Mechanism and effect of aescin intervention against brain injury in a rat model of intracerebral hemorrhage

doi:10.12307/2026.919

Abstract

Abstract: BACKGROUND: Neuronal apoptosis induced by endoplasmic reticulum stress following intracerebral hemorrhage is a significant cause of secondary brain injury. Aescin has been demonstrated to possess anti-inflammatory, antioxidant, and anti-apoptotic effects in brain injury models. However, whether it exerts neuroprotective effects by regulating endoplasmic reticulum stress-related pathways remains to be clarified.
OBJECTIVE: To investigate the effect of aescin on brain injury in rats with intracerebral hemorrhage and its potential mechanism of action.
METHODS: Fifty-four rats were randomly divided into a sham operation group, an intracerebral hemorrhage group, and an intracerebral hemorrhage+aescin group using a random number table method. An intracerebral hemorrhage rat model was established in the latter two groups using autologous blood injection. Rats in the sham operation group underwent the same stereotaxic puncture procedure but without blood injection. After successful modeling, the intracerebral hemorrhage+aescin group received intraperitoneal injection of aescin (10 mg/kg), while the sham operation group and intracerebral hemorrhage group received an equal volume of normal saline. Neurological function of rats was assessed using Longa score, rotarod test, forelimb placing test, and corner turn test. Pathological damage of the brain and brain edema were evaluated using hematoxylin-eosin staining and brain water content measurement. A cell injury model was established in nerve growth factor-incubated PC12 cells under oxygen-glucose deprivation. An endoplasmic reticulum stress cell model was established using tunicamycin, with the endoplasmic reticulum stress inhibitor 4-PBA serving as a positive control; cells were also treated with IXA4 (IRE1 agonist) or CCT020312 (PERK agonist). Cell viability was detected using the cell counting kit-8 assay, and apoptosis was detected using flow cytometry. Expression of endoplasmic reticulum stress-related proteins was detected by western blot. The mechanism of pathway involvement was verified using inositol-requiring enzyme 1 (IRE1) or protein kinase R-like endoplasmic reticulum kinase (PERK) small interfering RNA (IRE1 siRNA or PERK siRNA) and pathway agonists.
RESULTS AND CONCLUSION: (1) Compared with the sham operation group, rats in the intracerebral hemorrhage group showed significantly increased Longa scores (P < 0.05), significantly impaired sensorimotor function, obvious hemorrhagic foci, edema, and neuronal pyknosis in brain tissue, and significantly increased brain water content. In the aescin treatment group, these indicators were significantly improved from 12 hours to 5 days after surgery (P < 0.05). (2) Aescin intervention significantly reduced the expression levels of endoplasmic reticulum stress-related proteins (GRP78, CHOP, p-IRE1, p-PERK, etc.) in the brain tissue of intracerebral hemorrhage rats (P < 0.05). (3) Treatment with oxygen-glucose deprivation or the endoplasmic reticulum stress inducer tunicamycin significantly reduced the viability of nerve growth factor-incubated PC12 cells, increased the apoptosis rate (P < 0.05), and upregulated GRP78, CHOP, and the ratios of p-IRE1/IRE1 and p-PERK/PERK (P < 0.05). However, 10 and 20 μmol/L aescin significantly reversed these changes (P < 0.05). (4) Knockdown of IRE1/PERK produced effects consistent with aescin intervention, inhibiting the activation of the IRE1-XBP1 and PERK-eIF2α pathways induced by oxygen-glucose deprivation. Pathway agonists (IXA4 and CCT020312) reversed the protective effect of aescin (P < 0.05). (5) Correlation analysis showed that the expression of GRP78, p-IRE1, and p-PERK in rat brain tissue was positively correlated with brain edema volume and Longa score (P < 0.05). These findings suggest that aescin can alleviate endoplasmic reticulum stress by specifically blocking the IRE1/PERK signaling pathway, thereby ameliorating neurological impairment in rats with intracerebral hemorrhage and oxygen-glucose deprivation-induced injury in nerve growth factor-incubated PC12 cells.

