The Role Of Interferon Regulatory Factor 4 In Cerebral Ischemia-reperfusion Injury

BackgroundStroke is one of the most devastating and severe diseases worldwide. It is characterized by high morbidity, mortality and disability. In China, more than 2 million new-onset stroke patients were diagnosed a year. And more than 1.5 million people die each year because of stroke. Stroke has the highest morbidity and mortality our country. As Chinese epidemiological survey show, ischemic stroke is the predominant type of stroke(account for 66.4%). The ischemic stroke severely damages the neuronal cells and even results in cell death in a short time. After the onset of cerebral ischemia, the blood supply of brain tissue was interrupted, which leading to neural hypoxia-ischemia injury. So re-opening of the blocked cerebral vessels by thrombolysis is the most important therapeutic principle. But, the aggravated neuronal damage will be induced by cerebral ischemia-reperfusion. This is a cause of poor prognoses in stroke patients. The incidence of ischemia-reperfusion injury is increasing greatly, because of a growing number of stroke patients accept reperfusion therapy. Therefore, it is vital to prevent the ischemia-reperfusion injury. Neuroprotection could rescue the neurons from death. It is one of the important therapy for cerebral ischemia-reperfusion. Nevertheless, the clinical outcomes are unsatisfactory. Therefore, elucidate the molecular mechanisms of the cerebral ischemia-reperfusion injury and neuronal death may lead to novel strategy for ischemic stroke.Interferon regulatory factor 4(IRF4) belongs to Interferon regulatory factor family. There are nine family members in mammals. IRF4 possess an amino(N)-terminal DNA binding domain(DBD) which is common in all family members. This region could recognize the IFN-stimulated response element(ISRE) which is a consensus DNA sequence containing 5'-GAAA-3'core. The regulatory domain named IRF-associated domain(IAD1) is found in carboxy(C)-termial. This domain mediates interactions between IRF4 and other transcriptional factors(including other IRF family members). IRF4 is a very important transcription factor which plays a pivotal role in immune response. The B-cells, T lymphocytes and dendritic cells differentiation require IRF4. It is also participate in the regulation of autoimmune diseases and malignancy. Recent studies revealed that IRF4 plays role in regulating adipose metabolism and myocardial hypertrophy. Interestingly, IRF4 was involved in the ischemia/reperfusion induced renal injury. In this study, we observed the IRF4 was expressed by neurons and significant up-regulated after cerebral ischemia-reperfusion. Therefore, we speculated that IRF4 plays an important role in cerebral ischemia-reperfusion injury. So our study established the transient middle cerebral artery occlusion(MCAO) mice model to mimic the cerebral ischemia-reperfusion injury. Using neuron-specific IRF4 knockout and transgenic mice to elucidate the molecular biological mechanisms.AimsThe objective of this study was to evaluate the effect of IRF4 in cerebral ischemia-reperfusion injury, and elucidate the mechanisms.MethodsPart ?:Male C57BL/6 mice aged 11?12 weeks and weighted 25?30 g were subjected to transient middle cerebral artery occlusion(MCAO) for 45 minutes. Detected the IRF4 protein in mice brain tissues before MCAO,2 h,6 h,12 h,24 h or 72 h after MCAO respectively. In vitro manipulation, to obtain primary cortical neurons, the brain of Sprague Dawley rats within 1 day were removed. The neurons were subjected to transient oxygen and glucose deprivation(OGD) for 60 min. The IRF4 protein was examined before OGD,3 h,6 h,12 h or 24 h after OGD. Western Blot and immunofluorescence staining were used in this part.Part ?:The neuron-specific IRF4 konckout mice(IRF4-KO) were generated by crossing IRF4flox/flox mice with CaMKIIa-Cre mice. The WT or IRF4-KO mice(11-12 w, 25?30 g) were divided into Sham or MCAO group respectively. The neurological deficit scores, TTC staining and MRI were used for evaluating the level of impairment. The markers of apoptosis were evaluated by Western Blot and immunofluorescence staining. The cultured cortical neurons transfected with AdshRNA or AdshIRF4 were used to detected the cell viability and LDH release.Part ?:The full-length mouse IRF4 cDNA was inserted into the CAG-loxp-CAT-loxp cassette, and the chloramphenicol acetyltransferase (CAT) gene was flanked by loxP sites. The construct was microinjected into fertilized embryos (C57BL/6J background) to produce IRF4-floxed mice. IRF4-floxed mice crossing with CaMKII?