Use of ferulic acid in the preparation of a product for improving circadian rhythm disorders

Abstract

The present application relates to the application of ferulic acid in the preparation of products for improving biological clock disorders. Through a series of in vitro cell experiments and in vivo animal experiments, the present application systematically explores the regulatory effect of ferulic acid (FA) on biological clock disorders and the synaptic pruning abnormalities induced thereby, and clarifies the molecular mechanism. The present application first systematically reveals that ferulic acid directly targets Rev-erbα, thereby inhibiting complement C3-mediated neuroinflammation and pathological synaptic pruning, and ultimately improving biological clock disorders and the neurological and behavioral abnormalities caused thereby. Ferulic acid can be prepared into a drug or a functional product for improving biological clock disorders and the synaptic dysfunction and emotional disorders caused thereby. Meanwhile, ferulic acid has excellent safety and is suitable for long-term use. The present application provides a new direction for the subsequent research and development of new technologies, and has important scientific significance and application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of ferulic acid in the preparation of products that improve circadian rhythm disorders and / or regulate physiological indicators related to circadian rhythm disorders, and particularly to a novel application of ferulic acid in improving circadian rhythm disorders and the resulting neuroinflammation, abnormal synaptic pruning, and neurobehavioral abnormalities through the Rev-erbα-complement C3 pathway. Background Technology

[0002] The biological clock is an intrinsic timing mechanism developed by organisms to adapt to the Earth's rotation cycle, and it is crucial for maintaining physiological homeostasis. Modern lifestyles (such as staying up late, shift work, and sleep disorders) or genetic factors often lead to biological clock disruption, which in turn triggers a series of pathological changes, including inflammatory responses, metabolic disorders, cognitive impairment, and mood disorders (such as depression and anxiety). Studies have shown that biological clock disruption can drive peripheral and central nervous system inflammation, which in turn activates microglia and astrocytes, leading to excessive synaptic pruning. This is a key step in the early synaptic loss and cognitive decline in Alzheimer's disease and other neurodegenerative diseases.

[0003] REV-ERBα is a key negative transcription factor in the core regulatory network of the biological clock and also possesses potent anti-inflammatory effects. Complement C3 is a core component of the complement cascade, and its aberrant activation mediates astrocyte-microglia communication, driving pathological synaptic pruning. Therefore, targeting the Rev-erbα-complement C3 pathway is a potential strategy for intervening in circadian rhythm disorder-related encephalopathy.

[0004] Ferulic acid (FA) is a natural phenolic acid compound extracted from traditional Chinese medicines such as Angelica sinensis and Ligusticum chuanxiong. It is known to have antioxidant, anti-inflammatory, and neuroprotective effects. However, whether ferulic acid can improve circadian rhythm disorders and the resulting synaptic pruning abnormalities and neurobehavioral deficits by regulating the Rev-erbα-complement C3 pathway has not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to address the problems existing in the prior art, thereby providing the application of ferulic acid in the preparation of products that improve circadian rhythm disorders and / or regulate physiological indicators related to circadian rhythm disorders. Through extensive research, this invention has discovered that ferulic acid directly targets Rev-erbα, thereby inhibiting complement C3-mediated neuroinflammation and pathological synaptic pruning, ultimately improving circadian rhythm disorders and the resulting neurobehavioral abnormalities. This provides a new direction for the development of subsequent technologies and has significant scientific and application value.

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution.

[0007] The first aspect of this invention provides the use of ferulic acid in the preparation of products that improve circadian rhythm disorders and / or regulate physiological indicators related to circadian rhythm disorders.

[0008] Preferably, the ferulic acid improves circadian rhythm disorders and / or regulates physiological indicators related to circadian rhythm disorders through the Rev-erbα-complement C3 pathway.

[0009] Preferably, the product is selected from one or more of pharmaceuticals, food, and health products.

[0010] Preferably, the physiological indicators related to circadian rhythm disorder are selected from one or more of the following: oxidative damage, complement activation, astrocyte polarization, synaptic regulatory factor balance, cellular inflammation, circadian rhythm gene expression level, body weight, anhedonia caused by circadian rhythm disorder, depressive-like behavior caused by circadian rhythm disorder, and anxiety-like behavior caused by circadian rhythm disorder.

[0011] Preferably, the indicators related to oxidative damage are selected from one or more of SOD, GSH, and ROS.

[0012] Preferably, the complement is selected from C3.

[0013] Preferably, the indices related to astrocyte polarization are selected from one or more of S100A10 and GFAP.

[0014] Preferably, the indices related to the balance of synaptic regulatory factors are selected from one or more of Sparc, Sparcl1, Thbs1, Thbs2, and ApoE.

[0015] Preferably, the markers related to cellular inflammation are selected from one or more of IL-1β, IL-6, and TNF-α.

[0016] Preferably, the biological clock gene is selected from one or more of Bmal1, Rev-erbα, Cry1, Cry2, Per1, and Per2.

[0017] A second aspect of the present invention provides a composition for improving circadian rhythm disorder and / or regulating physiological indicators related to circadian rhythm disorder, comprising ferulic acid and a carrier.