Key words: aescin, intracerebral hemorrhage, apoptosis, endoplasmic reticulum stress, signaling pathway, PERK

CLC Number:

Ji Lingshan, Qin Hewei, Cheng Shuaifang, Zhao Jing. Mechanism and effect of aescin intervention against brain injury in a rat model of intracerebral hemorrhage[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(36): 9526-9537.

Figures/Tables 7

前肢放置次数比例均显著降低(P < 0.05)，向左侧转角次数比例明显升高(P < 0.05)；与脑出血组相比，七叶皂苷治疗组大鼠在术后12 h及1，3，5 d的旋转木马转棒停留时间和同侧肢体前肢放置次数比例均明显上升(P < 0.05)，而向左侧转角次数比例明显下降(P < 0.05)，见图2B-D。上述结果说明，七叶皂苷可改善脑出血大鼠的神经功能缺损情况。 2.3 七叶皂苷改善脑组织病理损伤 2.3.1 苏木精-伊红染色结果与假手术组相比，脑出血组大鼠脑组织在术后1，3，5 d明显出现出血灶，结构紊乱疏松，呈空泡状水肿，神经元可见大量核固缩等改变；七叶皂苷治疗组大鼠脑组织结构出血灶、胞浆内空泡以及核固缩等在第1天时有所减轻，术后3，5 d时损伤改善更为显著，见图3A。除术后第1天外，七叶皂苷处理在第3，5天可显著减少脑水肿体积，见图3B。 2.3.2 脑含水量测定结果与假手术组相比，脑出血大鼠在术后1，3，5 d的脑组织含水量显著升高；而同一时间点七叶皂苷治疗组的脑含水量较脑出血组显著降低，见图3C。 2.4 七叶皂苷对氧糖剥夺诱导神经生长因子-PC12细胞损伤和凋亡的保护作用 2.4.1 CCK-8检测细胞活力结果氧糖剥夺处理1，1.5，2，2.5 h后，神经生长因子-PC12细胞活力均显著降低(P < 0.05)，其中以2.5 h处理的抑制效应最显著，见图4A。与对照组相比，浓度为5，10，20 μmol/L 的七叶皂苷处理均不影响神经生长因子-PC12细胞活力(P > 0.05)，而浓度为40 μmol/L 和80 μmol/L 的七叶皂苷处理显著降低神经生长因子-PC12细胞活力(P < 0.05)，见图4B。与对照组相比，氧糖剥夺组细胞活力降低(P < 0.05)；与氧糖剥夺组相比，浓度为10 μmol/L和20 μmol/L的七叶皂苷处理可显著提高神经生长因子-PC12细胞活力(P < 0.05)，而40 μmol/L和80 μmol/L的七叶皂苷处理则不改变细胞活力(P > 0.05)，见图4C。 2.4.2 ELISA检测乳酸脱氢水平乳酸脱氢酶是一种稳定的细胞质酶，当细胞膜受损时乳酸脱氢酶会迅速释放到培养基中。与氧糖剥夺组相比，10 μmol/L和20 μmol/L的七叶皂苷处理可降低培养基中乳酸脱氢酶的释放量(P < 0.05)，而40 μmol/L和80 μmol/L的七叶皂苷处理则对乳酸脱氢酶的释放比没有显著影响(P > 0.05)，见图4D。基于这些结果，选择10 μmol/L和20 μmol/L的七叶皂苷剂量用于后续实验。 2.4.3 流式细胞术检测细胞凋亡情况与对照组相比，氧糖剥夺组细胞凋亡率显著升高(P < 0.05)；与氧糖剥夺组相比，10 μmol/L和20 μmol/L的七叶皂苷处理可显著降低细胞凋亡率(P < 0.05)，见图4E。上述结果表明，七叶皂苷对氧糖剥夺诱导的神经生长因子-PC12细胞损伤和凋亡具有保护作用。 2.5 七叶皂苷逆转氧糖剥夺对PC12细胞内质网应激相关蛋白水平的促进作用鉴于内质网应激是脑出血后诱导神经元凋亡的关键分子事件，随后探讨了七叶皂苷的保护作用是否与调控内质网应激及其下游未折叠蛋白反应(unfolded protein response，UPR)信号通路有关。内质网应激发生时，分子伴侣GRP78从UPR蛋白(IRE1、ATF6 和 PERK)中解离出来，进而激活这些通路以恢复稳态。然而，持续且强烈的内质网应激会导致UPR的促凋亡信号通路过度激活。已有研究表明，脑出血后的大面积缺氧会通过引发内质网应激和钙离子过载，导致神经元凋亡。靶向抑制IRE1/PERK通路对内质网应激介导的细胞凋亡具有较强的神经保护作用[20]。 2.5.1 七叶皂苷缓解内质网应激发挥保护作用 Western blot检测了内质网应激标志蛋白GRP78和促凋亡终末蛋白CHOP的表达，见图5A。结果显示，与对照组相比，氧糖剥夺处理显著上调了GRP78和CHOP的蛋白水平(P < 0.05)，证实氧糖剥夺成功"

