Repairing effect of linaloe essential oil on sympathetic neurons damaged by oxidative stress

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a repairing effect of eaglewood essential oil on sympathetic neurons damaged by oxidative stress. The technical scheme of the present application firstly constructs a kind of human induced pluripotent stem cell directional differentiation sympathetic neuron by using human induced pluripotent stem cells, and then constructs a sympathetic neuron model damaged by oxidative stress by using H2O2. Subsequently, in the oxidative stress model, the present application finds that the eaglewood essential oil can effectively promote the expression of neuron markers, repair the neurite morphology, and repair the number of neuron branch points, etc. The eaglewood essential oil can also effectively regulate the Ca² + / cAMP signal pathway of the oxidative stress model.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the repair effect of agarwood essential oil on sympathetic neurons damaged by oxidative stress. Background Technology

[0002] Oxidative stress is a key pathological factor in the development of many neurological diseases. It can disrupt intracellular redox homeostasis, leading to neuronal structural damage and functional disorders. Sympathetic neurons, as an important component of the autonomic nervous system, are highly sensitive to oxidative stress, and their damage is closely related to various autonomic nervous system dysfunctions. However, current research on oxidative stress-induced damage to sympathetic neurons remains relatively limited.

[0003] Currently, the main approaches to treating sympathetic neuronal oxidative stress injury include a comprehensive strategy encompassing antioxidant therapy, neurotrophic support, and etiological control, with antioxidant therapy and etiological control being the most important. For example, alpha-lipoic acid, centrally acting antihypertensive drugs, and Nrf2 pathway agonists are used for treatment; however, these drugs typically have significant side effects and are not suitable for long-term use.

[0004] The application of aromatic essential oils in improving oxidative stress damage to sympathetic neurons has attracted attention. Existing research shows that essential oils have antioxidant and autonomic nervous system regulating functions. For example, essential oils such as lavender, rosemary, and lemongrass, and their components (such as linalool and citral), can scavenge free radicals and upregulate antioxidant enzymes such as superoxide dismutase (SOD). However, these effects are mainly broad-spectrum cytoprotective, not specifically targeting sympathetic neurons, and there is currently a lack of clinical studies that clearly demonstrate their ability to repair oxidative stress damage to sympathetic neurons.

[0005] Agarwood belongs to the genus Aquilaria of the family Thymelaeaceae ( Aquilaria Lam.) or Aquilaria genus ( GyrinopsAgarwood essential oil (AEO) is an essential oil extracted from the resinous wood of the plant *Aquilaria sinensis*. The chemical composition of AEO is highly complex and diverse. Studies have shown that AEO is mainly composed of four classes of substances: sesquiterpenes, 2-(2-phenylethyl)chromones, low molecular weight aromatic compounds, and fatty acids. As a precious natural medicine, AEO exhibits significant characteristics of "multi-component, multi-target, and multi-pathway" pharmacological activity. Currently, research on the protective effects of AEO on the sympathetic nervous system is mostly limited to traditional animal models or tumor-derived cell lines. Due to species differences and cell line heterogeneity, these models have limitations in simulating the physiological and pathological conditions of the real human nervous system. For example, research on the effects of AEO on the nervous system mainly focuses on regulating neurotransmitter and inflammatory factor levels. Due to these limitations, the proposed protective mechanisms of AEO on the nervous system are not yet in-depth, and the effective application of AEO's medicinal value is still insufficient.

[0006] In summary, a novel application of agarwood essential oil in the protection of sympathetic neurons is proposed to supplement the shortcomings of existing technologies. Summary of the Invention

[0007] The purpose of this invention is to provide the repair or protection effect of agarwood essential oil on sympathetic neurons damaged by oxidative stress, and to partially solve or alleviate the above-mentioned deficiencies in the prior art. The invention specifically adopts the following technical solution.

[0008] One aspect of this invention is to provide a new use for agarwood essential oil.

[0009] Application of agarwood essential oil in the preparation of products that repair sympathetic neurons damaged by oxidative stress.

[0010] Furthermore, the agarwood essential oil is used to promote the synthesis of key functional proteins in the sympathetic neurons damaged by oxidative stress.

[0011] Furthermore, the agarwood essential oil is used to repair the neuronal structure of the sympathetic neurons damaged by oxidative stress.

