Application of lactobacillus muricatum in preparation of medicine for preventing and / or treating depression and medicine containing lactobacillus muricatum

Abstract

The invention discloses application of lactobacillus muricatum in preparation of a medicine for preventing and / or treating depression in the technical field of microorganisms, and the lactobacillus muricatum is preserved in the China Industrial Microbiological Culture Collection Center (CICC) with the preservation number of CICC23140. Research finds that lactobacillus muricatum which is preserved in the China Industrial Microbiological Culture Collection Center and has the preservation number of CICC23140 has a good effect of improving and / or preventing and / or treating cortisol-induced depression, and inactivated lactobacillus muricatum can effectively prevent hippocampal neuron damage, reduce expression of inflammatory factors in the brain, prevent and treat cortisol-induced depression, and can be used for preventing and treating cortisol-induced depression. Not only can the anxiety symptom of depression be improved, but also dyskinesia can be relieved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to an application of Lactobacillus murinus in preparing a drug for preventing and / or treating depression and a drug containing the Lactobacillus murinus. Background Art

[0002] Depression is different from normal mood changes and feelings about daily life. Depression can affect all aspects of life, including relationships with family, friends and other social groups. Depression may be caused by problems in work, study or life, or it may cause problems in work, study or life. Depression can happen to anyone, and people who have experienced abuse, major losses or other stressful events are more likely to suffer from depression. Women are more likely to suffer from depression than men, and about 280 million people in the world suffer from depression. The incidence of depression in women is about 50% higher than that in men. Worldwide, more than 10% of pregnant women and women who have just given birth suffer from depression. More than 700,000 people have life safety problems every year due to depression, most of whom are aged 15 to 29.

[0003] Although there are effective treatments for mental illness, more than 75% of patients in low- and middle-income countries do not receive effective treatment. Obstacles to effective treatment include lack of investment in mental health care and lack of trained health care providers. Therefore, identifying the causes of depression and finding effective treatment options remain urgent issues that need to be addressed.

[0004] Cortisol is a key glucocorticoid produced by the adrenal glands that plays a critical role in a variety of physiological processes. Its release is carefully orchestrated by the suprachiasmatic nucleus, which controls circadian rhythms and activates the intricate hypothalamic-pituitary-adrenal (HPA) axis, an important neuroendocrine system responsible for stress response and maintenance of homeostasis.

[0005] Inactivated probiotics are a type of probiotics. They have similar physiological functions to live probiotics and are safer and more stable than live probiotics, so they have received extensive attention and research. After being inactivated, probiotics release bacterial components with immunomodulatory and anti-pathogen properties. Many studies have shown that preparations of inactivated probiotics and their metabolites can also produce relevant biological responses and restore normal intestinal environmental stability. In most cases, their efficacy is similar to that of non-inactivated probiotics.

[0006] Lactobacillus murinus is a Gram-positive bacterium belonging to the family Lactobacillus in the phylum Firmicutes. It is isolated from the intestines of mice. Studies have reported that Lactobacillus murinus can repair damaged mucosa and enhance the intestinal barrier. It also has the functions of regulating intestinal microbiota and inhibiting systemic inflammatory markers. However, there are no reports on the effects of Lactobacillus murinus on cortisol-induced depression. Summary of the invention

[0007] The present invention aims to provide an application of Lactobacillus murinus in the preparation of a drug for preventing and / or treating depression and a drug containing the Lactobacillus murinus. The Lactobacillus murinus provided by the present invention can protect hippocampal neuron damage, improve anxiety and depression states, and has a good effect in preventing and treating depression.

[0008] In one aspect, the present invention provides a use of Lactobacillus murinus in the preparation of a drug for improving and / or preventing and / or treating anxiety and / or depression. The Lactobacillus murinus is deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC23140. The depression is preferably cortisol-induced depression.

