CN106619602A - Novel application of leonurine - Google Patents

Abstract

The invention belongs to the field of traditional Chinese medicine pharmacy and relates to the application of Leonurine in the preparation of drugs for preventing or treating depression. After the present invention induces depression-like symptoms in mice through a chronic mild stress (CMS) model, the motherwort is administered intragastrically, and the results show that it can significantly improve the depression-like behavior of CMS mice, increase 5‑HT, The content of NE and DA monoamine transmitters can improve the damage of neurons in the hippocampus, increase the number of hippocampal astrocytes, inhibit neuroinflammation in the brain, and increase the levels of neurotrophic factors BDNF and GDNF in the brain. It can be seen from the present invention that the motherwortine exerts antidepressant effects in various aspects, and is suitable for the preparation of drugs for preventing or treating depression.

Description

The New Application of Leonurine

technical field

The invention belongs to the field of traditional Chinese medicine preparations and relates to a new medicinal application of motherurine, in particular to the application of motherurine in the preparation of medicines for treating or preventing depression.

Background technique

Depression is a complex and long-lasting emotional disorder, also known as depressive disorder. Its main clinical manifestations are marked and persistent depression, loss of interest, emotional depression, and even pessimistic world-weariness, suicide attempts and other behaviors. According to statistics, since 2004, depression has become the third most persistent disease in human beings, and it is estimated that by 2030, depression may become the number one disease affecting human life.

A large number of studies have shown that in addition to many factors in the social environment, depression still has pathological hypotheses such as lack of monoamine neurotransmitters, hyperthyroidism of the thalamus-pituitary-adrenal cortex (HPA), lack of neurotrophic factors in the brain, and neuroinflammation. Clinical antidepressants mainly target the pathological mechanism of monoamine neurotransmitter deficiency, including monoamine oxidase inhibitors, selective 5-hydroxytryptamine (5-hydroxytryptamine, 5-HT) reuptake inhibitors, selective norepinephrine (Norepinephrine, NE) reuptake inhibitors and tricyclic antidepressants, etc. Although the above drugs have good therapeutic effects and have been widely used clinically, most of them have weak efficacy, slow onset, and adverse reactions. For example, the tricyclic antidepressant imipramine and the selective 5-HT reuptake inhibitor fluoxetine (FLX) are commonly used in clinical practice. To date, about one-third of people with depression do not respond to traditional antidepressant treatments. Elucidating new mechanisms of antidepressant action, finding new targets for antidepressant treatment, and developing new effective therapeutic drugs are still difficult and important tasks in neuroscience research. In recent years, the development of antidepressant preparations from traditional Chinese medicine/natural medicine is expected to become one of the breakthrough directions.

Leonurus japonicas houtts belongs to the genus Leonurus japonicas houtts of traditional Chinese medicine Lamiaceae. It is known as "a good recipe for economic production". Since 1990, motherwort has been included in the Pharmacopoeia of the People's Republic of China, and more than 300 prescriptions containing motherwort have been used to treat various diseases, and its pharmacological effects are mainly obstetrics, gynecology and cardiovascular protection. There are research reports that motherwort has protective effect on anxiety of pregnant women; motherwort mixed extract has protective effect on ischemic stroke model rats (Chinese patent application number: 20101087017.4). Leonurine (Leonurine) is the main effective alkaloid in Motherwort. Modern pharmacological research has proved that Leonurine can excite the uterus, dissolve thrombosis, anticoagulant, reduce blood viscosity, reduce lipid, reduce red blood cell aggregation, inhibit platelet aggregation, and improve microcirculation. , anti-oxidant free radicals, anti-inflammation, reducing intracellular calcium overload and many other pharmacological effects provide a reliable theoretical basis for its clinical application. In recent years, research reports have pointed out that motherwortine has many drug effects that are not different from traditional indications, such as anti-diabetes, cardiovascular protection, stroke treatment, and plays an important role in kidney damage.

