MicroRNA-206 down-regulated human umbilical cord mesenchymal stem cells alleviate cognitive decline in D-galactose-induced aging mice | Cell Death Discovery

Abstract

Background

Non-pathological cognitive decline is a neurodegenerative condition associated with brain aging due to epigenetic changes, telomere shortening, stem cell depletion, or altered differentiation. Human umbilical cord mesenchymal stem cells (hUCMSCs) have shown excellent therapeutic prospects on the hallmarks of aging. In this study, we aimed to elucidate the role of hUCMSCs with down-regulated miRNA-206 (hUCMSCs anti-miR-206) on cognitive decline and the underlying mechanism.

Methods

After daily subcutaneous injection of D-gal (500 mg / kg / d) for 8 weeks, 17-week-old male mice C57BL / 6 J were stem cells transplanted by lateral ventricular localization injection. During the 10-day rest period, the behavioral experiments applied to cognitive behavior in the hippocampus were tested. And then, the mice were sacrificed for sampling to complete the molecular and morphological experiments.

Results

Our behavioral experiments of an open field test (OFT), a new object recognition test (NOR), and a Y-maze revealed that D-galactose (D-gal) -induced aging mice treated with hUCMSCs anti-miR-206 had no evidence. spontaneous action. disorder and had recovery in learning and spatial memory ability compared with the PBS-treated group. The anti-miR-206 hUCMSCs reconstructed neuronal physiological function in the hippocampal regions of the aging mice with an increase in Nissl bodies and the overexpression of Egr-1, BDNF, and PSD-95.

Conclusion

This study first reports that hUCMSCs anti-miR-206 could provide a new stem cell-based anti-aging therapeutic approach.

Introduction

Aging is a gradual decrease in the biological functions of multiple organs in the body, among which the brain is one of the most susceptible [1, 2]. The non-pathological changes increase in the process of brain aging, characterized by a cognitive decline, manifested in the decline of learning, memory and recognition [3]. Although the neural aging mechanism remains transient, the hippocampus, which performs the learning and memory functions, is highly involved [4]. The hippocampus has a huge network of intermediate neurons. The main neurons in these regions are granular cells in the DG region and pyramidal cells in CA1 and CA3 of the Ammon’s horn, which form a one-way three-Synaptic pathway. This network has been shown to affect cognition, which in turn affects memory and mood. Hippocampal neurodegeneration is associated with decreased cellular transcription level, neuronal physiological function, synaptic plasticity [6, 7], and cognitive function [8,9,10,11,12]. Brain aging can be induced by D-gal, which causes oxidative stress-mediated superoxide free radicals. Studies have shown that long-term subcutaneous injection of high doses of D-gal in rodents has resulted in cellular swelling, metabolic disorders, and accelerated aging processes [13].

The unique tendency of MSCs to multiply and differentiate has been exploited in repair and transplantation of tissues / organs such as heart, bone and lung [14,15,16]. However, transplanted MSCs have shown low differentiation efficiency and survival rate decrease in the host [17] due to paracrine substances containing many cellular regulatory factors, and overexpressed exosomal microRNAs [18]. For example, paracrine function of stem cell therapy has been enhanced through microenvironmental control in vivo, immune regulation, inhibition of apoptosis, and nutritional support [19, 20]. Among differentially expressed microRNAs, miR-206 targets a brain-derived neurotrophic factor (BNDF), an important member of the neurotrophic factor family, mostly active in gray matter regions such as the cortex, hippocampus, and basal forebrain that protect the nervous system. Zhang et al. reported that MSCs with lower miR-206 showed improved survival in the infarcted heart [21]. In addition, J. Liu et al. showed that miR-206 overexpression reversed MSCs-Exo-mediated mitigation of chondrocyte injury [22].

Here, we explained that hUCMSCs with lower miR-206 improved neuroprotection and knowledge in a D-gal-induced aging mouse model, and behavioral tests showed upstream mechanisms by the expression of Egr-1, BDNF, and PSD-95 neurotrophic factors. in the hippocampus. . Overall, this study provides new insights into stem cell anti-aging therapy.