[0009] On the other hand, the present invention also provides a medicine, which has the function of improving and / or preventing and / or treating anxiety and / or depression, and the composition of the medicine includes the above-mentioned Lactobacillus murinus.

[0010] Furthermore, the Lactobacillus ratus is an inactivated Lactobacillus ratus.

[0011] Further, the concentration of inactivated Lactobacillus murinus in the drug is 10 6 ~10 12 cfu / ml, preferably 10 9 cfu / ml.

[0012] Furthermore, the composition of the drug also includes one or more of starch, maltodextrin, trehalose and lactose.

[0013] Furthermore, the dosage form of the drug is selected to be any one of a suspension, suppository, tablet, capsule, soft capsule, prefilled injection, effervescent tablet or gel.

[0014] The beneficial technical effect of the present invention is: the present invention has found that Lactobacillus murinus, which is deposited in the China Industrial Microbiological Culture Collection Management Center and has a deposit number of CICC23140, has a good effect on improving and / or preventing and / or treating cortisol-induced depression. The inactivated Lactobacillus murinus can effectively protect hippocampal neuron damage and reduce the expression of inflammatory factors in the brain, which can not only improve the anxiety symptoms of depression, but also relieve movement disorders. In addition, Lactobacillus murinus has many advantages such as excellent effect, high safety, good stability, mature production process, and wide source of raw materials, so it is suitable for promotion and use in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a statistical graph of rat weight; Figure 2 This is a statistical graph of sucrose preference in rats with depression; Figure 3 It is a bar chart of the proportion of time spent in the open arm of the elevated plus maze in depressed rats; Figure 4 This is the statistical graph of IL-6 protein expression in rat serum measured by ELISA; Figure 5 This is the statistical graph of IL-1β protein expression in rat serum measured by ELISA; Figure 6 It is the expression strip and bar chart of synaptophysin (Syn) protein in hippocampus tissue of rats with depression; Figure 7 The expression bands and statistical graphs of inflammatory factors COX2 and IL-18 proteins in the hippocampus of rats with depression; Meaning of the reference numerals in the drawings of the specification: Control group, sham operation group; HK Lm indicates sham operation + Lactobacillus murinus group; Cortisol indicates the model group; Cortisol+ HK Lm indicates the Cortisol model + Lactobacillus murinus group. DETAILED DESCRIPTION

[0016] The following is further described in detail through specific implementation methods: The present invention has found that Lactobacillus murinus, which is deposited in the China Industrial Microbiological Culture Collection Center and has a deposit number of CICC23140, can be used to prepare a drug for improving and / or preventing and / or treating cortisol-induced depression. The drug uses the Lactobacillus murinus (inactivated) as the main active ingredient and also includes pharmaceutically acceptable excipients. The excipients can play a role in improving patient compliance and improve the stability of the inactivated Lactobacillus murinus. The excipients can be selected from one or more of starch, maltodextrin, trehalose and lactose, preferably trehalose. The dosage forms of the drug include suspensions, suppositories, tablets, capsules, soft capsules, prefilled injections, effervescent tablets and gels. The present invention has no special requirements for the preparation method of the above-mentioned dosage form, and the preparation method well known in the art can be used.

[0017] In the present invention, the preparation method of the inactivated Lactobacillus murinus preferably comprises the following steps: The Lactobacillus murine is sterilized to obtain an inactivated Lactobacillus murine.

[0018] The present invention has no particular limitation on the sterilization method, and conventional sterilization methods may be used, such as inactivation at 121° C. for 20 to 30 min, more preferably inactivation at 121° C. for 20 min.

[0019] In the present invention, the concentration of the inactivated Lactobacillus murinus in the drug is preferably 10 6 ~10 12 CFU / ml is more preferably 10 9 cfu / ml. The inactivated Lactobacillus murinus of the above concentration can better play the role of preventing and treating depression induced by cortisol.