However, the effect of leonurine on depression has not been reported so far. The present invention confirms the possibility of the application of leonurine as a drug for preventing and treating depression from various aspects through relevant experimental research.

Contents of the invention

Purpose of the invention: In order to solve the above technical problems, the purpose of the present invention is to provide a new application of motherwort in pharmacy, specifically related to the application of motherwort in the preparation of drugs for the prevention or treatment of depression.

The leonurine of the present invention is obtained by chemical synthesis based on the structure of the active monomer leonurine of the Chinese herbal medicine Leonuri. Its molecular formula is C ₁₄ H ₂₁ N ₃ O ₅ , its molecular weight is 311.33, and its melting point is 238°C. The specific structural formula is as follows:

The present invention uses a chronic mild stress (CMS) model to induce depression-like behaviors in mice, and observes the effect of motherwort on the depression-like behaviors of CMS mice. The experimental results show that motherwort significantly improves the depression-like behaviors of CMS mice , showing a significant increase in the sugar preference rate (SPT), and a significant reduction in the immobility time of the mice in the forced swimming test (FST) and tail suspension test (TST).

The invention uses high-performance liquid chromatography to observe the effect of motherurine on monoamine neurotransmitters in the brain of CMS mice. The experimental results showed that motherwort significantly increased the content of monoamine neurotransmitters 5-HT, NE, and dopamine (Dopamine, DA) in the brains of CMS mice.

The invention uses Nissl staining and transmission electron microscope techniques to observe the influence of motherurine on hippocampal neurons of CMS mice. The results of Nissl staining showed that motherwortine significantly improved the morphology and structure of neurons in the hippocampus of CMS-depressed mice; the results of transmission electron microscopy showed that motherwortine significantly improved the damage of hippocampal neurons and the abnormal structure of neuron myelin in CMS mice.

The present invention uses immunofluorescence method to observe the effect of motherurine on hippocampal astrocytes of CMS mice. The experimental results show that motherurine significantly increases the number of hippocampal astrocytes of CMS mice, and has the possibility of improving the microenvironment in the brain .

The present invention uses the Western Blot method to detect the effect of motherurine on the hippocampal inflammation-related factors IL-1β, IL-6, TNF-α, NF-ΚB signaling pathways and neurotrophic factors BDNF and GDNF in CMS mice. The experimental results showed that motherurine significantly inhibited the protein expression of neuroinflammatory factors IL-1β, IL-6, and TNF-α in the brain of CMS mice and the activation of NF-κB signaling pathway, and increased the levels of neurotrophic factors BDNF and GDNF.

The leonurine described in the present invention includes but not limited to leonurine and its salts, including but not limited to the application in single-component and compound preparations. The dosage forms of the drug for preventing or treating depression in the present invention include but are not limited to tablets, capsules, sustained-release tablets, controlled-release tablets, oral liquids, syrups, dripping pills, injections, freeze-dried powders, etc.

It is known that the pathogenesis of depression is complex and may involve multi-system and multi-link dysfunction or obstacles. The invention uses multi-system and multi-angle research to comprehensively clarify the preventive and therapeutic effects and mechanism of the motherwortine on depression. From the description of the present invention, it can be seen that the effect of motherurine on improving the depression-like behavior of CMS mice, instead of increasing monoamine neurotransmitters in the brain, inhibiting neuroinflammation, increasing neurotrophic factors, regulating astrocytes, improving the microenvironment in the brain, etc. related.

The content of the present invention also includes the application of leonurine in constructing CMS animal models.

The content of the present invention also includes the application of motherwort in constructing a CMS mouse model.

As a preference, the daily dose of motherwort per kg of CMS mice in the present invention is 30 mg-60 mg.

According to the effective dose of motherwortine described in the present invention for preventing and treating depression in CMS mice, the daily dosage range for clinical adults is about 50-300 mg.

