Application of dendrobine and / or erianin in depression treatment

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of dendrobine and / or erianin in depression treatment. Specifically, tests prove that dendrobine and erianin can improve the depression-like behavior induced by LPS, and inhibit the activation of a TLR4 / PI3K / AKT / NF-kappa B pathway by targeting PDE4B, so that the oxidative stress level and neuroinflammatory response are reduced, and the symptoms are relieved by reversing the damage of neurotoxicity, apoptosis abnormality and synaptic plasticity. The dendrobine improves intestinal flora disorder and acts on a central nervous system through a microorganism-intestine-brain axis, so that the anti-depression effect of the dendrobine is superior to that of erianin. The invention opens up a new medicinal application for dendrobine and erianin, and also lays an experimental foundation and provides a new field of view for developing high-efficiency related medicines for treating depression.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the application of dendrobine and / or eranine in the treatment of depression. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Major depressive disorder (MDD) is a neurological disease characterized by persistent spontaneous mood disorders. Its pathogenesis is complex and involves neuroinflammation, oxidative stress, synaptic damage, intestinal flora, etc. Adolescent depression has become a hot topic of research in recent years. Previous studies have shown that many antidepressant drugs have low cure rates and have multiple side effects, such as potential damage to the liver and kidneys, which highlights the urgent need for new treatments for depression. At present, the hypotheses on 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 the key to treating adolescent depression.

[0004] Dendrobium officinale is well known for its important medicinal value and has been widely used to modulate immune responses and enhance gastrointestinal function. The main chemical components of Dendrobium officinale include polysaccharides, alkaloids, terpenoids, flavonoids, dibenzyl and phenanthrene. Among the alkaloids of Dendrobium officinale, dendrobine is its main bioactive component. Numerous studies have shown that dendrobine has pharmacological activities, including anti-inflammatory properties and anticancer activity. Erianin, another dibenzyl component of Dendrobium officinale, has been reported to have anti-inflammatory and protective effects in a variety of diseases. In addition, Chinese herbal medicines can exert their effects by regulating the intestinal flora, which can affect the central nervous system through the microbiome-gut-brain axis. Increasing evidence suggests that an imbalance in the intestinal flora may affect brain function and behavior and may play an important role in the development of depression. However, it is unclear whether dendrobine and eranin can improve depressive-like behaviors in adolescence by regulating the intestinal flora. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides the use of dendrobium and / or Erianin in the treatment of depression. Specifically, the present invention has proved through experiments that dendrobium and Erianin can improve LPS-induced depressive-like behavior, and inhibit the activation of the TLR4 / PI3K / AKT / NF-κB pathway by targeting PDE4B, thereby reducing the level of oxidative stress and neuroinflammatory response, reversing neurotoxicity, abnormal apoptosis and destruction of synaptic plasticity to alleviate symptoms. Dendrobium improves intestinal flora disorders and acts on the central nervous system through the microbial-intestinal-brain axis, making its antidepressant effect better than Erianin. Based on the above research results, the present invention is completed.

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

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

[0008] In the present invention, the depression may be severe depression, and may further be adolescent depression.

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

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

[0011] b) Improve LPS-mediated neuronal damage, synaptic plasticity disruption and cell apoptosis abnormalities;

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

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

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

[0015] f) Improve LPS-mediated intestinal flora imbalance.

[0016] According to the present invention, not only is the use of dendrobium and / or Erianin in preparing a drug for treating depression disclosed, but also it is disclosed that when dendrobium and / or Erianin are used in combination with at least one other active pharmaceutical ingredient, this effect can be enhanced. As a substitute or supplement for other active pharmaceutical ingredients, dendrobium and / or Erianin can also be used in combination with other non-active pharmaceutical ingredients.

[0017] The second aspect of the present invention provides a pharmaceutical composition for treating depression, wherein the pharmaceutical composition is composed of dendrobium alkaloids and / or Erianin and at least one other active pharmaceutical ingredient and / or at least one other non-active pharmaceutical ingredient.

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

[0019] The subject may be a human or a non-human mammal, and the non-human mammal may include mice, rats, guinea pigs, pigs, dogs, monkeys, orangutans, etc.

