Mixed gas as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antidepressant drugs, and particularly relates to mixed gas as well as a preparation method and application thereof. The invention provides a mixed gas which comprises hydrogen, oxygen and nitrogen, the volume concentration of the hydrogen is 2%-3%, the volume concentration of the oxygen is 21%, and the volume concentration of the nitrogen is 76%-77%. After the mixed gas disclosed by the invention is inhaled, the gas capable of penetrating through the blood brain barrier directly reaches central nervous unit cells, so that the mixed gas takes effect quickly; in addition, the hydrogen, the oxygen and the nitrogen in the mixed gas are wide in preparation source, low in cost, free of side effects on organisms, high in safety and free of influence on daily activities. The mixed gas can improve the mitochondrial function, improve neuron damage of the hippocampus and effectively prevent the progress of depression.

Description

Technical Field

[0001] The invention belongs to the technical field of antidepressant drugs, and in particular relates to a mixed gas and a preparation method and application thereof. Background Art

[0002] Major depressive disorder (MDD) is a systemic disease that crosses organelles, tissues, and organ systems. The main symptoms are emotional indifference, anhedonia, sleep disorders, and reduced social interaction. In severe cases, it can lead to suicidal behavior. The treatment of depression has always been a difficult problem to solve in the medical community. Currently, the treatment methods are limited, mainly including drug therapy and psychotherapy. Existing drugs cannot prevent the recurrence of depression, and the adverse reactions of antidepressants are large. Almost all antidepressant Western medicines have adverse reactions to varying degrees. Long-term use of drugs also has certain damage to the gastrointestinal tract and liver, which leads to the problem of difficulty in adhering to medication, and also affects the patient's work ability and quality of life. In addition, psychological counseling and other methods impose a large economic burden on patients and are prone to more psychological pressure. Therefore, there is an urgent need to seek a safe and effective antidepressant therapy. Summary of the invention

[0003] The purpose of the present invention is to provide a mixed gas and a preparation method and application thereof. The mixed gas of the present invention has a wide source of preparation and low cost. After inhalation, it can penetrate the blood-brain barrier and directly reach the central neuron cells, slowing down the pathological changes of depression, effectively preventing the progression of depression, and does not affect daily activities and has no side effects.

[0004] The present invention provides a mixed gas, which comprises hydrogen, oxygen and nitrogen. The volume concentration of the hydrogen is 2% to 3%, the volume concentration of the oxygen is 21%, and the volume concentration of the nitrogen is 76% to 77%.

[0005] Preferably, the volume concentration of the hydrogen is 2%.

[0006] The present invention also provides a method for preparing the mixed gas described in the above scheme, comprising the following steps: preparing oxygen by liquefaction separation method, preparing nitrogen by air separation method, and preparing hydrogen by water electrolysis method;

[0007] The hydrogen, oxygen and nitrogen are mixed uniformly in proportion to obtain a mixed gas.

[0008] The present invention also provides the use of the mixed gas described in the above scheme or the mixed gas obtained by the preparation method described in the above scheme in the preparation of antidepressant drugs.

[0009] Preferably, the antidepressant includes one or more of improving mitochondrial damage in the hippocampus, improving neuronal damage in the hippocampus, increasing the level of neurotransmitter secretion, inhibiting oxidative stress and inhibiting inflammatory response.

[0010] Preferably, the improvement of mitochondrial damage in the hippocampus includes increasing the number of mitochondria in the hippocampus and / or increasing the mitochondrial autophagy flux in the hippocampus.

[0011] Preferably, the increasing the number of mitochondria in the hippocampus includes increasing the expression of mitochondrial-related proteins; the mitochondrial-related proteins include one or more of the mitochondrial biogenesis regulatory factor PGC-1α, the mitochondrial fission protein Drp1, and the mitochondrial fusion protein Mfn2;

[0012] The increasing the mitochondrial autophagy flux in the hippocampus includes increasing the expression levels of mitochondrial autophagy-related proteins Pink1 and / or Parkin.

[0013] Preferably, the neurotransmitter comprises serotonin.

[0014] Preferably, the inhibition of inflammatory response includes reducing the expression of inflammatory factors and / or microglia.

[0015] Preferably, the inflammatory factors include tumor necrosis factor α and / or interleukin 1-β.