[0018] Preferably, the ferulic acid improves circadian rhythm disorders and / or regulates physiological indicators related to circadian rhythm disorders through the Rev-erbα-complement C3 pathway.

[0019] Preferably, the composition is selected from one or more of pharmaceuticals, foods, and health products.

[0020] Preferably, the physiological indicators related to circadian rhythm disorder are selected from one or more of the following: oxidative damage, complement activation, astrocyte polarization, synaptic regulatory factor balance, cellular inflammation, circadian rhythm gene expression level, body weight, anhedonia caused by circadian rhythm disorder, depressive-like behavior caused by circadian rhythm disorder, and anxiety-like behavior caused by circadian rhythm disorder.

[0021] Preferably, the indicators related to oxidative damage are selected from one or more of SOD, GSH, and ROS.

[0022] Preferably, the complement is selected from C3.

[0023] Preferably, the indices related to astrocyte polarization are selected from one or more of S100A10 and GFAP.

[0024] Preferably, the indices related to the balance of synaptic regulatory factors are selected from one or more of Sparc, Sparcl1, Thbs1, Thbs2, and ApoE.

[0025] Preferably, the markers related to cellular inflammation are selected from one or more of IL-1β, IL-6, and TNF-α.

[0026] Preferably, the biological clock gene is selected from one or more of Bmal1, Rev-erbα, Cry1, Cry2, Per1, and Per2.

[0027] Preferably, the carrier is selected from one or more of solvents, fillers, disintegrants, lubricants, adhesives, antioxidants, antibacterial agents, flavoring agents, fragrances, and chelating agents.

[0028] This invention systematically investigated the regulatory role of ferulic acid (FA) on circadian rhythm disruption and its induced synaptic pruning abnormalities through a series of in vitro cell experiments and in vivo animal experiments, and clarified its molecular mechanism. Specifically, FA alleviates oxidative stress through Rev-erbα: In an LPS-induced C8D1A astrocyte inflammation model, FA dose-dependently increased SOD activity and GSH levels while decreasing ROS levels, and this protective effect was significantly weakened after silencing Rev-erbα, indicating that FA exerts its antioxidant effect in dependence on Rev-erbα. FA inhibits complement C3 expression through Rev-erbα: FA significantly downregulated LPS-induced C3 mRNA and protein levels, and the inhibitory effect of FA was significantly weakened after silencing Rev-erbα, confirming that FA blocks complement cascade activation through the Rev-erbα-complement C3 pathway. FA regulates astrocyte polarization and synaptic regulatory factor balance: FA can reverse LPS-induced astrocyte polarization towards the A1 neurotoxic phenotype and restore the expression of the A2 protective phenotype marker S100A10. Simultaneously, FA can correct the LPS-induced downregulation of the "synaptic brake" molecule Sparc and the overexpression of pro-synaptic factors (Sparcl1, Thbs1, Thbs2, ApoE), and all of these regulatory effects depend on Rev-erbα. FA inhibits the release of inflammatory factors and restores circadian rhythms: FA can significantly reduce LPS-induced release of IL-1β, IL-6, and TNF-α, and effectively alleviate the circadian rhythm disorder of core circadian rhythm genes such as Bmal1, Rev-erbα, Cry1, Cry2, Per1, and Per2 caused by LPS. Molecular docking confirms direct binding of FA to REV-ERBα: Docking results show that FA and REV-ERBα have a stable binding conformation (binding energy -6.0 kcal / mol), providing a structural basis for the above functional mechanisms. Animal experiments validated the in vivo protective effects of FA: In a mouse model of circadian rhythm disorder (CRD), FA alleviated CRD-induced abnormal weight gain, anhedonia (reduced sucrose preference), depressive-like behavior (prolonged tail immobility time), and anxiety-like behavior (reduced entry into the central open field and decreased total movement distance). Simultaneously, FA reduced the levels of pro-inflammatory factors (IL-6, TNF-α, IL-1β) in the prefrontal cortex, restored Rev-erbα expression, inhibited complement C3 overexpression, and corrected the abnormal expression of A1 / A2 astrocyte markers and synaptic regulatory factors.

[0029] In summary, this invention is the first to systematically reveal that ferulic acid directly targets Rev-erbα, thereby inhibiting complement C3-mediated neuroinflammation and pathological synaptic pruning, ultimately improving circadian rhythm disorders and the resulting neurobehavioral abnormalities. Ferulic acid can be formulated into drugs or functional products to improve circadian rhythm disorders and their associated synaptic dysfunction and mood disorders; furthermore, ferulic acid exhibits excellent safety profiles and is suitable for long-term use. This invention provides a new direction for the development of subsequent technologies and has significant scientific and practical value. Attached Figure Description

[0030] Figure 1 This is a schematic diagram showing the effect of different concentrations of FA on cell viability.

[0031] Figure 2 This is a schematic diagram illustrating the efficiency of si-Rev-erbα knockdown of intracellular Rev-erbα.

[0032] Figure 3 This is a schematic diagram showing the effect of FA on intracellular SOD activity.

