蟾蜍灵对高糖诱导的肾小管上皮细胞细胞外基质合成的影响

doi:10.11958/20252443

天津医药 ›› 2025, Vol. 53 ›› Issue (10): 1009-1015.doi: 10.11958/20252443

• 细胞与分子生物学 • 下一篇

蟾蜍灵对高糖诱导的肾小管上皮细胞细胞外基质合成的影响

高辰(), 乔云阳, 季嘉玲, 王娥, 霍影, 张爱青^△()

南京医科大学第四附属医院儿科（邮编 210031）

收稿日期:2025-07-08 修回日期:2025-07-21 出版日期:2025-10-15 发布日期:2025-10-12
通讯作者: ^△E-mail：njaiqing@njmu.edu.cn
作者简介:高辰（1989），男，硕士在读，主要从事儿科肾脏病学方面研究。E-mail：945667129@qq.com
基金资助:
江苏省自然科学基金项目(BK20240297);南京市卫生科技发展专项资金项目(YKK23209)

The effect of bufalin on extracellular matrix synthesis in renal tubular epithelial cells induced by high glucose

GAO Chen(), QIAO Yunyang, JI Jialing, WANG E, HUO Ying, ZHANG Aiqing^△()

Department of Pediatrics, the Fourth Affiliated Hospital of Nanjing Medical University, Nanjing 210031, China

Received:2025-07-08 Revised:2025-07-21 Published:2025-10-15 Online:2025-10-12
Contact: ^△E-mail: njaiqing@njmu.edu.cn

高辰, 乔云阳, 季嘉玲, 王娥, 霍影, 张爱青. 蟾蜍灵对高糖诱导的肾小管上皮细胞细胞外基质合成的影响[J]. 天津医药, 2025, 53(10): 1009-1015.

GAO Chen, QIAO Yunyang, JI Jialing, WANG E, HUO Ying, ZHANG Aiqing. The effect of bufalin on extracellular matrix synthesis in renal tubular epithelial cells induced by high glucose[J]. Tianjin Medical Journal, 2025, 53(10): 1009-1015.

摘要/Abstract

摘要：

目的探讨蟾蜍灵调控铁死亡对高糖（HG）诱导的肾小管上皮细胞（RTECs）细胞外基质合成的作用和机制。方法体外使用HG干预RTECs，将细胞分为对照组、HG组、HG+二甲基亚砜（DMSO）组、HG+蟾蜍灵组、HG+铁抑素-1（Fer-1）组、HG+蟾蜍灵+DMSO组和HG+蟾蜍灵+铁死亡诱导剂（Erastin）组。采用蛋白免疫印迹法和实时定量PCR检测各组RTECs中纤维连接蛋白（FN）、Ⅰ型胶原蛋白（Col Ⅰ）、酰基辅酶A合成酶长链家族成员4（ACSL4）、溶质运载家族7成员11（SLC7A11）、谷胱甘肽过氧化物酶4（GPX4）的表达情况；采用SwissTargetPrediction数据库预测蟾蜍灵的作用靶点，使用Metascape分析功能并通过FerrDb进行基因集的铁死亡相关分析；采用微量法检测细胞亚铁离子（Fe²⁺）、丙二醛（MDA）和谷胱甘肽（GSH）水平。结果与对照组相比，HG组FN、Col Ⅰ、ACSL4的mRNA和蛋白相对表达量均升高，GPX4和SLC7A11 mRNA及蛋白表达量降低（P<0.05）；与HG+DMSO组相比，HG+蟾蜍灵组FN和Col Ⅰ、ACSL4 mRNA和蛋白表达量、Fe²⁺和MDA水平降低，GPX4和SLC7A11 mRNA和蛋白、GSH水平表达量升高（P<0.05），HG+Fer-1组GPX4和SLC7A11的mRNA及蛋白表达量升高，而ACSL4、FN和Col Ⅰ的mRNA和蛋白表达量降低（P<0.05）；SwissTargetPrediction数据库及Metascape分析功能显示蟾蜍灵下游功能与脂质代谢、炎症反应、细胞程序性死亡及铁死亡相关通路密切相关，FerrDb分析结果显示蟾蜍灵作用靶点与铁死亡标志物密切相关；与HG+蟾蜍灵+DMSO组相比，HG+蟾蜍灵+Erastin组GPX4和SLC7A11的mRNA和蛋白表达量降低，ACSL4、FN和Col ⅠmRNA和蛋白表达量升高（P<0.05）。结论蟾蜍灵通过抑制铁死亡减少HG诱导的RTECs细胞外基质合成。