[0012] Furthermore, the agarwood essential oil is used to regulate the Ca²⁺ levels of the sympathetic neurons damaged by oxidative stress. + / cAMP signaling pathway.

[0013] Furthermore, low-dose agarwood essential oil enhances cAMP signaling pathway activity, which can be used to further prepare neuroprotective drugs for treating depressive disorders. In some specific embodiments, the low-dose agarwood essential oil is 10 μg / mL.

[0014] Furthermore, high-dose agarwood essential oil inhibits cAMP signaling pathway activity, which can be used to further prepare drugs for treating sleep disorders or anxiety-related conditions. In some specific embodiments, the high-dose agarwood essential oil is 100 μg / mL.

[0015] Furthermore, the agarwood essential oil alleviates the calcium levels of the sympathetic neurons damaged by oxidative stress. 2+ The degree of inhibition of the signaling pathway.

[0016] Furthermore, the effective concentration range of the agarwood essential oil is 10 μg / mL to 100 μg / mL.

[0017] Another aspect of the present invention aims to provide a method for evaluating the effectiveness of agarwood essential oil.

[0018] A method for evaluating the efficacy of agarwood essential oil based on a sympathetic neuron model damaged by oxidative stress includes the following steps: S01: A sympathetic neuron model constructed based on human induced pluripotent stem cells; S02: Construct a sympathetic neuron model for oxidative stress injury based on the aforementioned sympathetic neuron model; S03: Apply agarwood essential oil to the sympathetic neuron model with oxidative damage, and evaluate the effectiveness of the agarwood essential oil based on the repair status of the sympathetic neuron model after administration; The evaluation indicators include neuronal marker expression, neurite morphology and number of neuronal branch points, and intracellular calcium in sympathetic neurons. 2+ The dynamic changes in cAMP levels and / or spontaneous calcium transient kinetic parameters (including calcium transient waveforms and their key parameters—amplitude, frequency, time to peak, and decay time constant).

[0019] When the agarwood essential oil can significantly promote the expression of neuronal markers, repair the morphology of neurites, and / or repair the number of neuronal branch points, the agarwood essential oil is judged to have high medicinal value.

[0020] When the agarwood essential oil can significantly alleviate the calcium deficiency of sympathetic neurons damaged by oxidative stress... 2+ When the signaling pathway is inhibited, it is determined that the agarwood essential oil has high medicinal value.

[0021] Furthermore, the sympathetic neuron model of oxidative stress injury was constructed by adding H2O2 to induce damage.

[0022] In some specific embodiments, the dosage of agarwood essential oil applied to the oxidatively damaged sympathetic neuron model ranges from 10 μg / mL to 100 μg / mL.

[0023] The present invention may also provide another specific method for evaluating the effectiveness of agarwood essential oil.

[0024] A method for evaluating the medicinal value of agarwood essential oil based on a sympathetic neuron model damaged by oxidative stress includes the following steps: S01: A sympathetic neuron model constructed based on human induced pluripotent stem cells; S02: Construct a sympathetic neuron model for oxidative stress injury based on the aforementioned sympathetic neuron model; S03: Apply agarwood essential oil to the sympathetic neuron model of oxidative damage, and evaluate the medicinal value of agarwood essential oil based on the changes in the cAMP signaling pathway of the sympathetic neuron model after administration. When enhanced cAMP signaling pathway activity was observed, the agarwood essential oil was evaluated as potentially useful for preparing neuroprotective drugs to treat depression-related disorders. When cAMP signaling pathway activity was inhibited, the agarwood essential oil was evaluated as potentially useful in the preparation of a drug for treating sympathetic overexcitation and stress-related neurological dysfunction.

[0025] Furthermore, the indications for the aforementioned sympathetic overexcitation and stress-related neurological dysfunction include sleep disorders or anxiety.