[0033] Figure 4 This is a schematic diagram showing the effect of FA on intracellular GSH levels.

[0034] Figure 5 This is a schematic diagram showing the effect of FA on intracellular ROS levels.

[0035] Figure 6 This is a schematic diagram showing the effect of FA on LPS-induced complement activation.

[0036] Figure 7 This is a schematic diagram showing the effect of FA on astrocyte polarization.

[0037] Figure 8 This is a schematic diagram illustrating the effect of FA on the balance of synaptic regulatory factors.

[0038] Figure 9 This is a schematic diagram showing the effect of FA on the levels of inflammatory factors in cells.

[0039] Figure 10 This is a schematic diagram illustrating the effects of FA on the level of circadian rhythm genes in cells with disrupted circadian rhythms.

[0040] Figure 11 This is a schematic diagram of a molecular docking simulation.

[0041] Figure 12 A schematic diagram of the process for constructing a mouse model of circadian rhythm disorder.

[0042] Figure 13 This is a schematic diagram showing the effect of FA on the body weight of mice with disrupted biological clock.

[0043] Figure 14 This is a schematic diagram illustrating the effect of FA on improving anhedonia caused by circadian rhythm disruption.

[0044] Figure 15 This is an example diagram of a tail suspension experiment.

[0045] Figure 16 This is a schematic diagram illustrating the effect of FA on improving depressive-like behavior after circadian rhythm disruption.

[0046] Figure 17 This is an example diagram of an open field experiment.

[0047] Figure 18 This is a schematic diagram illustrating the effects of FA on anxiety-like behaviors and spontaneous activities resulting from circadian rhythm disruption.

[0048] Figure 19 This is a schematic diagram showing the effect of FA on the secretion of inflammatory factors in the prefrontal cortex of mice with disrupted biological clocks.

[0049] Figure 20 This is a schematic diagram illustrating the regulatory effects of FA on circadian rhythm disruption and transcription factors related to synaptic plasticity.

[0050] Figure 21 This is a schematic diagram illustrating the regulatory effect of FA on complement system activation under circadian rhythm disorder.

[0051] Figure 22 This is a schematic diagram illustrating the effects of FA on the functional phenotype and synaptic regulation of astrocytes. Detailed Implementation

[0052] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0053] Unless otherwise specified, the experimental animals used in this invention were purchased from Guangdong Yaokang Biotechnology Co., Ltd., and were male 8-week-old C57BL / 6J SPF grade mice, housed in a sterile environment at the animal facility of South China Agricultural University; FA was purchased from Shanghai Maclean's Biochemical Technology Co., Ltd.; all other reagents used were commercially available. For animal experiments, the relevant procedures and methods complied with the requirements of South China Agricultural University's experimental animal and medical ethics guidelines, and were performed in accordance with their regulations. The experimental methods used in this invention, such as cell biology experiments, molecular biology experiments, and animal experiments, are all conventional methods and techniques in the field.

[0054] Representative results from biological experiments were selected from replicates and presented in the contextual figures. Data were displayed as mean ± SD and mean ± SEM as specified in the figures. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 8.0 or SPSS 22.0 software. Standard medical statistical methods such as t-tests, chi-square tests, and ANOVA were used to compare differences in means between two or more groups. p < 0.05 was considered statistically significant, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0055] Example 1 To evaluate the effects of ferulic acid (FA) and lipopolysaccharide (LPS) on the viability of C8D1A cells, cells were treated with different concentrations of FA and LPS to confirm their respective safe concentrations. The specific steps were as follows: C8D1A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and routinely cultured at 37°C in a 5% CO2 incubator. When the cells reached 80%-90% confluence, they were passaged using 0.25% trypsin. Cells in the logarithmic growth phase were harvested and cultured at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 μL in each well of a 96-well plate and pre-cultured in an incubator for 24 h until the cells were fully adhered.

[0056] A blank control group (containing no cells, only culture medium), a solvent control group (containing cells, with an equal volume of DMSO added to the culture medium, final concentration ≤0.1%), and different concentrations of FA treatment groups were set up. The original culture medium was discarded, and the FA groups were added with complete culture medium containing FA at final concentrations of 0, 5, 10, 15, 20, 50, and 100 μM, with 5 replicates per group. After culturing for 24 h, 20 μL MTT solution (5 mg / mL, prepared with sterile PBS) was added to each well, and the cells were incubated in the dark for 4 h. The supernatant was carefully aspirated, and 150 μL LDMSO was added to each well, shaken for 10 min to fully dissolve the crystals. The absorbance (OD value) of each well was measured at 490 nm using a microplate reader. Cells were also seeded in 96-well plates, with blank control, solvent control, and LPS treatment groups. The LPS groups were added with complete culture medium containing LPS at final concentrations of 0, 0.1, 1, 2, 5, 10, 20, and 50 μg / mL, with 5 replicates per group. After culturing for 24 h, the OD value was detected and the cell viability was calculated using the MTT method described above.

[0057] Data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was performed using GraphPad Prism 8.0, and *p < 0.05 was considered statistically significant.

