Application of IPA in chronic pressure-induced brain lesion
By studying the role of IPA in the mouse model of chronic stress, it was found that elevated IPA levels were related to brain region lesions, regulating microglia and inflammatory factors expression, and screening drugs that reduce IPA levels to treat chronic stress-induced brain region lesions, achieving the effect of improving chronic stress injury.
Patent Information
- Application Number
- CN202411660099.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-20
- Publication Date
- 2025-02-25
AI Technical Summary
Chronic stress can lead to brain lesions, and the existing technology has not yet effectively solved this problem.
By studying the role of IPA in chronic stress-induced brain region lesions, it was found that IPA levels were abnormally elevated in the hippocampus brain region and plasma in the chronic stress mouse model, and regulated the number of microglia and the expression of the inflammatory factor IL-1β, and then screened for drugs that reduce IPA levels to treat or prevent and treat chronic stress-induced brain region lesions.
Reducing IPA levels in the brain region and plasma, reducing the number of microglia in the brain and the expression of the inflammatory factor IL-1β, thereby improving chronic stress injury and providing new therapeutic strategies.
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Figure CN119510738A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical technology, and in particular to application of IPA in brain region lesions induced by chronic stress. Background Art
[0002] Chronic stress (CS) refers to stress that lasts for an abnormally long time, often recurring, and is caused by factors that are difficult to adapt to or overcome over the long term. The body is in a state of stress for a long time, and the stress system is activated for a long time, frequently, and repeatedly without rest, which puts the body in a chronic high load, affecting brain activity, neuroendocrine, nutrition, and the immune system, and can lead to serious psychological and physiological health disorders. Chronic stress is a long-term and continuous stress experience, and its sources mainly include emotional stress, environmental stress, work stress, and interpersonal relationship stress. Long-term and continuous stress can trigger an inflammatory response in the body, promote the occurrence and development of a variety of diseases, and thus damage health. If it is not promptly relieved and treated, it will have many negative effects on health and even cause a variety of diseases.
[0003] Indolepropionic acid (IPA), also known as indole-3-propionic acid, is a white flaky crystal. It is a plant growth regulator with auxin biological activity and can be absorbed by the roots, stems, leaves and flowers of plants. It has physiological effects such as promoting rooting and fruiting. In the prior art, IPA is mainly used as a pharmaceutical intermediate and plant growth stimulant. IPA is a product of the indole and aromatic hydrocarbon receptor pathway of tryptophan metabolism. Tryptophan metabolism can produce IPA; there is no report on the role of IPA in chronic stress. Summary of the invention
[0004] The main purpose of the present invention is to provide an application of IPA in brain lesions induced by chronic stress, aiming to determine the relationship between IPA and brain lesions induced by chronic stress, and then provide a drug for treating or preventing brain lesions induced by chronic stress, and to reduce the level of IPA in the brain and plasma, and reduce the number of microglia in the brain and the expression of the inflammatory factor IL-1β.
[0005] To achieve the above object, the present invention provides the following technical solutions:
[0006] Application of IPA in screening drugs for treating or preventing chronic stress-induced brain lesions.
[0007] Preferably, the drug for treating or preventing chronic stress-induced brain lesions refers to a drug that can reduce the level of IPA in the brain and plasma, reduce the number of microglia in the brain and the expression of the inflammatory factor IL-1β.
[0008] The present invention has found that by constructing a chronic stress mouse model using C57BL / 6 mice, the IPA level is abnormally elevated in the hippocampus and plasma of the mouse model of brain region lesions induced by chronic stress, while the plasma serotonin level is significantly reduced, and increasing the IPA level in the brain region of normal mice can significantly induce chronic stress-like depressive behaviors in normal mice; it can be seen that IPA can participate in the onset of chronic stress by regulating the number of microglia in the brain, and IPA can also affect the level of inflammatory factor IL-1β via the NFκB pathway by regulating the expression of MID1 protein in the brain region; the levels of IPA and MID1 in the body play an important role in improving the process of chronic stress brain region lesions, which provides a theoretical basis and clinical basis for the study of preventing and treating chronic stress-like depressive behaviors.
