Application of cystamine in the preparation of drugs for treating septic encephalopathy

Abstract

The present invention belongs to the field of medical technology and discloses the use of cystamine in the preparation of a drug for treating septic encephalopathy. The septic encephalopathy is simulated in an endotoxemia model or an in vitro reactive astrocyte model. The present invention is the first to discover that cystamine can inhibit the pathological progression of septic encephalopathy through multiple pathways, significantly improving mortality and cognitive dysfunction in mice modeled with septic encephalopathy. This invention provides new insights into the preparation of therapeutic products for septic encephalopathy and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the use of cystamine in preparing a drug for treating septic encephalopathy. Background Art

[0002] Sepsis, defined as life-threatening organ dysfunction caused by a dysregulated host response to infection, is a leading cause of death in critically ill patients. Sepsis is often accompanied by short-term, diffuse brain dysfunction and long-term, irreversible cognitive impairment, known as sepsis-associated encephalopathy (SAE). Clinically, SAE manifests as confusion, delirium, cognitive impairment, and even coma. A significant proportion of patients experience long-term cognitive impairment, severely impacting daily life and increasing the risk of mortality. Its pathogenesis is complex, involving a multifactorial interplay of factors, including neuroinflammation, blood-brain barrier disruption, metabolic disorders and mitochondrial dysfunction, and glial hyperactivation. The diagnostic criteria for SAE lack specificity and are a diagnosis of exclusion, requiring the exclusion of medications and other causes of encephalopathy. Currently, there is a lack of specific treatment for SAE. Existing treatments are mostly based on sepsis management (e.g., fluid resuscitation and antibiotics), but these are ineffective in reversing neuroinflammation and cognitive impairment. Early diagnosis and proactive treatment are crucial for prognosis. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a use of cystamine in the preparation of a drug for treating septic encephalopathy.

[0004] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0005] The invention discloses an application of cystamine in preparing a medicine for treating septic encephalopathy.

[0006] Cystamine, chemical name is 2,2'-dithiodiethylamine, and its chemical formula is C4H 12N2S2 (free base form, CAS number: 51-85-4) is a sulfur-containing organic compound. It typically exists as colorless crystals or white crystalline powder at room temperature and is readily soluble in water and polar organic solvents (such as ethanol). Its chemical properties include its ability to readily oxidize in acidic media to form stable disulfide bonds, leading to its widespread use as a reducing agent and antioxidant in biochemistry. Cystamine is an orally active transglutaminase (TGase) inhibitor. Cystamine also exhibits inhibitory activity against caspase-3. Furthermore, cystamine, as the disulfide form of the free thiol cysteamine, can release reactive sulfhydryl groups (-SH) under physiological conditions through dynamic disulfide exchange reactions, endowing it with significant biological activity and potential applications in the treatment of certain diseases, such as amyotrophic lateral sclerosis and oxidative stress-related diseases.

[0007] In the above application, further, the septic encephalopathy is septic encephalopathy simulated by an endotoxemia model or an in vitro reactive astrocyte model.

[0008] Furthermore, the symptoms of septic encephalopathy include at least one of cognitive dysfunction, neuroinflammation, astrocyte A1 polarization, and excessive mitochondrial fission.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] This study, published in Nature Communications, demonstrates for the first time that cystamine can inhibit the pathological progression of septic encephalopathy through multiple pathways, significantly improving mortality and cognitive impairment in mice modeled with sepsis. The specific mechanisms include inhibition of neuroinflammatory responses, suppression of A1 polarization of reactive astrocytes, and inhibition of excessive mitochondrial fission. This study provides a novel approach for the development of therapeutic products for septic encephalopathy and holds broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0012] Figure 1 This is the result of cystamine improving the survival rate of LPS model mice in the embodiment of the present invention;

[0013] Figure 2 This is the result that cystamine in the embodiment of the present invention improves the behavioral impairment of the open field test in LPS model mice;

[0014] Figure 3 This is the result that cystamine in the embodiment of the present invention improves the behavioral impairment of the cross-elevation test in LPS model mice;

[0015] Figure 4 This is the result of cystamine improving the behavioral impairment of the novel object recognition test in LPS model mice in the embodiment of the present invention;

[0016] Figure 5 This is the result that cystamine in the embodiment of the present invention improves the behavioral impairment of the Y maze test in LPS model mice;

[0017] Figure 6 The results of the present invention show that cystamine improves the upregulation of excessive inflammatory factors in LPS model mice. Figure A shows the changes in the levels of JAK2, STAT3 protein, iNOS, TNF-α, IL-1β, and β-Actin proteins in the hippocampus of LPS model mice. Figure B shows the statistical results of relative protein expression.