[0058] Experimental results are as follows Figure 1 As shown in the figure. The results showed that when the FA concentration was in the range of 0-20 μM, it had no significant inhibitory effect on cell viability; however, when the FA concentration was further increased, cell viability began to decrease. Specifically, the cell survival rate of the 20 μM FA treatment group was 91.3% ± 1.05%, indicating that this concentration had no significant cytotoxicity. Within the concentration range of 0–50 μg / mL, low concentrations of LPS had no significant effect on cell viability, but cell viability gradually decreased with increasing concentration. Therefore, 1 μg / mL LPS and 10, 15, and 20 μM FA were selected for subsequent experiments.

[0059] To investigate the role of Rev-erbα in FA-regulated LPS-induced inflammation in C8D1A cells, cells were transfected with si-Rev-erbα (forward sequence as shown in SEQ ID NO: 1, 5'-CCCACATACTTCCCACCATTT-3', reverse sequence as shown in SEQ ID NO: 2, 5'-ATGGTGGGAAGTATGTGGGTT-3') for 24 hours, with non-specific siRNA (si-Cont) used as a negative control. The experimental results are as follows: Figure 2 As shown in the figure. The results showed that, compared with the control group, the Rev-erbα mRNA expression level in the si-Rev-erbα group decreased to 28.45%±1.51% of that in the control group (p<0.05), indicating good knockdown efficiency; there was no significant difference between the si-Cont group and the control group, indicating that non-specific siRNA does not affect the basic expression of Rev-erbα.

[0060] To evaluate the protective effect of FA against LPS-induced oxidative damage in C8D1A cells, the SOD activity and GSH content of each group of cells were measured. The SOD activity results are shown below. Figure 3 As shown in the figure. The results showed that the SOD activity in the control group was 8.75±0.29 U / mg prot; compared with the control group, the SOD activity after LPS treatment was significantly reduced to 6.95±0.74 U / mg prot (p<0.05). After intervention with different concentrations of FA (10, 15, 20 μM), the SOD activity rebounded in a dose-dependent manner, reaching 7.71±0.07, 8.70±0.56, and 9.31±0.37 U / mg prot, respectively. Among them, the 20 μM FA group showed a statistically significant difference compared with the LPS group (p<0.05), and recovered to a level close to that of the control group.

[0061] GSH content results are as follows Figure 4As shown in the figure. The results showed that the control group had a GSH content of 97.17±5.20 μmol; compared with the control group, the GSH content decreased to 40.27±8.45 μmol after LPS treatment (p<0.001). After FA intervention, the GSH level increased sequentially to 62.75±2.61, 76.83±5.21 and 94.66±6.62 μmol, showing a clear dose-dependent relationship, with the 20 μM FA group showing a significant difference compared with the LPS group (p<0.01). These results indicate that LPS-induced oxidative stress in C8D1A cells leads to a significant decrease in SOD activity and GSH content; FA can dose-dependently restore these two antioxidant indicators and alleviate LPS-induced oxidative damage.

[0062] In addition, the ROS level detection results show (see...) Figure 5 The intracellular ROS level in the control group was 14.30% ± 1.53%; compared with the control group, the ROS level in the LPS-treated group was significantly increased to 54.25% ± 2.27% (p < 0.001). ROS levels decreased in a dose-dependent manner after FA intervention, decreasing to 26.92% ± 2.05% in the 20 μM FA group. Further silencing of Rev-erbα significantly weakened the ROS-reducing effect of FA compared with the 20 μM FA treatment group alone, with ROS levels recovering to 45.66% ± 3.09% (p < 0.05), indicating that the regulatory effect of FA on cellular redox balance depends on Rev-erbα.

[0063] To investigate the effect of FA on LPS-induced complement activation and to clarify whether Rev-erbα is involved in this process, qPCR and ELISA were used to detect the mRNA and protein expression levels of C3, respectively. The results are as follows: Figure 6As shown in the figure. The results showed that, compared with the control group (0.98±0.02), C3 mRNA was significantly increased after LPS treatment (1.83±0.09, p<0.01); after FA intervention, C3 mRNA significantly decreased to 1.15±0.05 (p<0.05). However, after silencing Rev-erbα, the downregulatory effect of FA on C3 was weakened, and C3 mRNA rebounded to 1.39±0.05, which was statistically significant compared with the FA-only treatment group (p<0.05). The ELISA results showed a consistent trend: compared with the control group (24.43±4.18 ng / mL), the C3 protein content in the LPS group was significantly increased (65.91±5.0 ng / mL, p<0.01); after FA treatment, it decreased to 54.29±0.87 ng / mL (p<0.05), and rebounded to 63.45±4.5 ng / mL after Rev-erbα silencing, which was statistically significant compared with the FA-only treatment group (p<0.05). These results indicate that the inhibitory effect of FA on LPS-induced C3 expression upregulation is Rev-erbα-dependent.

