Anti-inflammatory Effect And Mechanism Of ZC3H12D In Cerebral Ischemia-reperfusion Injury

Cerebral ischemia reperfusion injury(CIRI)is a secondary injury caused by Cerebral ischemia reperfusion.CIRI can cause edema and necrosis of nerve cells,and lead to severe neurological dysfunction.Saving nerve cells in time is the key to cure cerebral ischemia-reperfusion injury.ZC3H12D gene is a member of the CCCH type zinc finger protein family,which plays an important role in tumor and inflammation.In previous studies,we found throμgh high-throμgh put second-generation sequencing technology that the expression changes of ZC3H12D gene in CIRI may be involved in the occurrence and development of CIRI disease.while the role of ZC3H12D in CIRI is needs to be further studied.Therefore,we intend to establish in vitro and in vitro cerebral ischemia-reperfusion injury models and use biotechnological methods such as immunofluorescence co-staining,qRT-PCR,Western Blotting and ELISA to probe into the anti-inflammatory effect of ZC3H12D in CIRI from different aspects of phenomenon,function and mechanism.This study provides a theoretical basis for the clinical treatment of cerebral ischemia-reperfusion injury.PartⅠ Expression changes of ZC3H12D and P65 protein in brain tissue of rats with cerebral ischemia-reperfusion injuryObjectives:Morphological changes of cortical neurons in rats with MCAO/R(Middle Cerebral Artery Occlusion/Reperfusion)injury were observed,and the expression trends of ZC3H12D and P65 genes in the infarct area and brain tissue were detected.Methods:1.Establish cerebral ischemia-reperfusion injury model:30 male SD rats were randomly divided into 3 groups:control group(control group),sham operation group(sham group),cerebral ischemia-reperfusion injury(MCAO/R group).The Zea-longa suture method was used to establish a rat model of cerebral ischemia-reperfusion injury.The Zea-Longa grading score was used to screen the successfully modeled rats.2.After MCAO 2h/R 24h,the samples were collected and stained with TTC,and then the infarction of brain tissue was observed.3.Nissl staining(thionine)was used to observe the changes of the number of Nissl bodies in neurons.Immunofluorescence was used to detect neuronal apoptosis and cell type of ZC3H12D expression localization4.Observe the changes in the expression of ZC3H12D and P65 in the cerebral cortex after MCAO/R.Western Blotting detects the protein relative expression of ZC3H12D and P65.Results:1.MCAO/R cerebral ischemia-reperfusion injury model was successfully established in rats.A clear white infarct was observed on the right side of the brain after TTC staining.2.ZC3H12D was co-stained with cerebral cortical neurons by double immunofluorescence staining3.After MCAO/R,the Nissl bodies of cerebral cortex neurons decreased(P=0.0022)and the number of apoptotic cells increased significantly(P<0.0001).4.The expression levels of ZC3H12D and P65 in the cerebral cortex of MCAO/R increased significantly(P<0.05).Conclusions:1.ZC3H12D is expressed in cerebral cortical neurons.2.After MCAO/R,the number of cerebral cortex neurons is significantly reduced,and the number of apoptotic cells is significantly increased.3.MCAO/R increases the expression of ZC3H12D and P65 in the cerebral cortex.Part Ⅱ Effects of ZC3H12D on the inflammatory response and apoptosis of PC12 cells injured by oxygen and sugar deficiency/reoxygenationObjectives:To investigate the effects of ZC3H12D on the inflammatory response and apoptosis of PC 12 cells after OGD/R modeling.Methods:1.Clarify the time point of OGD/R.The optimal reoxygenation time point was determined by cell morphology observation,cell count,qRT-PCR detection of ZC3H12D mRNA expression,etc.2.The efficiency of lentivirus transfection m/sh-ZC3H12D was detected.The protein expression of transfected ZC3H12D gene was detected by immunofluorescence and Western Blotting to determine whether the transfection was successful.3.Detect cell viability after overexpression of