[0018] Figure 7 The results of the cystamine-treated LPS model mouse hippocampus inflammatory response storm test are as follows: Figures A to C show changes in the transcriptional levels of inflammatory factors IL-1β, IL-6, and iNOS in the hippocampus of the LPS model mouse; Figures D and E show changes in the transcriptional levels of inflammatory pathway transcription factors STAT1 and STAT3 in the hippocampus of the LPS model mouse; and Figure F shows changes in the transcriptional level of Gasdermin D, a pyroptosis-related molecule, in the hippocampus of the LPS model mouse.

[0019] Figure 8 These are the experimental results of the present invention showing that cystamine improves the hyperpolarization of type A1 astrocyte markers in LPS model mice, wherein: Figure A shows the C3 transcription level in the LPS model, Figure B shows the S100a10 transcription level in the LPS model, Figure C shows the changes in C3, TGM2, S100a10, and β-Actin protein levels in the LPS model, and Figure D shows the statistical results of relative protein expression.

[0020] Figure 9 These are the test results of cystamine improving mitochondrial function in LPS model mice in the examples of the present invention, wherein: Figure A shows the changes in protein levels of transamidase TGM2, mitochondrial fission protein p-Drp1 S616, Fis 1, apoptosis protein Caspase 3, mitochondrial oxidative respiratory chain protein Ndufs1, SDHB, ANT1, and β-Actin in hippocampal tissue of LPS model mice; Figure B shows the statistical results of relative protein expression;

[0021] Figure 10These are the test results of the embodiment of the present invention showing that cystamine improves the inflammatory response of primary astrocytes induced by LPS-MCM in an in vitro cell model, wherein: Figures AF are the changes in the levels of TGM2, IL-1β, IL-6, iNOS, TNF-α, and Caspase 3, respectively;

[0022] Figure 11 This is the experimental result of the embodiment of the present invention that cystamine improves the hyperpolarization of type A1 astrocytes induced by LPS-MCM in an in vitro cell model;

[0023] Figure 12 These are the experimental results showing that pre-administration of cystamine in an embodiment of the present invention can inhibit excessive mitochondrial fission induced by LPS-MCM in an in vitro cell model, wherein: Figure A is the immunofluorescence result, and Figure B is the WB result. DETAILED DESCRIPTION

[0024] To facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed form below in conjunction with the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0025] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0026] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0027] Example:

[0028] The invention discloses an application of cystamine in preparing a medicine for treating septic encephalopathy.

[0029] 1. Experimental Methods

[0030] 1.1 Establishment of a mouse model of sepsis

[0031] Endotoxemia (LPS) model preparation: LPS was pre-dissolved in normal saline until fully dissolved, to a final concentration of 2 mg / ml. C57 mice were fasted for 12 hours before surgery with free access to water. Mice were anesthetized with isoflurane beforehand, and the abdomen was disinfected. LPS was then injected intraperitoneally according to body weight. Care was taken to aspirate before administration to avoid damaging intraperitoneal blood vessels. The entire procedure was completed within 30 seconds. After administration, cotton swab pressure was applied for 1 minute, and the mice were placed on a heating pad to maintain warmth until they regained consciousness and resumed normal activity.

[0032] 1.2 Animal Behavior

[0033] All behavioral experiments in this study were conducted between 9:00 AM and 5:00 PM. Experimenters handled the mice for at least 7 days prior to the experiment to familiarize them with the experimenter. Mice were placed in the behavioral laboratory 3 hours before the experiment to allow them to acclimate. Experimenters avoided making excessive noises and strong odors during testing, as both noise and odors can cause anxiety in the mice and affect behavioral results.

[0034] 1.2.1 Open field test (OFT)

[0035] Experimental Procedure: The test mouse was removed from its cage and carefully placed in a corner of a 42 cm (length) × 42 cm (width) × 42 cm (height) open-top experimental box at the same position and angle. The operator left the behavioral laboratory and allowed the mouse to freely explore for 10 minutes. The mouse's movements were recorded using a video tracking system, and the time the mouse spent in the center area and the number of times it crossed the center area were counted. After the 10-minute test, the mouse was returned to its cage. After each experiment, the OFT apparatus was thoroughly wiped with 75% ethanol.

