Thousand And One Kinase 1 Protects MCAO-induced Cerebral Ischemic Infarction In Rats By Decreasing Apoptosis And Pro-inflammatory Factors

Backgroud:Cerebral infarction is one of the main diseases that threaten human physical and mental health.With the widespread application of neuroimaging diagnostic technology in the neonatal field,the incidence and diagnosis of neonatal cerebral infarction(NCI)have gradually increased.The incidence of NCI is about 1/2300 to 4000.A variety of neonatal diseases cause cerebral infarction,such as neonatal hypoxic-ischemic encephalopathy,central nervous system infection,polycythemia,and cerebral vascular developmental abnormalities.Cerebral infarction causes lifelong nerve damage,such as cerebral palsy,epilepsy,cognitive impairment and audiovisual dysfunction,which seriously affects the long-term prognosis and makes a huge burden on the family and society.In terms of treatment,due to the unique physiological and pathological characteristics of the neonatal nervous system,many mature treatment methods for adults or children,such as tissue plasminogen activator(t-PA)thrombolytic therapy,has rarely been used in newborns and the safety and effectiveness is unclear.Up to now,the treatment of cerebral infarction in neonates is still symptomatic and there is no effective treatment for the cause.Recently,numerous studies have confirmed that neural stem cell transplantation has huge therapeutic potential.Over the years,despite tremendous efforts to understand the underlying mechanisms of ischemic cerebral infarction,this has remained largely unknown.In the past 40 years,a variety of animal stroke models have been developed to determine the mechanism of cerebral ischemia and develop new stroke treatment drugs.In mammals,neural stem cells are mainly distributed in VZ,SVZ,striatum,dentate gyrus and spinal cord.Among them,SVZ is the main region producing new neurons.A large number of studies used SVZ of MCAO(middle cerebral artery occlusion)model to explore the potential mechanism and treatment of ischemic cerebral infarction.The middle cerebral artery(MCA)and its branches are the most common cerebral vessels in ischemic stroke,accounting for about 70%of the infarct area.Occlusion of this artery is closest to the manifestation of human ischemic stroke.MCAO model has little trauma and does not need craniotomy,thus avoiding the damage of skull structure.MCAO model has several advantages:first,it simulates human ischemic stroke,which usually originates from MCAO and presents a penumbra similar to human stroke.In addition,MCAO model has the characteristics of large infarct volume and high repeatability.The time of reperfusion and ischemia can be precisely controlled.In addition,the program is relatively easy to execute and time-consuming.MCAO model is considered to be suitable for replicating ischemic stroke and subsequent neuronal death,brain inflammation and blood-brain barrier(BBB)injury,and produces good results in behavioral tests.We use MCAO method to make ischemic stroke model,and detect the physiological indexes of the model to determine whether the model is successful,and then use the model to explore the molecular mechanism of ischemic stroke.TAOKs(thousand and one kinases)is one of the important members of the step 20p subfamily,which is composed of taokl,taok2 and taok3.Step 20p family contains a variety of protein kinases,some of which have been proved to act on the upstream of MAPKs.Tao activates MAPK signaling.TAOK1 and TAOK2 also induce apoptosis morphological changes by activating jnk-mapk and caspases.Other studies have shown that taokl induces microtubule instability by activating the phosphorylation of microtubule affinity regulated kinase and microtubule associated protein tau,and that taoks are phosphorylated and specifically activated during mitosis.In addition,taokl is involved in the activation of p38 and c-jun NH2 terminal kinase(JNK)on DNA damage and stress stimulation.Taokl can inhibit cell proliferation by directly phosphorylating T195,thus regulating the role of Hippo signaling pathway in development.In addition,it has been found that taokl plays a negative role in il-17-mediated signaling and inflammation.TAOK1 inhibited the expression of IL-17-induced inflammatory cytokines and chemokines.TAOK1 inhibited p38,JNK,ERK1/2 and p65 activated by IL-17 in HeLa cells.Taokl interacts with IL-17A to inhibit the formation of IL-17A-actl complex in a way unrelated to kinase activity,leading to the negative regulation of IL-17A-mediated inflammatory response.However,it is not clear whether taokl plays a role in the pathogenesis of ischemic stroke.Methods:The animal and cell model of ischemic stroke was established by MCAO and OGD.The expression of TAOK1 was detected by qRT-PCR,Western blot and immunofluorescence staining.The cell proliferation was determined by CCK8 assay.The cell apoptosis was examined by TUNEL staining,annexin V and hoechst33342 staining and the role of inflammatory factors(IL-1 β,IL-6 and IL-8)in ischemic stroke model in vivo was detected by ELISA under overexpression or suppression of TAOK1 conditions.The involvement of PI3K/Akt and MAPK signaling pathway was determined by Western blot.Results:The MCAO rat model was verified by examining the neurological deficits with Bederson/Longa scores at 24 hours after MCAO.Compared to sham animals,MCAO rats showed a significant increase of the neurological scores(P<0.001 vs sham group).The results from TTC staining showed an extensive infarction area in a large proportion of ipsilateral hemisphere of MCAO rats.In addition,compared with sham group,the MCAO rats exhibited obvious motor behavioral deficits in the average speed,nextime,distance,and revolving speed of rotarod test(P<0.001 vs sham group).These findings suggested that we have successfully established a model of ischemic cerebral