Beneficial effect: the motherwort described in the present invention exerts an antidepressant effect by adjusting multiple pathological links of depression, and has high safety (maximum tolerated oral dose>5g/kg), which is expected to exceed the current treatment acting on a single target drug.

Description of drawings

Figure 1: The effect of motherwort on the sugar water preference rate of CMS mice. The weekly sugar water preference rate during the experiment is expressed as mean ± standard error, n=11~13, ^* P<0.05, ^** P<0.01, compared with the control group at the same period; ^# P<0.05, ^## P<0.01 , compared with the model group of the same period. CON, normal mice; CMS, CMS mice; Leonurine, motherwort; FLX, fluoxetine;

Figure 2: The effect of motherwort on the forced swimming test of CMS mice. Values are expressed as mean ± standard error, n=8～12, ^* P<0.05, ^** P<0.01, compared with the control group at the same period; ^# P<0.05, ^## P<0.01, compared with the model group at the same period ; CON, normal mouse; CMS, CMS mouse; Leonurine, motherwort; FLX, fluoxetine;

Figure 3: Effects of motherwortine on the tail suspension test of CMS mice. Values are expressed as mean ± standard error, n=8～12, ^* P<0.05, ^** P<0.01, compared with the control group at the same period; ^# P<0.05, ^## P<0.01, compared with the model group at the same period ; CON, normal mouse; CMS, CMS mouse; Leonurine, motherwort; FLX, fluoxetine;

Figure 4: Effects of motherurine on the monoamine neurotransmitters 5-HT, NE, and DA in the brains of CMS mice (AF); values are expressed as mean ± standard error, n=7～9; Figures 4A-4C are the experimental results of 5-HT, NE, and DA in the hippocampus; Figure 4E-4F are the experimental results of 5-HT, NE, and DA in the prefrontal cortex; ^* P<0.05, ^** P<0.01, compared with the control group at the same period Comparison; ^# P＜0.05, ^## P<0.01, compared with the model group at the same period; CON, normal mice; CMS, CMS mice; Leonurine, motherwort; FLX, fluoxetine;

Fig. 5: Effect of motherurine on the morphology of hippocampal neurons in CMS mice (A-B) (×100); wherein Fig. 5A is the result of Nissl staining experiment in hippocampus DG area; Fig. 5B is the result of Nissl staining experiment in hippocampus CA3 area; n= 7-9, CON, normal mice; CMS, CMS mice; Leonurine, motherwort;

Figure 6: The effect of motherurine on hippocampal neuron damage in CMS mice; Figure 6 (A-C) is the experimental result of neuron transmission electron microscopy (×1900); Figure 6 (D-F) is the enlarged transmission electron microscope of the figure (A-C) Experimental results (×11000); n=3, mitochondria (arrows), CON, normal mice; CMS, CMS mice; Leonurine, motherwort;

Fig. 7: The effect of motherwortine on the myelin sheath of hippocampal neurons of CMS mice; Wherein Fig. 7A is transmission electron microscopy experiment result (× 1900); Fig. 7B is the G-ratio of each group namely the inner diameter/outer diameter of myelin sheath (n >100); CON, normal mice; CMS, CMS mice; Leonurine, motherwort;

Figure 8: Effect of motherurine on hippocampal astrocytes of CMS depression model mice (×100); where Figure 8A is the immunofluorescence experimental results of astrocyte-specific marker GFAP; Figure 8B is astrocytes The experimental results of GFAP-positive cell counts in plasma cells; ^* P<0.05, ^** P<0.01, compared with the control group at the same period; ^# P<0.05, ^## P<0.01, compared with the model group at the same period; n=5, CON, normal mice; CMS, CMS mice; Leonurine, motherurine;

Figure 9: Effects of motherurine on hippocampal inflammation-related factors IL-1β, IL-6, and TNF in CMS mice; where the data were compared with the target protein and the internal reference β-actin; ^* P<0.05, ^** P<0.01, Compared with the control group at the same period; ^# P<0.05, ^## P<0.01, compared with the model group at the same period; n=3, CON, normal mice; CMS, CMS mice; Leonurine, motherurine; FLX, fluoxetine Ting;

