Experimental Study On The Inhibition Of Ferroptosis In Mice With Spinal Cord Injury By Resveratrol

Objective(1)To establish a model of spinal cord injury(SCI)in mice and explore the expression and time point of ferroptosis in the spinal cord after SCI.(2)To explore the effect of resveratrol(Res)on ferroptosis of neurons and recovery of motor function in SCI mice.(3)To explore the mechanism of resveratrol inhibiting ferroptosis after SCI.Methods(1)The T10 SCI model of c57 mice was established by the spinal cord precision impactor(RWD68099 Ⅱ),and was randomly divided into 6 groups,including sham group,SCI-1d group,SCI-3d group,SCI-5d group,SCI-7d group,SCI-14d group.Western blotting(WB)was used to detect the expression level of ferroptosis-related proteins such as glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(xCT),iron transporter(FPN),ferritin heavy chain 1(FTH1)and acyl coenzyme A synthase long chain member 4(ACSL4)in the spinal cord of each group;The expression level of iron ions in the spinal cord of each group was detected by the enhanced staining of Perls blue DAB(diaminobenzidine).(2)The mice were randomly divided into 4 groups:sham operation group(sham group),SCI group,SCI+Vehicle group(SCI+V group),and SCI+Res(200mg/kg)group.Basso Mouse Scale score(BMS score)and footprint analysis were used to assess the recovery of hind limb motor function of mice in each group;The morphological changes of the injured spinal cord were observed by hematoxylin eosin(HE)staining;The expression of normal and degenerated neurons in the injured spinal cord was observed by Nissl staining and immunofluorescence(IF)staining.WB was used to detect the expression of ferroptosis related protein in spinal cord of each group.The content of glutathione(GSH),malonaldehyde(MDA),GPX4 activity and the concentration of ferrous ion(Fe2+)in the spinal cord of each group were detected by the kit.The morphology of neurons and mitochondria in the spinal cord were observed by transmission electron microscopy.Perls blue DAB enhanced staining was used to detect the deposition of iron ions in neurons of spinal cord lesions in each group.(3)The mice were randomly divided into two groups:SCI+Res group and SCI+Res+ML385 group(nuclear factor E2 related factor 2(Nrf2)inhibitor)(30mg/kg).WB was used to detect the expression of Nrf2/GPX4 pathway related proteins.Results(1)WB results showed that compared with sham group,the expression of xCT and GPX4 showed a downward trend after injury,significantly decreased on the third day(p<0.01),and then increased to varying degrees.The expression of ACSL4 decreased after injury,and then increased significantly on the 3rd day(p<0.01),reaching the peak.The expression of FTH1 increased after injury and significantly increased on the 5th day(p<0.01).The expression of FPN decreased significantly at all time points after injury(p<0.01).The Perls blue staining results showed that compared with the sham group,the iron ion deposition of neurons in the focal area of the spinal cord at each time point after injury increased to varying degrees,reaching the peak on the third day(p<0.01),and then showed a downward trend.(2)The BMS score showed that the score of SCI+Res treatment group was significantly higher than that of SCI+V group on the 14th,21st,28th and 35th day after SCI(p<0.05).Footprint test shall be conducted on the 35th day after injury.The results showed that compared with SCI and SCI+V group mice,the gait recovery and motor coordination ability of mice treated with Res were significantly improved(p<0.01).(3)HE staining results showed that Res alleviated the pathological morphological changes of the lesion area after injury.Nissl staining showed that Res could retain more surviving neurons than SCI group(p<0.01).The results of FJC staining showed that the neurons with degeneration in the focus area expressed the most after SCI 3 days(p<0.05).Compared with SCI+V group,Res could inhibit the degeneration of neurons(p<0.05).The IF results showed that Res inhibited neuronal death(p<0.05).(4)The transmission electron microscope observation showed that the mitochondria shrunk and the mitochondrial cristae decreased after SCI,while Res significantly improved the morphology of mitochondria.(5)Perls blue DAB enhanced staining showed that pathological brown iron ions appeared in the focus area after SCI.After the intervention with Res,the deposition of iron ions was significantly reduced(p<0.05).The expression of iron ions in the injured spinal cord was detected by iron ion kit.The results showed that Res reduced the expression of iron ions compared with SCI+V group(p<0.05).At the same time,Res can also reduce the expression of MDA,promote the activity of GPX4 and the expression of GSH.(6)WB results showed that compared with SCI+V group,Res intervention up-regulated the expression of ferroptosis related proteins such as GPX4,xCT,FPN,FTH1,and inhibited the upregulation effect of Res after the use of Nrf2 specific inhibitor ML385(p<0.05).Conclusions(1)The phenomenon of neuronal cell ferroptosis occurs at the early stage of SCI in mice and reaches the peak on the third day.(2)Res can improve the iron deposition of spinal cord neurons in the focus area after SCI in mice,and promote the recovery of nerve and motor functions after SCI.(3)Res may inhibit ferroptosis of neurons through Nrf2/GPX4 pathway,alleviate secondary injury of SCI,and promote functional recovery.