[0026] Beneficial technical effects: This invention innovatively proposes to evaluate the reparative and medicinal value of agarwood essential oil in a model of sympathetic neurons damaged by oxidative stress. The technical solution of this invention first constructs a type of sympathetic neuron derived from human induced pluripotent stem cells through directed differentiation, and then constructs a model of sympathetic neurons damaged by oxidative stress using H2O2 (hereinafter referred to as the oxidative stress model). Subsequently, in this oxidative stress model, this invention found that agarwood essential oil can effectively promote the expression of neuronal markers, repair neurite morphology, and repair the number of neuronal branch points; it can also effectively regulate the Ca²⁺ level in the oxidative stress model. + The cAMP signaling pathway. This invention is the first to discover that low-dose agarwood essential oil enhances the activity of the cAMP signaling pathway, and this enhanced activity has a positive effect on the treatment of depressive disorders. Therefore, this invention proposes that low-dose agarwood essential oil be developed into a neuroprotective drug for the treatment of depressive disorders, which is of guiding significance. High-dose agarwood essential oil inhibits the activity of the cAMP signaling pathway. The inhibition of excessively elevated cAMP signaling has a positive effect on the treatment of sympathetic overexcitation and stress-related neurological dysfunction. Therefore, this invention proposes that high-dose agarwood essential oil be developed into a drug for the treatment of sympathetic overexcitation and stress-related neurological dysfunction (such as sleep disorders, anxiety, etc.), which is of guiding significance. In summary, this invention is the first to propose that different doses of agarwood essential oil have two opposing effects on the regulation of the cAMP signaling pathway, which is of great significance for the development of agarwood essential oil into products or drugs with medicinal value.

[0027] On the other hand, this invention also provides a method for evaluating the efficacy of agarwood essential oil based on a sympathetic neuron model damaged by oxidative stress. This method can be used to screen agarwood essential oils with medicinal value. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0029] Figure 1 This is a bright-field plot of a mature hiPSC-SN model in one embodiment of the present invention; Figure 2 This is an immunofluorescence image of a 3D hiPSC-SN in one embodiment of the present invention (scale bar is 40 μm). Figure 3 Representative immunofluorescence images and analysis results of the oxidative stress and agarwood essential oil protective effects of hiPSC-SN in one embodiment of the present invention; Figure 4 This is the result of the effect of agarwood essential oil on calcium signal dynamics in hiPSC-SN in one embodiment of the present invention; Figure 5 This is the result of the effect of agarwood essential oil on hiPSC-SN cAMP signal transduction in one embodiment of the present invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0031] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0032] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0033] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0034] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0035] Detailed descriptions of some of the attached images: Figure 3 A: Confocal microscopy images of the control group, oxidative stress model group, low-dose AEO protection group, and high-dose AEO protection group, with merged channels TH (red), PRPH (green), and Hoechst (blue); B: TH channel images of each group; C: Skeletalized images showing neurites and branching points of each group; D: Average TH fluorescence intensity per field of view; E: Average neurite length per neuron; F: Number of branching points per neuron. Images are magnified 40 times, scale bar: 40 µm (A, B, C), normalized to 100% based on the control group (D, E, F).

[0036] Figure 4 A: Representative normalized intracellular calcium curve of hiPSC-SN; B: Calcium transient amplitude results for each group.

[0037] Figure 5 A: Representative normalized cAMP fluorescence resonance energy transfer curves of hiPSC-SN; B: cAMP signal transduction results for each group.

[0038] Definition of noun: AEO: Agarwood essential oil.

[0039] TH: Tyrosine hydroxylase.

[0040] PRPH: Peripherin.

[0041] Hoechst: A membrane-permeable fluorescent dye that can penetrate cell membranes.

[0042] hiPSC-SN: Human induced pluripotent stem cell-sympathetic neuron, a neuron derived from the directed differentiation of human induced pluripotent stem cells.

[0043] cAMP: Cyclic adenosine monophosphate.

[0044] The main materials involved in this invention are: The human induced pluripotent stem cell (hiPSC) cell line used in this invention was obtained by reprogramming dermal fibroblasts from healthy subjects. The healthy hiPSC cell line iPS-OX1-19 used for sympathetic neuron induction was obtained from Oxford Parkinson's Centre. The acquisition and use of cell samples from subjects were approved. All related experimental procedures strictly followed the relevant guidelines and regulations of the ethics committee.

[0045] The agarwood essential oil (AEO) used in this study was extracted from artificially cultivated agarwood. After inducing the production of agarwood using the whole-body resin formation technique, AEO was obtained by steam distillation and used in subsequent experiments.

[0046] Preparation of agarwood essential oil mother liquor: Take an appropriate amount of AEO and filter it using a 0.22 μm microporous membrane. Mix the filtered AEO with an equal volume of sterile DMSO in a sterile centrifuge tube, and homogenize the mixture using a vortex mixer until the two are fully mixed and homogeneous, finally obtaining an AEO stock solution with a concentration of 500 mg / mL.