[0020] Beneficial technical effects of one or more of the above technical solutions:

[0021] The above technical scheme has proved through experiments that the active ingredients of the traditional Chinese medicine Dendrobium officinale, dendrobium alkaloids and erlanin, can effectively alleviate the depressive-like behavior of adolescent mice induced by lipopolysaccharide (LPS), and the optimal dosage concentrations of the two have been determined. LPS treatment can lead to aggravated damage to the hippocampal and prefrontal neurons of mice, significant damage to synaptic structures, and abnormal cell apoptosis, while treatment with dendrobium alkaloids and erlanin effectively improved the above results. RNA-seq results show that dendrobium alkaloids and erlanin can target phosphodiesterase 4B (PDE4B), thereby alleviating the activation of the TLR4 / PI3K / AKT / NF-κB pathway, resisting the increased level of oxidative stress in the mouse brain, glial cell activation and excessive release of inflammatory factors, and improving the depressive-like behavior of adolescent mice. In addition, we found that dendrobium treatment was more effective than erythrine. Combined with the intestinal phenotype and 16s rRNA-seq results, dendrobium treatment effectively improved the increased intestinal permeability caused by LPS-induced intestinal flora disorders and intestinal epithelial cell damage in mice, indicating that dendrobium can further improve anxiety and depression symptoms through the microbiome-gut-brain axis.

[0022] In conclusion, the above technical scheme has opened up new medicinal uses for dendrobium and erythrin, and also laid an experimental foundation and provided new perspectives for the development of highly effective drugs for the treatment of depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1Dendrobium and Erianin in the present invention alleviated the anxiety-depressive-like behavior of adolescent mice induced by LPS. (A) Schematic diagram of the experimental process (8 male mice per group in the behavioral experiment). (B) Body weight change for 10 consecutive days. (C) Percentage of time spent in the central area of ​​OFT. (D) Total distance moved in OFT. (E) Movement trajectory of OFT. (F) Time spent on the open arms of EPM. (G) Movement trajectory of EPM. (H) SPT sucrose preference (%). (I) FST immobility time (s). (J) TST immobility 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 Dendrobium and Erianin can improve LPS-induced neuronal damage, synaptic plasticity destruction and cell apoptosis abnormalities in the examples of the present invention. (A, B) Western blot analysis of Nestin, NeuN, PSD95 and Syn protein expression in HIP compared with β-actin (n=4 per group). (C) HE and Nissl staining of HIP (DG and CA3 regions) and PFC (scale bar=100μm). (D) Representative images of Golgi apparatus and dendritic spine density statistics (n=5 per group) (scale bar=10μm). (E) Immunofluorescence staining of NeuN expression in HIP DG and CA3 regions (n=3 per group) (scale bar=100μm). (F, G) Transmission electron microscopy observation of the morphology of heterochromatin (10000x) and synapses (30000x and 50000x), as well as the number of synapses in each group (n=3 per group). (H) EdU experimental process. (I) Representative fluorescence images and EdU quantitative analysis of the HIP DG region (n = 3 per group) (scale bar = 100 μm). *p < 0.05, **p < 0.01, ***p < 0.001, compared with the LPS group; #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the CON group.

[0026] Figure 3Dendrobium and Erianin in the present invention can reduce LPS-induced neuroinflammation, glial cell activation and increased oxidative stress levels. (A) qPCR detection of IFN-γ, IL-1β, and IL-6 mRNA expression 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 quantitative analysis of TNF-α in HIP DG region (n=3 per group) (scale bar=100μm). (G) Representative fluorescence images and quantitative analysis of Iba-1 in HIP DG region (n=3 per group) (scale bar=100μm). (H) Representative fluorescence images and quantitative analysis of GFAP in HIP DG region (n=3 per group) (scale bar=100μm). (I) Sholl analysis of microglia morphology (n=3 per group). *p<0.05, **p<0.01, ***p<0.001, compared with the LPS group; #p<0.05, ##p<0.01, ###p<0.001, compared with the CON group.

[0027] Figure 4 In the present invention, dendrobium and Erianin down-regulate TLR4 expression, inhibit PI3K / AKT / NF-κB pathway activation, and alleviate depressive-like behavior. (A) Multi-component difference scatter plot shows differentially expressed genes in CON, LPS, dendrobium and Erianin 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 in the GO database. (F) Gene trend analysis, two significant difference groups are profile5 and profile14 (the inflection points from left to right represent the gene expression of CON, LPS, dendrobium and Erianin groups). (G) Difference Venn diagram. (H) Gene heat map shows 12 genes related to nerves and differentially expressed in the four groups. (I) qPCR detection of Hck, Lcn2, Csf1r, CYBB, TLR4 mRNA expression 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 with β-actin (n=4 per group). (K) Correlation analysis of TLR4 protein expression with behavioral data and inflammatory factor protein expression. *p<0.05, **p<0.01, ***p<0.001, compared with LPS group; #p<0.05, ##p<0.01, ###p<0.001, compared with CON group.