[0016] The present invention provides a mixed gas, which includes hydrogen, oxygen and nitrogen, wherein the volume concentration of the hydrogen is 2% to 3%, the volume concentration of the oxygen is 21%, and the volume concentration of the nitrogen is 76% to 77%. The mixed gas of the present invention is a gas that can cross the blood-brain barrier and directly reach the central neuron cells, and takes effect quickly; moreover, the hydrogen, oxygen and nitrogen in the mixed gas are stable in nature, and the preparation sources are wide, and they will not produce side effects on the body, and they are highly safe and will not affect daily activities. The mixed gas of the present invention can improve mitochondrial function, improve hippocampal damage, and effectively prevent the progression of depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 This is a graph showing the changes in rat body weight in Application Example 1. In the graph, **** indicates p < 0.0001, #### indicates p < 0.0001, and ns indicates no significant difference;

[0019] Figure 2 This is the data graph of the rat sucrose preference rate in Application Example 1. In the picture, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates no significant difference;

[0020] Figure 3 This is the behavioral diagram of the rat open box experiment in Application Example 1;

[0021] Figure 4 This is a graph of the immobility time of rats in the open box test in Application Example 1. In the graph, * indicates p < 0.05, **** indicates p < 0.0001, and ns indicates no significant difference;

[0022] Figure 5 This is the behavioral graph of the rat elevated plus maze test in Application Example 1;

[0023] Figure 6 This is a graph of the open arm / closed arm residence time of the rat elevated plus maze test in Application Example 1. In the graph, ** indicates p < 0.01, **** indicates p < 0.0001, and ns indicates no significant difference;

[0024] Figure 7 This is the Nissl staining image of rat in Application Example 1;

[0025] Figure 8 This is a graph of the neurotransmitter levels in rats in Application Example 2, where the left graph is 5-hydroxytryptamine and the right graph is tryptophan. In the graph, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no significant difference;

[0026] Fig. 9 This is the expression level of oxidative stress in rats in Application Example 2, where the left side is the hydrogen peroxide level, and the right side is the catalase level. In the figure, * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001, and ns indicates no significant difference;

[0027] Fig.10 This is a graph of the inflammatory factor content of rats in Application Example 2, where the left graph is tumor necrosis factor, and the right graph is interleukin-1β. In the graph, * indicates p < 0.05, and **** indicates p < 0.0001;

[0028] Fig.11 This is the protein expression diagram of iba-1, a rat microglia marker, in Application Example 2;

[0029] Fig.12 This is a schematic diagram of the relative expression of iba-1, a marker of rat microglia, in Application Example 2. In the figure, * indicates p < 0.05, and ns indicates no significant difference;

[0030] Fig.13 This is the transmission electron microscope image of rats in Application Example 2;

[0031] Fig.14This is the graph of adenosine triphosphate content in rats in Application Example 2. In the graph, **** indicates p < 0.0001, and ns indicates no significant difference;

[0032] Fig.15 This is the expression diagram of rat mitochondria-related proteins in Application Example 2, where the left figure is the mitochondrial biogenesis regulatory factor PGC-1α, and the right figure is the rat Bcl-2 homology domain protein Beclin-1. In the figure, * indicates p < 0.05, ** indicates p < 0.01, and ns indicates no significant difference;

[0033] Fig.16 This is the expression diagram of mitochondrial-related proteins in rats in Application Example 2, where the left diagram is mitochondrial dynamin-related protein Drp1, and the right diagram is mitochondrial fusion protein Mfn2. In the diagram, * indicates p < 0.05, and ns indicates no significant difference;

[0034] Fig.17 This is the expression diagram of rat mitochondria-related proteins in Application Example 2, where the left figure is the mitochondrial autophagy-related protein Pink1, and the right figure is the mitochondrial autophagy-related protein Parkin. In the figure, * indicates p < 0.05, and ns indicates no significant difference. DETAILED DESCRIPTION

[0035] The present invention provides a mixed gas, which comprises hydrogen, oxygen and nitrogen. The volume concentration of the hydrogen is 2% to 3%, the volume concentration of the oxygen is 21%, and the volume concentration of the nitrogen is 76% to 77%.

[0036] The volume concentration of hydrogen in the present invention is preferably 2%. After the mixed gas of the present invention is inhaled, the gas can cross the blood-brain barrier and directly reach the central neuron cells, and the effect is rapid; moreover, the hydrogen, oxygen and nitrogen in the mixed gas are prepared from a wide range of sources, will not produce side effects on the body, are highly safe, and do not affect daily activities.

