CN110693894A - Application of neuroprotective effect of dipsacoside VI in treatment of depression - Google Patents

Application of neuroprotective effect of dipsacoside VI in treatment of depression Download PDF

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CN110693894A
CN110693894A CN201911128827.XA CN201911128827A CN110693894A CN 110693894 A CN110693894 A CN 110693894A CN 201911128827 A CN201911128827 A CN 201911128827A CN 110693894 A CN110693894 A CN 110693894A
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张进强
周涛
江维克
郭兰萍
肖承鸿
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Guizhou University of Traditional Chinese Medicine
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Abstract

The invention relates to application of dipsacus asperoides VI in neuroprotection in depression treatment, wherein the dipsacus asperoides VI are used as main active ingredients of Chinese herbal medicines of dipsacus asperoides and have good pharmacological activity in neuroprotection, myocardial protection and the like. The nerve injury caused by neuroinflammation is considered as one of causes of depression, so that the invention has wide development prospect for exploring the effect of the neuroprotective effect of the teasel saponin VI in treating the depression.

Description

Application of neuroprotective effect of dipsacoside VI in treatment of depression
Technical Field
The invention relates to the field of medicine application, in particular to application of neuroprotective effect of dipsacoside VI in treatment of depression.
Background
Depression (Depression) is a disease mainly manifested by depressed mood, usually manifested as mood disorder, mainly characterized by low mood, lack of interest or fun, and persistent fatigue, and has the characteristics of high recurrence rate, high disability rate, high suicide rate, low recognition rate, low diagnosis rate, and low treatment rate, and it is predicted that Depression will become the second leading cause of cancer after 2020, which threatens human health, and therefore, the treatment and prevention of Depression is very important for improving human health and life quality.
① the cause of depression is that from the perspective of traditional Chinese medicine, liver qi deficiency, namely yang qi deficiency in liver qi, cannot cause agitation, excitation and promotion, leading to the appearance of yang deficiency symptoms which are mainly characterized by mood and will symptoms[3]② from the genetic point of view, depression is a highly inherited mental disorder, and due to its highly inherited characteristics, people in the family with a history of depression, particularly first-class relatives, tend to have a much greater chance of suffering depression than normal[4]③ from the neurotransmitter research perspective, it is now acknowledged that one of the important contributors to depression is the monoamine transmitter system[5]. The monoamine transmitter system mainly includes the dopamine system, (DA) 5-hydroxytryptamine system (5-HT) and norepinephrine system (NE). Studies suggest that monoamine transmitters in the brain play a critical role in the development, progression and treatment of depression. And when the depressive behavior occurs, the monoamine nuclei of the brainstem can also participate in the depressive behavior, because 5-HT can participate in the inhibition of autonomic activity and plays a role in inhibiting autonomic activity together with the NE system. In addition, the amino acid neurotransmitter system may also be involved in depressive behavior, the onset of which may be associated withThe interaction of gamma-aminobutyric acid (GABA), glutamic acid and the receptor thereof is closely related, which may become a new hot spot for researching the affective disorder medicament. In recent years, the ghrelin (intracerebral neuropeptide) theory and the serotonin-norepinephrine-glucocorticoid chain theory have attracted much attention. For example, Crespi found by studies that a decrease in ghrelin function could lead to the development of depression. The neuropeptides and their receptors are important mediators which cause mood disorders in patients at the onset of depression, schizophrenia, anxiety. In summary, the pathogenesis of depression may involve monoamine neurotransmitters and numerous neuropeptides, including amino acid neurotransmitters[4]④ from the aspect of neurobiochemistry, it was found that the pathogenesis of depression may be related to various neurobiochemical factors such as unbalanced neuroactive steroid level, altered adenosine and its receptor interaction and abnormal decrease of estrogen level, wherein neuroactive steroid has a very close relationship with psychotic disorder, depression and anxiety, and the like, and the neuroactive steroid plays a role in protecting neurons by reducing the toxic action of glucocorticoid on neurons and enhancing the function of central GABA neurons to play a role in antidepressant [4]⑤ from the view of social psychology, the occurrence, development and treatment process of depression are all closely related to individual personality characteristics, family environment, stressful life events, social support, coping style, etc. in the research process of personality and depression correlation, it is found that the occurrence and development of depression are established on a certain personality basis, people with different personality characteristics have great difference to the incidence rate of depression, for example, people with personality of nervousness and psychology have positive correlation in the occurrence and development of depression, people with personality of external personality have negative correlation in the occurrence and development of depression[6-7]。
Current therapeutic status of depression: the existing treatment modes for depression mainly comprise drug treatment, psychological treatment, combined treatment and physical treatment[8]. The treatment of depression with drugs can generally be divided into three stages, which are acute treatment, continued treatment and maintenanceThree treatment modes are kept. The drug therapy has better curative effect on most acute depression symptoms, the curative effect is approximately similar, but the side effect degrees are different. In terms of psychotherapy, the cognitive behavior therapy and the memorial training have better effect on treating depression for patients after acute phase, and are more effective on the problems appearing in the remission stage[9]. In the combination therapy, the combination therapy of psychological therapy and drug therapy is used more frequently at present, and the curative effect is better than that of the single drug therapy through the combination of the advantages of the psychological therapy and the drug therapy. Among the common physical therapies, Pannerong and Yang boys[10]The study of (2) considers that the electroconvulsive therapy for convulsion is a safe and effective method for treating depression, which is worth popularizing, and although the hospitalization rate and the hospitalization time of the depression patient can be reduced and reduced, the deficiency is more, for example, symptoms such as lethargy, muscle pain and nausea are accompanied, and even more serious patients can cause mild memory impairment. The research of repeated transcranial magnetic stimulation also shows that the rTMS treatment acting on the left prefrontal cortex every day is safe and effective, can improve the mood state of a patient and relieve the depression symptom of the patient, and can effectively treat refractory depression by combining with drug treatment[11]. Even so, rTMS has disadvantages of expensive use, difficult manipulation and the possibility of causing twitching[12]。
The inflammation hypothesis of depression, the clinical first-line antidepressant of depression, is mainly based on the drug of monoamine hypothesis, exerts antidepressant action by regulating monoamine transmitter, although the curative effect is definite, but there are a lot of defects such as delayed onset, low effective rate, many adverse reactions, etc. generally, the monoamine hypothesis can not explain all phenomena of depression. Therefore, in recent years, other hypotheses than the monoamine hypothesis have been increasingly emphasized, such as the inflammation hypothesis, which considers that stress stimuli trigger the inflammatory process, ultimately leading to the development of depression[13]。
The hypothesis that inflammation may be involved in pathophysiological processes in depression was first proposed by Smith in 1991[14]This hypothesis is also supported by a large number of research results① cancer patients with non-depressive disorders often show symptoms sufficient to satisfy the diagnostic criteria for depression after administration of proinflammatory cytokines, and the symptoms disappear upon cessation of administration of proinflammatory cytokines[15]The so-called pro-inflammatory cytokines (pro-inflammatory cytokines) are cytokines capable of promoting inflammation and belong to the class of pro-inflammatory cytokines, mainly including tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), IL-6, IL-8, etc., ② elevated levels of inflammatory biomarkers and pro-inflammatory factors in blood and cerebrospinal fluid of patients with depression[18-21]③ proinflammatory factors cause alterations in 5-HT levels[16]④ proinflammatory factors can explain the axial hyperactivity of the hypothalamus-pituitary-adrenal gland (HPA) in depression [17]⑤ Presence of proinflammatory factor polymorphism in depression patients to increase their susceptibility to inflammatory response[18]⑥ A change in the level of proinflammatory factor can predict the progression of the clinical course of depression[19]⑦ Depression has a higher co-prevalence with inflammation-mediated disorders[20]The inflammation hypothesis states that stress stimuli trigger inflammatory processes that lead to abnormal changes in the normal physiological functions of the 5-HT system and HPA axis, ultimately leading to the development of depression, and how does the inflammatory processes participate in the pathogenesis of depression [21]② Tryptophan metabolite (TRYCAT) pathway[22]③ reduction of omega-3 polyunsaturated fatty acids (PUFAs)[23]④ neurodegenerative changes and reduced neuronal regeneration[24]And the like.
