| 苗瑜,王桂岭,肖谦,等.安宫牛黄丸联合右美托咪定激活 PINK1/Parkin依赖性线粒体自噬减轻脑缺血再灌注大鼠海马神经元损伤[J].安徽医药,2026,30(3):497-503. |
| 安宫牛黄丸联合右美托咪定激活 PINK1/Parkin依赖性线粒体自噬减轻脑缺血再灌注大鼠海马神经元损伤 |
| Angong Niuhuang Pill combined with dexmedetomidine alleviates hippocampal neuronal via PINK1/Parkin-dependent mitophagy in rats with cerebral ischemia-reperfusion |
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| DOI：10.3969/j.issn.1009-6469.2026.03.014 |
| 中文关键词: 再灌注损伤 脑损伤 安宫牛黄丸 右美托咪定 神经元损伤 PINK1/Parkin通路 线粒体自噬 |
| 英文关键词: Reperfusion injury Brain injuries Angong Niuhuang Pill Dexmedetomidine Neuronal injury PINK1/Parkin pathway Mitophagy |
| 基金项目:衡水市科学技术局科技计划项目( 2020014082Z) |
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| 中文摘要: |
| 目的探讨安宫牛黄丸( ANP)联合右美托咪定( DEX)对脑缺血再灌注损伤(CIRI)大鼠海马神经元损伤的影响，并基于 PTEN诱导假定激酶 1(PINK1)/帕金蛋白( Parkin)介导的线粒体自噬探讨可能的作用机制。方法 2023年 10月至 2024年 4月，从 98只 8周龄无特定病原体( SPF)级雄性 SD大鼠中按照随机数字表法选取 18只大鼠作为假手术( Sham)组，其余大鼠采用大脑中动脉闭塞 /再灌注( MCAO/R)法建立 CIRI模型。采用随机数字表法将成功建模的 72只 MCAO/R大鼠分为四组: MCAO/R组(等量生理盐水)、 DEX组(腹腔注射 100 μg/kg DEX)、 ANP组(灌胃 0.25 g/kg ANP混悬液)、 DEX+ANP组(腹腔注射 100 μg/kg DEX+灌胃 0.25 g/kg ANP混悬液)，每组 18只。采用改良神经功能缺损评分( mNSS)评估神经功能， 2，3，5-氯化三苯基四氮唑(TTC)染色评估脑梗死体积，苏木精 -伊红( HE)染色、尼氏染色、 TUNEL染色评估海马组织神经元损伤，试剂盒检测海马组织中活性氧、丙二醛、白细胞介素 -6(IL-6)、肿瘤坏死因子 α(TNF-α)水平，透射电镜观察线粒体超微结构并检测钠钾 ATP酶活性，免疫荧光染色检测海马组织中线粒体外膜转位酶 20(Tom 20)和微管相关蛋白 1轻链 3(LC3)表达，蛋白质印迹法检测海马组织中 Tom 20、细胞色素 C氧化酶亚基 Ⅳ亚型 1(COX4I1)、 LC3Ⅱ/LC3Ⅰ、p62蛋白及 PINK1/Parkin通路蛋白表达。结果Sham组相比， MCAO/R组大鼠 mNSS［(14.40±0.86)分比( 0.00±0.00)分］、脑梗死体积百分比［(35.85±6.65)%比( 0.00±0.00)%］增与加( P<0.05)；海马组织神经元发生明显病理损伤，线粒体肿胀、嵴断裂；海马组织中活性氧、丙二醛、 IL-6、TNF-α水平及 p62蛋白表达均增加( P<0.05)钠钾 ATP酶活性［( 0.37±0.04)倍比( 1.00±0.05)倍］、 Tom 20+LC3+细胞比例［( 0.23±0.05)%比( 1.07±0.09)%］均降低( P<0.05)om 20、COX4I1、LC3Ⅱ/LC3Ⅰ、PINK1、Parkin蛋白表达均降低( P<0.05)。与 MCAO/R相比， DEX组、 ANP组和 DEX+ANP组mNSS［( 10.63±1.17)分、(8.53±1.60)分、(5.87±1.14)分比( 14.40±0.86)分］、脑梗死体积百分比［( 23.57±5.31)%、(17.64±3.49)%、(8.21±2.17)%比( 35.85±6.65)%］减少( P<0.05)；海马组织神经元病理损伤明显改善，线粒体肿胀、嵴断裂程度减轻；海马组织中活性氧、丙二醛、 IL-6、TNF-α水平及 p62蛋白表达均降低( P<0.05)钠钾 ATP酶活性［( 0.52±0.04)倍、(0.66±0.07)倍、(0.89±0.06)倍比( 0.37±0.04)倍］、 Tom 20+LC3+细胞比例［( 0.39±0.04)%±0.05)%、(0.73±T，大鼠，、(0.51，0.07)%比( 0.23±0.05)%］均升高( P<0.05)，Tom 20、COX4I1、LC3Ⅱ/LC3Ⅰ、PINK1、Parkin蛋白表达均升高( P<0.05)，且二者联合的改善效果更佳。结论 ANP联合 DEX能够改善 CIRI引起的神经功能损伤，修复线粒体功能障碍，这可能是通过激活 PINK1/Parkin通路介导的线粒体自噬来实现。 |
| 英文摘要: |
| Objective To investigate the effects of Angong Niuhuang Pill (ANP) combined with dexmedetomidine (DEX) on hippo. campal neuronal damage in rats with cerebral ischaemia-reperfusion injury (CIRI) and to explore potential mechanisms based on PTEN-induced putative kinase 1 (PINK1)/Park2 gene (Parkin) -mediated mitophagy. Methods From October 2023 to April 2024, 18 rats were randomly selected from 98 eight-week-old specific pathogen-free (SPF) male SD rats as the sham group using a random number ta.ble. The remaining rats underwent middle cerebral artery occlusion/reperfusion (MCAO/R) to establish a CIRI model. Using a randomnumber table, the 72 successfully modeled MCAO/R rats were divided into four groups: the MCAO/R group (equivalent saline solution),the DEX group (intraperitoneal injection