[0064] Example 2 Astrocytes can polarize into different phenotypes during neuroinflammation: type A1 is a neurotoxic phenotype (marker GFAP), and type A2 is a neuroprotective phenotype (marker S100A10). Simultaneously, synaptic regulatory factors derived from astrocytes play a crucial role in maintaining synaptic homeostasis: Sparc acts as a "synaptic brake" molecule, inhibiting excessive synapse formation, while Sparcl1, Thbs1, Thbs2, and ApoE are classic prosynaptic factors. To investigate the effects of fatty acids (FA) on LPS-induced astrocyte polarization and synaptic regulatory networks, and to clarify whether Rev-erbα is involved in this process, qPCR was used to detect the mRNA expression levels of the aforementioned genes, and gene silencing experiments were conducted using Si-Rev-erbα.

[0065] The results of the effect on astrocyte polarization are as follows: Figure 7As shown in the figure. The results showed that, compared with the control group (S100A10 = 1.00±0.05, GFAP = 1.00±0.06), the expression of the A2 marker S100A10 decreased to 0.57±0.09 after LPS treatment, while the expression of the A1 marker GFAP significantly increased to 1.66±0.08 (p<0.01), suggesting that LPS induces astrocyte polarization towards A1 type. After FA intervention, compared with the LPS group, S100A10 rebounded to 0.98±0.05, and GFAP decreased to 0.91±0.03 (p<0.05). After silencing Rev-erbα, compared with the FA treatment group alone, the regulatory effect of FA was weakened, with S100A10 decreasing to 0.52±0.10 and GFAP increasing to 1.32±0.11 (p<0.05). This indicates that FA partially reverses LPS-induced A1-type polarization and is dependent on Rev-erbα.

[0066] The results of the effect on the balance of synaptic regulatory factors are as follows Figure 8 As shown in the figure. The results showed that, compared with the control group (Sparc 1.01±0.12, Sparcl1 0.97±0.04, Thbs1 0.97±0.05, Thbs2 0.97±0.03, ApoE 1.01±0.03), the expression of the "synaptic brake" molecule Sparc decreased to 0.65±0.02 after LPS treatment, while the expression of the pro-synaptic factors Sparcl1 increased to 1.25±0.01, Thbs1 increased to 1.67±0.12, Thbs2 increased to 1.37±0.03, and ApoE increased to 1.54±0.14 (p<0.01). After FA intervention, compared with the LPS group, Sparc increased to 0.88±0.02, Sparcl1 decreased to 0.81±0.06, Thbs1 decreased to 1.12±0.05, Thbs2 decreased to 0.85±0.01, and ApoE decreased to 0.78±0.02 (p<0.05). After silencing Rev-erbα, compared with the FA-only treatment group, the regulatory effect of FA was weakened: Sparc further decreased to 0.72±0.02, while the pro-synaptic factors Sparcl1 increased to 1.11±0.05, Thbs1 increased to 1.47±0.08, Thbs2 increased to 1.28±0.01, and ApoE increased to 1.19±0.04 (p<0.05). This indicates that FA, through a Rev-erbα-dependent mechanism, blocks LPS-induced overexpression of pro-synaptic factors, restoring the balance of the synaptic regulatory network.

[0067] To further investigate the role of the core circadian clock gene Rev-erbα in FA's mitigation of LPS-induced cellular inflammatory responses, Rev-erbα gene expression was silenced using si-Rev-erbα, followed by FA treatment (20 μM). Cell samples were collected for ELISA and qPCR detection to measure the protein release levels and mRNA expression levels of inflammatory factors IL-1β, IL-6, and TNF-α, respectively. The experimental results are as follows: Figure 9 As shown in the figure, the results indicated that, compared with the control group, LPS treatment significantly upregulated the mRNA and protein expression levels of IL-1β, IL-6, and TNF-α (p < 0.01). After FA intervention, all three levels were significantly decreased compared with the LPS group (p < 0.05). After silencing Rev-erbα, the inhibitory effect of FA was significantly weakened compared with the FA treatment group alone, and the expression levels of each inflammatory factor recovered to near the LPS group level (p < 0.05). The results indicate that the alleviating effect of FA on LPS-induced inflammatory factor release is dependent on Rev-erbα.

[0068] A cell model of circadian rhythm disorder was established by treating C8D1A cells with LPS (specific method as in Example 1). After FA treatment, circadian rhythm gene expression was detected every 6 hours (18–42 hours). The experimental results are as follows: Figure 10 As shown in the figure, the results indicated that Bmal1, Rev-erbα, Cry1, Cry2, Per1, and Per2 all exhibited significant rhythmic oscillations in the control group. Compared with the control group, the rhythmicity of each gene was significantly disrupted after LPS treatment, manifested as phase shift and decreased amplitude. Compared with the LPS group, the rhythmic expression of the above genes tended to recover to the control group level after FA intervention. This indicates that FA can effectively alleviate LPS-induced disruption of the circadian rhythm of genes.

