Application of MSC-Exo in preparation of thermoplegia neuroprotective agent for regulating and controlling BV-2 cell polarization

Abstract

The invention relates to the technical field of medicines, in particular to application of a mesenchymal stem cell exosome in preparation of a thermoplegia neuroprotective agent. It is found through experiments that MSC-Exos can remarkably promote polarization conversion of microglial cells from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype, and therefore damage, caused by heat stroke, to a central nervous system is relieved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of MSC-Exo in the preparation of a neuroprotective agent for heatstroke that regulates BV-2 cell polarization. Background Technology

[0002] Heatstroke is a fatal acute illness caused by high temperatures, characterized by a sustained increase in core body temperature (usually exceeding 40°C) and an imbalance in the thermoregulatory center, accompanied by central nervous system abnormalities. The disease has a rapid onset and progression, and can trigger systemic inflammatory response syndrome, leading to multiple organ dysfunction or failure. Without timely treatment, the mortality rate is extremely high. With global warming, the intensity and frequency of heat waves are increasing, leading to a year-on-year rise in the number of people suffering from heat-related illnesses, with a significant increase in the incidence and mortality of heatstroke.

[0003] The incidence of central nervous system (CNS) dysfunction in Chinese heatstroke patients reaches 100%, with a long-term neurological dysfunction rate of 24.4%. When severe CNS damage and multiple organ dysfunction occur, the mortality rate and long-term sequelae rate of heatstroke patients will increase significantly.

[0004] Abnormal high signal intensity can be observed in the bilateral cortical and subhippocampal white matter of brain MRI in patients with heatstroke. Elevated levels of inflammatory factors such as IL-6 in brain tissue are significantly correlated with damage to the nervous system and neuronal death. These results suggest that a strong inflammatory response occurs in brain tissue during heatstroke.

[0005] The current treatment principles for heatstroke mainly follow the "ten early interventions and one prohibition," including: early cooling, early volume expansion, early blood purification, early sedation, early endotracheal intubation, early anticoagulation and anti-coagulation therapy, early anti-inflammatory treatment, early enteral nutrition, early dehydration, and early immune modulation; surgery is prohibited during the period of coagulation dysfunction. Current treatment principles primarily target the pathophysiological changes of heatstroke, lacking direct and effective interventions targeting neurological dysfunction. Summary of the Invention

[0006] To address the problems mentioned above, this invention provides the application of mesenchymal stem cell exosomes in the preparation of a neuroprotective agent for heatstroke. The drug reduces central nervous system damage caused by heatstroke by inducing a polarization shift of microglia from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype.

[0007] Furthermore, the drug is administered in the form of an injectable formulation.

[0008] In summary, the beneficial effects of the present invention are as follows:

[0009] Experiments have shown that mesenchymal stem cell exosomes can significantly reduce the levels of inflammation-related factors in the central nervous system of heatstroke rats, induce microglia to polarize to the M2 type, alleviate central nervous system damage, restore the number of neurons in heatstroke-damaged rats, prolong the survival time of heatstroke-damaged rats, and thus improve the therapeutic effect on central nervous system inflammatory response and functional damage caused by heatstroke.

[0010] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0011] Figure 1 To observe the morphology and structure of MSC-Exo using TEM;

[0012] Figure 2 For NTA and particle size analysis;

[0013] Figure 3 Detection of exosomal molecular marker proteins expressed by MSC-Exo;

[0014] Figure 4 The expression of IBA-1 in mice in the MSC-Exo treatment group;

[0015] Figure 5 To detect the expression of M1 and M2 markers in hippocampal microglia of heatstroke mice by PCR;

[0016] Figure 6 The levels of inflammatory factors in the culture supernatant of BV-2 cells were detected by ELISA.

[0017] Figure 7 To detect the levels of BV-2 cell markers in vitro using PCR;

[0018] Figure 8 To detect the expression levels of iNOS and Arg-1 proteins using Western blotting;

[0019] Figure 9 The expression levels of CD86 and CD206 in BV-2 cells were detected by flow cytometry. Detailed Implementation

[0020] To make the content of this invention easier to understand, the invention will be further described below with reference to specific embodiments and accompanying drawings.

[0021] This study was approved by the Animal Ethics Committee of the General Hospital of the Chinese People's Liberation Army. To ensure the scientific rigor of the experiment, the number of experimental animals used was minimized and the procedures were optimized to reduce animal suffering. Ninety 8-week-old male C57 / BL6J mice were used in the experiment. All animals were purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China). The animals were housed at the Experimental Animal Center of the General Hospital of the Chinese People's Liberation Army under an environment with a temperature of 20±2℃ and humidity of 50±5%, and had free access to food and water.

[0022] In this application, 90 eight-week-old male C57 / BL6J mice were randomly divided into 9 groups of 10 mice each. One group served as the control group, four groups served as the heatstroke group (HS group), and four groups served as the MSC-Exo treatment group (MSC-Exo group). The heatstroke group and the treatment group were further randomly divided into four subgroups according to the treatment time (3h, 12h, 24h and 72h).