关键词: 肾小管, 上皮细胞, 细胞外基质, 铁死亡, 蟾蜍灵, 高糖

Abstract:

Objective To investigate the effect and underlying mechanism of bufalin regulating ferroptosis on extracellular matrix synthesis in renal tubular epithelial cells (RTECs) under high glucose (HG) conditions. Methods RTECs were cultured in vitro and exposed to HG. The experimental groups included: the control group, the HG group, the HG + dimethyl sulfoxide (DMSO) group, the HG + bufalin group, the HG + ferrostatin-1 (Fer-1) group, the HG + bufalin + DMSO group and HG + bufalin + erastin group. The expression levels of fibronectin (FN), type Ⅰ collagen (Col Ⅰ), acyl-CoA synthetase long-chain family member 4 (ACSL4), solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were detected using Western blot assay and quantitative real-time PCR (qRT-PCR). The potential molecular targets of bufalin were predicted using SwissTargetPrediction, and functional enrichment analysis was conducted using Metascape. FerrDb was employed to analyze ferroptosis-related gene sets. The levels of ferrous ions (Fe²⁺), malondialdehyde (MDA) and glutathione (GSH) were measured using micro-methods to evaluate the occurrence of ferroptosis. Results Compared with the control group, the mRNA and protein relative expression levels of FN, Col Ⅰand ACSL4 were increased in the HG group, while the mRNA and protein expression levels of GPX4 and SLC7A11 were decreased (P<0.05). Compared with the HG + DMSO group, the mRNA and protein expression levels of FN, Col Ⅰand ACSL4, as well as levels of Fe²⁺ and MDA were decreased in the HG + bufalin group, while the mRNA and protein expression levels of GPX4 and SLC7A11, and the level of GSH were increased (P<0.05). In the HG + Fer-1 group, the mRNA and protein expression levels of GPX4 and SLC7A11 were increased, while the mRNA and protein expression levels of ACSL4, FN and Col Ⅰ were decreased (P<0.05). The SwissTargetPrediction database and Metascape analysis function showed that the downstream functions of bufalin were closely related to lipid metabolism, inflammatory response, programmed cell death and ferroptosis-related pathways. The FerrDb analysis results indicated that the target sites of bufalin were closely related to ferroptosis markers. Compared with the HG + bufalin + DMSO group, the mRNA and protein expression levels of GPX4 and SLC7A11 were decreased in the HG + bufalin + Erastin group, while the mRNA and protein expression levels of ACSL4, FN and Col Ⅰ were increased (P<0.05). Conclusion Bufalin attenuates extracellular matrix synthesis in HG-induced RTECs by inhibiting ferroptosis.

Key words: kidney tubules, epithelial cells, extracellular matrix, ferroptosis, bufalin, high glucose

中图分类号:

R692.39

图/表 12

参考文献 24

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基因名称	引物序列（5′→3′）	产物大小/bp
FN	上游：ATGTGGACCCCTCCTGATAGT 下游：GCCCAGTGATTTCAGCAAAGG	124
Col Ⅰ	上游：GCTCCTCTTAGGGGCCACT 下游：ATTGGGGACCCTTAGGCCAT	91
ACSL4	上游：CCTGAGGGGCTTGAAATTCAC 下游：GTTGGTCTACTTGGAGGAACG	91
SLC7A11	上游：GGCACCGTCATCGGATCAG 下游：CTCCACAGGCAGACCAGAAAA	100
GPX4	上游：TGTGCATCCCGCGATGATT 下游：CCCTGTACTTATCCAGGCAGA	89
β-actin	上游：GTGACGTTGACATCCGTAAAGA 下游：GCCGGACTCATCGTACTCC	245