Results

Characterization of hUCMSCs

hUCMSCs of human umbilical cord may multiply in culture plates (Fig. 1a). In vitro differentiation experiment showed that hUCMSCs were successfully differentiated into osteoblasts or adipocytes under the special induction medium (Fig. 1b, c). Flow cytometric sign identification provided confirmatory results that hUCMSCs showed positive for CD29 (99.87%), CD44 (99.31%), CD73 (94.41%), CD90 (99.17%) and CD105 (99.17%), and negative for CD34 (1.64%). ), CD45 (0.22%), CD31 (0.23%) and HLA-DR (0%), confirming the presence of human stem cells (Fig. 1d).

Spindle-shaped morphology of hUCMSCs (bar = 50 µm). b Osteogen-derived hUCMSCs (bar = 200 µm). c fat-derived hUCMSCs (bar = 50 µm). d Fluorescence activated cell sorting (FACS) analysis of hUCMSCs with positive CD44, CD90, CD105, CD29, CD73, and negative CD34, CD45, CD31, and HLA-DR (n = 6, p & lt; 0.05).

hUCMSCs improved the learning and spatial memory ability of mice

Prolonged subcutaneous injection of high-dose D-gal increases free radical medial oxidative stress in mice, causing premature brain aging, hippocampal damage, and possibly cognitive decline [13]. The effect of D-gal on cognitive behavior was analyzed by open-field activity (OFT), new object recognition test (NOR), and Y-maze) in the hippocampus during a 10-day rest period after treatment at 18 weeks. -old male C57BL / 6 J mice. The mice were randomly divided into four groups: normal, D-gal treated with PBS (D-gal + PBS), hucMSCs (Cell), and miR-206 down-regulated hucMSCs (Cell-anti-miR-206). After daily subcutaneous injection of D-gal (500 mg / kg / d) for 8 weeks, mice were injected into the lateral ventricle with untreated hucMSCs or pre-treated with Cell-anti-miR-206, separately. During the 10-day rest period, the OFT, NOR, and Y maze were completed. Mice were sacrificed on day 10 to remove the cerebral hippocampus for molecular and morphological subsequent experiments.

The OFT was performed to test the motor skills of mice. As shown in Figs. 2a, d, the results were not statistically different, indicating no obvious spontaneous activity disorder and anxiety. The NOR test was performed to observe the short-term learning and memory ability of mice. Compared with the normal group, the new object recognition index (NOI) of the D-gal group decreased and NOI increased significantly after cell transplantation (Fig. 2b). This showed that D-gal-induced aging mice decreased learning memory that recovered after cell intervention.

OFT experimentation of spontaneous mobility and anxiety of mice. b The learning and memory ability of mice tested by NOR. c The spatial memory capacity of mice tested by a Y maze. d Spontaneous movement of mice in an open field trajectory diagram. The results were expressed as the Mean ± SD, * p & lt; 0.05, versus D-gal + PBS groups, n = 6.

The Y-maze was made to observe the short-term spatial working memory of mice. The results showed that the spontaneous alternating rate of D-gal mice decreased, and after cell transplantation increased compared to the PBS control group (Fig. 2c). It is noteworthy that although there was no significant difference in OFT between the cell and the cell-versus-miRNA206 groups; difference was observed in NOR and more significant in Y-maze (Fig. 2b, c). Overall, the huMSCs improved the learning and spatial memory ability of mice.

hUCMSCs intervention through a paracrine mechanism

To track hUCMSCs, we stained hUCMSCs with DIR paint before transplanting. Immunofluorescence staining showed highly stained cells (Fig. 3a). After lateral ventricular injection transplantation, cells were monitored for 1, 5, and 10 days. The signal decreased progressively over time and there was no detectable trace of hUCMSCs in the hippocampus after 10 days (Fig. 3b), suggesting migration of hUCMSC through paracrine exosomes to stimulate treatment.

Detection of hUCMSCs-DiR and cell nucleus stained with DAPI in the lateral ventricle. b Detection of huCMSCs-DIR in the hippocampus, Bar = 50 µm, n = 6.

