Mesenchymal stem cell-derived exosome miR-542-3p

RESEARCH Open Access

Mesenchymal stem cell-derived exosomemiR-542-3p suppresses inflammation andprevents cerebral infarctionGuofeng Cai1*, Guoliang Cai2,3, Haichun Zhou1†, Zhe Zhuang1, Kai Liu1, Siying Pei1, Yanan Wang1, Hong Wang1,Xin Wang1, Shengnan Xu4, Cheng Cui4, Manchao Sun4, Sihui Guo4, Kunping Jia1, Xiuzhen Wang1 andDianquan Zhang5†

Abstract

Background: Cerebral infarction ranks as the second leading cause of disability and death globally, and inflammatoryresponse of glial cells is the main cause of brain damage during cerebral infarction.

Methods: Studies have shown that mesenchymal stem cells (MSCs) can secrete exosomes and contribute to cerebraldisease. Here, we would explore the function of MSC-derived exosome in cerebral infarction.

Results: Microarray indicated a decrease of miR-542-3p and an increase of Toll-Like Receptor 4 (TLR4) in middlecerebral artery occlusion (MCAO) mice comparing with sham mice. And luciferase and RIP analysis indicated a bindingof miR-542-3p and TLR4. Then, we injected AAV9-miR-542-3p into paracele of sham or MCAO mice. Functional analysisshowed that AAV9-miR-542-3p inhibited infarction area and the number of degenerating neurons and suppressedinflammatory factors’ expression and inflammatory cell infiltration. As well, transfection of miR-542-3p mimics intoHA1800 cells underwent oxygen and glucose deprivation (OGD). Similarly, overexpression of miR-542-3p alleviatedOGD induced cell apoptosis, ROS, and activation of inflammation response. Moreover, miR-542-3p could be packagedinto MSCs and secreted into HA1800 cells. The extractive exosome-miR-21-3p treatment relieved MCAO- or OGD-induced cerebral injury and inflammation through targeting TLR4.

Conclusion: These results confirmed that MSC-derived exosome miR-542-3p prevented ischemia-induced glial cellinflammatory response via inhibiting TLR4. These results suggest possible therapeutic strategies for using exosomedelivery of miR-542-3p to cure cerebral ischemic injury.

Keywords: Cerebral infarction, Inflammation, Exosome, MicroRNA, MSC

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* Correspondence: [email protected]†Dianquan Zhang and Haichun Zhou contributed equally to this studyas co-first authors.1Hanan Branch of Second Affiliated Hospital of Heilongjiang University ofTraditional Chinese Medicine, Harbin 150001, ChinaFull list of author information is available at the end of the article

Cai et al. Stem Cell Research & Therapy (2021) 12:2 https://doi.org/10.1186/s13287-020-02030-w

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BackgroundCerebrovascular disease is a kind of disease with acuteneurological disease and common system, which has thecharacteristics of high fatality rate, disability rate, and re-currence rate [1]. According to the data released by theWorld Health Organization, stroke has become the sec-ond leading cause of death worldwide in the past 10years from 2000 to 2012 [2]. Among them, ischemiccerebral infarction is the most common type of stroke,which occurs after cervical or cerebral blood flow isblocked [3]. Due to the active metabolism of the brain,more oxygen is needed. Once the blood flow is blockedand the perfusion cannot be restored in a short periodof time, the brain cells will die due to hypoxia, which inturn induces brain dysfunction [4]. Although the func-tion of surviving neurons can at least partially compen-sate for brain damage after ischemia, the repair ability ofhuman brain neurons is limited [5]. At present, thromb-olysis is the most effective method for the clinical treat-ment of ischemic stroke, but thrombolysis has a stricttreatment time window, and the vast majority of patientshave lost the opportunity of thrombolytic therapy whenthey are admitted to hospital [6]. Therefore, finding newtherapeutic targets is of great significance for the treat-ment of ischemic stroke.Inflammatory response is one of the important mecha-

nisms of dysfunction caused by cerebral infarction [7, 8].Ischemia induces the release of pro-inflammatory cyto-kines (TNF α, IL-1, IL-6, MCP1) and inflammatory me-diators (ROS and NOS) from glial cells and endothelialcells [9–11]. These cytokines recruit leukocytes andstimulate the production of adhesion molecules on leu-kocytes and endothelial cells, causing phagocytic activityand immune response [12]. It has been confirmed thatIL-1 releases arachidonic acid and increases the numberof TNFα and IL-1β, which leads to the accumulation ofinflammatory cells and aggravates brain injury [13].Therefore, the understanding of inflammatory responseis very important to understand the pathogenesis andexplore the treatment of hypoxic-ischemic brain injury.Exosomes originate from cellular polyvesicles [14].