[0037] The present invention also provides a method for preparing the mixed gas described in the above scheme, comprising the following steps: preparing oxygen by liquefaction separation method, preparing nitrogen by air separation method, and preparing hydrogen by water electrolysis method; mixing the hydrogen, oxygen and nitrogen uniformly in proportion to obtain a mixed gas. In the present invention, the uniform mixing method preferably includes charging the hydrogen, oxygen and nitrogen into a steel cylinder in proportion; the pressure of the mixed gas in the steel cylinder is preferably 9.5-10.5MPa, and more preferably 10MPa. The present invention preferably uses an industrialized liquefaction separation method to prepare hydrogen. The present invention prepares hydrogen, oxygen and nitrogen separately and then mixes them to prepare a mixed gas. Compared with the currently commonly used water decomposition hydrogen supply method, the cost is low, and the risk of explosion caused by excessive hydrogen concentration can be avoided, and the pressure of the steel cylinder is limited, which is conducive to the storage of the mixed gas.

[0038] The present invention also provides the use of the mixed gas described in the above scheme and the mixed gas obtained by the preparation method of the above scheme in the preparation of antidepressant drugs. The antidepressant of the present invention preferably includes improving mitochondrial damage in the hippocampus, improving neuronal damage in the hippocampus, increasing the level of neurotransmitter secretion, inhibiting oxidative stress and inhibiting inflammatory response. The improvement of hippocampal mitochondrial damage in the present invention preferably includes increasing the number of mitochondria in the hippocampus and / or increasing the mitochondrial autophagy flux; the increase in the number of mitochondria in the hippocampus preferably includes increasing the expression of mitochondrial related proteins; the mitochondrial related proteins preferably include one or more of mitochondrial biosynthesis regulatory factor PGC-1α, mitochondrial fission protein Drp1 and mitochondrial fusion protein Mfn2; the increase in hippocampal mitochondrial autophagy flux preferably includes increasing the expression of mitochondrial autophagy related proteins Pink1 and / or Parkin. The neurotransmitter of the present invention preferably includes 5-hydroxytryptamine; the inhibition of inflammatory response preferably includes reducing the expression of inflammatory factors and / or microglia; the inflammatory factors preferably include tumor necrosis factor α and / or interleukin 1-β. After the mixed gas in the present invention is inhaled, hydrogen can effectively improve the depressive-like behavior and hippocampal neuron damage of CUMS rats, and has an improving effect on the oxidative stress level and inflammatory response of CUMS rats; at the same time, hydrogen improves the number, morphological structure and functional damage of mitochondria in the hippocampus of CUMS rats, increases mitochondrial production capacity, and improves mitochondrial autophagy flux; it can restore the tryptophan and metabolite concentrations of CUMS rats, effectively regulate the disorder of neurotransmitter levels, and achieve anti-depression in a variety of ways.

[0039] In order to further illustrate the present invention, a mixed gas provided by the present invention and its preparation method and application are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] Beijing Xenon Heyu Gas Technology Co., Ltd. was commissioned to prepare oxygen using a liquefaction separation method; nitrogen using an air separation method; and hydrogen using a water electrolysis method. The hydrogen, oxygen, and nitrogen were charged into a 40L steel cylinder at a final volume concentration of 2% hydrogen, 21% oxygen, and 77% nitrogen, and mixed to obtain a mixed gas, the pressure of which was 10MPa.

[0042] Comparative Example 1

[0043] Oxygen is prepared by a liquefaction separation method; nitrogen is prepared by an air separation method; hydrogen is prepared by a water electrolysis method, and the hydrogen, oxygen and nitrogen are charged into a 40L steel cylinder at a final volume concentration of 1% hydrogen, 21% oxygen and 78% nitrogen, and mixed to obtain a mixed gas, the pressure of which is 10MPa.

[0044] Application Example 1

[0045] 1) Preparation of CUMS (Chronic Mild Unpredictable Stress) Model Rats

[0046] Experimental animals: 48 male SD rats, 6 weeks old, weighing 220±20 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd., Beijing, China. The breeding environment was constant temperature and humidity (temperature 22±2°C, humidity 50-60%), ventilation conditions were 8-15 times / h, and free food was available.

[0047] After one week of adaptive feeding, 8 SD rats were randomly selected as the control group and raised normally. Except for the control group, the remaining SD rats were raised in single cages and subjected to CUMS stress for 8 weeks. The CUMS stress included ① restraint for 2 hours; ② 6 foot shocks, 1.0 mA AC electric shock to the soles of the rats, each lasting 30 seconds, and 1 stimulation every 1 minute; ③ tail clamping for 1 minute; ④ wet bedding for 12 hours; ⑤ fasting for 12 hours; ⑥ water deprivation for 12 hours; ⑦ cage tilt for 12 hours; ⑧ continuous lighting overnight for 12 hours; ⑨ vinegar or pepper and other odor stimulation for 4 hours, a total of 9 stimulation methods, one of which was randomly selected every day and different for two consecutive days.

