目的 探讨原儿茶醛（protocatechuic aldehyde，PCA）对环磷酰胺（cyclophosphamide，CPA）所致急性肝损伤的预防作用及其机制。方法 分别以PCA（15、30 mg/kg）和甘草酸二铵（diammonium glycyrrhizinate，DG，67.5 mg/kg）ig小鼠，1次/d，连续14 d后，ip CPA（200 mg/kg）建立急性肝损伤小鼠模型，第2天收集小鼠血清和肝脏组织；采用生化法检测小鼠血清中丙氨酸氨基转移酶（alanine aminotransferase，ALT）和天冬氨酸氨基转移酶（aspartate aminotransferase，AST）水平以及肝脏组织匀浆中超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalase，CAT）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）和丙二醛（malondialdehyde，MDA）水平；采用苏木素-伊红（hematoxylin eosin，HE）染色观察各组小鼠肝组织病理变化；TUNEL染色检测小鼠肝脏细胞凋亡情况；采用超氧阴离子荧光探针检测小鼠肝细胞内活性氧（reactive oxygen species，ROS）的表达情况；采用免疫组织化学染色检测小鼠肝细胞中核因子-E2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）的易位情况；采用蛋白质免疫印迹法检测小鼠肝组织凋亡淋巴细胞瘤-2（B cell lymphoma 2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2-associated X protein，Bax）和半胱氨酸天冬氨酸蛋白酶-3（cysteine containing cysteinyl aspartate-3，Caspase-3）的表达，及氧化应激相关蛋白Kelch样ECH关联蛋白1（Kelch-like ECH-associated protein 1，Keap1）、Nrf2、血红素加氧酶（heme oxygenase，HO-1）、谷氨酸-半胱氨酸连接酶催化亚基（glutamate - cysteine ligase catalytic subunit，GCLC）、谷氨酸-半胱氨酸连接酶调节亚基（glutamate-cysteine ligase modifier subunit，GCLM）、醌氧化还原酶1 [NAD(P)H quinone oxidoreductase 1，NQO1]的表达情况。结果 PCA可以增加肝脏指数，降低血清ALT和AST水平，减少肝组织的细胞间隙和细胞肿胀，呈剂量相关性地减轻CPA诱导的肝损伤（P＜0.05、0.01、0.001）；PCA能明显降低肝组织TUNEL荧光染色强度（P＜0.005）。此外，蛋白质免疫印迹结果显示，PCA可以增加Bcl2的表达（P＜0.001），降低cleaved-Caspase3和Bax的表达（P＜0.05、0.001），表明PCA可以有效抑制肝细胞凋亡。氧化应激指标测定结果显示，PCA提高了肝脏SOD、CAT和GSH-PX酶活性（P＜0.05、0.01、0.001），降低了ROS和MDA水平（P＜0.01、0.001）。此外，PCA还能显著降低Keap1的表达（P＜0.01、0.001），引起Nrf2的核易位，并增加Nrf2及下游抗氧化蛋白的表达（P＜0.05、0.01、0.001）。结论 PCA可以预防CPA所致急性肝损伤，其作用机制可能与抑制氧化应激和细胞凋亡有关。

Objective To explore the prophylactic effect and mechanism of protocatechuic aldehyde (PCA) against cyclophosphamide (CPA)-induced acute liver injury. Methods Mice were orally treated with PCA at dosages of 15, 30 mg/kg or diammonium glycyrrhizinate (DG) at 67.5 mg/kg once daily for 14 days, followed by a single intraperitoneal injection of CPA at 200 mg/kg to establish an acute liver injury model. The serum and liver tissue were collected on the next day. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice serum and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in liver tissue homogenate were detected by biochemical kit. Hematoxylin eosin (HE) staining was used to observe the pathological changes of liver tissue. The apoptosis of liver cells was detected by dUTP nickel end-labeled fluorescent staining (TUNEL) mediated by terminal deoxynucleotide transferase. The expression of reactive oxygen species (ROS) in hepatocytes was detected by superoxide anion fluorescence staining. Immunohistochemical staining was used to detect the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in mouse hepatocytes. Western blotting was adopted to detect the protein expression of B cell lymphocyte 2 (Bcl-2), Bcl-2-associated X protein (Bax), cysteine containing cysteinyl aspartate-3 (Caspase-3), Kelch-like ECH-associated protein 1 (Keap1), Nrf2, heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM) and NAD(P)H quinone oxidoreductase (NQO1). Results PCA could increase liver index, decrease serum ALT and AST levels, reduce cell space and cell swelling in liver tissue, and alleviate CPA-induced liver injury in a dose-dependent manner (P < 0.05, 0.01, 0.001). PCA significantly decreased the intensity of TUNEL fluorescence staining (P < 0.001). In addition, western blotting results showed that PCA could increase the expression of Bcl-2 (P < 0.001) and decrease the expression of cleaved Caspase-3 and Bax (P < 0.05, 0.001), indicating that PCA could effectively inhibit hepatocyte apoptosis. The results of oxidative stress index showed that PCA increased the levels of SOD, CAT and GSH-Px (P < 0.05, 0.01, 0.001), and decreased the levels of ROS and MDA (P < 0.01, 0.001). It is worth noting that PCA could decreased Keap1 expression (P < 0.01, 0.001), induce nuclear translocation of Nrf2 and increase the expression of Nrf2 and downstream antioxidant proteins (P < 0.05, 0.01, 0.001). Conclusion PCA could prevent CPA-induced acute liver injury, which might be attributed to inhibiting oxidative stress and apoptosis.

R285.5

河北省自然科学基金中医药联合基金重点项目（H2023209038）;河北省自然科学基金中医药联合基金重点项目（H2022209087）;河北省高等学校科学技术研究项目（ZD2022141）