[0020] In the examples of the present invention, taking the cortisol-induced rat model as an example, the cortisol model showed depression-like symptoms, and the body weight decreased with the modeling time. The body weight of the cortisol-treated Lactobacillus rat group increased significantly; compared with the control group, the sucrose preference index of the cortisol-treated Lactobacillus rat group was significantly reduced, and compared with the cortisol group, the sucrose preference index of the cortisol-treated Lactobacillus rat group was significantly increased; compared with the control group, the proportion of the time the cortisol group stayed in the open arm of the elevated plus maze was significantly reduced, and compared with the cortisol group, the proportion of the time the cortisol-treated Lactobacillus rat group stayed in the open arm was significantly increased; the cortisol-treated inactivated Lactobacillus rat group had a significant improvement effect on depression.

[0021] The present invention takes cortisol-induced rats as an example, and the inactivated Lactobacillus murinus can rescue the synaptic damage of the rat hippocampus caused by cortisol. After exogenous intervention with inactivated Lactobacillus murinus, α-syn increased significantly. When used in the preparation of drugs, the dosage is preferably 10 6 ~10 12 cfu / ml, more preferably 10 9 cfu / ml.

[0022] The present invention also provides the use of inactivated Lactobacillus murinus in the preparation of a method for treating depression by reducing the expression level of related inflammatory factor proteins in hippocampal tissue and serum. The depression described in the present invention is preferably depression induced by cortisol. In the present invention, taking the rat model induced by cortisol as an example, inactivated Lactobacillus murinus can reduce the expression level of inflammatory factors IL-6 and IL-1β proteins in rat serum, and can also reduce the expression level of inflammatory factors COX2 and IL-18 proteins in rat hippocampal tissue.

[0023] experiment: 1 Experimental Materials and Instruments 1.1 Experimental animals SD male rats, weighing about 200 g, were purchased from the Experimental Animal Center of Zunyi Medical University and housed in the SPF animal room of the Key Laboratory of Basic Pharmacology of the Ministry of Education of Zunyi Medical University. Each group of experimental animals was kept under constant temperature and humidity conditions, with free access to clean drinking water and basic feed, and a circadian rhythm of 12 h.

[0024] 1.2 Main instruments and equipment Rat elevated plus maze, Bio-Rad chemiluminescence imaging system (Bio-Rad, USA), BSS-110 electronic analytical balance (Beijing Sartorius Electronic Balance Co., Ltd.), Milli QA pure water processor (Millipore).

[0025] 1.3 Main experimental reagents Lactobacillus murinus (Lactobacillus murinus, China Industrial Microbiological Culture Collection Center); cortisol (40 mg / kg); MRS medium: casein peptone 10.0 g, beef extract 10.0 g, yeast extract powder 4.0 g, glucose 20.0 g, sodium acetate 5.0 g, triammonium citrate 2.0 g, Tween 80 1.0 ml, K2HPO4 2.0 g, MgSO4·7H2O 0.2 g, MnSO4·H2O 0.05 g, distilled water 1000 mL (Solarbio); 6-hydroxydopamine (cortisol), purity ≥98% (Sigma); IL-6 ELISA kit, IL-1β ELISA kit (Solarbio); COX2, IL-18 rabbit polyclonal antibodies (Proteintech China).

[0026] 2. Experimental Methods 2.1 Experimental Animal Grouping Forty 6-week-old SD male rats weighing about 200 g were randomly divided into six groups, with 10 rats in each group, including sham operation (Control) group, sham operation (Control) + HK Lactobacillus murinus group, model (cortisol) group, and cortisol model + HK Lactobacillus murinus group.

[0027] 1) Sham operation group: After 4 weeks of drinking water treatment, 0.2% (DMSO, PEG300 and Tween 80 mixture) was injected subcutaneously and then drinking water treatment was continued for 3 weeks.

[0028] 2) Control + HK Lm group: After pretreatment with HK Lactobacillus murinus 109 cfu / day for 4 weeks, 0.2% (DMSO, PEG300 and Tween 80 mixture) was subcutaneously injected, and then HK Lactobacillus murinus 10 9 cfu / day for 3 weeks.

