| As a common cerebrovascular disease,cerebral hemorrhage has the characteristics of high incidence rate,high disability rate and high mortality,which seriously threatens human health.At present,there is no effective treatment for this subtype of stroke with high mortality and high disability rate.Controlling hypertension and other risk factors is the main method to prevent and treat cerebral hemorrhage in clinic,and relieving the compression of hematoma on brain tissue is the main treatment.However,there is no effective reversible treatment for intracerebral hemorrhage to improve its prognosis.Therefore,it is important to find new gene targets that play a role in the pathogenesis of intracerebral hemorrhage,which will require further exploration of the molecular mechanism of intracerebral hemorrhage.Casein kinase 2(CK2)is a serine/threonine kinase with hundreds of known substrates,which plays an important role in many diseases.CK2 also plays an important role in cell processes,including cell proliferation and growth,cell survival,cell cycle progression,cell apoptosis and transcription,cell differentiation and cell metabolism.However,the role of CK2 in cerebral hemorrhage is unclear.The purpose of this study is to explore the role and related mechanisms of CK2 in the neuronal apoptosis,inflammatory reaction and oxidative stress response induced by intracerebral hemorrhage,improve people’s understanding of the molecular mechanism of the pathogenesis of intracerebral hemorrhage,and provide certain theoretical basis for exploring new molecular targets for the treatment of intracerebral hemorrhage.Part One Expression of CK2 in intracerebral hemorrhage tissues and its role in hemorrhagic brain injury in ratsObjective: To explore the molecular regulatory mechanism of CK2 in inflammatory response,apoptosis and oxidative stress response after intracerebral hemorrhage.Methods:1.RT-PCR and Western Blot were used to detect the expression of CK2 in the brain tissue of patients with cerebral hemorrhage.2.The changes of CK2 activity in brain tissue of ICH rats were detected by enzyme activity assay.Western blot was used to detect the changes of CK2,NR2 B,and NR2 B phosphorylation levels in the brain tissue of rats with intracerebral hemorrhage.3.The effects of CK2 overexpression(pc-CK2)on neurobehavioral deficits and brain water content in ICH rats were observed.Western blot was used to detect the effects of CK2 overexpression on NR2 B,NR2B phosphorylation levels,and apoptotic protein caspase3 levels.Co-IP method was used to detect the effect of CK2 overexpression on the level of NR2BPSD95 complex.Results:1.The expression level of CK2 protein and m RNA in brain tissue of patients with intracerebral hemorrhage was significantly lower than that of control group(P<0.001).2.Changes of CK2 activity,protein expression and NR2 B phosphorylation in brain tissue of rats with intracerebral hemorrhageThe results of enzyme activity test showed that the activity of CK2 in both cerebral hemispheres of the sham-operated control group was similar.However,in the intracerebral hemorrhage group,the activity of CK2 in the ipsilateral hemisphere was significantly lower than that in the contralateral hemisphere(P<0.001).The results of Western blot showed that compared with the control group,the protein expression level of CK2 in the tissues around the hematoma of rats with intracerebral hemorrhage decreased at 12,24,48 and 72 hours after the establishment of the model,with the most significant at 24 hours(P<0.001).At the same time,the level of NR2 B and its phosphorylation sites proteins increased,the most significant at 24 h(P<0.001).3.Effect of CK2 overexpression on brain injury in rats with intracerebral hemorrhageWestern blot results confirmed that the expression level of CK2 was significantly increased after the lentivirus was transfected into the rat model of intracerebral hemorrhage(P<0.001).Bederson score,forelimb placement and angle test were used for neurological function score.The results showed that pc-CK2 treatment significantly alleviated the neurobehavioral disorder in rats with intracerebral hemorrhage(all P<0.001).The results of brain water content measurement showed that compared with sham operation group,the brain water content of rats in cerebral hemorrhage group increased(P<0.001).The brain water content of rats in pc-CK2 treatment group decreased(P<0.001).Western