Expression And Mechanism Of Cold-induced RNA-binding Protein During Cardiopulmonary Bypass

BACKGROUNDHypothermia provides neuroprotection and alleviates cerebral injury after cardiopulmonary bypass(CPB).The mechanism of cold-inducible RNA-binding protein(CIRP),which has been reported to be facilitated by hypothermia and act as a critical regulatory protein in the brain,remains unclear in CPB.Hence,the role of CIRP on hypothermia CPB-induced brain injury was investigated in a rat model.METHODSCirp-/-rats were generated using the transcription activator-like effector nucleases(TALEN)-based genome editing technique.CPB model was established in all rats after tail artery and right jugular vein cannulation.The angioplasty balloon catheter,which acted as an endoaortic crossclamp,was retrogradely inserted into the ascending aorta via the right common carotid artery.After cooling to a rectal temperature of 32℃,the endoaortic clamp was quickly inflated,while 0.9 ml of St Thomas’solution was concomitantly administered through the central lumen of the angioplasty catheter.After 30 minutes of cardioplegic arrest,the balloon was deflated and removed.CPB was discontinued when rats were rewarmed to a rectal temperature of 34.5℃ and the hemodynamics were stable.After ventilating for another 60 minutes,rats were sacrificed by decapitation.Arterial blood samples for blood gas analyses were collected just before CPB,before aortic cross-clamping,30 minutes after aortic cross-clamping,and immediately after the end of CPB.The animals were randomly allocated to three groups(n=5,each group):sham group,CPB group,and CB+Cirp-/-group(Cirp-/-group).Three biological replicates received RNA sequencing in the CPB and Cirpu-/-groups.The relative protein expression(CIRP,TGF-β1,MMP-9,TNF-α,IL-4,IL-5,IL-10,IL-13,MDA,Occ and ZO-1)of the hippocampus was detected.The integrity of the blood-brain barrier(BBB)was measured by transmission electron microscopy and IgG immunostaining.Glial fibrillary acidic protein(GFAP)in serum was detected.The brain was fixed for histopathological assessment(pathological score,surviving hippocampal neurons,and TUNEL-positive neurons).RESULTSCIRP protein significantly increased in the CPB group,when compared with the sham group(0.06 ± 0.01 vs 0.03±0.01;P=0.0008).More differentially expressed genes(DEGs)of BBB leakage were clustered functionally by GO and KEGG pathway analyses.TGF-β 1(273 ± 81 vs 169±65;P=0.018),MMP-9(0.62±0.12 vs 0.33±0.06;p<0.001),TNF-α(3.61±0.96 vs 2.12±0.35;P=0.003)and MDA(223 ± 43 vs 101 ±30;P=0.0001)in the hippocampus were higher in the Cirp-/-group,while IL-4 level was opposite(2.49±0.43 vs 3.69±0.73;P=0.002).Furthermore,more serious BBB disruption in the Cirp-/-group was demonstrated by transmission electron microscopy and IgG extravasation(6.36±0.68 vs 1.92±0.54;P<0.0001).Moreover,Cirp-/-enhanced the tight junction protein degradation(0.03±0.01 vs 0.08± 0.04 Occludin,and 0.04±0.01 vs 0.09±0.03 ZO-1;P=0.007,and 0.0058,respectively)and histopathologic injury in the hippocampus(3.0[2.0-3.0]vs 1.0[0.5-1.0]pathological score,and 802±216 vs 3369±564 Nissl staining,and 15.98±3.05 vs 0.70±0.14 TUNEL-positive cells;P<0.0001,<0.0001,and=0.0012,respectively).Therefore,CIRP significantly alleviated neurologic injury.CONCLUSIONCIRP exerted important neuroprotective effect by alleviating BBB breakdown which might be associated with TGF-β1-MMP-9 signals in hypothermia CPB.BACKGROUNDNeuroinflammation acts as a contributor to neurologic deficits following deep hypothermic circulatory arrest(DHCA).DHCA-induced microglial activation initiates the inflammatory process that aggravates neuronal damage.However,the molecular mechanism remains unclear.With Cirp knockout rats and Cirp small interfering RNA(siRNA)adenovirus vector,the present study is based on rat DHCA model and