[0028] Figure 5 Dendrobium and Erianin inhibit TLR4 / PI3K / AKT / NF-κB pathway by targeting PDE4B in the examples of the present invention. (A) PhamMapper and GeneCards were used to screen potential target proteins of dendrobium and Erianin. (B) Western blot analysis of PDE4B protein expression in HIP compared with β-actin (n=4 per group). (C, D) Molecular docking results of dendrobium, Erianin and PDE4B (minimum binding energy is 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 Dendrobium candidum alleviates LPS-induced intestinal pathological damage in the examples of the present invention. (A) Fecal moisture percentage (%) (n=3 per group). (B) Colon length (n=3 per group). (C, D) ELISA method was used to detect the expression of IL-1β and TNF-α in colon, serum and HIP (n=5 per group). (EG) qPCR was used to detect the expression of ZO-1, Claudin-1 and Occludin mRNA in colon (n=5 per group). (H, I) Western blot analysis of colon ZO-1 protein and β-actin expression (n=4 per group). (J) Representative fluorescence images and quantitative analysis of ZO-1 in colon (n=3 per group) (scale bar=100μm). (K) HE staining showed the colon tissue morphology and tissue damage score of each 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. Figure 7Dendrobium alkaloids in the present invention can alleviate the imbalance of intestinal flora induced by LPS. (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 between the three groups (n = 5 per group). (F) Fecal microbial community pattern of principal component analysis (PCoA). (G, H) Relative abundance of intestinal bacteria at the phylum level and genus level. (I) Circos diagram reflects the proportion of dominant species 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 microorganisms in different groups and the LEfSe multi-level species evolution diagram (K) Inter-group correlation analysis (the horizontal axis represents the key differential bacterial community, the vertical axis represents the data of the above experimental results, and blue to red represents the correlation from -1 to 1). DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0031] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0032] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out under conventional conditions or under conditions recommended by the sales company; the materials and reagents used in the examples, unless otherwise specified, can be purchased through commercial channels.

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

[0034] According to the invention, the concept of "treatment" means any relevant measure applicable to depression, either for the preventive treatment of the disease or the symptoms manifested, or for avoiding the recurrence of the disease, such as recurrence after the end of the treatment period or for treating the symptoms of the disease that has already occurred.

[0035] Specifically, the depression is severe depression, and may further be adolescent depression.

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

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

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

[0039] b) Improve LPS-mediated neuronal damage, synaptic plasticity disruption and cell apoptosis abnormalities;

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

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

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

[0043] f) Improve LPS-mediated intestinal flora imbalance.

[0044] Among them, in b), improving LPS-mediated neuronal damage, synaptic plasticity destruction and cell apoptosis abnormalities is specifically manifested as: restoring the LPS-induced decrease in NeuN, Nestin, PSD95 and Syn; alleviating LPS-induced neuronal morphological damage and abnormalities; alleviating the LPS-induced decrease in dendritic spine density and the decrease in the number of NeuN-positive cells; alleviating the LPS-induced increase in heterochromatin and the decrease in the number of synapses; and alleviating the decrease in EdU-positive cells in the DG region of the hippocampus.

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

[0046] In said e), the intestinal pathological damage is specifically manifested as: obvious destruction of the intestinal mucosa, sparse villi, accompanied by epithelial erosion, crypt deformation, and loss of colon tissue crypts and goblet cells.

[0047] According to the present invention, not only is the use of dendrobium and / or Erianin in preparing a drug for treating depression disclosed, but also it is disclosed that when dendrobium and / or Erianin are used in combination with at least one other active pharmaceutical ingredient, this effect can be enhanced. As a substitute or supplement for other active pharmaceutical ingredients, dendrobium and / or Erianin can also be used in combination with other non-active pharmaceutical ingredients.

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

[0049] The non-pharmaceutical active ingredient may be a pharmaceutically acceptable carrier or excipient, including a pharmaceutically acceptable material, composition or carrier suitable for administering the compound of the present invention to a mammal. The carrier includes a liquid or solid filler, diluent, excipient, solvent or encapsulating material that is involved in carrying the main substance or transferring it from one organ or part of the body to another organ or another part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients in the formulation and harmless 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 carboxymethylcellulose, ethylcellulose and cellulose acetate, powdered tragacanth, 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] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical compositions.

