Pharmaceutical composition for treating pulmonary fibrosis complicated with depression and application thereof

Abstract

The invention discloses a pharmaceutical composition for treating pulmonary fibrosis complicated with depression and application of the pharmaceutical composition, and belongs to the field of biological medicine. The pharmaceutical composition provided by the invention comprises harmine and deoxyvasicine, wherein the optimal mass ratio of harmine to deoxyvasicine is 1: 1.25. The curative effect of the pharmaceutical composition is verified by constructing a pulmonary fibrosis and depression combined mouse model, and experimental results show that the pharmaceutical composition provided by the invention can significantly improve the anti-fibrosis effect, can also effectively reduce the neurotoxicity of harmine, shows a synergistic effect, and can be used for preparing anti-fibrosis drugs for treating or preventing the fibrosis. And the curative effect and the safety are both improved. The invention lays a foundation for developing a safe and efficient novel medicine for treating pulmonary fibrosis combined with depression, and has important application value and wide application prospect in treatment of patients with pulmonary fibrosis combined with depression.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a pharmaceutical composition for treating pulmonary fibrosis combined with depression and an application thereof. Background Art

[0002] Pulmonary fibrosis (PF) is a severe and fatal interstitial lung disease characterized by progressive disease progression and acute exacerbations. In recent years, the number of new cases and incidence of PF has continued to increase worldwide, and the 5-year survival rate for patients diagnosed is less than 30%, making it a growing public health challenge. Furthermore, multiple studies have shown that depression is closely associated with dyspnea, cough, and the severity of lung dysfunction. Epidemiological studies have shown that the prevalence of depression in patients with PF ranges from 24.3% to 49.2%. Although significant advances have been made in the clinical management of PF, with antifibrotic drugs such as pirfenidone and nintedanib helping to slow the decline in lung function, effective preventive measures for PF and depression remain lacking. Studies have reported that patients with chronic lung disease who receive antidepressant treatment have improved medication adherence, improved quality of life, and reduced mortality and rehospitalization rates. Furthermore, increasing evidence supports that improving depression can protect lung function and may help delay the progression of PF at certain stages. Therefore, some studies have shown that central nervous system neuroinflammation is believed to play a role in the pathophysiology of depression, which may also interact with the persistent inflammatory response in the lungs in pulmonary fibrosis, forming a vicious cycle. Therefore, for patients with such comorbidities, there is an urgent need to develop dual-action therapies that can simultaneously target both pulmonary fibrosis pathology and central nervous system inflammation to more comprehensively improve patient prognosis and reduce the overall disease burden.

[0003] Peganum harmala L. is the whole herb of the genus Peganum harmala, a member of the Tribulus family. It is widely available in China, primarily distributed in Xinjiang, Gansu, Ningxia, and Tibet. It is highly adaptable and can survive in a variety of harsh deserts and sandy areas. Peganum harmala has a long history as a traditional Chinese medicine, known for its cough and asthma relief, rheumatism relief, and depression-relieving properties. It is commonly used to treat a variety of ailments, including rheumatic pain, cough and asthma relief, hemiplegia, amnesia, and dizziness and headaches. Currently, over ten Chinese herbal preparations are available, including compound Peganum harmala ointment and Pegan harmala tablets.

[0004] Phytochemical studies have revealed that Pegan harmala is rich in alkaloids, primarily two types: quinazoline and β-carboline alkaloids. β-carboline alkaloids include harmaline and dehydroharmine, while quinazoline alkaloids include vasicine and deoxyvasicine. Pegan harmala alkaloids exhibit a wide range of pharmacological effects, including anti-tumor, antibacterial, antiviral, antidepressant, anti-Alzheimer's, and analgesic properties. Quinazoline alkaloids (deoxyvasicine and vasicine) also exhibit potent cholinesterase inhibitory activity. Deoxyvasicine (DVAS) is a major representative active quinazoline alkaloid in Pegan harmala. It has a relatively simple structure and belongs to the pyrroloquinazoline class of alkaloids with diverse pharmacological activities. Modern research has revealed that deoxydivasine possesses antibacterial, antitussive, antiasthmatic, and insecticidal pharmacological effects. In recent years, it has also been shown to have a variety of pharmacological effects, including antitussive and expectorant, muscle relaxant, anti-inflammatory, antioxidant, and anti-drug addiction and mental disorders. In randomized, crossover, single-blind, placebo-controlled human clinical trials, DVAS was used to treat alcohol and tobacco addiction, with a bioavailability exceeding 50%. This suggests that DVAS is an alkaloid with great potential for development and application. However, research on DVAS's anti-pulmonary fibrosis effects has not yet been reported.

