三黄扶元汤治疗脾肾阳虚型慢性肾衰竭大鼠的分子机制

Molecular mechanisms of Sanhuang Fuyuan Decoction in the treatment of chronic renal failure rats with spleen-kidney yang deficiency pattern based on ferroptosis

关键词:  三黄扶元汤  脾肾阳虚型慢性肾衰竭  铁死亡  纤维化  铁代谢  ACSL4/SLC7A11/GPX4通路

Key words:  Sanhuang Fuyuan Decoction  chronic renal failure of spleen-kidney yang deficiency pattern  ferroptosis  fibrosis  iron metabolism  ACSL4/SLC7A11/GPX4 pathway

摘要:

[目的] 基于铁死亡探讨三黄扶元汤治疗脾肾阳虚型慢性肾衰竭（CRF）大鼠肾损伤的可能机制。[方法] 将48只大鼠随机均分为对照组、模型组、三黄扶元汤组、三黄扶元汤+铁死亡诱导剂（Erastin）组，除对照组外，其余组腺嘌呤灌胃建立脾肾阳虚型CRF模型大鼠。酶标法检测尿液中24 h尿蛋白、血清肌酐（Scr）和血尿素氮（BUN）水平；苏木精-伊红（HE）和Masson染色观察肾组织病理学变化及纤维化程度；生化法检测肾组织铁离子（Fe²⁺）、谷胱甘肽（GSH）、丙二醛（MDA）、超氧化物歧化酶（SOD）水平；透射电镜观察肾组织中线粒体超微结构；RT-qPCR法检测肾组织铁调节蛋白1（IRP1）、转运铁蛋白受体1（TFR1）、膜铁转运蛋白（FPN）mRNA表达变化；免疫组化染色检测肾组织中长链脂肪酸辅酶A连接酶4（ACSL4）、溶质载体家族7成员11（SLC7A11）、谷胱甘肽过氧化物酶4（GPX4）蛋白表达。[结果] 与对照组比较，模型组大鼠肾组织病理学损伤严重且线粒体超微结构受损，24 h尿蛋白、Scr、BUN、胶原纤维占比、Fe²⁺、MDA、TFR1、ACSL4水平均升高（P<0.05），GSH、SOD、IRP1、FPN、SLC7A11、GPX4水平均降低（P<0.05）；与模型组比较，三黄扶元汤组大鼠肾组织病理学损伤减轻且线粒体超微结构得到修复，24 h尿蛋白、Scr、BUN、胶原纤维占比、Fe²⁺、MDA、TFR1、ACSL4水平均降低（P<0.05），GSH、SOD、IRP1、FPN、SLC7A11、GPX4水平均升高（P<0.05）；而Erastin可减轻三黄扶元汤治疗效果（P<0.05）。[结论] 三黄扶元汤可有效提高脾肾阳虚型CRF大鼠肾功能，抑制肾组织病理损伤及铁死亡，其作用机制可能与改善肾组织铁代谢及调控ACSL4/SLC7A11/GPX4通路相关。

Abstract:

[Objective] To explore the potential mechanism of Sanhuang Fuyuan Decoction（SHFYD）in alleviating renal injury in rats with chronic renal failure（CRF）of spleen-kidney yang deficiency pattern，based on ferroptosis. [Methods] Forty-eight rats were randomly and equally divided into control，model，SHFYD，and SHFYD + ferroptosis inducer（Erastin）groups. Except for the control group，all groups received adenine gavage to establish the CRF model of spleen-kidney yang deficiency. Urinary 24-h protein，serum creatinine（Scr）and blood urea nitrogen（BUN）were determined by microplate assays；renal histopathology and fibrosis were evaluated by HE and Masson staining；renal levels of Fe²⁺，glutathione（GSH），malondialdehyde（MDA）and superoxide dismutase（SOD） were measured by biochemical methods；mitochondrial ultrastructure was observed under transmission electron microscopy；RT-qPCR was used to detect mRNA expression of iron regulatory protein 1（IRP1），transferrin receptor 1（TFR1）and ferroportin（FPN）；immunohistochemistry was employed to examine protein expression of long-chain- fatty-acid-CoA ligase 4（ACSL4），solute carrier family 7 member 11（SLC7A11）and glutathione peroxidase 4 （GPX4）. [Results] Compared with the control group，the model group showed severe renal pathological lesions and damaged mitochondrial ultrastructure，accompanied by increased 24-h proteinuria，Scr，BUN，collagen fraction， Fe²⁺，MDA，TFR1 and ACSL4（P<0.05），and decreased GSH，SOD，IRP1，FPN，SLC7A11 and GPX4（P<0.05）. Relative to the model group，SHFYD significantly attenuated renal injury，restored mitochondrial ultra structure， reduced 24-h proteinuria，Scr，BUN，collagen fraction，Fe²⁺，MDA，TFR1 and ACSL4（P<0.05），and elevated GSH， SOD，IRP1，FPN，SLC7A11 and GPX4（P<0.05）. These protective effects were partially reversed by Erastin（P<0.05）. [Conclusion] SHFYD effectively improves renal function and restrains pathological damage and ferroptosis in CRF rats with spleen-kidney yang deficiency，probably by modulating renal iron metabolism and regulating the ACSL4/SLC7A11/GPX4 pathway.