[0036] Experimental Principle: Mice prefer darkness, so they tend to move around the perimeter of the experimental chamber. However, healthy mice are curious and exploratory, leading them to explore the center. However, anxious mice exhibit less exploratory behavior, leading them to move less in the open center of the chamber and exhibit more stereotyped behaviors. The less time a mouse spends in the center of the chamber, the higher its anxiety level.

[0037] 1.2.2 Elevated plus maze (EPM)

[0038] The elevated plus maze consists of open arms and closed arms that cross in a cross shape. The middle area of ​​the cross is called the central area. The two arms are 10 cm wide and 35 cm long. The closed arms are 15 cm high. The maze is about 40 cm above the ground.

[0039] Experimental Principle: Mice's curiosity prompts them to explore new, brightly lit areas (open arms), but their natural preference for darkness leads them to prefer remaining in the closed arms. The height of the elevated maze is comparable to that of a human on the edge of a cliff, which can easily lead to fear and anxiety in the animals. The total number of entries into the open and closed arms, the percentage of each, and the duration of stay in the open and closed arms can reflect the mice's anxiety level. Experimental steps: a. Take the test mouse out of the cage and gently place it facing the open arm in the central area of ​​the plus maze from the same position and angle. After placing the mouse, the experimenter should quickly and quietly leave the behavior room; b. Record the whereabouts of the mouse through a video tracking system, and count the number of times the mouse enters the open arm (openarm entry OE), the number of times the mouse enters the closed arm (closed arm entry, CE), the active time in the open arm (openarm time, OT) and the active time in the closed arm (closedarm time, CT), the proportion of the number of times the mouse enters the open arm (OE%), that is, OE / (OE+CE), and the proportion of the active time in the open arm (OT%), that is, OT / (OT+CT). Each experimental time for each mouse is 5 minutes; c. After the experiment of each experimental mouse, wipe the maze with a paper towel containing 75% alcohol to remove the odor, urine and feces left by the mouse.

[0040] 1.2.3 Y-maze

[0041] The Y-maze consists of three closed arms, A, B, and C, of ​​equal length (40 cm long, 1 cm wide, and 6 cm high), with each arm angled 120 degrees. The frame is 40 cm above the ground. The Y-maze is widely used in research on functional impairment, tropism, and spatial working memory.

[0042] Experimental steps: a. On the first day, the mouse was gently placed in the center of the Y-maze from the same angle and position. The experimenter quietly left the behavior room and allowed the mouse to freely explore the maze and adapt to the maze environment for 8 minutes. b. The formal experiment was divided into two times. The above operation was repeated one hour after adapting to the two identical objects and on the second day. The whereabouts of the mouse were recorded by a video tracking system. The order in which the mouse entered the three arms A, B, and C was counted. The standard for entering an arm was that all four feet of the mouse entered an arm. Statistics: (1) Total number of entries m: the total number of times the mouse entered the three maze arms; (2) Number of valid behaviors n (analtermation): consecutively entering all three arms of the Y-maze was counted as a valid behavior; (3) Valid behavior score = n / (m-2) × 100%. The higher the valid behavior score, the stronger the mouse's working memory ability.

[0043] 1.2.4 New Object Recognition (NOR)

[0044] The experimental equipment includes: a set of equipment for open-field experiments: a square box (42 cm long, 42 cm wide, and 42 cm high), a video tracking system, and behavioral analysis software; three objects A, B, and C. Objects A and B are exactly the same, and object C is very different from objects A and B, but of similar size; mice generally need objects with a diameter of about 3 cm; it is better if the three objects are round, as mice tend to prefer cubes or rectangular prisms with corners.

[0045] Experimental principle: The experimental mice's natural curiosity towards novel things determines that they are more curious about new objects than familiar old objects. The cognitive memory ability of the experimental mice is evaluated by behavioral methods by evaluating the length of time the experimental mice explore familiar old objects and new unfamiliar objects.