基因名称	引物序列（5′→3′）	产物大小/bp
FN	上游：ATGTGGACCCCTCCTGATAGT 下游：GCCCAGTGATTTCAGCAAAGG	124
Col Ⅰ	上游：GCTCCTCTTAGGGGCCACT 下游：ATTGGGGACCCTTAGGCCAT	91
ACSL4	上游：CCTGAGGGGCTTGAAATTCAC 下游：GTTGGTCTACTTGGAGGAACG	91
SLC7A11	上游：GGCACCGTCATCGGATCAG 下游：CTCCACAGGCAGACCAGAAAA	100
GPX4	上游：TGTGCATCCCGCGATGATT 下游：CCCTGTACTTATCCAGGCAGA	89
β-actin	上游：GTGACGTTGACATCCGTAAAGA 下游：GCCGGACTCATCGTACTCC	245

组别	mRNA（n=4）		蛋白（n=3）
组别	FN	Col Ⅰ	FN	Col Ⅰ
对照组	1.00±0.21	1.00±0.17	1.00±0.24	1.00±0.27
HG组	1.90±0.21^a	2.09±0.15^a	2.38±0.26^a	2.45±0.07^a
HG+DMSO组	1.96±0.10	2.04±0.21	2.40±0.19	2.47±0.12
HG+蟾蜍灵组	1.43±0.12^b	1.48±0.26^b	1.33±0.07^b	1.32±0.20^b
F	28.831^＊＊	26.530^＊＊	38.606^＊＊	54.033^＊＊

组别	mRNA（n=4）		蛋白（n=3）
组别	FN	Col Ⅰ	FN	Col Ⅰ
对照组	1.00±0.21	1.00±0.17	1.00±0.24	1.00±0.27
HG组	1.90±0.21^a	2.09±0.15^a	2.38±0.26^a	2.45±0.07^a
HG+DMSO组	1.96±0.10	2.04±0.21	2.40±0.19	2.47±0.12
HG+蟾蜍灵组	1.43±0.12^b	1.48±0.26^b	1.33±0.07^b	1.32±0.20^b
F	28.831^＊＊	26.530^＊＊	38.606^＊＊	54.033^＊＊

组别	mRNA（n=4）
组别	ACSL4	SLC7A11	GPX4	FN	Col Ⅰ
对照组	1.00±0.25	1.00±0.16	1.00±0.13	1.00±0.13	1.00±0.27
HG组	2.77±0.17^a	0.24±0.05^a	0.30±0.03^a	1.99±0.10^a	2.00±0.10^a
HG+DMSO组	2.75±0.20	0.25±0.05	0.28±0.05	1.96±0.15	2.00±0.16
HG+Fer-1组	1.75±0.22^b	0.86±0.11^b	0.87±0.20^b	1.32±0.11^b	1.39±0.17^b
F	64.764^＊＊	59.404^＊＊	39.187^＊＊	61.903^＊＊	28.774^＊＊
组别	蛋白（n=3）
组别	ACSL4	SLC7A11	GPX4	FN	Col Ⅰ
对照组	1.00±0.24	1.00±0.16	1.00±0.14	1.00±0.18	1.00±0.20
HG组	2.32±0.08^a	0.19±0.02^a	0.23±0.02^a	2.24±0.08^a	2.06±0.15^a
HG+DMSO组	2.23±0.09	0.20±0.05	0.23±0.08	2.19±0.19	2.05±0.17
HG+Fer-1组	1.31±0.12^b	0.75±0.16^b	0.98±0.07^b	1.28±0.11^b	1.34±0.12^b
F	59.248^＊＊	36.726^＊＊	75.779^＊＊	54.794^＊＊	31.902^＊＊

蟾蜍灵对高糖诱导的肾小管上皮细胞细胞外基质合成的影响

The effect of bufalin on extracellular matrix synthesis in renal tubular epithelial cells induced by high glucose

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参考文献 24

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组别	Fe²⁺	MDA	GSH
对照组	1.00±0.08	1.00±0.18	1.00±0.19
HG组	2.26±0.10^a	3.06±0.25^a	0.39±0.11^a
HG+DMSO组	2.33±0.11	3.11±0.09	0.41±0.11
HG+蟾蜍灵组	1.62±0.08^b	1.93±0.22^b	0.97±0.19^b
F	170.965^＊＊	107.864^＊＊	18.859^＊＊

