| Stroke is highlighted as a main trigger of disability as well as the 2nd leading cause of mortality worldwide,of which ischemic stroke accounts for 87%.Mediation of the inflammatory cascade through neuroendocrine hormones and proinflammatory mediators might serve as participants of the disease process of ischemic brain injury(IBI).Neuroinflammation often follows upon the heels of cerebral ischemia,with characteristics of accumulation of inflammatory cells,resident brain cells(including astrocytes and microglia),and infiltrating immune cells(leukocytes)in the ischemic brain,which thereupon then result in inflammatory injury.Moreover,astrocytes are capable of not only supporting brain function under normal conditions,but also contributing to neurodegeneration when being reactive under pathological conditions including stroke.Previously,cortical neuron-derived exosomes have been suggested to be a possible candidate to block the activation of inflammasomes after central nervous system(CNS)injury through acting as a cargo to deliver si RNA in vivo.As vesicles that are enclosed by the phospholipid bilayer membrane,extracellular vesicles such as exosomes contain proteins,m RNA,and miRNA that are released from cells into extracellular tissue spaces as well as biological fluids,or in vitro into culture-conditioned medium.Exosomal micro RNA-17-92(miR-17-92)cluster harvested from mesenchymal stromal cells contributed to an enhancement of neural plasticity and functional recovery following a stroke event.miR-181 c has been reported to protect neurons against apoptosis that was mediated by oxygen and glucose deprivation(OGD)-activated microglia via suppression of tumor necrosis factor-?(TNF-?).Enrichment in miR-181 c expression has been found in cortical neurons,and miR-181 c is revealed to play a role as a modulator of the gene network in relation to cortical neuronal maturation.More importantly,miR-181c-3p signature in serum has been illustrated to serve as a noninvasive biomarker for detecting Alzheimer`s disease in early diagnosis.Bioinformatics analysis of the current study found(C-X-C motif)ligand 1(CXCL1)as a potential target gene of miR-181c-3p.CXCL1,a member of the C-X-C family,has the ability to attract neutrophils into the injured CNS.CXCL1 has been proven to be produced by astrocytes,whereas inhibited production of astrocytic CXCL1 decreased the number of infiltrated neutrophils into the brain,thereby alleviating CNS inflammation.Therefore,we put forward a hypothesis that the interaction between exosomal miR-181c-3p secreted by cortical neurons and CXCL1 produced by astrocytes might share association with neuroinflammation in IBI.Then,an OGD-stimulated IBI cell model was established to confirm this hypothesis.Collectively,exosomal miR-181c-3p derived from cortical neurons exerts protective effects on neuroinflammation in astrocytes via downregulation of CXCL1 in an IBI rat model.Part One After ischemic brain injury,CXCL1 is related to the expre-ssion of inflammatory factors and regulated by miR-181c-3pObjective: To find the important factors related to neuroinflammation and the micro RNA that can regulate the expression of these factors in the ischemic brain injury model,and to verify them in the MCAO model.Methods:1.The model of IBI was established by MCAO method,and the success of the model was verified by Morris water maze experiment and brain morphology.2.The expression of miR-181c-3p and CXCL1 in brain tissue of rats in sham operation group and MCAO operation group were detected by RT real-time PCR and Western blot respectively,and the levels of NF-?B,IL-8,IL-6 and TNF-? in serum were detected by ELISA.3.Predict the miRNA of CXCL1 by bioinformatics prediction website,and verify it in HEK293 cells by double luciferase reporter gene analysis.4.RT real-time PCR and Western blot were used to detect the effect of mir-181c-3p on the expression of CXCL1 m RNA and protein in astrocytes.Results:1.CXCL1 Is Identified as an Upregulated Gene in IBI and Regulated by miR-181c-3pDifferential analysis of GSE61616 and GSE78731 microarray data was processed,by which CXCL1 was found to be highly expressed in IBI.Bioinformatics online prediction websites DIANA TOOLS,miRWalk,and miRSearch were used to predict potential miRNAs that could bind to CXCL1,aiming to explore the underlying mechanism of CXCL1 in IBI.Consequently,miR-181a-1–3p,miR-181c-3p,and miR-711 were found to have binding sites to CXCL1.Additionally,miR-181 c conferred protection against