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

[0052] The subject may be a human or a non-human mammal, and the non-human mammal may include mice, rats, guinea pigs, pigs, dogs, monkeys, orangutans, etc.

[0053] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0054] Example

[0055] 1. Experimental Methods

[0056] 1.1 Animal Model

[0057] All animal care and experiments followed the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Shandong University. Four-week-old male C57BL / 6 mice (weight 18-20 g) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. All animals were housed under standard laboratory conditions, in ventilated cages, with a 12-h light-dark cycle (8 am and 8 pm), in a specific pathogen-free environment and with adequate food-water supply at room temperature (22 ± 1 °C). Mice were randomly divided into 8 groups (n=8 in each group): control group (mice in saline group), LPS group (mice in LPS group, 1 mg / kg, ip), dendrobium 5 group (mice treated with LPS and dendrobium, 5 mg / kg, ig), dendrobium 10 group (mice treated with LPS and dendrobium, 10 mg / kg, ig), dendrobium 20 group (mice treated with LPS and dendrobium, 20 mg / kg, ig), Erianin 2.5 group (mice treated with LPS and Erianin, 2.5 mg / kg, ig), Erianin 5 group (mice treated with LPS and Erianin, 5 mg / kg, ig), Erianin 10 group (mice treated with LPS and Erianin, 10 mg / kg, ig). LPS was from Escherichia coli strain O111:B4, Sigma, USA, #L2630, dissolved in 0.9% saline. From day 1 to day 7, mice in the control group and LPS group were given saline (8:00-9:00 am), and mice in the dendrobium group and erynthine group were given dendrobium and erynthine. In addition, from day 3 to day 5, 2 hours after the first gavage, mice in the control group were given saline again, and mice in the other 7 groups were intraperitoneally injected with LPS (1 mg / kg). Behavioral tests were performed over the next 7 days. The mice were then killed and samples were collected for subsequent experiments.

[0058] 1.2 Behavioral testing

[0059] The animal behavior tests were: open field test (OFT), elevated plus maze (EPM), tail suspension test (TST), forced swimming test (FST) and sucrose preference test (SPT). These experiments were conducted in a dedicated room isolated from external noise. The behavior experiments were recorded, measured and analyzed using the TopScan 3.0 tracking system.

[0060] 1.2.1 Open Field Test (OFT)

[0061] The mice were placed in an activity box of 35 cm × 35 cm × 25 cm, with a central area of ​​20 cm × 20 cm. The mice were allowed to explore freely in the box for 10 min to assess their motor ability. The total distance walked and the time spent in the central area were recorded for analysis 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 two trials. 1.2.2 Elevated plus maze (EPM)

[0062] The EPM apparatus consisted of two open arms and two closed arms, as well as a central area. The EPM was 50 cm above the floor, and mice were placed in the central area and allowed to freely explore the EPM for 5 min. The time the mice spent in the open arms was recorded and analyzed with a tracking system, expressed as a percentage, as an indicator of anxiety-like behavior. To prevent the spread of olfactory cues, the apparatus was cleaned with a 75% ethanol solution between tests.

[0063] 1.2.3 Tail suspension test (TST)

[0064] Depression-like behavior in mice was assessed using TST according to a previously established protocol. In this test, mice were individually suspended with tape, 1 cm from the tip of the tail. The test process was recorded in a video, lasting 6 minutes, and subsequently analyzed by a double-blind observer. Mice with a movement speed of less than 0.05 m / s were considered immobile, and their immobility time was recorded.

[0065] 1.2.4 Forced swim test (FST)

[0066] FST is a behavioral paradigm widely used to assess depressive-like behavior. In this experiment, mice were placed in a glass cylinder 25 cm high and 18 cm in diameter, filled with water at a height of 18 cm and maintained at a temperature of 22°C-25°C. Mice were forced to swim for 6 minutes. In order to prevent the behavioral effects of the animals, the water was changed after each trial. The analysis was performed by a double-blind observer, and mice with a movement speed of less than 0.05 m / s were considered immobile, and their immobility time was recorded.

