Use of shihubain and / or maoa in the treatment of depression

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to the application of dendrobium alkaline and / or pinoecorine in the treatment of depression. Specifically, the present application proves through experiments that dendrobium alkaline and pinoecorine can improve LPS-induced depressive behavior, and reduce the level of oxidative stress and neuroinflammatory response by targeting PDE4B to inhibit the activation of the TLR4 / PI3K / AKT / NF-κB pathway, thereby reversing the damage of neurotoxicity, apoptosis abnormality and synaptic plasticity to relieve symptoms. Dendrobium alkaline improves the intestinal flora disorder, and acts on the central nervous system through the microbe-gut-brain axis, so that the anti-depression effect of dendrobium alkaline is better than that of pinoecorine. The present application opens up a new drug use for dendrobium alkaline and pinoecorine, and lays an experimental foundation and provides a new perspective for the development of efficient drugs for treating depression.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of dendrobine and / or dendrobine in the treatment of depression. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Major depressive disorder (MDD) is a neurological disorder characterized primarily by persistent spontaneous mood disturbances. Its pathogenesis is complex, involving neuroinflammation, oxidative stress, synaptic damage, and gut microbiota. Adolescent depression has become a hot research topic in recent years. Previous studies have shown that many antidepressants have low cure rates and various side effects, such as potential liver and kidney damage, highlighting the urgent need for new therapies for depression. Currently, the hypotheses regarding the pathogenesis of adolescent depression mainly involve neuroinflammation, oxidative stress, and the kynurenine pathway. Therefore, improving the levels of oxidative stress and neuroinflammation in the brain may be key to treating adolescent depression.

[0004] Dendrobium officinale is renowned for its significant medicinal value and has been widely used to regulate immune responses and enhance gastrointestinal function. The main chemical components of Dendrobium officinale include polysaccharides, alkaloids, terpenoids, flavonoids, dibenzyl groups, and phenanthrene. Among the alkaloids of Dendrobium officinale, dendrobine is its main bioactive component. Numerous studies have shown that dendrobine possesses pharmacological activities, including anti-inflammatory and anticancer properties. Dendrobine, another dibenzyl component of Dendrobium officinale, has been reported to have anti-inflammatory and protective effects in various diseases. Furthermore, traditional Chinese medicine can exert its effects by regulating the gut microbiota, which can influence the central nervous system through the gut-microbe axis. Increasing evidence suggests that imbalances in the gut microbiota may affect brain function and behavior and may play an important role in the development of depression. However, whether dendrobine and dendrobine can improve adolescent depressive-like behaviors by regulating the gut microbiota remains unclear. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of dendrobine and / or dendrobine in the treatment of depression. Specifically, this invention demonstrates through experiments that dendrobine and dendrobine can improve LPS-induced depressive-like behavior and alleviate symptoms by targeting PDE4B to inhibit the activation of the TLR4 / PI3K / AKT / NF-κB pathway, thereby reducing oxidative stress levels, neuroinflammatory responses, and reversing neurotoxicity, apoptosis abnormalities, and synaptic plasticity disruption. Dendrobine, by improving gut microbiota dysbiosis, acts on the central nervous system through the gut-microbe axis, resulting in a superior antidepressant effect compared to dendrobine. Based on the above research findings, this invention is thus completed.

[0006] Specifically, the present invention relates to the following technical solutions:

[0007] A first aspect of the invention provides the use of dendrobine and / or dendrobine in the preparation of a drug for treating depression.

[0008] In this invention, the depression can be severe depression, or more specifically, adolescent depression.

[0009] The treatment of depression is manifested in having at least one or more of the following effects:

[0010] a) Alleviate LPS-mediated depressive-like behaviors;

[0011] b) Improves LPS-mediated neuronal damage, disruption of synaptic plasticity, and abnormal apoptosis;

[0012] c) Reduce LPS-mediated neuroinflammation, glial cell activation, and elevated oxidative stress levels;

[0013] d) Target PDE4B to inhibit the TLR4 / PI3K / AKT / NF-κB pathway;

[0014] e) Alleviate LPS-mediated intestinal pathological damage;

[0015] f) Improve LPS-mediated gut microbiota imbalance.

[0016] According to the present invention, not only is the use of dendrobine and / or dendrobine in the preparation of antidepressant drugs disclosed, but it is also disclosed that this effect can be enhanced when dendrobine and / or dendrobine is administered in combination with at least one other pharmaceutically active ingredient. Dendrobine and / or dendrobine can also be used in combination with other non-pharmaceutical active ingredients as a substitute or supplement to other pharmaceutically active ingredients.

[0017] A second aspect of the present invention provides a pharmaceutical composition for treating depression, said pharmaceutical composition comprising dendrobine and / or dendrobine with at least one other pharmaceutically active ingredient and / or at least one other non-pharmaceutical active ingredient.

[0018] A third aspect of the present invention provides a method for treating depression, the method comprising: administering to a subject the above-mentioned dendrobine and / or dendrobine or the above-mentioned pharmaceutical composition.

[0019] The subjects can be humans or non-human mammals, including mice, rats, guinea pigs, pigs, dogs, monkeys, and chimpanzees.