oxygen-glucose deprivation model and postulates that cold-inducible RNA-binding protein(CIRP),a newly discovered proinflammatory mediator,can promote DHCA-induced neuroinflammation.METHODSRats were randomly assigned into three groups(n=5,each group):sham group,DHCA group,and DHCA+Cirp-/-group(Cirp-/-group),Cardiopulmonary bypass(CPB)was established via the right external jugular vein-right atrium and tail artery.Rats were cooled to a rectal temperature of 18℃,followed by 30 minutes of DHCA with global ischemia.After rewarming for approximately 60 minutes,the animals were weaned from the CPB at 34℃.After ventilating for another 60 minutes,rats were sacrificed by decapitation.Arterial blood samples for blood gas analyses were collected just before CPB,before circulatory arrest,and at the end of CPB.The siRNA-CIRP or blank recombinant adenovirus were transfected into BV2 and subjected to 4-hour oxygen-glucose deprivation(OGD)after 48 hours.BV2 cells and culture supernatant in each group were harvested for further analysis.The conditioned medium was collected from the three groups of BV2 cells and added to SH-SY5Y cells.After 4-hour OGD,Annexin V-FITC/PI labeling was performed and the apoptotic cells were detected by flow cytometry.The brain was fixed for histopathological assessment(pathological score,surviving hippocampal neurons,and TUNEL-positiveneurons).The activation of microglia was evaluated by immunofluorescence staining of ionic calcium binding junction molecule(Iba)-1.The levels of tumor necrosis factor(TNF)-a,interleukin(IL)-5,IL-10 and IL-13 in hippocampal homogenate were detected by multi-factor kit.Three biological replicates received RNA sequencing in the DHCA and Cirp-/-groups.The mRNA content of Brd2 in hippocampus was measured with qPCR.The protein levels of CIRP,Brd2,NF-κB-p65 and I κBα in hippocampal homogenate and BV2 cell lysis were detected by Western Blot.The expression and distribution of CIRP and Brd2 in hippocampus and BV2 cells were observed by immunofluorescence.The content of TNF-α in the lysate and supernatant of BV2 cells was detected by enzyme-linked immunosorbent assay.The cytotoxicity of BV2 in each group was detected by MTT assay.RESULTSCIRP was elevated(0.42±0.04 vs 0.74±0.06,P<0.01)along with evident neuroinflammation and neuronal damage in rats exposed to DHCA.In Cirp-/-rats,histologic injury(3.00[Interquartile range,2.00-3.00]vs.1.00[Interquartile range,1.00-1.50]neuropathological score,260±53 vs 581±51 Nissl staining,and 21±12 vs 4±2 TUNEL-positive cells;P<0.001,=0.001,and 0.007,respectively)and microglial activation(40±4 vs.13±7 CA1 area,P<0.001)were alleviated after DHCA.With RNA-sequencing analysis,this associated with reduction of key pro-inflammatory cytokines induced by inhibiting Brd2-NF-κB signals(0.07±0.01 vs 0.03±0.01 Brd2,0.61±0.11 vs 0.43±0.08 NF-κ B-p65,and 0.05 ± 0.04 vs 0.21 ± 0.09 I κBα;p<0.001,=0.014,and 0.003,respectively).In BV2 cells treated with siRNA-CIRP,similar protective effects were observed,including decreased pro-inflammatory cytokines(73±11 vs 51±8 intracellular TNF-α,and 153± 25 vs 67±13 extracellular TNF-α;P=0.002 and<0.001,respectively)and cytotoxicity(1.58±0.15 vs 2.28±0.19;P<0.001).Brd2-NF-κ B signals were confirmed by the addition of Brd2 inhibitor JQ1(0.20±0.05 vs 0.33 ± 0.04 Brd2,0.53±0.16 vs 0.56±0.14 NF-κ B-p65,and 0.85±0.26 vs 0.82±0.27 I κ B α;P=0.213,0.827,and 0.749,respectively).Notably,the conditioned medium from BV2 cells transfected with siRNA-CIRP significantly reduced apoptosis in neural SH-SY5Y cells after OGD(40±8 vs 20±2;P<0.001),which was similar to that after JQ1 administration(40±8 vs 21±1;P<0.001).CONCLUSIONEnhanced CIRP in microglia aggravates neuronal injury by promoting the release of pro-inflammatory cytokines,which might be mediated through Brd2-NF-κ B signals during DHCA.