neuronal apoptosis and its expression was highly enriched in cortical neurons.Therefore,we hypothesized that miR-181c-3p could affect neuroinflammation in IBI by regulating CXCL1 expression in astrocytes.The Morris water maze test and observation of postoperative brain morphology were employed in order to verify whether the IBI rat model was successfully established.From the observation of postoperative brain morphology to compare the area of cerebral infarction after 24 h,it could be seen that the left and right brains were equal in size of the sham-operated rats,and there was no white ischemic trace.However,the right brain was swollen,and the right cerebral ischemia was obvious in the MCAO rats.According to the results of the Morris water maze test,the escape latency of MCAO rats significantly increased,while the frequency crossing the platform notably reduced compared with the rats receiving sham surgery.These results demonstrated the successful establishment of an IBI rat model.Further RT real-time PCR was conducted to determine the expression of miR-181c-3p and CXCL1 in brain tissues of sham-or MCAO-operated rats.It was found that the expression of miR-181c-3p was remarkably lower,while the expression of CXCL1 was significantly higher in MCAO rats than in sham-operated rats,which was consistent with the results of differential analysis of GSE61616 and GSE78731 microarray data.Since CXCL1 was a chemokine,the levels of inflammatory factors(NF-?B,IL-8,IL-6,and TNF-?)in serum of sham-or MCAO-operated rats were measured by ELISA.It was revealed that the levels of inflammatory factors were significantly higher in MCAO-operated rats than in sham-operated rats.From these results,we preliminarily judged that IBI could aggravate inflammatory response.2.CXCL1 Is a Target Gene of miR-181c-3pBioinformatics prediction websites were used to predict the binding sites between miR-181c-3p and CXCL1,and it was revealed that miR-181c-3p may specifically bind to CXCL1.Dual luciferase reporter gene assay was employed to verify their targeting relationship.The luciferase activity of HEK293 cells co-transfected with PGLO-CXCL1-Wt and miR-181c-3p mimic decreased,while it increased in cells co-transfected with PGLO-CXCL1-Wt and miR-181c-3p inhibitor.The luciferase activity did not differ in HEK293 cells co-transfected with PGLO-CXCL1-Mut and miR-181c-3p mimic or inhibitor.These results suggested that miR-181c-3p could specifically bind to CXCL1 and regulate its expression.Subsequently,the m RNA and protein expression of CXCL1 in astrocytes delivered with miR-181c-3p mimic was evaluated by RT real-time PCR and Western blot analysis,in order to further confirm their targeting relationship.It was shown that delivery of miR-181c-3p mimic into astrocytes resulted in decreased expression of CXCL1.Above all,CXCL1 was a target gene of miR-181c-3p and could be negatively regulated by it.Summary:1.CXCL1 is highly expressed in ischemic brain injury,which may be regulated by miR-181c-3p.2.CXCL1 is the target gene of miR-181c-3p and regulated by it.Part Two miR-181c-3p in exosomes of cortical neurons regulates the expression of CXCL1 in astrocytesObjective:To investigate the expression of CXCL1 in primary cultured astrocytes and cortical neurons.Methods:1.Astrocytes were extracted from fetal rat brain.The morphology of astrocytes and the expression of CXCL1 were observed by inverted microscope and immunofluorescence staining.2.The expression of CXCL1 was detected by RT real-time PCR and Western blot.3.Analyze the cell or extracellular vesicles with high expression of miR-181c-3p in RNAlocate and vesiclepedia database.4.Primary cultured cortical neurons were used to detect tau protein markers by immunofluorescence staining,and the presence of exosomes was determined by electron microscopy.5.The protein expression of tgs101,CD63 and GRP94 was detected by Western blot after 12 hours of OGD treatment.6.The exosomes were extracted by kit,and the expression of miR-181c-3p in cortical neurons and exosomes was detected by RT real-time PCR.Results:1.The ex CXCL1 Expression Might Be Regulated by Extracellular VesiclesThe astrocytes were extracted from brain tissues of fetal rats and then observed under the inverted microscope.The astrocytes had a more irregular cell shape than the oligodendrocytes,with abundant cell processes and branches.Immunofluorescence staining assay showed that the protein marker GFAP was abundantly expressed in astrocytes,and CXCL1 expression was also