诱导了细胞内质网应激。然而，七叶皂苷预处理显著抑制了氧糖剥夺诱导的GRP78和CHOP蛋白的上调(P < 0.05)，见图5A，提示七叶皂苷能够缓解氧糖剥夺诱导的内质网应激。 2.5.2 七叶皂苷对UPR三条核心分支通路的影响结果显示，与对照组相比，氧糖剥夺处理显著上调了p-IRE1/IRE1、 p-PERK/PERK和p-eIF2α/eIF2α比值及ATF6表达水平(P < 0.05)。与氧糖剥夺组相比，七叶皂苷预处理显著降低了p-IRE1、p-PERK和p-eIF2α的表达水平(P < 0.05)，但对ATF6的蛋白水平影响不大(P > 0.05)，见图5A。 2.5.3 七叶皂苷对PC12细胞的保护作用依赖于其对内质网应激的抑制采用内质网应激激动剂衣霉素建立内质网应激细胞模型，4-PBA被用作内质网应激抑制的阳性对照。与对照组相比，衣霉素处理可显著降低细胞活力(P < 0.05)，而七叶皂苷处理能显著逆转这一效应(P < 0.05)，见图5B。此外，与对照组相比，衣霉素触发了强烈的内质网应激反应，显著上调了GRP78、CHOP、p-IRE1、p-PERK和ATF6的表达水平(P < 0.05)；与衣霉素组相比，七叶皂苷处理显著下调CHOP、GRP78、p-IRE1及p-PERK蛋白水平(P < 0.05)，对IRE1/PERK通路的抑制模式与4-PBA一致，但七叶皂苷对衣霉素诱导的ATF6表达没有影响(P > 0.05)，见图5C，D。流式细胞术结果显示：与对照组相比，衣霉素组细胞凋亡率显著升高(P < 0.05)；与衣霉素组相比，七叶皂苷处理组的细胞凋亡率均显著降低(P < 0.05)，这与4-PBA处理组的结果一致，见图5E，F。透射电镜观察发现，衣霉素组细胞可见胞质内细胞器整体排布紊乱，局部区域因内质网扩张呈现疏松空泡样改变；七叶皂苷组与4-PBA组细胞胞质内疏松空泡样区域减少，细胞器排布更趋规整，见图5G。上述结果说明，七叶皂苷可阻断IRE1/PERK信号通路缓解氧糖剥夺和衣霉素诱导的细胞内质网应激损伤。 2.6 七叶皂苷通过抑制IRE1和PERK信号通路缓解氧糖剥夺诱导的内质网应激和细胞损伤为验证七叶皂苷的作用机制，分别利用IRE1或PERK的特异性siRNA进行基因敲低，并应用通路激动剂进行功能恢复实验。 2.6.1 IRE1信号通路方面与NC siRNA组相比，敲低IRE1显著降低细胞中IRE1蛋白表达水平，见图6A。与氧糖剥夺组相比，氧糖剥夺+IRE1 siRNA组与氧糖剥夺+七叶皂苷组细胞中内质网应激标志物GRP78、CHOP以及p-IRE1及其下游效应分子XBP1s的蛋白水平均显著降低(P < 0.05)，见图6B，提示敲低IRE1与七叶皂苷干预对IRE1-XBP1通路的抑制作用一致。"