[0005] However, β-carboline alkaloids themselves and their monomeric alkaloids have certain toxic effects. High doses can cause a series of side effects on the central nervous system, such as tremors and hallucinations, which limits their medical applications and clinical research. Therefore, there is an urgent need to provide a pharmaceutical composition that reduces the central nervous system toxicity of β-carboline alkaloids while enhancing their anti-pulmonary fibrosis activity. Summary of the Invention

[0006] The purpose of the present invention is to provide a pharmaceutical composition for treating pulmonary fibrosis combined with depression and its application, so as to solve the problems existing in the above-mentioned prior art. The pharmaceutical composition provided by the present invention can significantly improve the anti-fibrosis effect, and can also effectively reduce the neurotoxicity of dehydrogenated echinopsine, showing a synergistic effect, achieving a dual improvement in efficacy and safety, laying the foundation for the development of a new, safe and efficient drug for treating pulmonary fibrosis combined with depression, and has important application value and broad application prospects for the treatment of patients with pulmonary fibrosis combined with depression.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a pharmaceutical composition for treating fibrosis combined with depression. The pharmaceutical composition comprises dehydrogenated hyaluronidine and deoxyhyaluronidine.

[0009] Furthermore, the mass ratio of dehydrogenated harmaline to deoxygenated harmaline is (1-2.5):(2-3).

[0010] Furthermore, the mass ratio of dehydrogenated harmaline to deoxygenated harmaline is 1:1.25.

[0011] The present invention also provides a use of the above-mentioned pharmaceutical composition in preparing a medicine for treating fibrosis combined with depression.

[0012] Furthermore, the fibrosis is pulmonary fibrosis, liver fibrosis or kidney fibrosis.

[0013] Furthermore, the fibrosis is pulmonary fibrosis.

[0014] The present invention also provides a medicine for treating fibrosis combined with depression, which uses the above-mentioned pharmaceutical composition as a main active ingredient.

[0015] Furthermore, pharmaceutically acceptable excipients are also included.

[0016] Furthermore, the excipient is a filler, a carrier, a diluent, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

[0017] Furthermore, the dosage form of the drug is tablets, pills, powders, suspensions, emulsions, creams, granules, nanoparticles, gels, capsules, suppositories, injections, sprays, injections, oral liquids or pills.

[0018] The present invention discloses the following technical effects:

[0019] The pharmaceutical composition provided by the present invention comprises dehydrogenated echinopsine and deoxygenated echinopsine, and the combination of the two has a synergistic effect. Compared with the use of either drug alone, the daily dosage of dehydrogenated echinopsine is reduced by 50% of the original dosage, and the dosage of deoxygenated echinopsine is reduced to 37.5% of the original dosage, which greatly reduces the dosage; effectively reduces the central nervous system toxicity caused by the cumulative toxicity of long-term use of dehydrogenated echinopsine, as well as the side effects such as tremors and hallucinations caused. The pharmaceutical composition effectively improves the symptoms of depression in the process of treating pulmonary fibrosis, can improve the quality of life of patients, reduces adverse reactions during the treatment process, provides new ideas and methods for clinical treatment, and provides a more reliable treatment plan for patients with depression. The pharmaceutical composition provided by the present invention can effectively expand the user population of antidepressants and is of great significance in the clinical treatment of depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is the synergistic effect diagram of HM and DVAS on MRC-5 cells evaluated by the Synergy Finder website; A is the IC value of HM on MRC-5 cells evaluated by the Synergy Finder website 50 B is the result of evaluating the IC value of DVAS on MRC-5 cells using Synergy Finder website. 50 C and D are the results of the synergistic effect of HM and DVAS on MRC-5 cells evaluated by the Synergy Finder website;

[0022] Figure 2 Figure 2 is a graph showing the cell apoptosis rate of each group after combined administration of HM and DVAS; A is the blank control group; B is the (12.5+25) μg / mL HM and DVAS group; C is the (12.5+31.25) μg / mL HM and DVAS group; D is the (25+25) μg / mL HM and DVAS group; E is the (25+31.25) μg / mL HM and DVAS group; F is a statistical graph of the apoptosis rate of each group; *** indicates P < 0.001;

[0023] Figure 3 Comparison of cell migration between HM and DVAS; A is the comparison of cell migration between 0 h and 24 h after intervention with different drug groups; scale length is 500 μm; B is the statistical graph of cell migration rate in each group; *** indicates P < 0.001;

[0024] Figure 4 The graph shows the trend of behavioral indicators after drug administration in mice; A is the total distance the mice moved in the elevated plus maze; B is the total distance the mice moved in the open field test; C is the sugar consumption rate of the mice; D is the tail suspension immobility time of the mice; compared with the blank control group (Control), ***P<0.001; compared with the PF+DD model group, # P<0.05, ### P < 0.001;