[0029] 3) Cortisol group: Cortisol was dissolved in DMSO and injected subcutaneously at a dose of 40 mg / kg once a day for 28 days to establish a depression model.

[0030] 4) Cortisol + HK Lm group: HK Lactobacillus murinus 10 9 After 3 weeks of pretreatment with 40 mg / kg cortisol solution, the depression model was established. Then, H-KLactobacillus murinus was continuously administered for 10 9 cfu / day for 4 weeks.

[0031] 2.2 Lactobacillus murinus culture and administration Lactobacillus murine culture: First, activate the bacteria in the freeze-dried tube and perform subculture to restore the activity of Lactobacillus murine. Then, inoculate Lactobacillus murine in MRS medium and culture at 37°C for 8 h. Measure the OD value of the bacterial solution with an ultraviolet spectrophotometer at 600 nm. The OD value is about 0.8, and the total number of bacteria is 2.8×10 8 cfu / mL, centrifuge the suspension (4°C, 5000 rpm for 20 min). Discard the supernatant and adjust the bacterial solution concentration to 10 8 ~10 9 cfu / ml to obtain Live L. m, or to obtain Hk L. m after inactivation at 121℃ for 20 min. The total amount of bacteria administered to mice by gavage was 1.4× 109 cfu / ml.

[0032] 2.3 Behavioral testing Rats were tested for behavior before cortisol administration and at the first, second, third, and fourth weeks after administration, and the administration continued during the behavioral period.

[0033] 2.3.1 Weight Record The body weight of rats was recorded on days 0, 7, 14, 21 and 28 respectively: 2.3.2 Sucrose preference experiment Sugar water preference index can be used as an effective tool to assess the symptoms of depression in experimental animals. Animal adaptation period: Before the experiment, rats usually need to be housed individually for 3-7 days to eliminate the influence of social factors on the experimental results. During this period, the animals should be kept in a constant temperature, constant humidity and sufficient food environment. The first test requires sugar water adaptation, and the second and subsequent tests are directly formal experiments. Sugar water adaptation: 1% sucrose solution is provided to the animals 1 day before the experiment to allow them to adapt to the taste of sugar water. On the day of the experiment, sugar water and ordinary water are placed in two drinking bottles, and the weight of the drinking bottles is clearly marked and weighed. Drinking bottles of sugar water and ordinary water are placed in the animal cage at the same time, allowing rats to freely choose to drink. After 24 hours, the consumption of the two liquids is recorded by weighing the drinking bottles. The sugar water consumption ratio is calculated. Therefore, sugar water preference experiments were conducted on days 0, 7, 14, 21 and 28 after cortisol was given to rats.

[0034] 2.3.3 Elevated plus maze test Rats should be trained for adaptation before the experiment: the animals should be adapted to the laboratory environment for at least 30 minutes to reduce the stress response to the new environment. During the experiment, the animals were placed at the entrance of the maze, and the movement trajectory and movement data of the mice were automatically generated. The proportion of time spent in the open arm (OT%) was calculated, that is, the open arm stay time / (OT+CT)×100%. Each rat was tested for 10 minutes.

[0035] 2.4 Sampling and specimen preparation After the behavioral experiment, the rats were deeply anesthetized with 3% sodium pentobarbital, the abdominal cavity was cut open, the abdominal aorta was clamped, the chest cavity was cut open, and the heart was fully exposed. The perfusion needle was inserted into the aorta through the apex of the heart for 5 rats in each group, and the right atrial appendage was fixed with hemostats and cut open. PBS was perfused until the blood in the brain was washed clean, the whole brain was removed, fixed with formaldehyde solution and stored at room temperature, and paraffin sections (thickness 5 μm) were prepared. The whole brain of 5 rats in each group was removed, and the hippocampus and cortex were separated and stored at -80℃.

