| Background and objective:Subarachnoid haemorrhage is a type of haemorrhagic stroke with rapid onset,rapid progression,high mortality and disability rates.The current lack of effective treatments,the complexity of its pathogenesis and the poorly studied mechanisms of interaction with early brain injury and neurological impairment are the underlying causes of the low cure rate for this disease.Finally,neurological dysfunction such as behavioural abnormalities and cognitive dysfunction are the main pathologies of surviving SAH patients.Axon damage after SAH is one of the main causes of neurological disorders such as cognitive impairment.Transactive response DNA-binding protein of 43 k D(TDP-43)is a nuclear protein that regulates m RNA transport and stabilization,transporting m RNA to the cytoplasm and distal axon for local translation,thereby maintaining normal axon function.In pathological conditions,cytotoxicity from TDP-43 cytoplasmic aggregates is considered to be a pathological feature and the basis of many chronic neurodegenerative diseases,leading to neurological deficits such as cognitive dysfunction and dementia.However,no studies have been reported on whether TDP-43 cytoplasmic aggregates damage axons after SAH and its effects on neurological functions such as cognitive function.Heat shock proteins(HSP70),as important molecular chaperones,are widely involved in intracellular protein assembly,folding,transport and other important cellular activities,and have a role in refolding abnormal proteins,inhibiting abnormal protein aggregation and promoting abnormal protein degradation and clearance.In recent years,a large number of studies have found that HSP70 plays an important role in cerebrovascular diseases,amyotrophic lateral sclerosis and other neurological diseases.Research has reported that HSP70 can inhibit and clear TDP-43 cytoplasmic aggregation in a cell model of amyotrophic lateral sclerosis.At present,there is no research report on whether HSP70 can inhibit TDP-43 cytoplasmic aggregation and reduce neuronal axon damage in SAH models.Histone deacetylase 1(HDAC1)is an important member of the histone deacetylases(HDACs)family,plays an important role in regulating important life processes such as gene expression,cell division and differentiation,which can eventually reverse the acetylation modification of proteins by histone acetyltransferases.In recent years,HDAC1 has been widely studied as an important HDACs that can mediate the process of nervous system disease.Studies have shown that HDACs can target and regulate HSP participation in various disease processes by deacetylation of HSP.Suberoylanilide hydroxamic acid(SAHA)is a broad-spectrum histone deacetylases inhibitors(HDACi)that promotes protein acetylation modifications and has been reported to exert neuroprotective effects in stroke and neurodegenerative disease models by regulating gene expression,reducing microglia activation,anti-inflammation and antiapoptosis.There are no reports of the HDAC1/HSP70/TDP-43 signalling axis affecting neurological function after SAH,and few reports of SAHA exerting neuroprotective effects in SAH models.Here,based on the study that SAHA and TDP-43 have important biological functions in neurodegenerative diseases,with HDAC1 as an upstream regulator and TDP-43 as a downstream functional factor,this project aims to investigate the relationship between TDP-43 and SAH and its effects on axonal and neural functions after SAH,and to study the mechanism of the role of TDP-43 in nerve injury after SAH.The focus was on the mechanism of the HDAC1/HSP70/TDP-43 signalling axis in the process of SAH that affects neurological damage.Finally,it is clarified that SAHA exerts neuroprotective effects and related mechanisms on subarachnoid hemorrhage models by regulating the HDAC1/HSP70/TDP-43 axis,and provides new targets and strategies for the treatment of subarachnoid hemorrhage.Methods:(1).Cerebrospinal fluid was collected from 10 patients without SAH(Non-SAH)and 15 patients with aneurysmal SAH at 72 h after the onset of the disease,and TDP-43 protein expression levels were measured by ELISA and Western Blot.The rat model of SAH was constructed by intravascular puncture,and the TDP-43 overexpression lentiviral vector was constructed and transfected into rats by lateral ventricle injection.The rat model of SAH was established 5 days after lateral ventricle injection of lentiviral vector.Early neurological