SIRT1对大鼠脊髓损伤神经功能恢复的影响
摘要
目的：探索SIRT1与大鼠脊髓损伤后炎症因子，细胞凋亡及神经功能恢复的关系。
方法：采用改良Allen s法，建立大鼠脊髓钝挫伤模型（T10），112只SD大鼠被随机分成4组：假手术组（n=16只），模型组（n=32只），白藜芦醇组（n=32只），对照剂组（n=32只）。脊髓损伤术后立即给予腹腔注射白藜芦醇（resveratrol,RES），100mg/kg和聚乙二醇硬脂酸酯15(solutol HS 15,SOL)，100mg/kg.进行功能评分的大鼠术后每天给药一次，连续1周。分别采用QT-PCR方法、ELISA方法、TUNEL荧光标记法对4组（SHAM，RES，SOL，SCI）脊髓组织中各时间点SIRT1的表达活性、炎症因子变化和细胞凋亡情况进行检测，并对大鼠术前和术后1天，3天，1周，2周，3周，4周进行BBB scores功能评分
结果：所有引物都可以成功的扩增出目的片段。RES组，SCI组和SOL组3组SIRT1的表达都有一定升高，RES组SIRT1表达在4h明显增加，在8h达到高峰，与其他两组(SCI组, SOL组)比较在4h,24h,36h差别有统计学意义（P＜0.05）.除假手术组以外各组自8h后SIRT1表达下降。
SCI组大鼠损伤脊髓组织在损伤后4 h各炎症性细胞因子表达即明显升高,其中TNF-α和IL-6于损伤后8 h达峰值,IL-10在观察时间内呈持续性增高，IL-1β在24h达峰值。SOL组各炎症因子的变化规律与SCI组基本一致，RES组与前两组相比，浓度差别均有统计学意义（P＜0.01）。
与SHAM组相比，SCI组，RES组，SOL组术后1天凋亡细胞明显升高，统计学比较差异性有意义（p＜0.05）。在3天时，SCI组，RES组，SOL组凋亡细胞数持续升高分别达到54.82，38.49，53.24，与其他两组比较，RES组升高较低差异有统计学意义（p＜0.05），通过定位发现，凋亡主要存在于神经元。
术后1天除假手术组功能评分没有明显改变外，其他3组后肢功能完全丧失0分,从术后第三天到第3周呈现逐渐升高趋势。SCI组，RES组，SOL组比较，前3周功能评分差别没有统计学意义（p＞0.05）。在第四周时，RES组功能评分上升比较明显，与SCI组、SOL组比较差别有统计学意义（P＜0.05），可见通过上调SIRT1的表达活性，能够促进大鼠脊髓损伤后功能恢复。
结论: SIRT1可能是通过抗炎，抗凋亡作用，促进大鼠脊髓损伤后功能恢复,其具体机制需要进一步实验。
Objective: To explore the relationship among SIRT1 and inflammatory cytokines,apoptosis,neurological recovery after spinal cord injury in rats.
Methods: The SCI of animal model was made by using a modified Allen's method onT(10).One hundred twelve SD rats were randomly divided into four groups：SHAMgroup(n=16),SCI group(n=32), RES group(n=32),SOL group(n=32).Resveratrol(100mg/kg)and Solutol(100mg/kg) was gave respectively by intraperitoneal injection following SCI.The expresstion of SIRT1 were measured by reverse transcription-polymerase chainreaction (RT-PCR). we detected the inflammatory cytokines ( IL-1β，IL-6，IL-10，TNF-α)and apoptosis with ELISA,TUNNEL respectively. The BBB scores before surgery and at1d,3d,1 week,2 weeks,3 weeks and 4 weeks after the surgery were examined.
Results：All primers were amplified the target slices successfully.We have found that RESgroup ,SCI group and SOL group all raise up.But there was a remarkable increase in theSIRT1 level at 4 hours after the trauma and it got to the peak at 8h in RES group.Comparingto the other two groups(SCI group, SOL group), the expression of SIRT1 had significantlystatistical differences(p＜0.05) at 4h,24h,36h of RES group.Except the SHAM group,theSIRT1 expression level all descended after 8h.
The expression of inflammatory cytokines increased obviously at 4h after trauma in SCIgroup,and the IL-6 got to the peak at 8h as well as TNF-α.While the peak of IL-1βlevelwas at 24h after trauma.But the level of IL-10 increased by degrees.The change rule ofinflammation cytokines in SOL group was agreed with the SCI group.The level of everyinflammatory cytokine in RES group has statistical differences compared with the other twogroups(p＜0.01).
Compared with SHAM group，the number of cell apoptosis increased remarkably in SCIgroup，RES group，SOL group after one day injury,it had significantly statisticaldifferences(p＜0.05).and the number of cell apoptosis reached up to 54.82，38.49，53.24in SCI group,RES group，SOL group respectively.Compared with the other groups, the levelof RES group was lower,there were significantly statistical differences(p＜0.05).
There was no remarkable change in sham group ,while the function of hind limbs in otherthree groups was loss completely after one day spinal cord injury.Although there were anelevator tendency from 3th to 21th in SCI group,SOL group and RES group,there were no statistical significance（p＞0.05）.At 28th,compared with SCI group and SOL group,theBBB scores in RES group got heighten obviously and had statistical significance（P＜0.05）.it is thus evident that SIRT1 promote the functional rehabilation after spinal cord injury inrats.