-Cre mice to produce neuron-specific IRF4 transgenic mice(IRF4-TG). Divided NTG or IRF4-TG mice into Sham or MCAO group respectively. Evaluated the level of impairment through neurological deficit scores, TTC staining or MRI. Immunofluorescence staining and Western Blot were used for detecting protein expression. Cell viability and LDH release were examined in cultured cortical neurons transfected with AdGFP or AdIRF4.Part ?:After MCAO, brain tissues form WT or IRF4-KO mice were detected through the RAN-seq technology, and screened the downstream genes of IRF4. Then test again by RT-PCR and Western Blot. The AdshRAN, AdshRIR4, AdGFP or AdIRF4 transfected neurons underwent OGD. Then assessed the SRF promoter luciferase activity of these neurons by Dual-Luciferase Reporter Assay System. Built the mutant murine SRF promoter and detected its promoter luciferase activity. The IRF4 binding site were detected through Chromatin immunoprecipitation.Part V:The neurons were transfected by AdSRF, AdshSRF, AdshIRF4+AdSRF or AdIRF4+AdshSRF. Detected the cell viability and LDH release after OGD. The SRFflox/flox mice were crossed with CaMKII?-Cre mice to generate neuron-specific SRF knockout mice(SRF-KO), then crossed the SRF-KO mice with IRF4-TG mice to produce IRF4-TG/SRF-KO mice which knocked out SRF and over-expressed IRF4 both. After MCAO, the neurological deficit scores, TTC staining, Immunofluorescence staining and Western Blot were examined.ResultsPart I:The neuronal IRF4 expression was elevated both in vivo and in vitro. The mice suffered MCAO for 45 min, and recovered perfusion. The IRF4 protein expression was increased in a time-dependent manner from 2 h-72 h after MCAO. The IRF4 staining was stronger in the ipsilateral than contralateral, and the IRF4 was co-expressed with NeuN. Moreover, the ipsilateral cortex and striatum had more IRF4 positive neurons. But the IRF4 straining in bilateral hippocampal was no difference. The primary cortical neurons subjected to OGD for 1 h, and followed by normoxia culture. The IRF4 expression in neuronal was elevated gradually after OGD.Part II:The cerebral ischemia-reperfusion injury was aggravates by neuron-specific IRF4 deficiency. WT and IRF4-KO mice were subjected to MCAO(45 min). Neurological deficit scores and TTC staining of brain were evaluated at 24 h or 72 h. Compared with WT group, the IRF4-KO mice presented an increase in neurological deficit scores of 57.14%(24h) and 49.33%(72h), P<0.05. The infarct volumes were enlarged by 74.27%(24h) and 48.40%(72h), P<0.05. The MRI exhibited about 1.7 fold increase of infarct volume in IRF4-KO mice, P<0.05. Both Fluoro-Jade B and TUNEL positive cells significantly increased in IRF4-KO mice, P<0.05. In IRF4-KO mice the pro-apoptotic protein(Bax and Cleaved caspase3) were elevated, but the anti-apoptotic Bc12 was reduced markedly. Compared with cotrol group, AdshIRF4 rendered the neurons more prone to OGD induced death in vitro. The cell viability was decreased, but LDH release was increased significantly in AdshIRF4 group, P<0.05.Part ?:IRF4 overexpression attenuated post-ischemic injury. The IRF4-TG mice exhibited reduction of neurological deficit scores(38.10%at 24 h or 41.18%at 72 h after MCAO). The infarct volume in IRF4-TG mice was decreased by 52.71% at 24 h and 69.36% at 72 h after MCAO,P<0.05. The MRI of brain in IRF4-KO mice displayed an dramatically reduced infarct volume, P<0.05. IRF4 overexpression reduced the Fluoro-Jade B and TUNEL positive cell number, P< 0.05. The pro-apoptotic protein Bax and Cleaved caspase3 were decreased, but the anti-apoptotic Bc12 was increased dramatically in IRF4-TG mice P<0.05. In vitro study, AdIRF4 group's cell viability was declined, and the LDH release increased obviously in AdshIRF4 group, P<0.05.Part IV:IRF4 mediates neuroprotection through SRF. The RNA-seq revealed that the mRNA levels of BDNF, FosB, Egrl, et al were dramatically decreased in IRF4-KO mice after MCAO. Moreover, the SRF and downstream factors detected by RT-PCR in IRF4-KO mice were obviously lower than WT mice. The Western Blot and immunofluorescence analysis showed that SRF and downstream factors decreased in IRF4-KO mice and increased in IRF4-TG mice compared with their control groups respectively. After OGD, the neuronal SRF promoter luciferase activity was decreased in AdshIRF4 transfected cells or increased in AdIRF4 transfected cells, compared with each control groups, P<0.05. The mutant SRF promoter activity was significantly decreased in AdIRF4 transfected cells. ChIP assay demonstrated that IRF4 directly binded to P3 region of SRF promoter which contained 5'-GAAA-3'sequence.Part V:The neuroprotection of IRF4 is SRF dependent. AdSRF increased viability of OGD challenged neurons. When AdshIRF4 transfected neurons co-infected with AdSRF, the reduced of cell viability was negated by AdSRF. Conversely, the enhanced cell viability of AdIRF4 was reversed by AdshIRF4. The SRF-KO mice displayed an significantly increase in neurological deficit scores and infarct volume compared control group. Intriguingly, the IRF4-TG/SRF-KO mice and SRF-KO mice had similar neurological deficit scores and infarct volume(P>0.05).Conclusion1. The ipsilateral IRF4 expression is significantly increased after cerebral ischemia-reperfusion, and the IRF4 is induced in neurons.2. IRF4 overexpression is neuroprotective, and reduces the infarct volume after MCAO. whereas IRF4 deficiency aggravates cerebral damage.3. The cerebroprotective effects of IRF4 is dependent on SRF. The activation of SRF and its downstream factors protect the neuronal cells. While, SRF deficiency reverse the function of IRF4. So SRF is the key factor of IRF4 mediates neuronal protection.