针刺“百会”透“曲鬓”对脑出血大鼠线粒体分裂与融合相关蛋白表达水平的影响

Effects of acupuncture at "Baihui"(GV20) through "Qubin"(GB7) on the expression levels of mitochondrial fission and fusion-related proteins in rats with intracerebral hemorrhage

关键词:  脑出血  针刺  线粒体分裂  线粒体融合  百会  曲鬓

Key words:  intracerebral hemorrhage  acupuncture  mitochondrial fission  mitochondrial fusion  Baihui  Qubin

摘要:

目的 从线粒体分裂蛋白（Drp1及其活化形式、Fis1蛋白）和线粒体融合蛋白（Mfn1、Mfn2、OPA1）探讨针刺“百会”透“曲鬓”对脑出血（ICH）病变发挥的作用与机制。方法 30只SPF级雄性SD大鼠随机分为假手术组、ICH组和ICH+针刺组，每组10只。除假手术组外，其余大鼠使用自体血注入纹状体法造模，Berderson神经功能评分评估造模成功。造模1 h后进行针刺治疗，连续3 d，每24 h针刺1次，每次30 min。针刺结束1 d后进行神经功能评分，透射电镜观察针刺百会透曲鬓大鼠出血半暗带线粒体形态、大小和数量，实时聚合酶链锁反应（Realtime PCR）检测线粒体DNA表达水平，蛋白免疫印记法（Western blot）检测线粒体分裂蛋白p-Drp1（Ser637）、p-Drp1（Ser616）、mito-Drp1、cyto-Drp1、Fis1，线粒体融合蛋白Mfn1、Mfn2、OPA1，MAPK/Drp1通路p-p38α蛋白的表达。结果 与ICH组比较，ICH+Ac组大鼠针刺最后1 d Berderson神经功能评分降低（P＜0.000 1）；透射电镜观察可见ICH组大鼠线粒体形态明显异常，与ICH组相比，ICH+Ac组线粒体形态改善。与sham组相比，ICH组大鼠mtDNA表达水平降低（P＜0.000 1）。与ICH组相比，ICH+Ac组大鼠mtDNA表达水平提高（P＜0.000 1）。与sham组相比，ICH组大鼠p-Drp1（Ser637）、cyto-Drp1的表达下降（P＜0.000 1）；与ICH组相比，ICH+Ac组大鼠p-Drp1（Ser637）、cyto-Drp1的表达提高（P＜0.000 1）；与sham组相比，ICH组大鼠p-Drp1（Ser616）、mito-Drp1、Fis1的表达上升（P＜0.000 1）；与ICH组相比，ICH+Ac组大鼠p-Drp1（Ser616）、mito-Drp1、Fis1的表达下降（P＜0.000 1）；与sham组相比，ICH组Mfn1/2的表达水平下降（P＜0.000 1），ICH+Ac组Mfn1/2的表达水平上升（P＜0.000 1）；与sham组相比，ICH组OPA1 S/L的表达水平上升（P＜0.000 1），ICH+Ac组OPA1 S/L的表达水平下降（P＜0.000 1）。与sham组相比，ICH组p-p38α的表达水平上升（P＜0.000 1），ICH+Ac组p-p38α的表达水平下降（P＜0.000 1）。结论 针刺百会透曲鬓可能通过抑制ICH后线粒体的过度分裂，促进线粒体融合，维持线粒体动力学平衡，从而维持线粒体基因的完整性，改善ICH后神经损伤。

Abstract:

Objective To explore the effects and mechanisms of acupuncture at "Baihui"(GV20) through "Qubin"(GB7) on intracerebral hemorrhage(ICH) by examining mitochondrial fission proteins(Drp1, its activated forms, and Fis1) and mitochondrial fusion proteins(Mfn1, Mfn2, and OPA1).Methods Thirty SPF-grade male SD rats were randomly divided into a sham group, an ICH group, and an ICH + acupuncture group(n=10 per group). The ICH model was established in the latter two groups using the autologous blood injection method into the striatum, and successful modeling was assessed with the Bederson neurological score. One-hour post-modeling, acupuncture treatment was initiated for the ICH+acupuncture group, performed once daily for 30 minutes over 3 consecutive days. Neurological function was re-evaluated after the last acupuncture session, followed by tissue sampling. Transmission electron microscopy was used to observe the morphology, size, and quantity of mitochondria in the peri-hematoma region. Real-time PCR was employed to measure mitochondrial DNA(mtDNA) expression levels. Western blot analysis was conducted to detect the expression of mitochondrial fission proteins p-Drp1(Ser637), p-Drp1(Ser616), mito-Drp1, cyto-Drp1, Fis1, mitochondrial fusion proteins Mfn1, Mfn2, and OPA1, as well as p-p38α in the MAPK/Drp1 pathway.Results Compared with the ICH group, the ICH+acupuncture group exhibited significantly improved Bederson neurological scores on the final day of acupuncture(P < 0.000 1). Transmission electron microscopy revealed that mitochondrial morphology was markedly abnormal in the ICH group but improved in the ICH+acupuncture group. The mtDNA expression levels were significantly decreased in the ICH group compared to the sham group(P < 0.000 1) and significantly increased in the ICH + acupuncture group compared to the ICH group(P < 0.000 1). Expression of p-Drp1(Ser637) and cyto-Drp1 was reduced in the ICH group compared to the sham group(P < 0.000 1) but increased in the ICH+acupuncture group compared to the ICH group(P < 0.000 1). Conversely, expression of p-Drp1(Ser616), mito-Drp1, and Fis1 was elevated in the ICH group compared to the sham group(P < 0.000 1) but decreased in the ICH+acupuncture group compared to the ICH group(P < 0.000 1). Expression levels of Mfn1 and Mfn2 were significantly reduced in the ICH group compared to the sham group(P < 0.000 1) and significantly increased in the ICH+acupuncture group compared to the ICH group(P < 0.000 1). Expression levels of OPA1 S/L were elevated in the ICH group compared to the sham group(P < 0.000 1), but decreased in the ICH+acupuncture group compared to the ICH group(P < 0.000 1).Conclusion Acupuncture at Baihui through Qubin alleviates neurological damage following ICH by inhibiting abnormal mitochondrial fission, promoting mitochondrial fusion, and maintaining mitochondrial dynamics and genetic integrity.