[0069] Molecular docking is a computer simulation method based on structural bioinformatics, aiming to predict the three-dimensional binding modes of small molecules (ligands) and target proteins (receptors) and estimate binding affinity. Its core tasks include: pose sampling of the ligand in the receptor pocket; and evaluating the binding free energy (scoring) of each conformation. Through these steps, a reasonable binding mode of the ligand-receptor complex can be obtained, providing a testable structural hypothesis for experimental verification. Experimental results are shown below. Figure 11As shown in the figure, the results indicate that using AutoDock Vina to dock ferulic acid (FA) with the circadian rhythm inhibitor REV-ERBα (NR1D1) showed a minimum binding energy ΔG = –6.0 kcal mol⁻¹, suggesting that the two can form a stable complex; the key hydrogen bond distance was between 2.2 and 3.5 Å, meeting the criteria for drug-protein interactions. This also suggests that FA can stably bind to REV-ERBα to exert its effect. Functionally, previous studies have found that FA can downregulate C3 expression and inhibit synaptic pruning in A1 astrocytes. The docking results provide a structural basis for this: after FA binds to REV-ERBα, it blocks C3 expression, thereby inhibiting the complement cascade and reducing pathological excessive synaptic pruning.

[0070] Example 3 To further clarify the ameliorative effect and specific mechanism of ferulic acid (FA) on circadian rhythm disorder, a series of in vivo experiments were conducted. First, the effect of FA on the body weight of mice with circadian rhythm disorder was investigated. The mouse model of circadian rhythm disorder was established as follows: an 8-hour light phase shift method was used to establish a mouse model of circadian rhythm disorder. Specifically, each cycle consisted of 6 days. For the first 3 days (days 1-3) of each cycle, the light time was 8:00-20:00 and the dark time was 20:00-8:00 the next day. For the last 3 days (days 4-6), the light time was shifted forward by 8 hours, i.e., the light time on days 4-6 was 0:00-12:00 and the dark time was 12:00-24:00. After each cycle, the light start time for the next cycle was shifted forward by another 8 hours. The experimental results are as follows: Figure 12-13 As shown in the figure. The results showed no significant difference in body weight among the groups before the experiment. Starting from week 4, the body weight of mice in the CRD group was significantly higher than that in the CON group; with continued feeding until week 4, the average body weight of mice in the CRD group reached 25.60±1.71g, which was 1.12 times that of mice in the CON group. Rodents feed more frequently during their nocturnal active period, and CRD disrupted the timed feeding pattern, leading to weight gain. Compared with the CRD group, after gavage administration of different doses of FA, starting from week 4, the average body weight increase of mice in the FA group was slower than that of the CRD group, and was lower than that of the CRD group at week 8. This indicates that circadian rhythm disruption treatment causes weight gain in mice, and FA can effectively improve this situation.

[0071] To evaluate the effect of FA on improving anhedonia following circadian rhythm disruption, a sucrose preference test was used to detect the sucrose preference index in each group of mice. The experimental results are as follows: Figure 14As shown in the figure. The results showed that the saccharin preference index in the CON group was 78.74±4.67%. Compared with the CON group, the saccharin preference index in the CRD model group was significantly reduced to 57.08±16.92% (*p*<0.01), indicating significant anhedonia symptoms. Compared with the CRD group, the FA (10 mg / kg) group (66.36±3.81%), FA (20 mg / kg) group (68.28±5.62%), FA (40 mg / kg) group (72.06±9.30%), and FLX group (74.15±7.69%) all significantly increased the saccharin preference index (*p*<0.05), and there was no significant difference between the FA group and the CON group, indicating that FA can reverse CRD-induced anhedonia.

[0072] To evaluate the effect of FA on improving depressive-like behavior following circadian rhythm disruption, a tail suspension test was used to detect immobility time in each group of mice. The experimental results are as follows: Figure 15-16 As shown in the figure. The results showed that the immobility time of the tail in the CON group was 134.1±15.70 s. The immobility time in the CRD model group was significantly prolonged to 171.7±12.72 s (*p*<0.001), indicating that the depressive-like behavior model was successfully established. Compared with the CRD group, the immobility time in the FA (40 mg / kg) group was shortened to 128.4±12.36 s (*p*<0.001), and in the FLX group it was shortened to 128.1±27.44 s (*p*<0.001); although there was a certain decreasing trend in the FA (10 mg / kg) group (162.5±9.02 s) and the FA (20 mg / kg) group (154.1±18.61 s), the difference was not statistically significant. These results indicate that high-dose administration has a significant antidepressant-like effect.

[0073] To assess the effects of febrile acid (FA) on anxiety-like behavior and spontaneous activity induced by circadian rhythm disruption, an open field test was used to detect changes in activity levels in each group of mice. The experimental results are as follows: Figure 17-18As shown in the figure. The results showed that, in terms of the number of central zone entries, the CON group had 38.13±2.48 entries, while the CRD model group had a significantly reduced number to 15±8.32 entries (*p*<0.01); the FA (40 mg / kg) group (29.25±5.92 entries) and the FLX group (29.00±8.78 entries) had significantly increased numbers compared to the CRD group (*p*<0.05). In terms of total exercise distance, the CON group had 3584±655.1 cm, while the CRD model group had a significantly reduced distance to 2086±297.0 cm (*p*<0.01); the FA (20 mg / kg) group (2755±336.5 cm), the FA (40 mg / kg) group (3059±298.9 cm), and the FLX group (3258±444.0 cm) had significantly increased numbers compared to the CRD group (*p*<0.05). The results showed that FA could improve CRD-induced anxiety-like behavior and activity inhibition.

