To Explore The Molecular Mechanism Of CARD3-mediated Cerebral Ischemia-reperfusion Injury

Background Following cardiovascular disease and cancer,ischemic stroke is third causes that severely threaten public health and life.With the increasing aging population in China,the annual incidence of ischemic stroke is also increasing accordingly.Once attacked by ischemic stroke,neurons will encounter irreversible death in a short period,with leaving permanent sequelae or serious life-threatening.Therefore,to recover cerebral blood flow as soon as possible is the primary principle for treating ischemic stroke.However,ischemia-reperfusion(I-R)injury after restoring blood supply,usually aggravates neuron damage and worsen neurological dysfunction.Therefore,prevention of ischemia-reperfusion injury is critical to improve the prognosis of ischemic stroke.Related-studies have shown that ischemia-reperfusion injury is involved in a variety of complex processes,including inflammation,apoptosis,oxidative stress,cell excitotoxicity,acidosis,ion imbalance,and so on.CARD3 protein is a serine/threonine protein kinase,which is widely expressed in human tissues,such as spleen,peripheral blood leukocytes,placenta,brain and heart.The C-terminal CARD domain plays an important role in regulating other proteins through CARD-CARD interaction.Recent reports have demonstrated that CARD3 can activate NF-κB and MAPKs signaling pathways to regulate cellular inflammation and apoptosis,which plays an important role in innate and adaptive immunity,inflammatory diseases,tumors,and stroke.For example,durning myocardial ischemia-reperfusion injury,CARD3 may up-regulate the above pathways to induce myocardial death.Moreover,CARD3 also induces ischemia-anoxia injury in neurons through activation of caspase-1.Therefore,we proposed the hypothesis that CARD3 might exert an important role in cerebral ischemia-reperfusion injury.Objective By establishing t MCAO mouse model,we are going to explore the role of CARD3 in cerebral ischemia-reperfusion injury and its possible mechanisms in ischemia-reperfusion injury via using CARD3 transgenic mice(CARD3-TG)and CARD3 knockout mice(CARD3-KO),which will provide a new therapeutic target for prevention of cerebral ischemia-reperfusion injury.Method PartⅠ: Male C57BL/6 mice with 10-12 weeks were enrolled to establish a cerebral ischemia-reperfusion model by transiently occluding the left middle cerebral artery for 45 minutes and then restoring cerebral blood flow.In vivo,the CARD3 expression of CARD3 was determined by Western Blotting after reperfusion for 2h,6h,12 h,and 24 h.Immunofluorescence staining was performed to detect the CARD3 expression in cortex and straitum after I-R 24 h.In vitro,the primary cortex neurons,isolated from SD rat embyo,were subjected to oxygen glucose deprivation(OGD)stimuli for 1h,and then returned to normal culture.Western Blotting and immunofluorescence assays were also used to detect CARD3 expression in neurons.Part Ⅱ: 10-12 weeks of male CARD3 knockout mice(CARD3-KO)and wild type mice(WT)were enrolled in this part.All mice were randomly divided in diferent group: sham group and I-R group.The infarct volunmes after I-R 24 h or 72 h were analysized by TTC(2,3,5-triphenyltetrazolium chloride)staining,and neurological deficits scores were also evaluated.Fluoro Jade B staing and Tunel assays were used to determine the neuron damage.Q-PCR and Western Blotting were used to determin the expression level of apoptosis,inflammation and oxidative stress factors.In vitro,primary neurons,transfected with Adsh CARD3 or Adsh RNA were subjected to OGD,difference in LDH release and cell viability were compared in different group.Part Ⅲ: To generate CARD3-overexpression mice(CARD3-TG),full-length CARD3 c DNA was inserted into downstream of the neural-specific PDGF B-chain promoter and then micro-injected into fertilized egg.NTG mice were served as control group.CARD3-TG and NTG mice were randomly divided into sham group and I-R group and then enrolled to performed t MCAO model.The degree of I-R injury was measured by TTC staining and neurological dysfunction scores.Fluoro Jade B staing and Tunel assays were used to determine the neuron damage.Q-PCR and Western Blotting were used to determin the expression level of apoptosis,inflammation and oxidative stress factors.Part Ⅳ: The effects of CARD3 knockdown or overexpression on activation of JNK,p38,ERK signaling pathway and its upstream regulatory molecule TAK1 in MAPKs family after cerebral ischemia-reperfusion were detected by immunoblotting.Part Ⅴ: NTG and CARD3-TG mice were intravenously administrated with TAK1 inhibitor 5Z-7-oxozeaenol solution,and DMSO