Conclusions：SIRT1 can remarkably promote the functional rehabilation after spinal cordinjury in rats,by the mechanism of anti-inflammation and anti-apoptosis.The detailedmechanism needs further study.
方法：采用改良Allen s法，建立大鼠脊髓钝挫伤模型（T10），112只SD大鼠被随机分成4组：假手术组（n=16只），模型组（n=32只），白藜芦醇组（n=32只），对照剂组（n=32只）。脊髓损伤术后立即给予腹腔注射白藜芦醇（resveratrol,RES），100mg/kg和聚乙二醇硬脂酸酯15(solutol HS 15,SOL)，100mg/kg.进行功能评分的大鼠术后每天给药一次，连续1周。分别采用QT-PCR方法、ELISA方法、TUNEL荧光标记法对4组（SHAM，RES，SOL，SCI）脊髓组织中各时间点SIRT1的表达活性、炎症因子变化和细胞凋亡情况进行检测，并对大鼠术前和术后1天，3天，1周，2周，3周，4周进行BBB scores功能评分
结果：所有引物都可以成功的扩增出目的片段。RES组，SCI组和SOL组3组SIRT1的表达都有一定升高，RES组SIRT1表达在4h明显增加，在8h达到高峰，与其他两组(SCI组, SOL组)比较在4h,24h,36h差别有统计学意义（P＜0.05）.除假手术组以外各组自8h后SIRT1表达下降。
SCI组大鼠损伤脊髓组织在损伤后4 h各炎症性细胞因子表达即明显升高,其中TNF-α和IL-6于损伤后8 h达峰值,IL-10在观察时间内呈持续性增高，IL-1β在24h达峰值。SOL组各炎症因子的变化规律与SCI组基本一致，RES组与前两组相比，浓度差别均有统计学意义（P＜0.01）。
与SHAM组相比，SCI组，RES组，SOL组术后1天凋亡细胞明显升高，统计学比较差异性有意义（p＜0.05）。在3天时，SCI组，RES组，SOL组凋亡细胞数持续升高分别达到54.82，38.49，53.24，与其他两组比较，RES组升高较低差异有统计学意义（p＜0.05），通过定位发现，凋亡主要存在于神经元。
术后1天除假手术组功能评分没有明显改变外，其他3组后肢功能完全丧失0分,从术后第三天到第3周呈现逐渐升高趋势。SCI组，RES组，SOL组比较，前3周功能评分差别没有统计学意义（p＞0.05）。在第四周时，RES组功能评分上升比较明显，与SCI组、SOL组比较差别有统计学意义（P＜0.05），可见通过上调SIRT1的表达活性，能够促进大鼠脊髓损伤后功能恢复。
结论: SIRT1可能是通过抗炎，抗凋亡作用，促进大鼠脊髓损伤后功能恢复,其具体机制需要进一步实验。
Objective: To explore the relationship among SIRT1 and inflammatory cytokines,apoptosis,neurological recovery after spinal cord injury in rats.
Methods: The SCI of animal model was made by using a modified Allen's method onT(10).One hundred twelve SD rats were randomly divided into four groups：SHAMgroup(n=16),SCI group(n=32), RES group(n=32),SOL group(n=32).Resveratrol(100mg/kg)and Solutol(100mg/kg) was gave respectively by intraperitoneal injection following SCI.The expresstion of SIRT1 were measured by reverse transcription-polymerase chainreaction (RT-PCR). we detected the inflammatory cytokines ( IL-1β，IL-6，IL-10，TNF-α)and apoptosis with ELISA,TUNNEL respectively. The BBB scores before surgery and at1d,3d,1 week,2 weeks,3 weeks and 4 weeks after the surgery were examined.
Results：All primers were amplified the target slices successfully.We have found that RESgroup ,SCI group and SOL group all raise up.But there was a remarkable increase in theSIRT1 level at 4 hours after the trauma and it got to the peak at 8h in RES group.Comparingto the other two groups(SCI group, SOL group), the expression of SIRT1 had significantlystatistical differences(p＜0.05) at 4h,24h,36h of RES group.Except the SHAM group,theSIRT1 expression level all descended after 8h.
The expression of inflammatory cytokines increased obviously at 4h after trauma in SCIgroup,and the IL-6 got to the peak at 8h as well as TNF-α.While the peak of IL-1βlevelwas at 24h after trauma.But the level of IL-10 increased by degrees.The change rule ofinflammation cytokines in SOL group was agreed with the SCI group.The level of everyinflammatory cytokine in RES group has statistical differences compared with the other twogroups(p＜0.01).
Compared with SHAM group，the number of cell apoptosis increased remarkably in SCIgroup，RES group，SOL group after one day injury,it had significantly statisticaldifferences(p＜0.05).and the number of cell apoptosis reached up to 54.82，38.49，53.24in SCI group,RES group，SOL group respectively.Compared with the other groups, the levelof RES group was lower,there were significantly statistical differences(p＜0.05).
There was no remarkable change in sham group ,while the function of hind limbs in otherthree groups was loss completely after one day spinal cord injury.Although there were anelevator tendency from 3th to 21th in SCI group,SOL group and RES group,there were no statistical significance（p＞0.05）.At 28th,compared with SCI group and SOL group,theBBB scores in RES group got heighten obviously and had statistical significance（P＜0.05）.it is thus evident that SIRT1 promote the functional rehabilation after spinal cord injury inrats.
Conclusions：SIRT1 can remarkably promote the functional rehabilation after spinal cordinjury in rats,by the mechanism of anti-inflammation and anti-apoptosis.The detailedmechanism needs further study.
引文
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