BDNF is negatively regulated with miR-206

MicroRNA, as a post-transcriptional regulatory element, is involved in the post-transcriptional regulation of target gene expression [23]. The target Scan Human Database analysis showed that miR-206 targeted BDNF, suggesting that miR-206 may be associated with neuroprotection. To verify whether miR-206 regulates BDNF, we transfected hUCMSCs with miR-206 inhibitor and NC. RT-qPCR showed no significant difference in BDNF-mRNA expression (Fig. 4b). However, western blot showed that the protein expression level of BDNF was significantly increased (Fig. 4C, D). The results showed that the low expression of miR-206 promoted the high expression of BDNF in hUCMSCs, and BDNF was negatively regulated with miR-206, confirming that miR-206 could directly bind to the BDNF.

After the transfection of hUCMSCs with a miR-206 inhibitor, the expression level of miR-206 mRNA was detected by RT-qPCR. b After transfection with a miR-206 inhibitor, the expression level of BDNF-mRNA was detected by RT-qPCR. c After transfection of hUCMSCs with an miR-206 inhibitor, the expression level of BDNF protein was detected by western blot. d Quantitative analysis of BDNF protein levels. The results were expressed as the Mean ± SD, * p & lt; 0.05, against normal groups, n = 6.

hUCMSCs with down-regulated miR-206 achieve neuroprotective effects by targeting BDNF in vivo

The above results confirmed that BDNF is negatively regulated with miR-206. In addition, to study the effect of low miR-206 expression on brain aging, we performed immunofluorescence staining of anti-BDNF antibody and western blot in the hippocampus of mice. As shown in Figs. 5, the results showed that the low expression of miR-206 in hUCMSCs promoted the expression of BDNF in the damaged hippocampus. These results revealed that miR-206 subregulated hUCMSCs exhibited neuroprotection.

a BDNF protein levels were determined by western blotting. b Quantitative analysis of BDNF protein levels using ImageJ. c Representative micrographs for BDNF immunofluorescence staining of hippocampus of mice in four groups (n = 5 per group). d Quantification of BDNF-positive cells in four groups. The results were expressed as the Mean ± SD, * p & lt; 0.05, n.s., insignificant, bar = 50 µm, n = 6.

hUCMSCs with downregulated miR-206 promoted the physiological function of hippocampal neurons

Ten days after transplantation of hUCMSCs, mouse brain coronary sections were HE stained, which showed no mass cell shrinkage in the hippocampus of each group, and the cell structure was complete and orderly (Fig. 6a). The results showed that there was no large number of neuronal apoptosis that was consistent with non-pathological aging. However, more differences were observed in the Nissl staining. Nissl staining was performed to observe the changes in hippocampal CA1 and DG regions, involved in memory formation. The Nissl body staining was deeper, and neuron morphology was regular. In the D-gal + PBS group, the number of Nissl bodies decreased, the stain was superficial, and the morphology of neuronal cells was irregular. After transplantation of hUCMSCs with lower miR-206, the number of Nissl bodies increased, and the spot was deeper than that of the D-gal + Cell group (Fig. 6b, c). The increase in the Nissl bodies can be attributed to the reconstruction of protein synthesis in neurons and their normal physiological functioning, suggesting that hUCMSCs with lower miR-206 promoted the physiological function of hippocampal neurons.

a HE staining of the hippocampus of D-gal-induced aging mice. b Nissl staining of the hippocampal CA1 and DG regions of D-gal-induced aging mice. Bar = 200 µm. Enlarged images after the black frame. Bar = 50 µm. c Quantitative analysis of the number of neurons using ImageJ. The results are expressed as the Mean ± SD, * p & lt; 0.05, verses D-gal + PBS groups, #p & lt; 0.05, verses D-gal + Cell Groups (n = 6).

