Chemicals

The IronQ complex was synthesized according to our previous research work [25]. Shortly, 0.0050 mol of quercetin hydrate (Sigma, USA) was supplemented to 500 mL HPLC-methanol (Sigma, USA) with continued stir until complete dissolution of quercetin hydrate, showing the yellow solution. The pH of the solution was then slowly adjusted to 12 based on the addition of NaOH solution to obtain a deprotonated form of quercetin. 0.0025 mol Iron (III) chloride (Sigma, USA) in 500 mL ultrapure water (up water) was then blended with the above solution until the solution color turned to dark yellow, which was followed by incubation with a continuous stirring for 2 h at 60 °C. And then the final solution was purified and evaporated to dryness. The dark powder product was collected and stored it away from light at room temperature (RT).

Animals

One hundred wild-type C57BL/6 male mice, 8–9 weeks old, weighed between 22 and 25 g, were purchased from Chongqing Tengxin Biotechnology Co., Ltd (Chongqing, China). They were housed according to 5 mice per cage for acclimatization under the same animal care facility for three days. The animals were monitored by the research unit once daily. If the mice got the following symptoms, including lethargy, shortness of breath, skin discoloration or irregularity, enlargement of lymph nodes, and solid visible tumors under the subcutis, mice were euthanized directly. These morbid mice were documented as “adverse events”. The procedure for using the animals followed the Guidance Suggestions for the Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of China. Besides, it was approved (Approval No. 20211122–040) by the Animal Ethical Committee of the Animal Center of Southwest Medical University (Luzhou, Sichuan). The optimized experimental procedures minimized the number of experimental animals and alleviated their suffering. The mice (90 mice) were blindly and randomly divided into six groups (n = 15 mice per group): the sham operation group (Sham), the model group (Model), the quercetin gavage group (Quercetin), the MSCs transplantation group (MSCs), MSCs transplantation combined with IronQ group (MSCs + IronQ), and MSCs + IronQ control group, and then assigned to the corresponding experimental procedures (Fig. 1A).

Fig. 1

Conceptual diagram of the experimental protocols. A Brief schedule of experimental mouse grouping and treatment strategies and experimental procedures. B Schematic diagram of ICH mice model transplanted with MSCs and MSCs + IronQ

Intracerebral hemorrhage mice model

Mice were anesthetized intraperitoneally with 40 mg/kg of 1% pentobarbital sodium and fixed in a stereotaxic apparatus (supine position) with the anterior and posterior bregma kept at the same level. Incise the scalp sagittally about 1 cm and expose the anterior fontanelle with 30% H2O2. The mixture containing 1μL 0.15 U/μL collagenase I (C8140, Solarbio, China) and supplementary 0.9% normal saline was extracted with a 1μL microsyringe. Made an opening (1 mm) in the right skull, 2.5 mm lateral and 0.2 mm anterior. The needle was attached to the stereotaxic device, inserted it into the caudate nucleus (3 mm deep from the hole), and injected the mixture slowly (Fig. 1B). After injection, the drilled hole was closed with bone wax, and the scalp was sutured. The ICH mice were randomly and blindly separated into four ICH experimental groups. Among them, the mice in the quercetin group for intragastric administration with quercetin (50 mg/kg/day) after 24 h when successfully modeled were performed, referring to the previous study [23, 24]. The specific conceptual illustrations of the experimental protocols for the MSCs and MSCs + IronQ are given in Fig. 1. The mice in the sham operation group performed the same operation without the injection of the mixture, and the mice in the MSCs + IronQ control group were given the injection of the complex of MSCs with IronQ. After successfully modeled and treated with MSCs transplantation combined with IronQ, the sham operation, model, and MSCs + IronQ groups were visualized using 3.0 T MRI. Mice in all groups were euthanized by an overdose of intraperitoneal injection of 40 mg/kg of 1% pentobarbital sodium for harvesting the brain samples in the following experiments.

