0074] The present invention relates to the application of a reagent for detecting Elovl6 expression levels in the preparation of kits for diagnosing or prognostically assessing anxiety-related diseases.

[0075] Reagents for detecting Elovl6 expression levels include: primers, probes, or chips that specifically detect Elovl6 mRNA levels, and / or antibodies that specifically detect Elovl6 protein levels.

[0076] Example 3

[0077] The present invention relates to the application of a reagent that regulates the PPARα-Allopregnanolone signaling axis in the preparation of a medicament for treating anxiety disorders related to Elovl6 dysfunction.

[0078] This embodiment aims to verify the effectiveness and mechanism of action of agents that regulate the PPARα-Allopregnanolone signaling axis (PPARα agonists) in the preparation of drugs for treating anxiety disorders related to Elovl6 dysfunction.

[0079] The specific implementation process is as follows: Twelve 6-8 week old male Elovl6 gene knockout (Elovl6- / -) mice (to construct an Elovl6 dysfunction anxiety disease model) and 12 age- and sex-matched wild-type (WT) mice were selected as experimental animals. PPARα agonist GW7647, sterile saline, and intraperitoneal injection needles were prepared as experimental materials. The Elovl6- / - mice were randomly divided into a model control group and a drug administration group, with 6 mice in each group. WT mice served as the normal control group. The mice in the drug administration group were then intraperitoneally injected with GW7647 dissolved in sterile saline at a dose of 5 mg / kg. The model control group and the normal control group were injected with the same amount of sterile saline. The drugs were administered once daily for 7 consecutive days to ensure the drug's full effect. After drug administration, two classic anxiety behaviors were tested: the elevated cross maze test (mice were placed in the center of the maze, and the percentage of times they entered the open arms and the percentage of time they stayed within 5 minutes) and the bead burying test (10 marbles were placed in the mouse cages, and the number of marbles buried within 30 minutes was recorded). The improvement of anxiety-like behavior in mice was assessed using a scientific detection method. After the behavioral experiment, the mice were sacrificed, and their prefrontal cortex tissue was collected. The content of allopregnanolone in the tissue was quantitatively detected by liquid chromatography-mass spectrometry (LC-MS). The results showed that the proportion of mice in the model control group entering the open arm and the proportion of mice staying in the open arm were significantly lower than those in the normal control group, and the number of marbles buried was significantly higher than that in the normal control group, indicating that the model was successfully established. In contrast, the above-mentioned anxiety-related behavioral indicators in the drug-treated group were significantly improved (p<0.01 compared with the model control group), and the allopregnanolone content in the prefrontal cortex was restored to the physiological level of the normal control group (WT mice). This proves that PPARα agonists can improve anxiety-like behavior related to Elovl6 dysfunction by regulating the PPARα-Allopregnanolone signaling axis and restoring the level of allopregnanolone in the prefrontal cortex to the normal range, thus confirming that it can be used to prepare drugs for the treatment of this type of anxiety disorder.

[0080] Example 4

[0081] The present invention provides a pharmaceutical composition for treating anxiety associated with Elovl6 dysfunction, comprising an effective amount of a PPARα agonist and a pharmaceutically acceptable carrier.

[0082] The PPARα agonist is GW7647, with a dosage of 1-10 mg / kg, administered via intraperitoneal injection or oral administration.

[0083] It also contains auxiliary active ingredients for regulating fatty acid composition, which are selected from one or more of oleic acid, linoleic acid, or their derivatives.

[0084] Experimental Example 1

[0085] Research Methods:

[0086] 1.1 Laboratory Animals and Models

[0087] Male C57BL / 6J mice aged 6-8 weeks and Elovl6 knockout mice were used. A mouse anxiety model was established by chronic restraint stress (mice were restrained in 50 mL breathable centrifuge tubes for 4 hours each day for 21 days).

[0088] 1.2 Behavioral Testing

[0089] The study included open field (OFT) test, elevated cross maze (EPM) test, bead embedding test, self grooming test, and nesting test to comprehensively assess the anxiety behavior of mice.

[0090] Open field test: Mice were placed alone in the center of a 40cm×40cm×35cm square open box. They were allowed to explore freely for 5 minutes, and their activity was recorded by a camera. Observation indicators: ① Total movement distance (reflecting basic motor ability); ② Number of times and cumulative time entering the central area (reflecting anxiety level; shorter time and fewer times indicate greater anxiety).