Neuroinflammation refers to the interaction between the nervous system and the immune system. The nervous system is always in an immunologically sequestered state under the influence of the presence of the blood-brain barrier. How does peripheral inflammation affect depression occurring in the brain? The research shows that the autopsy of the depression patient finds that the mRNA level of the whole pro-inflammatory factor network is up-regulated, and TNF-alpha, IL-1 beta and the like are pre-regulatedAlthough the inflammatory factors are generated by peripheral immune cells and cannot pass through a blood brain barrier in a diffusion way, the inflammatory factors can enter the central nervous system to promote the synthesis of the central nervous system cytokines by ① entering a brain area with defect of the blood brain barrier or actively transferring into the central nervous system through an endothelial cell transporter, ② promotes blood brain barrier endothelial cells to generate Prostaglandin (PG) E2, PGE2 can activate neurons by activating microglia and astrocytes, ③ influences nerve afferent signals such as vagus nerves and the like associated with the cytokine receptors to inform the central nervous system of the existence of systemic inflammatory signals[18]Further activates the hypothalamus and other brain areas to participate in the disease process, activates macrophages and endothelial cells around the cerebral vessels, and promotes the release of proinflammatory factors. Microglia are the earliest cells that respond to peripheral inflammatory signals, initiating inflammatory cascades including cytokines, chemokines, reactive oxygen species, and reactive nitrogen species. Then astrocytes are activated to further amplify inflammatory signals, IL-1, IL-6, TNF-alpha, IFN-alpha and gamma can be induced to generate IDO, so that tryptophan is reduced, quinolinic acid is increased, neurotrophic factors are reduced, and glutamic acid excitotoxicity is enhanced[19]. Two neuroinflammatory hypotheses have been generated based on these phenomena: the first is the inflammation and neurodegenerative hypothesis[21]The hypothesis is that inflammation-induced neurotoxicity and neurodegeneration are one of the causes of depression; the second hypothesis is substantially similar to the first hypothesis, except that the initiating factors of psychological stress are emphasized, which is believed to lead to neuronal micro-damage, concomitant neuronal regeneration and reduced release of neurotrophic factors and enhanced neuroinflammatory activity, ultimately leading to the appearance of depressive symptoms[25]。
Recent studies indicate that fibroblasts, macrophages, dendritic cells and endothelial cells around the nerve membrane can be activated to generate immune substances such as cytokines, carbon monoxide and chemokines which induce immune cells in circulation to enter the nervous system to trigger immune response or inflammatory injury, and the major pathways include complement systems such as complement, immune cells and glial cells, and the like, and the immune substances are obtained by the complement systemsActivation of some complement systems is an important link in the mechanism of chronic neuroinflammation. A plurality of cells in a nervous system can express complement components and complement receptors, Complement Factor H (CFH) is an important regulatory factor in a complement activation system, researches show that the CFH protein level in peripheral blood of depression patients is obviously lower than that of normal healthy people, rs1061170 site of CFH gene is obviously associated with depression, eQTL analysis results show that the site obviously influences the CFH gene expression in infracerebral olivary nucleus and occipital cortex, and the CFH is predicted to be possibly related to depression[26]。
A recent data set encompassing 18 clinical studies, including meta-analysis of a number of patients with suicidal depression, patients without suicidal depression and healthy persons, showed that IL-1 β and IL-6 levels in peripheral blood and brain tissue were significantly higher in patients with suicidal depression than in patients without suicidal depression. In vitro experiments show that patients with suicidal depression who produce peripheral blood mononuclear cells IL-2 are significantly lower than those without suicidal depression. IL-8 levels in cerebrospinal fluid were significantly higher in normal controls than in patients with suicidal depression. Some studies have also shown that elevated IL-1 β and IL-6, as well as microglial proliferation and mononucleosis, are associated with suicidal behavior. In pharmacological studies, many antidepressants exhibit some anti-inflammatory effects, thus indirectly demonstrating that the inflammatory response is the underlying pathological mechanism of depression[27]。
① antidepressants have anti-inflammatory effects, and a great deal of clinical studies show that the antidepressants can inhibit the increase of the level of inflammatory mediators in peripheral blood, but the antidepressants have relatively less influence on the level of inflammatory mediators in cerebrospinal fluid and autopsy brain tissues, and Palhagen et al report that the SSRI antidepressant citalopram (citalopram) has no influence on the increase of IL-6 level in the cerebrospinal fluid of depression patients[28]. A large number of animal experiments show that the antidepressant can reduce the inflammatory reaction. Peripherally, clomipramine (clomipramine) and imipramine (imipramine) can inhibit the release of IL-1 beta and TNF-alpha from lymphocytes, citalopram can inhibit the release of IL-1 beta and TNF-alpha from activated monocytes, sertraline (sertraline) and paroxetine (parooxetine) canRemarkably reducing the release of TNF-alpha from T lymphocytes; in the central nervous system, desipramine (desipramine) can reduce LPS-induced increases in the levels of TNF-alpha, IL-1 beta, Inducible Nitric Oxide Synthase (iNOS) and nuclear transcription factor (NF-kB) in rat cortical tissues, and paroxetine can block IFN-alpha-induced increases in the level of IL-1 beta in the hypothalamus [29]② Effect of antidepressants on activated microglia, which play a role as a performer in central immune function and are also the major secretory cells of central inflammatory cytokines an important means to achieve interaction between the immune system and the central nervous system is the release of inflammatory cytokines studies have shown that microglia activation and inflammatory cytokine release are regulated by a variety of membrane surface receptors including TRPV1[30]. At present, although there is no direct evidence that the microglia participates in the pathogenesis of depression, research shows that the microglia hyperplasia in the brain of a suicide depression patient is obvious, psychological stress can promote the proliferation of the microglia by increasing glucocorticoid level, and various antidepressants can obviously inhibit the microglia activity enhancement induced by various stimuli[31]. For example, fluoxetine (fluoxetine) can significantly reduce the proliferation of microglia caused by cerebral ischemia, inhibit the increase of rat primary microglia TNF-alpha, IL-1 beta, cyclooxygenase 2(COX-2), NO and iNOS mRNA levels and the activation of NF-kappa B caused by LPS stimulation; paroxetine and sertraline can inhibit IFN-gamma induced 6-3 cell (mouse microglia) to produce TNF-alpha and NO, and reduce Ca2+Elevated level[32](ii) a The desipramine can inhibit LPS-induced microglial cell proliferation[33](ii) a The clomipramine and imipramine can obviously reduce the secretion of TNF-alpha, IL-1 beta and NO by BV2 cells (mouse microglia) caused by LPS stimulation and inhibit the activity of NF-kB signal channel[34](ii) a Moclobemide (moclobemide) is capable of dose-dependently reducing LPS-induced IL-1 beta and TNF-alpha release and NF-kappa B activation on cultured mixed glial cells[35](ii) a Venlafaxine (venlafaxine) also exhibits anti-inflammatory effects on mixed glial cells and is capable ofCan inhibit TNF-alpha and NO release induced by LPS[36]。
③ Effect of antidepressants on activated astrocytes, studies have shown that there is a significant reduction in the number of astrocytes in the prefrontal cortex of depressed patients, and in addition, there is a significant reduction in the number of astrocytes in the brain region of the hippocampus, as found in depressed patients and in tree shrews that are chronically stressed[26]. Oral fluoxetine administration for 28 days can significantly reverse the decrease in astrocyte counts caused by chronic stress[26]Can also increase the expression level of the rat hippocampal brain region astrocyte neurotrophin S100beta[24]. Amitriptyline (amitriptyline) can increase expression of astrocyte-derived neurotrophic factor (GDNF)[28]This effect may be characteristic of antidepressants, since neither antipsychotics nor anxiolytics induce GDNF release[33]. GDNF can further inhibit TNF-alpha and IL-1 beta release and NF-kappa B activation[26]. In addition, chronic stress can lead to impairment of astrocyte gap junction structure and function, while fluoxetine and duloxetine can significantly reverse these changes[22]。
① TNF-alpha antagonist, the best known report of Tyring et al, when studying the effect of TNF-alpha antagonist etanercept (entanercept) on psoriasis treatment, discovered that etanercept can also improve the patient's depressive symptoms, significantly lower the depression score compared to placebo, and its antidepressant effect is independent of its effect on psoriasis treatment[15]. The TNF-alpha monoclonal antibody infliximab (infliximab) also shows remarkable antidepressant effect on patients undergoing anti-inflammatory treatment[18]However, this effect is exhibited only when the levels of inflammatory markers such as TNF-alpha and C-reactive protein (CRP) are significantly increased, and when these inflammatory markers are at normal levels, no significant antidepressant effect is exhibited[24]. In addition, TNF- α antagonists may increase the risk of infection in patients during treatment [17]② non-steroidal anti-inflammatory drugsAcetyl-salicylic acid (ASA) can reduce prostaglandin and thromboxane by irreversibly inhibiting cyclooxygenase-1 (COX-1) and COX-2, thereby reducing TNF-alpha and IL-6 levels[24]. Researches show that ASA not only has antidepressant effect when being applied alone, but also can enhance the curative effect of SSRI and shorten the onset time when being used together with SSRI antidepressant drugs[28]. In addition, ASA and statins may also reduce the risk of depression. Although the role of ASA in the treatment of depression is not currently known, from existing and ongoing research, ASA holds great promise as a therapeutic or adjunctive therapeutic agent for depression. The COX-2 inhibitor celecoxib (celecoxib) serving as an adjuvant treatment drug can enhance the curative effect of first-line antidepressants such as fluoxetine and reboxetine (reboxetine) and improve cognitive dysfunction caused by depression, and the celecoxib has no obvious antidepressant effect when being applied alone[35]However, there are also studies to find that celecoxib can aggravate neuroinflammation and increase the risk of cardiovascular diseases, and is not suggested as an adjuvant treatment drug for antidepressants, so that the antidepressant adjuvant treatment effect of celecoxib still remains to be further studied ③ tetracyclic antibiotic, tetracyclic antibiotic minocycline (minocycline) has anti-inflammatory, anti-oxidative stress, neuroprotective effects, etc., can exert antidepressant effects through various routes, can significantly reduce LPS-induced depression-like behaviors, improve cognitive dysfunction due to depression, and is similar to the effect of lithium preparations, and thus may be effective for patients with bipolar depression[36]。
The active ingredients of the traditional Chinese medicine have the effect of resisting depression, ① omega-3 polyunsaturated fatty acid, and the omega-3 polyunsaturated fatty acid is unsaturated fatty acid which can not be synthesized by human head, and can reduce PGE2 level and reduce the production of proinflammatory factors by competing with Arachidonic Acid (AA) to combine COX enzymeThereby exerting an anti-inflammatory effect[37]. A large number of clinical, preclinical and epidemiological studies show that the omega-3 polyunsaturated fatty acid has remarkable antidepressant effect and good tolerance of patients. In addition, the omega-3 polyunsaturated fatty acid can be used as adjuvant therapeutic drug to enhance the curative effect of first-line antidepressant [38]② curcumin, curcumin extracted from rhizome of Curcuma longa has anti-inflammatory and antioxidant effects, and has been mainly used for supplementing replacement therapy[39]③ Salvianolic acid B, the research shows that the Salvianolic acid B shows the anti-depression characteristic in forced swimming, sugar water preference and tail suspension experiments, and does not influence the autonomous activity and weight change, and shows that the Salvianolic acid B is safe and effective, meanwhile, the Salvianolic acid B can reduce the stress-induced apoptosis and reduce the microglial cell-mediated neuroinflammation, and the correlation analysis result shows that the microglial cell activation is positively correlated with the apoptosis of hippocampus and cortex[40]。