[0020] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0021] The above-mentioned technical solutions have been experimentally demonstrated that dendrobine and dendrobine, the active components of the traditional Chinese medicine Dendrobium officinale, can effectively alleviate lipopolysaccharide (LPS)-induced depressive-like behavior in adolescent mice, and the optimal dosage of each component has been determined. LPS treatment leads to aggravated damage to hippocampal and prefrontal neurons, significant synaptic structural damage, and abnormal apoptosis in mice, while treatment with dendrobine and dendrobine effectively improved these results. RNA-seq results show that dendrobine and dendrobine can target phosphodiesterase 4B (PDE4B), thereby alleviating the activation of the TLR4 / PI3K / AKT / NF-κB pathway, resisting the increase in oxidative stress levels, glial cell activation, and excessive release of inflammatory factors in the mouse brain, and improving depressive-like behavior in adolescent mice. Furthermore, we found that dendrobine treatment was superior to daunoside. Combining intestinal phenotype and 16S rRNA-seq results, dendrobine treatment effectively improved LPS-induced intestinal flora dysbiosis and increased intestinal permeability caused by intestinal epithelial cell damage in mice, indicating that dendrobine can further improve anxiety and depression symptoms through the microbe-gut-brain axis.

[0022] In summary, the above-mentioned technical solutions open up new drug applications for dendrobine and dendrobine, and also lay an experimental foundation and provide new perspectives for the development of highly effective drugs for treating depression-related diseases. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1In this embodiment of the invention, dendrobine and dendrobine alleviated LPS-induced anxiety-depression-like behavior in adolescent mice. (A) Schematic diagram of the experimental process (8 male mice in each group of the behavioral experiment). (B) Weight change over 10 consecutive days. (C) Percentage of time spent in the central area of ​​OFT. (D) Total distance moved in OFT. (E) Movement trajectory in OFT. (F) Time spent on the open arm of EPM. (G) Movement trajectory of EPM. (H) SPT sucrose preference (%). (I) FST resting time (s). (J) TST resting time (s). *p<0.05,**p<0.01,***p<0.001, compared with the LPS group; compared with the CON group, #p<0.05,##p<0.01,###p<0.001.

[0025] Figure 2 In this embodiment of the invention, dendrobine and dendritic alkaloids can improve LPS-induced neuronal damage, synaptic plasticity disruption, and apoptosis abnormalities. (A, B) Western blot analysis of Nestin, NeuN, PSD95, and Syn protein expression in HIP compared to β-actin (n=4 per group). (C) HE and Nissl staining of HIP (DG and CA3 regions) and PFC (scale bar = 100 μm). (D) Representative images of the Golgi apparatus and dendritic spine density statistics (n=5 per group) (scale bar = 10 μm). (E) Immunofluorescence staining of NeuN expression in HIP DG and CA3 regions (n=3 per group) (scale bar = 100 μm). (F, G) Transmission electron microscopy observation of the morphology of heterochromatin (10000x) and synapses (30000x and 50000x), and the number of synapses in each group (n=3 per group). (H) EdU experimental procedure. (I) Representative fluorescence images of the HIP DG region and quantitative analysis of EdU (n=3 per group) (scale bar=100μm). *p<0.05,**p<0.01,***p<0.001, compared with the LPS group; compared with the CON group, #p<0.05,##p<0.01,###p<0.001.

[0026] Figure 3In this embodiment of the invention, dendrobine and dendrobine can alleviate LPS-induced neuroinflammation, glial cell activation, and elevated oxidative stress levels. (A) qPCR detection of IFN-γ, IL-1β, and IL-6 mRNA expression levels in HIP tissue (n=5 per group). (B, D) Western blot analysis of TNF-α and GFAP protein expression in HIP compared with β-actin (n=4 per group). (C, E) Western blot analysis of TNF-α and GFAP protein expression in PFC compared with β-actin (n=4 per group). (F) Representative fluorescence images and TNF-α quantitative analysis of the HIP DG region (n=3 per group) (scale bar=100μm). (G) Representative fluorescence images and quantitative analysis of Iba-1 in the HIP DG region (n=3 per group) (scale bar=100μm). (H) Representative fluorescence images and quantitative analysis of GFAP in the HIP DG region (n=3 per group) (scale bar=100μm). (I) Shore analysis of microglia morphology (n=3 per group). *p<0.05,**p<0.01,***p<0.001, compared with the LPS group; compared with the CON group, #p<0.05,##p<0.01,###p<0.001.

[0027] Figure 4 In this embodiment of the invention, dendrobine and dendrobine reduce depressive-like behavior by downregulating TLR4 expression, inhibiting the activation of the PI3K / AKT / NF-κB pathway. (A) Multi-component differential scatter plot showing differentially expressed genes in the CON, LPS, dendrobine, and dendrobine groups. (BD) KEGG pathway enrichment analysis (key pathways are marked with red boxes) (p<0.05). (E) Enrichment and classification of differentially expressed genes screened from the GO database. (F) Gene trend analysis, with two significant differential groups being profile5 and profile14 (the inflection points from left to right represent gene expression in the CON, LPS, dendrobine, and dendrobine groups). (G) Differential Venn diagram. (H) Gene heatmap showing 12 neurally related genes that are differentially expressed in the four groups. (I) qPCR detection of Hck, Lcn2, Csf1r, CYBB, and TLR4 mRNA expression levels in HIP (n=5 per group). (J) Western blot analysis of TLR4, p-PI3K, PI3K, p-AKT, AKT, p-P65, and P65 protein expression in HIP compared to β-actin (n=4 per group). (K) Correlation analysis of TLR4 protein expression with behavioral data and inflammatory cytokine protein expression. *p<0.05,**p<0.01,***p<0.001, compared with the LPS group; compared with the CON group, #p<0.05,##p<0.01,###p<0.001.