[0067] 1.2.5 Sucrose preference test (SPT)

[0068] The SPT was performed as in previous studies. Initially, mice were trained for 36 h to adapt to 1% sucrose solution (w / v) while minimizing the effect of bottle placement on their preference. During the adaptation phase, 2 bottles of 1% sucrose water were placed in each cage for 12 h, and then replaced with 1 bottle of pure water for 12 h. Then, the positions of the two bottles were exchanged for another 12 h. After this adaptation period, the mice were deprived of water for 12 h. The SPT was then performed by randomly placing two new bottles, one filled with 1% sucrose water and the other filled with pure water. The mice were allowed to drink water freely for 2 h, and the water bottles were weighed before and after the test. The sucrose preference rate was calculated as: (sucrose solution intake / total liquid consumption × 100%).

[0069] 1.3 Immunofluorescence staining

[0070] The mice were euthanized and perfused, and then the brain tissue was removed 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 freezing microtome (LEICA CM1850). The brain sections were then infiltrated with 0.1% Triton X-100PBS and blocked with 10% normal goat or donkey serum for 2 hours at room temperature. Subsequently, the sections were incubated with the primary antibody at 4°C overnight. After washing with PBS, the brain sections were incubated with secondary antibodies coupled to Alexa Fluor 488 or Alexa Fluor 594 (1:1000) at room temperature for 1 hour, and then washed three times with PBS. Before capturing fluorescent images, brain sections were stained with 2 μg / ml DAPI. Images were captured using a fluorescence microscope (Olympus BX51).

[0071] 1.4 Nissl staining and HE staining

[0072] The tissue was fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and then sectioned. The sections were dewaxed and rehydrated before staining. For Nissl staining, the sections were treated with 0.04% toluidine blue solution. For HE staining, the sections were stained with eosin and counterstained with hematoxylin for nuclei.

[0073] 1.5 EdU staining

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

[0075] 1.6 Golgi staining

[0076] Brain slices were completely immersed in Golgi-Cox solution in the dark for 14 days. Subsequently, the tissue was immersed in 80% glacial acetic acid overnight and then transferred to 30% sucrose and placed at 4°C for 72 hours. The brain was then cut into 100 μm slices using a freezing microtome. Each slice was placed on a gelatin-coated slide and dried overnight in the dark. Next, the slices were immersed in concentrated ammonia and acid hardening fixatives and washed with distilled water for 3 minutes each step. Finally, the slices were dehydrated, cleared, and mounted. Photos were taken using a fluorescence microscope (Olympus BX51). Neuronal apical dendrites were selected for analysis, and dendritic spines were quantified using ImageJ software. Golgi staining was performed using the FD Fast Golgi Staining Kit (Cat#PK401, FD Neurotechnologies Inc, USA) according to the manufacturer's instructions.

[0077] 1.7 Transmission electron microscopy (TEM)

[0078] The hippocampus tissue was removed immediately after sacrifice and cut into 1 mm 3 Tissue blocks were fixed in 2.5% glutaraldehyde at 4°C overnight. After washing three times with PBS, the samples were fixed in 1% osmium tetroxide and then dehydrated in a series of graded ethanol. The tissue was then infiltrated and immersed in propylene oxide overnight before being embedded in resin. 50 nm thick tissue sections were prepared using an ultrathin microtome (EMUC7, Leica, Germany) and stained with uranyl acetate. Micrographs 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). TM The concentration of total RNA was measured using a OneC spectrophotometer (Thermo Fisher Scientific, Waltham, MA). TM cDNA was synthesized using a cDNA synthesis kit (Thermo Fisher Scientific). qPCR was performed using SYBRGreen Realtime PCR Master Mix (TOYOBO CO, Ltd, Japan). Each sample was analyzed in duplicate. The expression of β-actin was used as a normalization control. -△△Ct The changes in gene expression levels were calculated.

[0081] 1.9 Western Blot

[0082] After the mice were killed, the tissues of each mouse were immediately isolated and stored on ice. Total protein was then extracted with lysis buffer. The protein concentration of the experimental samples was determined using the BCA Assay Kit (Beyotime, Shanghai, China). Electrophoresis and transfer were then performed, and the proteins were transferred to a PVDF membrane, followed by incubation with 5% skim milk at room temperature for 2 hours. After this, the membrane was incubated with the primary antibody at 4°C overnight while shaking. After removing the primary antibody, it was washed three times with 1x TBST and incubated with a horseradish peroxidase-conjugated secondary antibody for 1 hour. Proteins were detected using a chemical exposure instrument (Millipore, USA) and captured on film. ImageJ software was used to quantify band intensity. All experiments were repeated at least 3 times.