The teasel root saponin VI has the antidepressant prospect: radix Dipsaci is root of Dipsacus asperoides C.Y.ChengtT.M.ai of Dipsacaceae (Dipsaceae) genus (Dipsacus). The medicinal composition comprises 30 kinds of plants in the genus of Dipsacus of Dipsacaceae, 18 kinds of plants in the genus of Dipsacus of China, and 2 varieties, wherein 10 kinds and 2 varieties can be used for medicinal use, and the most widely distributed species are Japanese Dipsacus and Dipsacus asperoides, and the medicinal composition in China at present is mainly dry root of Dipsacus asperoides with mild nature, bitter, pungent, liver-returning and kidney channel[41]. In Shen nong Ben Cao JingIt is classified as the top grade, has effects of nourishing liver and kidney, strengthening tendons and bones, treating fracture, preventing metrorrhagia and metrostaxis, etc., and can be used for treating soreness of waist and knees, limb atrophy and paralysis, traumatic injury, tendon injury, femoral fracture, fetal movement, metrorrhagia, nocturnal emission, leukorrhagia, superficial infection, sore, etc[42]. In recent years, people have increasingly studied the chemical components, pharmacological action, clinical application and the like of teasel roots. The chemical components of the teasel root are relatively complex, and the main chemical components comprise volatile oil, alkaloids, iridoid and triterpenoid saponin[43]。
Dipsacasperoides VI (aspersion VI) also known as Akebia quinata saponin D, also known as Dipsacasperoides C. The Dipsacaceae Dipsacus asperoides saponin VI is a triterpenoid saponin compound extracted and separated from the root of Dipsacus asperoides of Dipsacus of Dipsacaceae, and is the main active component of traditional Chinese herbal medicine Dipsacus asperoides. Research shows that the incidence of Alzheimer's disease can be effectively reduced by inhibiting the inflammation generation way through the dipsacus asperoides VI. In the learning and cognitive dysfunction model of SD rats induced by injecting Abeta 1-42 into bilateral ventricles, the activation of Abeta-induced astrocytes and microglia is obviously inhibited by dipsacus asperoides VI, and the glia can also reduce the release of cytokines and inflammatory factors, such as the expression of tumor necrosis factor alpha (TNF-alpha), interleukin 6(IL-6) and cyclooxygenase-2 (COX-2), and can inhibit the generation of inflammatory reaction. When the dosage of the teasel root saponin VI is 90mg kg-1And 270 mg/kg-1The phosphorylation levels of IkB kinase (IKK) and protein kinase B (Akt) can be effectively reduced, so as to inhibit the activation of nuclear factor (blank FAB) and the nuclear transfer process, and inhibit the generation of inflammatory reaction[44]。
LPS induces rodent depressive-like behavior: non-clinical studies of depression are currently mainly studied in the way of establishing models in animals. The LPS model is a model in which Lipopolysaccharide (LPS) from escherichia coli is injected to induce immune stress in the body, thereby inducing the mice to develop disease behaviors including depressive-like behaviors. LPS is used as an immune activator and cytokine inducer in numerous studies related to inflammation. Peripheral LPS injection can be through TLR4(Tollike receptor)4) Activate the body's innate immune system and rapidly trigger the release of pro-inflammatory cytokines such as L-1 β, TNF-a, IL-6, and the like. There are successive differences in the central and peripheral immune responses due to different injection modes. Intraperitoneal injection is mainly used for activating the central nervous system after peripheral immune response is caused, and lateral ventricular injection can directly induce relatively long-lasting central immune response. The dose used will vary according to the purpose of the study, e.g., a high dose of 6mg/kg is used to study sepsis, while 0.83mg/kg is the dose commonly used to establish a depressive-like model. After the intraperitoneal injection of 0.83mg/kgLPS, the animals show a plurality of pathological behaviors due to the strong immune state of the body, including spontaneous pain, reduced sugar water preference, weight reduction, diet reduction, activity reduction and other pathological behaviors. The behaviors have higher surface effectiveness, structural effectiveness and predictive effectiveness with the depression behaviors of human beings, namely, the behaviors have similarity of symptoms, pathology and curative effect, namely, the mice induced by LPS show the pathological behavior syndrome with behavior similar to depression, such as reduced sugar water preference, diet reduction, weight reduction, disordered hair and lusterless hair, reduced desire for survival in tail suspension experiments and forced swimming experiments, and the like. In clinical studies, volunteers also induced symptoms similar to depression following LPS injection. These features also make the LPS model one of the common depression models. However, this LPS model also has certain limitations. The induced pathological behavioral syndrome is essentially an immune defense mechanism of the body with the main purpose of eliminating pathogens or dangerous signal sources and accompanied with fever and neuroendocrine disorder. It is an acute symptom caused by inflammation, which is not consistent with the characteristics of long cycle and complex action mechanism of depression[30]. Since the experiment explores the development prospect of the neuroprotective effect of the teasel saponin VI in the treatment of the depression, the LPS model is selected as the depression model to meet the requirement.
With the development of society, the number of stimulating factors inducing the onset of depression is increasing, and the depression becomes a common disease and an easily-occurring disease in our lives and is also a disease difficult to cure. Numerous research results have led us to believe that inflammation is one of the important causes of depression, however, if the inflammatory process does play an important role in the etiological and pathological mechanisms of depression, there are still many problems to be solved. For example, whether improvement of peripheral inflammation would also reduce inflammatory responses in the central system and whether there is a dose-effect relationship between inflammatory markers and changes in brain function in patients with depression. The inflammation hypothesis of depression not only bridges the understanding of the gap between the somatic symptoms and the mental symptoms of depression, but also provides a new thought and a new molecular target for improving the curative effect of the first-line clinical antidepressant and the research and development of the antidepressant. The teasel saponin VI is used as a main active ingredient of the Chinese herbal medicine teasel, and has good pharmacological activity in the aspects of neuroprotection, myocardial protection and the like. And nerve damage caused by neuroinflammation is considered as one of the causes of depression. Therefore, the exploration of the neuroprotective effect of the dipsacoside VI in the treatment of depression has wide development prospect.
Disclosure of Invention
The invention aims to solve the problems in the background art and provides application of neuroprotective effect of dipsacoside VI in treatment of depression.
The invention relates to application of dipsacus asperoides VI in the morphology of M1 microglia.
The dipsacus asperoides VI are applied to inhibiting inflammatory reaction mediated by M1 microglia.
The invention relates to application of dipsacus asperoides VI in inhibiting activation of hippocampus and cortical microglia.
The invention relates to application of dipsacus asperoides VI in inhibition of neuroinflammation induced by LPS.
The invention relates to application of dipsacoside VI in improving depression-like behaviors induced by LPS.
The invention can also be used for preparing a pharmaceutically acceptable preparation by adding the dipsacoside VI into pharmaceutically acceptable auxiliary materials and applying the pharmaceutically acceptable preparation to the treatment of depression.
The preparation provided by the invention is in the dosage forms of granules, capsules, powder, tablets, pills, injection preparations, freeze-dried powder injection and the like.
The pharmaceutically acceptable auxiliary materials are not limited, and can be one or more of common auxiliary materials in the field, such as a disintegrating agent, a filling agent, a lubricating agent, an antioxidant, an adhesive, a surfactant or a flavoring agent.
The problems in the prior art and the beneficial effects of the invention are as follows:
1. the problems of the prior art are as follows:
(1) the drug therapy has better curative effect on most acute depression symptoms, the curative effect is approximately similar, but the side effect degrees are different. Among the common physical therapies, Pannerong and Yang boys[10]The study of (2) considers that the electroconvulsive therapy for convulsion is a safe and effective method for treating depression, which is worth popularizing, and although the hospitalization rate and the hospitalization time of the depression patient can be reduced and reduced, the deficiency is more, for example, symptoms such as lethargy, muscle pain and nausea are accompanied, and even more serious patients can cause mild memory impairment. The research of repeated transcranial magnetic stimulation also shows that the rTMS treatment acting on the left prefrontal cortex every day is safe and effective, can improve the mood state of a patient and relieve the depression symptom of the patient, and can effectively treat refractory depression by combining with drug treatment[11]. Even so, rTMS has disadvantages of expensive use, difficult manipulation and the possibility of causing twitching[12]。
(2) Antidepressant effect of anti-inflammatory drugs: TNF-alpha antagonist, TNF-alpha monoclonal antibody infliximab (infliximab), also shows significant antidepressant effect on patients undergoing anti-inflammatory therapy[18]However, this effect is only exhibited when the levels of inflammatory markers such as TNF-alpha and C-reactive protein (CRP) are significantly increased, and when these inflammatory markers are at normal levels, no significant antidepressant effect is exhibited[24]. In addition, TNF- α antagonists may increase the risk of infection in patients during treatment[17]。
(3) Non-steroidal anti-inflammatory drugs: COX-2 inhibitor celecoxib (celecoxib) as adjuvant therapyThe composition can enhance the curative effect of first-line antidepressants such as fluoxetine, reboxetine and the like, and improve cognitive dysfunction caused by depression, while celecoxib alone has no obvious antidepressant effect[35]. However, the research also finds that the celecoxib can aggravate neuroinflammation and increase the risk of cardiovascular diseases, and the celecoxib is not suggested to be used as an auxiliary treatment drug of an antidepressant, so the antidepressant auxiliary treatment effect of the celecoxib is still to be further researched.
(4) The anti-depression effect of the active ingredients of the traditional Chinese medicine mainly comprises omega-3 polyunsaturated fatty acid, curcumin and salvianolic acid B which are used as antidepressant drugs, but the application of the neuroprotective effect of the teasel root saponin VI in the treatment of depression is not researched in a related way.
2. The invention has the advantages of
(1) The dipsacus asperoides VI are used as main active ingredients of the Chinese herbal medicine dipsacus asperoides, and have good pharmacological activity in the aspects of neuroprotection, myocardial protection and the like. And nerve damage caused by neuroinflammation is considered as one of the causes of depression. Therefore, the exploration of the neuroprotective effect of the dipsacoside VI in the treatment of depression has wide development prospect.
(2) The research shows that the antidepressant effect of ASP VI is stable and effective in experimental animals, and is related to the inhibition of microglial cell activation and central inflammatory reaction and the final protection of neurogenesis.
