The Role And Mechanism Of Osteocalcin In The Repair Of Traumatic Brain Injury

【Background】Traumatic brain injury（TBI）is a serious disease that threatens people’s life and health.More than 50 million people suffer from varying degrees of brain injury each year.In the application of military medicine,brain injury is an important part of war injuries,and its death rate,casualty rate and disability rate always rank the first in all parts of war injuries.Therefore,the basic research and clinical treatment of brain injury are of great significance to improve the combat effectiveness and the health level of soldiers.Ferroptosis is a distinct form of programmed cell death caused by the abnormal metabolism of lipids,Glutathione（GSH）and iron.At present,it has been found to be one of the main causes of central nervous system injury in Parkinson’s disease,cerebral hemorrhage and traumatic brain injury.Lipid peroxidation,mitochondrial dysfunction,and increased reactive oxygen species caused by increased iron accumulation in the brain can lead to neuronal damage.Neurons are the main cells in the brain,which have the functions of receiving,integrating,conducting and transmitting information.The pathological changes of neurons include acute necrosis of neurons,simple neuronal atrophy,dissolution of central Nissl bodies and formation of inclusion bodies.Cognitive function depends on the transmission of information by a variety of neurotransmitters released between neuronal synapses,and the release of neurotransmitters requires the triggering of calcium channels and a large amount of energy support.In the acute phase of traumatic brain injury,excessive neuroinflammatory response is not conducive to the survival and repair of neurons,thereby aggravating tissue damage.Therefore,reducing excessive accumulation of iron,inhibiting lipid peroxidation,maintaining mitochondrial homeostasis and regulating inflammatory response in the acute phase have become important strategies for nerve repair after traumatic brain injury.In recent years,the role of bone-derived endocrine factors in regulating the functions of multiple systems has attracted more and more attention.Osteocalcin specifically secreted by osteoblasts has been shown to regulate systemic glucose and energy metabolism,reproduction and neurocognitive functions.Hormonal changes in the brain are involved in the production of synaptic neurotransmission gene products and the remodeling of brain structure.Osteocalcin（OCN）is a bone-derived factor secreted by bone organs with important regulatory functions.OCN has two forms:fully carboxylated and undercarboxylated.The former is mainly deposited in the bone matrix,while the latter plays a regulatory role in multiple organs except bone through the circulatory system.OCN can regulate monoamine neurotransmitter levels through the blood-brain barrier,combine with midbrain,brainstem and hippocampus,maintain hippocampal neurogenesis,improve spatial learning and memory impairment,and play a protective role in diseases with cognitive decline as the main manifestation.Studies have reported that maternal uc OCN plays an important role in the neurogenesis and development of neonatal rats,but whether it is involved in the neural repair process after brain trauma in adults remains to be explored.Therefore,this study is the first to explore the role of OCN in ferroptosis,and to investigate the mechanism of OCN in the repair and protection of nerve in traumatic brain injury,so as to provide a new target for the treatment of traumatic brain injury.【Objective】To determine the expression of OCN in nerve injury after TBI;To understand the role of OCN in the regulation of neural repair after TBI;To explore the interaction between neural repair and local microenvironment after TBI and its mechanism.【Methods】Animal experiments:The animal model of traumatic brain injury in mice was established.The balance beam test,fatigue rotorcraft test,tail suspension test,forced swimming test,open field test,and water maze test were used to detect the motor balance and coordination ability,anxiety and depression like emotions,and spatial cognitive learning ability of mice after TBI.Western blot was used to detect the expression of OCN,Bcl2,NEUN,BDNF,PSD95 and ferroptosis-related protein GPX4at different time points.Nissl staining was used to detect neuronal apoptosis.HE staining was used to detect neuronal damage.Immunofluorescence was used to detect the activation of astrocytes and microglia.Immunofluorescence double staining was used to detect the colocalization of different types of neurons in OCN.q RT-PCR was used to detect the expression levels of inflammatory factors IL-1βand TNF-α.Cell experiments:Erastin-induced ferroptosis in HT22 cells.q RT-PCR was used to detect the expression levels of inflammatory factors IL-1β,TNF-α.Western blot was used to tested the expression of ferroptosis-related proteins GPX4 and SLC7A11.CCK-8 assay was used to detect the effects of different concentrations of Erastin on the survival rate of HT22 cells and the expression of OCN.si RNA transfection assay was used to detect the expression changes of ferroptosis-related proteins after knockdown of OCN in HT22 cells,and exogenous administration of uc OCN was used for recovery experiments.The interaction force between uc OCN and GPX4 was predicted by molecular docking.GPX4 si RNA transfection experiment was used to observe the effect of uc OCN after target interference.【Results】（1）The expression of OCN in the injured brain tissue of mice was dramatically improve after TBI.The expression of OCN protein in the injured brain tissue increased on day 1,3 and7 after TBI,and reached the peak on day 3 and continued to day 7.Among them,the results of fatigue rod-spinning test and balance beam test showed that compared with the sham group,the TBI mice spent less time on rotarod fatigue,more time on balance beam and decreased motor coordination and balance ability.The results of tail suspension test and forced swimming test showed the TBI group had added immobility time and depression-like behavior.The open field test showed that the time and distance in the center of the open field were significantly reduced,and the TBI mice showed anxiety-like behavior.The results of water maze test showed that the exploration time of mice was significantly increased and the spatial memory ability was decreased after TBI.q RT-PCR detection showed that the expression levels of inflammatory factors in the injured brain tissue of mice increased after TBI.Nissl staining was used to observe neuronal apoptosis in the injured brain tissue of mice after TBI.Immunofluorescence was used to detect the activation of astrocytes and microglia in the injured brain tissue of mice after TBI.