Protective Effect of Rehmannioside A on Podocyte Injury in Rats with Chronic Kidney Disease by Regulating SIRT1/Nrf2/GPX4 Ferroptosis Pathway
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摘要:
目的 探讨地黄苷A（rehmannioside A，ReA）对慢性肾脏疾病（chronic kidney disease，CKD）大鼠足细胞损伤的保护作用及机制。 方法 采用灌胃腺嘌呤构建CKD大鼠模型。将造模成功的大鼠随机分为模型组、ReA-L组（灌胃40.0 mg/kg/d的ReA）组、ReA-H组[灌胃80.0 mg/（kg·d）的ReA]组和ReA-H+Erastin组[灌胃80.0 mg/（kg·d）的ReA+腹腔注射10 mg/（kg·d）铁死亡激活剂Erastin]，每组12只，连续处理4周。另选取12只健康大鼠作为对照组。采集大鼠24 h尿液和腹主动脉血液用于肾功能和肝功能指标检测；苏木精-伊红（hematoxylin-eosin，HE）和马森（Masson）染色观察肾脏组织病理改变；定量逆转录聚合酶链式反应（quantitative reverse transcription polymerase chain reaction，qRT-PCR）检测肾脏组织中结蛋白（desmin）、肾足蛋白（Nephrin）、足蛋白（Podocin）、威尔姆斯肿瘤蛋白1（Wilms' tumor protein 1，WT1）mRNA表达；透射电镜观察足细胞超微结构；相应试剂盒检测大鼠肾脏组织中丙二醛（malondialdehyde，MDA）、谷胱甘肽（glutathione，GSH）、超氧化物歧化酶（superoxide dismutase，SOD）、4-羟基壬烯醛（4-Hydroxy-2-nonenal，4-HNE）、亚铁离子（ferrous ion，Fe2+）和前列腺素内过氧化物合酶2（prostaglandin-endoperoxide synthase 2，PTGS2）含量；蛋白质印迹（Western blot）检测肾脏组织铁蛋白重链1（ferritin heavy chain 1，FTH1）、脂酰辅酶A合成酶长链家族成员4（acyl-CoA synthetase long-chain family member 4，ACSL4）蛋白以及沉默信息调节因子1（silent information regulator 1，SIRT1）/核因子E2相关因子2（nuclear factor E2-related factor 2，NRF2）/谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）通路相关蛋白表达。 结果 与对照组相比，模型组大鼠肾小球基底膜增厚，可见大量蓝色胶原沉积，足突大部分融合，部分肥大，肾小球基底膜（glomerular basement membrane，GBM）增厚，肾小球硬化评分增加（P < 0.05），24 h尿蛋白定量（24-hour urine protein quantification，24 h UTP）、血尿素氮（blood urea nitrogen，BUN）、血清肌酐（serum creatinine，SCR）水平，desmin mRNA表达，MDA、4-HNE、Fe2+、PTGS2含量以及FTH1、ACSL4蛋白表达明显升高（P < 0.05），Nephrin、Podocin、WT1 mRNA表达，GSH、SOD含量以及SIRT1、p-NRF2、GPX4蛋白表达明显降低（P < 0.05）；与模型组相比，ReA-L组、ReA-H组和ReA-H+Erastin组足突大部分融合现象以及GBM增厚程度减轻，肾小球硬化评分减少（P < 0.05），24 h UTP、BUN、SCR水平，desmin mRNA表达，MDA、4-HNE、Fe2+、PTGS2含量以及FTH1、ACSL4蛋白表达明显降低（P < 0.05），Nephrin、Podocin、WT1 mRNA表达，GSH、SOD含量以及SIRT1、p-NRF2、GPX4蛋白表达明显升高（P < 0.05）；与ReA-H组相比，ReA-H+Erastin组部分逆转了ReA-H对CKD大鼠氧化应激、铁死亡及足细胞损伤的改善作用（P < 0.05）。各组间血清丙氨酸氨基转移酶（ALT）和天门冬氨酸氨基转移酶（AST）水平差异无统计学意义（P > 0.05）。 结论 ReA能够抑制氧化应激和铁死亡，减轻CKD大鼠足细胞损伤，其机制可能与SIRT1/NRF2/GPX4通路激活有关。 -
关键词:
- 地黄苷A /
- SIRT1/NRF2/GPX4信号通路 /
- 铁死亡 /
- 慢性肾脏疾病 /
- 足细胞
Abstract:Objective To investigate the protective effect and mechanism of rehmannioside A （ReA） on podocyte damage in rats with chronic kidney disease （CKD）. Methods A CKD rat model was established by administering adenine via gavage. Rats with successful modeling were randomly divided into the model group, a ReA-L group [administered 40.0 mg/（kg·d） of ReA by gavage], a ReA-H group [administered 80.0 mg/（kg·d） of ReA by gavage], and a ReA-H+Erastin group [administered 80.0 mg/（kg·d） of ReA by gavage and 10 mg/（kg·d） of the ferroptosis activator