[0025] Figure 5Fig. 1 is a graph of the changes in lung function, lung system number and hydroxyproline content of mice; wherein A is respiratory rate; B is minute ventilation; C is tidal volume; D is forced breathing gap; E is lung system number; F is hydroxyproline content; compared with the control group (control), *P<0.05, **P<0.01, ***P<0.001; compared with the pulmonary fibrosis complex depression model group (PF+DD model), # P<0.05, ## P<0.01, ### P<0.001;

[0026] Figure 6 Fig. 3 is a lung HE and MASSON pathological graph;

[0027] Figure 7 Fig. 4 is a liver and kidney HE pathological graph;

[0028] Figure 8 Fig. 5 is a statistical graph of the contents of 5-HT, CORT and dopamine in the hippocampus of mice and the contents of IL6, IL-1β and TNF-α in the brain tissue; wherein A is the content of 5-HT in the hippocampus; B is the content of dopamine in the hippocampus; C is the content of CORT in the hippocampus; D is the content of IL6 in the brain tissue; E is the content of IL-1β in the brain tissue; F is the content of TNF-α in the brain tissue; compared with the control group (control), *P<0.05, **P<0.01, ***P<0.001; compared with the pulmonary fibrosis complex depression model group (PF+DD model), # P<0.05, ## P<0.01, ### P<0.001. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present application will now be described in detail with reference to the drawings. The detailed description is not intended to limit the present application, but to explain certain aspects, features and embodiments of the present application.

[0030] It should be understood that the terms used in the present application are merely used to describe particular embodiments and are not intended to limit the present application. In addition, for the numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within the stated range and between any stated value or intermediate value within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] In previous studies, the inventors observed that treatment with the β-carboline alkaloid compound harmaline significantly improved pulmonary fibrosis pathology in patients with parasitic infections. This led the inventors to further explore the potential of various individual alkaloid compounds from the total harmal alkaloids in the treatment of pulmonary fibrosis, aiming to identify more effective alkaloids as preferred anti-fibrotic agents. To this end, the inventors constructed a pulmonary fibrosis model and conducted a pharmacodynamic comparison of β-carboline alkaloids (such as harmaline, harmalin, harmaline, and halol) and quinazoline alkaloids (e.g., trochophorine and deoxytrochophorine). The results showed that both β-carboline and quinazoline alkaloids improved pulmonary fibrosis, with harmaline and harmaline showing the most significant improvement. However, the central nervous system toxicity of β-carboline alkaloids has limited their clinical application, making it difficult to balance efficacy and safety. Therefore, the inventors used dehydrogenated harmaline combined with deoxy-merapidine to screen the appropriate ratio of the two drugs through in vitro cell experiments and in vivo animal models, verifying their synergistic and toxicity-reducing effects, ensuring that while effectively combating pulmonary fibrosis, the potential toxicity to the central nervous system was significantly reduced, so as to achieve the maximum efficacy and minimum side effects of the drug combination, and ultimately form a systematic treatment plan, which provides strong support for the clinical treatment of patients with comorbid pulmonary fibrosis and depression, and lays a solid foundation for the development of new anti-pulmonary fibrosis drugs.

[0035] The molecular formula of harmine (HM) of the present invention is C 13 H 12N2O, molecular weight is 212.25, CAS number is 442-51-3.

[0036] The molecular formula of deoxyvasicine is C 11 H 12 N2, molecular weight is 172.23, CAS number is 495-59-0.

[0037] Example 1 Cultivation of human embryonic lung fibroblasts (MRC-5) and detection of synergistic related indicators

[0038] 1. Experimental Materials

[0039] Human embryonic lung fibroblast MRC-5 cells (Pnosai Life Science Co., Ltd., Wuhan, China), fetal bovine serum (Thermo Fisher, USA), trypsin (Sigma-Aldrich, USA), PBS, MEM cell culture medium (Gibco, USA), TGF-β1 (Yun-Clone Biotechnology Co., Ltd., Wuhan, China).

[0040] 2. MRC-5 cell culture and stimulation experiments

[0041] MRC-5 cells were cultured in MEM medium containing 1% double antibody and 10% fetal bovine serum in a 37°C constant temperature incubator. When the cell confluence was about 80%, the cells were passaged at a ratio of 1:2.

[0042] After conventional culture of MRC-5 cells, cells in logarithmic growth phase were collected and 1×10 4 Cells were seeded in 96-well culture plates, and a culture medium group (Control) and a 5 ng / mL TGF-β1 stimulation group (model group) were set up for 24 h drug response, and photos were taken and recorded.

