基于HIF-1α/BNIP3信号通路调控线粒体自噬探讨通脉开窍丸治疗血管性痴呆大鼠的机制

Tongmai Kaiqiao Pills Treat Vascular Dementia in Rats by Regulating Mitochondrial Autophagy via HIF-1α/BNIP3 Signaling Pathway

摘要:

目的 观察通脉开窍丸对血管性痴呆（VD）大鼠海马体缺氧诱导因子-1α（HIF-1α）/腺病毒E1B19kD相互作用蛋白3（BNIP3）信号通路及线粒体自噬的影响。方法 将90只雄性SD大鼠适应性喂养1周后，随机抽选10只作为假手术组，仅分离颈总动脉不结扎，剩余大鼠将采用颈总动脉分次结扎法制作VD大鼠模型。剔除死亡和造模不成功的大鼠后，将造模成功的大鼠随机分为模型组、通脉开窍丸高、中、低剂量组（27.6、13.8、6.9 g·kg^-1）、盐酸多奈哌齐组（0.45 mg·kg^-1）、联合组（通脉开窍丸高剂量27.6 g·kg^-1+HIF-1α抑制剂YC-1 2.5 mg·kg^-1），各组对应治疗4周后取材。尼氏染色法观察海马区神经元丢失情况；苏木素-伊红（HE）染色观察海马区病理学变化；蛋白免疫印迹法（Western blot）检测海马组织HIF-1α、BNIP3、自噬效应蛋白-1（Beclin-1）、微管相关蛋白1轻链3B（LC3B）蛋白表达水平；透射电镜观察海马组织线粒体超微结构及自噬小体数量；免疫荧光（IF）观察海马组织HIF-1α、BNIP3、LC3B的荧光强度。结果 与假手术组比较，模型组大鼠逃避潜伏期延长（P<0.01），穿越平台次数减少（P<0.01），海马区神经元层次减少，数量骤减，结构排列不齐，尼氏小体数量减少，丢失严重，线粒体嵴紊乱，线粒体双模结构破坏，损伤加重，自噬小体数量增加，HIF-1α、BNIP3、Beclin-1、LC3B蛋白表达增加（P<0.05，P<0.01），HIF-1α、BNIP3、LC3B的荧光强度增强（P<0.05，P<0.01）；与模型组比较，通脉开窍丸各治疗组、盐酸多奈哌齐组大鼠寻找平台时间缩短（P<0.01），穿越平台次数增加（P<0.01），HE染色显示，海马神经元细胞数量增多，形态正常，排列整齐，尼氏小体丰富，丢失减少，损伤减轻，自噬小体数量增多，HIF-1α、BNIP3、Beclin-1、LC3B蛋白表达明显增加（P<0.05，P<0.01），HIF-1α、BNIP3、LC3B的荧光强度增强（P<0.05，P<0.01）。与通脉开窍丸高剂量组比较，联合组大鼠逃避潜伏期显著延长（P<0.01），穿越平台次数显著降低（P<0.01），海马神经元细胞数量减少，损伤加重，尼氏小体数量下降，自噬小体数量有所减少，HIF-1α、BNIP3、Beclin-1、LC3B蛋白表达显著降低（P<0.01），HIF-1α、BNIP3、LC3B的荧光强度显著下降（P<0.01）。结论 通脉开窍丸可能通过激活HIF-1α/BNIP3信号通路，促进线粒体自噬的发生，清除功能受损的线粒体，为健康细胞提供能量，减少神经细胞死亡，促进脑功能的恢复，从而减轻大鼠海马组织的缺血性损伤，提高大鼠学习记忆能力，从而发挥对VD的治疗作用。

Abstract:

Objective To observe the effects of Tongmai Kaiqiao pills on the hypoxia-inducible factor-1α （HIF-1α）/adenovirus E1B 19 kD-interacting protein 3 （BNIP3） signaling pathway and mitochondrial autophagy in the hippocampus of the rat model of vascular dementia （VD）.Method Ninety male SD rats underwent adaptive feeding for one week before the study. Ten rats were randomly assigned to the sham group， where the common carotid artery was isolated without ligation. The remaining rats were subjected to sequential ligation of the common carotid artery for the modeling of VD. The successfully modeled rats were randomly assigned into the following groups： model， high-， medium-， and low-dose （27.6， 13.8， 6.9 g·kg^-1， respectively） Tongmai Kaiqiao pills， donepezil hydrochloride （0.45 mg·kg^-1）， and combination （27.6 g·kg^-1 Tongmai Kaiqiao pills + 2.5 mg·kg^-1 HIF-1α inhibitor YC-1） groups. After 4 weeks of treatment， samples were collected. Nissl staining and hematoxylin-eosin staining were performed to observe the loss of neurons and pathological changes， respectively， in the hippocampal region. Western blot was employed to determine the protein levels of HIF-1α， BNIP3， Beclin-1， and microtubule-associated protein 1 light chain 3B （LC3B） in the hippocampal tissue. Transmission electron microscopy was used to observe the mitochondrial ultrastructure and the number of autophagosomes in the hippocampal tissue. Immunofluorescence was employed to observe the fluorescence intensity of HIF-1α， BNIP3， and LC3B in the hippocampal tissue.Result Compared with the sham group， the model group showed prolonged escape latency （P<0.01）， decreased number of platform crossings （P<0.01）， reduced and disarranged neuronal layers in the hippocampal region， decreased number of Nissl bodies， disrupted mitochondrial cristae， damaged mitochondrial double-membrane structures， increased number of autophagosomes， upregulated expression of HIF-1α， BNIP3， beclin1， and LC3B （P<0.05， P<0.01）， and enhanced fluorescence intensity of HIF-1α， BNIP3， and LC3B （P<0.05， P<0.01）. Compared with the model group， Tongmai Kaiqiao pills and donepezil hydrochloride shortened the searching time for the platform （P<0.01） and increased the number of platform crossings （P<0.01）. Moreover， the drugs increased the number of neurons with normal morphology and orderly arrangement and the number of Nissl bodies， alleviated the damage， increased the number of autophagosomes， upregulated the expression of HIF-1α， BNIP3， Beclin1， and LC3B （P<0.05， P<0.01）， and enhanced the fluorescence intensity of HIF-1α， BNIP3， and LC3B （P<0.05， P<0.01）. Compared with high-dose Tongmai Kaiqiao pills， the combination group prolonged the escape latency （P<0.01）， reduced the number of crossing platforms （P<0.01）， decreased the number of hippocampal neurons， aggravated the damage， decreased the number of Nissl bodies and autophagosomes， downregulated the expression of HIF-1α， BNIP3， beclin1， and LC3B （P<0.01）， and decreased the fluorescence intensity of HIF-1α， BNIP3， and LC3B （P<0.01）.Conclusion Tongmai Kaiqiao pills may activate the HIF-1α/BNIP3 signaling pathway to promote the occurrence of mitochondrial autophagy， clear damaged mitochondria， provide energy for healthy cells， reduce neuronal cell death， and restore the brain function， thereby reducing ischemic damage to the hippocampal tissue， improving learning and memory abilities， and exerting therapeutic effects on VD in rats.