[0047] After the prepared AEO mother liquor is sealed with sealing film, it is stored at room temperature away from light for later use.

[0048] Example 1 Culture of human induced pluripotent stem cells and differentiation of sympathetic neurons Experimental methods: Cell model construction: Refer to Winbo A, Ramanan S, Eugster E, et al. Functional coculture of sympathetic neurons and cardiomyocytes derived from human- induced pluripotent stem cells[J]. American Journal of Physiology-Heart and Circulatory Physiology, 2020, 319(5): H927-H937 The method utilizes hiPSCs to achieve directed differentiation into sympathetic neurons (hiPSC-SN) through in vitro directed differentiation technology.

[0049] (1) After three consecutive passages, hiPSCs were seeded at an appropriate density in Matrigel-coated 12-well plates and routinely maintained using mTeSR medium. When the cell confluence reached approximately 80%, small molecule compounds (see Table 1) were sequentially added to the medium to regulate multiple signaling pathways, including Wnt, TGF-β, Hedgehog, AKT, Notch, and BMP, thereby inducing the production of neural mesodermal progenitor cells and neural crest cells.

[0050] (2) Starting from day 4 of differentiation, mTeSR medium was gradually replaced with N2 medium to promote the development of sympathetic neurons. N2 medium consisted of: Neurobasal Plus medium, 2 mM L-glutamine, B-27 Plus, N-2 supplements, 0.2 mM ascorbic acid, 0.2 nM dbcAMP, 10 ng / mL NGF, 10 ng / mL BDNF, and 10 ng / mL GDNF. On day 12 of differentiation, cells were collected by Accutase digestion at a concentration of 1×10⁻⁶. 6 Re-seed at a density of cells / mL in 12-well plates.

[0051] (3) On day 12 of differentiation, the cells were dissociated into a single-cell suspension using Accutase. The cells were then introduced at a density of 2000-3000 cells per well (approximately 3 × 10⁶ cells per well for a 24-well plate). 6 HiPSC-SN cells were seeded in AggreWell 800 microplates, centrifuged at 200 × g for 5 min, and then incubated at 37°C in a 5% CO2 incubator. After 24 h of incubation, the ROCK inhibitor (10 µM Y-27632) was removed, and the cells were cultured for another 24–48 h to promote neural microtissue maturation. The matured hiPSC-SN cells were used for subsequent experiments.

[0052] Table 1 Information on small molecule compounds used for hiPSC-SN differentiation To further verify that the cells differentiated from hiPSCs possess the characteristics of sympathetic neurons, immunofluorescence staining was performed to detect the expression of neuronal-related biomarkers. The biomarkers selected included TH and PRPH.

[0053] The results are as follows Figure 1 and Figure 2 As shown. The obtained hiPSC-SN model is shown in [the image / document]. Figure 1As shown in the figure, the differentiated hiPSC-SNs are uniformly attached to the bottom surface of the culture medium, and the cells form a dense neural-like network structure through protrusions, initially exhibiting the typical morphological characteristics of peripheral neurons.

[0054] Figure 2 The study revealed significant positive signals of TH and PRPH in the 3D sympathetic nerve microtissue. PRPH formed a dense cellular scaffold, with nerve fibers interwoven into a network; TH was expressed in both the cell bodies within the neurosphere and in the outward-growing nerve bundles. The fluorescence signal exhibited a strong sense of spatial hierarchy, not only present within the cell clusters but also distributed along the outward-extending neurites, displaying a relatively complex three-dimensional neural network structure. This demonstrates that the hiPSCs in this experiment have successfully differentiated into sympathetic neurons.

[0055] Example 2 Establishment of a hiPSC-SN oxidative stress and agarwood essential oil protection model Experimental methods: An oxidative stress injury and AEO protection model was established in the hiPSC-SN model constructed in Example 1. Specific grouping and treatment protocols are shown in Table 2. After the hiPSC-SN cells matured and reached the required experimental density, they were subjected to appropriate drug treatments and incubated at 37°C in a 5% CO2 incubator for 48 h. Cells were then collected for subsequent experiments.