After combining with the plasma membrane, exosomescan be secreted out of cells. Almost all types of cells cansecrete exosomes, which are widely distributed in bodyfluids, in which there are intracellular sources of lipidsand RNA and other bioactive substances [15]. Exosomescan mediate intercellular communication, and it hasbeen confirmed in recent studies that exosomes can par-ticipate in the remodeling process after cerebral infarc-tion by mediating nucleotide and protein affectingangiogenesis and neuroglia [16, 17]. At present, exo-somes in the treatment of cerebral infarction is still lim-ited to clinical research, mainly in stem cell expressionof related genes and transport-related regulatory

proteins. In addition, as a drug delivery carrier, thera-peutic drugs are transferred into the exosomes by meansof electroporation to effectively achieve the purpose oftreatment [18]. When exosomes played a role in stemcell expression of related genes and transport-relatedregulatory proteins, the motor function of exosome-related model mice derived from stem cell (MSCs) re-covered significantly. Exosomes on the basis of MSCs-mediated miR-133b can effectively improve the neuriteremodeling of ischemic model mice and restore thefunction of model mice [19]. In addition, exosomes canalso play a full role on the basis of transport-relatedregulatory proteins. Exosomes released by MSC havebeen found to contain 1927 proteins [20]. Proteomicanalysis has proved that exosomes released by MSC havethe effect of angiogenic paracrine, which indicates thatexosomes released by MSC have the role and great po-tential in the treatment of ischemic tissue-related dis-eases [21, 22].In recent years, related studies have shown that miR-

NAs play an important role in all stages of the processof cerebral ischemic injury and can aggravate or alleviatebrain injury in a variety of ways [23]. Chi et al. have con-firmed that overexpression of miR-134 aggravates celldeath and apoptosis in vivo and in vitro [24]. WhenmiR-134 was deficient, the level of HSPA128 protein in-creased in N2A cells treated with oxygen and glucosedeprivation and ischemic brain tissue. MiR-134 defi-ciency can reduce the cerebral infarction area and nervecell injury and improve the neurological function scorein mice. Sun et al. found that overexpression of miR-124significantly increased the infarct size of the mousemodel of middle cerebral artery ischemia, while knock-out of miR-124 significantly reduced the infarct size[25]. MiR-124 plays a neuroprotective role by inhibitingapoptosis in the pathological process of ischemic stroke.MiR-542-3p is mainly studied in tumors, also expressedin brain tissues [26]. And He et al. indicated that miR-542-3p might regulate pathological processes of ischemicstroke [27]. However, the role of miR-542-3p in cerebralinfarction is poorly identified. Herein, we explored theeffect of miR-542-3p in cerebral infarction-induced glialcell inflammation and injury and clarify whether miR-542-3p could be packaged into exosomes to treat cere-bral infarction.

MethodsAnimal experimentsMiddle cerebral artery occlusion (MCAO) of mice usedfor cerebral infarction model. As previously described[28], left distal middle cerebral artery was occluded afterthe mice were completely anesthetized. The mice in thesham group only received vascular separation withoutligation. One hundred-microliter AAV9-miR-542-3p/

Cai et al. Stem Cell Research & Therapy (2021) 12:2 Page 2 of 12

AAV9-miR-NC (2.0 × 1011 GC/ml) was injected intoparacele of mice at 1 h after MCAO or sham operation.Neurological assessment used to determine neurologicaldeficiency of mice at 3 days after MCAO operation [29].The left hemispheres of the mice were extracted, andthe brain tissue water content was detected as previouslydescribed [30]. The research protocol of this study wasapproved by the Animal Care and Use Committee of theSecond Affiliated Hospital of Heilongjiang University ofTraditional Chinese Medicine.

Cell culture and treatmentThe MSCs and HA1800 cells (human glial cells) werepurchased from the Science Cell Laboratory. Cell lineswere cultured in DMEM (Thermo-life, USA) with 10%FBS (Thermo Fisher, USA) and 100 μL/mL penicillinand streptomycin (Beyotime, China) and placed at 37 °Cwith 5% CO2. The HA1800 cells were plated until thecell density reached 80% confluency of dishes to trans-fect. MiR-542-3p mimics and Toll-Like Receptor 4(TLR4) plasmid were constructed by Genechem (Shang-hai, China) and transfected into cells with Lipofectamine2000 (Invitrogen, Carlsbad, CA). For oxygen and glucosedeprivation (OGD), HA1800 cells were seeded in Hank’smedium and cultured at 37 °C with 5% CO2 and 95%N2 for 2 h.