[0023] Experiment 1: Isolation and Culture of Umbilical Cord Mesenchymal Stem Cells

[0024] Experimental methods

[0025] 1. Extraction and purification of human umbilical cord mesenchymal stem cells (HUCMSCs): Freshly collected umbilical cord mesenchymal stem cells were washed three times with phosphate-buffered saline (PBS). After removing two arteries and one vein, the umbilical cord was cut into pieces approximately 1 mm in size. 3 Small pieces of umbilical cord tissue were cultured in DMEM (Gibco, USA) containing F12 and 10% fetal bovine serum (FBS; Gibco) at 37°C and 5% CO2. When the cells on the umbilical cord tissue pieces reached 80% confluence, they were stratified and cultured with the medium changed every 2 days. The cells used in the following examples were all passage 5 HUCMSCs.

[0026] 2. Isolation of exosomes: Exosomes were isolated from the culture medium of human umbilical cord mesenchymal stem cells using the ExoQuick TC kit (SBI, USA). The structure of exosomes was identified by transmission electron microscopy (TEM), the number and size of exosomes were detected by nanoparticle tracking analysis (NTA), and the expression of exosome protein markers CD9 (Affinity, AF5139), CD63 (Affinity, AF5117), and CD81 (Affinity, DF2306) was determined by Western blotting.

[0027] Experimental results:

[0028] like Figure 1 As shown, TEM observations revealed that MSC-Exo exhibits a typical spherical morphology and a bilayer membrane structure.

[0029] like Figure 2 As shown, the particle diameter of MSC-Exo is mainly concentrated between 80-150 nm.

[0030] Western Blot results are as follows: Figure 3 As shown, the MSC-Exo used in this study expresses exosomal molecular marker proteins including CD9, CD63, and CD81.

[0031] The above results indicate that the MSC-Exo used in this study meets the exosome criteria.

[0032] Experiment 2: Model Building

[0033] 1. Methods for establishing animal models

[0034] (1) HS group: The heatstroke group mice were placed in a simulated climate chamber, and the temperature was gradually increased from 25°C to 39.5°C until the rectal temperature reached 42.7°C. Then, the heatstroke group mice were injected with physiological saline (200 μg).

[0035] (2) HS+MSC-Exo group: The mice in the treatment group were placed in a simulated climate chamber, and the temperature was gradually increased from 25℃ to 39.5℃ until the rectal temperature reached 42.7℃. Then, the mice in the treatment group were injected with MSC-Exo (200μg).

[0036] (3) Control group: The mice in the control group did not require any treatment.

[0037] Sample collection: After the models were established, samples were collected from the four subgroups of the HS group and the MSC-Exo group at four time points: 3h, 12h, 24h, and 72h. All mice were anesthetized intraperitoneally with sodium pentobarbital (60mg / kg) and sacrificed by cervical dislocation.

[0038] Experiment 3: Regulatory effect of MSC-Exo on microglia activation and polarization in heatstroke mice

[0039] Experimental methods:

[0040] 1. Immunofluorescence staining: Hippocampal tissue from mice in each group was fixed with 4% paraformaldehyde. The fixed hippocampal tissue was then dehydrated using a sucrose gradient treatment, embedded in OCT, and frozen sectioned (40 μm). The sections were washed three times (5 min) with PBS, permeabilized with 0.5% Triton X-100 (15 min), and then washed three times (5 min) with PBS. The tissue sections were blocked with 10% normal goat serum (NGS) at 37°C for 30 min, and then incubated overnight at 4°C with IBA1 antibody (Iba-1, 1:100, Servicebio, China). The sections were washed three times with PBS for 5 min each time, and then incubated for 1 hour at room temperature (22-25°C) in the dark with HRP-labeled anti-rabbit IgG secondary antibody (1:100, Servicebio, China). The slides were washed three times with PBS for 5 minutes each time, followed by staining with DAPI to block cell nuclei. The stained slides were visualized and scanned using a Pannoracic MIDI CaseViewer 2.0 system (3DHISTECH Ltd.).

[0041] 2. Flow cytometry: CD86 is a surface marker for M1 microglia, and CD206 is a surface marker for M2 microglia. Microglia from the hippocampus of mice in each group were collected, and approximately 1 × 10⁵ co-cultured BV-2 cells were centrifuged at 1000g for 2 minutes, followed by resuspending in 100 μl of PBS. The BV-2 cells were then incubated with CD86, CD206, and fluorescent antibodies at room temperature for 30 minutes. Flow cytometry (BD FACSCALIBUR) was used to quantify the percentage of M1 / M2 microglia.

[0042] Experimental results:

[0043] like Figure 4 Immunofluorescence results showed that the level of IBA-1, a marker of microglia activation in the hippocampus, was significantly increased after heatstroke modeling in mice, indicating excessive microglia activation. The IBA-1 level in the MSC-Exo treatment group was lower than that in the model group, with the most significant differences observed at 3h and 24h.

[0044] like Figure 5 PCR results showed that the levels of M1 microglia markers (MCP-1, iNOS, CD86) in the hippocampus of heatstroke mice were significantly upregulated, and these marker levels were significantly reduced after MSC-Exo treatment. Furthermore, the levels of M2 microglia markers (Arg-1, Fizz-1, CD206) were reduced in the hippocampus of heatstroke mice, but these marker levels were significantly increased after MSC-Exo treatment.