Figure 10: The effect of motherurine on the NF-ΚB signaling pathway in the hippocampus of CMS mice; the data are compared with the target protein and the internal reference β-actin; ^* P<0.05, ^** P<0.01, compared with the control group at the same period; ^# P ＜0.05, ^## P<0.01, compared with the model group at the same period; n=3, CON, normal mice; CMS, CMS mice; Leonurine, motherwort; FLX, fluoxetine;

Figure 11: The effect of motherurine on hippocampal neurotrophic factors BDNF and GDNF in CMS mice; the data are compared with the target protein and the internal reference β-actin; ^* P<0.05, ^** P<0.01, compared with the control group at the same period; ^# P<0.05, ^## P<0.01, compared with the model group at the same period; n=3, CON, normal mice; CMS, CMS mice; Leonurine, motherwort; FLX, fluoxetine.

detailed description:

Experimental materials and instruments:

Experimental material: Leonurine, white powder, purity>99.0%, provided by School of Pharmacy, Fudan University. Fluoxetine (fluoxetine) was purchased from sigma company. The use of standard products is as follows: norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT), purchased from Sigma Corporation of the United States. Antibody usage is as follows: rabbit anti-Bax, rabbit anti-Bcl-2, rabbit anti-Phospho-IKKα/β(Ser176/180)(16A6), rabbit anti-IKKβ(D30C6), rabbit anti-Phospho-NF-κB p65(Ser536)( 93H1), rabbit anti-NF-κB p65 (D14E12) was purchased from Cell Signaling Technology Company of the United States; goat anti-IL-1β, mouse anti-β-actin were purchased from American Sigma Company, rabbit anti-TNF-a, rabbit anti-IL-6 were purchased from Rabbit anti-BDNF (N-20) was purchased from abcam Company, and rabbit anti-GDNF were purchased from Santa Cruz Biotechnology Company.

Experimental equipment: Forced Swim ScanTM (Clever Sys Inc., VA, USA); Tail Susp ScanTM (Clever Sys Inc., VA, USA), Thermo ultimate 3000 high performance liquid chromatography (Thermo, USA), ImageQuant LAS 4000 chemiluminescence Image analyzer (GE, USA), transmission electron microscope (JEM-1010, Tokyo, Japan), stereology system (MBF, USA).

Source of mice: male C57BL/6J mice, weighing 18-22 g, aged 2-3 months, were purchased from the Comparative Medicine Center of Yangzhou University (animal production license number is SCXR (Su) 20120004).

30mg/kg/day or 60mg/kg/day in all the examples of the present invention means that the daily dose per kg of mice is 30mg or 60mg.

Example 1: Improvement effect of motherwortine on depression-like behavior in CMS mice

The present invention adopts the CMS depression model recognized by the international academic circle to prepare rodent depression. Male C57BL/6J mice, weighing 18-22 g, aged 2-3 months, were purchased from the Comparative Medicine Center of Yangzhou University (animal production license number is SCXR (Su) 20120004). After one week of adaptive feeding, the mice in the normal control group ate and drank normally without any stimulation, and the stressed mice received two or three random, mild and unpredictable stimuli every day until depression-like symptoms appeared. The CMS method includes a series of unpredictable chronic mild stresses including: tail pinching, circadian reversal, 45° inclined cage (6h), restraint (12h), wet cage (12h), empty cage (10-14h), water deprivation (15h), pairing (2h), changing bedding, strobe lighting for 12h, etc. The same stimulus cannot be given continuously. The sugar water preference rate was measured weekly for each mouse. The mice were subjected to chronic mild stimulation until the 6th week, and were given leonurine (30 mg/kg/day or 60 mg/kg/day) by intragastric administration for 4 weeks. One hour after the last administration, sugar water preference, forced swimming and tail suspension tests were performed to evaluate the effect of motherwort on the behavior of CMS mice. The experimental results were analyzed by one-way analysis of variance.