Erastin by intraperitoneal injection], with 12 rats in each group, continuous treatment for 4 weeks. Another 12 healthy rats were selected as the control group. Rat 24-hour urine and abdominal aortic blood were collected for testing renal and liver function indicators. Hematoxylin-eosin （HE） and Masson staining were used to observe renal histopathological changes. Quantitative reverse transcription polymerase chain reaction （qRT-PCR） was used to detect the mRNA expression of desmin, nephrin, podocin, and Wilms' tumor protein 1 （WT1） in kidney tissue. Transmission electron microscopy was used to observe podocyte ultrastructure. Corresponding kits were used to detect the contents of malondialdehyde （MDA）, glutathione （GSH）, superoxide dismutase （SOD）, 4-hydroxy-2-nonenal （4-HNE）, ferrous ion （Fe2+）, and prostaglandin-endoperoxide synthase 2 （PTGS2） in rat renal tissues. Western blot was used to detect the protein expression of ferritin heavy chain 1 （FTH1）, acyl-CoA synthetase long-chain family member 4 （ACSL4）, and silent information regulator 1 （SIRT1）/nuclear factor E2-related factor 2 （NRF2）/glutathione peroxidase 4 （GPX4） pathway-related proteins in renal tissues. Results Compared with the control group, the model group rats showed thickening of the glomerular basement membrane, massive blue collagen deposition, extensive fusion of foot processes, partial hypertrophy, thickening of the glomerular basement membrane （GBM）, and increased glomerulosclerosis score （P < 0.05）. 24-hour urine protein quantification （24 h UTP）, blood urea nitrogen （BUN）, serum creatinine （SCR） levels, desmin mRNA expression, MDA, 4-HNE, Fe2+, PTGS2 content, and FTH1, ACSL4 protein expression were significantly increased （P < 0.05）, while the expression of Nephrin, Podocin, and WT1 mRNA, the content of GSH and SOD, as well as the expression of SIRT1, p-NRF2, and GPX4 proteins were significantly decreased （P < 0.05）. Compared with the model group, the ReA-L group, ReA-H group, and ReA-H Erastin group showed a reduction in most fusion phenomena of podocytes, and the degree of GBM thickening, and the glomerulosclerosis score decreased （P < 0.05）, and the levels of 24 h UTP, BUN, and SCR, desmin mRNA expression, MDA, 4-HNE, Fe2+, PTGS2 content, as well as FTH1 and ACSL4 protein expression were significantly reduced （P < 0.05）, while Nephrin, Podocin, WT1 mRNA expression, GSH and SOD content, as well as SIRT1, p-NRF2, and GPX4 protein expression were significantly increased （P < 0.05）. Compared with the ReA-H group, the ReA-H+Erastin group partially reversed the improvement effect of ReA-H on oxidative stress, ferroptosis, and podocyte injury in CKD rats （P < 0.05）. There was no statistically significant difference in serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） levels among the groups （P > 0.05）. Conclusion ReA can inhibit oxidative stress and ferroptosis and alleviate podocyte damage in CKD rats, and its mechanism may be related to the activation of the SIRT1/NRF2/GPX4 pathway. -