[0048] Four weeks after modeling, the sucrose preference test (SPT) was used to screen the CUMS model rats with successful modeling. The method was referred to [Chen B, Jin K, Dong J, Cheng S, Kong L, Hu S, Chen Z, Lu J.Hypocretin-1 / HypocretinReceptor 1Regulates Neuroplasticity and Cognitive Function throughHippocampal Lactate Homeostasis in Depressed Model. Adv Sci (Weinh). 2024Aug 9:e2405354.doi:10.1002 / advs.202405354.Epub ahead of print.PMID:39119889.].

[0049] 2) Group processing

[0050] Forty CUMS model rats were randomly divided into model group, hydrogen group, hydrogen control group 1, hydrogen control group 2 and positive drug group, with 8 rats in each group. The rats in each group were placed in an animal gas control system box (BD1202390003, Huayue Medical Technology Co., Ltd., Guangzhou, China). While the stress continued, corresponding treatment measures were given.

[0051] Hydrogen group, denoted as CUMS+H 2(4h, 2%): The mixed gas prepared in Example 1 was introduced from the gas inlet of the gas control box, and the discharged gas was collected and processed from the gas outlet. A portable hydrogen detector (Huayue Medical Technology Co., Ltd., Guangzhou, China) was placed in the gas control box. The gas flow rate was controlled by a pressure reducing valve with a pressure gauge. The hydrogen concentration was monitored to maintain it at 2%. The pressure reducing valve was adjusted at any time to control the gas flow rate to maintain the hydrogen concentration. The treatment lasted for 4 weeks, and the mixed gas was inhaled for 4 hours every day during the treatment.

[0052] Hydrogen control group 1, denoted as CUMS+H 2 (2h, 2%): The treatment method is the same as the hydrogen group, except that the mixed gas is inhaled for 2h every day during the treatment period.

[0053] Hydrogen control group 2, denoted as CUMS+H 2 (4h, 1%): The treatment method was the same as that of the hydrogen group, except that the mixed gas inhaled during the treatment was prepared in Comparative Example 1, and the hydrogen concentration was monitored to maintain it at 1%.

[0054] The positive drug group, denoted as CUMS+FLU, was treated with fluoxetine (FLU) by intragastric administration, with a daily intragastric dose of 10 mg / kg body weight for 4 weeks.

[0055] Model group, denoted as CUMS: normal feeding.

[0056] The control group, denoted as Control: normal feeding.

[0057] 3) Behavioral experiments

[0058] ①Weight measurement

[0059] Starting from model establishment, the body weight of the rats was measured once every two weeks for a total of 6 times to assess the general condition of the animals. The measurement results are shown in Tables 1 and Figure 1 According to Table 1 and Figure 1 It can be seen that compared with the Control group, the weight gain of rats in the CUMS group was slow (p<0.0001); 2 The weight gain of rats in the (4h, 2%) group recovered (p<0.0001); 2 (2h, 2%) group, CUMS+H 2 The weight gain of rats in the (4h, 1%) group recovered slightly, but the difference was not significant.

[0060] Table 1 Average body weight of rats in each treatment group

[0061]

[0062] ②Sucrose preference test (SPT)

[0063] The sucrose preference test (SPT) was used to evaluate the anhedonia symptoms of rats, using 1% (w / v) sucrose solution as the sugar water. First, a 48-hour adaptation training was carried out. In the first 24 hours, two bottles of sugar water were placed in each cage; in the second 24 hours, one bottle of ordinary water and one bottle of sugar water were placed in each cage, and the positions of the water bottles were swapped every 4 hours. After the training, the rats were fasted and deprived of water for 12 hours. Then, a bottle of 500g ordinary water and a bottle of 500g sugar water were placed in each cage. The positions were swapped after 1 hour, and the consumption for 2 hours was recorded. The sugar water preference rate of each group of rats was calculated according to the sugar water preference rate calculation formula. The results are shown in Tables 2 and Figure 2 .

[0064] Sugar water preference rate = sugar water intake / (sugar water intake + normal water intake) × 100%.

[0065] Table 2 Average sugar water preference rate of rats in each treatment group

[0066]

[0067] According to Table 2 and Figure 2 It can be seen that compared with the Control group, the rats in the CUMS group had a more serious anhedonia (p<0.001), and the CUMS+H 2 The sucrose preference rate of rats in the (4h, 2%) group was restored (p<0.001); 2 (2h, 2%) group, CUMS+H 2 The rats in the (4h, 1%) group recovered slightly, but the difference was not significant.