function was assessed using the beam balance test and modified Garcia behavioral score,and balanced and coordinated motor function was measured using the Rotarod test.Morris Water maze test was used to detect long-term cognitive learning and memory function.TUNEL and Neu N double staining method was used to detect neuronal apoptosis.immunohistochemistry was used to detect APP expression,and transmission electron microscopy was used to evaluate the degree of axonal injury.q RT-PCR and Western blot were used to detect the m RNA and protein expression levels of TDP-43,and immunofluorescent double-label staining to detect the expression and localization of TDP-43 in different neuronal cells.(2).Primary neurons were isolated from fetal rat brain tissue and induced by 20 μM Oxyhemoglobin(Oxy Hb)to construct a SAH cell model in vitro,and TDP-43 silencing and HDAC1 silencing lentiviral vectors were constructed and transfected into cells.After 48 hours of lentivirus transfection,20 μ M Oxy Hb treatment was used to establish a SAH cell model,CO-IP assay was used to test the binding and interaction of HDAC1,HSP70 and TDP-43,q RT-PCR was used to detect the m RNA expression level of HDAC1,Western blot to test the protein expression levels of HDAC1,HSP70,TDP-43,acetyl-K,Dynactin,NFL and Apo E,nucleoplasmic separation assay to detect the expression level of TDP-43 in the cytoplasmic and nucleus,transmission electron microscopy to test the damage of neuron nuclear membrane,flow cytometry to detect the level of neuronal apoptosis.(3).HDAC1 silencing lentiviral vector was constructed and transfected into rats by lateral ventricular injection,and a rat SAH model was established 5 d after lateral ventricular injection of lentiviral vector.SAHA(50 mg/kg)or 5% DMSO saline was injected into the lateral ventricle 1 h before SAH modeling.Then,the beam balance test and modified Garcia behavioral score were used to assess early neurological function,balance and coordinated motor function was assessed using the Rotarod rotating stick test,the Morris water maze test for long-term cognitive learning and memory function,q RT-PCR for HDAC1 m RNA levels,Western blot for HDAC1,Acetyl-K,HSP70 and TDP-43 protein expression levels,and TUNEL and Neu N double-staining method to detect apoptosis in neuronal cells,immunohistochemistry to detect APP expression,and transmission electron microscopy to assess the extent of axonal damage.Result:(1).TDP-43 protein expression in cerebrospinal fluid was significantly higher in SAH patients than in Non-SAH patients(p<0.05).Compared with the Sham group,the beam balance test and modified Garcia score of rats in the SAH group were significantly decreased,indicating severe early neurological impairment(p<0.05),and the residence time on the rotating rod was significantly shortened,showing significant impairment of balance and coordination motor function(p<0.05),in the water maze,the escape latency and swimming distance were significantly prolonged,and the time spent in exploring the target quadrant without underwater platform was shorter,which showed that the long-term cognitive learning and memory function was significantly impaired(p<0.05),a significant increase in the number of apoptotic cells in Neu N+Tunel+ double-positive neurons(p<0.05),a significant local accumulation of positive APP in the white matter region of the brain(p<0.05),the presence of multiple ultrastructural features of axonal injury,including axonal enlargement,nerve fibre compaction.TDP-43 expression gradually increased after SAH,with peak expression levels at 72 h after SAH(p<0.05),and the fluorescence intensity of TDP-43 expression in neuronal cells was significantly higher than that in microglia and astrocytes(p<0.05).Compared with the SAH+NC group,the m RNA level of TDP-43 was significantly higher in the SAH+TDP-43 group(p<0.05),the protein expression of TDP-43 was significantly increased(p<0.05),the early neurological impairment was significantly aggravated(p<0.05),and the balance coordination motor function and long-term cognitive learning memory function were significantly impaired(p<0.05)in the SAH+TDP-43 group,at the same time,a significant increase in the number of apoptotic cells in Neu N+Tunel+ double positive neurons(p<0.05),a significant increase in the number of APP positive cells(p<0.05)and a significant increase in the number of swollen and dystrophic axons were also observed.