[0028] Figure 5 In this embodiment of the invention, dendrobine and dendrobine inhibit the TLR4 / PI3K / AKT / NF-κB pathway by targeting PDE4B. (A) PhamMapper and GeneCards screened potential target proteins of dendrobine and dendrobine. (B) Western blot analysis of PDE4B protein expression in HIP compared to β-actin (n=4 per group). (C, D) Molecular docking results of dendrobine, dendrobine, and PDE4B (minimum binding energy marked). (E) CETSA model diagram. (F, G) CETSA test results (n=3 per group). (H) Correlation analysis of PDE4B protein expression with p-P65, p-PI3K, and p-AKT protein expression. *p<0.05,**p<0.01,***p<0.001, compared with the LPS group; compared with the CON group, #p<0.05,##p<0.01,###p<0.001.

[0029] Figure 6 This invention illustrates how dendrobine alleviates LPS-induced intestinal pathological damage. (A) Fecal water percentage (%) (n=3 per group). (B) Colon length (n=3 per group). (C, D) ELISA detection of IL-1β and TNF-α expression in colon, serum, and HIP (n=5 per group). (EG) qPCR detection of colonic ZO-1, Claudin-1, and Occludin mRNA expression levels (n=5 per group). (H, I) Western blot analysis of colonic ZO-1 protein and β-actin expression (n=4 per group). (J) Representative fluorescence images and quantitative analysis of ZO-1 in the colon (n=3 per group) (scale bar = 100 μm). (K) HE staining showing colonic tissue morphology and tissue damage score in each group (scale bar = 100 μm). *p<0.05,**p<0.01,***p<0.001, compared with the LPS group; compared with the CON group, #p<0.05,##p<0.01,###p<0.001. Figure 7In this embodiment of the invention, dendrobine can alleviate LPS-induced gut microbiota imbalance. (A) Chao1 index (n=5 per group). (B) Shannon index (n=5 per group). (C) Sob index (n=5 per group). (D) Simpson dilution curve (n=5 per group). (E) Overlapping OTUs among the three groups (n=5 per group). (F) Principal component analysis (PCoA) of fecal microbial community pattern. (G, H) Relative abundance of gut bacteria at the phylum and genus levels. (I) Circos plot reflects the proportion of dominant species composition in each sample and the distribution ratio of each dominant species among groups. (J) Linear discriminant analysis (LDA) and (LEfSe) analysis (LDA>3.2) were used to determine the differential classification of different groups of microorganisms and the LEfSe multi-level species evolution map. (K) Intergroup correlation analysis (the horizontal axis represents key differentially expressed microbial groups, the vertical axis represents the data of the above experimental results, and blue to red represents the correlation from -1 to 1). Detailed Implementation

[0030] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0031] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0032] The present invention will be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the selling company. Materials and reagents used in the examples, unless otherwise specified, are commercially available.

[0033] In a typical embodiment of the present invention, the use of dendrobine and / or dendrobine in the preparation of a drug for treating depression is provided.

[0034] According to the present invention, the concept of "treatment" means any relevant measures applicable to depression, or preventive treatment of the disease or its symptoms, or prevention of recurrence of the disease, such as recurrence after the end of a treatment period or treatment of symptoms of an already occurring disease.

[0035] Specifically, the depression referred to is severe depression, and further, it can be adolescent depression.

[0036] More specifically, the depression is mediated by lipopolysaccharide (LPS).

[0037] The treatment of depression is manifested in having at least one or more of the following effects:

[0038] a) Alleviate LPS-mediated depressive-like behaviors;

[0039] b) Improves LPS-mediated neuronal damage, disruption of synaptic plasticity, and abnormal apoptosis;

[0040] c) Reduce LPS-mediated neuroinflammation, glial cell activation, and elevated oxidative stress levels;

[0041] d) Target PDE4B to inhibit the TLR4 / PI3K / AKT / NF-κB pathway;

[0042] e) Alleviate LPS-mediated intestinal pathological damage;

[0043] f) Improve LPS-mediated gut microbiota imbalance.

[0044] Specifically, in b), improving LPS-mediated neuronal damage, synaptic plasticity disruption, and apoptosis abnormalities manifests as follows: restoring LPS-induced reductions in NeuN, Nestin, PSD95, and Syn; alleviating LPS-induced neuronal morphological damage and abnormalities; mitigating LPS-induced decreases in dendritic spine density and the number of NeuN-positive cells; mitigating LPS-induced increases in heterochromatin and decreases in the number of synapses; and mitigating the reduction in EdU-positive cells in the hippocampal DG region.

[0045] In c), the reduction of LPS-mediated neuroinflammation, glial cell activation and oxidative stress levels is specifically manifested in: reversing LPS-induced increases in IFN-γ, IL-1β and IL-6 expression levels; alleviating LPS-induced increases in GFAP and TNF-α; reducing LPS-induced increases in the number of microglia and astrocytes; and inhibiting LPS-induced increases in microglia cell body size and branching complexity.