Neurological Function Assessment by modified Neurologic Severity Score (mNSS) Test

The mNSS was operated as previously reported [34]. Neurologic deficits were performed by evaluating abnormal movements, including motor, sensory, and reflex deficits, at 24 h after ICH induction using the mNSS system (18-point neurological deficit scale, normal score, 0; maximal score, 18), which is extensively administrated to evaluate the degree of ICH-induced nerve damage. The higher score represents the more severe neurological damage. After 24 h of the ICH model constructed by the right caudate nucleus injection of collagenase, mice were evaluated and scored blindly. The criteria are 13–18 points, representing severe damage; 7–12 points, representing moderate damage; 1–6 points, representing slight damage. When assessing neurological function, one point is awarded for the inability to complete the test or lack of relevant reflexes. That is to say, the more elevated the score, the more severe the injury. This study used ICH mice with moderate damage for the subsequent experiments.

Extraction, Passage, and Identification of MSCs

Obtaining the MSCs is followed by the methodology as previously described [35]. Briefly, the femurs and tibias were isolated from the male healthy C57BL/6 mice (total = 10 mice) on the asepsis condition after intraperitoneal injection anesthesia. Then, the muscles were carefully removed on the bone with a scissor. Their medullary cavities were several washed by Dulbecco’s modified eagle medium (DMEM, Gibco, USA) to acquire a uniform suspension with mixed cells. Centrifuged the cells and removed the red blood cells with red blood cell lysate (R1010, Solarbio, China). The cells were collected and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin (C0222, Beyotime Biotechnology, China) at 37 °C and 5% CO2 in a humidified environment. Cells from the third passage were harvested, and 1 × 106 single cells were incubated with the primary antibodies against CD90, CD29, and CD45 (Bioscience, USA) for 30 min at 37 °C. Flow cytometry accomplished cell identification (BD FACSCanto II, BD Biosciences, USA).

MTT assay

The effect of IronQ on cell proliferation was demonstrated by utilizing the MTT assay. Shortly, MSCs were seeded at a density of 2 × 103 cells/well in a 96-well plate and cultivated in DMEM (Gibco, USA) with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (C0222, Beyotime Biotechnology, China). After 24 h of incubation, MSCs were treated with IronQ at different concentrations (0, 200, 600, 1000, and 2000 μg/mL) and incubated for 24 h. The cells were washed twice with PBS and added 200 μL DMEM + 20 μL 5 mg/mL MTT (Amresco, USA) solution to continue incubation for 4 h at 37 °C. Then, the medium was discarded and replaced with 200 μL DMSO to dissolve the MTT formazan crystals. The absorbance at 490 nm of the solution was measured using the SYNERGY2 microplate reader (BioTek, USA).

IronQ labeling of MSCs and determination of labeling efficiency by Prussian blue

MSCs (1 × 106 cells, 5 mL) were seeded in a 6-well plate with the DMEM (Gibco, USA) accompanied by 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (C0222, Beyotime Biotechnology, China). After MSCs adherence, IronQ solution (1000 μg/mL, IronQ dissolved with sterile up water) was added to the six-well plate at diverse concentrations (0, 50, 100, 200, and 400 μg/mL). Cells were then continued to be cultivated for 2 days at 37 °C with 5% CO2 in a humidified incubator. After the indicated time, the cells were washed 3 times with PBS to remove the unbound IronQ. Prussian blue stain confirmed the intracellular uptake of IronQ by MSCs. In brief, the cells were fixed with 4% paraformaldehyde for 20 min in a humidified incubator at 37 °C. Then, the fixation reagent was replaced with 1 mL of pearl blue reaction solution (G1422, Solarbio, China) in each well and re-incubated for 30 min. After that, cell morphology and the blue-stained positive cells were visualized and photographed using a Nikon-inverted fluorescence microscope (ECLIPSE Ti, Nikon, Japan) with a Nikon DS-Ri2 camera, and images were captured using Nikon software NIS-Elements F.4.30.01, at magnification 100× based on an objective lens of 10× and eyepiece of 10×. The images were processed using image processing software such as Adobe illustrator 2021.

Transplantation of MSCs and MSCs combined with IronQ

Relative experiments were performed by MSCs and the combination of MSCs with IronQ at passages 3–6. The cells were harvested and adjusted the cell concentration to 5 × 107 cells/mL. Extracted cell suspension (20 μL) by a microsyringe was injected into the point (position: the right of bregma: 3 mm; front of bregma: 0.2 mm; depth: 3 mm) of mouse brains for MSCs, MSCs + IronQ, and MSCs + IronQ control groups, respectively, at 2 μL/minute 24 h after successfully modeling. Then, the pinhole was sealed with bone wax and sutured and sterilized the skin.

