The Effect And Mechanism Of RCAN1.4 In Post-ischemic Inflammatory Response

BackgroudStroke is a leading cause of human death and long-term disability worldwide.Nearly 70%of stroke scenarios are classified as ischemic,as the consequence of sudden obstruction of criminal arteries,leading to the ischemia and hypoxia of involved brain tissue.As the population ages in the wordwide,the burden of ischemic stroke will be substantially increased on individuals and families.The timely restoration of blood supply in the acute phase is the most efficacious therapeutic strategy in contemporary clinical practice of ischemic stroke.However,the narrow therapeutic window and side effects severely restrict clinical application.Despite intensive researches carried out on the neuroprotective agents in recent years,unfortunately,none has been validated as beneficial in clinical application.Therefore,a thorough understanding of the underlying mechanisms about ischemic stroke becomes urgent,and a more potent treatment scheme is badly needed.The pathophysiology in ischemic stroke is complicated,with changes in multiple aspects such as energy failure,loss of cell ion homeostasis,acidosis,excitotoxicity,free radical-mediated toxicity,complement activation,activation of glial cells,disruption of the blood-brain barrier(BBB),infiltration of leukocytes and so on.These changes are interrelated and coordinated events,which can lead to ischemic injury.It is generally accepted that the postischemic inflammation induced by ischemic stroke contributes to brain damage.The acute stage of ischemic stroke lasts approximately 7 days and is characterized by infiltration of immune cells into the brain parenchyma and robust secretion of pro-inflammatory cytokines,which promotes activation of astrocyte and microglia in the brain and leads to ischemic injury.An ischemic stroke begins with a disruption in blood supply to a region of the brain,which results in an ischemic penumbra,an area of hypoxia and glucose deprivation.In the absence of reperfusion,the cells in this area die,leading to the development of the ischemic core.Dead and dying cells release a variety of damage-associated molecular patterns,DAMPs,which stimulate production of inflammatory cytokines and chemokines,recruiting leukocytes to the brain and worsening neurological outcome.The production of inflammatory cytokines and chemokines will activate the astrocyte and microglia in the brain.Activated microglia and astrocytes produce additional cytokines and chemokines,leading to a positive feedback and severe inflammatory cascade reaction.An inflammatory response can alleviate the brain damage in ischemic stroke by clearing dead cells and cell fragments,but excessive inflammatory reaction is harmful.Severe inflammatory cascade reaction lead to the destruction of blood-brain barrier in the brain,aggravation of brain edema,damage of vascular endothelial cells,downregulation of tight junction proteins,upregulated expression of adhesion molecules and increased vascular permeability.These events indicate that further studies are extremely needed to find more potent treatments to alleviate the severe inflammatory response in ischemic stroke.The regulator of calcineurin 1 gene(RCAN1)was first identified as the Down syndrome critical region 1(DSCR1)gene on chromosome 21q22,consisting of two major isoforms:the RCAN1 isoform 1(RCAN1.1)and the RCAN1 isoform 4(RCAN1.4).The typical role of RCAN1.1 in the nervous system has been proven.RCAN1.1 has been found to be increased in the brain of Down syndrome and Alzheimer’s disease,leading to neuronal apoptosis through the cascade3 and cascade 9 pathway.RCAN1.1 could stimulate the N-glycosylation in the endoplasmic reticulum.RCAN1.4 has also been recognized as playing important roles in diverse biological and pathological conditions.According to our previous studies,RCAN1.4 suppresses lymphoma growth in mice.In addition,RCAN1.4,which is downregulated in hepatocellular carcinoma,prevents the proliferation,migration,and invasion activity of cancer cells and the growth of orthotopic tumors by inhibiting nuclear translocation of