blot results showed that compared with ICH+pc-NC group,the expression level of NR2 B protein in ICH+pc-CK2 group decreased(P=0.0342),and the expression level of NR2 B phosphorylated protein at S1480 site increased(P=0.0259).In addition,the level of NR2B-PSD95 complex decreased(Co-IP method,P=0.0163),and the level of apoptotic protein caspase3 decreased(P=0.0017).Conclusions:1.Compared with the control brain tissue,the expression of CK2 in patients with intracerebral hemorrhage is decreased,and the difference is statistically significant.2.Compared with sham operation group,the activity of CK2 enzyme and the expression of CK2 protein in brain tissue of rats with intracerebral hemorrhage decreased.The expression level of NR2 B and NR2 B phosphorylation sites S1480,S1303 and Tyr1472 increased.3.Overexpression of CK2 can alleviate brain injury in rats with intracerebral hemorrhage.Overexpression of CK2 can significantly reduce the neurological dysfunction of rats with intracerebral hemorrhage.CK2 may down-regulate the expression of NR2 B by phosphorylating NR2 B at S1480,interfere with the interaction between NR2 B and PSD 95,and reduce the neuronal apoptosis of rats with intracerebral hemorrhage.Thus,the nerve function damage of rats with intracerebral hemorrhage can be alleviated.Therefore,CK2 may be a new potential therapeutic target for intracerebral hemorrhage.Part Two Effects of CK2 on apoptosis and oxidative stress response of neurons after hemorrhagic injury and on inflammatory response of astrocytesObjective: To explore the effect of CK2 on apoptosis and oxidative stress response of neurons after hemorrhagic injury,and the effect on inflammatory response of astrocytes,and preliminarily explore the mechanism of CK2.Methods:1.Embryonic rat neurons and neonatal rat astrocytes were cultured and treated with hemin to establish an in vitro cell model of ICH.Western blot and enzyme activity assay were used to detect the expression of CK2 in the in vitro cell model of ICH.2.The efficiency of CK2 overexpression was detected by RT-q PCR and Western Blot.CCK8 method was used to detect the effect of CK2 overexpression on neuronal survival and astrocyte proliferation.TUNEL method was used to detect the effect of CK2 overexpression on neuronal apoptosis.3.ELISA was used to detect the effect of CK2 overexpression on the inflammatory response of astrocytes.Fluorescence probe method and colorimetric method were used to detect the effect of CK2 overexpression on neuronal oxidative stress.Results:1.The expression of CK2 in the cell model of intracerebral hemorrhage in vitroIn order to further study the molecular mechanism of intracerebral hemorrhage,we cultured neurons and astrocytes in vitro.CCK8 experiment showed that with the extension of culture time,the OD value of cells increased,and the proliferation ability of astrocytes reached its peak on the 14 th day.In our culture,the purity of neurons is more than 95%,and the positive expression of glial fibrillary acidic protein(GFAP)in astrocytes is more than 95%.Then,neurons and astrocytes were treated with hemin to establish an cell model of intracerebral hemorrhage in vitro.In hemin treated cells,the expression of CK2 was lower than that of the control group(P=0.0065;P=0.0439),and the enzyme activity was lower than that of the control group(P<0.001;P=0.0011).2.Effect of CK2 overexpression on proliferation and activation of astrocytes and apoptosis of neuronsThe results of RT-q PCR and Western blot confirmed that the expression of CK2 was significantly increased after lentivirus transfection into neurons and astrocytes(P<0.001;P=0.0052,P=0.0014).CCK8 results showed that the proliferation of astrocytes in the Hemin+pc-CK2 group was significantly enhanced compared with that in the Hemin+pc-NC group(P<0.001).CCK8 was used to detect the survival rate of CK2 overexpression neurons,and TUNEL was used to detect the apoptosis of CK2 overexpression neurons.The results showed that compared with Hemin+pc-NC group,the survival rate of neurons in Hemin+pc-CK2 group was significantly increased(P<0.001),and the apoptosis rate was significantly decreased(P<0.001).In short,CK2 overexpression enhanced the proliferation and activation of astrocytes and inhibited neuronal apoptosis.3.Effects of CK2 overexpression on astrocyte inflammatory response and neuronal oxidative stressELISA results showed that compared with the cells treated with