(3) It has been shown by studies that microglial activation and inflammatory cytokine release are regulated by various membrane surface receptors including TRPV 1. Microglial activation is usually accompanied by inflammatory factor changes, especially after LPS stimulation, the microglial cells are usually produced with a large amount of proinflammatory cytokines (such as IL-1 beta, iNOS, TNF-alpha, IL-6 and the like), and the microglial cells are generally called classically activated microglial cells, also called M1 type microglial cells. In the research, LPS is used for treating microglia cultured in vitro, the microglia is induced to be polarized to M1 phenotype, different concentrations of dipsacus asperoides VI are used for intervening the activated microglia, and the results show that 100 mu M and 200 mu M dipsacus asperoides VI stem pre-set can obviously inhibit the cell expansion of the microglia, increase the phagolysosome and inhibit the expression of IL-1 beta, iNOS, TNF-alpha and IL-6 compared with an LPS model set. The dipsacoside VI is shown to be capable of obviously inhibiting the activation of microglia, so that the microglia-mediated inflammatory reaction is improved.
(4) The research researches the antidepressant effect of ASP VI on mice of LPS depression models from phenomena to mechanisms by combining two major experiments of animal behaviors and biochemistry, and further researches the relation between the antidepressant effect of ASP VI and activation of hippocampus and cortical microglia, central inflammatory reaction and neurogenesis. The experimental results show that:
① in weight, forced swimming and tail suspension experiments, the 40mg/kg ASPPI VI pretreatment group shows significant antidepressant effect;
② ASPPI VI pretreatment group significantly inhibited the level of activation of microglial cells and central inflammatory response in hippocampus and cortical tissues of the brain of LPS mice.
Drawings
Fig. 1 statistical chart of microglial purity [ microglia labeled with Iba1 antibody, astrocytes labeled with GFAP antibody, nuclei labeled with DAPI, scale: 50 μm ].
Fig. 2 difference of M1 type microglia morphology affected by dipsacoside VI [ (a) microglia immunofluorescence staining pattern, CD68 labeling phagolysosome (green), IBA1 labeling microglia (red), DAPI labeling nucleus (blue), large indicates superposition of the three (orange); (B) and (C) statistical plots of the positive areas of microglia CD68 and IBa1, the areas of CD68 and IBA1 positive stains were counted using ImageJ software, the statistical results were plotted using GraphPad software, and significant difference analysis was performed using one-way anova LSD post hoc,. P <0.005 vsControl; # P <0.01, # # P <0.005vs LPS; n-4-5, scale: 25 μm ].
Fig. 3 differential expression of M1 type microglia-mediated inflammatory cytokines after teasel saponin VI treatment of mRNA expression levels of proinflammatory cytokines (IL-1 β, iNOS, TNF- α, IL-6) in primary cultured microglia cells [ (a) - (D) were plotted using GraphPad software and significant differential analysis using LSD post hoc of one-way variance analysis, { P <0.01, { P <0.005vs Control; (ii) a # P <0.05, # P <0.01, # P <0.005 vsLPS; n-4-5 ].
FIG. 4 influence of Dipsacus asperoides VI on the activation of microglia in mouse hippocampus [ (A) and (B) qPCR assay for phenotypic markers of microglial activation, which predominantly assay for CD11B and Iba1 in mouse hippocampus; (C) microglial immunofluorescent staining pattern, IBA1 labeled microglial cells (red); (D) and (E) statistical plots of the areas of positive microglia Iba1 and microglia branches, the areas of IBA1 positive staining were counted using ImageJ software, the statistical results were plotted using GraphPad software, and significant difference analysis was performed using one-way anova LSD posthoc,. P <0.005vs Control; # P <0.01, # # P <0.005vs LPS; n-4-5, scale: 25 μm ].
FIG. 5 influence of Dipsacus asperoides VI on the activation of mouse cortical microglia [ (A) and (B) qPCR assay phenotypic markers of cortical microglia activation, which predominantly assay CD11B and Iba1 of mouse cortex; (C) microglial immunofluorescent staining pattern, IBA1 labeled microglial cells (red); (D) and (E) statistical plots of the areas of positive microglia Iba1 and microglia branches, the areas of IBA1 positive staining were counted using ImageJ software, the statistical results were plotted using GraphPad software, and significant difference analysis was performed using the one-way anova LSDpost hoc,. about.P <0.005vs Control; # P <0.01, # # P <0.005vs LPS; n-4-5, scale: 25 μm ].
Figure 6 differential expression of inflammatory cytokines induced by LPS in hippocampus after treatment of dipsacus saponin VI mRNA expression levels of proinflammatory cytokines (IL-1 β, iNOS, TNF- α, IL-6) of inflammatory mice under intervention of dipsacus saponin VI [ (a) - (D) and results were plotted using GraphPad software and analyzed for significant differences by LSD post hoc with one-way variance analysis, { P <0.01, { P <0.005vs Control; (ii) a # P <0.05, # P <0.01, # P <0.005vs LPS; n-4-5 ].
Figure 7 differential expression of inflammatory cytokines induced by LPS in the cortex after treatment of dipsacus asperoides saponin VI the expression levels of the mRNA of proinflammatory cytokines (IL-1 β, iNOS, TNF- α, IL-6) of inflammatory mice under intervention of dipsacus asperoides saponin VI (a) - (D) were plotted with GraphPad software and analyzed for significant differences using LSD post hoc with one-way variance analysis, < P <0.01, < P <0.005vs Control; (ii) a # P <0.05, # P <0.01, # P <0.005vs LPS; n-4-5 ].
FIG. 8 forced swim test [ (A) schematic of forced swim test; (B) a latency period statistical chart of an inflammatory mouse under the intervention of dipsacoside VI in a forced swimming test; (C) statistical chart of immobility time of inflammatory mice under the intervention of dipsacoside VI in forced swimming test. The results were plotted using GraphPad software and analyzed for significant differences using one-way anova LSD post hoc, # P <0.01, # P <0.005vs Control; (ii) a # P <0.05, # P <0.01vs LPS; n-8-12 ].
FIG. 9 shows a schematic representation of the tail suspension experiment [ (D); (E) a latency period statistical chart of an inflammatory mouse under the intervention of dipsacoside VI in a tail suspension test; (F) statistical chart of immobility time of inflammatory mice under the intervention of dipsacoside VI in tail suspension test. The results were plotted using GraphPad software and analyzed for significant differences using one-way anova LSD post hoc, # P <0.01, # P <0.005vs Control; (ii) a # P <0.05, # P <0.01, # P <0.005 vsLPS; n-8-10 ].
Figure 10 effect of dipsacoside VI and LPS on mouse food consumption [ (a) effect of inflammatory mice under dipsacoside VI intervention on food consumption; (B) statistical graph of percentage change in inflammatory mice relative to food consumption with teasel saponin VI intervention ].
Figure 11 effect of dipsacoside VI and LPS on mouse body weight [ (C) effect of dipsacoside VI on inflammatory mouse body weight under intervention; (D) statistical graph of percentage change of inflammatory mice relative to body weight under teasel saponin VI intervention ].
FIG. 12 Effect of Dipsacus asperoides VI and LPS on autonomic activity in mice [ (A) schematic representation of open field experiments; (B) recording activity track thermograms of mice treated differently in an open field by software; (C) and (3) a statistical graph of immobility time of an inflammatory mouse under the intervention of dipsacoside VI in an open field experiment. (D) A motion time statistical chart of an inflammatory mouse under the intervention of dipsacoside VI in an open field experiment; (E) statistics of on-center duration of the inflammatory mice under dipsacoside VI intervention in open field experiments; (F) a movement distance statistical chart of an inflammatory mouse under the intervention of dipsacoside VI in an open field experiment; the results were plotted using GraphPad software and analyzed for significant differences using one-way anova LSD post hoc, # P <0.01, # P <0.005vs Control; (ii) a # P <0.05, # P <0.01vs LPS; n-8-12 ].
FIG. 13 is a schematic diagram of an elevated plus experiment [ (A) an elevated plus maze experiment; (B) recording the activity track thermogram of the mice treated differently in the elevated plus maze by software; (C) the number of open-arm probes of the inflammatory mice under the intervention of dipsacoside VI in the elevated plus maze. (D) A percentage statistical chart of the times of inflammatory mice under the intervention of dipsacoside VI entering an open arm in an elevated plus maze; (E) a percentage statistical chart of the time of inflammatory mice under the intervention of dipsacoside VI entering an open arm in an elevated plus maze; the results were plotted using GraphPad software and analyzed for significant differences using one-way anova LSD post hoc, # P <0.01, # P <0.005vs Control; (ii) a # P <0.05, # P <0.01vs LPS; n-8-12 ].
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1: taking the teasel saponin VI pieces as raw material medicines, adding 1/20 starch, and granulating to obtain granules.
Example 2: taking the teasel root saponin VI as a raw material medicine, adding 1/20 starch, mixing uniformly, and encapsulating to obtain the capsule.
Example 3: taking radix Dipsaci saponin VI as raw material medicine, adding dextrin 1/20, mixing, drying, and making into pill.
Example 4: taking the teasel saponin VI as a raw material medicine, adding 1/15 starch, granulating, tabletting and preparing into tablets.
Example 5: taking the teasel root saponin VI as a raw material medicine, adding 12 times of water for injection, soaking for 6-8 hours, filtering, and sterilizing to obtain the injection.
Example 6: taking dipsacoside VI as a raw material medicine, adding 13 times of injection water, soaking for 6-8 hours, filtering, and freeze-drying to obtain freeze-dried powder.
Example 7 Dipsacasperosaponin VI inhibits inflammatory response mediated by microglia type M1
Introduction to 1
Dipsacaceae saponin VI, also known as Akebia saponin D, is the main active ingredient of Chinese traditional Chinese herbal medicine Dipsacus asperoides. The teasel saponin VI has wide pharmacological actions, and the pharmacological activities thereof in the aspects of neuroprotection, myocardial protection, apoptosis resistance, pain relief and the like attract the attention of researchers, but the neuroprotection mechanism of the teasel saponin VI is not clear up to now. Numerous studies have shown that proinflammatory (type M1) activation of microglia cells often causes nerve damage, a process that is associated with many neurodegenerative diseases. The study aims to explore the inhibition effect of the dipsacoside VI on M1 microglia, and lays a foundation for researching the neuroprotective mechanism of the dipsacoside VI.
2 method of experiment
2.1 Experimental animals
C57BL/6J suckling mice 0-3 days after birth (purchased from Tian Zhi Biotechnology Ltd, Changsha, Hunan).
