甘草次酸的差向异构体对顺铂诱导H9c2心肌细胞损伤的保护机制

doi:10.11958/20220987

天津医药 ›› 2022, Vol. 50 ›› Issue (12): 1264-1269.doi: 10.11958/20220987

甘草次酸的差向异构体对顺铂诱导H9c2心肌细胞损伤的保护机制

马治¹(), 王欣爽¹, 刘玥², 魏丽萍², 齐新²^,^△()

1 天津中医药大学研究生院（邮编301617）
2 天津市人民医院心脏科

收稿日期:2022-06-24 修回日期:2022-07-22 出版日期:2022-12-15 发布日期:2022-12-30
通讯作者: 齐新 E-mail:476997137@qq.com;qixinx2011@126.com
作者简介:马治（1990），女，硕士，主要从事中西医结合防治心血管的研究。E-mail：476997137@qq.com
基金资助:
天津市卫生健康委员会-天津市中医药管理局-中医中西医结合科研课题(2021028);天津市卫生健康委员会-天津市中医药管理局-中医中西医结合科研课题(2021155)

The protective mechanism of differential isomers of glycyrrhetinic acid on cisplatin-induced H9c2 cardiomyocyte injury

MA Zhi¹(), WANG Xinshuang¹, LIU Yue², WEI Liping², QI Xin²^,^△()

1 Graduate School, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China
2 Department of Cardiology, Tianjin Union Medical Center

Received:2022-06-24 Revised:2022-07-22 Published:2022-12-15 Online:2022-12-30
Contact: QI Xin E-mail:476997137@qq.com;qixinx2011@126.com

马治, 王欣爽, 刘玥, 魏丽萍, 齐新. 甘草次酸的差向异构体对顺铂诱导H9c2心肌细胞损伤的保护机制[J]. 天津医药, 2022, 50(12): 1264-1269.

MA Zhi, WANG Xinshuang, LIU Yue, WEI Liping, QI Xin. The protective mechanism of differential isomers of glycyrrhetinic acid on cisplatin-induced H9c2 cardiomyocyte injury[J]. Tianjin Medical Journal, 2022, 50(12): 1264-1269.

摘要/Abstract

摘要：

目的探究甘草次酸（GA）的差向异构体18α-GA和18β-GA对顺铂（CDDP）诱导H9c2心肌细胞损伤的保护机制。方法采用CCK-8法检测细胞活力，筛选CDDP诱导细胞损伤的浓度，18α-GA和18β-GA安全浓度以及18α-GA与18β-GA改善CDDP致心肌细胞活力下降的有效浓度；将细胞分为对照组、CDDP组、18α-GA组和18β-GA组，使用Hoechst染色检测各组细胞凋亡情况；流式细胞术检测活性氧（ROS）水平；Mito-Tracker Red CMXRos染色评估线粒体活性；Western blot法检测各组细胞剪切化胱天蛋白酶3（C-Caspase3）、B-淋巴细胞瘤基因-2（Bcl-2）、Bcl-2相关X蛋白（Bax）以及细胞色素C（Cyt-c）的蛋白表达。结果 CCK-8实验结果显示，CDDP在20 μmol/L时可使H9c2心肌细胞活力显著下降（P<0.01）；18α-GA和18β-GA浓度小于100 μmol/L时，对心肌细胞无明显影响，且50和100 μmol/L时均可改善CDDP导致的心肌细胞活力的降低（P<0.01）。与对照组相比，CDDP组细胞凋亡明显，ROS水平升高，心肌细胞内线粒体足长度和具有网状结构的线粒体降低，C-Caspase3、Cyt-c的蛋白表达升高，Bcl-2/Bax的蛋白表达降低（P<0.05）；与CDDP组相比，18α-GA组和18β-GA组细胞凋亡情况改善，ROS水平降低，心肌细胞内线粒体足长度和具有网状结构的线粒体增加，C-Caspase3、Cyt-c的蛋白表达水平降低，Bcl-2/Bax的蛋白表达水平升高（P<0.05）。结论 18α-GA和18β-GA可通过抑制ROS水平以及保护线粒体功能减少CDDP诱导的H9c2心肌细胞凋亡。

关键词: 顺铂, 心脏毒性, 甘草次酸, 活性氧, 线粒体, 差向异构体

Abstract:

Objective To explore the protective mechanism of 18α-glycyrrhetinic acid (GA) and 18β-GA, the differential isomers of glycyrrhetinic acid, on cisplatin (CDDP) -induced H9c2 cardiomyocyte injury. Methods CCK-8 assay was used to detect cell viability and the concentration of CDDP-induced cell injury. The safe doses of 18α-GA and 18β-GA, and the effective concentrations of 18α-GA and 18β-GA to improve the decrease of cardiomyocyte viability induced by CDDP were screened. The cells were divided into the control group, the CDDP group, the 18α-GA group and the 18β-GA group. Hoechst staining was used to detect apoptosis in each group. The level of reactive oxygen species (ROS) was detected by DCFH-DA. Mito-Tracker Red CMXRos staining was used to assess mitochondrial activity. The expressions of C-Caspase3, B-lymphocytoma gene-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cytochrome C (Cyt-c) were detected by Western blot assay in each group. Results The results of CCK-8 assay showed that CDDP at 20 μmol/L significantly decreased the viability of H9c2 cardiomyocytes (P<0.01). When the concentrations of 18α-GA and 18β-GA were less than 100 μmol/L, there were no significant effects on cardiomyocytes, and 50 and 100 μmol/L could improve the decrease of cardiomyocyte viability caused by CDDP (P<0.01). Compared with the control group, the apoptosis was obvious in the CDDP group, the ROS level was increased, the mitochondrial foot length and mitochondria with reticular structure were decreased, the protein expression levels of C-Caspase3 and Cyt-c were increased, and the protein expression of Bcl-2/Bax was decreased (P<0.05). Compared with the CDDP group, cell apoptosis was improved in the 18α-GA group and the 18β-GA group, ROS level decreased, mitochondrial foot length and mitochondria with reticular structure increased, protein expression levels of C-Caspase3 and Cyt-c decreased, and protein expression levels of Bcl-2/Bax increased (P<0.05). Conclusion 18α-GA and 18β-GA can reduce the apoptosis of H9c2 cardiomyocytes induced by CDDP by inhibiting ROS level and protecting mitochondrial function.

Key words: cisplatin, cardiotoxicity, glycyrrhetinic acid, reactive oxygen, mitochondria, species differential isomers

中图分类号:

R285.5

图/表 9

图1 CDDP对H9c2心肌细胞活力的影响 a与0 μmol/L比较，P<0.05。

Fig.1 Effects of CDDP on viability of H9c2 cardiomyocytes

图2 18α-GA和18β-GA对H9c2心肌细胞活力的影响 A~G分别为0、3.125、6.25、12.5、25、50和100 μmol/L CDDP。

Fig.2 Effects of 18α-GA and 18β-GA on the viability of H9c2 cells

图3 18α-GA及18β-GA低、中、高剂量对CDDP诱导心肌细胞活力的影响 A：对照组；B：CDDP组；C：低剂量组；D：中剂量组；E：高剂量组；a与对照组比较，b与CDDP组比较，P<0.05。

Fig.3 Effects of low, medium and high doses of 18α-GA and 18β-GA on cisplatin-induced myocardial cell viability

图4 18α-GA及18β-GA对CDDP诱导的心肌细胞凋亡的影响（Hoechst染色，×400）

Fig.4 Effects of 18α-GA and 18β-GA on CDDP-induced apoptosis of cardiomyocytes (Hoechst staining, ×400)

图5 18α-GA和18β-GA对顺铂诱导心肌细胞ROS含量的影响

Fig.5 Effects of 18α-GA and 18β-GA on ROS content in cardiomyocytes induced by cisplatin

图6 18α-GA和18β-GA对CDDP诱导心肌细胞线粒体活性的影响（×400）

Fig.6 Effects of 18α-GA and 18β-GA on mitochondrial activity of cardiomyocytes induced by CDDP (×400)

表1 各组细胞线粒体足长度和具有网状结构的线粒体数量比较（n=6）

Tab.1 Comparison of mitochondrial foot length and number of mitochondria with reticular structure between the four groups

组别	线粒体足长度	具有网状结构的线粒体数量
对照组	1.00±0.20	0.94（0.75，1.22）
CDDP组	0.53±0.03^a	0.38（0.38，0.47）^a
18α-GA组	0.79±0.07^b	0.75（0.75，0.84）^b
18β-GA组	0.87±0.21^b	0.75（0.75，0.75）^b
F或H	34.748^＊＊	13.320^＊＊

表2 各组C-Caspase3、Bcl-2/Bax、Cyt-c的蛋白表达水平比较（n=3, $\bar{x}\pm s$）

Tab.2 Comparison of protein expression levels of C-Caspase3, Bcl-2/Bax and Cyt-c between the four groups

组别	C-Caspase3	Bcl-2/Bax	Cyt-c
对照组	1.06±0.40	1.00±0.15	0.97±0.41
CDDP组	2.15±0.40^a	0.50±0.04^a	2.66±0.22^a
18α-GA组	1.30±0.28^b	0.97±0.18^b	1.11±0.25^b
18β-GA组	1.30±0.35^b	0.88±0.09^b	0.57±0.04^b
F	10.321^＊＊	9.768^＊＊	36.039^＊＊

图7 18α-GA和18β-GA对CDDP诱导的心肌细胞线粒体相关凋亡蛋白表达的影响 A：对照组；B：CDDP组；C：18α-GA 组；D：18β-GA组。

Fig.7 Effects of 18α-GA and 18β-GA on CDDP-induced mitochondrial-related apoptostic protein expression in cardiomyocytes

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甘草次酸的差向异构体对顺铂诱导H9c2心肌细胞损伤的保护机制

The protective mechanism of differential isomers of glycyrrhetinic acid on cisplatin-induced H9c2 cardiomyocyte injury

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