[0009] Therefore, IPA caused chronic stress-like depressive behaviors in normal mice, IPA significantly increased the number of microglia in the brains of normal mice, and IPA increased the level of inflammatory factor IL-1β in the prefrontal cortex of normal mice through MID1 and NFκB pathway-related proteins. IPA can be used to screen drugs for the treatment or prevention of chronic stress-like depressive behaviors; at the same time, it can also affect the level of inflammatory factor IL-1β through the NFκB pathway by regulating the expression of MID1 protein in the brain, thereby providing new therapeutic drugs for the treatment of chronic stress-like depressive behaviors.
[0010] The beneficial technical effects of the present invention are:
[0011] 1. The present invention found that IPA levels were abnormally elevated in the hippocampus and plasma of the chronic stress mouse model; and increasing the IPA level in the brain of normal mice can significantly induce chronic stress-like depressive behaviors in normal mice, and IPA may play an important role in the pathogenesis of chronic stress; IPA can participate in the pathogenesis of chronic stress by regulating the number of microglia in the brain; IPA can also affect the level of inflammatory factor IL-1β through the NFκB pathway by regulating the expression of MID1 protein in the brain. Therefore, the number of microglia in the brain and the expression of inflammatory factor IL-1β can be reduced by reducing the level of IPA, thereby achieving the important role of improving chronic stress damage and protecting the host. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 Schematic diagram of the expression levels of IPA and 5-hydroxytryptamine (Serotonin) in the hippocampus and plasma of chronic stress model mice of the present invention;
[0013] Figure 2 This is a graph of normal mice of the present invention injected with IPA through the tail vein, and the IPA content in the hippocampus and prefrontal cortex was detected by liquid chromatography-mass spectrometry;
[0014] Figure 3 Schematic diagram of the effect of IPA of the present invention on chronic stress-like depression-like behavior in normal mice, wherein Figure 3 A is the time spent in the center area and the total distance walked in the open field test (OFT), **P<0.01, ***P<0.001; Figure 3 B is the number and time of stereotyped grooming behavior (Grooming), **P<0.01;
[0015] Figure 4 Schematic diagram of the effect of IPA of the present invention on the number of microglia in the brain of normal mice, Nissl staining of pathological sections, ****P<0.0001;
[0016] Figure 5 This is a schematic diagram of the effect of IPA of the present invention on the expression of MID1 and NFκB pathway-related proteins in the prefrontal cortex of normal mice, *P<0.05, ***P<0.001. DETAILED DESCRIPTION
[0017] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.
[0018] The terms "first", "second" and "third" in this application are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, the features defined as "first", "second" and "third" can explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. All directional indications in the embodiments of the present application (such as up, down, left, right, front, back...) are only used to explain the relative position relationship, movement, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication also changes accordingly. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not limited to the listed steps or units, but optionally also includes steps or units that are not listed, or optionally also includes other steps or units inherent to these processes, methods, products or devices.
[0019] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.
[0020] The C57BL / 6J mice used in the following examples of the present invention were purchased from Beijing Sibeifu Experimental Animal Co., Ltd. Unless otherwise specified, the reagents and materials used in the following examples were commercially available.
[0021] Example 1
[0022] (1) Construction of chronic stress mouse model: After 20 8-week-old male C57BL / 6 mice were adapted to the breeding environment (temperature 22-24°C, day and night cycle 12h, humidity controlled at 45-55%) for 7 days, the C57BL / 6 mice were randomly divided into 2 groups of 10 mice each, namely the control group (Control) and the chronic stress model group (Model). ① The mice in the control group (Control) did not receive any treatment or stimulation, and were placed in the normal breeding environment, and the food, water source, lifestyle, temperature, humidity, and lighting conditions were normal for 28 days. ② The mice in the chronic stress model group (Model) were subjected to irregular stress stimulation such as restraint, fasting, water deprivation, humidity, irregular day and night reversal, tail clamping, ice packs, rat bedding, tilting, etc., with 3 random different stimulation methods every day to prevent the animals from predicting the stimulation method in advance, and the stimulation was continuous for 28 days. After the experiment, the mice in the control group and the model group underwent behavioral testing (open field test). The mice in the model group showed chronic stress-like depression-like behavior, indicating that the model was successfully established. Mice were bled from their eyeballs, killed, and their brain tissues were obtained, and all animal experiments were performed in accordance with the rules of the Animal Ethics Committee.