[0056] Table 2 Grouping and Drug Administration To identify the effects of H2O2 and AEO intervention on the morphology and biomarker expression of sympathetic neurons, and thus assess the damaging effects of oxidative stress on sympathetic neurons and the protective effect of AEO, immunofluorescence staining was performed on cells in each group according to standard experimental procedures. Fiji (Image J) software was used for quantitative analysis of the stained images. The number of neurons in each field of view was determined by counting Hoechst-positive cell nuclei co-localized with TH-positive cell bodies; TH-positive cell bodies after background subtraction were used as regions of interest (ROIs), and the average fluorescence intensity of TH was measured; TH or PRPH channel images were thresholded and binarized, and skeletonization analysis was performed using the AnalyzeSkeleton plugin to quantify the total protrusion length and branch point number of each neuron. Data for each group were first averaged within a single image, and then aggregated for inter-group difference analysis.

[0057] See results Figure 3 . Figure 3 A shows the morphology of each hiPSC-SN group and the details of its network layer under a confocal microscope. Figure 3(B, D) show that in the oxidative stress model group, the average TH fluorescence intensity of cells was significantly reduced, reaching the lowest level among all groups. This indicates that 200 μM H2O2-induced oxidative stress significantly reduced the expression level of TH protein, resulting in a significant inhibitory effect on the functional phenotype of sympathetic neurons. In contrast, when AEO was added to treat the oxidative stress model, both concentrations of AEO could restore TH fluorescence intensity to some extent, showing a significant recovery trend compared to H2O2 treatment alone. This suggests that AEO may partially alleviate the damage to key functional proteins of sympathetic neurons by reducing oxidative stress damage, and has a potential protective effect on the functional integrity of sympathetic neurons. Figure 3 (C, E) show that in the oxidative stress model group, the average neurite length of a single neuron was significantly shortened, significantly lower than that in the control group, indicating that oxidative stress leads to damage and collapse of the neuronal cytoskeleton structure and causes neurite retraction. In contrast, when combined with AEO treatment, neurite length showed a significant recovery, exhibiting a certain dose-dependent trend. Specifically, high concentrations of AEO largely inhibited neurite retraction, restoring the average neurite length to near the control group level, suggesting that AEO has a certain protective and restorative effect on oxidative stress-induced neurite structural damage. Figure 3 (F) shows that the number of branch points in the oxidative stress model group was significantly less than that in the control group, reflecting the reduced complexity of the neuronal network caused by oxidative stress. In contrast, when combined with AEO, both low-dose and high-dose AEO could partially restore the branching ability of neurons, with the high-dose AEO group showing a more significant effect, restoring the complexity of neuronal dendritic branching to a level close to that of the control group, further confirming the protective effect of AEO on the structure and network integrity of sympathetic neurons.

[0058] Example 3 Agarwood essential oil on Ca 2+ The influence of the cAMP signaling pathway In the regulation of neuronal function, the intracellular second messenger system plays a central role. Among them, calcium ions (Ca) 2+ α-Cyclic adenosine monophosphate (cAMP) and cyclic adenosine monophosphate (cAMP) are the two most crucial signaling molecules, participating in the regulation of various physiological processes such as neuronal excitability, synaptic transmission, and gene expression. 2+ The signaling pathway primarily undergoes dynamic changes through voltage-gated calcium channels and intracellular calcium storage release; its abnormal fluctuations are closely related to neurodegenerative diseases and stress injury. Meanwhile, the cAMP signaling pathway participates in regulating cellular metabolism, ion channel activity, and transcriptional regulation through the synergistic regulation of adenylate cyclase (AC) and phosphodiesterase (PDE). Previous studies have shown that Ca... 2+There is a complex interactive regulatory relationship between cAMP signaling and cAMP signaling, which together constitute the intracellular signaling network and are of great significance for maintaining neuronal functional homeostasis.

[0059] Experimental methods: 1. Calcium imaging Intracellular calcium concentration in each model constructed in Example 2 was detected using the live-cell calcium indicator Fura-2 AM. hiPSC-SN cells differentiated to approximately day 35 were re-seeded onto glass coverslips, and calcium imaging experiments were performed between days 40 and 65 of differentiation. During the experiments, the coverslips were placed in 5 μM Fura-2 AM and incubated at 37°C for 30 min. Subsequently, the coverslips were washed three times with Tyrode solution. The composition of the Tyrode solution is shown in Table 3, and the pH of the solution was adjusted to 7.4 with NaOH.