[0074] Neuroinflammation may be one of the causes of depressive changes resulting from circadian rhythm disruption. To investigate this, we examined the effects of FA (fiber-dependent inflammatory factor) on the secretion of inflammatory factors in the prefrontal cortex of mice with circadian rhythm disruption. The experimental results are as follows: Figure 19 As shown in the figure. The results showed that, by qPCR detection, compared with the CON group (IL-6: 0.98±0.04, TNF-α: 1.00±0.06, IL-1β: 1.00±0.03), the mRNA expression levels of IL-6 (2.19±0.14), TNF-α (2.58±0.28), and IL-1β (3.14±0.27) in the prefrontal cortex of the CRD model group were significantly increased (**p<0.01); after administration of FA (40 mg / kg), the mRNA levels of the above inflammatory factors decreased to IL-6: 1.36±0.05, TNF-α: 1.58±0.08, and IL-1β: 1.64±0.04, respectively (*p<0.05). The ELISA results were consistent with the trends observed by qPCR: in the CON group, the protein concentrations of IL-6, TNF-α, and IL-1β were 169.9±9.04 pg / mL, 546.6±27.15 pg / mL, and 122.2±1.65 pg / mL, respectively; in the CRD group, these concentrations increased to 207.0±6.63 pg / mL, 819.0±66.6 pg / mL, and 162.0±7.79 pg / mL, respectively (**p<0.01); and in the FA group, the protein concentrations significantly decreased to IL-6: 184.2±7.41 pg / mL, TNF-α: 688.6±44.47 pg / mL, and IL-1β: 131.0±2.06 pg / mL (*p<0.05). These results suggest that depressive-like behavior caused by circadian rhythm disruption may be related to neuroinflammation. FA can improve neuroinflammation by reducing the level of pro-inflammatory factors, thereby improving depressive-like behaviors caused by circadian rhythm disorders.

[0075] To further clarify the regulatory role of FA on circadian rhythm disruption and synaptic plasticity-related transcription factors, the expression level of REV-ERBα mRNA was detected, and the results are as follows: Figure 20 As shown in the figure. The results showed that the expression level in the CON group was normalized to 1.07±0.12. The Rev-erbα mRNA expression level in the CRD model group was significantly reduced to 0.55±0.03 (**p<0.01). Compared with the CRD group, the Rev-erbα mRNA expression level in the FA (40 mg / kg) administration group was significantly restored to 0.80±0.08 (*p<0.05), indicating that FA can restore REV-ERBα expression, regulate the biological clock, neuroinflammation and synaptic plasticity, and improve the neurological dysfunction induced by biological clock disorder.

[0076] To investigate the regulatory role of FA on complement system activation under circadian rhythm disruption, qPCR was used to detect the expression level of complement C3 in the brain tissue of mice in each group. Results are as follows: Figure 21 As shown, the expression level in the CON group was normalized to 0.99±0.025. The C3 mRNA expression level in the brains of mice in the CRD model group was significantly increased to 1.69±0.18 (***p<0.001). Compared with the CRD group, the C3 mRNA expression level in the FA (40 mg / kg) administration group was significantly decreased to 0.82±0.02 (***p<0.001), and returned to the level of the normal control group, indicating that FA can inhibit the overexpression of complement C3 induced by circadian rhythm disorder and block the abnormal synaptic phagocytosis process mediated by the complement system.

[0077] To investigate the intervention effects of febrile alpha (FA) on astrocyte functional phenotype and synaptic regulation, qPCR was used to detect the mRNA expression levels of A1 / A2 astrocyte markers and synaptic regulatory factors in the prefrontal cortex of mice in each group. The results are as follows: Figure 22As shown in the figure. The results showed that, regarding astrocyte phenotypic markers, the expression level in the CON group was normalized to 0.98±0.04. In the CRD model group, the expression of the A2 protective marker S100A10 mRNA decreased to 0.82±0.02 (*p<0.05), while the expression of the A1 toxic marker GFAP mRNA increased to 1.90±0.13 (**p<0.01). The FA (40 mg / kg) group reversed the above abnormalities, with S100A10 rising back to 0.92±0.08 and GFAP decreasing to 0.74±0.02 (*p<0.05). Regarding synaptic regulatory factors, the expression of the "synaptic brake" molecule Sparc in the CRD model group decreased to 0.72±0.05, while the expression of pro-synaptic factors Sparcl1, Thbs1, Thbs2, and ApoE increased to 2.04±0.21, 2.84±0.10, 2.43±0.15, and 1.21±0.02, respectively (**p<0.01). FA treatment significantly inhibited the overexpression of pro-synaptic factors, with Sparcl1 decreasing to 1.39±0.15, Thbs1 decreasing to 1.29±0.12, Thbs2 decreasing to 1.20±0.34, and ApoE decreasing to 0.77±0.09 (*p<0.05), while maintaining normal expression of Sparc (1.21±0.11). This indicates that FA can block the pathological synapse occurrence induced by circadian rhythm disorder and restore synaptic regulatory balance.