Exosome isolation and identificationExosomes in supernatant of MSCs transfected with miR-542-3p/miR-NC were isolated, and several centrifuga-tions were performed to purify exosomes [31]. Trans-mission electron microscopy (TEM) was used to identifyexosomes structure. The diameter of exosome was ex-amined by particle size analysis, and exosome markerprotein CD63, CD9, and GM130 were tested by westernblot.

MTT assayHA1800 cells were plated in 96-well plates, and we usedMTT assay to detect the cell viability. MTT (0.5 mg/mL;Beyotime Biotechnology, China) was added to every wellafter treatment and incubated for 3 h at 37 °C. And150 μL DMSO was added and incubated for 15 min. Wemeasured the absorbance by Spectrophotometer (Tecan,Austria) at 493 nm.

ROS assayReactive oxygen species (ROS) detection was performedaccording to the procedures (Beyotime, China). Briefly,HA1800 cells were plated in 12-well plates and ROS so-lution was added into cells for 20 min. After fixation in4% paraformaldehyde and PBS washing solution, thecells were incubated in DAPI for 10 min. Fluorescencewas observed by fluorescence microscope.

qRT-PCRRNA extraction was performed using trizol reagent.NanoDrop 8000 (Thermo Scientific, Waltham, MA,USA) was used to detect the concentration and purity ofRNA. The single-stranded cDNAs were synthesized from1 μg of RNA. The expression of mRNAs and miRNAswere quantified by RT-PCR with SYBR Green I (ThermoFisher Scientific, Inc).

Western blotTissues, cells, or exosomes were harvested at the indi-cated times, prepared with SDS lysis buffer (Beyotime,China), and centrifuged at 14,000×g at 4 °C. Proteinswere separated using 10% or 15% polyacrylamide gelsand transferred onto 0.22-μm PVDF membranes (MerckMillipore, USA). The total amount of protein samples is50 μg, 30 μg, and 60 μg for exosome, cell, and tissue pro-tein detection, respectively. The membrane was blockedwith 5% BSA for 1.5 h. The first incubation and secondincubation were carried out according to the operationsteps. The expression of the protein was expressed bythe gray value. Primary antibodies list: CD63 (ab134045,Abcam), CD9 (ab92726, Abcam), GM130 (ab52649,Abcam), GADPH (ab181602, Abcam), TLR4 (ab22048,Abcam), TNFα (17590-1-AP, Proteintech), IL 6 (66146-1-Ig, Proteintech). The secondary antibodies IRDye700/800 Mouse or Rabbit were produced by LICOR (Lincoln,Nebraska, USA).

Luciferase assayHEK293 cells were co-transfected with 20 mmol/L miR-542-3p mimic or miR-NC together with WT-TLR4/Mut-TLR4. Luciferase activity was measured with DualLuciferase Reporter Assay Kit (Transgene, China) onGloMax20/20 at 48 h after the transfection.

RIPWe used RIP assay to determine the binding betweenTLR4 and miR-542-3p using Magna RIP™ RNA-BindingProtein Immunoprecipitation Kit (Millipore) as previousstudy [32]. Briefly, HA1800 cells were transfected withbiotinylated TLR4, and the mRNA level of miR-542-3pwas detected using qRT-PCR.

H&E stainingThe brain tissues were gathered and fixed in 4% parafor-maldehyde for 24 h. Then, the fixed tissues were embed-ded in paraffin. Next, Paraffin slicer machine was usedto cut slices (5-mm cross-sectional). H&E staining wasused to evaluate pulmonary morphology. Liver sectionswere dewaxed with xylene and treated with ethanol atdifferent concentrations for 5 min. Hematoxylin stainingfor 5 min, 5% acetic acid treatment for 1 min, waterrinse. Dye with eosin for 1 min, rinse with running


water. Dehydrate in 70%, 80%, 90%, 100% ethanol for 10s, xylene for 1 min. Drizzle with neutral gum and seal.

TTC stainingTTC staining was used to evaluate the infarcted size ofthe brain. The brain sections were placed in the smallestvessel containing 2% TTC staining solution (G3005,Solarbio) and incubated at 37 °C in darkness for 15–30min. The color changes of the samples were observedwhile incubating. The excess staining solution on the tis-sue surface was rinsed off with PBS and photographswere taken.