[0091] Elevated Cross Maze: The maze is 50cm above the ground and consists of two open arms (without sidewalls) and two closed arms (with high sidewalls). Mice are placed in the central area of ​​the maze, head facing one of the open arms, and allowed to explore freely for 5 minutes. Observation indicators: ① Percentage of mice entering the open arms; ② Percentage of mice spending time in the open arms (the lower the percentage, the higher the anxiety level).

[0092] Bead embedding experiment: Twenty clean glass marbles were evenly placed in a cage lined with 5cm of bedding. Mice were placed in the cage and allowed to move freely for 30 minutes. Observation indicators: After the experiment, the number of marbles covered by more than 2 / 3 of the bedding was counted. A higher number of embedded marbles indicated more significant anxiety-like behavior.

[0093] Nesting experiment: Mice were housed individually in cages, with eight 5cm x 5cm square pieces of paper placed in one corner of the cage. Nesting behavior was observed and recorded at fixed time points over six consecutive days. Observation indicators: Mice were scored from 1 to 4 points based on whether the paper pieces were torn, tidied, and piled up in the corner (1 point: paper pieces were not tidied and scattered; 4 points: paper pieces were severely torn and piled up to form a nest). Lower scores indicated poorer social behavior and survival skills.

[0094] 1.3 Molecular Biology Experiments

[0095] Tissue collection: After perfusion, prefrontal cortex, hippocampus, and amygdala were isolated from mice. RT-qPCR and Western Blot: mRNA and protein expression of Elovl6, PPARα, and Allo synthesis genes (StAR, Cyp11a1, 5α-RI) were detected. Gas chromatography analysis: The relative content of various fatty acids in the prefrontal cortex was detected. ELISA: Allo content in tissues and cells was determined.

[0096] 1.4 Cell Experiments

[0097] Human neuroblastoma SH-SY5Y cell line was used. Cells were treated with palmitic acid (PA), oleic acid (OA), linoleic acid (LA), and the PPARα agonist GW7647. Cell viability was assessed using CCK-8 assays, and changes in the PPARα-Allo pathway were detected using the aforementioned molecular biological methods.

[0098] 1.5 In vivo pharmacological intervention

[0099] For Elovl6 - / - Mice were injected intraperitoneally with the PPARα agonist GW7647 (5 mg / kg, once daily for seven days), and changes in behavior and brain Allo levels were observed.

[0100] 1.6 Statistical Analysis

[0101] Data are expressed as mean ± standard deviation. Intergroup comparisons were performed using t-tests or one-way ANOVA, and p < 0.05 was considered statistically significant.

[0102] Experimental Example 2

[0103] Elovl6 knockout mice exhibited specific anxiety-like behaviors and social behavioral abnormalities.

[0104] Behavioral experiments showed that, compared with wild-type mice, Elovl6- / - mice exhibited a significantly reduced number of times and duration of entry into the central region during open field experiments. Figure 1 A- Figure 1 C); The number of times and time spent entering the open arm in the elevated cross maze were significantly reduced. Figure 1 D- Figure 1 F). The number of marbles buried in the marble burying experiment increased significantly ( Figure 1 G- Figure 1 H). The time and frequency of grooming in the self-grooming experiment increased significantly ( Figure 1 I- Figure 1 J). Scores in the nesting experiment were significantly reduced (J). Figure 1 K- Figure 1 L).

[0105] Experimental Example 3

[0106] Elovl6 knockout leads to dysbiosis of fatty acid composition in the prefrontal cortex and inhibition of the PPARα-Allo pathway.

[0107] Mechanistic investigation revealed significant changes in the fatty acid composition of the prefrontal cortex in Elovl6- / - mice: the content of saturated fatty acid PA increased, while the content of unsaturated fatty acids OA and LA decreased. Figure 2 A). This fatty acid disorder is accompanied by the following changes: 1) The expression level of PPARα is significantly downregulated ( Figure 2 B); 2) The expression levels of key Allo biosynthetic genes (StAR, Cyp11a1, 5α-RI) were significantly reduced. Figure 2 C); 3) Ultimately, the content of Allo in the prefrontal cortex decreased significantly ( Figure 2 D).

[0108] Test Example 4

[0109] Validating the regulation of the PPARα-Allo pathway by fatty acids at the cellular level

[0110] In SH-SY5Y cells, PA treatment significantly inhibited cell viability, PPARα protein expression, Allo synthesis gene expression, and Allo levels. OA, LA, or the PPARα agonist GW7647 effectively reversed the aforementioned inhibitory effects induced by PA. Figure 3 A- Figure 3 D).