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组别		mRNA（n=4）
组别		ACSL4		SLC7A11	GPX4
对照组		1.00±0.16		1.00±0.17	1.00±0.20
HG组		2.69±0.12^a		0.25±0.07^a	0.26±0.09^a
HG+DMSO组		2.82±0.28		0.22±0.07	0.25±0.10
HG+蟾蜍灵组		1.55±0.12^b		0.95±0.15^b	0.96±0.18^b
F		93.737^＊＊		46.731^＊＊	30.760^＊＊
组别	蛋白（n=3）
组别	ACSL4		SLC7A11			GPX4
对照组	1.00±0.21		1.00±0.09			1.00±0.20
HG组	2.16±0.17^a		0.22±0.03^a			0.27±0.04^a
HG+DMSO组	2.15±0.08		0.21±0.04			0.23±0.04
HG+蟾蜍灵组	1.29±0.04^b		0.64±0.08^b			0.86±0.07^b
F	52.292^＊＊		100.954^＊＊			39.744^＊＊

组别		mRNA（n=4）
组别		ACSL4		SLC7A11	GPX4
对照组		1.00±0.16		1.00±0.17	1.00±0.20
HG组		2.69±0.12^a		0.25±0.07^a	0.26±0.09^a
HG+DMSO组		2.82±0.28		0.22±0.07	0.25±0.10
HG+蟾蜍灵组		1.55±0.12^b		0.95±0.15^b	0.96±0.18^b
F		93.737^＊＊		46.731^＊＊	30.760^＊＊
组别	蛋白（n=3）
组别	ACSL4		SLC7A11			GPX4
对照组	1.00±0.21		1.00±0.09			1.00±0.20
HG组	2.16±0.17^a		0.22±0.03^a			0.27±0.04^a
HG+DMSO组	2.15±0.08		0.21±0.04			0.23±0.04
HG+蟾蜍灵组	1.29±0.04^b		0.64±0.08^b			0.86±0.07^b
F	52.292^＊＊		100.954^＊＊			39.744^＊＊

组别		mRNA（n=4）
组别		ACSL4		SLC7A11		GPX4		FN		Col Ⅰ
HG+蟾蜍灵+ DMSO组		1.00±0.31		1.00±0.23		1.00±0.27		1.00±0.28		1.00±0.24
HG+蟾蜍灵+ Erastin组		3.02±0.32		0.23±0.07		0.22±0.09		3.04±0.25		2.56±0.31
t		8.977^＊＊		6.369^＊＊		5.493^＊＊		10.940^＊＊		7.941^＊＊
组别	蛋白（n=3）
组别	ACSL4		SLC7A11		GPX4		FN		Col Ⅰ
HG+蟾蜍灵+ DMSO组	1.00±0.19		1.00±0.22		1.00±0.21		1.00±0.15		1.00±0.25
HG+蟾蜍灵+ Erastin组	2.22±0.20		0.23±0.08		0.23±0.06		2.26±0.29		2.30±0.44
t	7.578^＊＊		5.782^＊＊		6.097^＊＊		6.780^＊＊		4.466^＊

组别		mRNA（n=4）
组别		ACSL4		SLC7A11		GPX4		FN		Col Ⅰ
HG+蟾蜍灵+ DMSO组		1.00±0.31		1.00±0.23		1.00±0.27		1.00±0.28		1.00±0.24
HG+蟾蜍灵+ Erastin组		3.02±0.32		0.23±0.07		0.22±0.09		3.04±0.25		2.56±0.31
t		8.977^＊＊		6.369^＊＊		5.493^＊＊		10.940^＊＊		7.941^＊＊
组别	蛋白（n=3）
组别	ACSL4		SLC7A11		GPX4		FN		Col Ⅰ
HG+蟾蜍灵+ DMSO组	1.00±0.19		1.00±0.22		1.00±0.21		1.00±0.15		1.00±0.25
HG+蟾蜍灵+ Erastin组	2.22±0.20		0.23±0.08		0.23±0.06		2.26±0.29		2.30±0.44
t	7.578^＊＊		5.782^＊＊		6.097^＊＊		6.780^＊＊		4.466^＊