The Role Of Ferroptosis In Paclitaxel-induced Neuropathic Pain In Rats

Objective We established paclitaxel-induced neuropathic pain(PINP)model in rats by intraperitoneal injection of paclitaxel,and explored the role of ferroptosis in PINP through behavioral test and molecular biological methods,so as to provide a new idea for the treatment of PINP.Methods1.The role of ferroptosis inducer in normal rats.Fifty male Sprague-Dawley(SD)rats were randomly divided into different groups.The effects of single intrathecal injection of Ras-selective lethal small molecule3(RSL3)on mechanical paw withdrawal threshold(MPWT)in normal rats were evaluated by Von Frey filaments before and 0.5,1,2 and 4 h after administration.RSL3 were given once daily from day 1 to day 5 to examine the effects of repeated administration of RSL3 on MPWT in normal rats,and the rats were anesthetized at 0.5h after administration of RSL3 on day 5 to detect the expression of glutathione peroxidase 4(GPX4)by Western blot. To investigate the effect of ferroptosis inhibitor Liproxstatin-1(Lip-1)on RSL3-induced mechanical allodynia in normal rats,Lip-1 was injected 30 min before the single injection of RSL3.2.The role of ferroptosis in PINP rats.Eighty-eight male Sprague-Dawley(SD)rats were randomly divided into different groups.The PINP model was established by intraperitoneal injection of paclitaxel(2mg/kg)on 0d,2d,4d and 6d.The MPWT of bilateral hind paws was evaluated at baseline and days 3,7,14 and 21.The lumbar enlargement segments of the spinal cord in Vehicle group were extracted at 21 d,and those in the PINP group were extracted at 3d,7d,14 d,and 21 d,respectively.The expression of GPX4 was detected by Western blot.Rats in PINP group were anesthetized by intraperitoneal injection of 1% sodium Pentobarbital at a dose of 60mg/kg on 21 d,then the lumbar enlargement segments of the spinal cord were removed after perfusing with paraformaldehyde(PFA),and the distribution and cell localization of GPX4 were detected by immunofluorescence.The effects of single and repeated administration of different dose of Lip-1 on MPWT of PINP rats were detected at 14 d and 14 to 18 d,respectively.After intrathecal injection of Lip-1 40μ g for 5 consecutive days from 14 to 18 days in PINP rats,the spinal lumbar enlargement segments were obtained at 1 hour after the administration on 18 days,and the expression of GPX4 was detected by Western blot.LIP-1 was given intrathecally from day 0 to day 6 for 7 consecutive days to detect the prophylactic effect of ferroptosis inhibitor on MPWT of PINP rats.Results1.Single and repeated intrathecal injection of RSL3 markedly induced mechanical allodynia in normal rats,and the protein expression level of spinal GPX4 in normal rats was significantly decreased.Intrathecal injection of Lip-1 in advance could notably reverse the mechanical allodynia induced by RSL3 in normal rats.2.The MPWT in PINP group significantly decreased at 7 days after modeling and lasted at least 21 days.Meanwhile,the expression level of spinal GPX4 was significantly decreased.The fluorescence intensity of GPX4 in PINP group decreased significantly,and GPX4 was mainly expressed in neurons of spinal dorsal horn.Single and repeated intrathecal injection of Lip-1 significantly increased the MPWT in PINP rats and reversed the decreased expression of spinal GPX4 in PINP rats.Preventive intrathecal injection of Lip-1 only increased the MPWT of PINP rats on day 7,but it had no significant effect on the MPWT of PINP rats at day 14 and day21.Conclude Ferroptosis inducer can induce mechanical allodynia in normal rats.Ferroptosis inhibitor can significantly alleviate the established PINP and delay the occurrence of PINP.Ferroptosis is involved in the occurrence and development of PINP.