ACSL4 Regulates Neuronal Ferroptosis In Traumatic Spinal Cord Injury Via V-ATPase:a Mechanistic Study

Objective:One of the serious consequences of secondary injury in traumatic spinal cord injury（TSCI）is ferroptosis.ACSL4 is a key enzyme in the production of lipid peroxides and one of the markers of ferroptosis,which can affect the sensitivity of cells to ferroptosis.The role and mechanism of ACSL4 in ferroptosis of TSCI neurons is not clear.Therefore,in this study,we used in vitro experiments to explore the effect and mechanism of ACSL4 on ferroptosis of neurons,and combined with in vivo experiments to verify these effects and mechanisms.Methods:In this study,the mouse TSCI model was constructed,the expression level of ACSL4was detected by immunohistochemical technique,and the expression of ACSL4 was localized by immunofluorescence double staining technique.The results of bioinformatics analysis of ACSL4-related genes showed that there was a high correlation between ACSL4 and v-ATPase.We used the mouse hippocampal neuron stable transition cell line with overexpression of ACSL4（LV-ACSL4-HT22）in our laboratory as the research object.The model of ferroptosis of neurons was established by erastin,and RNAi interference and drug inhibition were used to inhibit ACSL4.Then CCK-8,Western Blotting,q PCR and fluorescence probe techniques were used to study the role and mechanism of ACSL4 in ferroptosis of neurons.In the mouse TSCI model,the expression of ferroptosis markers was detected,the survival of neurons was detected by immunohistochemical method,the recovery of motor function was evaluated by BMS score,and the expression levels of ACSL4 and ATP6V1A protein were detected by Western Blotting technique.Results:（1）The results of immunohistochemical staining and immunofluorescence staining showed that the expression of ACSL4 was up-regulated in the spinal cord of TSCI mice,and the expression of ACSL4 was high in neurons.（2）The results of bioinformatics analysis showed that ATP6V1C2 expression was similarly elevated when the expression was elevated in ACSL4,which is closely related to the v-ATPase composed of ATP6V1C2 subunits.（3）In vitro experiments showed that RNAi interference and drug inhibition of ACSL4 could inhibit neuronal ferroptosis;after RNAi interference and drug inhibition of ACSL4 expression,ATP6V1A expression decreased,Lyso Sensor fluorescence probe detection results showed that RNAi interference and drug inhibition of ACSL4 could inhibit the function of v-ATPase;Rho Nox-1 divalent iron fluorescence probe detection results showed that RNAi interference and drug inhibition of ACSL4 decreased the concentration of Fe²⁺in neuronal ferroptosis.（4）In vivo experiments validate the role and mechanism of ACSL4 in neuronal ferroptosis.In a mouse TSCI model,inhibition of ACSL4 was able to suppress ferroptosis and improve neuronal survival and promote motor function recovery in TSCI mouse,similarly,inhibition of ACSL4 was able to suppress ATP6V1A protein expression levels in a mouse model of TSCI.Conclusions:（1）ACSL4 is highly expressed in spinal neurons after TSCI in mouse.（2）Inhibition of ACSL4 in TSCI mouse can inhibit neuronal ferroptosis and improve the recovery of motor function after TSCI.（3）In the process of neuronal ferroptosis,ACSL4 affects the function of v-ATPase by regulating ATP6V1A,which leads to the accumulation of Fe²⁺in neurons and finally leads to ferroptosis of neurons.