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

doi:10.11958/20252443

Abstract

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

CLC Number:

R692.39

Figures/Tables 12

References 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^＊＊