Mechanisms Of HIPK2 In Regulating The Neurotoxicity Induced By Sevoflurane In Immature Rat Hippocampus

In China,millions of children undergo surgery every year.Most operations need to be performed under general anesthesia,which can help to offer children safe and comfortable treatments.While the impact of general anesthetics on the development of children’s nervous system has become a common public health concern for parents,anesthesiologists and medical management agencies.Regarding the mechanism of neurodevelopmental toxicity caused by general anesthetics in the critical period of brain development,most of previous rodent and non-primate studies focused on the effects of general anesthetics on the proliferation,differentiation and apoptosis of neurons during developmental stages.In recent years,it has been gradually recognized that abnormalities in synaptic structure and function may be the main mechanisms of cognitive impairment caused by general anesthetics.Homeodomain-interacting protein kinase 2(HIPK2)is a Ser/Thr protein kinase discovered in recent years,which can interact with homeobox proteins and is widely involved in transcriptional regulation.As a transcription factor,HIPK2 mediates a variety of biological processes,such as proliferation,differentiation,maturation and apoptosis.Therefore,this topic focuses on the molecular mechanism of hippocampal neurotoxicity induced by sevoflurane during developmental stages with HIPK2 as a breakthrough.The study consists of the following three parts:Part one: Distribution and subcellular localization of HIPK2 in the brain of SD rats.Currently,although changes in the expression of HIPK2 mRNA in mouse brain have been identified,the distribution and subcellular localization of HIPK2 remain unclear.Therefore,this study used immunofluorescence staining and Western blot to explore the distribution of HIPK2 in the central nervous system,laying the foundation for the following work.Objective: To observe the changes and distribution of HIPK2 in the developmental brain.Methods: Immunofluorescence histochemistry and Western blot were used to observe the expression changes,distribution and subcellular localization of HIPK2 in the central nervous system of SD rats at different time points from birth to adult.Result: HIPK2 was widely distributed in the central nervous system of SD rats.It was mainly expressed in the nucleus of neurons,and a small part was expressed in oligodendrocytes.No expression in astrocytes and microglia was observed.From birth to adulthood,the expression of HIPK2 in the olfactory bulb,prefrontal cortex and hippocampus gradually decreased.Conclusion: HIPK2 is widely distributed in the central nervous system and is mainly located in the nucleus.Part two: Effects of HIPK2 on the apoptosis of primary hippocampal neurons after sevoflurane exposure.Recent studies have observed that general anesthetics affect the survival of neurons through several aspects,such as proliferation,differentiation and apoptosis.The molecular mechanism for this change is less clear.As tumor suppressor and DNA damage monitoring kinase,HIPK2 mediates the expression of apoptosis and genes expression related to DNA repair through phosphorylation and activation of P53 at serine 46,thereby promoting apoptosis.Therefore,in this study,the effect of HIPK2 gene on hippocampal neuron apoptosis induced by sevoflurane was investigated by silencing the HIPK2 gene,so as to provide a new therapeutic strategy for the neurotoxicity caused by general anesthesia.Objective: Observe the expression of HIPK2 after Sevoflurane exposure.And construct shHipk2 lentiviral expression vector and explore the role of HIPK2(homologous domain interacting protein kinase 2)in the apoptosis of primary neurons after Sevoflurane exposure.Methods: The lentivirus against HIPK2 target gene was designed,constructed,and then infected to primary hippocampal neurons.The infection efficiency was detected by immunofluorescence,and the interference effect was verified by RT-PCR.Three days after the infection,the corresponding detection of the treatment group was done six hours after the exposure of 4.1% sevoflurane.Cell activity was detected by CCK8 colorimetric assay,cytotoxicity was detected by LDH,and apoptosis was detected by FITC/PI flow cytometry and Western blot.Result: RT-PCR showed that compared with Scr group and Con group,the level of HIPK2 mRNA in the shHIPK2 group decreased obviously,with the interference efficiency being75.29 %,which suggested that the lentivirus shRNA expression vector of target silent HIPK2 was successfully constructed.Compared with Scr group,cell activity decreased and cytotoxicity increased in the Scr+Sev group.While there was no significant difference in the cell viability and cytotoxicity between shHIPK2+Sev group and shHIPK2 group.The proportion of apoptotic cells in theScr+Sev group was significantly higher than that of Scr group(P<0.05),but there was no significant difference between shHIPK2+Sev group and shHIPK2 group(P>0.05).The results of Western blot showed that the level of Cl-caspase3 protein in the Scr+Sev group was significantly higher than that in the Scr group(P<0.05),and there was no statistical difference between the group of shHIPK2+Sev and the shHIPK2(P>0.05).Conclusion: HIPK2 could promote the apoptosis of hippocampal neurons induced by sevoflurane.Part three: Involvement of HIPK2-JNK/c-Jun cascade in the long term synaptic toxicity and cognition impairment induced by neonatal Sevoflurane exposureObjective: Investigated the roles of HIPK2 and its downstream signaling in the long term cognition dysfunction and synapse toxicity of neonatal Sevoflurane exposure.Methods: P6 rats were subjected to 3 times of Sevoflurane exposure.Cognition function,synaptic morphology and related proteins were evaluated in adult.Apoptosis,expression of HIPK2 and JNK/c-Jun were analyzed shortly after Sevoflurane exposure.Antagonist of HIPK2(A64)and JNK signaling(SP600125)were administered to block the function of HIPK2 and JNK.Result: Neonatal Sevoflurane exposure results in decrease of spatial memory,increase of anxiety,less number of synapse and lower levels of synaptic proteins in hippocampus,anterior cingulate cortex(ACC)and caudate putamen(CPu)in adult.Increase of apoptosis was observed mainly in non-neuron cells.Up-regulation of HIPK2 and JNK/c-Jun was found in neurons in the ACC,CPu and hippocampus.HIPK2 antagonist A64 significantly rescued the cognition impairment,synapse toxicity and the activation of JNK/c-Jun signaling induced by Sevoflurane.Further,JNK antagonist SP600125 partially restored synapse development and cognition function.Conclusions: HIPK2-JNK/c-Jun signaling may account for the long term synaptic toxicity and cognition impairment of neonatal Sevoflurane exposure.Interfering HIPK2-JNK/c-Jun cascade may be utilized to minimize the synaptic toxicity of Sevoflurane.