Protective Effect Of Carnosic Acid On Spinal Cord Injury Via Inhibiting Ferroptosis

Objective: Spinal cord injury(SCI)can result in diversified cascades of pathology at the site of injury,inducing a series of cell death.In turn,multiple danger signals released by dead cells can further aggravate the secondary spinal cord injury,consequently,becoming a vicious circle.Ferroptosis is a newly identified form of cell death,and widely involved in the occurrence and development of diseases in central nervous system.Carnosic acid(CA)is a poly-phenolic diterpene,possessing antioxidant and neuroprotective activities.However,few studies had investigated the protective effects of CA on SCI.Therefore,the purposes of this study are to: 1)investigate the appearance of ferroptosis after spinal cord injury via establishing SCI model of SD rats;2)look into whether CA could inhibit the occurrence of ferroptosis in PC12 cells induced by erastin;3)explore the mechanism of carnosic acid inhibiting ferroptosis in vitro;4)investigate whether CA can improve neurological function and its specific mechanism in vivo.Methods: 1)SD rats were randomly divided into six groups: Sham group,SCI-6h group,SCI-1d group,SCI-3d group,SCI-7d group,and SCI-14 d group.Modified Allen's method was used to establish the SCI model of rats.Common targets between ferroptosis and spinal cord injury were analyzed by bioinformatics.Specific ferroptotic genes PTGS2,RPL8 F,HMGB1,ATP5G3,IREB2,and CS were analyzed by RTq PCR.WB was utilized to detect the protein expression of ACSL4,FPN,FTH1,GPX4,and x CT for each group.Iron level was measured by Perl's staining and Iron Assay Kit,and GPX4 activity was measured by a commercial kit.The denatured neurons were analyzed By FJB staining.2)CCK-8 was used to detect the effect of different concentrations of CA and erastin on the viability in PC12 cells.The experiment included six groups: Control group,Model group,Positive control group(Fer-1 group),Low-dose CA group,Middledose CA group,and high-dose CA group.Commercial kits were used to detect MDA contents,GSH levels,iron levels,and GPX4 activity levels in PC12 cells.Flow cytometry was used to detect the effect of CA on cellular ROS levels.The influence of CA on mitochondrial membrane potential and morphology was detected by Mito-Tracker Red probe and double-stained with uranyl acetate and lead citrate.3)The experiment contained six groups: Control group,Model group,Low-dose CA group,Middle-dose group,Highdose group,and Nrf2 inhibitor group.WB and Immunofluorescence(IF)staining were used to detect the effect of CA on expression level and location of Nrf2.RT-q PCR and WB were used to measure the m RNA and protein expression level regarding x CT,GCLM,GCLC,GSS,GPX4,FTH1,and FPN,respectively.Commercial kits were utilized to measured cellular MDA contents,GSH levels,iron levels,and GPX4 activity in PC12 cells.4)SD rats were randomly divided into four groups: Sham group,SCI group,SCI+L-CA group,and SCI+H-CA group.Neurological function was assessed using the Basso-BeattieBresnahan(BBB)open-field locomotor scale and inclined-plane test.The morphologic changes of spinal cord for each group were observed by HE staining.The influence of CA on neurons after spinal cord injury was assessed by FJB staining and Nissl staining.Commercial kits were used to detect the effect of CA on MDA contents,GSH levels,Iron levels,and GPX4 activity after spinal cord injury.RT-q PCR and WB were utilized to analyze the effect of CA on m RNA level and protein level for ferroptosis-related targets.Results: Results from RT-q PCR showed that the m RNA levels of PTGS2,RPL8 F,and HMGB1 after spinal cord injury were higher than those in sham group(P<0.01).Among them,the m RNA level of PTGS2 was highest at 6 hours and RPL8 F and HMGB1 reached their peaks at 3th day after spinal cord injury.The results of WB demonstrated that the protein level of ACSL4 was significantly higher compared to sham group at 3th day after operation(P<0.01).The protein level of FPN decreased significantly after spinal cord injury(P<0.01),and reached the lowest value at 3th day after operation.The protein level of FTH1 increased significantly since the third day after operation(P< 0.01).Compared to sham group,the protein level of GPX4 and x CT decreased significantly at first day after spinal cord injury.The results showed that iron level in spinal cord tissue significantly increased at 6 h after spinal cord injury(P< 0.01),and reached the highest value at 3th day after operation.Compared to sham group,GPX4 activity significantly decreased at first day following spinal cord injury,and reached the lowest value at 3th day after operation.The results from FJB staining showed that the denatured neurons increased obviously at 6 h after operation(P<0.01),and had peaked at 3th day after spinal cord injury.2)The results from CCK-8 demonstrated that administration of ? 20 ? M CA had no significant cytotoxic effect on PC12 cells compared with control group(P>0.05),and 1,5,and 10 ?M CA were selected for further experiments.Data showed that the decreased cell viability of PC12 cells caused by erastin was rescued by CA.Stimulus with erastin can decrease cellular GSH levels and GPX4 activity levels and increase MDA contents(P<0.01)in PC12 cells,which can be reversed by treatment with CA.The results from flow cytometry showed that erastin could significantly increase cellular ROS levels in PC12 cells(P<0.01),and treatment with CA can reduce cellular ROS levels in erastin-treated PC12 cells(P<0.01).Data from our study showed that erastin can decrease mitochondrial membrane potential,and lead to shrunken mitochondria and decreased or complete absence of mitochondria cristae in PC12 cells,and obvious improvement of mitochondrial morphology could be observed after treatment with CA.3)The results from RT-q PCR showed that the m RNA levels of Nrf2 had no significant change in PC12 cells exposed to erastin with or without CA.However,CA can significantly increase cellular Nrf2 protein level and promote its translocation into cell nucleus compared to erastin group.The results from RT-q PCR and WB demonstrated that CA strikingly upregulated the m RNA and protein expression levels in term of x CT,GCLM.GCLC,GSS,GPX4,FTH1,and FPN,moreover,ML385,a specific Nrf2 inhibitor,could weaken the protective effect of carnosic acid.4)BBB scale and Inclined-plane test indicated that administration of CA could significantly promote motor function recovery.Compared to sham group,CA can relieve the histopathological changes of spinal cord,reduce the number denatured neurons(P < 0.01),and increase the number of surviving neurons(P<0.01).And,CA also can reverse the increased m RNA level of PTGS2,protein level of ACSL4,MDA contents,and iron level,as well as the decreased GSH level and GPX4 activity.The results from RT-q PCR and WB showed that CA could regulate the m RNA and protein expression levels of Nrf2,x CT,GCLC,GCLM,GSS,GPX4,FPN,and FTH1 compared to SCI group.Conclusion: 1)Ferroptosis was involved in the process of secondary spinal cord injury and appeared at the early stage of injury(6 h),and it was much obvious at 3th day after operation;2)Carnosic acid could inhibit the occurrence of ferroptosis in PC12 cells induced by erastin;3)Carnosic acid could upregulate GSH-GPX4 axis and increase the expression levels of FTH1 and FPN via activating Nrf2 pathway,consequently improving cellular endogenous antioxidant capacity and inhibiting the occurrence of ferroptosis;4)Carnosic acid could inhibit the occurrence of ferroptosis,increase the survival of neurons,and reduce spinal cord injury via activating the Nrf2 pathway,thus promoting the recovery of neurological function.