[0046] Experimental Procedure: The novel object recognition (NOR) experiment consists of three phases: acclimation, familiarization, and testing. a. Phase I - Acclimation: Place the mouse in the center of the experimental chamber for 10 minutes, without any objects placed in it. b. Phase II - Familiarization: Place two identical objects (A and B) in the experimental chamber (ensuring they are odorless and immovable). Place the objects 10 cm from the chamber wall. Place the mouse equidistant from both objects. Place the mouse with its back facing the objects. The experimenter quietly leaves the chamber, allowing the mouse to explore freely. A video tracking system records the amount of time the mouse explores each object over a 5-minute period (valid exploration is defined as the time the mouse's mouth or nose comes within 2-3 cm of the object). c. Phase III - Testing: This phase is conducted 1 hour after the end of Phase II. Object B is replaced with object C (AC) of a different shape and color. Ensure that object C is immovable. Gently place the mouse in the same position. Observe and record the amount of time the mouse explores objects A and C over a 5-minute period. After each mouse completed an exploration, the experimental box and the surfaces of the objects were wiped with a paper towel containing 75% alcohol to remove the odor left by the mouse. T1 is the total time the mouse spent exploring object C, and T2 is the total time the mouse spent exploring object A.

[0047] 1.3 Primary glial cell mixed extraction and culture method

[0048] (1) Sterilize the operating instruments and ddH2O one day in advance, add 10 mL of PDL diluted with sterile ddH2O (final concentration 10 g / pL, prepared immediately before use) to the T75 cell culture flask, incubate at 37°C for 6-8 hours, and wash with sterile ddH2O 6 times, with an interval of 1 hour between each wash.

[0049] (2) Pregnant mice on day 16-18 of gestation were killed by cervical dislocation, and the abdomen was sprayed with alcohol. The fetuses were placed in pre-cooled 1× HBSS, transferred to a clean bench, and decapitated and placed in a small dish containing pre-cooled 1× HBSS;

[0050] (3) Through a microscope, insert the two ends of a pair of sharp forceps into the fetal mouse's eyeballs to fix the head, and use another pair of sharp forceps to peel off the skin and skull of the fetal mouse to expose the brain;

[0051] (4) Place a 15 mL centrifuge tube filled with 1× HBSS on ice; tear off the vascular membrane on the surface of the brain and remove structures such as the olfactory bulb and midbrain, remove obvious bleeding spots, and collect the cortex in a centrifuge tube;

[0052] (5) Wash three times with pre-cooled 1× HBSS, discard the 1× HBSS in the centrifuge tube, add 200 μL of 2.5% trypsin and 2 mL of pre-heated serum-free DMEM along the tube wall, shake gently to mix, and then insert it obliquely in a 37°C water bath for digestion for 20 minutes. Take it out and mix it every 5 minutes;

[0053] (6) After digestion, wash three times with preheated 1× HBSS, taking care not to pour out the clumps of tissue;

[0054] (7) Pipette 5 mL of glial cell culture medium (added with pyruvate and GM-CSF) into a centrifuge tube and blow with an electric gun about 20 times until no large tissue clumps are visible to the naked eye;

[0055] (8) Filter the cell suspension through a 70 μm filter into a 50 mL centrifuge tube, fill the tube with 12 mL of culture medium per T75 flask, and seed the tubes into T75 cell culture flasks;

[0056] (9) After seeding, the medium was changed every 4 days. After 10 days, glial cells were observed to grow in layers, with translucent microglia on the top and astrocytes attached to the bottom.

[0057] (10) After 14 days of inoculation, aspirate part of the medium in the T75 cell culture flask, wrap with plastic wrap, and place on a flatbed shaker at 120 rpm. After 1 hour, collect the cell suspension in a 15 mL centrifuge tube. The remaining adherent cells in the T75 cell culture flask are astrocytes. Centrifuge at 1000 x g for 5 minutes at room temperature, pipette and mix with 1 mL of microglial cell-specific medium, count, and seed into well plates according to experimental requirements.

[0058] 1.4 In vitro reactive astrocyte model

[0059] Primary microglia were stimulated with 1 μg / mL LPS. 12 hours later, the cell supernatant was collected as LPS-microglia conditioned medium (LPS-MCM) and used to stimulate primary astrocytes to differentiate into type A1 reactive astrocytes. A blank control group was also given blank microglia medium (Blank-MCM).

[0060] 2. Test results

[0061] 2.1 Cystamine improves the survival rate of sepsis model mice

[0062] The inventors established a sepsis mouse model by intraperitoneal injection of 20 mg / kg lipopolysaccharide (LPS). Compared with the saline control group (Saline), Figure 1 As shown in the figure, the seven-day survival rate of mice in the LPS group was significantly decreased, and intraperitoneal injection of 25 mg / kg Cystamine 1 hour before modeling could significantly improve the seven-day survival rate of mice in the LPS group.