(2).CO-IP assay showed that HDAC1,HSP70 and TDP-43 combined and interacted with each other.Compared with the Control group,the m RNA expression level of HDAC1 in Oxy Hb group was significantly increased(p<0.05),and the protein expression levels of HDAC1,HSP70,TDP-43,Dynactin and NFL were significantly increased(p<0.05),the protein expression levels of acetyl-K and Apo E were decreased(p<0.05),TDP-43 expression levels were significantly increased in the cytoplasm(p<0.05),and neuronal cell nuclei had irregular morphology and nuclear membrane invagination,the number of apoptotic cells in neurons was significantly increased(p<0.05),while silencing TDP-43 significantly alleviated the abnormal nuclear membrane morphology and decreased the number of apoptotic cells in neurons(p<0.05).Compared with the Oxy Hb+sh-NC group,the m RNA expression level of HDAC1 in the Oxy Hb+sh-HDAC1 group was significantly decreased(p<0.05),and the protein expression levels of HDAC1,TDP-43,Dynactin and NFL were significantly decreased(p<0.05),the protein expression levels of acetyl-K and Apo E were increased(p<0.05),but there was no significant difference in the level of HSP70.The expression of TDP-43 in the cytoplasm was significantly decreased(p<0.05),at the same time,the cell morphology was round and regular,the nuclear envelope morphology was basically normal,and the number of apoptotic cells in neurons was significantly decreased(p<0.05).(3).Compared with Sham group,the early neurological function of rats in SAH+sh-NC group was significantly impaired(p<0.05),the balance and coordination motor function and long-term cognitive learning and memory function were significantly deteriorated(p<0.05),and the m RNA level of HDAC1 was significantly increased(p<0.05),the protein expression levels of HDAC1,HSP70 and TDP-43 were significantly increased(p<0.05),the acetyl-K level was significantly decreased(p<0.05),the number of Neu N+Tunel+ double positive cells was significantly increased(p<0.05),and the number of APP positive cells and swollen and dystrophic axons were significantly increased(p<0.05).Compared with SAH+sh-NC group,the rats in SAH+sh-HDAC1 group had significantly improved early neurological impairment(p<0.05),significantly improved balance and coordination motor function and long-term cognitive learning and memory function impairment(p<0.05),and significantly decreased HDAC1 m RNA level(p<0.05),the protein expression levels of HDAC1 and TDP-43 were significantly decreased(p<0.05),the acetyl-K level was significantly increased(p<0.05),the HSP70 level had no significant difference,and the number of Neu N+Tunel+ double positive cells,APP positive cells and swollen and dystrophic axons were significantly decreased(p<0.05).Compared with the SAH+DMSO group,the early neurological impairment was significantly improved in the SAH+SAHA group(p<0.05),and the balance and coordination motor function and longterm cognitive learning and memory function were also significantly improved in the SAH+SAHA group(p<0.05),there was no significant difference in the HDAC1 m RNA level,Acetyl-k level was significantly increased(p<0.05),HDAC1 and HSP70 levels had no significant difference,TDP-43 level was significantly decreased(p<0.05),the number of Neu N+Tunel+ double positive cells,APP positive cells and swollen and dystrophic axons were significantly decreased(p<0.05).Conclusion:(1)TDP-43 is highly expressed in SAH patients,SAH rats and cell models.In SAH cell model,TDP-43 is highly expressed in neuronal cells and accumulates in cytoplasm,which leads to the damage of neuronal axon and nuclear envelope morphology,and promotes neuronal apoptosis.(2)HDAC1 silencing may promote HSP70 acetylation by regulating HDAC1/HSP70/TDP-43 axis,inhibited TDP-43 cytoplasmic aggregation,and promoted TDP-43 degradation,finally alleviated neuronal axon damage and nuclear envelope abnormality in SAH cell model,reduced axonal injury in rats after SAH,reduced neuronal apoptosis,and improve early and long-term neurological impairment and cognitive dysfunction in rats after SAH.(3)SAHA may play a neuroprotective role by inhibiting HDAC1,regulating HDAC1/HSP70/TDP-43 axis,promoting HSP70 acetylation and TDP-43 degradation,alleviating neuronal apoptosis and axonal injury in rats after SAH,and improving early and long-term neurological damage and cognitive dysfunction in rats after SAH.Figures 12,Tables 11,References 129... |
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