2.2 Experimental reagents
LPS (Sigma, usa), dipsaposide VI (sichuan biotechnology limited), protein lysate (beijing solibao biotechnology limited), BCA kit (wuhan doctor de biotechnology limited), ELISA kit (wuhan doctor de biotechnology limited), reverse transcription kit (Takara bioscience), trypsin, upstream and downstream primers (biol limited), fetal bovine serum (Gibico) DMEM-F12 complete medium (Gibico), donkey serum (Gibico), DAPI (Cell Signaling Technology, Inc), trikara bioscience, Iba-1(Abcam), GFAP (Cell Signaling Technology, Inc), CD68(Abcam), luciferase (Takara bioscience).
2.3 Experimental instruments
C1000 Touch PCR instrument (BIO-RAD), TE200-S inverted fluorescence microscope (Nikon), SIM-F124 ice maker (SANYO), EL104 electronic analytical balance (Mettler scientific instruments Co.), H fluorescence enzyme labeling instrument, Centrifuge 5810R refrigerated Centrifuge (eppendorf), UPT-II-20T ultrapure water machine (Sa' an Yopu instruments and equipments Co., Ltd.), 7500RealTime PCR System (USA applied biosystem), DW-40L508 medical low temperature storage box (Qingdao Haier Special electric appliances Co., Ltd.).
2.3 Primary microglia culture
Killing C57/BL6 suckling mice by means of spondylolisthesis in 0-3 days, soaking the suckling mice in 75% alcohol for 3-5S, disinfecting, inserting scissors from the upper part of the neck, cutting the suckling mice to the eyes, and cutting the two sides. The whole brain was taken out into sterile PBS, the cerebellum and olfactory bulb were removed, and the meninges were peeled off with sharp forceps. And blowing the tissue blocks by using a pipette to make the tissue blocks as small as possible, then transferring the tissue blocks into a 50mL centrifuge tube for centrifugation for 5min at 800g, reserving precipitates after the centrifugation is finished, abandoning the supernatant, and carefully operating to avoid shaking the precipitates as much as possible. Adding 5mL of trypsin, placing in a water bath at 37 deg.C for digestion for 3-5min, shaking until jelly begins to appear, adding DMEM-F12 complete culture medium containing 10% fetal calf serum in the same volume to stop digestion, and blowing and beating for multiple times. The 70 μm cell screen was placed in a new 50mL centrifuge tube port, the cell suspension after termination of digestion was transferred to the filter using a pipette gun, and the liquid was filtered and centrifuged at 1200g for 10 min. Discarding the supernatant, wherein the bottom precipitate which can be clearly observed is the cell, adding a certain amount of sterile PBS for washing, blowing the precipitate by a pipette gun until the precipitate is dissolved, and centrifuging for 10min at 1200g again. The supernatant was discarded, 10-15mL of microglial cell culture medium (45mL of DMEM, 5mL of FBS, 100. mu.L of double antibody) was added, and single cells were blown. Finally, the cells are added into a square bottle for cell culture by an electric gun, the placing direction of the square bottle is noticed, and a big belly pipette is not needed to be placed too deeply so as to prevent the cells from being polluted. After 24h, the microglial cell culture medium was replaced and used for subsequent experiments after two weeks of culture. After 14 days, the microglia cells after mixed culture are separated and purified by a shaking separation method.
2.4 drug treatment
Culturing the purified microglia in a 24-well plate, pretreating the microglia for 30min by using 10 mu M, 50 mu M, 100 mu M and 200 mu M teasel saponin VI respectively, adding 100ng/mL LPS to induce M1 type microglia, and performing q-PCR analysis and morphological identification after 24 h.
2.5 q-PCR detection of inflammatory factor expression
1mL Trizol was added to each well of cells, and the cells were aspirated with a 5mL syringe until no clumps were present and allowed to stand at room temperature for 5 min. Adding 200 μ L chloroform, shaking vigorously to extract protein in tissue, standing at room temperature for 5min, 12000g, centrifuging at 4 deg.C for 15min, and taking care of tube placing direction. Sucking supernatant, transferring into a new 1.5mL centrifuge tube, collecting supernatant as RNA, removing excess, cutting, precipitating at lower part, adding isopropanol with the same volume as supernatant, shaking, mixing, incubating at 15-30 deg.C for 10min, precipitating RNA, 12000g, and centrifuging at 4 deg.C for 10 min. And (3) sucking the supernatant by using a pipette gun to see that white precipitate exists at the bottom, adding 500 mu L of ethanol treated by 75% DEPC, shaking up and down violently, mixing the sample, suspending the white precipitate in the liquid, 7500g, centrifuging at 4 ℃ for 5min, wherein the RNA can not be completely dried, and otherwise, the solubility of the RNA can be greatly reduced. Discard the supernatant, open the tube lid, put into a 4 ℃ refrigerator, air dry, add 30 μ L of DEPC treated water, put into-80 ℃ for storage, and do not keep to the door of the refrigerator. The extracted RNA was prepared using a reverse transcription kit supplied by TaKaRa, following the protocol of the protocol. Firstly, 5 XgDNA Eraser Buffer (2. mu.L), gDNA Eraser (1. mu.L) and total RNA (1. mu.g) are sequentially added into a 200. mu.L DEPC soaked EP tube, and finally RNase Free dH2O is added to the total volume of 10. mu.L, the mixture is uniformly mixed, the mixture is centrifuged to deposit at the bottom of the EP tube, and the mixture is heated at 42 ℃ for 2 min. Then, Reaction solution from Step1 (10. mu.L), 5 XPrimeScript buffer 2(for Real Time) (4. mu.L), PrimeScript RT Enzyme Mix1 (1. mu.L), RTPrime Mix (1. mu.L), and RNase Free dH2O (4. mu.L) were sequentially added thereto to obtain 20. mu.L of a mixed solution, and the mixture was centrifuged again, incubated at 30 ℃ for 15min, and heated at 85 ℃ for 5S to terminate the Reaction. mu.L of the resulting cDNA sample was added with 5. mu.L of MasterMix and 1. mu.L of each of the upstream and downstream primers, and DEPC water was added to the 10. mu.L system. After mixing, the mixture was placed in a 7500Real Time PCR System, pre-denatured at 95 ℃ for 2s, annealed at 70 ℃ for 5s, and extended, and the process was repeated for 35 cycles, 3 for each sample. The internal reference gene is beta-actin, and the expression of the related gene is calculated according to the method of 2-delta ct.
2.6 immunohistochemical staining
Soaking the glass slide in alcohol, sterilizing with fire, spreading on the bottom of 24-well plate, culturing separated microglia on the glass slide, treating with medicine for 24 hr, fixing cells with 4% PFA for 30min, washing with PBS for 3 times, and storing at 4 deg.C. The preserved cell slide was left at room temperature for 5min, washed with PBS 3 times for 5 min/time, the residual liquid was completely blotted in the last time, washed with 0.5% Triton X-100 through 15min, washed with 10% and then PBS 3 times for 5 min/time. The brain slices were blocked with 10% donkey serum in PBS for 2h at room temperature and incubated overnight at 4 ℃ with primary antibody. The next day, brain slice was left at room temperature for 10min, washed with PBS 3 times, 5 min/time, and incubated for a second antibody 2h in the dark, after which brain slice was washed with PBS 3 times, 5min each time. Finally, DAPI stained nuclei were added and the brain slices were observed by microscopic examination.
2.7 statistical analysis
Graghpad prism software was used for all chart generation and statistical analyses, all data were presented by Mean + -SEM, analysis was by the Bonnferoni post hoc test in one-way ANOVA, and P <0.05 indicated significant differences.
3 results
3.1 Effect of Dipsacus asperoides Saponin VI on the morphology of M1 microglia
This study cultured primary microglia for neuroinflammation, which were isolated from the brain of mice and cultured in cell culture plates. The microglia is purified by a shaking separation method, the purity of the microglia is detected by immunohistochemistry, the Iba1 marks the microglia, the GFAP marks the astrocytes, the DAPI marks the cell nucleus, the detection result is shown in figure 1, 99.6% of cells are the microglia, and only 0.4% of the cells are the astrocytes. In the research, the purity of the microglia can reach more than 95 percent generally and can be used for experimental research, so that the purified microglia in the research has higher purity and can be used for subsequent research.
The method comprises the steps of culturing primary microglia, activating the microglia cultured in vitro by LPS treatment, establishing an inflammation model of the microglia, and carrying out pharmaceutical intervention by teasel saponin VI with different concentrations. The immunohistochemical staining results are shown in fig. 2, and compared with the blank group, the LPS model group had enlarged microglia, increased surface area, reduced branching, and higher activation degree. CD68 is used as a marker for microglial activation and is mainly used for marking the phagolysosome of microglia, and the lysosome gradually expands along with the increase of the activation degree of the microglia in the process of the microglial activation. After LPS treatment, the area of positive staining for CD68 increased significantly (P <0.005), indicating significant activation of microglia. After different teasel root saponin VI are used for intervening activated microglia, the area of CD68 positive staining and the area change of the microglia are respectively counted, and the statistical result shows that the 10 mu M teasel root saponin VI intervention group has no statistical difference (P >0.05) in the area of CD68 positive staining and no significant difference (P >0.05) in the area of the microglia compared with the LPS model group. The 50 μ M dipsacoside VI dried group significantly reduced the area of positive staining of CD68 (P <0.01) compared to the LPS model group, with no statistical difference in the area of microglia (P > 0.05). Compared with the LPS model group, the Dipsacus asperoides VI dry pre-treated group with 100 mu M significantly reduced the area of positive staining of CD68 (P <0.005), and had no statistical difference (P >0.05) although there was a reduction in the area of microglia. Compared with the LPS model group, the dipsacoside VI dry-control group with 200 mu M significantly reduces the area of positive staining of CD68 (P <0.005) and also significantly reduces the relative area of microglia (P < 0.005).