NFAT1.RCAN1.4 displays antiangiogenic properties via an NFATc1-dependent pathway in ischemic retinal ganglion cells and RCAN1.4 helps to maintain a more fused mitochondrial network in cardiomyocytes during ischemia/reperfusion(I/R)damage.Nonetheless,the expression and distribution of RCAN1.4 in the brain have not been characterized in detail,and whether it plays a role in ischemic stroke has yet to be clarified.In the present study,the animal and cellular model of ischemic stroke were conducted to investigated the effects of RCAN1.4 on inflammatory response in ischemic stroke and the mechanisms involved,so as to provide a new target for ischemic stroke.Research Objectives1.To determine the expression changes of RCAN1.4 in the animal and cellular model of ischemic stroke.2.Experiments in vitro and in vivo were performed to investigate the function of RCAN1.4 on ischemic stroke and the mechanisms involved.3.Identify the upper key molecules related to the upregulation of RCAN1.4 in ischemic stroke.Materials and methods1.The expression of RCAN1.4 in the animal and cellular model of ischemic stroke The mouse model of middle cerebral artery occlusion(MCAO)was conducted to mimic ischemic stroke.2,3,5-triphenyltetrazolium chloride(TTC)staining and the laser speckle contrast imaging(LSCI)were performed to confirm the MCAO model.The ischemic brain was harvested at 12h and 24h after MCAO surgery.Primary astrocytes were isolated and the cellular model of oxygen and glucose deprivation(OGD)was conducted.Astrocytes were harvested at different indicated times after OGD treatment.Real-time quantitative PCR and western blotting were performed to detect the RCAN1.4 mRNA and protein in ischemic stroke.2.Effects of RCAN1.4 on the inflammatory response in the OGD-induced primary astrocytes.Recombinant lentiviruses Lenti-RCAN1.4-GFP,Lenti-shRCAN1.4-GFP,and their control viruses were conducted.Primary astrocytes were infected with the lentiviruses for 5 days,respectively.Infected cells were treated with OGD 6h and moved to normal condition for another 12h.Real-time quantitative PCR was used to detect the tumor necrosis factor α(TNFα)/Interleukin 1β(IL1β)/Interleukin 6(IL6)mRNA.Enzyme linked immunosorbent assay(ELISA)was performed to detect the secretion of TNFα/IL1β/IL6 in the supernatant and western blotting was also performed to detect the expression of COX2 and iNOS in the infected cells.3.Effects of RCAN1.4 on the nuclear translocation of NF-κB/p65 in the OGD-induced primary astrocytes.Primary astrocytes were infected with the lentiviruses for 5 days,respectively.Infected cells were treated with OGD 6h and moved to normal condition for another 12h.Western blotting and immunofluorescence analysis were performed to detect the effects of RCAN1.4 on the nuclear translocation of NF-KB/p65 in the OGD-induced primary astrocytes.To confirm the function of RCAN1.4 on NF-κB/p65,dual luciferase activity controlled by NF-κB was assessed under normal and OGD conditions.4.Effects of RCAN1.4 on the degradation rate of IκBα in the OGD-induced primary astrocytes.Primary astrocytes were infected with the lentiviruses for 5 days,respectively.Infected cells were treated with OGD 6h and moved to normal condition for another 12h.Western blotting was performed to detect the effects of RCAN1.4 on the protein levels of IKKβ,pIKKβ,Iκβα,pIκβα-S32/36 in the infected primary astrocytes.5.The function of RCAN1.4 in ischemic stroke was confirmed in vivo.Recombinant AAV6-related viruses,AAV6-GFAP-RCAN1.4 and AAV6-GFAP-CON,were conducted.We injected AAV6-GFAP-RCAN1.4/AAV6-GFAP-CON into the right brain of the mice by the stereotaxic apparatus.ELISA and western blotting were conducted to investigate the of RCAN1.4 on the post-ischemic stroke inflammation.TTC staining was used to assess the effect of RCAN1.4 on the infarct volume induced by MCAO.TUNEL staining was performed to detect the cell apoptosis in the MCAO model and neurological scores was also assessed to confirm the function of RCAN1.4 in ischemic stroke was confirmed in vivo.6.Regulation of HIF1α on the RCAN1.4 promoter.To uncover the molecular mechanism of RCAN1.4 gene