Immunohistochemistry (IHC) stainingParaffin sections of brain tissues from different groupswere dewaxing to water in xylene and descending seriesof ethanol. We penetrated sections using 0.5% Triton X-100. After 3 times wash, we blocked sections with 50%goat serum. Then, sections were incubated with VEGFAantibody (ab51745, Abcam) overnight. We incubated thesections using secondary antibody. The sections werephotographed by light scope under an IX73 fluorescencemicroscope (Olympus, Valley, PA).

Fluoro-Jade C (FJC) stainingFJC staining was used to determine the degeneratingneurons in brain tissues. Brain sections were stainedusing Fluoro-Jade® C kit (TR-100-FJ, Biosensis) accord-ing to the protocol and previous study [33].

TUNELWe used the in situ Cell Death Detection Kit (TUNELfluorescence FITC kit, Roche, Germany) to detect apop-totic. We used DAPI to stain nuclei. We used IX73fluorescence microscope (Olympus, Valley, PA) toanalyze fluorescence staining. We used Image-J to countthe total cells and TUNEL-positive cells numbers.

Statistical analysisData were shown as mean ± SD. Student’s t test or one-way ANOVA was used to compare the groups. P < 0.05was considered significance.

ResultsMiR-542-3p was downregulated during cerebral infarctionWe first established mouse model of MCAO at 12 h,1 days, 3 days, 5 days, and 7 days. Then, FJC staining wasused to determine degenerating neurons. FJC staining datashowed that the number of degenerating neurons was in-creased at 12 h and peaked at 3 days after MCAO oper-ation (Fig. 1a), which suggested that we successfullyestablished a cerebral infarction model. Then, we per-formed microarray analysis, and the data showed the dif-ferentially expressed miRNAs in sham and MCAO

operation of brain tissues, which showed a decrease ofmiR-542-3p in infarcted brain tissues (Fig. 1b). FollowingqRT-PCR analysis indicated that miR-542-3p was de-creased after MCAO operation (Fig. 1c). Moreover, micro-array analysis showed a significantly increase of TLR4 inMCAO mice comparing with sham mice (Fig. 1d). Then,qRT-PCR also suggested that TLR4 was induced in braintissues after MCAO surgery (Fig. 1e). Meanwhile, bio-informatics analysis of the 3′ UTR of TLR4 predicted aconserved binding site for miR-542-3p. To verify thatmiR-542-3p interacts with the TLR4 mRNA, luciferase ac-tivity was performed in HEK293T cells. Cells transfectedwith miR-542-3p mimic decreased the luciferase activityin WT-TLR4 not in mutant type, which indicated the rela-tionship miR-542-3p with TLR4 (Fig. 1f). And endogenousTLR4 was enriched in biotinylated miR-542-3p trans-fected HA1800 cells, which reveals a direct binding ofTLR4 with miR-542-3p (Fig. 1g). The abnormal expres-sion of miR-542-3p and TLR4 may be novel target forcerebral infarction.

Overexpression of miR-542-3p attenuated MCAO inducedcerebral injuryFor further research, we constructed adeno-associatedvirus 9 (AAV9) miRNA for overexpression of miR-542-3p (AAV9-miR-542-3p, AAV9-miR-NC was indicated asa control group) and injected into paracele of mice thatunderwent with sham or MCAO operation (Fig. 2a).TTC staining showed that higher infarction volume in-duced by MCAO operation was significantly reduced byAAV9-miR-542-3p administration (Fig. 2b). The brainwater content in the MCAO mice was lower afterAAV9-miR-542-3p treatment (Fig. 2c). Neurologicalgrading scores of the MCAO mice reduced after theAAV9-miR-542-3p injection (Fig. 2d). FJC stainingshowed that AAV9-miR-542-3p, but not AAV9-miR-NC, decreased FJC-positive cell numbers upon MCAOsurgery (Fig. 2e). Moreover, H&E staining showed thatMCAO surgery caused resulted in obvious swelling ofthe cells, large intercellular space and infiltration of neu-trophils, while AAV9-miR-542-3p significantly alleviatedthe MCAO-induced brain edema and the infiltration ofinflammatory cells (Fig. 2f). Considering the importanceof revascularization for infarcted brain, VEGFA (themain angiogenic factor) level in brain tissues was testedusing IHC. And data showed that MCAO surgery in-creased VEGFA expression, while AV9-miR-542-3p in-jection further significantly promoted the expression ofVEGFA (Fig. 2g). And qRT-PCR showed that AAV9-miR-542-3p reduced MCAO-induced increase of inflam-matory factors TNFα and MCP-1 (Fig. 2h). As well,MCAO operation induced TNFα and IL 6 protein ex-pression, while AAV9-miR-542-3p reversed the effect ofMCAO operation (Fig. 2i). Besides, AAV9-miR-542-3p


injection inhibited TLR4 expression both in sham andMCAO mice (Fig. 2i). These results indicated that miR-542-3p protected against MCAO-induced cerebral injuryand inflammation.