（2）OCN knockout resulted in anxiety-depression-like behaviors and impaired spatial memory ability in mice.The gene knockout mice were constructed,and the same lot of WT and OCN^-/-mice were simultaneously subjected to TBI.The results of fatigue rotorcraft test and balance beam test showed that compared with the WT group,OCN knockout did not affect the motor balance ability of the mice,and there was no significant difference in the motor balance and coordination ability between the OCN^-/-TBI group and the WT TBI group.The results of tail suspension test and forced swimming test showed the immobility time of OCN knockout mice was prolonged compared with WT mice,indicating that OCN knockout could lead to depressive-like behavior in mice,which was aggravated after TBI.The results of the open field test showed that compared with the WT group,the OCN knockout mice showed significantly reduced time and distance in the center of the open field and anxiety-like behavior.The results of water maze test showed that OCN knockout affected the exploration time and spatial memory ability of mice compared with the WT group,and the OCN^-/-group had the longest TBI exploration time.（3）Neurons are the key cells that play a role in nerve repair of OCN in the injured brain tissue after TBI.The brain tissue of TBI mice was taken for frozen section,and the co-localization results of immunofluorescence staining showed that OCN was mainly expressed in neurons,and there was no co-localization with GFAP,and there was partial co- localization with microglia,indicating that neurons were the key cells for OCN to play a role in nerve repair in the injured brain tissue of mice after TBI.q RT-PCR and Western blot were used to detect the changes in the expression of NEUN,neuropeptide NPY,neurotrophic factor BDNF,postsynaptic density protein PSD95 and ferroptosis key protein GPX4 in the injured brain tissue of mice at different time points after TBI.（4）The effects of OCN knockout on ferroptosis,immune microenvironment and related pathways in TBI mice.Immunofluorescence assay showed that compared with the WT group,the OCN^-/-Sham group had no significant changes,while the OCN^-/-TBI group had significant activation of astrocytes and microglia.The results showed that compared with the WT group,the OCN^-/-sham group had no significant change,while the OCN^-/-mice had significantly increased inflammatory factors in the brain tissue around TBI.The results of Nissl staining showed that compared with the WT group,the distribution of Nissl bodies in the OCN^-/-sham group was uneven,the damaged neurons shrank and the cytoplasm staining was deepened,while the number of neurons in the OCN-/-TBI group was significantly lower than that in the WT TBI group.Western blot was used to detect the expression of ferroptosis-related protein GPX4 in OCN knockout mice.Compared with the WT group,the expression level of GPX4 in OCN^-/-sham group was reduced,and the expression of GPX4 in OCN-/-TBI group was the lowest.The changes of related pathways in the injured brain tissues of OCN knockout mice in different groups were detected,and the results showed that OCN may further play a protective role on neurological injury after TBI by regulating Akt-m TOR and Sirt1/PGC1a pathways.（5）Interference of OCN expression aggravates ferroptosis in HT22 neurons HT22cells were treated with different concentrations of Erastin to induce ferroptosis.The results showed that the cell growth was significantly inhibited after 30μmol/l Erastin treatment for 24h,and the cell death rate was close to 50%.Optical microscope observation showed that HT22 cells treated with Erastin for 24h had widened intercellular space and decreased adhesion ability,showing the morphological performance of cell damage.After adding 5ng/ml uc OCN,the floating cells in the monolayer culture cells were significantly reduced,the adhesion ability was enhanced,and the intercellular space was restored,which effectively alleviated the cell damage,indicating that uc OCN had a certain protective effect on the cells.Western blot analysis showed that the expression of GPX4 and SLC7A11 proteins in HT22 cells was further reduced after OCN knockdown compared with Eratin treatment alone,indicating that the presence of OCN in the cells contributes to the maintenance of the stability of the key proteins of ferroptosis in neurons.The results showed that the expression levels of ferroptosis-related proteins GPX4 and SLC7A11 were restored after uc OCN treatment, further corroborating the protective effect of uc OCN on HT22 cells.Western blot showed that OCN could alleviate ferroptosis in HT22 cells by regulating Akt-m TOR and Sirt1/PGC1a pathways in vitro.【Conclusions】The expression of OCN in the injured brain tissue of mice is significantly increased after TBI.OCN knockout can lead to anxiety-depression-like behaviors and affect the spatial memory ability of mice.Neurons are the main cells that play a role in nerve repair of OCN in the injured brain tissue after TBI.OCN knockout leads to the increase of inflammatory factors in the immune microenvironment of TBI mice,aggravate the activation of glial cells and the occurrence of ferroptosis.Interfering the expression of OCN promotes ferroptosis of HT22 neurons,and OCN alleviates ferroptosis of HT22neurons probably by regulating Akt-m TOR and Sirt1/PGC1a pathways.The results not only reveal the role of OCN in the neurological damage process after TBI,but also provide potential intervention targets and therapeutic strategies for the treatment of traumatic brain injury.