[0068] ③ Open box test (OFT) and elevated plus maze test (EPM)

[0069] Behavioral experiments were performed to determine whether the rat model was successful and to test the therapeutic effect. All behavioral tests were performed in a soundproof environment.

[0070] The open-field test (OFT) and elevated plus maze test (EPM) were used to assess the anxiety-like behavior and exploratory activities of rats. The open-field test was 100 cm × 100 cm × 100 cm in size, the elevated plus maze was purchased from Shanghai Jiliang Software Technology Co., Ltd. (Shanghai, China), and the universal spontaneous activity (open field) video analysis system was purchased from Shanghai Jiliang Software Technology Co., Ltd. (Shanghai, China).

[0071] In OFT, before the experiment begins, each group of rats is placed in the experimental area to adapt for 5 minutes in advance, and then the rats are placed in a nine-square open box to monitor the rats' autonomous activities for 5 minutes, one rat at a time, and the camera automatically records for 5 minutes each time. Keep the surroundings quiet during the experiment. Each rat needs to clean the experimental device with 75% alcohol before and after the experiment to avoid the influence of residual odor on the experimental results. Figure 3The results are shown in Table 3 and Figure 4 .

[0072] Table 3 Average immobility time of rats in each treatment group

[0073]

[0074] According to Table 3 and Figure 4 It can be seen that compared with the Control group, the immobility time of rats in the CUMS group was prolonged (p<0.0001); 2 The immobility time of rats in the (4h, 2%) group was shortened (p<0.0001); 2 (2h, 2%) group, CUMS+H 2 The immobility time of rats in group (4h, 1%) recovered slightly, but the significance was poor.

[0075] In EPM, rat cages were placed in the experimental area to adapt for 5 minutes before the experiment began, and then rats were placed in the center of the maze facing the open arm, one at a time, and the camera automatically recorded each time for 5 minutes, and recorded the time they stayed in the closed arm and the open arm respectively. The surroundings were kept quiet during the experiment. Each rat needed to clean the experimental device with 75% alcohol before and after the experiment to avoid the influence of residual odor on the experimental results. Figure 5 The results are shown in Table 4 and Figure 6 .

[0076] Table 4 Average open arm / closed wall immobility time of rats in each treatment group

[0077]

[0078] According to Table 4 and Figure 6 It can be seen that compared with the Control group, the ratio of open arm / closed arm time in the CUMS group was shortened (p<0.0001); 2 The time ratio of rats in the (4h, 2%) group increased (p<0.0001); CUMS+H 2 (2h, 2%) group, CUMS+H 2 The immobility time of rats in group (4h, 1%) recovered slightly, but the significance was poor.

[0079] ④ Nissl staining

[0080] The rats in the control group, model group, positive drug group and hydrogen group were killed, their heads were cut off and their brains were removed. They were rinsed with ice saline and immediately fixed in 4% paraformaldehyde solution. The tissues were dehydrated with gradient ethanol, embedded in wax and then sliced. The slices were 5 μm thick and stained with Nissl staining kit (Biyuntian, Shanghai, China). After staining, the slices were observed under a microscope (Zeiss, Germany). The results are shown in Figure 7 .

[0081] According to the above results, the weight gain of the model group was slower than that of the control group (see Figure 1 ), showed obvious anxiety and depression behaviors in behavioral experiments, such as the number of rats crossing the grid decreased, the total movement distance decreased, and the immobility time increased; and the ratio of the time the rats stayed in the open arms to the closed arms increased significantly. After inhaling hydrogen, the weight gain of the rats gradually converged to that of the control group, and the symptoms of anhedonia in the rats were significantly improved (see Figure 3 to Figure 6 ). The results showed that molecular hydrogen had an improving effect on the depressive symptoms of CUMS rats. Moreover, the therapeutic effect of hydrogen inhalation for 4 hours at a concentration of 2% was better than that of inhalation for 2 hours at a concentration of 2% and 4 hours at a concentration of 1%.

[0082] Compared with the control group, the neurons in the CUMS group were disordered, with reduced density, obvious neuronal loss, and residual vacuoles. However, the degree of hippocampal tissue damage in CUMS rats treated with hydrogen was significantly restored. These results suggest that hydrogen can effectively protect the hippocampal tissue of rats from stress damage (see Figure 7 ).

[0083] Application Example 2

[0084] 1) Preparation of CUMS (Chronic Mild Unpredictable Stress) Model Rats

[0085] The operation method is the same as step 1 of Example 1).