MiR-542-3p alleviated OGD induced glial cell damageIn vitro, we cultured HA1800 cells treated with OGD tomimic in vivo MCAO operation. And miR-542-3p wastransfected into HA1800 cells to force miR-542-3p ex-pression (Fig. 3a). MTT results showed that OGD de-creased cell viability, while miR-542-3p recovered cellviability (Fig. 3b). In addition, TUNEL analysis indicatedthat OGD promoted cell apoptotic numbers, while miR-542-3p decreased apoptotic cell numbers (Fig. 3c). AndROS assay showed that OGD increased ROS production,while miR-542-3p inhibited ROS level in HA1800 cells

(Fig. 3d). Moreover, miR-542-3p remitted the promotingeffect of OGD on inflammation of HA1800 cells, as wellas the expression of TLR4 (Fig. 3e, f). Together, miR-542-3p alleviated OGD-induced glial cell injury.

MiR-542-3p was packaged into MSC exosomesMiRNA in exosomes has attracted much attention due tothe protection role of exosomes, and MSCs are a mainsource of exosomes [34]. In present study, MSCs weretransfected with miR-542-3p mimic or miR-NC, and theexpression level of miR-542-3p was detected by qRT-PCR(Fig. 4a). Exosomes isolated from supernatant of MSCswere identified by TEM (Fig. 4b), and particle size analysis(Fig. 4c). As well, exosome markers (CD63 and CD9) weredetected using western blot (Fig. 4d). Then, the expressionof miR-542-3p in isolated exosomes was assessed, which

Fig. 1 The expression of miR-542-3p and TLR4 in brain tissues during cerebral infarction. Mice suffered from sham or MCAO operation with different timepoints (12 h, 1 days, 3 days, 5 days, and 7 days). a FJB staining was used to detect the degenerating neurons in brain tissues. b MicroRNA expression profiles insham and MCAO (3 days) mice. Scale bar 20μm. c qRT-PCR analyzed the expression of miR-542-3p in brain tissues of different groups. d MiRNA expressionprofiles in sham and MCAO (3d) mice. e TLR4 level in brain tissues of different groups was examined. f The pairing bases between TLR4 and miR-542-3p, andluciferase assay was used to determine the binding of TLR4 and miR-542-3p in HEK293 cells. g RIP assay for the binding of TLR4 and miR-542-3p in HA1800cells. Data are mean± SD; *P<0.05, **P<0.01. All experiments were repeated in triplicate


showed an increase of miR-542-3p after miR-542-3ptransfection of MSCs (Fig. 4e). In summary, miR-542-3pcan be secreted by MSCs via exosomes.

Exo-miR-542-3p protected against MCAO-inducedcerebral injuryTo illustrate the role of exosomal miR-542-3p (exo-miR-542-3p), we injected exo-miR-542-3p from Fig. 4d intoparacele of mice accompanied with or without MCAO.And miR-542-3p in brain tissues was calculated; exo-miR-542-3p injection promoted miR-542-3p expression(Fig. 5a). Following function experiments showed thatexo-miR-542-3p decreased infarcted area, brain watercontent, neurological grading scores, and FJC-positivecell numbers during MCAO surgery (Fig. 5b–e). Inaddition, exo-miR-542-3p inhibited MCAO-induced

brain edema and neutrophils infiltration brain damageand inflammatory response (Fig. 5f–i).

Exo-miR-542-3p inhibited inflammation and injury viaregulating TLR4 in glial cellsWe then treated HA1800 cells with exo-miR-542-3p andOGD and transfected with TLR4 plasmid at the sametime. Exo-miR-542-3p treatment increased miR-542-3pexpression and inhibited TLR4 level, while TLR4 trans-fection reversed exo-miR-542-3p effects (Fig. 6a). Func-tional analysis has been suggested that exo-miR-542-3pincreased cell viability and inhibited cell apoptosis, ROSproduction, and inflammation (Fig. 6b–f). But, TLR4transfection removed the benefit role of exo-miR-542-3pon HA1800 cells during OGD exposure (Fig. 6b–f).Taken together, exo-miR-542-3p from MSCs inhibitedischemic injury of brain via modulating TLR4.