3.2 Dipsacasperosaponin VI inhibits inflammatory reaction mediated by M1 microglia
Microglial activation is usually accompanied by inflammatory factor changes, especially after LPS stimulation, the microglial cells are usually produced with a large amount of proinflammatory cytokines (such as IL-1 beta, iNOS, TNF-alpha, IL-6 and the like), and the microglial cells are generally called classically activated microglial cells, also called M1 type microglial cells. In the research, the microglia cultured in vitro is treated by LPS to induce the microglia to be activated to M1 type, the activation phenotype of the microglia detects the expression of inflammatory cytokines by q-PCR, the detection result is shown in figure 3, and compared with a blank group, the contents of IL-1 beta, iNOS, TNF-alpha and IL-6 proinflammatory cytokines in an LPS model group are obviously increased (P is less than 0.05); the LPS can successfully induce the microglia to be activated to the M1 phenotype, and the inflammation model can be used for subsequent drug intervention research.
After different concentrations of dipsacus asperoides VI are used for intervening activated microglia, gene expression changes of IL-1 beta, iNOS, TNF-alpha and IL-6 are respectively detected, and statistical results show that compared with an LPS model group, a dipsacus asperoides VI intervention group with 10 mu M has no statistical difference (P >0.05) in the mRNA expression levels of IL-1 beta, iNOS, TNF-alpha and IL-6 and no significant difference (P >0.05) in the area of microglia. Compared with the LPS model group, the 50 mu M Dipsacus asperoides VI intervention group remarkably reduces the mRNA expression levels of IL-1 beta and IL-6 (P <0.05), and the mRNA expression levels of iNOS and TNF-alpha are reduced but have no statistical difference (P > 0.05). There was no statistical difference in the area of microglia (P > 0.05). Compared with the LPS model group, the Dipsacus asperoides VI intervention groups with the concentration of 100 mu M and 200 mu M remarkably reduce the mRNA expression levels of IL-1 beta, iNOS, TNF-alpha and IL-6 (P is less than 0.05).
4. Summary of the invention
Microglia are immune cells present in the brain, which play a role as executives in central immune functions and are also the major secretory cells of central inflammatory cytokines. An important means of achieving interaction between the immune system and the central nervous system is the release of inflammatory cytokines. Studies have shown that microglial activation and inflammatory cytokine release are regulated by a variety of membrane surface receptors including TRPV 1. Microglial activation is usually accompanied by inflammatory factor changes, especially after LPS stimulation, the microglial cells are usually produced with a large amount of proinflammatory cytokines (such as IL-1 beta, iNOS, TNF-alpha, IL-6 and the like), and the microglial cells are generally called classically activated microglial cells, also called M1 type microglial cells. In the research, LPS is used for treating microglia cultured in vitro, the microglia is induced to be polarized to M1 phenotype, different concentrations of dipsacus asperoides VI are used for intervening the activated microglia, and the results show that 100 mu M and 200 mu M dipsacus asperoides VI stem pre-set can obviously inhibit the cell expansion of the microglia, increase the phagolysosome and inhibit the expression of IL-1 beta, iNOS, TNF-alpha and IL-6 compared with an LPS model set. The dipsacoside VI is shown to be capable of obviously inhibiting the activation of microglia, so that the microglia-mediated inflammatory reaction is improved.
Example 8: dipsacus asperoides saponin VI for inhibiting neuroinflammation and improving depression
Introduction to 1
Depression, also known as depressive disorder, is the major type of mood disorder. Clinically, the depressed mood is not matched with the situation, the depressed mood can be from sultriness to sadness, and there are suicide attempts or behaviors in the depressed self-being and even pessimistic boredom. With the increasing social competition, the incidence of depression tends to increase year by year and the age of the incidence is becoming younger. At present, depression is ascending as the fourth disease type in the world, and the depression is initially estimated to become the second disease by 2020. However, the curative effect of the existing antidepressant is not very ideal, the treatment period is long, and the antidepressant has no effect on part of patients and has strong side effect. Therefore, the development of a safe and effective antidepressant is a problem to be solved urgently.
The asperosaponin VI is the main active ingredient of Chinese traditional Chinese herbal medicine asperosa himalaica. The teasel saponin VI has wide pharmacological action, and the pharmacological activity of the teasel saponin VI in the aspects of neuroprotection, myocardial protection, apoptosis resistance, pain relief and the like draws wide attention of scholars. Nerve damage caused by neuroinflammation is considered to be one of causes of depression. The experiment aims to explore the significance and the action mechanism of the neuroprotective effect of the teasel saponin VI in the antidepressant.
2 method of experiment
2.1 Experimental animals
The experimental animals used in this study were 7-week-old C57/BL6 male mice (purchased from Tianli Biotechnology Co., Ltd., Changsha, Hunan). The experimental animals are all kept in the same daily feeding condition, which comprises the following steps: the lighting time and the dark time are both 12 hours (light is turned on at 7 am and turned off at 7 pm), the temperature is 25 +/-1 ℃ and the room temperature is 75% of air humidity, standard feed and drinking water are fed in a single cage, the animals move freely, and the method is suitable for experimental study after two weeks. In the whole experimental process, the relevant laws and relevant regulations of animal ethics committees are strictly followed, and animal behavior regulations are met.
2.2 Experimental reagents
LPS, dipsacus asperoides VI, protein lysate, BCA kit (Strobilanthes, Wuhan, Germany Co., Ltd.), ELISA kit (Strobilants, Wuhan), reverse transcription kit (Takara treasure, Inc.), upstream and downstream primers (Biotechnology, Inc.), trypsin, fetal bovine serum, DMEM-F12 complete medium, donkey serum, DAPI (Cell Signaling Technology, Inc), Trizol (Takara treasure, Inc.), Iba-1(Abcam), GFAP (Cell Signaling Technology, Inc), CD68(Abcam), and luciferase (Takara treasure, Inc.).
2.3 Experimental instruments
C1000 Touch PCR instrument (BIO-RAD), fluorescence microscope, SIM-F124 ice machine (SANYO), EL104 electronic analytical balance (Mettler scientific instruments Co.), microplate reader, Centrifuge 5810R refrigerated Centrifuge (eppendorf), UPT-II-20T ultrapure water machine (Sigan Yopu instruments Co., Ltd.), 7500Real Time PCR System (USA applied biosystems Co., Ltd.), DW-40L508 medical low temperature preservation box (Qingdao Haier special electric appliances Co., Ltd.), mouse activity experiment box (Chengdu Tai union software Co., Ltd.)
2.3 establishment of models of Depression inflammation and drug intervention
The experiment was started after 7 week old mice were acclimated in a new environment for one week. The control group was injected with 10mg of normal saline intraperitoneally, the experimental group was injected with 10mg of LPS intraperitoneally, and the drug intervention group was administered with 10mg/kg, 20mg/kg, 40mg/kg and 80mg/kg of Dipsacus asperoides saponin VI before LPS injection, respectively. Behavioral changes were examined 24h after injection.
2.4 behavioural testing
2.4.1 elevated Cross maze
Mice were placed in the maze from the central grid towards the closed arms at the beginning of the experiment and activity was recorded within 5 minutes. The observation indexes comprise the number of times of entering the open arm (two front melons need to enter the arm), the dwell time of the open arm, the number of times of entering the closed arm and the dwell time of the closed arm. And calculating the proportion of the residence time of the open arm, the proportion of the entering times of the open arm and the total entering times in the elevated plus maze. After the experiment was completed, the mouse was taken out, the two arms were cleaned, and alcohol was sprayed to remove odor.
2.4.2 open field experiments
Open field experiments in mice were performed in a quiet environment. The animals were placed in the center of the bottom of an open field reaction chamber (length 50 cm. times. width 50 cm. times. height 30cm), and the images were taken and the time was counted. And stopping shooting for 5min after observing for a certain time. Designing different observable parameters according to computer software, wherein the observable parameters comprise the residence time of an animal in a central compartment in unit time, the number of compartments crossed by a certain limb is horizontal score (horizontal), the number of hindlimb standing times is vertical score (vertical), the number of decoration times and the number of urination and defecation times; speed of movement, distance of movement, time of rest, distance of movement along the edge, distance of movement in the center, etc. And the sensitivity of the mouse to the smell is considered, so that after each experiment is finished and the record is finished, the experimental instrument must be cleaned, and the influence of the smell of the previous mouse on the experimental mouse is avoided. 2.4.3 Forced Swimming Test (FST)
The day before the experiment, each mouse was pre-tested and placed in a 21X 12cm glass beaker with water depth of 12cm, water temperature of 22 + -1 deg.C, and swimming experiment was carried out for 10 min. The experiment is formally carried out on the next day, all experimental mice keep swimming for 6min, and the whole experiment process is recorded by a digital camera. Thereafter, the hopeless immobility time 4min after 6min and the first struggle abandoning time 6min were counted using the double blind method.
2.4.4 Tail suspension experiment (TST)
The tip of the tail of each experimental mouse is fixedly hung at a height of 30cm above the ground by using an adhesive tape, each experimental mouse needs to be separated by a certain distance, the middle of each two experimental mice is separated by using a black paperboard, the experiment lasts for 6min, the experiment process is recorded for 6min by using a digital camera, and then the time for the mice to move still within 6min and the time for the mice to struggle for the first time are counted by using a double-blind method.
2.4.5 mouse food consumption test
At the beginning of the experiment, each mouse was given the same volume and weight of food per group after dosing. After 24h, the food consumption of each mouse was measured and recorded.
2.4.6 mouse weight Change test
At the beginning of the experiment, the body weight of each mouse in each group was measured and recorded. After 24h of administration, the change in body weight of each mouse was measured and recorded.