[0060] The temperature of the perfusion system was set at 37°C using a temperature controller. After the system reached equilibrium, the coverslip was transferred to the perfusion chamber, and Tyrode solution was used for continuous perfusion at a flow rate of 2 mL / min. The imaging system consisted of an inverted microscope equipped with a 40× oil immersion objective and a CCD camera.

[0061] After recording began, a stable baseline signal was first acquired. Then, 1 μM acetylcholine (ACh) was rapidly applied via the perfusion system to acutely stimulate neurons and induce calcium transients. Once the fluorescence signal reached its peak, the perfusion solution was immediately switched back to Tyrode solution. During the experiment, the excitation light was alternated between 340 nm and 380 nm to acquire the emission fluorescence signal.

[0062] After subtracting background fluorescence intensity, the intracellular calcium response level was quantified as a 340 / 380 ratio and normalized relative to the baseline. To ensure data quality, signal values ​​with a signal-to-noise ratio <3, unstable baseline before stimulation (coefficient of variation >20%), and coverslip samples with a neuron ratio below 20% were discarded.

[0063] Table 3. Composition and concentration of Tyrode solution. 2. cAMP fluorescence resonance energy transfer Using Epac-S H187 A high-sensitivity cAMP sensor was used to monitor intracellular cAMP levels in hiPSC-SN cells in real time. Before the experiment, monolayer neurons seeded on glass-bottomed culture dishes were combined with cells carrying Epac-S... H187The adenovirus vector encoding the sequence was incubated overnight at 37°C with 5% CO2 for transfection. The viral volume was adjusted according to the number of cells. After transfection, cells were cultured for another 2-3 days to ensure adequate expression of the FRET sensor within the cells.

[0064] FRET measurements were performed in Tyrode solution. Cells were immersed in Tyrode solution to reach a stable physiological baseline state before stimulation was applied.

[0065] The imaging system employed an inverted microscope equipped with a 40× oil immersion objective, connected to a Cairn Research dual-channel OptoLED light source and a Prime BSI optical camera. During the experiment, excitation was performed every 15 seconds (exposure time 100 ms). The excitation wavelength was set to 430 nm, and the emission fluorescence intensity at 480 nm (blue-green fluorescent protein) and 535 nm (yellow fluorescent protein) was recorded simultaneously.

[0066] To induce cAMP production, hiPSC-SN was stimulated with the cAMP agonist Forskolin (FSK). Before the end of recording, 25 μM FSK and 100 μM nonselective phosphodiesterase inhibitor IBMX were used in combination to induce intracellular cAMP saturation.

[0067] After subtracting background fluorescence, the FRET ratio was calculated as the ratio of CFP emission intensity to YFP emission intensity (CFP / YFP). To facilitate comparisons between groups, the original ratios were normalized relative to the baseline mean ratio (R0). Drug-induced changes in cAMP were expressed as a percentage of saturated cAMP levels (i.e., under FSK+IBMX), and peak differences after stimulation were compared between groups.

[0068] The effect of agarwood essential oil on calcium signaling dynamics in hiPSC-SN is shown in Figure 4 .like Figure 4 As shown in Figure A, all groups of sympathetic neurons exhibited significant calcium transient amplitude responses after ACh stimulation. This result is consistent with the cholinergic sensitivity shown by hiPSC-SN, indicating that it possesses mature neuronal functional characteristics. In this embodiment, the strong calcium response amplitudes exhibited by each group of sympathetic neurons indicate that the model differentiation state is stable and can effectively reflect the physiological function of the sympathetic nervous system. Under H2O2-induced oxidative stress conditions, the calcium of sympathetic neurons... 2+ The transient amplitude was significantly lower than that of the control group (see...) Figure 4 B) suggests that oxidative stress inhibits neuronal calcium signaling. This phenomenon may be related to impaired function of voltage-gated calcium channels or intracellular calcium... 2+This is related to buffer system disorder, which weakens stimulus-induced Ca2+. 2+ The influx capacity was further verified to validate the reliability of the model in simulating oxidative damage. Subsequently, the protective effect of AEO against oxidative stress damage was evaluated. Results showed that under AEO intervention conditions, the Ca2+ levels in the 10 μg / mL and 100 μg / mL treatment groups were significantly reduced. 2+ The transient amplitude showed a certain upward trend compared to the H2O2 group, and overall approached the control group level. This indicates that AEO can alleviate the effects of oxidative stress on Ca to some extent. 2+ The inhibition of signaling pathways may work by protecting calcium channel function or regulating intracellular calcium homeostasis.