Fig. 2 AAV9-miR-542-3p injection attenuated MCAO induced injury. AAV9-miR-542-3p and AAV9-miR-NC were injected into paracele of mice at 1h after MCAO or sham operation. Brain tissues were collected after 3 days of operation. a The expression of miR-542-3p in brain tissues after AAV9injection was tested using qRT-PCR. b TTC staining indicated the infarcted area of brain tissues. c The brain water content in brain was detected.d Neurological grading scores of mice were evaluated. e FJB staining was used to detect the degenerating neurons. Scale bar 20 μm. f H&Estaining showed the structure of brain in different groups. Scale bar 20 μm. g IHC staining for VEGFA expression in brain tissues. Scale bar 50 μm.h Inflammatory factors TNFα and MCP-1 were detected using qRT-PCR. i Western blot was used to test the protein expression of TLR4, TNFα, andIL 6. Data are mean ± SD; *P < 0.05, **P < 0.01. All experiments were repeated in triplicate


DiscussionCerebral infarction is a series of cascade injury reactionscaused by the damage of neurovascular componentsupon ischemia and hypoxia [35]. Cerebral infarction ismainly treated with tissue-type plasminogen activatorthrombolytic therapy, but few patients actually recovertheir neurological function clinically. Exosomes are 40–100 nm in diameter, vesicles encapsulated by lipid bi-layer membranes released from cells into the extracellu-lar space [36]. Exosomes and miRNAs can effectivelyrespond to the pathophysiological state of cells, and theyhave great potential in the diagnosis and treatment oftumors and cardiovascular and other systemic diseases[37]. Present study showed a downregulation of miR-542-3p and upregulation of TLR4 in infarcted brain tis-sues, and miR-542-3p interacted with TLR4 in HA1800

cells. Functionally, miR-542-3p protected against ische-mic injury of brain tissue and nutritional deprivationdamage of HA1800 cells. Interestingly, miR-542-3p canbe packaged and secreted by MSCs. And exo-miR-542-3p from MSCs also showed a protective effect forMCAO-treated brain tissues or OGD-exposed HA1800cells via modulating TLR4 level.MiRNAs is a highly conserved non-coding RNA in eu-

karyotes [38]. By complementary pairing with the 3′UTR of the target mRNA molecule, miRNA regulatesthe expression of target genes at the post-transcriptionallevel, leading to the degradation or inhibition of thetranslation of the target mRNA molecule [39]. Throughthis regulatory relationship, miRNA affects many aspectsof life activities, such as early embryonic differentiation,development, cell proliferation, differentiation, apoptosis,

Fig. 3 MiR-542-3p alleviated OGD induced glial cell damage. MiR-542-3p or miR-NC was transfected into HA1800 cells; then, cells were treatedwith or without OGD for 2 h. a MiR-542-3p expression in HA1800 cells was detected using qRT-PCR after transfection or OGD treatment. b Cellviability was tested by MTT. c TUNEL assay was used to calculate cell apoptotic level. Scale bar 20 μm. d ROS assay was used to determine theproduction of ROS in HA1800 cells. Scale bar 25 μm. e Inflammatory factors TNFα and MCP-1 were detected using qRT-PCR. f Western blot wasused to test the protein expression of TLR4, TNFα, and IL 6. Data are mean ± SD; *P < 0.05, **P < 0.01. All experiments were repeated in triplicate


inflammation, and so on [40]. MiRNA also plays mul-tiple roles in various aspects of cerebral ischemia, regu-lating apoptosis, differentiation, vascular inflammatoryresponse, ischemia-reperfusion injury, and so on.Ouyang et al. found that the target protein of miR-181 isa classic marker of GRP78, endoplasmic reticulum stress.The expression of miR-181 decreases and GRP78 in-creases during cerebral ischemia, which mediates apop-tosis and promotes the development of brain injury [41].Other studies have confirmed that miR-181a inhibitortreatment can significantly reduce the area of cerebralinfarction, reduce the loss of neurological function, re-duce the activation of NF-κB, reduce leukocyte infiltra-tion, and protect against cerebral ischemic injury for along time [42]. To explore the mechanism of ischemicinjury of the brain, we constructed the mouse MCAOmodel, which is a commonly used animal model to de-tect cerebral ischemic diseases [43]. And FJC staining,TTC staining, and neurological grading scores were usedto confirm the success of the MCAO model. To identifythe regulating miRNA in cerebral infarction, we per-formed microarray and found a significant decrease ofmiR-542-3p in ischemic tissues. And the low expressionof miR-542-3p in ischemic brain tissue has been show inthe previous study [44]. Interestingly, TLR4, which