[0086] 2) Group processing

[0087] CUMS+H 2 Group: The treatment method is the same as that of the hydrogen group CUMS+H in step 2) of application example 1 2 (4h, 2%) treatment.

[0088] CUMS+FLU group: The treatment method is the same as that of the positive drug group in step 2) of Application Example 1.

[0089] CUMS group: The processing method is the same as the model group processing in step 2) of application example 1.

[0090] Control group: The treatment method is the same as that of the control group in step 2) of Application Example 1.

[0091] ① Neurotransmitter detection

[0092] Rat hippocampus samples were obtained, homogenized and centrifuged. The supernatant was subjected to UPLC-MS / MS analysis to determine the levels of 5-hydroxytryptamine and tryptophan. The SCIEX OS-MQ software (Sciex, USA) was used with default parameters to automatically identify and integrate each MRM transition, and manual inspection was assisted. The results are shown in Figure 8 and Table 5.

[0093] The chromatographic conditions were as follows: injection volume: 5 μL; flow rate: 0.3 mL / min; mobile phase: A (0.1% (v / v) formic acid-water solution), B (0.1% (v / v) formic acid-methanol); gradient elution procedure: 0 min A / B (99:1, V / V), 1 min A / B (99:1, V / V), 6 min A / B (5:95, V / V), 7 min A / B (5:95, V / V), 7.01 min A / B (99:1, V / V), 8 min A / B (99:1, V / V).

[0094] The mass spectrometer works as follows: curtain gas: 30 (psi); collision-activated dissociation (CAD) parameters: medium; positive ion spray voltage: 5500 V; negative ion spray voltage: -4500 V; ion source temperature: 450°C; column temperature: 40°C; spray gas (Gas1): 50 (psi); auxiliary heating gas (Gas2): 50 (psi).

[0095] Table 5 Average neurotransmitter levels in rats in each treatment group

[0096] Group <![CDATA[CUMS+H 2 ]]> CUMS+FLU CUMS Control 5-HT (ng / g) 15428.903 14439.707 12748.230 40625.960 Tryptophan (ng / g) 17.209 20.220 18.637 29.807

[0097] The content of central tryptophan and its metabolite 5-hydroxytryptamine and other neurotransmitters was compared between different groups to observe the effect of hydrogen on neurotransmitters. The results showed that compared with the control group, the content of central 5-hydroxytryptamine and its precursor tryptophan in the model group rats showed a significant deficiency, which is consistent with the characteristics of depression. According to Table 5 and Figure 8 It can be seen that CUMS+H 2 The neurotransmitter content of rats in the (4h, 2%) group recovered, indicating that hydrogen has an antidepressant effect.

[0098] ② Detection of oxidative stress product levels, inflammatory factor levels, and ATP generation

[0099] Kits (MB-1588A rat interleukin 1β (IL-1β) ELISA research kit, MB-1721A rat tumor necrosis factor α (TNF-α) ELISA research kit, ELISA; A007-1-1 catalase (CAT) assay kit, A064-1-1 hydrogen peroxide (H 2 O 2 ) assay kit, Nanjing Jiancheng) to assess the levels of inflammatory factors, hydrogen peroxide, and catalase in hippocampal tissue. The experiment was performed according to the manufacturer's instructions, and the results are shown in Fig. 9 , Fig.10 and Table 6.

[0100] The relative expression of microglial markers (Ionized Calcium Binding Adaptor Molecule 1, iba-1) was detected by Western blotting. The results are shown in Fig.11 , Fig.12 and Table 6.

[0101] Table 6 Average levels of oxidative stress products and inflammatory factors in rats in each treatment group

[0102]

[0103]

[0104] According to Table 6 and Fig. 9 It can be seen that compared with the Control group, the catalase content in the CUMS group was reduced (p<0.0001); 2 The catalase content of rats in the (4h, 2%) group recovered (p < 0.05). This shows that the oxidative stress level in the model group increased sharply, and hydrogen intervention significantly restored this abnormality. In order to further detect the inflammatory response, Elisa was used to detect the levels of tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β) in brain tissue. Fig.10 It can be seen that compared with the Control group, the CUMS group had increased tumor necrosis factor and interleukin 1-1β (p<0.0001); the CUMS+H2 (4h, 2%) group had restored the content of inflammatory factors (TNF-α, p<0.0001), (IL-1β, p<0.05). This shows that hydrogen intervention resisted the abnormal changes in the secretion of inflammatory factors. In addition, the expression of microglia in the hippocampal tissue that released inflammatory factors was also observed by WB. Fig.11 and Fig.12 It can be seen that compared with the control group, the expression of microglial marker (iba-1) in the model group was increased (p < 0.05), while the inhalation of hydrogen restored the normal microglial expression (p < 0.05).