[0046] In e), the specific manifestations of intestinal pathological damage are: significant destruction of the intestinal mucosa, sparse villi, accompanied by epithelial erosion, crypt deformation, and loss of colonic crypts and goblet cells.

[0047] According to the present invention, not only is the use of dendrobine and / or dendrobine in the preparation of antidepressant drugs disclosed, but it is also disclosed that this effect can be enhanced when dendrobine and / or dendrobine is administered in combination with at least one other pharmaceutically active ingredient. Dendrobine and / or dendrobine can also be used in combination with other non-pharmaceutical active ingredients as a substitute or supplement to other pharmaceutically active ingredients.

[0048] In another specific embodiment of the present invention, a pharmaceutical composition for treating depression is provided, the pharmaceutical composition comprising dendrobine and / or dendrobine with at least one other pharmaceutical active ingredient and / or at least one other non-pharmaceutical active ingredient.

[0049] The non-pharmaceutical active ingredient may be a pharmaceutically acceptable carrier or excipient, including pharmaceutically acceptable materials, compositions, or carriers suitable for administering the compounds of the present invention to mammals. The carrier includes liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials that participate in carrying the main substance or transferring it from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of compatibility with other components in the formulation and harmlessness to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers or excipients include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate, powdered tragacanth gum, malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; buffers such as magnesium hydroxide and aluminum hydroxide; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0050] The pharmaceutical composition may also contain wetting agents, emulsifiers and lubricants such as sodium dodecyl sulfate and magnesium stearate, as well as colorants, releasing agents, coating agents, sweeteners, flavoring agents and aroma agents, preservatives and antioxidants.

[0051] In another specific embodiment of the present invention, a method for treating depression is provided, the method comprising: administering to a subject the above-mentioned dendrobine and / or dendrobine or the above-mentioned pharmaceutical composition.

[0052] The subjects can be humans or non-human mammals, including mice, rats, guinea pigs, pigs, dogs, monkeys, and chimpanzees.

[0053] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0054] Example

[0055] 1. Experimental Methods

[0056] 1.1 Animal Model

[0057] All animal care and experiments were conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals" and approved by the Institutional Animal Care and Use Committee of Shandong University. Four-week-old male C57BL / 6 mice (weighing 18-20g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals were housed under standard laboratory conditions in ventilated cages with a 12-hour light-dark cycle (8:00 AM and 8:00 PM), and with an adequate food-water supply (22±1℃) at a specific pathogen-free environment and room temperature. Mice were randomly divided into 8 groups (n=8 per group): control group (mice treated with saline), LPS group (mice treated with LPS, 1 mg / kg, ip), dendrobine group 5 (mice treated with both LPS and dendrobine, 5 mg / kg, ig), dendrobine group 10 (mice treated with both LPS and dendrobine, 10 mg / kg, ig), dendrobine group 20 (mice treated with both LPS and dendrobine, 20 mg / kg, ig), dendrobine group 2.5 (mice treated with both LPS and dendrobine, 2.5 mg / kg, ig), dendrobine group 5 (mice treated with both LPS and dendrobine, 5 mg / kg, ig), and dendrobine group 10 (mice treated with both LPS and dendrobine, 10 mg / kg, ig). LPS was obtained from *Escherichia coli* strain O111:B4, Sigma, USA, #L2630, and dissolved in 0.9% saline. From day 1 to day 7, mice in the control and LPS groups were given saline (8:00-9:00 AM), while mice in the dendrobine and dendrobine groups were given dendrobine and dendrobine, respectively. Additionally, from day 3 to day 5, two hours after the first gavage, mice in the control group were given saline again, while the other seven groups were intraperitoneally injected with LPS (1 mg / kg). Behavioral tests were performed over the next seven days. Mice were then sacrificed, and samples were collected for subsequent experiments.

[0058] 1.2 Behavioral Testing

[0059] The animal behavioral tests were conducted in the following order: Open Field Test (OFT), Elevated Cross Maze (EPM), Suspended Tail Test (TST), Forced Swimming Test (FST), and Sucrose Preference Test (SPT). These tests were performed in a dedicated room isolated from external noise. The TopScan 3.0 tracking system was used to record, measure, and analyze the behavioral experiments.

[0060] 1.2.1 Open Field Experiment (OFT)

[0061] Mice were placed in an activity box measuring 35cm × 35cm × 25cm, with a central area measuring 20cm × 20cm. Mice were allowed to explore freely within the box for 10 minutes to assess their motor abilities. Total walking distance and time spent in the central area were recorded as indicators of motor ability and anxiety-like behavior. To prevent the spread of olfactory cues, the box was cleaned with 75% ethanol solution between trials. 1.2.2 Elevated Cross Maze (EPM)

[0062] The EPM device consists of two open arms and two closed arms, as well as a central area. The EPM is positioned 50 cm above the floor. Mice are placed in the central area and allowed to freely explore the EPM for 5 minutes. The time spent in the open arms is recorded and analyzed as a percentage, serving as an indicator of anxiety-related behaviors. To prevent the spread of olfactory cues, the instrument is cleaned with a 75% ethanol solution between tests.

