Mechanism Of Ferroptosis Of Spinal GABAergic Neurons In Mice With Bone Cancer Pain

Objective: In this study,a mouse with bone cancer model was constructed to observe the effect of bone tumor progression on ferroptosis of GABAergic intermediate inhibitory neurons.By interfering with the occurrence of ferroptosis,the effects on pain behavior,neurotransmitter expression and ferroptosis-related protein expression changes in mice were observed.To elucidate the relationship between ferroptosis of spinal GABAergic neurons and bone cancer pain in mice,and then explore methods that can effectively improve the treatment of bone cancer pain.Methods:1.fifty-two 8-week-old male C57BL/6 mice in total,weighing about20-25 g,were divided into 2 groups according to the random number table:bone cancer pain group(BCP group,n=40)and control group(Sham group,n = 12).In the BCP group,the model was established by injecting a medium containing tumor cells;in the control group,after incision of the ipsilateral knee joint,only an equal volume of medium containing inactivated tumor cells was injected.The behavioral changes of the hindlimbs on the modeling side of the mice in each group were measured before modeling and on the5 th,10th,15 th,and 20 th days after modeling(n=8),including: paw withdrawal mechanical threshold(PWMT),paw withdrawal thermal latency(PWTL),mechanical withdrawal frequency,and spontaneous lift of the surgical side within 10 minutes.The growth of bone tumors was observed by in vivo imaging techniques and HE staining of bone tissue sections.Spine and lumbar spine augmentation were performed on the 5th,10 th,15th,and20 th days after modeling in the BCP group,and on the corresponding 20 th day in the Sham operation group.The expression of GABA protein in the spinal cord of each group was detected by Western blot(n=6);the distribution of GABA in the bilateral spinal cord of the mice in each group was observed by immunofluorescence method(n=4).On the 20 th day,ELISA was used to detect the level of oxidative stress in spinal cord neurons in each group;the changes of spinal GABAergic neurons and mitochondria in each group were observed by immunoelectron microscope(n=2).2.Another thirty 8-week-old male C57BL/6 mice were randomly divided into 3 groups(n=10): vehicle group(BV group),control group(SV group)and inhibitor group(BF group).After the bone cancer pain model was successfully established,ferrostain-1 was injected intraperitoneally at a dose of 10 mg/kg in the BF group,an equal volume of mixed DMSO solvent was intraperitoneally injected in the BV group,and the SV group was intraperitoneally injected with mixed DMSO solvent on the basis of Sham operation,continuously for 20 days.PWMT,PWTL,mechanical foot withdrawal frequency and spontaneous foot withdrawal times within 10 minutes were measured in each group before and on the 5th,10 th,15th,and20 th days after modeling(n=8).On the 20 th day after operation,the spinal cord lumbar enlargement in each group was collected.;Western blot was used to detect changes of GABA protein,COX-2 protein and ferroptosisrelated protein(n=6);immunofluorescence was used to observe changes of spinal cord GABA protein in each group(n=4),and immunoelectron microscopy was used to observe ultrastructural changes of cells and mitochondria(n=2)3.Another thirty-two 8-week-old male C57BL/6 mice were randomly divided into 4 groups(n=8): after the bone cancer pain modeling was completed by the above method,they were divided into vehicle group(BCP+Vehicle group)and inhibitor group(BCP+FER-1 group),and then each group was divided into parecoxib group(parecoxib group)and normal saline group(NS group)according to the different injected drugs.The parecoxib group was intraperitoneally injected with 10 mg/kg parecoxib on the 20 th day after modeling,and the NS group was intraperitoneally injected with an equal volume of normal saline on the 20 th day.After the injection was completed,the behavior of the mice in each group was measured.Results:1.Compared with the control group,the bone cancer pain model mice showed a significant decrease in the PWMT and PWTL on the 5th day after the operation,and lasted until the 20 th day(p<0.05).The frequency of mechanical foot withdrawal and the number of spontaneous foot withdrawal within 10 minutes increased(p<0.05);in vivo imaging technology observed that the femoral tumor of the mouse gradually increased,and HE staining of the femoral section showed tumor cells in the bone marrow cavity.The above results indicate that the model is established success.The results of Western blot and immunofluorescence showed that spinal GABA protein decreased on the 5th day and continued to the 20 th day(p<0.05).The levels of malondialdehyde(MDA)and reactive oxygen species(ROS)in the spinal cord of mice with bone tumors were increased by enzyme-linked immunosorbent assay(ELISA)(p<0.05),while the level of superoxide dismutase(SOD)and glutathione(GSH)levels decreased(p<0.05).Immunoelectron microscopy showed that the mitochondria of GABAergic neurons in the spinal cord of the mice in the bone cancer pain group shrunk and the double-layer membrane density increased.2.Compared with the control group(SV group),the PWMT and PWTL were decreased(p<0.05)while the frequency of mechanical foot withdrawal and the number of spontaneous foot withdrawal were increased(p<0.05)in the vehicle group(BV group).Compared with the BV group,the PWMT and PWTL increased from the 5th day after inoculation(p<0.05)while the frequency of mechanical foot withdrawal and the number of spontaneous foot withdrawal decreased in the inhibitor group(BF group)(p<0.05).The in vivo imaging results showed that FER-1 did not affect tumor growth(p>0.05,BF VS BV).Western blot showed that compared with the SV group,the expressions of GPX4 and GABA in the spinal cord of the mice in the BV group were significantly down-regulated(p<0.05),while the expressions of4-HNE and p-ERK were up-regulated(p<0.05);compared with the BV group,the expressions of GPX4 and GABA were up-regulated(p<0.05)and the expressions of 4-HNE and p-ERK were down-regulated(p<0.05)in BF group.The results of immunofluorescence showed that compared with the SV group,the GABA distribution in the spinal cord of the BV group was decreased(p<0.05),while the GABA distribution in the BF group was increased compared with the BV group(p<0.05).Immunoelectron microscopy showed that the mitochondrial contraction and double membrane density of GABAergic neurons in BV group were more obvious than those in SV group;the number of GABA neurons characterized by ferroptosis was decreased in BF group compared with BV group.3.In the vehicle group,compared with the injection of NS,there was no significant difference in the PWMT,PWTL,mechanical foot withdrawal frequency and the number of spontaneous foot withdrawal within 10 minutes of the mice injected with parecoxib(p>0.05).;In the inhibitor group,compared with NS injection,mice injected with parecoxib increased the PWMT and PWTL(p<0.05),while the frequency of mechanical paw withdrawal(p<0.05)and spontaneous 10 minutes The number of foot withdrawal was significantly reduced(p<0.01).Conclusion:1.Bone tumors in mice can cause ferroptosis in spinal GABAergic neurons and induce hyperalgesia2.The use of ferroptosis inhibitors can relieve bone cancer pain by inhibiting ferroptosis of GABAergic neurons.3.Inhibition of ferroptosis in GABAergic neurons can enhance the analgesic effect of parecoxib in mice with advanced bone cancer pain.