Hemin+pc-NC,the TNF-α and IL-6 levels of the cells treated with Hemin+pcCK2 were significantly decreased(all P<0.001).The ROS content measured by fluorescence probe showed that the fluorescence intensity of neurons treated with hemin significantly decreased after CK2 overexpression(P<0.001).The results of the determination of GSH,SOD and MDA content by colorimetry showed that compared with the cells treated with Hemin+pc-NC,the levels of GSH and SOD in the cells treated with Hemin+pc-CK2 increased and the levels of MDA decreased(P<0.001;P=0.0016;P<0.001).Overexpression of CK2 alleviated the inflammatory and oxidative stress response of cell hemorrhagic injury.Conclusions:1.In the cell model of intracerebral hemorrhage in vitro,the activity of CK2 enzyme and the expression of CK2 protein were significantly lower than those in the control group.2.Lentivirus transfection pc-CK2 can significantly increase the expression of CK2 m RNA and protein in neurons and astrocytes.Overexpression of CK2 significantly enhanced the proliferation and activation of astrocytes,improved the survival rate of neurons,and inhibited the apoptosis of neurons.Overexpression of CK2 significantly reduced the inflammatory response of astrocytes after hemorrhagic injury and the oxidative stress response of neurons after hemorrhagic injury.Part Three CK2 reduces inflammatory reaction,apoptosis and oxidative stress reaction after intracerebral hemorrhage by regulating the phosphorylation of NR2BObjective: To explore the molecular regulatory mechanism of CK2 in inflammatory response,apoptosis and oxidative stress response after intracerebral hemorrhage.Methods:1.Western blot was used to detect the effect of CK2 overexpression on NR2 B and NR2 B phosphorylation levels.Co-IP method was used to detect the effect of CK2 overexpression on the level of NR2B-PSD95 complex.2.The efficiency of NR2 B overexpression was detected using RT-q PCR and Western Blot.TUNEL method was used to detect the effect of NR2 B overexpression on pc-CK2 attenuated neuronal apoptosis.ELISA was used to detect the effect of NR2 B overexpression on pc-CK2 alleviated astrocyte inflammatory response.Fluorescence probe method was used to detect the effect of NR2 B overexpression on pc-CK2 alleviated neuronal oxidative stress.Results:1.Effect of CK2 overexpression on NR2 B phosphorylation and NR2BPSD95 complex levelWestern blot results showed that the expression of NR2 B and its sites phosphorylated protein in Hemin-treated cells was higher than that in control cells(all P<0.001).Compared with the neurons and astrocytes in the Hemin+pcNC treatment group,the expression level of NR2 B protein in the neurons and astrocytes treated with Hemin+pc-CK2 group decreased(P=0.0414,P=0.0049),while the phosphorylation level of NR2 B at the S1480 site increased(P=0.0084,P<0.001).Co-IP results showed that the level of NR2B-PSD95 complex in Hemin+pc-CK2 group was lower than that in Hemin+pc-NC group(P=0.0040,P=0.0155).2.Overexpression of CK2 reduces cell inflammation,apoptosis and oxidative stress by affecting the level of NR2 B phosphorylation and NR2BPSD95 complexThe results of RT-q PCR and Western blot confirmed that the expression level of NR2 B in the cells transfected with lentivirus was significantly increased(P<0.001;P=0.0046,P=0.0029).ELISA results showed that compared with the cells treated with Hemin+pc-CK2,the level of TNF-α and IL-6 in the cells treated with Hemin+pc-CK2+pc-NR2 B increased(P<0.001).TUNEL apoptosis staining showed that compared with the neurons treated with Hemin+pc-CK2,the apoptosis rate of neurons treated with Hemin+pc-CK2+pcNR2B was higher(P<0.001).The ROS content of cells treated with Hemin+pcCK2+pc-NR2 B was higher than that of cells treated with Hemin+pc-CK2(P=0.0083).In short,NR2 B reversed the inflammatory,apoptotic and oxidative stress responses alleviated by CK2 overexpression after hemorrhagic injury.Conclusions:CK2 can regulate the phosphorylation level of NR2 B at S1480,regulate the expression of NR2 B protein,and interfere with the interaction between NR2 B and PSD95,so as to reduce the inflammatory response of astrocytes after hemorrhagic injury,and reduce the apoptosis and oxidative stress response of neurons after hemorrhagic injury.Therefore,it is speculated that CK2 may become a new target for the treatment of intracerebral hemorrhage. |
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