HUCMSCs with down-regulated miR-206 promoted the expression of learning and memory-related proteins

Postsynaptic density-95 (PSD-95) is the main component of the excitatory postsynaptic membrane is dense and plays an important role in synaptic signaling, learning, and memory. Studies have shown that PSD-95 can promote synaptic maturation and improve synaptic plasticity [24]. Therefore, a PSD-95 protein expression level was detected, which was significantly upregulated in tissue samples from an anti-miR-206 group (Fig. 7a, b). We then detected Egr-1, an immediate early gene and one of the important molecules involved in the formation and consolidation of long-term memory by synaptic plasticity of neurons, [25] and its decline impairs spatial learning and memory [26]. Interestingly, our immunohistochemical staining results showed Egr-1 expression upregulated in the dental gyrus in the anti-miR-206 group (Fig. 7c, d).

a The protein levels of PSD-95 were determined by western blotting. b Quantitative analysis of the protein levels of PSD-95 and using ImageJ. c Egr-1 in the hippocampus was detected by immunohistochemistry, Bar = 50 µm. d Quantitative analysis of the Egr-1 protein levels of ImageJ. The results were expressed as the Mean ± SD, * p & lt; 0.05, against D-gal + PBS groups (n = 6).

Discussion

Aging is an inevitable process. With the deterioration of the aging population, minimizing or mitigating aging is essential to possibly slow down age and age-related diseases. Stem cell therapy promises anti-aging due to its rapid and effective therapeutic effect, precision delivery and safety.

MicroRNAs are a class of endogenous small non-coding RNAs responsible for post-transcriptional regulation of gene expression. Recent studies have shown that miR-206 targets BDNF, which regulates anxiety-related behaviors and neuropathic pain induced by chronic contraction injury [28, 29]. The miR-206 knockdown exosomes therapy has been more effective in reducing neuronal death and improving neurobehavioral scores during early brain injury (EBI) [30]. We hypothesize that microRNAs play a key role in communication between MSCs and parenchymal cells. In this study, we focused on the relationship between miR-206 abundance in paracrine hUCMSCs and neuroprotection. Decreasing miR-206 expression from hUCMSCs, cells were transplanted into D-gal-induced mouse aging. Our results showed that the BDNF of hUCMSCs was increased after the subregulation of miR-206. Because microRNA is responsible for the post-transcriptional regulation of gene expression, we detected no difference in the mRNA level of BDNF, but the protein expression was upregulated. This is why we know that miR-206 targets BDNF and is negatively regulated. In addition, we detected that after subregulation of miR-206, BDNF expression in the mouse hippocampus was significantly increased. And Nissl staining showed that the state of neurons in the DG and CA1 regions of the mouse hippocampus was restored to varying degrees after stem cell intervention. Nissl staining may reflect the reproductive and synthetic capacity of nerve cells, indicating that the physiological state of nerve cells in the hippocampus has been better improved. Suggesting that miR-206 may be a key regulator of intervention in D-gal-induced aging.

Recently, proteins containing the PDZ motif have been proposed as a molecular scaffold of receptors and synaptic cytoskeletal elements. The prototype PDZ protein, PSD-95 / SAP-90, is a membrane-related guanylate kinase (MAGUK) concentrated at glutamatergic synapse. PSD-95 is the main component of excitatory postsynapses, which plays an important role in the formation and maturation of synapses and synaptic signaling, learning and memory functions. Meanwhile, D-gal as a common model of aging research, induced aging has been widely used in pharmacological studies. Our results also showed that after D-gal-induced aging, the protein expression level of PSD-95 in the hippocampal tissues of mice decreased significantly, which was recovered after stem cell transplantation. Egr-1 is also involved in learning and memory and synaptic plasticity, memory formation, and consolidation. We detected that Egr-1 expression was downregulated after D-gal-induced aging, then up-regulated after treated hUCMSCs with lower miR-206. Therefore, we believe that hUCMSCs with down-regulated miR-206 have a better neuroprotective effect targeting BDNF in D-gal-induced aging mice.