The results of the sugar water preference experiment (Table 1, accompanying drawing 1) showed that the sugar water preference rate of the CMS-stimulated mice was reduced by 20.14% ± 1.08% in the 6th week, which was significantly different from the normal control group (P<0.001). The sugar preference rate of the mice in each group increased week by week. At the 4th week, the sugar preference rate of the mice in the 60mg/kg/day Leonurine group increased by 8.12%±0.87% compared with the CMS group, with a significant difference (P<0.05 ). The results of the mouse forced swimming test (table 2, accompanying drawing 2) show: given CMS stimulation, the immobility time of mice forced to swim increases by 174.55% ± 4.53%, has significant difference (P<0.001), and motherwortine shortens the time of CMS mice Immobility time, wherein the immobility time of 60mg/kg/day motherwortine mice shortens 31.85% ± 2.13%, has significant difference (P<0.05); Mouse tail suspension test result (table 2, accompanying drawing 3) shows : Given CMS stimulation, the tail-suspension time of mice increased by 83.11%±3.77%, with a significant difference (P<0.01). The immobility time shortened by 28.93%±0.31%, with significant difference (P<0.05). It is suggested that motherwortine can improve the depressive-like behavior of CMS mice.

Table 1 Mouse sugar water preference rate (%)

Note: ^* p<0.05, ^** p<0.01, ^*** p<0.001vs normal control group, ^# p<0.05, ^## p<0.01vs CMS model group Mean±SEM

Table 2 The immobility time of mice forced swimming and tail suspension tests

Note: ^*** p<0.001vs normal control group, ^# p<0.05, ^## p<0.01vs CMS model group Mean±SEM

Example 2: Effects of Leonurine on Monoamine Neurotransmitters in the Brain of CMS Mice

In this part, the CMS depression model described in Example 1 was used. Up to the sixth week, leonurine (30 mg/kg/day or 60 mg/kg/day) was given by intragastric administration for 4 weeks. 1 hour after the last administration, the mice were deeply anesthetized, and the brain tissue was quickly peeled off, and the hippocampus and prefrontal cortex were separated, and the homogenate (0.1M HClO ₄ , 0.1mM EDTA) was added to the homogenate, centrifuged (20,000rpm, 30min), and the supernatant was taken. Thermo ultimate 3000 high-performance liquid chromatography (HPLC) was used to detect the contents of monoamine neurotransmitters 5-hydroxytryptamine (5-HT), norepinephrine (NE) and dopamine (DA) in each sample.

As shown by the assay results of monoamine transmitters in the hippocampus (Table 3, accompanying drawings 4A-4C), compared with the normal control group, the contents of 5-HT, NE, and DA in CMS stress mice decreased by 35.73%, 27.91%, and 37.38%, respectively. %, with significant difference (P<0.001, P<0.01, P<0.05); the contents of hippocampus 5-HT, NE, and DA in mice given 60mg/kg Leonurine group for 4 weeks were respectively increased by 40.99% compared with CMS group , 35.50%, and 52.42%, with significant differences (P<0.05). As shown in the results of the determination of monoamine transmitters in the prefrontal cortex (Table 4, accompanying drawings 4D-4F), the contents of 5-HT, NE, and DA in CMS stress mice were reduced by 42.55%, 37.68%, and 57.31%, respectively, which was significant Difference (P<0.001, P<0.001, P<0.01), the content of 5-HT, NE, DA in the administration group increased by 21.37%, 28.65%, 68.30% respectively compared with the CMS group, there was a significant difference (P<0.05 , P<0.01, P<0.05). It suggested that motherwort increased the content of monoamine neurotransmitters such as 5-HT, NE and DA in the brain of CMS mice, and improved the abnormality of monoamine neurotransmitters. Table 3 Monoamine neurotransmitter content in mouse hippocampus (ng/g wet tissue)