[0023] The hippocampus and plasma of mice in the control group and model group were taken, and the levels of IPA and 5-HT were detected by liquid chromatography tandem mass spectrometry (LC-MS / MS). Sample pretreatment: ①Take out the sample and thaw it on ice (subsequent operations were all performed on ice); ②After the sample was thawed, weigh 50 mg into a centrifuge tube; ③Add 500 μL of methanol solution (containing 20 μL of internal standard working solution with a concentration of 250 ng / mL) to the centrifuge tube, vortex for 3 minutes to mix, and then stand in a -20℃ refrigerator for 30 minutes; ④Centrifuge at 12000r / min for 10 minutes at 4℃, and transfer 250 μL of supernatant to a new centrifuge tube; ⑤After centrifugation again, transfer 150 μL of supernatant to a sample injection bottle and temporarily store it in a -20℃ refrigerator for LC-MS / MS analysis. Chromatographic mass spectrometry acquisition conditions: ① Liquid phase conditions mainly include: Waters ACQUITY UPLC HSS T3 C18 column (1.8μm, 100mm×2.1mm id), mobile phase A phase ultrapure water (containing 0.1% formic acid) and phase B phase acetonitrile (containing 0.1% formic acid), flow rate 0.35mL / min column temperature 40℃ injection volume 2μL, etc. ② Mass spectrometry conditions: electrospray ion source temperature 550℃, mass spectrometry voltage 5500V in positive ion mode, mass spectrometry voltage -4500V in negative ion mode, curtain gas 35psi. In Q-Trap 6500+, each ion pair is scanned and detected according to the optimized declustering voltage and collision energy.
[0024] Schematic diagram of the expression levels of IPA and 5-hydroxytryptamine (Serotonin) in the hippocampus and plasma of chronic stress model mice Figure 1 As shown by Figure 1 It can be seen that the hippocampus and plasma IPA levels of mice in the chronic stress model group were significantly increased compared with those in the control group, while the plasma serotonin level was significantly decreased.
[0025] (2) Ten 8-week-old male C57BL / 6 mice were injected with IPA (40 mg / kg) via the tail vein, and ten 8-week-old male C57BL / 6 mice were injected with an equal amount of saline via the tail vein as a control group (Control). The mice were sacrificed after behavioral tests and brain tissues were obtained. LC-MS / MS was used to detect IPA levels in the hippocampus and prefrontal cortex of each group of mice. Figure 2 As shown by Figure 2 It can be seen that the IPA levels in the hippocampus and prefrontal cortex were significantly increased. It can be seen that the IPA levels in the hippocampus and prefrontal cortex of mice can be significantly increased by tail vein injection.
[0026] (3) The open field test (OFT) is a widely used behavioral testing method, which is usually used to evaluate the movement and exploratory behavior of mice at different time periods. Specific operation: Place the mice in a pre-set 50×50 open field area. Before placing the mice, the mice are prohibited from observing the open field area in advance to prevent them from having pre-perceptual behavior. The movement trajectory of the mice in the next 2-10 minutes is recorded by a camera, and the activity behavior of 0-2 minutes is regarded as the process of the mice adapting to the open field environment and is not counted. By observing the movement trajectory of the mice in the open field and the time they stay in the central area, the adaptability, exploratory behavior and activity of the mice can be evaluated, and then their anxiety level can be understood. Stereotyped grooming behavior is an important feature of anxiety and depression in mice. Therefore, the duration and number of stereotyped grooming behaviors of mice were recorded in detail in the mine field experiment. Combined with the results of the mine field experiment, the anxiety behavior, despair state and depression level of the mice were comprehensively evaluated to detect whether IPA would cause chronic stress-like depression in mice.
[0027] Schematic diagram of the effect of IPA on chronic stress-like depressive behaviors in normal mice Figure 3 As shown, compared with the control group, IPA significantly increased the time that normal mice stayed in the center area of the open field and significantly increased the distance they walked in the open field ( Figure 3 A); Compared with the control group, IPA significantly increased the frequency and duration of stereotyped grooming behavior in normal mice ( Figure 3 B). It can be seen that IPA causes chronic stress-like depression-like behavior in normal mice.