Upstream CREB regulates Egr-1. When p-CREB, the activated form of CREB, is continuously enhanced in the hippocampus, the expression of downstream Egr-1 will be promoted, thus influencing learning and memory ability [31]. CREB is a key transcription factor in the activation signal of TrkB, which is the binding receptor of BDNF. Overregulation of BDNF / TrkB / CREB this classical neuroprotective pathway signaling prevents neuronal apoptosis in neurodegenerative diseases [32]. Therefore, we consider that miR-206 can enhance aging knowledge by the BDNF / TrkB / CREB signaling pathway targeting BDNF in D-gal-induced aging mice.

In our study, the tracking of stem cells in the brain showed no cells in the lateral ventricle after 10 days. We also tested three short-term memory behavioral experiments related to episodic and spatial learning. However, it is also necessary to explore long-term learning and memory ability of cognitive ability, which can be explored by other related behavioral tests in future studies. In addition, we have shown that miR-206 targets BDNF to enhance cognitive decline by investigating key intermediate molecules associated with the signaling pathway, suggesting further studies on the regulatory mechanism of its internal signaling pathway.

Conclusion

Our results first report that miR-206 targets BDNF and negatively regulates BDNF expression. hUCMSCs with down-regulated miR-206 can enhance the physiological function of nerve cells in the hippocampus and promote the expression of PSD-95 and Egr-1 in relation to learning and memory. The data propose that the neuroprotective role of hUCMSCs with subregulated miR-206 is one strategy to improve the cognitive decline of aging.

Methods

Animals

In this experiment, a total of 60 C57BL / 6 J mice (Hunan Slack Jingda Experimental Animal Co., Ltd, Changsha, China) weighing 25–30 g at 9 weeks in this experiment were used. The animal model was established for eight weeks, to ensure the physical health of the animals, we selected a 9-week-old in young mice. Mice were bred in the Department of Laboratory Animal Science (Central South University, Changsha, Hunan, China) and housed individually in separate cages (SPF + IVC) and had free access to food and water and a 12 h light-dark cycle for seven days. . before the experiment. All animal experiments were in accordance with the “Guide for the Care and Use of Laboratory Animals, 8th ed., 2010” (National Institutes of Health, Bethesda, MD) and were approved by the Institutional Animal Care and Use Committee of Central South University (Changsha, China; License Number: 2020sydw0913).

hUCMSCs Culture

hUCMSCs were from the company HYS Cell Gene Engineering (Changsha, Hunan, China). The hUCMSCs were cultured using F12-DMEM (DMEM, Gibco, Grand Island, NY, USA), which contained 10% FBS (FBS, Gibco, Grand Island, NY, USA) for the primary culture (37 ° C, 5% CO2). ). ), and then passed after reaching 80-90% confluence. P5 hUCMSCs were used in all experiments and counted by hemocytometer. Cell phenotypes were analyzed by flow cytometry. The P5 hUCMSCs were characterized using CD34, CD44, CD45, CD29, CD31, CD73, CD90, CD105, and HLA-DR antibodies (Biolegend, Way San Diego, CA, USA). When hUCMSCs reached an 80–90% confluence, hUCMSCs were seeded in a six-well plate with approximately 5 × 106 cells per well. After 24 h, Mesenchymal Stem Cell Osteogenic Differentiation Kit (5011-024-K, Trevigen, MD, USA) was used to induce osteogenic differentiation of hUCMSCs. Calcium nodules formed after osteogenic induction were stained with Alizarin Red. The hUCMSCs were induced by mesenchymal stem cell adipogenic differentiation kit (5010–024-K, Trevigen, MD, USA) to stimulate adipogenic differentiation and stained with red O oil.

Cell transfection

The miR-206 inhibitors and negative control (NC) (GenePharma, Shanghai, China) were transfected into hUCMSCs at a final concentration of 50 nmol / L. After recovery, P5 hUCMSCs were cultured in the 6-well plates until their density reached 80–90%. The miR-206 or NC inhibitors and Lipofectamine 3000 Transfection Reagent (L3000001, Thermo Fisher Scientific, MA, USA) were diluted with Opti-MEM (31985062, Gibco, Grand Island, NY, USA) and cells were cultured with Opti-MEM . After 6 hours of transfection, the hUCMSCs were transferred to DMEM medium supplemented with 10% FBS for 48 h before post-transfection performance measurement by RT-PCR or Western blot.