Note: ^* p<0.05, ^** p<0.01, ^*** p<0.001vs normal control group, ^# p<0.05, ^## p<0.01vs CMS model group Mean±SEM

Table 4 Mouse prefrontal cortex monoamine neurotransmitter content (ng/g wet tissue)

Note: ^** p<0.01, ^*** p<0.001vs normal control group, ^# p<0.05, ^## p<0.01vs CMS model group Mean±SEM

Example 3: Effects of Leonurine on Neurons and Glial Cells in the Brain of CMS Mice

In this part, the CMS depression model described in Example 1 was adopted, and until the sixth week, leonurine (30 mg/kg/day or 60 mg/kg/day) was given by intragastric administration for 4 weeks. 1 hour after the last administration, the mice were deeply anesthetized, perfused with 4% paraformaldehyde, and dehydrated with gradient sucrose. The brain tissue was frozen and sectioned, and the hippocampal brain slices were selected according to the brain atlas for Nissl staining to observe the morphology and structure of hippocampal neurons; in addition, at 4 % (mass percentage) after paraformaldehyde perfusion, the isolated hippocampus was fixed in 2.5% (mass percentage) glutaraldehyde, the electron microscope specimen was prepared, and the ultrastructure of the hippocampal neurons was observed under the transmission electron microscope; the immunofluorescence method was used to detect Astrocytes in the hippocampus, combined with the MBF stereology system to count the number of GFAP-positive cells.

The results of Nissl staining (Figure 5) showed that in the normal control group, the neurons in the dentate gyrus (DG) and CA3 area of the mouse hippocampus were closely arranged, the neuron cell bodies were larger, the nuclei were large and round, and Nissl bodies were abundant; the CMS model group The pyramidal cell layer of neurons in the DG and CA3 areas of the mouse hippocampus became thinner, the intercellular space was large, the arrangement was loose, the cell volume became smaller, and the Nissl bodies decreased; The arrangement of neurons, the size of the cell body, the shape of the nucleus and the number of Nissl bodies were all close to normal (Fig. 6C). It is suggested that motherwortine can improve the morphology of neurons in the brain of CMS mice.

The results of transmission electron microscopy (Figure 6) showed that the normal control group had normal cell body size, smooth surface, oval nucleus, clear two-layer membrane structure, uniform distribution of nuclear chromatin, abundant organelles in the cytoplasm, and complete structure. Mitochondria are round or oval, and the inner cristae are clearly visible (6A, 6D); the cell bodies of neurons in the CMS model group become smaller, and the nuclear heterochromatin increases, which is blocky and gathers towards the edge; the perinuclear space is widened; Mitochondria were swollen, with a small amount of vacuoles; some inner cristae of mitochondria were blurred (6B, 6E). In the motherurine (60mg/kg/day) group, the neuron cell bodies were basically normal, the heterochromatin in the nucleus was slightly increased, the perinuclear space was basically normal, and the nuclear margin was smooth; the mitochondrial structure was basically complete, and mitochondrial swelling was rare (Fig. 6C, Fig. 6F) . It suggested that motherwort improved the structure of neurons in the hippocampus of CMS mice and alleviated neuron damage.

Myelin sheath is a key mediator of the interaction between neurons. Abnormal myelin sheath structure indirectly leads to neuron loss or death, affects brain synaptic structure and spatial memory, and is the structural basis of hippocampal function. The present invention uses transmission electron microscopy to detect the structure of hippocampal myelin, and the results are shown in Figure 7A. The shape and structure of myelin in the hippocampus of the normal control group are normal, with dense plasma membrane rings; The signs of tissue damage such as loose arrangement of sheaths and thinning of myelin sheaths, while the structure of myelin sheaths tended to be normal in the leonurine (60mg/kg/day) group. The ratio (G ratio) of the inner diameter of the myelin sheath to the total outer diameter was used for statistical analysis, and the results showed (Table 5, Fig. 7B) that the CMS model group increased by 25.37% compared with the normal control group, and there was a significant difference (P<0.001); Motherwort The alkali (60mg/kg/day) group was similar to the normal control group, and the G ratio was 15.48% lower than that of the CMS group, which was significantly different (P<0.01). It is suggested that motherwort can improve the structure of neurons in the brain of CMS mice.