potentially binds with miR-542-3p, was increased in in-farcted brain tissue. And we performed luciferase assayand RIP assay to confirm the relation between miR-542-3p and TLR4. Our data indicated an interaction of miR-542-3p and TLR4 in glial cells. TLR4 is a key receptor ofinnate immunity, which mediates inflammatory responsein multiple organs [45].Then, we aimed to determine the role of miR-542-3p in

cerebral infarction. AAV9-miR-542-3p was constructed toforce miR-542-3p level in brain tissues. Adeno-associatedvirus vector is a safe, durable, and efficient gene manipula-tion tool, which has been widely used in the field of neuro-biology [46]. The AAV9 subtype is a common type ofbrain tissue infection [47]. And in the present study, miR-542-3p level in brain tissues was prominently increasedafter AAV9-miR-542-3p injection. Then, we evaluated thefunction of miR-542-3p in infarcted brain, and our datashowed that AAV9-miR-542-3p injection decreased in-farcted size, reduced the number of degenerating neurons,and inhibited brain water content and neurological grad-ing scores. Considering the key role of inflammation incerebral infarction [48], we detected inflammatory re-sponse. Our results suggested that MCAO operation pro-moted the expression of inflammatory factors, andinfiltration of inflammatory cells, while AAV9-miR-542-

Fig. 4 MiR-542-3p can be packaged into MSC exosomes. a MiR-542-3p was transfected into MSCs, and the expression of miR-542-3p wasdetermined. Exosomes in supernatant were isolated, and b TEM was used to show the morphology of exosomes. c Particle size analysis showedthe size of exosomes. d Exosome markers (CD63 and CD9) and MSC marker (GM130) were detected using western blot. e The expression of miR-542-3p in isolated exosomes was determined. Data are mean ± SD; **P < 0.01. All experiments were repeated in triplicate


3p injection suppressed MCAO-induced inflammation inbrain tissues. Furthermore, human glial cell line HA1800cells were treated with OGD to mimic brain ischemia, andHA1800 cells were transfected with miR-542-3p mimics.In consistent with the results of in vivo experiments, miR-

542-3p mimics inhibited OGD-induced cell apoptosis,ROS production, and inflammation.Exosomes are vesicles secreted by cells with phospho-

lipid bilayers. Different from other extracellular vesicleswith a diameter of 100~1000 nm, exosomes originate

Fig. 5 Exosomal miR-542-3p remitted MCAO induced inflammation. MiR-542-3p was transfected into MSCs; exosomes in supernatant were isolatedand injected into paracele of mice with or without MCAO. Brain tissues were collected after 3 days of operation. a MiR-542-3p expression in braintissues was tested using qRT-PCR. b TTC staining for infarcted area of brain tissues. c The brain water content in brain was detected. d Neurologicalgrading scores of mice were evaluated. e FJB staining was used to detect the degenerating neurons. Scale bar 20 μm. f H&E staining showed thestructure of brain in different groups. Scale bar 20 μm. g IHC staining was used to detect VEGFA level brain in different groups. Scale bar 50 μm. hInflammatory factors TNFα and MCP-1 were detected using qRT-PCR. i Western blot tested the protein expression of TLR4, TNFα, and IL 6. Data aremean ± SD; *P < 0.05, **P < 0.01. All experiments were repeated in triplicate


directly from the cell membrane. Exosomes originatefrom the endosomal system of cells [45]. Exosomesmainly carry endogenous proteins, lipids, nucleic acids,and other substances, which are transmitted betweencells through target cell internalization, receptor-ligandinteraction, or lipid membrane fusion [49]. In 2007,Valadi et al. confirmed for the first time the existence ofRNA, in exosomes, including mRNA, miRNA, and somenon-coding RNA [50]. Exosomes can also carry proteinsand peptides. Through proteome analysis, Laura Otero-Ortega’s team has identified 2416 proteins in exosomesderived from MSCs, which are fully involved in molecularregulation, binding, and catalytic functions [51]. Inaddition, exosomes contain proteins involved in brain re-pair, including synaptic transmission, neural differenti-ation of neural stem cells, angiogenesis, neural projection,synaptic growth and so on. Studies have shown that afterMSCs treatment, miR-133b levels are increased, andMSCs transmit miR-133b through exosomes to regulatenerve growth. MiR-133b overexpressed in exosomes