observed.The above results suggested that astrocytes were successfully extracted from brain tissues of fetal rats,and CXCL1 was located in the astrocytes.The astrocytes were randomly assigned into two groups,with one group treated with OGD for 12 h to simulate IBI and the other without any treatment(control).The astrocytes following OGD treatment did not show differences in expression of GFAP and CXCL1 compared with those without any treatment.These results led to a conclusion that astrocytes did not cause significant changes in the expression of CXCL1,which might be potentially regulated by extracellular vesicles.2.OGD-Treated Cortical Neurons Exhibit Inhibited Secretion of Exosomal miR-181c-3pIt has been previously suggested that the expression of miR-181c-3p was highly enriched in cortical neurons,and subsequent bioinformatics analysis of the RNALocate database and the Vesiclepedia database found that miR-181c-3p was expressed in exosomes.The following steps were done in order to analyze whether cortical neurons could release exosomes.Immunofluorescence staining assay was performed for the detection of the presence of cortical neuron marker Tau protein in the cortical neurons collected from fetal rats,aiming to verify whether the obtained cells were cortical neurons.It could be seen that the Tau protein was expressed in the cytoplasm,which demonstrated that cortical neurons were successfully extracted.Following extraction and purification of cortical neuron-released exosomes,TEM(Transmission Electron Microscope)was used for observation in order to ascertain the existence of exosomes.Consequently,some compact solid bodies shaped like a disk or cup,which had a typical two-layer membrane structure and an average diameter of 100 nm,could be seen in the supernatant of cell culture medium,suggesting that exosomes were released by cortical neurons.After cortical neurons were subjected to a 12-h OGD treatment for in vitro stimulation of IBI,Western blot analysis was adopted to confirm whether the release of exosomes from cortical neurons would be affected by OGD.When comparing to cells without any treatment,OGD-treated cells has no significant difference in protein expression of TGS101,CD63,and GRP94.These results suggested that OGD treatment did not affect the secretion of exosomes from cortical neuron cells.The expression of miR-181c-3p in cortical neurons and the exosomes extracted from cortical neurons was evaluated by means of RT real-time PCR.The cortical neurons following OGD treatment showed a low expression of miR-181c-3p compared with those without any treatment.Similarly,the expression of miR-181c-3p was significantly downregulated in exosomes extracted from OGD-treated cortical neurons.These results suggested that IBI might lead to an inhibited release of exosomal miR-181c-3p from cortical neurons.Summary:1.Extracellular factors can regulate the expression of CXCL1 in astrocytes.2.The inhibitory effect of OGD treated cortical neurons on the secretion of miR-181c-3p by exosomes.Part Three miR-181c-3p in exosomes released by cortical neurons inhi-bits the expression of CXCL1 and its downstream inflamm-atory factors in astrocytesObjective: To investigate the regulatory mechanism of miR-181c-3p in exosomes secreted by cortical neurons and astrocytes on the expression of CXCL1 in astrocytes.Methods:1.The cortical neurons and astrocytes were cultured for 48 hours.After PKH67 was used to label exosomes,the absorptive capacity of astrocytes to exosomes was observed by inverted fluorescence microscope.RT real-time PCR,Western blot and ELISA were used to observe the expression of inflammatory factors and CXCL1.2.The expression of CXCL1 was detected by RT real-time PCR and Western blot.3.Astrocytes were transfected with mimic,inhibitor or si RNA of miR-181c-3p.The expression of inflammatory factors in astrocytes or cell culture supernatant was detected by RT real-time PCR,Western blot and ELISA.Results:1.Cortical Neuron-Released Exosomes Suppress Inflammation and CXCL1 Expression in AstrocytesAfter being labeled by PKH67,the exosomes extracted from normal cortical neurons were co-cultured with astrocytes under OGD treatment for 48 h.Then the inverted fluorescence microscope was adopted to observe the absorption ability of astrocytes to exosomes.Green fluorescence was found in astrocytes,indicating that exosomes were up-taken by astrocytes.GW4869 was a type of extracellular vesicle inhibitor that inhibited miRNA vesicle trafficking.Therefore,the