Table 5 The myelin sheath g-ratio (inner diameter/outer diameter of the myelin sheath) of mice in each group

Note: ^*** p<0.001vs normal control group, ^## p<0.01vs CMS model group Mean±SEM

Astrocytes are an important part of the brain parenchyma. They not only provide neurons with a variety of nutritional support substances, but also play a role in synaptic remodeling, nerve cell regeneration, and regulation of nerve extracellular ions, neurotransmitters and metabolites. Plays an important role, and astrocytes in the hippocampus are significantly reduced in patients with depression. This embodiment uses immunofluorescence to detect the influence of motherurine on the astrocytes of CMS mice, and the results (Table 6, Figure 8) show that: compared with the normal control group, the astrocytes in the DG region of the hippocampus of the mice in the CMS model group Cell expression decreased (8A), and the number of glial fibrillary acidic protein (GFAP) (astrocyte-specific marker) positive cells decreased by 36.09% (8B), with a significant difference (P<0.01); Leonurine (60mg /kg/day) group mouse astrocytes increased by 31.20% compared with the model group, with a significant difference (P<0.05). It is suggested that motherwort has the effect of increasing astrocytes in the brain and significantly improving the microenvironment of neuron survival.

Table 6 The number of astrocytes in DG area of hippocampus of mice in each group

Note: ^** p<0.01vs normal control group, ^# p<0.05vs CMS model group Mean±SEM

Example 4: Effects of Leonurine on Neuroinflammation in the Brain of CMS Mice

In this part, the CMS depression model described in Example 1 was used. Up to the sixth week, leonurine (30 mg/kg/day or 60 mg/kg/day) was given by intragastric administration for 4 weeks. 1 hour after the last administration, the mice were deeply anesthetized, and the hippocampal tissue was quickly separated, and protein lysate was added to extract the tissue protein. Western blot was used to detect the protein expressions of neuroinflammation-related factors IL-1β, IL-6, and TNF in the mouse hippocampus and NF - Changes in the κB signaling pathway. After weighing the hippocampal tissue, add RIPA lysate at 1:10 (w/v), homogenate the tissue, lyse on ice for 30 min, 16000 rpm×4°C×15 min, and absorb the supernatant. Take 1 μL for BCA protein quantification (Beiyuntian, Shanghai), and the remaining supernatant protein was added to 5X loading buffer according to the volume ratio, denatured at 95°C for 5min, and stored at -20°C after aliquoting. According to the quantitative results of BCA, 40 μg sample/swimming lane was loaded, and the concentration was selected according to the molecular weight of the protein to prepare SDS-PAGE gel for constant voltage (80-120V) gel electrophoresis separation, and electroporation at a constant current of 300mA for 70-120min to PVDF membrane (Millipore, USA). 5% (mass percent) TBST (pH7.4, 10mM Tris-HCl, 150mM NaCl, 0.1% Tween-20) of skimmed milk powder was blocked at room temperature for 1 hour, and the primary antibody prepared by adding 5% (mass percent) BSA-TBST: rabbit Anti-Phospho-IKKα/β(Ser176/180)(16A6)(1:1000, CST, 2697), Rabbit Anti-IKKβ(D30C6)(1:1000, CST, 8943), Rabbit Anti-Phospho-NF-κB p65(Ser536 )(93H1)(1:1000, CST, 3033), rabbit anti-NF-κB p65(D14E12) (1:1000, CST, 8242S), rabbit anti-Bcl-2 (1:1000, CST, 2876), rabbit anti- Bax (1:1000, CST, 2772), goat anti-IL-1β (1:1000, sigma, 13767,), rabbit anti-TNF-a (1:1000, Abcam, ab9739), rabbit anti-IL-6 (1: 1000, Abcam, ab83339), rabbit anti-BDNF (N-20) (1:1000, Santa Cruz, sc-546), rabbit anti-GDNF (1:1000, Santa Cruz, sc-328), mouse anti-β-actin (1:1000, Sigma, A5441). Incubate overnight at 4°C. Wash with TBST for 10 min 3 times, add horseradish peroxidase-labeled secondary antibody: goat anti-rabbit-HRP (1:800, KPL), goat anti-mouse -HRP (1:800, KPL), rabbit anti-goat-HRP (1:800, KPL), incubate at room temperature for 60min, add ECL (Pierce) chemiluminescence substrate for color development. Develop with Image Quant LAS 4000 chemiluminescence imaging analyzer Analysis; the gray value of phosphorylated protein kinase was compared with the gray value of total protein kinase to determine the degree of phosphorylation, and the ratio of the gray value of the target protein to the gray value of the respective internal reference β-actin was used for semi-quantitative analysis (Image J. ). The above 1:1000 refers to the dilution of 1000 times.

