Wang Dejian, Huang Yuanfang, Li Qiaoyun, Xu Shijun, Liu Xiaokang
Department of Pharmacology, College of Basic and Forensic Medicine, Sichuan University, Chengdu Sichuan, 610041, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2010 Sep;24(9):1107-12.
Ginsenoside Rgl could increase the tolerance of neural hypoxia and ischemia under stress, and play an anti-apoptotic effect in hypoxia ischemia brain damage (HIBD). To investigate the effects of ginsenoside Rgl on neural apoptosis and recovery of neurological function in neonatal rats with HIBD, and to explore the possible mechanism.
Fifty-four 10-day-old SD rats (weighing 16-22 g) were randomly allocated into sham-operation group (Sham group, n=6), HIBD model group (HIBD group, n=24), and ginsenoside Rg1 treatment group (Rg1 group, n=24). SD rats in HIBD group and Rg1 group were made the models of HIBD by ligation of the right common carotid artery (CCA) and subsequently hypoxic ventilation (8% O2 plus 92% N2) for 2.5 hours; and in Sham group, the right CCA was only exposed without ligation of CCA and hypoxic ventilation. Intraperitoneal injection of 0.1 mL normal saline (NS) containing 40 mg/kg Rgl was given immediately after operation in Rg1 group, intraperitoneal injection of 0.1 mL pure NS was given in both HIBD group and Sham group and was repeated every 24 hours. The general state of SD rats was monitored after operation, and Longa scores were recorded to evaluate the neurological function at 4, 8, 24, and 72 hours after HIBD. Western blot and immunohistochemistry staining were used to detect protein expressions of both hypoxia inducible factor 1alpha (HIF-1alpha) and cleaved caspase 3 (CC3). TUNEL staining was used to evaluate neural apoptosis in situ.
All rats survived to the end of the experiment. Neurological dysfunction was observed in both HIBD group and Rg1 group, showing significant difference in Longa score when compared with that in Sham group (P < 0.05). There was significant difference in Longa score between Rg1 group and HIBD group at 72 hours after HIBD (P < 0.05). Western blot showed that the protein expressions of both HIF-1alpha and CC3 were observed at every time point in every group. The expressions of HIF-1alpha protein in HIBD group and Rg1 group were significantly higher than those in Sham group at 4, 8, 24, and 72 hours (P < 0.05), and the expressions in Rg1 group were significantly higher than those in HIBD group (P < 0.05). The expressions of CC3 protein in HIBD group were significantly higher than those in Sham group at 4, 8, 24, and 72 hours (P < 0.05), and significant difference was found between Rg1 group and Sham group only at 4 hours (P < 0.05). Immunohistochemistry staining demonstrated that HIF-1alpha and CC3 protein mainly distributed in nucleus and cytoplasma, the results of HIF-1alpha and CC3 protein expression were similar to the results by Western blot. TUNEL staining showed that the positive cells were characterized by yellow or brown particle confined within nucleus. The number of apoptotic cells at every time point in HIBD group was significantly higher when compared with that in Sham group (P < 0.05), and the number of apoptotic cells in Rg1 group was significantly lower when compared with that in HIBD group at 8, 24, and 72 hours (P < 0.05).
Rg1 could inhibit Caspase 3 activation by strengthening and stabilizing HIF-1alpha signal pathway, and plays a role of anti-apoptosis in neonatal rats with HIBD.
人参皂苷Rg1可增强应激状态下神经细胞对缺氧缺血的耐受性，对缺氧缺血性脑损伤（HIBD）发挥抗凋亡作用。本研究旨在探讨人参皂苷Rg1对新生大鼠HIBD神经细胞凋亡及神经功能恢复的影响，并探索其可能机制。
将54只10日龄SD大鼠（体重16 - 22 g）随机分为假手术组（Sham组，n = 6）、HIBD模型组（HIBD组，n = 24）和人参皂苷Rg1治疗组（Rg1组，n = 24）。HIBD组和Rg1组大鼠采用结扎右侧颈总动脉（CCA）并随后进行低氧通气（8% O₂加92% N₂）2.5小时的方法制备HIBD模型；Sham组仅暴露右侧CCA，不进行CCA结扎和低氧通气。Rg1组术后立即腹腔注射0.1 mL含40 mg/kg Rg1的生理盐水（NS），HIBD组和Sham组均腹腔注射0.1 mL纯NS，每24小时重复1次。术后监测SD大鼠的一般状态，并记录Longa评分以评估HIBD后4、8、24和72小时的神经功能。采用蛋白质免疫印迹法（Western blot）和免疫组织化学染色检测缺氧诱导因子1α（HIF - 1α）和裂解的半胱天冬酶3（CC3）的蛋白表达。采用TUNEL染色原位评估神经细胞凋亡。
所有大鼠均存活至实验结束。HIBD组和Rg1组均出现神经功能障碍，与Sham组相比，Longa评分差异有统计学意义（P < 0.05）。HIBD后72小时，Rg1组与HIBD组Longa评分差异有统计学意义（P < 0.05）。Western blot结果显示，各组各时间点均观察到HIF - 1α和CC3蛋白表达。HIBD组和Rg1组在4、8、24和72小时的HIF - 1α蛋白表达均显著高于Sham组（P < 0.05），且Rg1组的表达显著高于HIBD组（P < 0.05）。HIBD组在4、8、24和72小时的CC3蛋白表达均显著高于Sham组（P < 0.05），Rg1组与Sham组仅在4小时差异有统计学意义（P < 0.05）。免疫组织化学染色显示，HIF - 1α和CC3蛋白主要分布于细胞核和细胞质，HIF - 1α和CC3蛋白表达结果与Western blot结果相似。TUNEL染色显示，阳性细胞表现为细胞核内黄色或棕色颗粒。HIBD组各时间点的凋亡细胞数均显著高于Sham组（P < 0.05），Rg1组在8、24和72小时的凋亡细胞数显著低于HIBD组（P < 0.05）。
Rg1可通过增强和稳定HIF - 1α信号通路抑制半胱天冬酶3激活，对新生大鼠HIBD发挥抗凋亡作用。
