十二井穴放血对不同程度颅脑创伤大鼠脑水肿及线粒体生物合成的影响

Neuro-protective effect of blood-letting punctures at twelve Jing-Well points in rat model of traumatic brain injury

摘要:

目的 探讨十二井穴放血对颅脑创伤（TBI）大鼠脑水肿及线粒体生物合成的影响。方法 将56 只 SD 大鼠随机等分为7 组，即轻度TBI 组、轻度TBI 加井穴放血组、中度TBI 组、中度TBI 加井穴放血组、重度 TBI 组、重度TBI 加井穴放血组和对照组，每组8 只。应用电子控制性脑皮质撞击仪，轻度、中度、重度TBI 组 的打击深度分别为1、3 和4 mm。对照组仅开骨窗后缝合皮肤，不进行打击。井穴放血通过1 ml 注射器针头于 大鼠双侧前肢趾端的十二井穴点刺出血完成，出血量为每穴10 μl，每12 h 进行1 次放血。手术后72 h，进行神 经功能损伤评分（mNSS），随后取10 mg 损伤周围脑组织，qRT-PCR 检测过氧化物酶体增殖物激活受体γ 共激活因子1α（PGC-1α）、线粒体转录因子A（TFAM）基因的表达和线粒体DNA（mtDNA）拷贝数，剩 余脑组织进行脑含水量测量。结果 TBI 后大鼠的mNSS 评分均高于对照组，且随着TBI 严重程度的增加，评分 依次增高（P <0.05），且轻、中度TBI 组大鼠在应用井穴放血疗法后mNSS 评分与相应的单纯损伤组比较降低 （P <0.05）；TBI 模型大鼠的PGC-1α 和TFAM 基因表达水平以及mtDNA 拷贝数均高于对照组（P <0.05）； 轻度TBI 组大鼠在应用井穴放血疗法后，mtDNA 拷贝数高于相应的未应用放血疗法的大鼠（P <0.05），中度 TBI 组大鼠在应用放血疗法后PGC -1α 基因表达水平和mtDNA 拷贝数升高（P <0.05），轻、重度TBI 组大鼠在应 用井穴放血疗法后，虽然PGC -1α 和TFAM 基因表达水平均有升高趋势，但差异无统计学意义（P >0.05）； TBI 后各组大鼠脑组织含水量与对照组比较均增高，且中度TBI 组大鼠在应用井穴放血疗法后脑组织含水量与 相应的单纯损伤组大鼠比较降低（P <0.05）。结论 井穴放血可能通过激活PGC 及下游通路，促进mtDNA 的生 物合成，从而增强损伤脑组织的能量供应，进而改善脑水肿程度，发挥脑保护作用。

Abstract:

Objective To investigate the effect of blood-letting punctures at twelve Jing-Well points on cerebral edema and mitochondrial biogenesis in rat model of traumatic brain injury (TBI). Methods A total of 56 male SD rats were randomly divided into 7 groups: mild TBI group, mild TBI + blood-letting group, moderate TBI group, moderate TBI + blood-letting group, severe TBI group, Severe TBI + blood-letting group and sham group. (8 for each group). TBI were conducted by controlled technic of cortical impact injury with impacting depth of 1mm (mild TBI), 3 mm (moderate TBI) and 4 mm (severe TBI). Rats in the sham group underwent the same operation except for cortical impacting. Blood-letting at Jing-Well points was performed by pricking the bilateral forelimb toes of the rats with 1ml syringe needle every 12 h with bleeding amount of 10 μl per point each time. The modified neurological severity score (mNSS) was recorded 72 h post operation. Brain tissue surrounding the lesion was harvested for analysis of PGC- 1α, TFAM and mtDNA copy number.Brain water was measured. Results mNSS in 3 TBI groups were significantly higher compared with sham group, which increased along with the severity of TBI (P < 0.05). Treatment of bloodletting decreased mild and moderate TBI-induced mNSS (P < 0.05). Expression levels of PGC-1α, TFAM gene and the mtDNA copy in TBI group were significantly upregulated compared with Sham group (P < 0.05). Treatment of bloodletting in mild TBI+blood-letting group aggrandized increase of mtDNA copy number when compared with TBI only groups (P < 0.05). Expression of PGC-1α was upregulated by blood-letting therapy in moderate TBI group while no significant differences were observed in mild and severe TBI groups in terms of PGC-1α and TFAM gene expressions (P > 0.05). Water content in brain tissue of TBI group was significantly increased when compared with Sham group, which was decreased with the application of bloodletting therapy (P < 0.05). Conclusion Neuroprotective effect of blood-letting punctures at twelve Jing-Well pointsis achieves probably due to the fact that it enhances energy supplement, ameliorates brain edema through activation of PGC mediated signal pathways and biosynthesis of mtDNA.