人参皂苷Rg1拮抗亚砷酸钠诱导C57BL/6小鼠肾毒性研究

doi:10.11958/20221834

摘要/Abstract

摘要：

目的探讨人参皂苷Rg1对亚砷酸钠（SA）诱导小鼠肾脏毒性的干预效应。方法 20只雄性健康C57BL/6小鼠采用随机数字表法均分为对照组（给予去离子水灌胃）、SA染毒组（10.0 μg/g SA进行灌胃）、人参皂苷Rg1+SA染毒组（20.0 μg/g人参皂苷Rg1在SA染毒前8 h腹腔注射+10.0 μg/g SA灌胃）、人参皂苷Rg1对照组（20.0 μg/g人参皂苷Rg1腹腔注射）。以上各组均隔天给予相应处理1次，持续14 d。HE染色观察肾组织病理改变并进行肾小管损伤（TI）评分；酶联免疫吸附试验（ELISA）检测血清肌酐（Scr）和肾组织谷胱甘肽（GSH）、血红素加氧酶-1（HO-1）、丙二醛（MDA）含量；免疫印迹试验检测肾组织HO-1、磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）、泛素结合蛋白P62（SQSTM1/p62）、unc-51样激酶-1（ULK1）和微管相关蛋白轻链3B（LC3-B）表达水平；免疫荧光染色检测LC3-B水平。结果与对照组相比，SA染毒组小鼠TI评分、Scr和肾组织MDA、ULK1和LC3-B表达水平升高，肾组织GSH和HO-1、p-mTOR和SQSTM1/p62表达水平降低（P<0.05），呈红色斑点的LC3-B染色强度增强、增多；与SA染毒组相比，人参皂苷Rg1+SA染毒组TI评分、Scr和肾组织MDA、ULK1和LC3-B表达水平降低，而GSH、HO-1、p-mTOR和SQSTM1/p62表达水平升高（P<0.05），LC3-B免疫荧光染色强度减弱、减少。结论人参皂苷Rg1拮抗SA诱导的小鼠肾毒性，可能与HO-1信号激活和细胞自噬抑制有关。

关键词: 人参皂苷Rg1, 砷中毒, 血红素加氧酶-1, 自噬, 肾毒性

Abstract:

Objective To investigate the intervention effect of ginsenoside Rg1 (Rg1) against sodium arsenite (SA) induced nephrotoxicity in mice. Methods Twenty healthy male C57BL/6 mice were randomly divided into the control group (given deionized water by gavage), the SA exposure group (10.0 μg/g SA by gavage), the Rg1 intervention+SA exposure group (20.0 μg/g Rg1 was injected intraperitoneally 8 hours before SA exposure+10.0 μg/g SA gavage) and the Rg1 control group (20.0 μg/g Rg1 intraperitoneal injection). All of groups were given corresponding treatment once every other day for 14 days. HE staining was performed to observe pathological changes of renal tissue and renal tubular injury (TI) score. Serum creatinine (Scr) and renal glutathione (GSH), heme oxygenase-1 (HO-1) and malondialdehyde (MDA) were detected by enzyme-linked immunosorbent assay (ELISA). The expression levels of HO-1, phosphorylated mammalian target of rapamycin (p-mTOR), ubiquitin-binding protein P62 (SQSTM1/p62), unc-51-like kinase-1 (ULK1) and microtubule-associated protein light chain 3B (LC3-B) in renal tissue were detected by Western blot assay. LC3-B levels were detected by immunofluorescence staining. Results Compared with the control group, the TI score, Scr and expression levels of MDA, ULK1 and LC3-B in renal tissue were increased in the SA group, while expression levels of GSH and HO-1, p-mTOR and SQSTM1/p62 in renal tissue were decreased (P<0.05). The staining intensity of red spot LC3-B was enhanced and increased. Compared with the SA group, TI score, Scr and expression levels of MDA, ULK1 and LC3-B in renal tissue were decreased in the Rg1 +SA group, while expression levels of GSH, HO-1, p-mTOR and SQSTM1/p62 were increased (P<0.05). The immunofluorescence staining intensity of LC3-B was weakened and decreased. Conclusion Rg1 antagonizes SA-induced nephrotoxicity in mice, which may be associated with the activation of HO-1 signal and the inhibition of autophagy.

Key words: Ginsenoside Rg1, arsenic poisoning, heme oxygenase-1, autophagy, nephrotoxicity

中图分类号:

R114

图/表 6

图1 SA染毒及人参皂苷Rg1预处理干预后肾脏组织病理学特征（HE染色，×100）

Fig.1 Renal histopathological characteristics after SA exposure and Rg1 pretreatment (HE stanining, ×100)

表1 各组血清肌酐、TI评分和MDA、GSH、HO-1含量比较（n=5，$\bar{x}±s$）

Tab.1 Comparison of serum creatinine, TI score and contents of MDA, GSH and HO-1 in kidney tissue between four groups

组别	Scr/（μmol/L）	TI评分/%	MDA/（nmol/g）	GSH/（ng/g）	HO-1/（ng/g）
对照组	204.86±14.47	1.60±0.48	35.18±2.61	213.07±12.08	88.61±3.13
SA染毒组	293.60±30.94^a	38.40±5.62^a	52.12±4.15^a	173.34±9.73^a	72.97±4.42^a
人参皂苷Rg1对照组	208.38±9.74	2.20±0.64	33.67±3.18	214.76±12.35	90.19±6.18
人参皂苷Rg1+SA染毒组	226.95±10.87^b	24.21±3.56^b	41.24±3.93^b	189.54±10.40^b	81.50±3.37^b
F	24.674^＊＊	143.096^＊＊	28.257^＊＊	15.817^＊＊	15.675^＊＊

图2 各组肾组织HO-1和p-mTOR免疫印迹特征 A：对照组；B：SA染毒组；C：Rg1干预+SA染毒组；D：Rg1对照组。

Fig.2 Western blot assay of renal tissue HO-1 and p-mTOR in each group

图3 各组肾组织SQSTM1/p62、ULK1和LC3-B免疫印迹图 A：对照组；B：SA染毒组；C：Rg1干预+SA染毒组；D：Rg1对照组。

Fig.3 Western blot assay of SQSTM1/p62, ULK1 and LC3-B in each group

表2 各组肾组织HO-1、p-mTOR、SQSTM1/p62、ULK1和LC3-B水平比较（n=5，$\bar{x}±s$）

Tab.2 Comparison of levels of HO-1, p-mTOR, SQSTM1/p62, ULK1 and LC3-B in renal tissue between four groups

组别	HO-1	p-mTOR/mTOR	SQSTM1/p62	ULK1	LC3-B
对照组	0.52±0.06	0.40±0.11	0.76±0.12	0.13±0.04	0.08±0.02
SA染毒组	0.14±0.04^a	0.12±0.05^a	0.22±0.05^a	0.43±0.09^a	0.30±0.07^a
人参皂苷Rg1对照组	0.56±0.13	0.50±0.14	0.71±0.07	0.09±0.04	0.07±0.03
人参皂苷Rg1+SA染毒组	0.73±0.11^b	0.47±0.12^b	0.45±0.04^b	0.27±0.05^b	0.16±0.05^b
F	36.655^＊＊	12.970^＊＊	55.626^＊＊	36.758^＊＊	26.192^＊＊

图4 肾组织自噬标志物LC3-B的免疫荧光染色图（免疫荧光染色，×400）红色斑点为自噬标记物LC3-B，散布于蓝色染色的细胞核周围。

Fig.4 Fluorescence staining images of autophagy marker LC3-B in kidney tissue (immunofluorescence staining, ×400)

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