Key words:
- Rehmannioside A /
- SIRT1/NRF2/GPX4 signaling axis /
- Ferroptosis /
- Chronic kidney disease /
- Podocytes
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图 1 各组大鼠肾脏和肝脏功能相关指标检测结果（n = 12，$\bar x \pm s $）
A：24 h UTP水平；B：BUN水平；C：SCR水平；D：ALT水平；E：AST水平；与对照组相比，*P < 0.05；与模型组相比，#P < 0.05；与ReA-L组相比，&P < 0.05；与ReA-H组相比，△P < 0.05。
Figure 1. Detection results of kidney and liver function-related indicators in rats of each group （n = 12，$\bar x \pm s $）
图 2 ReA对肾脏组织病理损伤的影响（n = 12，$\bar x \pm s $）
A：各组大鼠肾脏组织HE染色结果（×400，红色箭头指示肾小球）；B：各组大鼠肾小球硬化评分；C：各组大鼠肾脏组织Masson染色结果（×200）。与对照组相比，*P < 0.05；与模型组相比，#P < 0.05；与ReA-L组相比，&P < 0.05；与ReA-H组相比，△P < 0.05。
Figure 2. Effect of ReA on renal tissue pathological damage （n = 12，$\bar x \pm s $）
图 3 各组大鼠足细胞损伤相关标志物水平（n = 12，$\bar x \pm s $）
A：desmin mRNA相对表达水平；B：Nephrin mRNA相对表达水平；C：Podocin mRNA相对表达水平；D：WT1 mRNA相对表达水平；与对照组相比，*P < 0.05；与模型组相比，#P < 0.05；与ReA-L组相比，&P < 0.05；与ReA-H组相比，△P < 0.05。
Figure 3. Levels of podocyte injury-related markers in rats of each group （n = 12，$\bar x \pm s $）
图 4 足细胞电镜超微结构（×5 000）
A：对照组；B：模型组；C：ReA-L组；D：ReA-H组；E：ReA-H+Erastin组；红色箭头表示足突，绿色箭头表示GBM。
Figure 4. Electron microscopic ultrastructure of podocytes （× 5 000）
图 5 各组大鼠氧化应激和铁死亡相关指标结果（n = 12，$\bar x \pm s $）
A：MDA含量；B：GSH含量；C：SOD含量；D：4-HNE含量；E：Fe2+含量；F：PTGS2含量；G：FTH1、ACSL4蛋白电泳图；H：FTH1蛋白表达定量分析；I：ACSL4蛋白表达定量分析；与对照组相比，*P < 0.05；与模型组相比，#P < 0.05；与ReA-L组相比，&P < 0.05；与ReA-H组相比，△P < 0.05。
Figure 5. Results of oxidative stress and ferroptosis-related indicators in rats of each group （n = 12，$\bar x \pm s $）
图 6 Western Blot检测各组大鼠肾脏组织SIRT1/NRF2/GPX4通路相关蛋白表达（n = 12，$\bar x \pm s $）
A：SIRT1、p-NRF2、NRF2、GPX4蛋白电泳图；B：SIRT1蛋白表达定量分析；C：p-NRF2蛋白定量分析；D：GPX4蛋白定量分析；与对照组相比，*P < 0.05；与模型组相比，#P < 0.05；与ReA-L组相比， & P < 0.05；与ReA-H组相比，△P < 0.05。
Figure 6. Western Blot detection of the expression of SIRT1/NRF2/GPX4 pathway-related proteins in kidney tissues of rats in each group （n = 12，$\bar x \pm s $）
表 1 引物序列
Table 1. Primer sequences
基因 引物序列（5'-3'） desmin F：CTGAGCAAAGGGGTTCCGA
R：GTGTGACATCCGAGAGTGGANephrin F：CTGGGGGACAGTGGATTGAC
R：CCACCAACTGCAAAGAGCACWT1 F：TGCGTCCTATCAGGTTTGCC
R：CCCAGCGAAATGAGCACAAGPodocin F：GCTGTCTGCTACTACCGCAT
R：GTGAGGGATCGATGTGCCAAGAPDH F：TGGGTGTGAACCACGAGAA
R：GGCATGGACTGTGGTCATGA
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