[0063] 2.2 Cystamine improves cognitive dysfunction in septic encephalopathy model mice

[0064] Cognitive dysfunction caused by sepsis-induced brain damage is becoming increasingly prominent in clinical practice, manifesting as decreased memory function and anxiety. This reduces patients' quality of life, increases the cost of social medical care and patient care, and places a significant burden on society and families. Therefore, it is of great significance to attach importance to the prevention and treatment of septic encephalopathy and sepsis-induced cognitive dysfunction. The inventors used the open field test and the cross-elevated test to detect anxiety-like behaviors in cognitive dysfunction in LPS-induced sepsis mice, and used the Y-maze test and the novel object recognition test to detect memory-impaired behaviors in cognitive dysfunction in LPS-induced sepsis mice. The specific methods are described in the Methodology section.

[0065] like Figure 2 As shown in the open field test, the results showed that the time the mice in the LPS group stayed in the central area of ​​the open field was significantly reduced, suggesting that they had anxiety-like behavior, and the administration of cyclamine could significantly reverse the cognitive dysfunction phenomenon of the mice in the LPS group.

[0066] like Figure 3 As shown in the cross-elevated test, the time that the mice in the LPS group stayed in the open arm was significantly reduced, suggesting that they had anxiety-like behavior, and cyclamine administration could significantly reverse this cognitive dysfunction phenomenon in the mice in the LPS group.

[0067] like Figure 4As shown in the results of the novel object recognition test, the LPS group mice had significantly reduced ability to recognize novel objects, suggesting that they had impaired memory, and cyclamine administration could significantly reverse this cognitive dysfunction in the LPS group mice.

[0068] like Figure 5 As shown in the Y-maze test results, the LPS group mice had a significantly reduced novel arm rotation ratio (effective exploration times) in the Y-maze test, suggesting that they had memory impairment, and Cystamine administration could significantly reverse this cognitive dysfunction phenomenon in the LPS group mice.

[0069] 2.3 Cystamine inhibits neuroinflammation in a mouse model of septic encephalopathy

[0070] The hippocampus of the brain is considered to be an important brain area for performing working memory and cognitive functions. Clinical studies have shown that compared with non-septic patients, septic patients have defects in memory and language learning, and their hippocampal volume is significantly reduced, indicating that hippocampal lesions are closely related to cognitive dysfunction in sepsis. Therefore, the present invention mainly studies the effect of Cystamine on the hippocampus of mice in the acute phase of sepsis. 24 hours after LPS modeling, the mice were killed and the hippocampal tissues were taken. RNA was extracted from one side of the hippocampus, and protein was extracted from the other side of the hippocampus. Real-time fluorescence quantitative PCR (Quantitative Real-time polymerase chain reaction, Q-PCR) and protein immunoblotting (Western Blot, WB) were used to simultaneously detect the expression levels of inflammatory factors. Figure 6 As shown, WB results showed that Cystamine inhibited the activation of the inflammatory pathway JAK2-STAT3 pathway and reduced the expression levels of inflammatory factors iNOS, TNF-α, and IL-1β proteins.

[0071] like Figure 7 As shown, Q-PCR revealed that cystamine reduced the upregulation of inflammatory factors IL-1β (Panel A), IL-6 (Panel B), and iNOS (Panel C) in the hippocampus of LPS-induced mice; the transcription factors STAT1 / 3 (Panels D and E) in the inflammatory pathway; and the pyroptosis-related molecule Gasdermin D (GSDMD) (Panel F). This suggests that TGM2 inhibitors can inhibit the upregulation of inflammatory factors in the hippocampus of mice induced by LPS, demonstrating a certain degree of anti-inflammatory effect.

[0072] 2.4 Cystamine inhibits A1 polarization of astrocytes in septic encephalopathy model mice

[0073] Astrocytes are the most abundant glial cells in the brain, accounting for approximately 20–50% of the total number of cells in the central nervous system. They participate in a variety of structural, metabolic, and homeostatic functions. Furthermore, astrocytes play a crucial role in the progression of septic encephalopathy. In 2017, Liddelow's group first reported in Nature that LPS and middle cerebral artery occlusion induce two distinct types of reactive astrocytes: type A1 (neurotoxic) and type A2 (neuroprotective). Genes such as C3, H2-T23, H2-D1, and Serping1 are markers of type A1 reactive astrocytes, while genes such as S100a10, CD109, EMP1, and TGM1 are markers of type A2 reactive astrocytes.