transcription in ischemic stroke,a 1200-bp fragment from the 5’UTR of RCAN1.4(pRCAN 1.4 luc-Long)was amplified by PCR,cloned into the pGL3-basic vector and functionally analyzed.The cloned fragment was transfected into HEK293 cells,which were subjected to OGD for 0h,3h,and 6h after 48 h transfection.To further confirm the relationship between HIF1αand RCAN1.4 gene transcription,pRCAN1.4 luc-Long was also co-transfected into HEK293 cells together with siHIF1α or siCON.Western blotting and dual luciferase assay were conducted to investigate the regulation of HIF1αon the RCAN1.4 promoter.7.Identify the specific HRE on the RCAN1.4 promoterFirstly,a series of RCAN1.4 promoter truncations were conducted by subcloning the RCAN1.4 promoter deletions into the pGL3-Basic vector.Dual luciferase assay was uesd to detect the transcriptional regulation of these truncations by HIF1α.The JASPAR database was used to predict the possible HRE on the RCAN1.4 promoter.And the mutant truncation of the possible HRE on the RCAN1.4 promoter was also conducted to confirm the specific HRE.Electrophoretic Mobility Shift Assay(EMSA)and Chromatin Immunoprecipitation(CHIP)were performed to further confirm the functional HRE on the RCAN1.4 promoter.ResultsSection 1 RCAN1.4 alleviates the inflammatory response in ischemic stroke via inhibition of the NF-κB signaling pathway1.RCAN1.4 expression was significantly increased in both animal and cellular models of ischemic stroke.The mouse model of MCAO and the cellular model of OGD were conducted to mimic ischemic stroke.The ischemic brain and cell were harvested to investigate the changes of the RCAN1.4 RNA and protein.In the results,we found that RCAN1.4 expression was significantly increased in both animal and cellular models of ischemic stroke and RCAN1.4 was mainly expressed in astrocytes in the brain.2.RCAN1.4 alleviated the inflammatory response in the OGD-induced primary astrocytes.We found that the mRNA levels of TNFα,IL1β,and IL6 increased significantly at 12h after OGD treatment,and these effects were markedly alleviated by RCAN1.4 overexpression.Furthermore,elevated secretion of TNFα,IL1β,and IL6 in the supernatant at 12h after OGD treatment was found by ELISA,which was also inhibited by overexpression of RCAN1.4.Expression of COX2 and iNOS in unstimulated and OGD-treated primary astrocytes was detected by western blotting and the results showed that under OGD conditions,expression of COX2 and iNOS in Lenti-RCAN1.4-GFP-treated astrocytes was much lower than that in control(Lenti-CON-GFP)-treated astrocytes.Opposite results were obtained in the Lenti-shRCAN1.4-GFP-treated astrocytes.These results indicated that RCAN 1.4 alleviated the inflammatory response in the OGD-induced primary astrocytes.3.RCAN1.4 inhibits the nuclear translocation of NF-κB/p65 in the OGD-induced primary astrocytes.The results showed that the nuclear NF-κB/p65 was sharply increased after OGD treatment,compared with that in the control group(non-OGD treatment)and RCAN1.4 overexpression inhibited the NF-κB/p65 nuclear translocation in astrocytes subjected to OGD,while RCAN 1.4 knockdown promoted the NF-κB/p65 nuclear translocation.Additionally,immunofluorescence analysis of NF-κB/p65 in primary astrocytes was also performed to consolidate the conclusion.To confirm the function of RCAN1.4 on NF-κB/p65,dual luciferase activity controlled by NF-κB was assessed under normal and OGD conditions,and similar results were obtained.These results indicated that nuclear NF-κB/p65 was significantly inhibited by RCAN1.4 overexpression.4.RCAN 1.4 inhibits the degradation of IκBα in the OGD-induced primary astrocytes.The increased protein levels of pIKKβ and pIκBα-S32/36 and enhanced degradation of IκBα(an inhibitor protein of NF-κB/p65)were found in the OGD-induced primary astrocytes,compared with that in the control group(non-OGD treatment).Overexpression of RCAN1.4 inhibited the OGD-upregulated pIKKβ and p IκBα-S32/36,leading to decreased degradation of IκBα in primary astrocytes and the opposite effect was observed when RCAN1.4 was knocked down.5.RCAN1.4 alleviated the inflammatory response