[0028] (4) Nissl staining of brain tissue (Nissl copper-hematoxylin staining). The steps are as follows: First, dewax the paraffin sections, soak the sections in environmentally friendly dewaxing solution I for 20 minutes, then soak them in environmentally friendly dewaxing solution II for 20 minutes, then soak them in anhydrous ethanol I for 5 minutes, soak them in anhydrous ethanol II for 5 minutes, and finally soak them in 75% alcohol for 5 minutes, and then wash them thoroughly with tap water. Next, take the frozen sections out of the -20℃ refrigerator, return to room temperature, fix them with tissue fixative for 15 minutes, and then rinse them with running water. Immerse the tissue sections in Nissl staining solution for 2 to 5 minutes, wash them with water after staining, use 0.1% glacial acetic acid for slight differentiation, and wash them again with pure water to terminate the reaction. After controlling the degree of differentiation under a microscope, wash them with pure water, and then place the sections in an oven to dry. Finally, transparentize the sections in clean xylene for 10 minutes and seal them with neutral gum. Observe the pathological changes of brain tissue under a microscope, and perform image acquisition and analysis.
[0029] Schematic diagram of the effect of IPA on the number of microglia in the brain of normal mice Figure 4As shown by Figure 4 It can be seen that IPA significantly increases the number of microglia in the brains of normal mice.
[0030] (5) Protein immunoblotting. The steps are as follows: extract the prefrontal cortex brain region tissue protein, prepare a 5mL centrifuge tube, add 40mg pre-cooled prefrontal cortex and 320μL of prepared tissue lysis solution containing 1% protease inhibitor, use a tissue disruptor to perform low-temperature homogenization, and centrifuge the homogenate at 10000rpm in a 4℃ centrifuge for 10min. After centrifugation, aspirate the supernatant, repeat centrifugation 3 times, and obtain fresh prefrontal cortex tissue protein supernatant. Then use the BCA protein concentration detection kit to measure the protein concentration. After preparing the protein sample, gel preparation and gel electrophoresis, PVDF membrane protein transfer, blocking and primary and secondary antibody incubation, signal detection and protein band recording are performed to analyze the expression of related proteins in the prefrontal cortex brain region tissue of each group of mice.
[0031] Schematic diagram of the effect of IPA on the expression of MID1 and NFκB pathway-related proteins in the prefrontal cortex of normal mice Figure 5 As shown by Figure 5 It can be seen that IPA increased the expression of MID1 and NFκB pathway-related proteins in the prefrontal cortex of normal mice. The protein immunoblotting method was used to detect the prefrontal cortex of mice and found that compared with the control group, the expression of MID1 protein in the IPA group was significantly increased, the phosphorylation level of JUN protein was significantly increased, the phosphorylation level of JNK protein was significantly increased, the phosphorylation level of NFκB protein was significantly increased, and the expression of inflammatory factor IL-1β was significantly increased. Therefore, IPA increased the level of inflammatory factors in the prefrontal cortex of normal mice through MID1 and NFκB pathway-related proteins.
[0032] The above results show that IPA levels are abnormally elevated in the hippocampus and plasma of the chronic stress mouse model, and increasing the IPA level in the brain of normal mice can significantly cause chronic stress-like depressive behavior in normal mice, suggesting that IPA plays an important role in the onset of chronic stress, which depends on the regulatory effect of IPA on the number of microglia and the levels of inflammatory factors in the brain. This provides a theoretical basis and clinical basis for the study of the prevention and treatment of chronic stress-like depressive behavior. Targeted reduction of IPA levels in brain regions can become the research and development direction of drugs for the prevention and treatment of chronic stress-like depressive behavior, thereby providing a new treatment strategy for the treatment of chronic stress-like depressive behavior diseases.
[0033] The specific implementation methods of the invention are described in detail above, but they are only examples, and the invention is not limited to the specific implementation methods described above. For those skilled in the art, any equivalent modification or substitution of the invention is also within the scope of the invention, therefore, the equalization, modification, improvement, etc. made without departing from the spirit and principle of the invention should be included in the scope of the invention.
Claims
1. Application of IPA in screening drugs for treating or preventing chronic stress-induced brain lesions.
2. The use according to claim 1, characterized in that: The drug for treating or preventing chronic stress-induced brain lesions refers to a drug that can reduce the IPA level in the brain and plasma, reduce the number of microglia in the brain and the expression of the inflammatory factor IL-1β.