MRI of MSCIronQ transplant in ICH Mice

Mice were anesthetized as the above modeling method throughout the MRI examination. MRI was performed using a 3-Tesla MRI scanner (Siemens, Germany). T1 fast spin-echo (repetition time/echo time = 500/11 ms) was used to acquire a field of view of 16 × 16 mm2, matrix of 256 × 256, and 0.75-mm thick for coronal, axial, and sagittal plane. The single image was preserved as 1019 × 602-pixel picture for T1 lesion evaluation.

Hematoxylin–eosin Staining and Nissl staining

Perfusion-fixed brain tissue was further fixed by soaking overnight in 4% formaldehyde, as previously described [36]. The tissue was then dehydrated through a series of graded alcohols followed by xylene for 30 min each. The brain tissue was then embedded in paraffin and cut into 4-µm-thick sections frontally using a microtome (RM 2245, Leica, Germany). Tissue sections were dewaxed and rehydrated for staining. Hematoxylin–eosin (HE) staining and Nissl staining were performed, referring to standard procedures and the manufacturer’s instructions. Brain tissue slides were observed under a Leica optical microscope (DM500, Leica, Japan) equipped with a Leica ICC50W camera, and images were magnified 200 × based on an objective lens of 20 × and eyepiece of 10 × using Leica Software LAS X. The images were processed using image processing software such as Adobe illustrator 2021.

BWC Examination

BWC was measured as previously reported [37]. In short, brain samples were harvested from anesthetized animals and immediately weighed to determine the wet weight via a precise electronic scale. After drying at 100 ℃ in the thermostat for 24 h, brain samples were reweighed to get their dry weight. The formula of BWC is calculated as [(wet weight – dry weight)/wet weight] × 100%.

Immunofluorescence

Immunofluorescence staining was carried out as previously reported [37]. Under deep anesthesia of the mice, the heart was perfused with 0.9% normal saline and 4% paraformaldehyde with 0.01 M phosphate-buffered saline (PBS, pH7.4) successively. Brain samples were collected, fixed in 4% paraformaldehyde for 24 h at 4 °C, and dehydrated in the 30% sucrose solution (prepared in PBS) for an additional 24 h until the samples sank to the bottom of the sucrose solution. The samples in optimal cutting temperature compound (OCT) were sectioned coronally for 4 μm at the level of basal ganglion by using the freezing microtome (CM1950, Leica, Germany). After washing three times with PBS, tissue sections were blocked for 1 h at RT with 5% BSA and incubated with primary antibodies, including mouse anti-IL-6 (sc32296, Santa Cruz, USA, dilution 1:100), mouse anti-TNF-α (sc52746, Santa Cruz, USA, dilution 1:100), mouse anti‐Mincle (sc390806, Santa Cruz, USA, dilution 1:100), rat anti‐F4/80 (sc52664, Santa Cruz, USA, dilution 1:100), rabbit anti-GFAP (16825-1-AP, Proteintech, USA, dilution 1:100), rabbit anti-MBP (10458-1-AP, Proteintech, USA, dilution 1:100), and rabbit anti-NeuN (12943, CST, USA, dilution 1:100) overnight at 4 °C. The following day, tissue sections were washed three times with PBS for 5 min each and then treated with Alexa Fluor® 555 conjugated anti‐mouse secondary antibody (A21424, Life Technologies, USA, dilution 1:500), Alexa Fluor™ 555 conjugated anti‐rabbit second antibody (A21428, Invitrogen, USA, dilution 1:1000), Alexa Fluor™ 488 conjugated anti‐mouse secondary antibody (A11001, Invitrogen, USA, dilution 1:500), and Alexa Fluor™ 488 conjugated anti‐rabbit secondary antibody (A11034, Invitrogen, USA, dilution 1:500) incubation for 1 h at RT. Images were recorded by a Leica Fluorescence orthotopic microscope (DM4B, Leica, Germany) equipped with a Leica DMC6200 camera using the software LAS X, at a magnification of 200 × based on an objective lens of 20 × and an eyepiece of 10 × . The images were processed using image processing software such as Adobe illustrator 2021.