infarction in rats and caused motor dysfunction.Compared to sham group,the relative mRNA expression of TAOK1 was significantly reduced in the SVZ of MCAO rats(P<0.01).Western blotting results demonstrated a significant downregulation of TAOK1 protein expression in the SVZ of MCAO rats compared to sham animal.The results from immunofluorescence staining showed that compared with the sham group,the staining intensity of TAOK1 in SVZ of MCAO group was significantly weakened.In addition,we also determined the distribution of neural stem cells in brain sections by immunofluorescence staining.We found that TAOK1 and Nestin were widely distributed in the SVZ of brain.TUNEL assay was carried out to detect the apoptosis of neural stem cells of SVZ brain slice in sham and MCAO rats.The results showed a significant increase in the apoptotic rate in the SVZ brain slice of MCAO rats when compared with sham group.In addition,ELISA was performed to evaluate the production of IL-1β,IL-6;and IL-8,and the results showed that their expression levels were remarkably increased in the blood samples collected from MCAO rats when compared to blood samples collected from sham rats(P<0.01).An in vitro model of ischemic stroke was established using cortical neural stem cells collected from fetal rats at 18 days of gestation.The cultures showed an obvious Nestin positive signal and GFAP/Tuj1 negative signal,suggesting that the majority of the cells in the cultures were neural stem cells.TAOK1 mRNA and protein expression in OGD-treated neural stem cells were significantly decreased when compared with normal neural stem cells(P<0.001).Immunofluorescent staining of TAOK1 in OGD-treated cells showed remarkably attenuated signals compared to normal cells.In addition,immunofluorescence assay showed that compared with the normal group,OGD significantly weakened TAOK1 immunofluorescence staining in neural stem cells.To investigate the effect of TAOK1 on OGD-induced ischemic cerebral infarction cell model,shRNA or TAOK1 recombinant plasmid was used to knock out or overexpress TAOK1 in neural stem cells induced by OGD.Compared with normal group,OGD-treated group showed a significant suppression of cell proliferation;compared with OGD+NC treated group,TAOK1 blocked cells under OGD(OGD+shRNA group)aggravated the OGD-induced proliferation suppression,whereas TAOK1 overexpressed cells under OGD(OGD+TAOK1 group)have partially reversed the OGD-induced proliferation suppression(P<0.001 vs normal group,P<0.001 vs OGD+NC group).The cell number of OGD-treated group showed a significant upregulation in G0/G1 phase and a downregulation in S phase when compared with normal group.The cell number of OGD+shRNA group showed a remarkable upregulation in G0/G1 phase,and a downregulation in S phase,whereas the OGD+TAOK1 group showed opposed trend with OGD+shRNA group when compared with OGD+NC group.The cell apoptotic rate was significantly higher in OGD-treated group than normal group.Compared to OGD+NC group,the cell apoptosis was markedly aggravated in OGD+shRNA group,whereas the cell apoptotic rate in OGD+TAOK1 group was significantly inhibited.In addition,Bax,cleaved-caspase-3,and p-21 expressions were significantly upregulated,whereas Bcl-2 and CyclinD1 expressions were significantly downregulated in OGD group compared to normal group.Compared to OGD+NC group,Bax,cleaved-caspase-3,and p-21 expressions in OGD+shRNA group were higher,whereas their expression in OGD+TAOK1 group were lower;Bcl-2 and CyclinDl expressions in OGD+shRNA and OGD+TAOK1 groups exhibited opposing trends with Bax,Cleaved-caspase-3,and p-21 expression.The significant up-regulation of Bcl-2,downregulation of Bax and cleaved-caspase-3,which were mediated by overexpression of TAOK1 in OGD-induced cortical neural stem cells,were significantly weakened after inactivation of the intrinsic kinase activity.Besides,we revealed that the reduction of apoptosis mediated by TAOK1 overexpression could be prevented by the inactivation of the intrinsic kinase activity.Moreover,TUNEL results showed that inactivation of endogenous kinase activity prevented TAOK1 overexpression-mediated reduction in apoptosis.Compared to normal group,OGD treatment significantly increased the production of IL-1p,IL-6,and IL-8(**P<0.001,***P<0.001 vs normal group).Compared to OGD+NC group,IL-1β,IL-6,and IL-8 production were significantly increased in OGD+shRNA group,whereas their productions were remarkably downregulated in OGD+TAOK1 group(*P<0.05,**P<0.01 vs OGD+NC group).We further found that p-AKT and PI3K expressions were significantly reduced in OGD group compared to normal group.However,p-ERK and p-P38 expressions in OGD group showed opposing expression trends with p-AKT and PI3K.Compared to OGD+NC group,p-AKT and PI3K expressions were lower in OGD+shRNA group,whereas higher in OGD+TAOK1 group.However,p-ERK and p-P38 expressions were higher in OGD+shRNA group,whereas lower in OGD+TAOK1 group.These data indicated that PI3K/AKT and MAPK signaling pathways were involved in the regulation of TAOK1 in ischemic cerebral infarction.Conclusion:Our results showed that TAOK1 expression be significantly downregulated,cell apoptosis and IL-1β,IL-6 and IL-8 expression were upregulated in MCAO rat model and OGD-induced neural stem cell model of ischemic stroke.In in vitro cell model,TAOK1 partly reversed the OGD-induced cell injury,and upregulated IL-1β,IL-6 and IL-8.In mechanism,the PI3K/AKT and MAPK signaling pathways were involved in the induction of TAOK1 effects in ischemic stroke.However,this study is not perfect due to time and funding,we only investigated the expression level of TAOK1 in SVZ of brains.In the future,we will further investigate the levels of TAOK1 in cortical and striatal regions,and explore the function and possible mechanism of TAOK1 on the neural stem cells in cortical and striatal regions.