[0069] The effect of agarwood essential oil on hiPSC-SN cAMP signal transduction is shown in Figure 5 . Figure 5 As shown in Figure A, after FSK stimulation, all groups showed a significant increase in cAMP levels, indicating that cAMP was effectively activated and the cAMP signal increased. After combined use of FSK and IBMX at the end of the recording period, the cAMP level further increased and tended to saturate, verifying the reliability of the sensor response in the hiPSC-SN model and the functional integrity of the cAMP production and degradation mechanism. Under H2O2-induced oxidative stress, the cAMP signal of hiPSC-SN changed significantly. Figure 5 As shown in Figure B, compared with the control group, the cAMP levels of sympathetic neurons in the oxidative stress model group were significantly increased after FSK stimulation. This result indicates that oxidative stress can alter the regulatory state of the intraneuronal cAMP signaling pathway, possibly by affecting the activity of AC or inhibiting the PDE-mediated cAMP degradation process, thereby leading to enhanced cAMP accumulation and disrupting the intraneuronal cAMP homeostasis.

[0070] Co-treatment with AEO significantly regulated the abnormal increase in cAMP induced by oxidative stress, with different concentrations of AEO exhibiting dose-dependent regulation of cAMP signaling. In the low-dose (10 µg / mL) AEO treatment group, the increase in cAMP was further increased compared to the oxidative stress group, indicating that low-dose AEO may enhance the activity of the cAMP signaling pathway; enhanced cAMP signaling pathway activity has a positive effect on the treatment of depressive disorders. Conversely, the high-dose (100 µg / mL) AEO treatment group effectively inhibited excessive cAMP increases, bringing the overall level close to that of the control group; the inhibition of excessive cAMP signaling pathway increases has a positive effect on the treatment of sympathetic hyperexcitability and stress-related neurological dysfunction. These results suggest that AEO has a dose-dependent bidirectional regulatory effect on cAMP signaling, potentially restoring intracellular signal homeostasis disrupted under oxidative stress by regulating the dynamic balance between cAMP production and degradation.

[0071] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0072] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. Application of agarwood essential oil in the preparation of products for repairing sympathetic neurons damaged by oxidative stress.

2. The application as described in claim 1, characterized in that, The agarwood essential oil is used to promote the synthesis of key functional proteins in the sympathetic neurons damaged by oxidative stress.

3. The application as described in claim 1, characterized in that, The agarwood essential oil is used to repair the neuronal structure of the sympathetic neurons damaged by oxidative stress.

4. The application as described in claim 1, characterized in that, The agarwood essential oil is used to regulate the Ca²⁺ levels of the sympathetic neurons damaged by oxidative stress. + / cAMP signaling pathway.

5. The application as described in claim 4, characterized in that, Low-dose agarwood essential oil enhances the activity of the cAMP signaling pathway.

6. The application as described in claim 4, characterized in that, High doses of agarwood essential oil inhibit the activity of the cAMP signaling pathway.

7. The application as described in claim 4, characterized in that, The agarwood essential oil alleviates the calcium levels of the sympathetic neurons damaged by oxidative stress. 2+ The degree of inhibition of the signaling pathway.

8. The application as described in any one of claims 1-7, characterized in that, The effective concentration range of the agarwood essential oil is 10 μg / mL to 100 μg / mL.

9. A method for evaluating the efficacy of agarwood essential oil based on a sympathetic neuron model damaged by oxidative stress, characterized in that, Includes the following steps: S01: A sympathetic neuron model constructed based on human induced pluripotent stem cells; S02: Construct a sympathetic neuron model for oxidative stress injury based on the aforementioned sympathetic neuron model; S03: Apply agarwood essential oil to the sympathetic neuron model with oxidative damage, and evaluate the effectiveness of the agarwood essential oil based on the repair status of the sympathetic neuron model after administration; The evaluation indicators include neuronal marker expression, neurite morphology and the number of neuronal branch points, and / or intracellular calcium in sympathetic neurons. 2+ The dynamic changes in cAMP levels.

10. The method as described in claim 9, characterized in that, The sympathetic neuron model of oxidative stress injury was constructed by adding H2O2 to induce damage.

Images