[0105] ③ Transmission electron microscopy (TEM), ATP production

[0106] Part of the hippocampus tissue was cut longitudinally into approximately 1 mm × 1 mm × 1 mm size, immediately fixed in 4% (w / v) glutaraldehyde solution, and stored at 4°C. After being fully washed with PBS, the tissue was post-fixed with 1% (w / v) osmium acid, then fully washed with PBS, dehydrated in acetone gradients, embedded in epoxy resin mixture, and ultrathin sections of the hippocampus were prepared using an ultrathin sectioning machine (ultra-low temperature UCT; Leica) with a thickness of 50 nm. After double staining with uranyl acetate-lead citrate, the tissue was observed using a transmission electron microscope (Hitachi transmission electron microscope HT7700 Exalens). The results are shown in Fig.13 .

[0107] The level of ATP production in rat hippocampal mitochondria was assessed using a kit (S0027 ATP assay kit, Bio-Tech). The results are shown in Table 7. Fig.14 .

[0108] Table 7 Average ATP production of rats in each treatment group

[0109] Group <![CDATA[CUMS+H 2 ]]> CUMS+FLU CUMS Control ATP 5991.6 5963.6 4426.4 7738.8

[0110] according to Fig.13 It can be seen that compared with the control group, transmission electron microscopy observed that the number of mitochondria in the hippocampal tissue of the model group was reduced, the outer membrane was incomplete, most of the inner cristae were blurred and disordered or dissolved, the matrix was loose, and some mitochondria were swollen; according to Fig.14 It can be seen that the ATP content in the model group was significantly reduced (p < 0.0001), and after hydrogen treatment, the mitochondrial ATP production capacity was significantly improved. In addition, more swollen mitochondria appeared in the neurons of rats in the CUMS group, with flocculent amorphous substances, short and unclear cristae structures, and spherical vacuolated and swollen mitochondria. The degree of mitochondrial swelling in the neurons of rats in the positive drug and hydrogen inhalation groups was reduced, and the number of mitochondria with damaged cristae structures was significantly reduced. These results show that hydrogen can improve the damage of neurons and their mitochondria in the rat hippocampus caused by CUMS exposure.

[0111] ④Western blotting

[0112] Protein was extracted from hippocampal tissue using RIPA (Radio-Immunoprecipitation Assay, China) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentration was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). The supernatant was diluted according to the results of the BCA assay, and the loading buffer was added and boiled for 10 min. The protein samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane. After that, the PVDF membrane was blocked with 5% skim milk for 1 h at room temperature, incubated with the primary antibody at 4°C overnight, eluted with TBST, incubated with the corresponding secondary antibody at room temperature for 1 hour, and finally developed using ECL luminescent solution (Pulile, China). Evaluation was performed using the ChemiDoc XRS+ gel imaging system (Bio-Rad Laboratories, Inc.) and Image Lab (version 6.1.0; Bio-Rad Laboratories, Inc.) was used. Antibodies were purchased from Wuhan Sanying Biotechnology Co., Ltd., China: PGC-1α (Cat No. 66369-1-Ig), MFN2 (Cat No. 12186-1-AP), DRP1 (Cat No. 12957-1-A), PINK1 (Cat No. 23274-1-AP), Parkin (Cat No. 14060-1-AP), IBA-1 (Cat No. 10904-1-AP), GAPDH (Multi-rAb HRP-goat anti-rabbit recombinant secondary antibody (Cat No. RGAR001-100ul). Goat anti-rabbit IgG (Alexa Fluor H&L 594 (Cat No. ab150080 purchased from abcam, UK). Figure 15 to Figure 17 and Table 8.

[0113] Table 8 Average relative expression of mitochondrial related proteins in rats in each treatment group

[0114] Group <![CDATA[CUMS+H 2 ]]> CUMS+FLU CUMS Control PGC-1α 0.55 0.54 0.32 1 Drp1 1.04 1.45 1.48 1 Mfn2 1.11 1.09 1.33 1 Pink1 1.11 0.99 0.76 1 Parkin 0.85 0.64 0.18 1 Beclin-1 1.02 1.06 1.54 1

