Preliminary Study On The Mechanism Of Ferroptosis In Neuropathic Pain

Objective:Establish a rat neuropathic pain(neuropathic pain,NP)model to detect Ferroptosis in the spinal cord.Through the administration of Ferroptosis inhibitors and agonists,to investigate whether inhibiting the occurrence of Ferroptosis can alleviate NP,and provide new ideas for the study of NP mechanism and the exploration of treatment methods.Methods:1 Forty rats were divided into 5 groups by random number table method,which were 1 group of sham operation group(Sham)and 4 groups of rats with chronic sciatic nerve ligation(Chronic constriction injury of the sciatic nerve,4 groups).The CCI components of the 4 groups were 1 day(1d group),3 days(3d group),7 days(7d group),and 14 days group(14d group),with 8 rats in each group.Mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were used to detect changes in mechanical pain threshold and thermal pain threshold in rats.Detection of Ferroptosis and oxidative stress in CCI model: Western blot and immunofluorescence were used to detect Ferroptosis specific protein Glutathione peroxidase 4(GPX4),long Expression of Long chain acyl Co A synthetase 4(ACSL4).Changes of mitochondria in spinal dorsal horn of rats in Sham group and 7d group were observed with electron microscope.Corresponding kits were used to detect changes in iron ion concentration,reactive oxygen species(ROS),malondialdehyde(MDA),and glutathione(GSH)content in the spinal cord of each group of rats.2 32 SD rats will be randomly divided into four groups : Sham group,CCI group,Ferrostatin-1 group(CCI + F group),Ferroptosis agonist Erastin group(CCI + E group),after intraperitoneal injection of different drugs before surgery,observe behavioral changes and changes in Ferroptosis-related proteins GPX4,ACSL4 expression at 7 days after operation,and detect changes in iron ion concentration,ROS,MDA content,and GSH content.Explore the role of Ferroptosis in NP.The difference was statistically significant with P <0.05.Results:1 Modeling situationNo rats died during the entire modeling process.Rats in each group did not eat well within 2 days after surgery,and gradually recovered afterwards.The incision healed well without redness,swelling,or inflammatory exudation.CCI model rats had lameness around 3 days after operation.The affected hindfoot showed mild valgus,licking,hanging and other painful behaviors.The sham-operated model rats had no pain behaviors such as foot contraction and valgus foot.2 Ferroposis in spinal dorsal horn of CCI model mice2.1 Behavioral changes in painCompared with the Sham group,the MWT and TWL of the rats in the CCI group decreased after 3 days(P<0.05).2.2 Ferroptosis-related proteins in spinal dorsal horn of CCI model ratsCompared with the Sham group,the expression level of GPX4 in the spinal dorsal horn of the CCI group was significantly decreased from 3 to 14 days after surgery(P<0.05),and the expression level of ACSL4 was significantly increased(P<0.05).2.3 Localization of Ferroptosis-related proteins GPX4 / ACSL4 in the spinal dorsal horn of CCI model ratsFerroptosis related proteins GPX4 and ACSL4 are mainly expressed in the cytoplasm of neurons.2.4 Mitochondrial changes in the spinal dorsal horn of CCI model ratsIn the CCI model,the mitochondria in the spinal cord dorsal horn cells show characteristic changes in Ferroptosis: mitochondrial membrane thickening,mitochondrial shrinkage,and mitochondrial crest disappearance.In the Sham group,the mitochondria in the cells at the dorsal horn of the spinal cord were normal,and no characteristic changes in Ferroptosis occurred.2.5 Changes of iron content,ROS,MDA,GSH in spinal cord of CCI model ratsCompared with the Sham group,the iron content in the spinal cord tissue of the rats in the CCI group increased from 3 to 14 days after the operation(P<0.05),the oxidative stress response increased,ROS(P <0.05)and MDA(P<0.05))increased,GSH decreased(P<0.05).3 The effects of regulating the occurrence of Ferroptosis on CCI model rats3.1 Regulation of the occurrence of Ferroptosis on allodynia and hyperalgesia in CCI model ratsCompared with the CCI group,the Ferroptosis inhibitor Ferrostatin-1 significantly reduced the abnormal pain(P<0.05)and thermal hyperalgesia(P<0.05)in the CCI + F group rats.Compared with the CCI + F group,the Ferroptosis agonist Erastin treatment significantly increased the abnormal pain(P<0.05)and thermal hyperalgesia(P<0.05)in the CCI + E group.3.2 Effect of regulating the occurrence of Ferroptosis on the expression of iron-related proteins in the spinal dorsal horn of CCI model ratsCompared with the CCI group,the expression of GPX4 protein in the spinal dorsal horn of the CCI + F group was increased(P<0.05),and the expression of ACSL4 protein was decreased(P<0.05);Compared with the CCI + F group,CCI + E Group GPX4 protein expression decreased(P<0.05),ACSL4 protein expression increased(P<0.05).3.3 Effect of regulating the occurrence of Ferroptosis on iron content,ROS,MDA,and GSH content in spinal cord tissue of CCI model ratsCompared with the CCI group,the iron content in the spinal cord tissue of the rats in the CCI + F group decreased(P<0.05);the iron content in the spinal cord tissue of the rats in the CCI + E group increased(P<0.05).Compared with the CCI group,the oxidative stress response in the spinal cord of the rats in the CCI + F group was reduced,and ROS(P<0.05),MDA(P<0.05)decreased,and GSH(P<0.05)increased.Compared with the CCI + F group,the oxidative stress response in the spinal cord of the rats in the CCI + E group increased,and ROS(P<0.05),MDA(P<0.05)increased,and GSH decreased(P<0.05).Conclusion:1 Ferroptosis can occur in NP.2 Inhibition of Ferroptosis can reduce the abnormal pain and hyperalgesia in rats with neuropathic pain,and alleviate the occurrence of oxidative stress in spinal cord of CCI rats,and have a therapeutic effect on neuropathic pain.