[0043] 2.1 Using CCK-8 assay to detect the optimal concentration of drug

[0044] Cells were plated at 5 × 10 3The cells were inoculated in 96-well plates and cultured at 37°C in a 5% CO2 cell incubator for 12 h to adhere to the wall. The cells were divided into five groups, namely a control group (containing only complete culture medium), a TGF-β1 group (5 ng / mL TGF-β1 for cell induction), a TGF-β1 + HM group (5 ng / mL TGF-β1 induction + HM at concentrations of 0, 1.0, 10.0, 20.0, 40.0, 60.0, and 80.0 μg / mL, respectively), a TGF-β1 + DVAS group (5 ng / mL TGF-β1 induction + DVAS at concentrations of 0, 1.0, 10.0, 20.0, 40.0, 60.0, and 80.0 μg / mL, respectively), and different concentration groups of HM and DVAS for determining the optimal application concentration of dehydropeimine combined with deoxyvibrin. In addition, blank wells without cells were set as a blank group. Dehydropeimine and deoxyvibrin were dissolved in dimethyl sulfoxide (DMSO) to prepare a 5 mg / mL stock solution. In the experiment, the dehydropeimine and deoxyvibrin stock solution was diluted with MEM complete medium to the required concentration, 100 μL of the drug was added to each well for 24 h, 10 μL of CCK-8 solution was added to each well for 4 h, and the optical density (OD) value at 450 nm wavelength was determined by an enzyme-labeled instrument. The cell viability was calculated according to the following formula:

[0045] Cell viability (%) = (OD of experimental group 450 - OD of blank group 450 ) / (OD of control group 450 - OD of blank group 450 ) x 100%.

[0046] In the above formula, the experimental group is the model group and the drug administration group. The above values were introduced into the Synergy Finder website for analysis of the combined application of the two drugs.

[0047] According to the half-inhibitory concentration value and the analysis of the combined application of HM and DVAS by the Synergy Finder website, the synergistic concentrations of (12.5 + 25), (12.5 + 31.25), (25 + 25), and (25 + 31.25) μg / mL of HM and DVAS in the synergistic ratio were selected for the study of TGF-β1-induced MRC-5 cell apoptosis and migration.

[0048] The results showed that TGF-β1 stimulation was effective, and the presence of TGF-β1 was beneficial to cell proliferation; compared with the TGF-β1-induced model group, the proliferation rate of the model cells in each drug administration group gradually decreased with the increase of the concentration of HM and DVAS, indicating that the proliferation of MRC-5 cells was inhibited in the presence of HM and DVAS, and the IC 50The values ​​were 28.11μg / mL and 32.16μg / mL, respectively, and this inhibition occurred in a concentration-dependent manner. Figure 1 A and B.

[0049] Different concentrations of HM and DVAS were added to TGF-β1-induced MRC-5 cells simultaneously. After 24 hours of treatment, the cell viability was detected using CCK-8. The above values ​​were imported into the Synergy Finder website and found that the ZIP score of the two drugs was 16.114 (>10), indicating that the combined use of HM and DVAS has a synergistic effect. When the two drugs are used in combination, the optimal concentrations of HM and DVAS are 12.5-31.25μg / mL and 25-37.25μg / mL, respectively. Figure 1 C and D).

[0050] 2.2 Flow cytometry (Annexin / PI method) to verify the cell apoptosis rate after combined administration

[0051] The synergistic concentrations of HM and DVAS at (12.5+25), (12.5+31.25), (25+25), and (25+31.25) μg / mL in the above synergistic ratios were selected to intervene in MRC-5 cells induced by TGF-β1 for 24 hours. The MRC-5 cells in each group were washed with pre-cooled PBS, centrifuged at 613g for 10 minutes at 4°C, and the supernatant was discarded. Binding Buffer (500 μL) was added to suspend the cells, and 5 μL Annexin V-FITC and 5 μL PI were added in turn. After thorough mixing, the cells were incubated in the dark for 30 minutes, and the apoptosis rate of each group was detected by flow cytometry.

[0052] The experimental results showed that after the intervention of MRC-5 cells with the synergistic concentrations of HM and DVAS (12.5+25), (12.5+31.25), (25+25), and (25+31.25) μg / mL, the combination of HM and DVAS at the four synergistic concentrations had a significant inhibitory effect on the proliferation of MRC-5 cells (all P<0.001), among which the combined apoptosis rate of (25+31.25) μg / mL was the highest (87.22%). Figure 2 The results showed that the synergistic effect of dehydrogenated echinopsine and deoxygenated echinopsine was the best at a ratio of 1:1.25, significantly reducing the survival rate of model cells.