2.5 brain extraction method
2.5.1 perfusion brain extraction
Immediately after anaesthesia, the mice were fixed on a foam plate with a needle (the limb was inserted with the needle). The skin of the chest was pulled up with forceps and the skin and ribs of the chest were cut with scissors with the other hand, exposing the heart and liver. The injection needle was inserted into the left ventricle of the mouse while the liver of the mouse was removed to allow blood to flow out. The normal saline is infused for about 10min, and the limbs, liver and tongue become white after blood is removed. After the mouse limbs, liver and tongue are whitened, perfusion fixation with 4% PFA is performed, and when PFA flows to the brain, the mouse tail may be slightly reflected (and sometimes may not be reflected), the perfusion speed can be adjusted downwards to ensure more complete fixation. The total PFA perfusion time was about 20 min. (fixation principle: protein cross-linking can be achieved by paraformaldehyde) the initial end of the catheter is taken out from PFA, after the liquid in the catheter flows out, the needle head on the heart is pulled out, and if the fixation is good, the eyeball of the mouse can be found to be white. The blood in the tray is poured into a waste liquid barrel and is ready for the next brain taking operation. Taking brains and dehydrating: the skin of the head was cut open, exposing the white skull. The cartilage coated on the medulla oblongata was cut open and excess connective tissue was removed. It should be noted that the eyes are cut by scissors and cannot be pulled directly because the posterior part of the eyes is connected with the optic nerve, and if the eyes are pulled, other brain tissues such as suprachiasmatic nucleus and the like can be damaged. When the skull is carefully stripped to expose the white brain, care is taken to strip the olfactory bulb, and if necessary, the broken bones at the front part of the olfactory bulb are left for further fixation and then removed. After the skull is stripped, the nerves connected with the lower part of the brain are cut short one by one (optic nerve crossing can be seen), after the nerves are completely cut off, the whole brain is stripped, and the stripped brain is soaked in 4% PFA and is fixed overnight. The PFA solution is replaced by 30% sucrose for dehydration (to prevent ice crystal and holes from forming during cryo-slicing), the brain will float on the sucrose during initial dehydration, and the brain will sink after complete dehydration. At this time, the brain can be stored in a refrigerator at-20 deg.C.
2.5.2 taking brain from neck
After the head is broken, skin and muscle tissues on the surface of the skull are cut off, scissors are used to vertically and deeply insert into the back edge of the parietal bone, the parietal bone is moved to two sides, cerebral hemispheres on two sides are exposed, and then redundant temporal bones and frontal bones are carefully removed by hemostatic forceps, and brain tissues are exposed. Cutting lateral cranial nerve and dura mater of brain stem and cerebellum with scissors, picking up brain with small spoon, cutting off cranial base trigeminal nerve and optic nerve, scooping out whole brain, separating hippocampus and cortex, and storing in-80 deg.C refrigerator.
2.6q-PCR detection of Gene expression
Hippocampus and cortex of mice of each group were isolated, placed in a sterile 1.5mL centrifuge tube, 1mL Trizol was added to each tube, cells were aspirated with a 5mL syringe until no clumps were present, and left at room temperature for 5 min. Adding 200 μ L chloroform, shaking vigorously to extract protein in tissue, standing at room temperature for 5min, 12000g, centrifuging at 4 deg.C for 15min, and taking care of tube placing direction. Sucking supernatant, transferring into a new 1.5mL centrifuge tube, collecting supernatant as RNA, removing excess, cutting, precipitating at lower part, adding isopropanol with the same volume as supernatant, shaking, mixing, incubating at 15-30 deg.C for 10min, precipitating RNA, 12000g, and centrifuging at 4 deg.C for 10 min. And (3) sucking the supernatant by using a pipette gun to see that white precipitate exists at the bottom, adding 500 mu L of ethanol treated by 75% DEPC, shaking up and down violently, mixing the sample, suspending the white precipitate in the liquid, 7500g, centrifuging at 4 ℃ for 5min, wherein the RNA can not be completely dried, and otherwise, the solubility of the RNA can be greatly reduced. Discard the supernatant, open the tube lid, put into a 4 ℃ refrigerator, air dry, add 30 μ L of DEPC treated water, put into-80 ℃ for storage, and do not keep to the door of the refrigerator. The extracted RNA was prepared using a reverse transcription kit supplied by TaKaRa, following the protocol of the protocol. Firstly, 5 XgDNA Eraser Buffer (2. mu.L), gDNA Eraser (1. mu.L) and total RNA (1. mu.g) are sequentially added into a 200. mu.L DEPC soaked EP tube, and finally RNase Free dH2O is added to the total volume of 10. mu.L, the mixture is uniformly mixed, the mixture is centrifuged to deposit at the bottom of the EP tube, and the mixture is heated at 42 ℃ for 2 min. Then, Reaction solution from Step1 (10. mu.L), 5 XPrimeScript buffer 2(for Real Time) (4. mu.L), PrimeScript RT Enzyme Mix1 (1. mu.L), RT Prime Mix (1. mu.L), and RNaseFree dH2O (4. mu.L) were sequentially added thereto to obtain 20. mu.L of a mixed solution, and the mixed solution was centrifuged again, incubated at 30 ℃ for 15min, and heated at 85 ℃ for 5S to terminate the Reaction. mu.L of the resulting cDNA sample was added with 5. mu.L of MasterMix and 1. mu.L of each of the upstream and downstream primers, and DEPC water was added to the 10. mu.L system. After mixing, the mixture was placed in a CFX96 fluorescent quantitative real-time PCR instrument, pre-denatured at 98 ℃ for 2s, annealed at 70 ℃ for 5s, and extended, and this process was repeated for 35 cycles, 3 for each sample. The internal reference gene is beta-actin, and the expression of the related gene is calculated according to the 2-delta ct calculation method.
2.7 immunohistochemical staining
Mice were anesthetized with 1% pentobarbital (50mg/kg. i.p.), perfused with 4% paraformaldehyde (0.1M phosphate buffer in PBS) for 10min, the whole brains of mice were removed and placed in 4% paraformaldehyde for post-fixation for 48 hours, and then placed in 30% sucrose for dehydration for 48 hours. Frozen sections were 30 μm and stored at 4 ℃. The preserved brain slices are placed at room temperature for 5min, washed with PBS for 3 times and 5 min/time, the residual liquid is completely sucked off in the last time, washed thoroughly with 0.5% Triton X-100 for 15min, washed with 10% and then washed with PBS for 3 times and 5 min/time. The brain slices were blocked with 10% donkey serum in PBS for 2h at room temperature and incubated overnight at 4 ℃ with primary antibody. The next day, brain slice was left at room temperature for 10min, washed with PBS 3 times, 5 min/time, and incubated for a second antibody 2h in the dark, after which brain slice was washed with PBS 3 times, 5min each time. Finally, DAPI stained nuclei were added and the brain slices were observed by microscopic examination.
2.8 statistical analysis
Graghpad prism software was used for all chart generation and statistical analyses, all data were presented by Mean + -SEM, analysis was LSDpost hoc test in one-way ANOVA, and P <0.05 indicated significant differences.
3 results
3.1 Dipsacus asperoides saponin VI inhibits activation of mouse hippocampal and cortical microglia
An animal model of neuroinflammation is established by injecting LPS into the abdominal cavity of a mouse, and medicinal intervention is carried out by using teasel saponin VI with different concentrations. The instinct study firstly uses qPCR to detect the phenotype marker of microglia activation, the phenotype marker of the microglia mainly detects the mRNA level of CD11B and Iba1 of mouse hippocampus, the detection result is shown in FIGS. 4A and 4B, the mRNA level of CD11B and Iba1 of hippocampus is obviously increased in LPS treated mice (P < 0.01); the teasel saponin VI with different concentrations is used for intervening normal mice, and the gene expression of CD11b and Iba1 of hippocampus of the mice is not influenced (P is more than 0.05); compared with the LPS model group, the Dipsacus asperoides VI intervention group of 10mg/kg has no statistical difference in gene expression of CD11b and Iba1 (P > 0.05); compared with the LPS model group, the 20mg/kg Dipsacus asperoides VI intervention group significantly reduces the gene expression level of Iba1 (P <0.05), and has no statistical difference (P >0.05) in the gene expression of CD11b although the gene expression is reduced; compared with the LPS model group, the Dipsacus asperoides VI intervention group with the concentration of 40mg/kg significantly reduces the gene expression level of Iba1 (P <0.05), and has no statistical difference (P >0.05) to the gene expression of CD11 b; compared with the LPS model group, the Dipsacus asperoides VI intervention group with the concentration of 80mg/kg remarkably reduces the gene expression of CD11b (P <0.05) and also remarkably reduces the gene expression of Iba1 (P < 0.005).
The morphological detection of the microglia adopts immunohistochemical staining, and the result 4C-4E shows that compared with the blank group, the microglia of the hippocampus in the LPS model group is enlarged, the branches are reduced, and the activation degree is higher. By counting the relative areas and branches of microglia, it was found that Iba1 positive staining area significantly increased in hippocampus (P <0.05) while microglia branches significantly decreased (P <0.05) after LPS treatment, indicating significant activation of microglia. After different teasel root saponins VI are used for intervening and activating, the areas of the Iba1 positive staining and the branch changes of microglia in the hippocampus are respectively counted, and the statistical result shows that the areas of the Iba1 positive staining and the branch number of the microglia in the hippocampus of the mouse are not influenced by the use of the teasel root saponins VI with different concentrations for intervening and activating the normal mouse; compared with the LPS model group, the Dipsacus asperoides VI dry group at 10mg/kg has no statistical difference (P >0.05) in the area of Iba1 positive staining of hippocampus and no significant difference (P >0.05) in the branch number of microglia. The 20mg/kg Dipsacasperoides VI dried group significantly reduced the area of Iba1 positive staining of hippocampus (P <0.05) compared to the LPS model group, and there was no statistical difference in the number of branches of microglia (P > 0.05). Compared with the LPS model group, the Dipsacus asperoides VI dried group at 40mg/kg and 80mg/kg remarkably reduces the Iba1 positive staining area of hippocampus (P <0.05), and also remarkably increases the branch number of microglia (P < 0.005).
In addition to testing the hippocampus of mice, phenotypic markers of microglia in the cerebral cortex of mice were also tested in this study, mainly for mRNA levels of CD11B and Iba1, as shown in fig. 5A and 5B, with significantly elevated mRNA levels of CD11B and Iba1 in the cortex (P <0.01) in LPS-treated mice; the teasel saponin VI with different concentrations is used for intervening normal mice, and the gene expression of CD11b and Iba1 of the cortex of the mice is not influenced (P is more than 0.05); compared with the LPS model group, the Dipsacus asperoides VI intervention group of 10mg/kg has no statistical difference in gene expression of CD11b and Iba1 in the cortex (P > 0.05); compared with the LPS model group, the 20mg/kg Dipsacus asperoides VI intervention group significantly reduces the gene expression level of Iba1 in the cortex (P <0.05), and has no statistical difference (P >0.05) in the CD11b gene expression; compared with the LPS model group, the Dipsacus asperoides VI intervention group with the concentration of 40mg/kg remarkably reduces the gene expression level of cortical Iba1 (P <0.05), and has no statistical difference (P >0.05) to the gene expression of CD11 b; compared with the LPS model group, the 80mg/kg Dipsacus asperoides VI intervention group remarkably reduces the gene expression of cortical CD11b (P <0.05), and also remarkably reduces the gene expression of Iba1 (P < 0.005).