[0045] The above results indicate that MSC-Exo can inhibit the excessive activation of microglia caused by heat stress and regulate the polarization of microglia from M1 type to M2 type.

[0046] Experiment 4: Regulatory effects of MSC-Exo on inflammation and polarization in BV-2 cells under heat stress.

[0047] Experimental methods:

[0048] 1. In vitro experiments: A heatstroke cell model was established using BV-2 cells subjected to heat shock. The BV-2 mouse microglia cell line was purchased from Interlab Cell Line Collection (ICLC, Genova, Italy).

[0049] (1) Cell Culture

[0050] BV-2 microglia were cultured in DMEM medium (Gibco, New York, USA) containing 10% fetal bovine serum (FBS, Gibco, New York, USA) at an ambient temperature of 37°C and a carbon dioxide concentration of 5%.

[0051] (2) Experimental grouping

[0052] Control group: No treatment was given to BV-2 microglia.

[0053] Heatstroke group (HS group): BV-2 cells were placed in a 42°C heat stress incubator for 2 hours and then recovered at a normal growth temperature of 37°C for 6 hours.

[0054] Treatment group (HS+MSC-Exo group): BV-2 cells were placed in a 42℃ heat stress incubator for 2 hours, followed by recovery at a normal growth temperature of 37℃ for 6 hours, and then co-cultured with MSCs.

[0055] 2. ELISA: Enzyme-linked immunosorbent assay (ELISA) was performed on the supernatant of BV-2 cells in each group. Then, the concentrations of TNF-α, IL-1β, IL-6, IL-10 and TGF-β were determined using an ELISA kit (Shanghai Biotech Co., Ltd.) according to the manufacturing process.

[0056] 3. PCR detection of BV-2 cell marker levels.

[0057] 4. Western blot: BV-2 cells from each group were cultured in six-well plates and lysed on ice with RIPA buffer (Servicebio, G2002). Protein concentration was determined using a BCA protein assay kit (Servicebio, G2026). Each sample was analyzed by SDS-PAGE (Servicebio, G2003) and then transferred to a PVDF membrane (Servicebio, G6015-0.45). Subsequently, the cells were diluted 1-fold with specific primary antibodies for iNOS (Affinity, AF0199), Arg1 (Affinity, DF6657), Per2 (Affinity, DF12304), and β-actin (KANGCHENG, KC-5A08), respectively, and then blocked with Tween-melamine buffered saline (TTBS) containing 5% skim milk at room temperature for 2 hours. All primary antibodies were diluted 1:2000 and incubated with the membrane overnight at 4°C. Wash the membrane at least three more times with TTBS and incubate with secondary antibody (1:5000) at room temperature for 2 hours.

[0058] 5. Flow cytometry (BD FACSCALIBUR) is used to quantify the percentage of M1 / M2 microglia. The indicators measured include: M1 polarization marker: CD86 (pro-inflammatory phenotype), and M2 polarization marker: CD206 (anti-inflammatory phenotype).

[0059] Experimental results

[0060] like Figure 6 ELISA results showed that pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in BV-2 cells were significantly elevated after heat shock, and the levels of pro-inflammatory cytokines were significantly reduced after co-culturing BV-2 cells with MSCs in the heatstroke group. Anti-inflammatory cytokines such as IL-10 and TGF-β were significantly downregulated after heat shock, but MSCs were able to increase the levels of these anti-inflammatory cytokines.

[0061] like Figure 7 PCR results showed that the transcriptional levels of M1 markers (iNOS, MCP-1, CD45) in BV-2 cells were significantly upregulated after heat shock, but co-culturing with MSCs significantly reduced the levels of M1 markers. The transcriptional levels of M2 markers (Arg-1, Fizz-1, Ym-1) were highest in the control group, and significantly decreased after heat shock; however, MSCs significantly upregulated the transcriptional levels of Arg-1 and Ym-1.

[0062] like Figure 8Western blot results showed that iNOS protein levels were upregulated after heat shock, while MSCs could reduce iNOS protein levels. Arg-1 protein expression levels decreased in BV-2 cells after heat shock, but Arg-1 protein expression levels increased after co-culturing BV-2 cells with MSCs in the heatstroke group.

[0063] like Figure 9 Flow cytometry results showed that CD86 expression was significantly upregulated in BV-2 cells after heat shock, and significantly decreased after co-culturing with MSCs. CD206 levels in BV-2 cells were significantly reduced after heat shock, while MSCs significantly increased CD206 expression in BV-2 cells.

[0064] The embodiments described above are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and modifications made by those skilled in the art based on the invention shall fall within the scope of protection of the present invention.

Claims

1. The application of mesenchymal stem cell exosomes in the preparation of neuroprotective agents for heatstroke, characterized in that, The drug reduces central nervous system damage caused by heatstroke by inducing a polarization shift in microglia from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype.

2. The application according to claim 1, characterized in that, The drug is administered in the form of an injectable formulation.

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