[0053] 2.3 Scratch assay to verify the effect of drugs on cell migration rate

[0054] MRC-5 cells in the logarithmic growth phase were collected and 1×10 4Cells were inoculated into 12-well plates with 10 μL of PBS per mL and cultured for 24 h. When the cells were fused to 80% or more, they were vertically streaked with a 10 μL pipette tip and washed three times with PBS. 2 mL of serum-free culture medium was added and the cells were treated with TGF-β1 (5 ng / mL) and a final concentration of 25 μg / mL HM, 31.25 μg / mL DVAS, and a synergistic concentration of (25+31.25) μg / mL HM and DVAS. The migration status and scratch status of each group of cells at 0 h and 24 h after scratching were recorded under a microscope. Image J software was used to analyze the scratch area and calculate the cell migration rate. The cell migration rate was calculated according to the following formula:

[0055] Cell migration rate (%) = (0 h scratch area - 24 h scratch area) / 0 h scratch area × 100%.

[0056] The results are as follows Figure 3 As shown in Figures A and B, under the induction of TGF-β1, MRC-5 cells were treated with the optimal HM concentration obtained from the CCK-8 assay. Cell scratches were observed 24 hours after treatment. After 24 hours of TGF-β1 induction, cells showed significant migration, with shortened scratch spacing and a migration rate of 61.34% (P < 0.001). However, after treatment with HM, DVAS, or HM+DVAS, cell migration was inferior to that in the TGF-β1 group, with wider scratch spacing compared to the TGF-β1 group. The 24-hour cell migration rate decreased by 20.06%, 14.34%, and 43.07%, respectively, compared to the TGF-β1 group (all P < 0.001). These results indicate that all treatments significantly inhibited the migration of the model cells, with HM+DVAS providing the greatest effect.

[0057] Example 2 Animal experiment verifies the effect of a mixed drug of dehydrogenated hyaluronic acid and deoxyhyaluronic acid on the intervention of pulmonary fibrosis combined with depression

[0058] 1. Experimental Materials

[0059] 1.1 Experimental Animals

[0060] 30 SPF male C57BL / 6 mice, 5 weeks old, weighing (20±2) g, were provided by the Animal Experiment Research Center of Xinjiang Medical University, with production license number SCXK(Xin)2018-0002. They were raised in single cages at a temperature of 22-24°C and a humidity of 50%±10%, with free access to water and food. The light / dark cycle followed a circadian light cycle of 12h / 12h, and the experiment began after the mice were adapted to feeding for 1 week. The animal experiments in this invention were formally approved by the Ethics Committee of the Animal Experiment Research Center of Xinjiang Medical University (approval number IACUC-20230321-54), and the experimental operations complied with animal welfare and ethical standards.

[0061] 1.2 Instruments

[0062] 1658001 electrophoresis apparatus, electrotransformation apparatus (Bio-Rad, USA); GelDoc XR gel imaging system (Bio-Rad, USA); Open field test (Model ZH-OFT), Tail suspension test (Model TST-100) instruments purchased from Anhui Zhenghua Biological Instrument Equipment Co., Ltd.;

[0063] 1.3 Reagents

[0064] Cell lysis solution (batch number A027241016), PMSF (batch number 20240116), BCA protein quantification kit (batch number WH333438), Marker (batch number MPC2310002), goat anti-mouse IgG (batch number BB12191995), goat anti-rabbit IgG (batch number AI09236261), beta-Actin (batch number BJ11164652) were purchased from Beijing Boao Sun Biotechnology Co., Ltd.; ECL chemiluminescence substrate kit (ultra-sensitive) (batch number 30424029AZ).

[0065] 2. Construction of a disease animal model of pulmonary fibrosis combined with depression

[0066] Male C57BL / 6 mice were randomly divided into 6 as a blank control group (control) without stimulation and 24 for pulmonary fibrosis combined with depression modeling. Pulmonary fibrosis combined with depression modeling first used 5 μg / kg bleomycin intranasal instillation to construct a pulmonary fibrosis model; 2 days after bleomycin intranasal instillation, a chronic unpredictable mild stress (CUMS) method was used to establish a depression animal model, and the specific method was as follows: C57BL / 6 mice were adaptively fed in the laboratory for 1 week, and then single-caged and given different stress stimuli to the mice every day. The stress stimuli included: ① intermittent electric stimulation (voltage 36 V, current 0.1 A, 5 min); ② tail clamping (wooden clamp clamping tail, 1 cm from tail end, lasting 2 min); ③ oscillation for 5 min; ④ light stimulation (strong light frequency flash, lasting 10 min); ⑤ fasting for 24 h; ⑥ water deprivation for 24 h; ⑦ day-night reversal (light blocking for 12 h during the day, lighting for 12 h at night); ⑧ sound stimulation, intermittent noise stimulation (80 dB); ⑨ wet bedding for 24 h. Nine kinds of stimulation, each mouse was randomly given one kind of stimulation every day, and the order was randomly arranged every week, and each kind of stimulation was used no more than 5 times in total. The stimulation was carried out at 10:00-14:00 every day, and the continuous stress stimulation lasted for 4 weeks. The blank control group mice did not receive stress treatment.

