目的 探讨甘草多糖（Glycyrrhiza polysaccharide，GCP）对顺铂诱导的急性肾损伤（cisplatin-induced acute kidney injury，CI-AKI）模型大鼠和人肾小管上皮细胞HK-2损伤模型的保护作用及作用机制。方法 设置对照组、单纯GCP高剂量（100 mg/kg）组、模型组和GCP低、高剂量（50、100 mg/kg）组。ip顺铂建立大鼠CI-AKI模型，给予GCP干预后，测定血清肌酐（serum creatinine，SCr）、血尿素氮（blood urea nitrogen，BUN）、炎症因子及肾组织还原性谷胱甘肽（glutathione，GSH）、丙二醛（malondialdehyde，MDA）、亚铁离子（Fe²⁺）水平；苏木素-伊红（hematoxylin-eosin staining，HE）和过碘酸雪夫（periodic acid-schiff，PAS）染色观察肾脏病理形态；TUNEL法观察肾组织细胞凋亡情况；Western blotting测定肾组织中肾损伤分子-1（kidney injury molecule-1，KIM-1）、中性粒细胞明胶酶相关脂质运载蛋白（neutrophil gelatinase-associated lipocalin，NGAL）、B淋巴细胞瘤-2（B-cell lymphoma-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）、剪切型半胱氨酸天冬氨酸蛋白酶-3（cleaved cysteine-asparate protease-3，cleaved Caspase-3）、Kelch样环氧氯丙烷相关蛋白1（Kelch-like ECH-associated protein 1，Keap1）、核因子E2相关因子2（nuclear factor E2 related factor 2，Nrf2）、还原型烟酰胺腺嘌呤二核苷酸磷酸醌脱氢酶1（nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1，NQO1）、血红素加氧酶-1（heme oxygenase-1，HO-1）、谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）、溶质载体家族7成员11（solute carrier family 7 member 11，SLC7A11）、酰基辅酶A合成酶长链家族成员4（acyl-CoA synthetase long-chain family member 4，ACSL4）、铁蛋白重链1（ferritin heavy chain 1，FTH1）、铁蛋白轻链（ferritin light chain，FTL）蛋白表达。顺铂处理HK-2细胞建立肾小管损伤模型，设置对照组、模型组、GCP（800 μg/mL）组、GCP＋Nrf2抑制剂ML385（2 μmol/L）组，并通过测定细胞活力、活性氧（reactive oxygen species，ROS）、Fe²⁺水平、Nrf2核转位情况以及Bcl-2、Bax、Nrf2、NQO1、HO-1、GPX4、SLC7A11、ACSL4蛋白表达，进一步明确Nrf2在GCP干预CI-AKI中的确切作用。结果 GCP可显著降低CI-AKI模型大鼠SCr、BUN水平（P＜0.001），减轻顺铂引起的肾脏病理损伤、恢复肾小管形态，降低KIM-1和NGAL的蛋白表达（P＜0.01、0.001）；降低CI-AKI大鼠血清中炎症因子水平（P＜0.01、0.001），抑制炎症反应；上调Bcl-2/Bax比值（P＜0.05），下调cleaved Caspase-3的蛋白表达（P＜0.01、0.001），减轻肾小管上皮细胞凋亡。GCP在体内外中能够诱导Keap1与Nrf2解离，促进Nrf2核转位（P＜0.01），上调下游蛋白HO-1、NQO1、GPX4、SLC7A11表达（P＜0.05、0.01、0.001），提高GSH活性同时下调ACSL4表达（P＜0.05、0.01），降低ROS、MDA和Fe²⁺含量（P＜0.05、0.01、0.001），改善CI-AKI模型大鼠脂质过氧化水平、降低铁沉积，抑制铁死亡，而ML385可逆转上述保护作用（P＜0.05、0.01、0.001）。结论 GCP能够改善CI-AKI，该保护作用可能通过激活Nrf2信号通路抑制肾脏细胞铁死亡发挥作用。

Objective To investigate the protective effect and mechanism of Glycyrrhiza polysaccharide (GCP) on cisplatin-induced acute kidney injury (CI-AKI) in model rats and human tubular epithelial cell HK-2 injury model. Methods Control group, GCP high-dose (100 mg/kg) alone group, model group, GCP low-and high-dose (50, 100 mg/kg) groups were set up. A rat CI-AKI model was established via intraperitoneal injection (ip) of cisplatin. After GCP intervention, levels of creatinine (SCr), blood urea nitrogen (BUN), inflammatory factors in serum, and glutathione (GSH), malondialdehyde (MDA), ferrous ion (Fe²⁺) in renal tissue were measured; Hematoxylin-eosin staining (HE) and periodic acid-Schiff (PAS) staining were used to observe the pathological morphology of kidneys; TUNEL method was used to observe the apoptosis of renal tissue cells; Western blotting was used to measure expressions of kidney injury molecule-1 (KIM-1), neutrophil gelatinase associated lipocalin (NGAL), B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cleaved cysteine aspartate protease-3 (cleaved Caspase-3), Kelch-like ECH-associated protein 1 (Keap1), nuclear factor E2 related factor 2 (Nrf2), nicotinamide adenine dinucleotide phosphate quinone dehydrogenase 1 (NQO1), heme oxygenase-1 (HO-1), glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), acyl-CoA synthase long-chain family member 4 (ACSL4), ferritin heavy chain 1 (FTH1) and ferritin light chain (FTL) protein in renal tissue. A renal tubular injury model was established by treating HK-2 cells with cisplatin. Control group, model group, GCP (800 μg/mL) group, and GCP + Nrf2 inhibitor ML385 (2 μmol/L) group were set up. Cell viability, reactive oxygen species (ROS), Fe²⁺ levels, Nrf2 nuclear translocation, and protein expressions of Bcl-2, Bax, Nrf2, NQO1, HO-1, GPX4, SLC7A11 and ACSL4 were measured to further clarify the exact effect of Nrf2 in GCP intervention in CI-AKI. Results GCP significantly reduced the levels of SCr and BUN in CI-AKI model rats (P < 0.001), alleviated cisplatin-induced renal pathological damage, restored renal tubular morphology, reduced the protein expressions of KIM-1 and NGAL (P < 0.01, 0.001), reduced the levels of inflammatory factors in serum of CI-AKI rats (P < 0.01, 0.001), inhibited the inflammatory response, up-regulated Bcl-2/Bax ratio (P < 0.05), down-regulated protein expression of cleaved Caspase-3 (P < 0.01, 0.001), alleviated apoptosis in renal tubular epithelial cells. GCP induced the dissociation of Keap1 and Nrf2 in vitro and in vivo, promoted Nrf2 nuclear translocation (P < 0.01), up-regulated the expressions of downstream proteins HO-1, NQO1, GPX4 and SLC7A11 (P < 0.05, 0.01, 0.001), increased GSH activity while down-regulated ACSL4 expression (P < 0.05, 0.01), reduced ROS, MDA and Fe²⁺ levels (P < 0.05, 0.01, 0.001), improved lipid peroxidation levels, reduced iron deposition, and inhibited ferroptosis in CI-AKI model rats, while ML385 could reverse the above protective effects (P < 0.05, 0.01, 0.001). Conclusion GCP could improve CI-AKI, and this protective effect may be achieved by activating the Nrf2 signaling pathway to inhibit ferroptosis in renal cells.

R285.5

国家自然科学基金项目（82374132）；山东省自然科学基金资助项目（ZR2023LZY001）