[0036] 2.5 ELISA was used to detect the activity of inflammatory factors IL-6 and IL-1β in rat serum.

[0037] Take rat serum and dispense about 230 μL per tube. After the ELISA kit was balanced at room temperature for 10 min, the standard was diluted, 100 μL of protein sample and standard was added to each well, incubated at 37 °C for 2 h, and then 100 μL of detection solution A working solution was added after drying, incubated at 37 °C for 1 h, the liquid in the well was discarded and washed 3 times, 100 μL of detection solution B working solution was added to each well, incubated at 37 °C for 30 min, and the plate was washed 5 times, 90 μL of substrate solution was added to each well, and color was developed at 37 °C in the dark. After adding the stop solution, the absorbance value of each well was detected at a wavelength of 450 nm, and the concentration of the corresponding protein was calculated according to the standard curve. The results are expressed in ng / μg protein.

[0038] 2.6 Western blotting Weigh an appropriate amount of hippocampal tissue, grind it on ice, add RIPA lysis buffer (containing PMSF), and let it stand for 40 min; use an ultra-low temperature centrifuge to centrifuge at 12000 rpm for 10 min, and collect the supernatant; use the BCA protein quantification kit to quantify, and prepare the loading system, mix well, and denature at 100℃ for 10 min; load 10 µL of sample into each well, and electrophoresis for 2 h (voltage 70-110V); use the wet transfer method to transfer the membrane, put it into the electrotransfer box, set the current to 300 mA, and transfer for 50 min; after the end, use 5% skim milk to block for 2 h, wash 3 times with TBST, 10 min / time; add primary antibody at 4℃ overnight, wash 3 times with TBST; 10 min / time; add secondary antibody to incubate at 4℃ for 40 min, wash 3 times with TBST; 10 min / time; add exposure agent and expose.

[0039] 2.7 Statistical analysis of data The experimental data were statistically analyzed using Graphpad prism statistical software. All data were expressed as (mean ± standard deviation). One-way analysis of variance (ANOVA) was used to compare the group differences in mean values. The LSD method was used for the homogeneity of variance test. Dunnett's T3 was used to compare the group differences in mean values. P <0.05 was statistically significant.

[0040] 3. Experimental results 3.1 Effects of cortisol and inactivated Lactobacillus murinus on body weight in rats The rats were weighed on days 0, 7, 14, 21 and 28 after subcutaneous injection of cortisol. Figure 1As shown. Two weeks after the injection of cortisol, compared with the control group, the weight of the cortisol rats decreased with the extension of the modeling time, and the difference was statistically significant (P<0.05); compared with the cortisol group, the cortisol-treated inactivated Lactobacillus murine group was significantly increased. And it was statistically significant ( p <0.05).

[0041] 3.2 Effects of cortisol and inactivated Lactobacillus murinus on sucrose preference in rats Sucrose preference test was performed on rats on days 0, 7, 14, 21 and 28 after subcutaneous injection of cortisol. The sucrose preference index was Figure 2 Compared with the control group, the sugar preference index of rats with cortisol decreased significantly with the extension of modeling time, and the difference was statistically significant (P<0.05); compared with the cortisol group, the cortisol-treated inactivated Lactobacillus murine group increased significantly. And it was statistically significant (P<0.05).

[0042] 3.3 Effects of cortisol and inactivated Lactobacillus murinus on anxiety and depression in rats On the 28th day of the establishment of the cortisol model, all rats were subjected to an elevated plus maze test to understand the anxiety and depression state of the rats. As shown in the figure ( Figure 3 ), after 28 days of depression modeling, compared with the Control group, the proportion of the number of times the cortisol group entered the open arm and the proportion of the time they stayed in the open arm were significantly reduced, and the difference was statistically significant (P<0.05); compared with the cortisol group, the cortisol inactivated Lactobacillus murinus group was significantly increased, and the difference was statistically significant (P<0.05). This shows that the depression model was successfully established, and inactivated Lactobacillus murinus can improve the anxiety and depression behaviors induced by cortisol.