[0067] 3. Behavioral experiments

[0068] The 24 modeling mice were randomly divided into four groups, each consisting of six mice (n=6): a PF+DD model group, a 25 mg / kg HM group, a 31.25 mg / kg DVAS group, and a 25 mg / kg HM + 31.25 mg / kg DVAS group. Except for the PF+DD model group, all drug-treated groups received oral administration of the corresponding drug according to the dose. During the experiment, the body weight, diet, and activity of all mice were recorded daily, and their mental state and behavioral changes were observed.

[0069] 3.1 Sucrose preference test (SPT)

[0070] After the CUMS stress, the mice in each group were subjected to SPT, and the volumes of sucrose solution and pure water consumed were recorded. The consumption and proportion of sucrose solution were analyzed, and the sugar preference index was calculated according to the following formula:

[0071] Sugar water preference index = sugar water consumption / (sugar water consumption + pure water consumption) × 100%.

[0072] 3.2 Open field test (OFT)

[0073] After placing the mouse in the center of the open field, the mouse's activity was observed within 5 minutes, and the total distance the mouse moved in the open field was analyzed.

[0074] 3.3 Elevated plus maze (EPM)

[0075] The mice were placed into the maze from the central grid facing the open arms, and their activities were recorded within 5 minutes, and the total distance moved by the mice in the open arms was analyzed.

[0076] 3.4 Tail suspension test (TST)

[0077] The posterior 1 / 3 of the mouse's tail was fixed with tape and hung on a stand. The mouse's activity was observed within 5 minutes and the immobility time was analyzed.

[0078] 4. Experimental Results

[0079] like Figure 4As shown in Figures AD, after model establishment and drug treatment, compared with the blank control group, the total distance moved in the elevated plus maze, the total distance moved in the open field test, and the sugar water consumption rate of mice in the pulmonary fibrosis combined depression model group were significantly decreased (P < 0.001), and the tail suspension immobility time of mice was significantly increased (P < 0.001). Compared with the pulmonary fibrosis combined depression model group, the sugar water consumption rate, the total distance moved in the elevated plus maze, and the total distance moved in the open field test of mice in the 25mg / kg HM group, the 31.25mg / kg DVAS group, and the 25mg / kg HM + 31.25mg / kg DVAS group were significantly increased (P < 0.001); the tail suspension immobility time was significantly decreased (P < 0.01, P < 0.001). These experimental results show that the 25mg / kg HM group and the 25mg / kg HM + 31.25mg / kg DVAS group can significantly improve the depressive behavior of mice.

[0080] Example 3 Pulmonary function test, lung coefficient and lung tissue hydroxyproline level test of mice

[0081] 1. Animal Grouping and Dosing Schedule

[0082] Mice were randomly divided into five groups: a control group, a pulmonary fibrosis combined with depression model group (PF+DD model, modeling method as in Example 2), a 25 mg / kg HM group, a 31.25 mg / kg DVAS group, and a 25 mg / kg HM+31.25 mg / kg DVAS group, with 6 mice in each group (n=6). HM was dissolved in 0.5% CMC-Na (sodium carboxymethylcellulose) solution. The control group and the PF+DD model group were given an equal amount of 0.5% CMC-Na solution for 14 days. The drug-treated group was orally administered with the corresponding dose of drug for 14 consecutive days. Body weight was measured every 3 days. All operations strictly adhered to ethical standards.

[0083] 2. Pulmonary function test

[0084] On day 14 of the experiment, six mice from each group were placed in the plethysmograph chamber of a noninvasive pulmonary function testing system for three consecutive days. All procedures were performed according to the EMMSLink WBP system's operating standards. Pulmonary function parameters for each mouse, including respiratory rate (frequenc, f), tidal volume (TVb), minute ventilation (MVb), and forced breathing gap (Penh), were averaged over the three days for statistical analysis.

[0085] 3. Lung coefficient test

[0086] After three consecutive days of lung function testing (day 17), mice were weighed after fasting for 12 hours. Blood was collected from the eyeballs and the lungs were harvested and weighed. The lung coefficient was calculated according to the following formula, and the harvested organs were stored in liquid nitrogen until further use.

[0087] Lung coefficient = lung mass / mouse body mass before the last administration × 100%.