[0063] 1.2.3 Tail Suspension Test (TST)

[0064] Following the previously established protocol, the TST was used to assess depressive-like behaviors in mice. In this experiment, mice were suspended individually with tape 1 cm from the tip of their tails. The testing process was recorded on video for 6 minutes, and subsequently analyzed by double-blind observers. Mice moving at a speed less than 0.05 m / s were considered immobile, and the duration of immobility was recorded.

[0065] 1.2.4 Forced Swimming Test (FST)

[0066] FST is a widely used behavioral paradigm for assessing depressive-like behaviors. In this experiment, mice were placed in a glass cylinder 25 cm high and 18 cm in diameter, filled with water to a height of 18 cm, and the temperature was maintained at 22℃-25℃. The mice were forced to swim for 6 minutes. To prevent the influence of animal behavior, the water was changed after each test. Analysis was performed by double-blind observers; mice with a movement speed less than 0.05 m / s were considered immobile, and their immobility time was recorded.

[0067] 1.2.5 Sucrose Preference Test (SPT)

[0068] SPT was conducted as previously studied. Initially, mice were trained for 36 hours to acclimatize to a 1% sucrose solution (w / v) while minimizing the influence of bottle placement on their preferences. During the acclimatization phase, two bottles of 1% sucrose solution were placed in each cage for 12 hours, followed by one bottle of purified water for 12 hours. Then, the positions of the two bottles were swapped for another 12 hours. After this acclimatization period, the mice were deprived of water for 12 hours. SPT was then conducted by randomly placing two new bottles, one filled with 1% sucrose solution and the other filled with purified water. Mice were allowed free access to water for 2 hours, and the water bottles were weighed before and after the experiment. The sucrose preference rate was calculated as: (sucrose solution intake / total fluid consumption × 100%).

[0069] 1.3 Immunofluorescence staining

[0070] Mice were euthanized and perfused, and brain tissue was harvested and fixed in 4% paraformaldehyde for 24 hours. The tissue was then infiltrated with 30% sucrose. Coronal brain sections (20 μm) were cut using a cryostat (LEICA CM1850). The sections were then infiltrated with 0.1% Triton X-100 PBS and blocked with 10% normal goat or donkey serum at room temperature for 2 hours. The sections were then incubated overnight with primary antibody at 4°C. After washing with PBS, the brain sections were incubated with secondary antibody conjugated to Alexa Fluor 488 or Alexa Fluor 594 (1:1000) at room temperature for 1 hour, followed by three washes with PBS. The brain sections were stained with 2 μg / ml DAPI before capturing fluorescence images. Images were captured using a fluorescence microscope (Olympus BX51).

[0071] 1.4 Nissl staining and HE staining

[0072] Tissues were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and then sectioned. Sections were dewaxed and rehydrated before staining. Nissl staining was performed using 0.04% toluidine blue solution. HE staining was performed using eosin, with hematoxylin counterstaining of cell nuclei.

[0073] 1.5 EdU staining

[0074] To measure cell proliferation in S phase, mice were intraperitoneally injected with 50 mg / kg (EdU) 2 hours before sacrifice. EdU labeling was detected using the Cell-Light EdU Apollo 567 kit (Ribobio, Guangzhou, China) according to the manufacturer's instructions.

[0075] 1.6 Golgi staining

[0076] Brain sections were completely immersed in Golgi-Cox solution in the dark for 14 days. Subsequently, the tissue was immersed in 80% glacial acetic acid overnight, then transferred to 30% sucrose and incubated at 4°C for 72 hours. The brain was then sectioned into 100 μm slices using a cryostat. Each slice was placed on a gelatin-coated slide and dried in the dark overnight. Next, the slices were immersed in concentrated ammonia and acidic hardening fixative, washed with distilled water for 3 minutes at each step. Finally, the slices were dehydrated, cleaned, and mounted. Images were taken using a fluorescence microscope (Olympus BX51). Dendritic spines at the apex of neurons were selected for analysis, and their quantification was performed using ImageJ software. Golgi staining was performed using the FD Rapid Golgi Staining Kit (Cat#PK401, FD Neurotechnologies Inc, USA) according to the manufacturer's instructions.

[0077] 1.7 Transmission Electron Microscopy (TEM)

[0078] Immediately after the animal was killed, the hippocampus tissue was removed and cut into 1mm pieces. 3 Tissue blocks were fixed in 2.5% glutaraldehyde overnight at 4°C. After washing three times with PBS, the samples were fixed in 1% osmium tetroxide, followed by a series of gradient ethanol dehydration processes. The tissues were then infiltrated and soaked in propylene oxide overnight before being embedded in resin. 50 nm thick tissue sections were prepared using an ultramicrotome (EMUC7, Leica, Germany) and stained with uranyl acetate. Photomicrographs were taken using a transmission electron microscope.

[0079] 1.8 RNA extraction and qPCR

[0080] Total RNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). Then, NanoDrop was used. TM The concentration of total RNA was measured using a OneC spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Then, RevertAid was used. TM cDNA was synthesized using a cDNA synthesis kit (Thermo Fisher Scientific). qPCR was performed using a SYBRGreen Realtime PCR Master Mix (TOYOBO CO,Ltd, Japan). Each sample was analyzed in duplicate. β-actin expression was used as a normalization control. -△△Ct The method calculates changes in gene expression levels.