Animal model experimentation and cell transplantation

D-gal (500 mg / kg / d) was subcutaneously injected into the back of each C57 mouse once a day for 8 weeks. The normal group injected the same volume of salt. Mice were anesthetized by intraperitoneal injection of pentobarbital (ZaoZhuang, Shandong, China) at approximately 10 mg / kg. According to the mouse brain brain stereotypical map, the anterior fontanelle is 2.0 mm backward, the sagittal suture (middle, ML) was 1.5 mm left and right, and the insertion depth (dorsoventral, DV) was 2.5 mm. 10 µl of PBS or MSCs cell suspension was slowly injected into the lateral ventricle with microliter injectors and stopped for 2 min after each injection of 2 µl. After completion, the syringe was stopped for 5 min.

The mice were randomly assigned to 4 groups: normal (n = 15) control group, D-gal + PBS (n = 15), D-gal + Cell (n = 15), D-gal + Cell-anti-miR-206 (n = 15).

After the operation, 10 days after injection of MSCs, all mice were sacrificed and the hippocampus and entire brain were removed.

Tracers after cell transplantation

P5 hUCMSCs were collected and covered in the 6-well plates or circular microscope cover. When it reaches 80–90%, cells were stained with DIR Iodine (DiIC18) (Maokangbio, Shanghai, China) at 37 ° C for 20 min according to the manufacturing protocol (7). The cells were revived, washed and collected using PBS. Then, the glass slide was DAPI stained for nuclear observation under fluorescence microscopy. The collected cells were injected into the lateral ventricle of the mouse brain, and the post-OCT embedded brain was cut into serial 15-μm-thick coronal sections and DAPI stained for fluorescence microscopy.

Behavioral experiment

The open-field test (OFT): a 40 × 40 × 40 cm cube box was used. The bottom of the box was divided into 25 squares of equal size, of which nine squares in the center were defined as the central region and the rest as the peripheral region. Each mouse in turn was placed in the central area and left to explore for 10 min.

The new object recognition test (NOR): it is divided into three periods: adaptation, training and testing. The first day was the adaptation period. The mice were positioned in the middle of the open field in turn and explored freely for 5 min. Twenty-four hours later, entering the training period, two identical objects were placed in the open field with an interval of 20 cm or 10 cm between the two objects and the side wall. Each mouse was placed from the center line attached to the wall on the side of the non-object area of ​​the open field and was left to explore for 5 min. After a break of 1 h, entering the test period, one of the objects in the open field was removed and replaced with a new object with the same material but different colors and sizes. Then, the mice were placed in the same position in the box in turn and left to explore freely for 5 min. Then calculate according to the formula: new object recognition index (NOI) = explore new object time / (exploring new object time + exploring old object time) × 100% [34, 35].

The Y-maze: three identical 30 × 5 × 15 cm gray closed arms with a 120 ° angle between the three arms (new arm, different arm and initial arm), and a triangle middle joint. There are two periods: the training period and the testing period. In the training period, the new arm was closed, and the mice were placed in the initial arm turning to the middle articular part and left to explore for 10 minutes. After a 1 h rest, open the new arm, and the mice were tested for free exploration for 5 minutes in a row. When the mouse limbs fully entered the arm, it was assessed as an arm entry. When the third bracing was different from the first two, it was considered the correct bracing. Then calculate according to the formula: spontaneous alternation percentage% = (Number of Alternations / [Total number of arm entries-2]) × 100% [36].

In all three behavioral experiments, the mice were placed in a behavioral room an hour earlier to acclimatize to their environment. Prior to the experiment, even after each mouse completed the test, the devices were cleaned and wiped with 70% alcohol and kept dry, eliminating the interference of biases and olfactory signals. Each mouse completed the test and was placed in a separate cage to avoid interaction with the untested mice.

The hippocampal-involved behavioral experiments were completed in the Department of Laboratory Animal Science (Central South University, Changsha, China). An infrared camera was installed on top of the site and connected to a computer to track the mouse. The experimental data were collected and analyzed using the Smart3.0 software system.