[0088] 4. Detection of lung tissue hydroxyproline levels by alkaline hydrolysis

[0089] The hydroxyproline (HYP) content in lung tissue was determined by alkaline hydrolysis using a commercially available hydroxyproline assay kit. According to the instructions, approximately 80 mg (wet weight) of fresh left upper lung lobe of mice was accurately weighed and placed in a test tube. Four mice were included in each group. 1 mL of hydrolysis solution was added to the test tube and boiled at 100°C for 20 minutes to perform the hydrolysis reaction. The pH of the hydrolyzate was then adjusted to 6.0-6.8 and centrifuged at 5000 g for 10 minutes. 1 mL of the supernatant was added to a new test tube, and chloramine-T and dimethylaminobenzaldehyde were added successively. Subsequently, the hydroxyproline content in the lung tissue was assessed by recording the absorbance at 550 nm. The hydroxyproline content in the mouse lung tissue was calculated, and the results were expressed as μg HYP / mg wet lung.

[0090] 5. Experimental Results

[0091] The main characteristics of PF are the abnormal accumulation of fibrous tissue in the lung parenchyma, the replacement of healthy tissue with altered extracellular matrix, and the destruction of alveolar structure, leading to decreased lung function and ultimately respiratory failure and death.

[0092] like Figure 5 As shown in Figures AD, the results showed that multiple lung function indicators were decreased in mice with bleomycin-induced pulmonary fibrosis. Compared with the control group, the PF+DD model group had significantly decreased tidal volume (TV) and minute expiratory volume (MV), and significantly increased respiratory rate (f) and forced respiratory interval (Penh) (P<0.001). After treatment with HM, DVAS, or HM+DVAS, the respiratory function of mice was significantly improved. Compared with the PF+DD model group, each drug group had significantly increased tidal volume (TV) and minute expiratory volume (MV), and decreased respiratory rate (f) and forced respiratory interval (Penh).

[0093] Changes in lung coefficients during the study period Figure 5As shown in Figure E, compared with the control group, the lung coefficient of mice in the PF+DD model group was significantly increased (P<0.001). Compared with the PF+DD model group, the 25mg / kg HM group, the 31.25mg / kg DVAS group, and the 25mg / kg HM+31.25mg / kg DVAS group all significantly reduced the lung coefficient of mice (P<0.01 or P<0.001), with the 25mg / kg HM+31.25mg / kg DVAS group showing a more pronounced effect. This indicates that drug treatment can effectively improve the increased lung coefficient in mice with pulmonary fibrosis and alleviate BLM-induced pulmonary fibrosis in mice.

[0094] The results of the determination of hydroxyproline content in mouse lung tissue are as follows Figure 5 As shown in Figure F, compared with the control group (0.67±0.03μg / mg), the hydroxyproline content in the lung tissue of mice in the PF+DD model group (1.31±0.05μg / mg) was significantly increased (P<0.001). After treatment, the hydroxyproline content in the 25mg / kg HM group, the 31.25mg / kg DVAS group, and the 25mg / kg HM+31.25mg / kg DVAS group were 1.05±0.11μg / mg, 1.18±0.07μg / mg, and 0.83±0.04μg / mg, respectively. Compared with the PF+DD model group, all drug-treated groups were able to significantly reduce the hydroxyproline content (P<0.001), with the 25mg / kg HM+31.25mg / kg DVAS group having a more pronounced effect. This shows that HM combined with DVAS can effectively reduce the collagen content in lung tissue.

[0095] The above results indicate that HM treatment can significantly improve the decreased lung function of PF mice and effectively reduce the collagen content in lung tissue.

[0096] Example 4 Pathological Observation of Mouse Visceral Tissue

[0097] The mice in each group in Example 3 were killed, and the right upper lobe of the lung, liver, and kidney of the mice were selected, with 3 mice in each group. They were immediately immersed in 4% paraformaldehyde and fixed for 24 hours, dehydrated, transparent, and paraffin-embedded. The slices were cut into 4 μm thick sections, and then fixed on slides and stained with hematoxylin-eosin (HE) and Masson, and the sections were mounted for pathological observation under a light microscope.

[0098] HE and Masson staining results of lungs Figure 6As shown. HE staining results showed that the alveolar structure of the control group was clear and complete, the alveolar wall was smooth and not thickened, and there was no inflammatory cell infiltration in the pulmonary interstitium; compared with the control group, the lung tissue structure of the mice in the model group was obviously disordered, collapsed or fused, the alveolar wall was significantly thickened, and inflammatory cell infiltration was visible in the interstitium; after drug intervention in each group, bleomycin could still induce the above-mentioned damage to the lung tissue, but the degree was significantly reduced. Masson staining results showed that the blue part of the model group had significantly more collagen fibers than the control group, and the collagen fiber deposition was significantly reduced after drug administration. The results showed that the 25mg / kg HM+31.25mg / kg DVAS group could significantly improve the pathological damage of mice with pulmonary fibrosis.

