| Objective:To study whether metformin can regulate the 5hmC level of DNA through the Tet2-Foxo3 a pathway,thereby regulating spinal cord injury and improving the recovery of nerve function.Methods:The 9-11 lamina of the thoracic vertebrae of the rat was excised to fully expose the spinal cord,and the SCI model was established by a NYU II-generation striker using a 10 g weight to freely fall at a height of 6 cm from the plane of the rat spinal cord.Twenty rats were selected and randomly divided into four groups.SCI+MET group:After spinal cord injury,rats were injected with metformin(200 mg/kg)intraperitoneally from the first day.SCI+NS group: rats were injected with the same volume of normal saline after spinal cord injury.C+MET group: Only the lamina was removed without damaging the spinal cord tissue,and metformin(200 mg/kg)was injected intraperitoneally from the first day.C+NS group: Only the lamina was removed,no damage of spinal cord tissue.Intraperitoneal injection of the same volume of physiological saline.In 1,3,7,14,21 and 28 days cant BBB score and hind legs movement function test evaluation.Twenty-four rats were selected and randomly divided into four groups.The treatments were the same as above.Surgery on the seventh day of rats,complete separation of the damaged spinal cord tissue,by TTC staining to observe spinal cord tissue necrosis area and carry on quantitative analysis.Twenty-four rats were selected and randomly divided into four groups.The treatments were the same as above.At 24 h after surgery,the spinal cord tissue was isolated,DNA was extracted for DOT-blot analysis and immunohistochemistry,RNA was extracted for qPCR and RT-PCR,and protein was extracted for western blot and immunoprecipitation.Eighteen rats were selected and randomly divided into three groups.SCI+MET+SC-1 group: 10 ul of Tet2 inhibitor SC-1 was injected intrathecally half an hour before modeling.The SCI+MET group and SCI+NS group were treated as before,and 10 ul normal saline was injected intrathecally.Surgery on the seventh day of rats,complete separation of the damaged spinal cord tissue,by TTC staining to observe spinal cord tissue necrosis area and carry on quantitative analysis.Eighteen rats were selected and randomly divided into three groups.The treatment was the same as above.After 24 hours,the spinal cord tissue was separated,DNA was extracted for Dot-blot analysis,RNA was extracted for qPCR,and protein was extracted for western blot.Results:1.Metformin promotes functional recovery in SCI ratsTo determine whether metformin affects apoptosis after SCI,we injected metformin directly into the abdominal cavity of rats.At 7 day after SCI,Spinal cord tissues injured were quickly removed and then cut into 0.1-cm thick slices.Slices were incubated with 0.2% TTC at 37°C for 30 min and fixed in 4% paraformaldehyde.The results showed that metformin could reduce the necrotic volume of spinal cord tissue.These results suggest that metformin plays a key role in regulating apoptosis after SCI.To determine the effect of metformin on motor recovery after SCI,BBB score was used to evaluate the functional recovery at 4 weeks after injury.During the whole experiment,we found that the BBB score of the control group was normal and the motor function of the hind limbs of the rats was not affected,while the BBB score of the injury group increased gradually over time but was still lower than the normal level.BBB scores were significantly higher on days 14(9.6± 1.02vs6.8±1.33,p =0.0101),21(14.8±1.33vs10.2±1.72,p = 0.0029)and 28(15±1.10vs12±0.63,p = 0.0015)after injury in metformin treated animals when compared to untreated animals.These findings showed that metformin treatment after SCI promoted the recovery of motor function in rats.Similarly,rats treated with metformin showed the same trend in the incline test scores after injury.2.Metformin can increase global DNA 5hmC modificationTo determine whether metformin affects global DNA 5hmC modification after SCI,we examined 5hmC levels in spinal cord tissues at 24 h after SCI.In this SCI rat model,global 5hmC levels were increased at 24 h after SCI,while the global 5hmC was further increased after metformin was used.This was confirmed by quantitative analyses(P <0.05).Meanwhile,fluorescent staining demonstrated an increase in 5hmC at 24 h after SCI compared with control group,and a further increase of 5hmC after metformin was used.These results suggest that Metformin alters the dynamics of cytosine methylation.3.Tet2 is involved in the anti-apoptotic effect of metformin after SCIWe further examined the levels Tet2 by Western blot at 24 h after SCI.We found that Tet2 levels were increased at 24 h after SCI,while the Tet2 was further increased after metformin was used.In order to further prove that Tet2 is involved in the regulation of metformin in SCI,we used a previously reported Tet2 inhibitor,SC-1(Pluripotin),and injected it into the spinal cord tissue of rats.The results showed that the application of SC-1 reduced the level of 5hmC and inhibited the protective effect of metformin on spinal cord in the SCI model.4.Foxo3 a is involved in the anti-apoptotic effect of metformin after SCIIn order to observe the metformin Tet2 and Foxo3 a changes before and after the treatment.We then tested whether Foxo3 a influenced the anti-apoptotic effect of metformin.Our results showed that metformin increased the interaction of Tet2 with Foxo3 a.In addition,we examined the mRNA levels of Foxo3 a by quantitative polymerase chain reaction(qPCR)at 24 h after SCI.We found no significant change in Foxo3 a mRNA level after SCI.5.The mRNA and the 5hmC levels in genes downstream of Foxo3ATo further prove that the Tet2-Foxo3 a axis is involved in the regulation of metformin.We also detected the expression of apoptotic genes downstream of Foxo3 A after SCI and the changes in the expression after metformin treatment by the quantitative RT-PCR analysis,such as Bim,P27 kip,Bax and Fas L.We found that the mRNA levels of Bim,P27 kip,Bax significantly increased after SCI.After treatment with metformin,the mRNA levels of Bim,Bax decreased compared with that of the SCI group.The mRNA level of Fas L showed no obvious change with different treatments.in addition,Hydroxymethylated DNA was used for qPCR analysis,and the results showed that the relative 5hmC levels in Bim,P27 kip,Bax significantly increased after SCI injury.Compared with the SCI group,the relative 5hmC levels in Bim and Bax decreased,while in P27 kip genes the 5hmC levels increased after metformin treatment.the relative 5hmC levels in Fas L did not change appreciably.Conclusion:Metformin can promote the interaction between Tet2 and Foxo3 a,increase the5 hmC level of the overall DNA,thereby inhibit the expression of related apoptotic genes,and improve tissue damage and nerve function recovery after spinal cord injury. |
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