目的:大花红景天(钩。f.etThoms。)H.Ohba(R.齿状),一种著名而有特色的藏药,已被证明在治疗高原缺氧疾病中具有突出的脑保护作用。然而,R.screnulate对高原缺氧性脑损伤(HHBI)的代谢作用仍未完全了解。在这里,通过体内和体外实验探讨了小齿的抗缺氧作用和相关机制。
方法:使用动物低压低氧舱建立HHBI小鼠模型。R.齿状提取物(RCE,0.5、1.0和2.0g/kg)和红景天苷(萨尔,25、50和100mg/kg)通过管饲法给予7天。用H&E和TUNEL染色评价小鼠海马和皮质的病理变化和神经元凋亡,分别。通过Evans蓝染色和NIR-II荧光成像检测RCE和Sal对血脑屏障(BBB)通透性的影响。同时,用透射电镜(TEM)观察BBB超微结构和脑血管损伤。免疫荧光法检测紧密连接蛋白Claudin-1,ZO-1和occludin的水平。此外,使用UHPLC-MS和MALDI-MSI分析测定小鼠血清和脑中的代谢物。通过细胞计数试剂盒-8研究了Sal在CoCl2诱导的低氧HT22细胞上的细胞活力。LDH的含量,MDA,SOD,使用商业生化试剂盒检测GSH-PX和SDH。同时,细胞内ROS,通过相应的特异性标记探针测定Ca2和线粒体膜电位。通过Q300试剂盒的靶向代谢组学分析来进行HT22细胞的细胞内代谢物。YO-PRO-1/PI检测细胞凋亡和坏死,膜联蛋白V/PI和TUNEL染色。此外,线粒体形态通过Mito-tracker红色与共聚焦显微镜和TEM测试。实时ATP生产,耗氧率,和质子流出率使用海马分析仪测量。随后,MCU,OPA1,p-Drp1ser616,p-AMPKα,通过免疫荧光和蛋白质印迹分析确定p-AMPKβ和Sirt1。
结果:结果表明,小齿和Sal发挥抗缺氧脑保护作用,抑制神经元凋亡,保持BBB完整性,增加紧密连接蛋白Claudin-1,ZO-1和occludin,改善线粒体形态和功能。机械上,R.齿状和Sal通过增强三羧酸循环来缓解HHBI,以满足大脑对能量的需求。此外,体外实验证实,Sal可以改善HT22细胞的凋亡,通过增强线粒体呼吸和糖酵解改善线粒体形态和能量代谢。同时,Sal介导的MCU抑制Drp1的激活并增强OPA1的表达以维持线粒体稳态,以及激活AMPK和Sirt1以增强ATP的产生。
结论:总的来说,研究结果表明,RCE和Sal可能通过激活AMPK/Sirt1信号通路平衡MCU介导的线粒体稳态,维持BBB完整性,改善能量代谢,从而对HHBI提供保护性干预.
OBJECTIVE: Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba (R. crenulate), a famous and characteristic Tibetan medicine, has been demonstrated to exert an outstanding brain protection role in the treatment of high-altitude hypoxia disease. However, the metabolic effects of R. crenulate on high-altitude hypoxic brain injury (HHBI) are still incompletely understood. Herein, the anti-hypoxic effect and associated mechanisms of R. crenulate were explored through both in vivo and in vitro experiments.
METHODS: The mice model of HHBI was established using an animal hypobaric and hypoxic chamber. R. crenulate extract (RCE, 0.5, 1.0 and 2.0 g/kg) and salidroside (Sal, 25, 50 and 100 mg/kg) was given by gavage for 7 days. Pathological changes and neuronal apoptosis of mice hippocampus and cortex were evaluated using H&E and TUNEL staining, respectively. The effects of RCE and Sal on the permeability of blood brain barrier (BBB) were detected by Evans blue staining and NIR-II fluorescence imaging. Meanwhile, the ultrastructural BBB and cerebrovascular damages were observed using a transmission electron microscope (TEM). The levels of tight junction proteins Claudin-1, ZO-1 and occludin were detected by immunofluorescence. Additionally, the metabolites in mice serum and brain were determined using UHPLC-MS and MALDI-MSI analysis. The cell viability of Sal on hypoxic HT22 cells induced by CoCl2 was investigated by cell counting kit-8. The contents of LDH, MDA, SOD, GSH-PX and SDH were detected by using commercial biochemical kits. Meanwhile, intracellular ROS, Ca2+ and mitochondrial membrane potential were determined by corresponding specific labeled probes. The intracellular metabolites of HT22 cells were performed by the targeted metabolomics analysis of the Q300 kit. The cell apoptosis and necrosis were examined by YO-PRO-1/PI, Annexin V/PI and TUNEL staining. In addition, mitochondrial morphology was tested by Mito-tracker red with confocal microscopy and TEM. Real-time ATP production, oxygen consumption rate, and proton efflux rate were measured using a Seahorse analyzer. Subsequently, MCU, OPA1, p-Drp1ser616, p-AMPKα, p-AMPKβ and Sirt1 were determined by immunofluorescent and western blot analyses.
RESULTS: The results demonstrated that R. crenulate and Sal exert anti-hypoxic brain protection from inhibiting neuronal apoptosis, maintaining BBB integrity, increasing tight junction protein Claudin-1, ZO-1 and occludin and improving mitochondrial morphology and function. Mechanistically, R. crenulate and Sal alleviated HHBI by enhancing the tricarboxylic acid cycle to meet the demand of energy of brain. Additionally, experiments in vitro confirmed that Sal could ameliorate the apoptosis of HT22 cells, improve mitochondrial morphology and energy metabolism by enhancing mitochondrial respiration and glycolysis. Meanwhile, Sal-mediated MCU inhibited the activation of Drp1 and enhanced the expression of OPA1 to maintain mitochondrial homeostasis, as well as activation of AMPK and Sirt1 to enhance ATP production.
CONCLUSIONS: Collectively, the findings suggested that RCE and Sal may afford a protective intervention in HHBI through maintaining BBB integrity and improving energy metabolism via balancing MCU-mediated mitochondrial homeostasis by activating the AMPK/Sirt1 signaling pathway.