[0083] 1.10 Statistical analysis

[0084] The data were expressed as mean ± SEM, with significance set at P < 0.05. The statistical methods used were t-test, one-way analysis of variance or two-way analysis of variance, and SPSS software was used for statistical analysis.

[0085] 2 Experimental results

[0086] 2.1 Dendrobium and Erianin alleviated LPS-induced depressive-like behavior in adolescent mice

[0087] LPS can induce acute inflammatory response and depressive-like behavior in mice. Dendrobium alkaloids (5, 10, 20 mg / kg) and Erianin (2.5, 5, 10 mg / kg) were gavaged for 7 consecutive days, and LPS (1 mg / kg) was injected intraperitoneally for 3-5 days. The body weight was recorded continuously for 10 days. The data showed that both dendrobium alkaloids and Erianin could slightly improve the weight loss of mice induced by LPS ( Figure 1 A, B). The anxiety-like behavior of mice was evaluated by open field test (OFT) and elevated plus maze (EPM). We found that the three concentrations of dendrobium and eranine could alleviate the LPS-induced decrease in the time mice spent in the center area and the time they spent in the open arms (%). Figure 1 CG). Depressive-like behaviors were assessed by sucrose preference test (SPT), forced swim test (FST), and tail suspension test (TST). We found that dendrobium and eranine at three administration concentrations could alleviate the prolongation of immobility time and the decrease of sucrose preference rate ( Figure 1 HJ). It is worth noting that the therapeutic effect of 10 mg / kg of dendrobium and Erianin was the most significant. Therefore, we selected the optimal concentration for subsequent experiments. The above results show that dendrobium and Erianin can alleviate LPS-induced anxiety-depression-like behaviors in adolescent mice.

[0088] 2.2 Dendrobium and Erianin can improve LPS-induced neuronal damage, synaptic plasticity disruption and apoptosis abnormalities

[0089] Studies have shown that depression is often associated with neural damage, disruption of synaptic plasticity, and abnormal apoptosis. Two specific brain regions were selected for the study: the hippocampus (HIP) and prefrontal cortex (PFC). Western blot data showed that dendrobium alkaloids and Erianin restored the LPS-induced decrease in NeuN, Nestin, PSD95, and Syn ( Figure 2 A, B). HE and Nissl staining showed that dendrobium and Erianin alleviated LPS-induced neuronal morphological damage and abnormalities ( Figure 2 C). Golgi staining showed that dendrobium and eranine significantly alleviated the decrease in dendritic spine density induced by LPS ( Figure 2 D). Immunofluorescence staining showed that LPS reduced the number of NeuN-positive cells. However, these changes were effectively reversed by dendrobium and erythrin ( Figure 2 E). By transmission electron microscopy (TEM), we found that dendrobium and Erianin significantly alleviated the increase in heterochromatin and decrease in synapse number induced by LPS ( Figure 2 F, G). In addition, EdU staining showed that dendrobium and Erianin alleviated the decrease of EdU-positive cells in the DG region of the hippocampus, indicating that dendrobium and Erianin rescued the LPS-induced cell proliferation disorder in the DG region ( Figure 2 H, I).

[0090] 2.3 Dendrobium and Erianin can alleviate LPS-induced neuroinflammation, glial cell activation and increased oxidative stress levels

[0091] Previous studies have shown a strong association between neuroinflammation and depression. In the present study, dendrobium and erythrin reversed the LPS-induced increase in IFN-γ, IL-1β, and IL-6 mRNA levels ( Figure 3 A). Western blot was used to detect the expression levels of GFAP and TNF-α in HIP and PFC tissues. Our data showed that dendrobium and Erianin significantly alleviated the increase of GFAP and TNF-α induced by LPS ( Figure 3 BE). In addition, the results of TNF-α immunofluorescence staining also showed the same trend ( Figure 3 F). Neuroinflammation is characterized by the activation of microglia and astrocytes. To visualize glial cells, we performed immunofluorescence staining for GFAP and Iba-1 in HIP and PFC. We found that dendrobium and Erianin reduced the LPS-induced increase in the number of microglia and astrocytes ( Figure 3G, H). In addition, Sholl analysis results showed that dendrobium and eranine inhibited the LPS-induced enlargement of microglia cell bodies and the reduction of branching complexity ( Figure 3 I). Therefore, our results suggest that dendrobine and eriocin can alleviate LPS-induced neuroinflammation and glial cell activation.