[0099] The above research results show that HM alleviates bleomycin-induced pulmonary fibrosis in mice by improving lung function and reducing tissue fibrosis.

[0100] The results of liver HE staining are as follows Figure 7 As shown in the data, the model mice had structural abnormalities compared with the control group, with infiltration of inflammatory cells in the liver portal area and congestion in the portal area. After treatment with the 25 mg / kg HM group, the 31.25 mg / kg DVAS group, and the 25 mg / kg HM + 31.25 mg / kg DVAS group, the degree of inflammatory response in the liver tissue showed a downward trend, congestion in the portal area was alleviated, and the liver cell structure gradually recovered. The effect of the 25 mg / kg HM + 31.25 mg / kg DVAS group was more obvious, indicating that the combination of HM and DVAS can effectively alleviate liver tissue damage.

[0101] Kidney HE staining results are as follows Figure 7 As shown in the data, compared with the control group, the model group had abnormal kidney structure, obvious congestion of glomeruli and renal interstitium, and a large number of pathological inflammatory cells infiltrated; after drug treatment in the 25mg / kg HM group, 31.25mg / kg DVAS group, and 25mg / kg HM+31.25mg / kg DVAS group, there were no obvious lesions in the glomeruli and renal tubules of the mice in the treatment group, and there was no interstitial congestion or bleeding. The effect of the 25mg / kg HM+31.25mg / kg DVAS group was significant, indicating that the combination of HM and DVAS has a significant therapeutic effect in protecting kidney structure and reducing inflammatory response.

[0102] Example 5 Kit method was used to detect the levels of 5-HT, CORT, dopamine, IL6, IL-1β and TNF-α in the hippocampus tissue of each group of mice

[0103] 1. Experimental Methods

[0104] Take the hippocampal tissue of each group of mice in Example 3, 6 mice per group, weigh them and place them in EP tubes, add 9 times the weight of the tissue in normal saline, grind with a high-speed low-temperature tissue ball mill, obtain hippocampal tissue homogenate, and let the homogenate sample stand for 1 hour and centrifuge. The protein concentration was determined using a BCA protein quantification kit, and the content of 5-HT, CORT, DA, IL6, IL-1β and TNF-α was detected according to the kit instructions. The operation steps were carried out according to the kit instructions. The OD value of each well was read on a microplate reader with a wavelength of 450nm. The experiment was repeated 3 times and the average value was calculated.

[0105] 2. Experimental Results

[0106] The results are as follows Figure 8 As shown in Figures AF, compared with the control group, the IL6, IL-1β and TNF-α contents in the brain tissue of mice in the PF+DD model group were increased (P<0.001), the CORT content in the hippocampus of mice was significantly increased (P<0.001), and the 5-HT and dopamine contents in the hippocampus of mice were significantly decreased (P<0.001); compared with the PF+DD model group, the CORT content in the hippocampus of mice was significantly decreased in the 25 mg / kg HM group, the 31.25 mg / kg DVAS group, and the 25 mg / kg HM+31.25 mg / kg DVAS group after drug treatment, and the IL6, IL-1β and TNF-α contents in the brain tissue of mice were decreased, and the effect was most significant in the 25 mg / kg HM+31.25 mg / kg DVAS group, indicating that the combination of HM and DVAS has a significant therapeutic effect in protecting brain neural activity and alleviating inflammatory response.

[0107] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A pharmaceutical composition for treating fibrosis combined with depression, characterized in that: The pharmaceutical composition comprises dehydrogenated hyaluronic acid and deoxyhyaluronic acid.

2. The pharmaceutical composition according to claim 1, wherein The mass ratio of dehydroharmine to deoxyharmine is (1-2.5):(2-3).

3. The pharmaceutical composition according to claim 2, wherein The mass ratio of dehydroharmaline to deoxymerpine is 1:1.

25.

4. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a medicament for treating fibrosis combined with depression.

5. The use according to claim 4, characterized in that The fibrosis is pulmonary fibrosis, liver fibrosis or kidney fibrosis.

6. The use according to claim 5, characterized in that The fibrosis is pulmonary fibrosis.

7. A drug for treating fibrosis combined with depression, characterized in that: The pharmaceutical composition according to any one of claims 1 to 3 is used as the main active ingredient.

8. The drug according to claim 7, wherein Pharmaceutically acceptable excipients are also included.

9. The drug according to claim 8, wherein The auxiliary materials are fillers, carriers, diluents, adhesives, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

10. The drug according to claim 7, wherein The dosage form of the drug is tablets, pills, powders, suspensions, emulsions, creams, granules, nanoparticles, gels, capsules, suppositories, injections, sprays, injections, oral liquids or pills.

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