与氧糖剥夺+七叶皂苷组相比，加入IRE1激动剂IXA4后，细胞中GRP78、CHOP、p-IRE1与XBP1s蛋白水平显著升高(P < 0.05)，见图6B，表明IXA4可通过激活IRE1通路，逆转七叶皂苷对该通路的抑制作用。此外，与氧糖剥夺组相比，氧糖剥夺+IRE1 siRNA组与氧糖剥夺+七叶皂苷组的细胞活力均明显升高(P < 0.05)、细胞凋亡率均降低(P < 0.05)，见图6C，D，提示IRE1的基因敲低与七叶皂苷的保护效应一致。与氧糖剥夺+七叶皂苷组相比，氧糖剥夺+七叶皂苷+ IXA4组的细胞活力显著下降(P < 0.05)，凋亡率显著升高(P < 0.05)，见图6C，D，表明IRE1的激活逆转了七叶皂苷对细胞损伤的保护作用。 2.6.2 PERK信号通路方面观察到了与1IRE1信号通路高度一致的结果。与NC siRNA组相比，敲低PERK显著降低了细胞中PERK蛋白表达水平，见图7A。敲低PERK与七叶皂苷处理同样有效地抑制了氧糖剥夺诱导的GRP78、CHOP表达以及p-PERK及其下游靶点eIF2α的磷酸化(P < 0.05)(图7B)，并改善了细胞存活(P < 0.05)(图7C)、抑制了凋亡(P < 0.05)(图7D)。同样地，PERK激动剂CCT020312的共处理也成功恢复了PERK-eIF2α通路的活性、并逆转了七叶皂苷对细胞的保护作用(P < 0.05)(图7B-D)。以上结果表明，七叶皂苷可通过特异性抑制IRE1-XBP1与PERK-eIF2α信号通路，缓解氧糖剥夺诱导的内质网应激，提高细胞活力、减少细胞凋亡。 2.7 七叶皂苷通过阻断IRE1和PERK信号缓解脑出血后脑水肿与神经功能缺损为验证七叶皂苷对脑出血大鼠内质网应激的保护作用，构建了脑出血大鼠模型并检测其对脑出血大鼠脑组织中内质网应激相关蛋白表达水平的影响。 2.7.1 Western blot实验结果与假手术组相比，脑出血大鼠脑组织中CHOP、GRP78、p-IRE1、p-PERK和ATF6表达水平显著升高(P < 0.05)；与脑出血组相比，七叶皂苷治疗组大鼠脑组织中CHOP、GRP78、p-IRE1和p-PERK表达水平显著降低(P < 0.05)，而ATF6表达水平无显著变化(P > 0.05)，见图8A。 2.7.2 相关性分析结果为直接验证七叶皂苷的疗效与其机制的相关性，对七叶皂苷治疗组大鼠进行了相关性分析。结果显示，GRP78、p-IRE1和PERK的蛋白表达水平与脑水肿体积呈显著正相关(r=0.957，P=0.003；r=0.939， P=0.005；r=0.983，P=0.000)，同时与神经功能Longa评分亦呈显著正相关(r=0.865，P=0.026；r=0.847，P=0.033；r=0.892，P=0.017)，见图8B-D。这表明，七叶皂苷对p-IRE1和PERK通路的抑制作用越强，其减轻脑水肿和改善神经功能的效果越显著。"

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