of 100 μg/kg DEX), the ANP group (oral administration of 0.25 g/kg ANP suspension), and theDEX+ANP group (intraperitoneal injection of 100 μg/kg DEX + oral administration of 0.25 g/kg ANP suspension), with 18 rats pergroup. Neurological function was assessed using the modified neurological deficit score (mNSS). Cerebral infarction volume was evaluat.ed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Hematoxylin-eosin (HE) staining, Nissl staining, and TUNEL staining wereused to assess neuronal damage in hippocampal tissue. Levels of reactive oxygen species (ROS), malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) in hippocampal tissue were detected using assay kits. Mitochondrial ultrastructure wasobserved by transmission electron microscopy, and Na./K.-ATPase activity was detected. Immunofluorescence staining was performedto detect the expression of translocase of outer mitochondrial membrane 20 (Tom 20) and microtubule-associated protein 1 light chain 3(LC3) in hippocampal tissue. Western blotting was used to detect the expression of Tom 20, cytochrome c oxidase subunit Ⅳ isoform 1(COX4I1), the LC3Ⅱ/LC3Ⅰ ratio, p62, and PINK1/Parkin pathway proteins in hippocampal tissue.Results Compared with the sham group, rats in the MCAO/R group showed increased mNSS scores [(14.40±0.86) vs. (0.00±0.00)] and percentage of cerebral infarct vol. ume [(35.85±6.65)% vs. (0.00±0.00)%] (P<0.05). Hippocampal neurons exhibited significant pathological damage, with mitochondrialswelling and cristae rupture. Levels of reactive oxygen species, malondialdehyde, IL-6, TNF-α, and p62 protein expression in hippo. campal tissue were increased (P<0.05); Na+/K+-ATPase activity [(0.37±0.04) -fold vs. (1.00±0.05) -fold] and the proportion of Tom 20+LC3+ cells [(0.23±0.05)% vs. (1.07±0.09)%] were decreased (P<0.05). The expression of Tom 20, COX4I1, the LC3Ⅱ/LC3Ⅰ ratio, PINK1, and Parkin proteins were also decreased (P<0.05). Compared with the MCAO/R group, rats in the DEX, ANP, and DEX+ANPgroups showed reduced mNSS scores (10.63±1.17, 8.53±1.60, 5.87±1.14 vs. 14.40±0.86) and percentages of cerebral infarct volume [(23.57±5.31)%, (17.64±3.49)%, (8.21±2.17)% vs. (35.85±6.65)%] (P<0.05). Pathological damage to hippocampal neurons was signifi.cantly ameliorated, with reduced mitochondrial swelling and cristae rupture. Levels of ROS, MDA, IL-6, TNF-α, and p62 protein ex. pression in hippocampal tissue were decreased (P<0.05). Na./K.-ATPase activity [(0.52±0.04)-fold, (0.66±0.07)-fold, (0.89±0.06)-fold vs. (0.37±0.04)-fold] and the proportion of Tom 20+LC3+ cells [(0.39±0.04)%, (0.51±0.05)%, (0.73±0.07)% vs. (0.23±0.05)%] were in. creased (P<0.05). The expression of Tom 20, COX4I1, the LC3Ⅱ/LC3Ⅰ ratio, PINK1, and Parkin proteins was increased (P<0.05), with the combined treatment showing superior effects.Conclusion The combination of ANP and DEX can ameliorate CIRI-induced neurological impairment and restore mitochondrial dysfunction, which may be achieved by activating PINK1/Parkin pathway mediatedmitophagy. |
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