[0043] 3.4 Expression of inflammatory factors IL-6 and IL-1β in rat serum As shown in the figure ( Figure 4 and Figure 5 ), inactivated Lactobacillus murinus can reduce the expression of IL-6 and IL-1β protein levels in the serum of rats with cortisol-induced depression.

[0044] 3.5 Effect of inactivated Lactobacillus murinus on synaptic damage in a rat model of depression induced by cortisol Compared with the Control, the expression level of Syn protein in the cortisol group was significantly reduced, and the difference was statistically significant (P<0.05); compared with the cortisol group, the expression level of Syn protein in the cortisol-treated inactivated Lactobacillus murinus group was significantly increased, and the difference was statistically significant (P<0.05). Figure 6 .

[0045] 3.6 Effects of inactivated Lactobacillus murinus on inflammatory factors COX2 and IL-18 in a rat model of depression induced by cortisol Compared with the Control, the expression levels of COX2 and IL-18 proteins in the cortisol group were significantly reduced, and the difference was statistically significant (P<0.05); compared with the cortisol group, the expression levels of COX2 and IL-18 proteins in the cortisol-treated inactivated Lactobacillus murinus group were significantly increased, and the differences were statistically significant (P<0.05). Figure 7 .

[0046] Summary: From the perspective of the overall experiment, the present invention verifies that inactivated Lactobacillus murinus can delay or treat depression caused by long-term use of cortisol and improve depressive behavior through methods such as sugar water preference. This proves that inactivated Lactobacillus murinus has a therapeutic effect on depression and can delay its occurrence and development.

[0047] The invention is beneficial in that: the invention provides an application of inactivated Lactobacillus murinus, that is, the inactivated Lactobacillus murinus is used in the preparation of a drug for preventing or treating depression induced by cortisol. The inactivated Lactobacillus murinus has a preventive and therapeutic effect on depression induced by long-term use of cortisol.

[0048] (1) It can improve rats' sucrose preference to a certain extent. Emotional disorders are significantly reduced after inactivating Lactobacillus murineus, and it can improve depression caused by long-term use of cortisol.

[0049] (2) It plays a protective role against cortisol-induced synaptic damage in the hippocampus of rats with depression.

[0050] (3) It can reduce the expression level of inflammatory factors in the hippocampus of rats with cortisol-induced depression.

[0051] Inactivated Lactobacillus murinus can improve the anxiety-like depressive behaviors of depression and delay the onset of depression. It not only has a certain therapeutic effect on the symptoms of depression, but also can delay or improve depression induced by long-term use of cortisol to a greater extent. It is suitable for promotion and use in clinical practice.

Claims

1. A method for preparing a drug for improving and / or preventing and / or treating anxiety and / or depression, wherein the Lactobacillus murine is deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC23140.

2. The use according to claim 1, characterized in that: The depression is preferably Cortisol-induced depression.

3. A drug, characterized in that The medicine has the function of improving and / or preventing and / or treating anxiety and / or depression, and the composition of the medicine includes the Lactobacillus murine as described in claim 1.

4. A medicine according to claim 3, characterized in that: The Lactobacillus mouse is an inactivated Lactobacillus mouse.

5. A medicine according to claim 4, characterized in that: The concentration of inactivated Lactobacillus murinus in the drug is 10 6 ~10 12 cfu / ml.

6. A medicine according to claim 4, characterized in that: The concentration of the inactivated Lactobacillus murinus in the drug is preferably 10 9 cfu / ml.

7. A drug according to any one of claims 3 to 6, characterized in that: The composition of the medicine also includes one or more of starch, maltodextrin, trehalose and lactose.

8. A drug according to any one of claims 3 to 6, characterized in that: The dosage form of the drug is selected from any one of suspension, suppository, tablet, capsule, soft capsule, prefilled injection, effervescent tablet or gel.

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