[0092] 2.4 Dendrobium and Erianin down-regulate TLR4 expression, inhibit PI3K / AKT / NF-κB pathway activation, and alleviate depressive-like behavior

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

[0094] 2.5 Dendrobium and Erianin inhibit TLR4 / PI3K / AKT / NF-κB pathway by targeting PDE4B

[0095] To further elucidate the mechanism of action of dendrobine and eriocin and determine their binding sites, we used the PharmMapper and GeneCards databases to identify proteins that may interact with dendrobine and eriocin. Using the PharmMapper database, we identified 346 target proteins that may interact with dendrobine and 372 target proteins that may interact with eriocin and prioritized them using Z-scores to indicate the strength of potential interactions. Searching for depression in GeneCards generated 5,650 related genes, which were screened according to the following criteria: (1) genes encoding proteins, (2) genes with contributions greater than the median, and (3) genes with correlations higher than the median. Intersecting the three datasets resulted in 33 common targets, and we screened out four targets that were closely related to depression and neuroinflammation and had high z-scores. Among these targets, we focused on PDE4B ( Figure 5 A). PDE4B is an important phosphodiesterase that plays an important role in promoting inflammation. Western blot results showed that the expression of PDE4B increased in the LPS group, while the expression of PDE4B decreased in the dendrobium and eryngium groups ( Figure 5 B). Molecular docking results showed that the minimum binding energy of dendrobium and eranine with PDE4B was lower than -5 kcal / mol, indicating that there was 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 dendrobium and eringin could interact with PDE4B and promote the degradation of PDE4B ( Figure 5 EG). Previous studies have shown that inhibition of PDE4B can reduce the activation of the TLR4 / PI3K / AKT / NF-κB pathway. Correlation analysis showed that the expression of PDE4B was positively correlated with the activation of the PI3K / AKT / NF-κB pathway. These findings suggest that key transcription factors, especially NF-κB, may be potential targets of PDE4B ( Figure 5 H). Therefore, our experiments showed that dendrobium and eringin could inhibit the TLR4 / PI3K / AKT / NF-κB pathway by targeting PDE4B.

[0096] 2.6 Dendrobium officinale alleviates LPS-induced intestinal pathological damage

[0097] In the above study, we found that the therapeutic effect of dendrobium was generally better than that of eryngine. Therefore, we speculated that dendrobium may relieve depressive-like behavior in other ways. Recent studies have shown that the active ingredients in traditional Chinese medicine can regulate the central nervous system through the gut-brain axis. We found that dendrobium significantly alleviated the loose stools and reduced colon length caused by LPS, while eryngine did not show any such effect ( Figure 6 A, B). Next, ELISA results showed that dendrobium officinale significantly reduced the elevated IL-1β and TNF-α levels in HIP, serum, and colon ( Figure 6 C, D). In addition, we evaluated the expression of tight junction proteins. The results showed that dendrobium alkaloids could reduce the levels of ZO-1 and Occludin mRNA in the colon, while there was no significant change in the expression of Claudin-1 mRNA ( Figure 6 EG). Western blot data showed that dendrobium alkaloids could alleviate the increase of colon ZO-1 induced by LPS ( Figure 6 H, I), immunofluorescence staining results are the same as protein results ( Figure 6 J). Finally, we used HE staining to determine whether dendrobium attenuated intestinal damage. The LPS group showed significant mucosal destruction, sparse villi, accompanied by epithelial erosion, crypt deformation, and loss of crypts and goblet cells in colonic tissue. As expected, these manifestations could be effectively reversed by dendrobium ( Figure 6 K). In summary, dendrobium can effectively alleviate LPS-induced intestinal pathological damage.

[0098] 2.7 Dendrobium officinale can alleviate LPS-induced intestinal flora imbalance