ZC3H12D.CCK8 method was used to detect cell viability after over-expression of ZC3H12D.4.The effect of overexpression of ZC3H12D on NF-icB P65 nucleoprotein was tested.Detect the expression level of ZC3H12D protein in each group by Western Blotting.5.Detect the effect of overexpression of ZC3H12D on inflammatory factors.Detect the expression levels of inflammatory factors mRNA and molecules by qRT-PCR and ELISA.6.Detect the effect of overexpression of ZC3H12D on cell apoptosis and apoptotic proteins.The apoptosis rate of each group was detected by flow cytometry,and the expression of anti-apoptotic protein Bcl-2 and apoptosis protein Bax and Caspase-3 were detected by Western Blotting.Results:1.Compared with the Control group,the expression level of ZC3H12D mRNA at 3h after OGD/R reoxygenation was more significant than that at 6h;2.CCK8 detection found that cell viability increased after lentivirus transfection and overexpression of ZC3H12D(P=0.0003);3.Overexpression of ZC3H12D can reduce the expression of nuclear protein NF-κB P65 in cells,and reduce the expression of inflammatory factors IL-1β,IL-6,TNF-α and NF-κB;4.Overexpression of ZC3H12D can reduce the rate of cell apoptosis and increase the anti-apoptotic protein Bcl-2 and reduce the expression of apoptotic proteins Bax and Caspase-3.Conclusions:1.Overexpression of ZC3H12D may reduce the expression of inflammatory factors IL-1β,IL-6,TNF-α and NF-κB by inhibiting the NF-κB signaling pathway,thereby protecting cells from OGD/R injury;2.Overexpression of ZC3H12D can reduce the rate of cell apoptosis after OGD/R,increase the expression of anti-apoptotic proteins and inhibit apoptotic albumin.It shows that ZC3H12D can also protect cells throμgh anti-apoptotic effects.Part Ⅱ ZC3H12D plays an anti-inflammatory role in cerebral ischemia-reperfusion injury by regμLating the NF-κB signaling pathway and decreasing the mRNA stability of inflammatoryfactorsObjectives:To observe the effect of NF-κB signaling pathway on the expression of inflammatory factors and effect of ZC3H12D interference on the mRNA stability of inflammatory cytokines.Methods:1.After TNF-α activates the NF-κB signaling pathway,we observe the activation of P65 nucleoprotein and nuclear expression:we detect the relative amount of P65 protein by Western Blotting,and detect NF-Activation of κB signaling pathway;2.P65 nuclear expression:immunofluorescence detection of P65 and nuclear co-staining,fluorescence detection to determine the nuclear expression of P65 nucleoprotein in each group and compare them;3.Observe the influence of TNF-α on the expression of inflammatory factors after activating the NF-κB signaling pathway:we analyze the changes of inflammatory factors IL-1β,IL-6,TNF-α and NF-κB in each group by qRT-PCR and ELISA;4.Observe the effects of overexpression of ZC3H12D and inhibition of ZC3H12D expression on the stability of inflammatory factors mRNA:qRT-PCR was used to detect the inflammatory factors IL-1β,IL-6,and TNF-α in each group at 0 h,1 h,3 h,and 6 h And NF-κB mRNA levels.Results:1.TNF-α activates the NF-κB signaling pathway and increases the expression of P65 nucleoprotein.Overexpression of ZC3H12D can partially reverse the effect of TNF-α(P<0.05);2.TNF-α can increase the amount of P65 that enters the nucleus,and overexpression of ZC3H12D can reduce the amount of P65 in the nucleus(P<0.05).3.Compared with TNF-α+ overexpression ZC3H12D group,overexpression ZC3H12D group inflammatory factors IL-1β,IL-6,TNF-α and NF-κB mRNA levels and molecμLar levels decreased(P<0.05);4.Compared with the interfering ZC3H12D group,overexpression of ZC3H12D gradually reduced the ratio of inflammatory factors IL-1β,IL-6,TNF-α and NF-κB mRNA levels over time(P<0.05).Conclusions:1.Overexpression of ZC3H12D can reduce the expression of inflammatory factors by inhibiting the activation of NF-κB signaling pathway;2.Overexpression of ZC3H12D can inhibit the level of inflammatory response by reducing the stability of inflammatory factor mRNA.