influence of exosomes on the expression of CXCL1 and inflammatory factors(NF-?B,IL-6,IL-8,and TNF-?)in astrocytes was evaluated by treatment with PBS + dimethyl sulfoxide(DMSO),cortical neuron-released exosomes(CN-Exo)+ DMSO,or CN-Exo + GW4869.According to the results of RT real-time PCR and Western blot analysis,the expression of CXCL1 was significantly decreased in astrocytes with treatment of CN-Exo + DMSO compared to those with treatment of PBS + DMSO.Astrocytes treated with CN-Exo + GW4869 had increased expression of CXCL1 in contrast to those with treatment of CN-Exo + DMSO.These results suggested that cortical neuron-released exosomes downregulated the expression of CXCL1 in astrocytes.Based on the results of RT real-time PCR,Western blot analysis,and ELISA,the expression of inflammatory factors decreased in astrocytes treated with CN-Exo + DMSO compared to those with treatment of PBS + DMSO,whereas it increased in astrocytes treated with CN-Exo + GW4869 compared to those with treatment of CN-Exo + DMSO.All in all,these results demonstrated that cortical neuron-derived exosomes could inhibit inflammation and CXCL1 expression in astrocytes.2.Cortical Neuron-Released Exosomal miR-181c-3p Inhibits CXCL1 Expression in AstrocytesAs we have claimed above,CXCL1 was verified to be a target gene of miR-181c-3p.Subsequently,with the aim of exploring the effects of exosomal miR-181c-3p on CXCL1 expression in astrocytes,the astrocytes were treated with CN-Exo,miR-181c-3p mimic,and miR-181c-3p inhibitor.RT real-time PCR and Western blot analysis were employed to determine the m RNA and protein expression of CXCL1.Compared with astrocytes co-treated with negative control(NC)mimic + PBS,a significant decline in CXCL1 expression was found in astrocytes co-treated with NC mimic + CN-Exo and with miR-181c-3p mimic + PBS.There was no significant difference regarding CXCL1 expression among astrocytes co-treated with NC mimic + CN-Exo and those treated with miR-181c-3p mimic + PBS.Moreover,CXCL1 expression was notably lower in astrocytes co-treated with CN-Exo + miR-181c-3p mimic than that in astrocytes co-treated with NC mimic + PBS,NC mimic + CN-Exo,or miR-181c-3p mimic + PBS.The astrocytes in the presence of CN-Exo + miR-181c-3p inhibitor showed that inhibition of miR-181c-3p could effectively reverse the increase of CXCL1 expression in astrocytes caused by CN-Exo.These results evidenced that cortical neuron-released exosomes could carry miR-181c-3p to inhibit the expression of CXCL1 in astrocytes.3.miR-181c-3p Inhibits Inflammation in Astrocytes via CXCL1 DownregulationThe expression of inflammatory factors was decreased in the OGD-treated astrocytes by co-treating with cortical neuron-released exosomes.In the next step,in order to understand whether inflammation in astrocytes could be inhibited by lowering CXCL1 expression,the astrocytes were transfected with miR-181c-3p mimic,inhibitor,or si RNA against CXCL1.Both RT real-time PCR and Western blot analysis were conducted to assess the expression of inflammatory factors in astrocytes,and ELISA was used to determine the expression of inflammatory factors in the supernatant of cell culture medium.A significant increase in the expression of inflammatory factors was found in astrocytes delivered with miR-181c-3p inhibitor.The presence of miR-181c-3p mimic or si RNA against CXCL1 in astrocytes led to a notable downregulation of inflammatory factors.Moreover,the expression of inflammatory factors was significantly decreased in astrocytes co-transfected with miR-181c-3p mimic and si RNA against CXCL1,but still significantly higher than those transfected with si RNA against CXCL1.To conclude,miR-181c-3p could suppress inflammatory response in astrocytes by inhibiting the expression of CXCL1.Summary:1.Exosomes released by cortical neurons inhibit the expression of astrocyte inflammatory factors and CXCL1.2.miR-181c-3p released by cortical neurons inhibited the expression of CXCL1 in astrocytes.3.miR-181c-3p inhibits astrocytic inflammation by down regulating the expression of CXCL1.Conclusions:1.After ischemic brain injury,the expression of CXCL1 and its downstream inflammatory factors in astrocytes increased.2.Cortical neurons can communicate with each other by secreting exosomes and endocytosis by astrocytes.3.Cortical neurons regulate the expression of CXCL1 and its downstream inflammatory factors by releasing miR-181c-3p in exosomes. |
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