Western Blot

Mice were decapitated under deep anesthesia, and brain samples were quickly collected and separated into ipsilateral brain hemispheres. Proteins were uniformly extracted with RIPA lysis buffer (Beyotime, China) and incubated at 13,000 rpm at 4 °C, and the supernatant was collected. Then, the total protein concentration in each sample group was determined by bicinchoninic acid (BCA) protein assay (Beyotime, China). Equal amounts of protein samples (50 μg) were blotted to nitrocellulose (NC) membranes by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Membranes were then incubated with the following primary antibodies against IL-6 (sc32296, Santa Cruz, USA, dilution 1:1000), TNF-α (sc52746, Santa Cruz, USA, dilution 1:1000), NeuN (12943, CST, USA, dilution 1:1000), MBP (10458-1-AP, Proteintech, USA, dilution 1:1000), Mincle (sc390806, Santa Cruz, USA, dilution 1:1000), syk (13198, CST, USA, dilution 1:1000) and p-syk (2710, CST, USA, dilution 1:1000), NFκB-p65 (sc8008, Santa Cruz, USA, dilution 1:1000), p-NFκB-p65 (3033, CST, USA, dilution 1:1000), and GAPDH (Abcam, dilution 1:10,000) overnight at 4 °C. After incubated with Alexa Fluor™ 790 conjugated anti‐mouse secondary antibody (A11359, Invitrogen, USA, dilution 1:3000), Alexa Fluor™ 680 conjugated anti‐mouse secondary antibody (A21109, Invitrogen, USA, dilution 1:3000), and Alexa Fluor™ 680 conjugated anti-rat secondary antibody (A21096, Invitrogen, USA, dilution 1:3000) for 2 h at RT, the blots were exposed under a Far Infrared Laser Imaging System (Amersham Typhoon, USA). Protein content was analyzed by the corresponding amount of GAPDH using Image J software.

Real‐Time Polymerase Chain Reaction (qPCR) Analyses

Total RNA was extracted from mouse brain tissue using TRIzol reagent (11596026, Invitrogen, USA) according to the manufacturer’s protocol. One μg of RNA was removed from each sample and reverse-transcribed into complementary DNA for qPCR using HiScript III RT SuperMix (+ gDNA wiper) (No. R323-01, Vazyme, China). A fluorescent dye ChamQ Universal SYBR qPCR Master Mix (No. Q711-02/03, Vazyme, China) was used for the reaction in Mastercycler ep Realplex2 real‐time PCR system (Eppendorf, Germany). Specific primers for qPCR were purchased from Sangon Biotech (Shanghai) Co., Ltd, as shown in Table 1.

Table 1 List of primers sequenceBV2 cell culture and experiments

Murine BV2 microglial cells were purchased from China Infrastructure of Cell Line Resource (Beijing, China) and cultured in DMEM (glucose containing 4.5 g, Gibco, Invitrogen) spiked with 10% FBS (Gibco, Invitrogen) and 1% penicillin/streptomycin (C0222, Beyotime Biotechnology, China) in a 5% CO2 humidified incubator at 37 °C. The cells were incubated with LPS (200 ng/mL) and treated with quercetin (Q), the conditioned medium of MSCs (M), and the conditioned medium of MSCs + IronQ (M + Q) for 6 and 24 h to check mRNA and protein expression levels, respectively. Then, the trehalose-6,6-dibehenate (TDB) (66758-35-8, Invivogen, USA), a Mincle agonist, was utilized to activate Mincle including its mRNA and protein expression. According to the manufacturer’s illustration, TDB was dissolved and made the concentration at 1 mg/mL (DMSO/PBS:1/9) for the store. BV2 cells were incubated with LPS (200 ng/mL) and TDB (70 μg/mL) in the absence of M + Q for 6 and 24 h. Then, BV2 cells were collected for the following qPCR and western blot experiments.

Statistical analysis

Parametric data were analyzed by GraphPad Prism 8 software and presented as the mean ± standard error of the mean (SEM). Statistical differences among multiple groups were analyzed by one-way analysis of variance (ANOVA), and P < 0.05 was considered statistically significant.