Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Changle West Road 127, Xi'an, Shaanxi, China.
Mol Neurobiol. 2016 Jan;53(1):728-743. doi: 10.1007/s12035-014-9033-x. Epub 2015 Jan 31.
Diabetes mellitus substantially increases the risk of stroke and enhances brain's vulnerability to ischemia insult. In a previous study, Chikusetsu saponin IVa (CHS) pretreatment was proved to protect the brain from cerebral ischemic in normal stroke models. Whether CHS could attenuate cerebral ischemia/reperfusion (I/R) injury in diabetic mice and the possible underlying mechanism are still unrevealed. Male C57BL/6 mice were injected streptozotocin to induce diabetes. After that, the mice were pretreated with CHS for 1 month, and then, focal cerebral ischemia was induced following 24-h reperfusion. The neurobehavioral scores, infarction volumes, and some cytokines in the brain were measured. Apoptosis was analyzed by caspase-3, Bax, and Bcl-2 expression. Downstream molecules of adiponectin (APN) were investigated by Western blotting. The results showed that CHS reduced infarct size, improved neurological outcomes, and inhibited cell injury after I/R. In addition, CHS pretreatment increased APN level and enhanced neuronal AdipoR1, adenosine monophosphate-activated protein kinase (AMPK), and glycogen synthase kinase 3 beta (GSK-3β) expression in a concentration-dependent manner in diabetic mice, and these effects were abolished by APN knockout (KO). In vitro test, CHS treatment also alleviated PC12 cell injury and apoptosis, evidenced by reduced tumor necrosis factor alpha (TNF-α), malondialdehyde (MDA) and caspase-3 expression, and Bax/Bcl-2 ratio in I/R injured cells. Moreover, CHS enhanced AdipoR1, AMPK, and GSK-3β expression in a concentration-dependent manner. Likewise, short interfering RNA (sinRNA) knockdown of liver kinase B1 (LKB1), an upstream kinase of AMPK, reduced the ability of CHS in protecting cells from I/R injury. Furthermore, this LKB1-dependent cellular protection resulted from AdipoR1 and APN activation, as supported by the experiment using sinRNA knockdown of AdipoR1 and APN. Thus, CHS protected brain I/R in diabetes through AMPK-mediated phosphorylation of GSK-3β downstream of APN-LKB1 pathway.
糖尿病显著增加中风的风险，并增强大脑对缺血性损伤的易感性。在之前的研究中，已证实柴胡皂甙 IVa (CHS) 预处理可保护正常中风模型的大脑免受脑缺血。CHS 是否可以减轻糖尿病小鼠的脑缺血/再灌注 (I/R) 损伤以及可能的潜在机制仍未被揭示。雄性 C57BL/6 小鼠注射链脲佐菌素以诱导糖尿病。之后，小鼠用 CHS 预处理 1 个月，然后在 24 小时再灌注后诱导局灶性脑缺血。测量神经行为评分、脑梗死体积和脑内某些细胞因子。通过 caspase-3、Bax 和 Bcl-2 表达分析细胞凋亡。通过 Western blot 检测脂联素 (APN) 的下游分子。结果显示，CHS 减少梗死面积，改善神经功能，抑制 I/R 后的细胞损伤。此外，CHS 预处理以浓度依赖性方式增加糖尿病小鼠的 APN 水平并增强神经元 AdipoR1、腺苷单磷酸激活蛋白激酶 (AMPK) 和糖原合酶激酶 3β (GSK-3β) 的表达，这些作用在 APN 敲除 (KO) 后被消除。体外试验表明，CHS 处理还减轻了 PC12 细胞损伤和凋亡，表现为 I/R 损伤细胞中肿瘤坏死因子-α (TNF-α)、丙二醛 (MDA) 和 caspase-3 表达以及 Bax/Bcl-2 比值降低。此外，CHS 以浓度依赖性方式增强 AdipoR1、AMPK 和 GSK-3β 的表达。同样，LKB1 是 AMPK 的上游激酶，其短发夹 RNA (sinRNA) 敲低降低了 CHS 保护细胞免受 I/R 损伤的能力。此外，这种依赖于 LKB1 的细胞保护归因于 APN-LKB1 通路下游的 AdipoR1 和 APN 的激活，这一点得到了使用 AdipoR1 和 APN 的 sinRNA 敲低实验的支持。因此，CHS 通过 APN-LKB1 通路下游的 AMPK 介导的 GSK-3β 磷酸化来保护糖尿病中的脑 I/R。