[0099] The close connection between intestinal microbiota and psychosocial stress prompted us to investigate the effect of dendrobium on the intestinal microbiota of LPS mice. First, α diversity analysis showed that the α diversity of the LPS group was significantly lower than that of the CON group, and the Chao1, Shannon, and Sob indices all decreased ( Figure 7 AC). However, dendrobium alkaloids improved the low level of microbiota diversity in the LPS group. Rarefaction curves confirmed the suitability of the sequencing data volume ( Figure 7 D). The Venn diagram showed that a total of 1739 OTUs were detected in the CON, LPS, and dendrobium groups, with 409, 418, and 401 independent OTUs, respectively ( Figure 7 E). In addition, the microbial communities of the CON and dendrobium groups clustered together, while those of the LPS group were clearly separated. To identify samples with similar microbial community structure and composition, we performed multidimensional cluster analysis based on the Bray-Curtis distance, and the results showed that the CON and dendrobium groups clustered together, while they were clearly separated from the LPS group ( Figure 7F). Next, we analyzed the relative abundance of various types of microorganisms at specific taxonomic levels. Our results showed that the proportion of Firmicutes decreased significantly, while that of Proteobacteria increased in the LPS group. Notably, dendrobium reversed this trend ( Figure 7 G). To further distinguish species between groups, we used linear discriminant analysis to identify biomarkers ( Figure 7 HI). Interestingly, our data showed that LPS significantly reduced the abundance of Lactobacillaceae and Akkermansia, while promoting the growth of potential pathogens such as Klebsiella and Ureaplasma. This is consistent with the effect of remodeling on microbial diversity, with dendrobium treatment enhancing the abundance of Lactobacillaceae and inhibiting the expansion of Klebsiella and Ureaplasma ( Figure 7 HI). The above results indicate that dendrobium can effectively alleviate LPS-induced intestinal flora imbalance. In addition, we also evaluated the relationship between key differential intestinal flora, behavioral indicators, neural injury markers, oxidative stress and neuroinflammatory indicators, and intestinal integrity indicators at the species level. Bacteria that negatively regulate the intestinal microenvironment, such as Proteobacteria, are significantly positively correlated with depression-related factors. In contrast, healthy intestinal flora, such as Akkermansia, showed a significant positive correlation with antidepressant-related phenotypes ( Figure 7 K). These findings suggest that dendrobium can modulate brain function and behavior via the microbiome-gut-brain axis.

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

[0101] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of dendrobium alkaloids and / or eranine in the preparation of drugs for treating depression.

2. The use according to claim 1, characterized in that The depression is severe depression, and further is adolescent depression.

3. The use according to claim 1, characterized in that: The depression is mediated by lipopolysaccharide.

4. The use according to claim 1, characterized in that: The treatment of depression is at least manifested in having any one or more of the following effects: a) Alleviate LPS-mediated depressive-like behavior; b) Improve LPS-mediated neuronal damage, synaptic plasticity disruption and cell apoptosis abnormalities; c) reduce LPS-mediated neuroinflammation, glial cell activation, and elevated oxidative stress levels; d) Targeting PDE4B to inhibit TLR4 / PI3K / AKT / NF-κB pathway; e) Alleviate LPS-mediated intestinal pathological damage; f) Improve LPS-mediated intestinal flora imbalance.

5. The use according to claim 4, characterized in that: In the above b), the improvement of LPS-mediated neuronal damage, synaptic plasticity destruction and cell apoptosis abnormalities is specifically manifested as: restoring the LPS-induced decrease in NeuN, Nestin, PSD95 and Syn; alleviating the LPS-induced neuronal morphological damage and abnormalities; alleviating the LPS-induced decrease in dendritic spine density and the decrease in the number of NeuN-positive cells; alleviating the LPS-induced increase in heterochromatin and the decrease in the number of synapses; and alleviating the decrease in EdU-positive cells in the DG region of the hippocampus.

6. The use according to claim 4, characterized in that: In the above c), the reduction of LPS-mediated neuroinflammation, glial cell activation and increased oxidative stress levels is specifically manifested as: reversing the LPS-induced increase in IFN-γ, IL-1β and IL-6 expression levels; alleviating the LPS-induced increase in GFAP and TNF-α; reducing the LPS-induced increase in the number of microglia and astrocytes; and inhibiting the LPS-induced enlargement of microglial cell bodies and the reduction in branching complexity.

7. The use according to claim 4, characterized in that: In said e), the intestinal pathological damage is specifically manifested as: obvious destruction of the intestinal mucosa, sparse villi, accompanied by epithelial erosion, crypt deformation, and loss of colon tissue crypts and goblet cells.

8. A pharmaceutical composition for treating depression, characterized in that: The pharmaceutical composition consists of dendrobium alkaloids and / or Erianin and at least one other pharmaceutically active ingredient and / or at least one other non-pharmaceutically active ingredient.

9. The pharmaceutical composition according to claim 8, characterized in that The non-drug active ingredient is a pharmaceutically acceptable carrier or excipient.

10. A method for treating depression, characterized in that: The method comprises: administering the dendrobine and / or Erianin or the pharmaceutical composition according to any one of claims 8 to 9 to a subject.

Images