The result shows (accompanying drawing 9, figure 10). Compared with the normal mouse group, the protein expressions of inflammatory factors IL-1β, IL-6, and TNF-α in the hippocampus of CMS mice (Fig. 9) and the phosphorylation levels of IKKβ and P65 (Fig. 10) were significantly increased. The protein levels of hippocampal inflammatory factors IL-1β, IL-6, TNF-α and the phosphorylation levels of IKKβ and P65 in the leonurine group were lower than those in the CMS group, and the 60mg/kg/day leonurine group had a significant difference (P<0.01) . It is suggested that motherwort inhibits the activation of NF-κB signaling pathway in the brain of CMS mice, thereby inhibiting the inflammatory response.

Example 5: Effects of Leonurine on Neurotrophic Factors in the Brain of CMS Mice

In this part, the CMS depression model described in Example 1 was adopted, and until the sixth week, leonurine (30 mg/kg/day or 60 mg/kg/day) was given by intragastric administration for 4 weeks. 1h after the last administration, the mice were deeply anesthetized, the hippocampal tissue was rapidly separated, protein lysate was added, the tissue protein was extracted, and the protein levels of neurotrophic factors BDNF and GDNF in the mouse hippocampus were detected by Western blot method (same as Example 4).

The results are shown in Figure 11, the protein levels of neurotrophic factors BDNF and GDNF in the hippocampus of CMS mice were significantly reduced, and the protein levels of BDNF and GDNF in the Leonurine (60mg/kg/day) group were significantly higher than those in the CMS group (P<0.05 ). It is suggested that motherwort can significantly restore the level of neurotrophic factors in the brains of CMS mice.

Claims (8)

1. leonurine is preparing treatment or the application in prevention of depression medicine.

2. application according to claim 1, it is characterised in that comprising but be not limited only to leonurine and its salt.

3. application according to claim 1, it is characterised in that the treatment or prevention of depression medicine are included but not only limited In one-component or compound preparation.

4. application according to claim 1, it is characterised in that the formulation of the treatment or prevention of depression medicine be comprising But it is not limited only to tablet, capsule, sustained release tablets, controlled release tablet, oral liquid, syrup, dripping pill, parenteral solution formulation or freeze-dried powder formulation.

5. application of the leonurine described in claim 1 in CMS animal models are built.

6. application of the leonurine described in claim 1 in CMS mouse models are built.

7. application according to claim 6, it is characterised in that the agent of the daily leonurine of every kilogram of CMS mouse Measure as 30mg ~ 60mg.

8. the application according to any one of Claims 1 to 4, it is characterised in that the daily adult administration of the leonurine Dosage is 50～300mg.