The morphological detection of the microglia in the cortex area adopts immunohistochemical staining, and the result 5C-5E shows that the microglia in the cortex in the LPS model group expands, the branches are reduced and the activation degree is higher compared with that in the blank group. By counting the relative areas and branches of microglia, it was found that Iba1 positive staining area significantly increased in hippocampus (P <0.05) while microglia branching significantly decreased (P <0.05) after LPS treatment, indicating that the microglia of cortex were significantly activated. After different teasel root saponins VI are used for intervention and activation, the areas of the Iba1 positive staining and the branch changes of microglia in the cortex are respectively counted, and the statistical result shows that the areas of the Iba1 positive staining and the branch numbers of the microglia of the cortex of the mouse are not influenced by the intervention of the teasel root saponins VI with different concentrations in a normal mouse; compared with the LPS model group, the 10mg/kg Dipsacasperoides VI intervention group has no statistical difference (P >0.05) in the area of Iba1 positive staining of the cortex and no significant difference (P >0.05) in the branch number of the microglia. The 20mg/kg Dipsacasperoides VI dried group significantly reduced the area of Iba1 positive staining of the cortex (P <0.05) compared to the LPS model group, and there was no statistical difference in the branch number of microglia (P > 0.05). Compared with the LPS model group, the Dipsacus asperoides VI dried group at 40mg/kg and 80mg/kg remarkably reduces the area of Iba1 positive staining of the cortex (P <0.05), and also remarkably increases the branch number of microglia (P < 0.005).
3.2 Dipsacasperoides VI inhibit LPS-induced neuroinflammation
After the teasel saponin VI is found to be capable of inhibiting the activation of microglia induced by LPS, the influence of the teasel saponin VI on neuroinflammation induced by LPS is further detected by the research, q-PCR is used for detecting the expression of inflammatory cytokines in hippocampus, the detection result is shown in figure 6, and compared with a blank group, the contents of IL-1 beta, iNOS, TNF-alpha and IL-6 proinflammatory cytokines in an LPS model group are obviously increased (all P is less than 0.05); the LPS can successfully induce the microglia to be activated to the M1 phenotype, and the inflammation model can be used for subsequent drug intervention research. After the teasel root saponin VI with different concentrations is used for intervention and stimulation, the gene expression changes of IL-1 beta, iNOS, TNF-alpha and IL-6 are respectively detected in a hippocampus, and the statistical result shows that the teasel root saponin VI with different concentrations is used for intervention and stimulation of a normal mouse, and the expression of IL-1 beta, iNOS, TNF-alpha and IL-6 cytokines of the hippocampus of the mouse is not influenced; compared with the LPS model group, the Dipsacus asperoides VI intervention group with the concentration of 10mg/kg has no statistical difference (P is more than 0.05) in the mRNA expression levels of IL-1 beta and IL-6, but can obviously inhibit the gene expression of iNOS and TNF-alpha (P is less than 0.05). Compared with an LPS model group, 20mg/kg, 40mg/kg and 80mg/kg of dipsacoside VI intervention groups can obviously reduce the mRNA expression levels of IL-1 beta, iNOS and TNF-alpha (P <0.05), and only 80mg/kg of dipsacoside VI can obviously inhibit the mRNA expression level of IL-6 (P < 0.05).
The expression of inflammatory cytokines in the cerebral cortex of mice was measured by q-PCR, with results similar to those measured in the hippocampus, but the change in the cortex was more pronounced than in the hippocampus, in fold expression of inflammatory cytokines. The detection results are shown in FIG. 7, compared with the blank group, the contents of IL-1 beta, iNOS, TNF-alpha and IL-6 proinflammatory cytokines in the LPS model group are obviously increased (all P is less than 0.05); the LPS can successfully induce the microglia to be activated to the M1 phenotype, and the inflammation model can be used for subsequent drug intervention research. After the teasel root saponin VI with different concentrations is used for intervention and stimulation, the gene expression changes of IL-1 beta, iNOS, TNF-alpha and IL-6 are respectively detected in the cortical areas, and the statistical result shows that the teasel root saponin VI with different concentrations is used for intervention and stimulation of normal mice, and the expression of IL-1 beta, iNOS, TNF-alpha and IL-6 cytokines of mouse cortex is not influenced; compared with the LPS model group, the Dipsacus asperoides VI intervention group with the concentration of 10mg/kg has no statistical difference (P is more than 0.05) in the mRNA expression levels of IL-1 beta and IL-6 in the cortical area, but can obviously inhibit the gene expression of iNOS and TNF-alpha (P is less than 0.05). Compared with the LPS model group, 20mg/kg, 40mg/kg and 80mg/kg of dipsacoside VI intervention groups can obviously reduce the mRNA expression level (P <0.05) of IL-1 beta, iNOS and TNF-alpha, and only 80mg/kg of dipsacoside VI can obviously inhibit the mRNA expression level (P <0.05) of cortical region IL-6. 3.3 Dipsacus asperoides Saponin VI improves LPS-induced depressive-like behavior 3.3.1 forced swimming test results
Analysis of the data of the forced swimming experiment shows that: compared with the blank group, the mice in the LPS model group have shortened latency period of forced swimming and prolonged accumulative immobilization time (P < 0.05); compared with the LPS model group, after the intervention of simultaneously administering the dipsacoside VI, the forced swimming latency of the mice is prolonged, and the cumulative immobility time is shortened, wherein the effect of the 40mg/kg dose group is the best (P is less than 0.05) (see figure 8).
3.3.2 Tail overhang test results
The analysis result of the tail suspension experiment data shows that: compared with a blank group, the incubation period of mice in an LPS model group is shortened, and the accumulated immobility time is obviously prolonged (P is less than 0.05); compared with LPS model group, the mice have obviously prolonged incubation period and obviously reduced accumulative immobility time after simultaneously taking intervention of teasel saponin VI, wherein the three dosage groups of 20mg/kg, 40mg/kg and 80mg/kg have better effect (P <0.05) (see figure 9).
3.3.3 mouse food Change results
The results of the one-way anova show that: the LPS model group had significantly reduced food consumption (P <0.05) compared to the blank group (fig. 10).
3.3.4 mouse weight Change results
The results of the one-way anova show that: LPS model group weight reduction compared to blank group (P < 0.05); compared with LPS model group, the weight of the mice is obviously increased (P <0.05) after the intervention of the simultaneous administration of the teasel saponin VI (see figure 11).
3.4 Effect of Dipsacus asperoides VI on the LPS-induced anxiety-like behavior of mice
3.4.1 autonomic Activity test results
The analysis result of the autonomic activity test data shows that: compared with the blank group, the resting time of the mice in the autonomic activity box is increased, and the movement time, the central time and the total movement distance of the mice are obviously reduced (P <0.05) (figure 12).
3.4.2 elevated plus maze test result
The analysis result of the data of the elevated plus maze experiment shows that: the LPS model group showed a significant reduction in the number of open arm entries (P <0.05) compared to the blank group, with no significant effect on the percentage of open and closed entry times and the open arm entry time (P >0.05) (see fig. 13).
To summarize:
depression is a common mental disorder disease, has the characteristics of high recurrence rate, high disability rate, high suicide rate, low recognition rate, low diagnosis rate and low treatment rate, and becomes a main factor for causing global disease burden. However, due to its strong heterogeneity, the cause and pathogenesis of the disease are not clear at present, so that safe and effective anti-depression treatment means and treatment drugs are clinically lacked at present. In recent years, researches show that microglial cells in brains of suicide depression patients are remarkably proliferated, the microglial cells release cytokines after activation, the cytokines can cause neurogenesis disorder, and the neurogenesis rate is considered to be directly related to depression. Research shows that in a learning and cognitive dysfunction model of SD rats induced by injecting Abeta 1-42 into bilateral ventricles, the activation of Abeta-induced astrocytes and microglia is obviously inhibited by dipsacus asperoides VI, and the glia cells can also reduce the release of cytokines and inflammatory factors, such as the expression of tumor necrosis factor alpha (TNF-alpha), interleukin 6(IL-6) and cyclooxygenase-2 (COX-2), so that the generation of inflammatory reaction can be inhibited.
The research researches the antidepressant effect of ASP VI on mice of LPS depression models from phenomena to mechanisms by combining two major experiments of animal behaviors and biochemistry, and further researches the relation between the antidepressant effect of ASP VI and activation of hippocampus and cortical microglia, central inflammatory reaction and neurogenesis. The experimental results show that: 1. in weight, forced swimming and tail suspension experiments, the 40mg/kg ASPPI VI pretreatment group shows obvious antidepressant effect; ASP VI pretreatment group significantly inhibited the level of activation of microglial cells and central inflammatory response in hippocampus and cortical tissues of LPS mice brain. The anti-depression effect of ASP VI is shown to be stable and effective in experimental animals, and is related to the inhibition of microglial cell activation and central inflammatory reaction and the final protection of neurogenesis.
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Claims (8)

1. Application of neuroprotective effect of Dipsacus asperoides saponin VI in treating depression is provided.
2. The use of claim 1, wherein the dipsacoside vi is used to inhibit M1 microglial cell morphology.
3. The use of claim 1, wherein the dipsacoside vi is used to inhibit M1 microglia-mediated inflammatory response.
4. The use of claim 1, wherein said dipsacoside VI is used for inhibiting activation of hippocampal and cortical microglia.
5. The use according to claim 1, wherein the dipsacoside VI is used for inhibiting LPS-induced neuroinflammation.
6. The use according to claim 1, characterized in that the dipsacoside VI is used for ameliorating LPS-induced depressive-like behavior.
7. The use of claim 1, wherein the dipsacoside VI is added to a pharmaceutically acceptable adjuvant to prepare a pharmaceutically acceptable preparation for use in the treatment of depression.
8. The use of claim 7, wherein the formulation is a granule, a capsule, a powder, a tablet, a pill, an injection, a lyophilized powder for injection.
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