[0081] 1.9 Protein Immunoblotting

[0082] After mouse sacrifice, tissues from each mouse were immediately isolated and preserved on ice. Total protein was then extracted using lysis buffer. Protein concentration in the experimental samples was determined using the BCA Assay Kit (Beyotime, Shanghai, China). Electrophoresis and membrane transfer were then performed, transferring proteins to a PVDF membrane, which was then incubated with 5% skim milk at room temperature for 2 hours. Following this, the membrane was incubated overnight at 4°C with primary antibody and agitated. After primary antibody removal, the membrane was washed three times with 1x TBST and incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour. Proteins were detected and captured on film using a chemical exposure analyzer (Millipore, USA). Band intensity was quantified using ImageJ software. All experiments were performed in at least triplicate.

[0083] 1.10 Statistical Analysis

[0084] Data are expressed as mean ± SEM, with significance set at P < 0.05. Statistical methods employed include t-tests, one-way ANOVA, or two-way ANOVA, and SPSS software was used for statistical analysis.

[0085] 2 Experimental Results

[0086] 2.1 Dendrobine and dendrobine alleviated LPS-induced depressive-like behavior in adolescent mice.

[0087] LPS induced an acute inflammatory response and depressive-like behavior in mice. Mice were administered dendrobine (5, 10, 20 mg / kg) by gavage and dendrobine (2.5, 5, 10 mg / kg) by intragastric administration for 7 consecutive days, followed by intraperitoneal injection of LPS (1 mg / kg) on ​​days 3–5. Body weight was recorded for 10 consecutive days. Data showed that both dendrobine and dendrobine slightly improved LPS-induced weight loss in mice. Figure 1 A, B). Anxiety-like behaviors in mice were assessed using the open field test (OFT) and the elevated cross maze (EPM). We found that all three concentrations of dendrobine and dendrobine could alleviate the LPS-induced reduction in time spent in the central region and the reduction in time spent in the open arm (%) in mice. Figure 1 CG). Depressive-like behaviors were assessed using the sucrose preference test (SPT), forced swimming test (FST), and tail suspension test (TST). We found that all three dosage concentrations of dendrobine and dendrobine alleviated the prolongation of resting time and the decrease in sucrose preference rate. Figure 1 (HJ). Notably, 10 mg / kg of dendrobine and dendrobine showed the most significant therapeutic effects. Therefore, we selected the optimal concentration for subsequent experiments. The above results indicate that dendrobine and dendrobine can alleviate LPS-induced anxiety-depression-like behavior in adolescent mice.

[0088] 2.2 Dendrobine and dendrobine can improve LPS-induced neuronal damage, synaptic plasticity disruption, and abnormal apoptosis.

[0089] Studies have shown that depression is often associated with neurological damage, disruption of synaptic plasticity, and abnormal apoptosis. This study selected two specific brain regions: the hippocampus (HIP) and the prefrontal cortex (PFC). Western blot data showed that dendrobine and dendrobine restored LPS-induced reductions in NeuN, Nestin, PSD95, and Syn. Figure 2 A, B). HE and Nissl staining showed that dendrobine and dendrobine alleviated LPS-induced neuronal morphological damage and abnormalities. Figure 2 C). Golgi staining showed that dendrobine and romaine significantly alleviated the LPS-induced decrease in dendritic spine density. Figure 2 D). Immunofluorescence staining showed that LPS reduced the number of NeuN-positive cells. However, these changes were effectively reversed by dendrobine and dendrobine. Figure 2 E). Transmission electron microscopy (TEM) revealed that dendrobine and dendrobine significantly alleviated LPS-induced heterochromatin increase and synapse number reduction. Figure 2 F, G). Furthermore, EdU staining showed that dendrobine and dendrobine alleviated the reduction of EdU-positive cells in the hippocampal DG region, indicating that dendrobine and dendrobine rescued LPS-induced DG region cell proliferation impairment (F, G). Figure 2 H, I).

[0090] 2.3 Dendrobine and dendrobine can alleviate LPS-induced neuroinflammation, glial cell activation, and elevated oxidative stress levels.

[0091] Previous studies have shown a strong association between neuroinflammation and depression. In this study, dendrobine and dendrobine reversed LPS-induced elevations in IFN-γ, IL-1β, and IL-6 mRNA levels. Figure 3 A). The expression levels of GFAP and TNF-α in HIP and PFC tissues were detected by Western blot. Our data showed that dendrobine and dendrobine significantly alleviated the LPS-induced increase in GFAP and TNF-α. Figure 3 BE). Furthermore, TNF-α immunofluorescence staining results also showed the same trend ( Figure 3 F). Neuroinflammation is characterized by the activation of microglia and astrocytes. To observe glial cells, we performed immunofluorescence staining for GFAP and Iba-1 in HIP and PFC. We found that dendrobine and dendrobine reduced the LPS-induced increase in the number of microglia and astrocytes (F). Figure 3G, H). Furthermore, Sholl analysis showed that dendrobine and dendrobine inhibited LPS-induced microglial cell body enlargement and decreased branching complexity. Figure 3 (I). Therefore, our findings suggest that dendrobine and dendrobine can alleviate LPS-induced neuroinflammation and glial cell activation.

