Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (2): 177-183.doi: 10.12307/2022.1007
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Zhong Jinglin, Cao Huimin, Pan Yaru, Jian Wenxuan, Wang Qi
Received:
2022-02-07
Accepted:
2022-03-02
Online:
2023-01-18
Published:
2022-06-20
Contact:
Wang Qi, Master, Professor, Doctoral supervisor, Science and Technology Innovation Center, Guangzhou University of Chinese Medicine, Guangzhou 510000, Guangdong Province, China
About author:
Zhong Jinglin, Master candidate, Science and Technology Innovation Center, Guangzhou University of Chinese Medicine, Guangzhou 510000, Guangdong Province, China
Supported by:
CLC Number:
Zhong Jinglin, Cao Huimin, Pan Yaru, Jian Wenxuan, Wang Qi. Ginsenoside Rg3 protects PC12 cells against oxygen-glucose deprivation/reoxygenation-induced damage[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(2): 177-183.
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2.1 人参皂苷Rg3对氧糖剥夺/复糖复氧损伤PC12细胞的保护作用 图1A是人参皂苷Rg3的结构式,使用1-100 nmol/L的Rg3处理受到不同时间氧糖剥夺损伤的PC12细胞,如图1B所示,在不同损伤时间内,Rg3在10-100 nmol/L浓度范围时,都可以不同程度地升高细胞活力。通过测定乳酸脱氢酶的释放来评估PC12细胞受损的程度,如图1C所示,在氧糖剥夺2 h时,Rg3在25-100 nmol/L时均可以明显减少乳酸脱氢酶的释放(P < 0.05)。因为在0.5,1,1.5,2 h时,细胞活力都出现明显下降,分别为81%,75%,55%,51%,为保证模型的稳定性与损伤的效果,后续实验使用氧糖剥夺2 h,复糖复氧16 h进行造模。"
2.2 人参皂苷Rg3对氧糖剥夺/复糖复氧损伤诱导的细胞凋亡的影响 Calcein-AM/PI双染色结果显示Rg3可以减少氧糖剥夺/复糖复氧损伤诱导的细胞凋亡,见图2A。利用Western blot检测凋亡相关蛋白BAX、ProCaspase-3、Pro-Caspase-9、CleavedCaspase-3、CleavedCaspase-9的表达水平,与模型组相比,Rg3在100 nmol/L时可以明显降低BAX蛋白的表达(P < 0.01)。意外的是,与对照组相比,模型组Cleaved-Caspase-9活性升高,Cleaved-Caspase-3活性降低,而Pro-Caspasse-9和Pro-Caspase-3蛋白的活性无明显变化;在使用不同浓度的Rg3处理后,Cleaved-Caspase-9表达降低,Cleaved-Caspase-3表达明显升高,对Pro-Caspasse-9和Pro-Caspase-3的表达无明显影响,见图2B。"
2.4 LY294002对氧糖剥夺/复糖复氧损伤PC12细胞的保护作用 为明确PI3K/AKT信号通路在氧糖剥夺/复糖复氧损伤模型中发挥的作用,使用特异性PI3K/AKT通路抑制剂LY294002进行阳性对照实验。在氧糖剥夺2 h的条件下,单独使用LY294002预处理细胞,结果表明不同浓度的LY294002均能够减少乳酸脱氢酶的释放,见图4A。 Calcein-AM/PI染色和流式结果均表明,LY294002能够抑制氧糖剥夺/复糖复氧损伤介导的细胞凋亡,Western blot结果发现LY294002能够明显降低炎症相关因子NLRP3、白细胞介素1β、肿瘤坏死因子α和凋亡蛋白BAX的表达水平,并且随着LY294002浓度的升高,其对PI3K和AKT的磷酸化抑制程度逐渐增强,见图4B-F。 "
2.5 拮抗Rg3对PI3K/AKT信号通路调节作用后对氧糖剥夺/复糖复氧损伤PC12细胞的影响 为进一步探索Rg3对PI3K/AKT信号通路的调节作用,将Rg3与特异性AKT激活剂SC79联合使用。乳酸脱氢酶释放实验结果显示,与模型组相比,单独使用SC79的乳酸脱氢酶释放率无明显差异,而Rg3和SC79联合使用与单独使用Rg3相比,乳酸脱氢酶释放会增多,但无统计学差异,见图5A。Calcein-AM/PI染色和流式结果均表明,SC79能够促进细胞凋亡,升高细胞凋亡率,Western blot结果表明SC79能恢复炎症相关因子NLRP3、白细胞介素1β、肿瘤坏死因子α和凋亡蛋白BAX的水平,并且能够明显促进AKT的磷酸化,图5B-F。"
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