secreted by MSCs can promote the recovery of nervefunction and increase nerve plasticity by stimulating sec-ondary secretion of exosomes from astrocytes [52]. MiR-17-92 derived from exosomes of MSCs promotes nerverecovery and neuroplasticity by activating PI3K/Akt/mTOR/GSK-3β signaling pathway [53]. These evidencesindicate the important role of MSC exosome-derived miR-NAs in cerebral ischemia, and we wondered whether miR-542-3p can be enveloped in MSCs and then secreted viaexosomes. Exosomes were isolated from MSCs transfectedwith miR-542-3p, and we found a high expression of miR-542-3p in exosomes of MSCs supernatant, which revealedthat miR-542-3p was packaged into exosomes and se-creted into supernatant by MSCs. Moreover, exo-miR-542-3p exerted a protective role in MCAO-induced injuryand OGD-treated HA1800 cells via modulating TLR4.It has been proved that MSCs from different tissue

sources can enhance the recovery of neurological func-tion in animal models of stroke. Despite these findings,the difficulty of treatment in patients with acute stroke,

Fig. 6 MiR-542-3p alleviated OGD induced glial cell damage. HA1800 cells were incubated with exo-miR-NC/exo-miR-542-3p and treated with orwithout OGD for 2 h and transfected with TLR4 plasmid at the same time. a MiR-542-3p expression in HA1800 cells was detected using qRT-PCR.b MTT assay for cell viability. c TUNEL assay for cell apoptotic level. Scale bar 20 μm. d ROS assay for the production of ROS in HA1800 cells. Scalebar 25 μm. e Inflammatory factors TNFα and MCP-1 were detected using qRT-PCR. f Western blot for the protein expression of TLR4, TNFα, and IL6. Data are mean ± SD; *P < 0.05, **P < 0.01. All experiments were repeated in triplicate


the risk of MSCs transplantation, and the high cost ofmaintaining stem cell activity limit the use of MSCs. Inrecent years, exosome has attracted wide attention be-cause of its unique characteristics in the diagnosis andtreatment of stroke. Because there is not enough clinicaldata to support the clinical application of exosome ther-apy, the diagnosis and treatment of exosomes is still inthe theoretical and experimental stage.

ConclusionIn summary, MSC-derived exosome miR-542-3p pre-vented ischemia-induced glial cell inflammatory re-sponse via inhibiting TLR4. These results suggestpossible therapeutic strategies for using exosome deliv-ery of miR-542-3p to cure cerebral ischemic injury.

AbbreviationsMSCs: Mesenchymal stem cells; TLR4: Toll-Like Receptor 4; MCAO: Middlecerebral artery occlusion; OGD: Oxygen and glucose deprivation;TEM: Transmission electron microscopy; ROS: Reactive oxygen species;FJC: Fluoro-Jade C; AAV9: Adeno-associated virus 9

AcknowledgementsNot applicable.

Authors’ contributionsDianquan Zhang, Guoliang Cai, Haichun Zhou, Zhe Zhuang, Kai Liu, andSiying Pei performed the majority of experiments and analyzed the data;Yanan Wang, Hong Wang, Xin Wang, and Shengnan Xu performed themolecular investigations; Cheng Cui, Manchao Sun, and Sihui Guo designedand coordinated the research; Kunping Jia, Xiuzhen Wang and Guofeng Caiwrote the paper. The authors read and approved the final manuscript.

FundingThis study was supported by Natural Science Foundation of HeilongjiangProvince (H2018066), Scientific research fund of Heilongjiang University oftraditional Chinese Medicine (2019MS19), and State Administration ofTraditional Chinese Medicine of Heilongjiang Province, No. ZHY16-027.

Availability of data and materialsThe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable request.

Ethics approval and consent to participateThe research protocol of this study was approved by the Animal Care andUse Committee of the Second Affiliated Hospital of Heilongjiang Universityof Traditional Chinese Medicine.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Hanan Branch of Second Affiliated Hospital of Heilongjiang University ofTraditional Chinese Medicine, Harbin 150001, China. 2Postdoctoral ResearchWorkstation of Harbin Sport University, Harbin, China. 3Department of SportScience and Health, Harbin Sport University, Harbin 150008, China.4Heilongjiang University of Traditional Chinese Medicine, Harbin 150001,China. 5Department of Rehabilitation Medicine, Shenzhen Longhua DistrictCentral Hospital, Shenzhen, Guangdong Province, China.

Received: 26 October 2020 Accepted: 16 November 2020

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Mesenchymal stem cell-derived exosome miR-542-3p