[0078] In summary, this invention systematically investigated the regulatory role of ferulic acid (FA) on circadian rhythm disruption and its induced synaptic pruning abnormalities through a series of in vitro cell experiments and in vivo animal experiments, and clarified its molecular mechanism. Specifically, FA alleviates oxidative stress through Rev-erbα: In an LPS-induced C8D1A astrocyte inflammation model, FA dose-dependently increased SOD activity and GSH levels while decreasing ROS levels, and this protective effect was significantly weakened after silencing Rev-erbα, indicating that FA exerts its antioxidant effect in dependence on Rev-erbα. FA inhibits complement C3 expression through Rev-erbα: FA significantly downregulated LPS-induced C3 mRNA and protein levels, and the inhibitory effect of FA was significantly weakened after silencing Rev-erbα, confirming that FA blocks complement cascade activation through the Rev-erbα-complement C3 pathway. FA regulates astrocyte polarization and synaptic regulatory factor balance: FA can reverse LPS-induced astrocyte polarization towards the A1 neurotoxic phenotype and restore the expression of the A2 protective phenotype marker S100A10. Simultaneously, FA can correct the LPS-induced downregulation of the "synaptic brake" molecule Sparc and the overexpression of pro-synaptic factors (Sparcl1, Thbs1, Thbs2, ApoE), and all of these regulatory effects depend on Rev-erbα. FA inhibits the release of inflammatory factors and restores circadian rhythms: FA can significantly reduce LPS-induced release of IL-1β, IL-6, and TNF-α, and effectively alleviate the circadian rhythm disorder of core circadian rhythm genes such as Bmal1, Rev-erbα, Cry1, Cry2, Per1, and Per2 caused by LPS. Molecular docking confirms direct binding of FA to REV-ERBα: Docking results show that FA and REV-ERBα have a stable binding conformation (binding energy -6.0 kcal / mol), providing a structural basis for the above functional mechanisms. Animal experiments validated the in vivo protective effects of FA: In a mouse model of circadian rhythm disorder (CRD), FA alleviated CRD-induced abnormal weight gain, anhedonia (reduced sucrose preference), depressive-like behavior (prolonged tail immobility time), and anxiety-like behavior (reduced entry into the central open field and decreased total movement distance). Simultaneously, FA reduced the levels of pro-inflammatory factors (IL-6, TNF-α, IL-1β) in the prefrontal cortex, restored Rev-erbα expression, inhibited complement C3 overexpression, and corrected the abnormal expression of A1 / A2 astrocyte markers and synaptic regulatory factors.

[0079] In summary, this invention reveals for the first time that ferulic acid, through the Rev-erbα-complement C3 signaling pathway, inhibits astrocyte-mediated neuroinflammation and pathological synaptic pruning, restoring the balance of the synaptic regulatory network, thereby improving neurobehavioral abnormalities induced by circadian rhythm disruption. This invention provides new theoretical and experimental evidence for the practical application of ferulic acid in circadian rhythm-related mood disorders and the treatment or improvement of neurodegenerative diseases (such as Alzheimer's disease). Furthermore, its high safety profile makes it suitable for long-term use, providing a new direction for the development of subsequent technologies.

[0080] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. Application of ferulic acid in the preparation of products that improve circadian rhythm disorders and / or regulate physiological indicators related to circadian rhythm disorders.

2. The application according to claim 1, characterized in that, The product is selected from one or more of the following: pharmaceuticals, food, and health products.

3. The application according to claim 1, characterized in that, The physiological indicators related to circadian rhythm disorder are selected from one or more of the following: oxidative damage, complement activation, astrocyte polarization, synaptic regulatory factor balance, cellular inflammation, circadian rhythm gene expression level, body weight, anhedonia caused by circadian rhythm disorder, depressive-like behavior caused by circadian rhythm disorder, and anxiety-like behavior caused by circadian rhythm disorder.

4. The application according to claim 3, characterized in that, Indicators related to oxidative damage are selected from one or more of SOD, GSH, and ROS.

5. The application according to claim 3, characterized in that, The complement is selected from C3.

6. The application according to claim 3, characterized in that, Indicators related to astrocyte polarization were selected from one or more of S100A10 and GFAP.

7. The application according to claim 3, characterized in that, The indicators related to the balance of synaptic regulatory factors are selected from one or more of Sparc, Sparcl1, Thbs1, Thbs2, and ApoE.

8. The application according to claim 3, characterized in that, Indicators related to cellular inflammation are selected from one or more of IL-1β, IL-6, and TNF-α.

9. The application according to claim 3, characterized in that, The biological clock gene is selected from one or more of Bmal1, Rev-erbα, Cry1, Cry2, Per1, and Per2.

10. A composition for improving circadian rhythm disorder and / or regulating physiological indicators related to circadian rhythm disorder, characterized in that, This includes ferulic acid and its carrier.

Images