[0115] Mitochondrial homeostasis involves the dynamics of mitochondrial biogenesis, mitophagy, and mitochondrial fission / fusion. 2The dynamic changes of each link in the model group were investigated, and the mitochondrial related proteins in the hippocampus were detected. First, compared with the control group, the CUMS group significantly downregulated the mitochondrial biogenesis regulatory factor (Peroxisomeproliferator activated receptorγcoactivator-1α, PGC-1α, p < 0.01), while hydrogen treatment restored the PGC-1α content to a certain extent (p < 0.05); secondly, the mitochondrial fission protein (Dynamin-relatedprotein1, Drp1) and mitochondrial fusion protein (Mitochondrial fusion protein 1, Drp1) in the hippocampus of rats in the model group were significantly increased. The expression of Drp1 and Mfn2 in the hydrogen group was significantly increased (p<0.01, p<0.001), indicating that CUMS damaged the mitochondrial fission process, while the expression of Drp1 and Mfn2 in the hydrogen group was lower than that in the model group, but the difference was not significant (ns), which may indicate that the mitochondrial fusion process was not affected. Finally, autophagy-related proteins (Atg6 / Beclin1) were detected. Beclin-1 is an important ATG protein that participates in the initiation and regulation of autophagy. It can play a dual regulatory role in the formation and maturation of autophagosomes by forming different complexes. The results showed that CUMS treatment reduced the degree of autophagy (p<0.05), and hydrogen treatment restored the autophagic flux (p<0.05). On this basis, the expression of mitochondrial autophagy-related proteins Pink1 (PTEN-induced kinase 1) and Parkin (Parkin RBRE3 ubiquitin-protein ligase) was further explored. Hydrogen treatment increased the expression of Pink1 and Parkin proteins that were reduced in CUMS compared with the control group (p<0.05), and restored Beclin-1 expression to a certain extent (p<0.05). The restored autophagic flux can effectively remove damaged mitochondria, block the occurrence of oxidative stress from the source, and then play an anti-inflammatory role. This shows that hydrogen treatment can restore mitochondrial autophagic flux, block the occurrence of oxidative stress, and play an anti-inflammatory role.

[0116] In summary, the mixed gas of the present invention is inhaled for 4 hours every day, and the treatment method is safe and painless, does not affect daily activities; and takes effect quickly, hydrogen can cross the blood-brain barrier and directly reach central neuron cells; the therapeutic effect is stable, and inhaling hydrogen while under continuous stress significantly slows down the occurrence of pathological changes. The mixed gas of the present invention can play a role in assisting the improvement of depression symptoms and effectively preventing the progression of depression.

[0117] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A mixed gas, characterized in that: The mixed gas includes hydrogen, oxygen and nitrogen, the volume concentration of the hydrogen is 2% to 3%, the volume concentration of the oxygen is 21%, and the volume concentration of the nitrogen is 76% to 77%.

2. The mixed gas according to claim 1, characterized in that: The volume concentration of the hydrogen is 2%.

3. The method for preparing a mixed gas according to claim 1 or 2, characterized in that: The following steps are involved: Oxygen is produced by liquefaction separation, nitrogen is produced by air separation, and hydrogen is produced by water electrolysis; The hydrogen, oxygen and nitrogen are mixed uniformly in proportion to obtain a mixed gas.

4. Use of the mixed gas according to claim 1 or 2 or the mixed gas obtained by the preparation method according to claim 3 in the preparation of antidepressant drugs.

5. The use according to claim 4, characterized in that: The antidepressant comprises one or more of improving mitochondrial damage in the hippocampus, improving neuronal damage in the hippocampus, increasing the secretion level of neurotransmitters, inhibiting oxidative stress and inhibiting inflammatory response.

6. The use according to claim 5, characterized in that: The improving of mitochondrial damage in the hippocampus includes increasing the number of mitochondria in the hippocampus and / or increasing the mitochondrial autophagy flux in the hippocampus.

7. The use according to claim 6, characterized in that: Increasing the number of mitochondria in the hippocampus includes increasing the expression of mitochondrial-related proteins; the mitochondrial-related proteins include one or more of the mitochondrial biogenesis regulatory factor PGC-1α, the mitochondrial fission protein Drp1, and the mitochondrial fusion protein Mfn2; The method of increasing the mitochondrial autophagy flux in the hippocampus includes increasing the expression levels of mitochondrial autophagy-related proteins Pink1 and / or Parkin.

8. The use according to claim 5, characterized in that: Such neurotransmitters include serotonin.

9. The use according to claim 5, characterized in that: The inhibition of inflammatory response includes reducing the expression of inflammatory factors and / or microglia.

10. The use according to claim 9, characterized in that: The inflammatory factors include tumor necrosis factor α and / or interleukin 1-β.

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