[0074] The present invention uses Q-PCR and WB to simultaneously detect the expression levels of TGM2, A1 type astrocyte marker C3, and A2 type astrocyte marker S100a10. Figure 8 As shown, Q-PCR results showed that cystamine administration reduced C3 transcription levels in the LPS model (Panel A) and increased S100a10 transcription levels (Panel B). Cystamine also acts as a transglutaminase (Tgase) inhibitor. Western blot results also demonstrated that cystamine administration reduced transglutaminase 2 (TGM2), C3, and TGM2 protein expression, and increased S100a10 protein expression in the LPS model (Panel C). This suggests that cystamine promotes the transformation of astrocytes from type A1 to type A2 in the hippocampus of LPS-induced mice.

[0075] 2.5 Cystamine improves mitochondrial fission in septic encephalopathy model mice

[0076] Mitochondria are a type of organelle surrounded by two membranes that exist in most cells. They are structures that produce energy in cells and are also the main site for cells to carry out aerobic respiration. The outer mitochondrial membrane contains many integral proteins called "porins" and has high permeability, which can transport small molecules such as adenosine 5'-triphosphate (ATP); the inner mitochondrial membrane is the site of oxidative phosphorylation where the respiratory chain is located, contains a series of enzyme complexes, and has poor permeability. Mitochondria in living cells exist in a dynamic cyclic homeostasis of fusion and fission. Studies have shown that the balance of fusion and fission directly affects the morphology of the mitochondrial network and thereby affects ATP homeostasis and cell function, playing a very important role in the physiological and pathological processes of cells. The effect of intraperitoneal injection of Cyatamine on mitochondrial-related proteins in the hippocampus of LPS model mice was further tested at the animal level. Figure 9As shown, Western blot results showed that CTM inhibited the LPS-induced upregulation of mitochondrial fission proteins p-Drp1 S616 and Fis 1, apoptosis protein Caspase 3, and transamidase TGM2 protein levels, and inhibited the LPS-induced downregulation of mitochondrial oxidative respiratory chain proteins Ndufs1 and ANT1 protein levels. This suggests that cystamine can improve mitochondrial dysfunction in the hippocampus of LPS-induced mice.

[0077] 2.6 Cystamine improves inflammatory response, A1 polarization, and mitochondrial dysfunction in primary astrocytes in vitro

[0078] An in vitro reactive astrocyte model was established by stimulating primary mouse astrocytes with LPS-activated microglial conditioned medium (LPS-MCM). Cystamine (10 μM) was pretreated for 1 hour and then LPS-MCM was continued for 24 hours. Figure 10 As shown, Q-PCR showed that compared with the blank control microglial cell-conditioned medium (Blank-MCM) not activated by LPS, LPS-MCM could induce a strong inflammatory response in astrocytes, and cystamine administration could significantly reverse the upregulation of TGM2 (Figure A), IL-1β (Figure B), IL-6 (Figure C), iNOS (Figure D), TNF-α (Figure E), and Caspase 3 (Figure F).

[0079] The mouse primary astrocyte model cultured in vitro was pretreated with Cystamine (10 μm) for 1 hour and then stimulated with LPS-MCM for 24 hours. Figure 11 As shown in the figure, Q-PCR results showed that cyclamine could inhibit the upregulation of A1 type astrocyte markers H2-T23, H2-D1, Serping1, and C3 and the downregulation of A2 type astrocyte markers S100a10 and EMP1 induced by LPS-MCM, suggesting that cyclamine can reverse the A1 type polarization of astrocytes induced by LPS-MCM in vitro.

[0080] like Figure 12 As shown, the immunofluorescence results showed that pretreatment with Cystamine (10 μm) for 1 hour could inhibit the upregulation of the fluorescence intensity of mitochondrial fission protein p-Drp1 S616 induced by LPS-MCM (Figure A). The WB results showed that Cystamine could inhibit the upregulation of mitochondrial fission proteins Drp1, p-Drp1 S616, and Fis1 induced by LPS-MCM (Figure B).

Claims

1. A use of cystamine in preparing a drug for treating septic encephalopathy, characterized in that: The cystamine is 2,2'-dithiodiethylamine.

2. The use according to claim 1, characterized in that The septic encephalopathy is septic encephalopathy simulated by an endotoxemia model or an in vitro reactive astrocyte model.

3. The use according to claim 1, characterized in that The symptoms of septic encephalopathy include at least one of cognitive dysfunction, neuroinflammation, astrocyte A1 polarization, and excessive mitochondrial fission.

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