[0092] 2.4 Dendrobine and dendrobine reduce depressive-like behavior by downregulating TLR4 expression, inhibiting PI3K / AKT / NF-κB pathway activation.

[0093] To further investigate the mechanisms by which dendrobine and dendrobine alleviate depressive-like behaviors, we extracted total RNA from HIP and performed RNA-seq on the CON, LPS, dendrobine, and dendrobine groups. Volcano plots and multi-component differential scatter plots showed the differential expression of genes in the four groups. Compared with the LPS group, differential gene expression analysis showed 354 and 723 differentially expressed genes in the dendrobine and dendrobine groups, respectively. Figure 4 A). KEGG pathway enrichment analysis showed that differentially expressed genes were significantly enriched in pathways closely related to neuroinflammation, oxidative stress, and depression, including the Toll-like receptor signaling pathway, NF-κB signaling pathway, TNF signaling pathway, and PI3K-AKT signaling pathway. Figure 4 BD). GO pathway enrichment analysis showed significant enrichment of differentially expressed genes in pathways related to cellular life processes. Figure 4 E). To identify specific differentially expressed genes, we performed a trend analysis on 255 differentially expressed genes from the intersection of the three comparison groups. The trend analysis showed that these genes were divided into 20 gene modules, with two modules showing significant differences (E). Figure 4 F and G). These two gene modules were selected as key modules, containing a total of 236 genes. Among them, we screened and identified 5 differentially expressed genes closely related to the nervous system. The trends of qPCR results and RNA-seq results were similar (F, G). Figure 4 H, I). TLR4 is an important receptor for LPS. Activation of TLR4 stimulates the PI3K / AKT / NF-κB pathway, leading to the upregulation of pro-inflammatory factors. Western blot data showed that dendrobine and dendrobine reduced the LPS-induced increase in TLR4 protein expression. In addition, compared with the LPS group, the phosphorylation levels of NF-κB subunit P65, PI3K, and AKT were significantly reduced in the dendrobine and dendrobine groups. Figure 4 (J). Subsequent correlation assessments aimed to describe the relationship between TLR4 expression levels and behavioral performance. Our results showed that elevated TLR4 expression was significantly correlated with behavior and inflammatory factors (J). Figure 4Therefore, the above data indicate that dendrobine and dendrobine alleviate depressive-like behavior by downregulating TLR4 expression and inhibiting the activation of the PI3K / AKT / NF-κB pathway.

[0094] 2.5 Dendrobine and dendrobine inhibit the TLR4 / PI3K / AKT / NF-κB pathway by targeting PDE4B.

[0095] To further elucidate the mechanisms of action of dendrobine and dendrobine and determine their binding sites, we used the PharmMapper and GeneCards databases to identify proteins that may interact with dendrobine and dendrobine. Using the PharmMapper database, we identified 346 potential target proteins that may interact with dendrobine and 372 potential target proteins that may interact with dendrobine, prioritizing them using Z-scores to indicate the strength of potential interactions. A search for depression in GeneCards yielded 5650 related genes, which were screened according to the following criteria: (1) genes encoding proteins, (2) genes contributing more than the median, and (3) genes with a correlation higher than the median. Intersecting these three datasets yielded 33 common targets, from which we screened four targets closely associated with depression and neuroinflammation and exhibiting high Z-scores. Among these targets, we focused on PDE4B (… Figure 5 A). PDE4B is an important phosphodiesterase that plays a crucial role in promoting inflammation. Western blot results showed that PDE4B expression was increased in the LPS group, while it was decreased in the dendrobine and dendrobine groups. Figure 5 B). Molecular docking results showed that the minimum binding energies of dendrobine and dendrobine to PDE4B were both below -5 kcal / mol, indicating a potential binding possibility between them. Figure 5 C, D). We performed a protein thermal shift assay (CETSA) using mouse HIP tissue. The thermal shift curves showed that both dendrobine and dendrobine interact with PDE4B and promote its degradation. Figure 5 Previous studies have shown that inhibiting PDE4B reduces the activation of the TLR4 / PI3K / AKT / NF-κB pathway. Correlation analysis showed a positive correlation between PDE4B expression and the activation of the PI3K / AKT / NF-κB pathway. These findings suggest that key transcription factors, particularly NF-κB, may be potential targets of PDE4B. Figure 5 Therefore, our experiments show that dendrobine and dendrobine can inhibit the TLR4 / PI3K / AKT / NF-κB pathway by targeting PDE4B.