RNA isolation and RT-qPCR

Total RNA was extracted from cells using Trizol (Thermo, USA) according to the manufacturing protocol. RNA was inversely transcribed into cDNA using a cDNA synthesis kit (Cwbio, Beijing, China). Subsequently, the cDNA product was used as a template for PCR using SYBR green mixture (Cwbio, Beijing, China). The tests were performed in triplicate, and data were analyzed using the 2-ΔΔCt method. U6 and Actin were used as internal controls. The U6, Actin, and BDNF introductions were designed as shown in the following table.

Western blot analysis

Total protein was extracted from cultured cells or tissue using RIPA (CWBIO, Shanghai, China) and a protease inhibitor (CWBIO, Shanghai, China). Protein concentration was determined by BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of proteins were electrophoresed in 10% SDS-PAGE and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA), and sealed with 5% skim milk for 2 h at room temperature and incubated with anti -BDNF antibody (1: 1000, Abcam, USA) or anti-PSD95 antibody (1: 1000, Proteintech, Wuhan China) overnight at 4 ° C. After washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. Protein bands were detected by enhanced chemiluminescent gear (Thermo Scientific, Rockford, IL, USA). Image J software (National Institutes of Health, USA) was used to quantify the band densities.

Morphology staining

After mouse intracranial infusion with PBS followed by 4% paraformaldehyde (PFA), the whole brain was fixed with PFA solution (Sinopharm Chemical Reagent Co. Ltd, Shanghai, China) t.e. 4% PFA in 0.1 M PBS (Solarbio, Beijing, China). ) at 4 ° C for 24 h. Subsequently, it was dehydrated with 15% sucrose solution at 4 ° C for 24 h and replaced with 30% sucrose solution for 24 h the next day. Brains were then cut into serial 15-μm-thick crown sections (Leica, Wetzlar, Germany) and mounted on glass slides. Tissue sections were stained with Hematoxylin Eosin (HE) (Beyotime, Shanghai, China) or with a Nissl body staining Kit (Meilun, Dalian, China) and ultimately studied using light microscopy. Images were captured with a Nikon confocal microscope (Nikon Instruments, Inc., Japan).

Immunohistochemistry and immunofluorescence

For immunohistochemical staining, tissue sections were incubated in 0.3% hydrogen peroxide / PBST (Solarbio, Beijing, China) for 30 min. The sections were then sealed with normal horse serum (Beyotime, Shanghai, China) / PBST (1: 200) for 2 h. Subsequently, they were incubated with anti-Egr1 antibody (1: 100, PTG, USA) overnight at 4 ° C, washed with PBS, and then incubated with HRP-labeled Goat Anti-Rabbit IgG antibody (1: 500). , ZSGB-BIO, Beijing China) at 37 ° C for 2 h. Finally, the slices were washed with PBS and sealed with the lid with neutral resin (Sinopharm Chemical Reagent Co. Ltd, Shanghai, China).

For immunofluorescence staining, the sections were blocked in normal azense serum / PBST (1: 200) for 2 h. Subsequently, they were incubated with anti-BDNF antibody (1: 1000, Abcam, USA) at 37 ° C for 1 h and washed with PBS. Subsequently, the sections were incubated with secondary antibodies (Alexa Fluor Cy5, 1: 800; APExBIO, USA). Cell cores were stained with DAPI (1: 1000, DAPI, Sigma Aldrich, MO, USA) and images were captured with a Nikon confocal microscope (Nikon Instruments, Inc., Japan). Finally, the slices were sealed with the lid with glycerin (Solarbio, Beijing, China).

Statistical Analysis

The data are expressed as Mean ± SD. The Prism Graph Pad software (version 7.0, La Jolla, CA) was used to perform the statistical analysis. One-way ANOVA and the Turkey test (analysis of variance) were used to check the differences between groups. p & lt; 0.05 was considered statistically significant.

Data availability

All relevant data and materials are available from the authors upon acceptable request.

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