[0111] Experimental Example 5

[0112] Activation of PPARα can reverse the anxiety phenotype in Elovl6 knockout mice.

[0113] After intraperitoneal injection of GW7647 into Elovl6- / - mice: 1) the Allo content in the prefrontal cortex returned to normal levels. Figure 4 A); 2) Behavioral tests showed that mice spent more time and made more frequent explorations of open arms in the elevated cross maze. Figure 4 B, Figure 4 C), both self-grooming behavior and bead-burying behavior were significantly reduced. Figure 4 D- Figure 4 F), anxiety-like behaviors were comprehensively improved.

[0114] Discussion and Summary: This invention systematically elucidates the key role and mechanism of Elovl6 in the regulation of anxiety-like behavior. The results show that Elovl6 dysfunction can cause fatty acid metabolism disorders in the prefrontal cortex, specifically manifested as an increase in the "anxiety-promoting" saturated fatty acid PA and a decrease in the "anxiety-resistant" unsaturated fatty acids OA and LA. This imbalance in fatty acid composition further inhibits the activity of the nuclear transcription factor PPARα and downregulates the biosynthesis of its downstream neurosteroid Allo. Since Allo is an important positive allosteric regulator of GABAA receptors, its reduced levels directly weaken inhibitory neurotransmission, ultimately contributing to anxiety-like behavior.

[0115] The core discovery of this invention lies in the first-ever revelation of a signaling axis running through the “Elovl6-fatty acid-PPARα-Allo” pathway. This pathway directly links fatty acid metabolism with mood regulation, providing a new molecular perspective for understanding the mechanisms of metabolic and mental health comorbidities. Furthermore, the study confirmed that targeting downstream components of this pathway using pharmacological methods (such as the PPARα agonist GW7647) can effectively reverse pathological phenotypes induced by upstream gene defects. This result not only validates the biological importance of this signaling axis but also provides crucial evidence for its translational medical potential, suggesting that PPARα may serve as a potential therapeutic target for anxiety disorders—especially subtypes accompanied by fatty acid metabolism disorders.

[0116] This invention deepens our understanding of the neurobiological mechanisms of anxiety disorders and provides new directions and theoretical basis for the development of anti-anxiety drugs.

[0117] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. The application of Elovl6 as a drug target in the preparation or screening of drugs for the treatment of anxiety or depression-related disorders.

2. The application according to claim 1, characterized in that: The Elovl6 includes the Elovl6 gene or the Elovl6 protein, wherein the nucleotide sequence of the Elovl6 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Elovl6 gene is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that: The drug works through at least one of the following mechanisms: (1) Upregulate the expression level or activity of Elovl6; (2) Adjust the fatty acid composition of the prefrontal cortex, reduce the relative content of palmitic acid and / or increase the relative content of oleic acid and linoleic acid; (3) Activate the PPARα-Allopregnanolone signal axis; (4) Increase the biosynthesis of Allopregnanolone.

4. The use of a reagent for detecting Elovl6 expression levels in the preparation of kits for the diagnosis or prognostic assessment of anxiety-related disorders.

5. The application according to claim 4, characterized in that: The reagents for detecting Elovl6 expression levels include: primers, probes, or chips that specifically detect Elovl6 mRNA levels, and / or antibodies that specifically detect Elovl6 protein levels.

6. The application of a drug that regulates the PPARα-Allopregnanolone signaling axis in the preparation of a drug for treating anxiety disorders related to Elovl6 dysfunction.

7. The application according to claim 6, characterized in that: The drug that regulates the PPARα-Allopregnanolone signaling axis is a PPARα agonist; The drug improves anxiety-like behaviors by restoring allopregnanolone levels in the prefrontal cortex to normal range.

8. A pharmaceutical composition for treating anxiety associated with Elovl6 dysfunction, characterized in that: It contains an effective amount of PPARα agonist and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that: The PPARα agonist is GW7647, with a dosage of 1-10 mg / kg, administered via intraperitoneal injection or oral administration.

10. The pharmaceutical composition according to claim 8, characterized in that: It also contains an auxiliary active ingredient for regulating the fatty acid composition, said auxiliary active ingredient being selected from one or more of oleic acid, linoleic acid, or their derivatives.

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