serving as control solution begore t MCAO procedures.TTC staing and neurological deficits scores were performed to evaluate the role of TAK1 inhibitor on CARD3-mediating I-R injury.After reperfusion for 6 hours,the protein levels of inflammation,apoptosis,and oxidative stress were analysized with Western Blotting.Results Part Ⅰ: In vivo,the expression of CARD3 was significantly increased in a time-dependent manner.Immunofluorescence staings showed that the number of CARD3-positive cells in cortex and striatum of the ischemia hemisphere were much more than that in the contralateral hemisphere,p<0.05.And in vitro,the expression of CARD3 in primary neurons was also significantly increased after OGD stimulation in a time-dependent manner.Part Ⅱ: CARD3 gene knockout reduces cerebral ischemia-reperfusion injury in mice.TTC staining at 24 h and 72 h after ischemia-reperfusion showed that compared with WT group,the infarct size of the CARD3-KO group decreased by 42.18% and49.27% at 24 h and 72 h,respectively,p<0.05,and the neurological function score declined by 40.91% and 31.43% in CARD3-KO mice,p<0.05.The Fluoro Jade B and Tunel staining for 24 h after infarction showed that the number of Fluoro Jade B and Tunel positive cells in CARD3-KO group was significantly lower than that in WT group,p<0.05.In vitro,compared with neurons transfected Adsh RNA,the primary neurons transfected with Adsh CARD3 group showed decreasing LDH release and increasing cell viability after OGD stimulation for 12 h.The results of Q-PCR and Western Blotting showed that the expression of Bax,Bid and caspase-3 in the CARD3-KO group was lower than that in the WT group;however,the expression of Bcl2 was higher than that in WT group;the level of p-IKKβ and p-p65 was significantly lower in the CARD3-KO group;otherwise,the degradation of IKBa was slightly lower than that in the WT group.The anti-oxidants HO-1,Nrf-2,and SOD1/2were significantly elevated in CARD3-KO.And the pro-oxidants p47-phox,p67-phox,and gp91-phox were down-regulated in the CARD3-KO group.Part Ⅲ: Overexpression of CARD3 aggravated cerebral ischemia-reperfusion injury in mice.TTC staining at 24 h and 72 h after ischemia-reperfusion showed that the infarct areas of the CARD3-TG mice were increased by 67.01% and 58.04% at 24 h and 72 h,respectively,compared with NTG group,and the neurological function score increased by 32.26% and 29.79,respectively,p<0.05.Fluoro Jade B and Tunel staining showed that the number of Fluoro Jade B and Tunel positive cells in the CARD3-TG group was significantly higher than that in the NTG group,p<0.05.The expressions of pro-apoptosis-related proteins Bax,Bid,and c-caspase-3 was higher in CARD3-TG group than that in NTG group.The expression of Bcl2 was lower than that in NTG group.The levels of p-IKKβ and p-p65 were significantly higher in the CARD3-TG group than in the NTG group,while the degradation of IKBa was larger than NTG mice.In addition,the anti-oxidants HO-1,Nrf-2,and SOD1/2 were significantly down-regulated in CARD3-TG;while the pro-oxidants,including p47-phox,p67-phox,and gp91-phox were up-regulated in the CARD3-TG group.Part Ⅳ: Western Blotting results showed that phosphorylation levels of p38 and JNK were significantly decreased in the CARD3-KO group compared with WT group;whereas ERK did not change significantly.In contrast,when CARD3 was overexpressed,p38 and JNK phosphorylation levels significantly enhanced.The expression of p-TAK1 was also inhibited in the CARD3-KO group but extensively enhanced in CARD3-TG.Part Ⅴ: Intravenous injection of 5Z-7-oxozeaenol before t MCAO procedure significantly inhibited the expression of p-TAK1.TTC staings results showed that TAK1 inhibitors significantly reduced the enlargement of cerebral infarction caused by CARD3 overexpression compared with DMSO.Mechanistically,the upregulated phosphorylation levels of JNK and p38 caused by CARD3 overexpression were significantly inhibited when pre-treated with 5Z-7-oxozeaenol other than DMSO.The same results were represent in the inflammation-related factors p-IKKβ,p-p65 and pro-oxidants p47-phox and P67-phox,while anti-peroxidase HO-1 and SOD1 expression were significantly up-regulated after 5Z-7-oxozeaenol administration.Conclusion(1)CARD3 is involved in cerebral ischemia-reperfusion injury,and its expression is up-regulated after ischemia-reperfusion injury in a time-dependent manner.(2)CARD3 knockout extenuate apoptosis,inflammation and oxidative stress after ischemia-reperfusion injury.(3)Overexpression of CARD3 aggravates apoptosis,inflammation and oxidative stress induced by I-R injury.(4)CARD3 promotes cerebral ischemia-reperfusion injury via activation of TAK1.