and the infarct volume in the MCAO model of ischemic stroke.To further confirm the functional role of RCAN1.4 in ischemic stroke in vivo,we injected the AAV6-GFAP-RCAN1.4/AAV6-GFAP-CON into the right brain of the mice by the stereotaxic apparatus.We observed that the decreased expression of TNFα and IL1β in the ischemic brain was found by ELISA after RCAN1.4 overexpression.Similar results were obtained in the expression of COX2 and iNOS protein.We also found that RCAN1.4 markedly reduced the infarct size induced by MCAO as shown by TTC staining.Furthermore,RCAN1.4 alleviated cell apoptosis at 24 h after MCAO as shown by TUNEL staining.In addition,the downregulation of neurological scores was also observed in the group of RCAN1.4,comparing to the control group.Collectively,our results indicated that RCAN1.4 played a critical role in regulating the post-ischemic inflammatory response and thus alleviated the cerebral ischemic injury in vivo.Section 2 HIF1α upregulates the transcriptional activity of RCAN1.41.HIF1α specifically activates the RCAN1.4 promoter.In the section 1,we found that the RCAN1.4 expression was significantly upregulated both animal and cellular models of ischemic stroke.To uncover the molecular mechanism of RCAN1.4 gene transcription in ischemic stroke,a 1200-bp fragment from the 5’ UTR of RCAN1.4(pRCAN1.4 luc-Long)was amplified by PCR,cloned into the pGL3-basic vector and functionally analyzed.The dual luciferase assay revealed that the relative luciferase activity(RLU)of pRCAN1.4 luc-Long was also increased by OGD,consistent with results of HIF1α protein.The RLU of pRCAN1.4 luc-Long was significantly decreased when the HIF1α protein was knocked down.The above results suggest that HIF1α specifically activates the RCAN1.4 promoter under OGD conditions.2.-254bp～-245bp of the RCAN1.4 promoter was a HRE site.To further investigate the location of the HRE in the RCAN1.4 promoter,a series of truncation plasmids were constructed by deletion of pRCAN1.4 luc-Long,as follows:pRCAN1.4 luc-B(-860 to-15 bp),pRCAN1.4 luc-C(-576 to-15 bp),and pRCAN1.4 luc-D(-315 to-15 bp).The dual luciferase assay showed that the region-315 to-15 bp of the RCAN1.4 promoter contains the HRE site.Computer-based sequence analysis of this-315 to-15 bp fragment showed a putative HRE site at-254 to-245 bp.To verify this result,the mutation of pRCAN1.4 luc-D(pRCAN1.4 luc-Dmut)were constructed by substitution of the HRE wild-type sequence with an HRE mutant sequence.Compared with the empty vector,the RLU of pRCAN1.4 luc-D,but not that of pRCAN1.4 luc-D mut,was significantly elevated by HIF1α.These results demonstrate that the HRE at-254 to-245 bp is the functional HRE site in the RCAN1.4 promoter.3.EMSA assay and ChIP assay were conducted to confirm that the functional HRE was located between-254～-245 bp in the RCAN1.4 promoter region.ConclusionsAccording to the results above,we clarified the RCAN1.4 expression and the molecular mechanism of regulating the inflammatory response in ischemic stroke and drew the following conclusions:1.RCAN1.4 expression was significantly increased in both animal and cellular models of ischemic stroke.2.RCAN1.4 exerts protective effects in ischemic stroke by alleviating the inflammatory response via inhibition of the NF-κB signaling pathway.3.RCAN1.4 transcription was specifically activated by HIF1α through a specific HRE in the RCAN1.4 promoter.The functional HRE was located between-254 and-245 bp in the RCAN1.4 promoter region.SignificanceIn the study,we prove that RCAN1.4 is markedly upregulated in both animal and cellular model of ischemic stroke.RCAN1.4 exerts protective effects in ischemic stroke by alleviating the inflammatory response via inhibition of the NF-κB signaling pathway.Mechanistically,RCAN1.4 transcription is specifically activated by HIF1α through a specific HRE in the RCAN1.4 promoter.The functional HRE is located between-254 and-245 bp in the RCAN1.4 promoter region.These results indicate the important role of RCAN1.4 in alleviating the inflammatory response and RCAN1.4 could be a potential therapeutic target for the treatment of ischemic stroke.