[0096] 2.6 Dendrobine alleviates LPS-induced intestinal pathological damage

[0097] In the aforementioned study, we found that dendrobine's therapeutic effect was generally superior to that of romaine. Therefore, we hypothesize that dendrobine may alleviate depressive-like behaviors through other mechanisms. Recent studies have shown that active ingredients in traditional Chinese medicine can regulate the central nervous system through the gut-brain axis. We found that dendrobine significantly reduced loose stools and decreased colon length caused by LPS, while romaine did not show any such effect. Figure 6 A, B). Next, ELISA results showed that dendrobine significantly reduced elevated IL-1β and TNF-α levels in HIP, serum, and colon. Figure 6 C, D). Furthermore, we assessed the expression of tight junction proteins. The results showed that dendrobine reduced colonic ZO-1 and Occludin mRNA levels, while Claudin-1 mRNA expression remained largely unchanged. Figure 6 EG). Western blot data showed that dendrobine can alleviate LPS-induced elevation of colonic ZO-1 ( ). Figure 6 H, I), the immunofluorescence staining results were the same as the protein results. Figure 6 J). Finally, we used HE staining to determine whether dendrobine alleviated intestinal damage. The LPS group showed significant mucosal destruction, sparse villi with epithelial erosion, crypt deformities, and loss of colonic crypts and goblet cells. As expected, these findings were effectively reversed by dendrobine. Figure 6 In conclusion, dendrobine can effectively alleviate LPS-induced intestinal pathological damage.

[0098] 2.7 Dendrobine can alleviate LPS-induced gut microbiota imbalance.

[0099] The close link between gut microbiota and psychosocial stress prompted us to investigate the effects of dendrobine on the gut microbiota of LPS mice. First, α-diversity analysis showed that the α-diversity in the LPS group was significantly lower than that in the CON group, with decreases in Chao1, Shannon, and Sob indices. Figure 7 AC). However, dendrobine improved the low level of microbial diversity in the LPS group. Dilution curves confirmed the appropriateness of the sequencing data volume ( Figure 7 D). The Venn diagram shows that a total of 1739 OTUs were detected in the CON, LPS, and dendrobine groups, with 409, 418, and 401 independent OTUs, respectively. Figure 7 E). Furthermore, the microbial communities of the CON and Dendrobium alkaloid groups clustered together, while the microbial community of the LPS group was clearly separated. To identify samples with similar microbial community structure and composition, we performed multidimensional clustering analysis based on Bray-Curtis distance. The results showed that the CON and Dendrobium alkaloid groups clustered together, while significantly different from the LPS group (E). Figure 7F). Next, we analyzed the relative abundance of various microbial types at specific taxonomic levels. Our results showed that in the LPS group, the proportion of Firmicutes decreased significantly, while the proportion of Proteobacteria increased. Notably, dendrobine reversed this trend ( Figure 7 G). To further distinguish species among different groups, we used linear discriminant analysis to identify biomarkers (G). Figure 7 Interestingly, our data showed that LPS significantly reduced the abundance of Lactobacillus and Akkermania species while promoting the growth of potential pathogens such as Klebsiella and Ureaplasma. This is consistent with the effects of remodeling on microbial diversity, where dendrobine treatment enhanced the abundance of Lactobacillus and inhibited the spread of Klebsiella and Ureaplasma. Figure 7 HI). The above results indicate that dendrobine can effectively alleviate LPS-induced gut microbiota dysbiosis. Furthermore, we assessed the relationships between key differentially expressed gut microbiota, behavioral indicators, neurodegenerative markers, oxidative stress and neuroinflammatory markers, and gut integrity indicators at the species level. Microbiota that negatively regulate the gut microenvironment, such as Proteobacteria, showed a significant positive correlation with factors related to depression. Conversely, healthy gut microbiota, such as Akkermansia, showed a significant positive correlation with antidepressant phenotypes. Figure 7 These findings suggest that dendrobine can regulate brain function and behavior through the microbe-gut-brain axis.

[0100] In summary, dendrobine and dendrobine can alleviate anxiety and depression-like behaviors in adolescent mice by targeting PDE4B, thereby inhibiting TLR4 expression and reducing the activation of the PI3K / AKT / NF-κB pathway. Furthermore, dendrobine can also inhibit intestinal pathological damage and improve gut microbiota dysbiosis through the microbe-gut-brain axis, thus further improving brain function and behavior.

[0101] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of dendriticil in the preparation of antidepressant drugs, characterized in that, The depression mentioned is adolescent depression; the depression is mediated by lipopolysaccharide; the treatment of depression has the following effects: a) Alleviate LPS-mediated depressive-like behaviors; b) Improves LPS-mediated neuronal damage, disruption of synaptic plasticity, and abnormal apoptosis; c) Reduce LPS-mediated neuroinflammation, glial cell activation, and elevated oxidative stress levels; d) Target PDE4B to inhibit the TLR4 / PI3K / AKT / NF-κB pathway.

2. The application as described in claim 1, characterized in that, In b), the improvement of LPS-mediated neuronal damage, synaptic plasticity disruption, and apoptosis abnormalities is specifically manifested in: restoring LPS-induced reductions in NeuN, Nestin, PSD95, and Syn; alleviating LPS-induced neuronal morphological damage and abnormalities; mitigating LPS-induced decreases in dendritic spine density and the number of NeuN-positive cells; mitigating LPS-induced increases in heterochromatin and decreases in the number of synapses; and mitigating the reduction in EdU-positive cells in the hippocampal DG region.

3. The application as described in claim 1, characterized in that, In c), the reduction of LPS-mediated neuroinflammation, glial cell activation and oxidative stress levels is specifically manifested in: reversing LPS-induced increases in IFN-γ, IL-1β and IL-6 expression levels; alleviating LPS-induced increases in GFAP and TNF-α; reducing LPS-induced increases in the number of microglia and astrocytes; and inhibiting LPS-induced increases in microglia cell body size and branching complexity.

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