目的 探讨黄芪甲苷IV通过调控肿瘤进展位点2（tumor progression locus 2，TPL2）-结缔组织生长因子（connective tissue growth factor，CTGF）信号通路减轻肾缺血再灌注损伤（ischemia-reperfusion injury，IRI）后的纤维化损伤，并阐明其对急性肾损伤（acute kidney injury，AKI）向慢性肾脏病（chronic kidney disease，CKD）进展的干预作用。方法 采用单侧肾动脉夹闭法建立小鼠IRI模型，设置假手术组、模型组和黄芪甲苷IV低、高剂量（40、80 mg/kg）组。分别于术后1、28 d取材，考察黄芪甲苷IV对肾缺血再灌注急性期和肾缺血再灌注后纤维化损伤小鼠的影响。通过检测血清肌酐（serum creatinine，Scr）和血尿素氮（blood urea nitrogen，BUN）评估肾功能；采用苏木素-伊红（hematoxylin-eosin staining，HE）染色、过碘酸雪夫（periodic acid-schiff，PAS）染色、Masson染色、天狼星红（Sirius red）染色观察肾脏组织病理损伤及纤维化程度。建立转化生长因子-β1（transforming growth factor-β1，TGF-β1）诱导的人肾皮质近曲小管上皮细胞（human kidney-2，HK-2）体外损伤模型，采用TPL2的小干扰RNA（small interfering RNA，siRNA）转染HK-2细胞进行分子机制研究；采用Western blotting检测小鼠肾脏和HK-2细胞纤维化标志物[α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、I型胶原蛋白（collagen I，COL1）、TGF-β1]及TPL2、CTGF和下游炎症因子肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）的蛋白表达。结果 在IRI后的肾脏急性损伤期，黄芪甲苷IV显著降低Scr和BUN水平（P＜0.05、0.01、0.001），缓解肾小管扩张及刷状缘脱落。在肾脏纤维化进展期，黄芪甲苷IV持续抑制Scr和BUN的升高（P＜0.05、0.01、0.001），同时减少肾间质胶原纤维沉积（P＜0.001）。Western blotting结果显示，黄芪甲苷IV呈剂量相关性地下调TPL2蛋白表达及其下游效应分子CTGF表达水平（P＜0.001），并显著抑制肾间质纤维化相关蛋白的表达水平（P＜0.05、0.01、0.001）。体外实验结果显示，加入黄芪甲苷IV后，TGF-β1诱导的TPL2、CTGF、α-SMA以及TNF-α高表达水平均被显著抑制（P＜0.01、0.001）；转染si-TPL2，继续给予黄芪甲苷IV处理后TPL2、CTGF、α-SMA、TNF-α表达水平较黄芪甲苷IV单独处理组无显著差异。结论 黄芪甲苷IV通过抑制TPL2-CTGF通路，减轻IRI后肾炎症反应与纤维化进程，阻断AKI向CKD转化，为临床防治缺血性肾损伤提供潜在治疗策略。

Objective To investigate astragaloside Ⅳ (AS-Ⅳ) alleviates renal fibrosis following ischemia-reperfusion injury (IRI) by modulating tumor progression locus 2 (TPL2)-connective tissue growth factor (CTGF) signaling pathway and elucidate its intervention in preventing the progression from acute kidney injury (AKI) to chronic kidney disease (CKD).Methods A IRI mice model was established via unilateral renal artery clamping. Sham group, model group, AS-Ⅳ low- and high-dose (40, 80 mg/kg) groups were set up. Samples were collected on postoperative days 1 and 28 to investigate the effect of AS-Ⅳ on acute renal ischemia-reperfusion injury and fibrosis in mice after renal ischemia-reperfusion injury. Renal function was assessed by measuring serum creatinine (Scr) and blood urea nitrogen (BUN). Renal histopathological injury and fibrosis were evaluated using hematoxylin-eosin (HE) staining, periodic acid-schiff (PAS) staining, Masson staining and Sirius red staining. An in vitro injury model was established using human kidney-2 (HK-2) cells induced by transforming growth factor-β1 (TGF-β1), and molecular mechanisms were explored by transfecting HK-2 cells with small interfering RNA (siRNA) targeting TPL2. Western blotting was used to detect the protein expression levels of fibrosis markers including α-smooth muscle actin (α-SMA), collagen I (COL1), TGF-β1, as well as TPL2, CTGF, and the downstream inflammatory factor tumor necrosis factor-α (TNF-α) in kidney of mice and HK-2 cells.Results In the acute phase of renal injury post-IRI, AS-Ⅳ significantly reduced Scr and BUN levels (P < 0.001), alleviated tubular dilation and brush border loss. During the fibrotic progression phase, AS-Ⅳ persistently suppressed the elevation of Scr and BUN (P < 0.05, 0.01, 0.001), while reduced collagen fiber deposition in the renal interstitium (P < 0.001). Western blotting results showed that AS-Ⅳ dose-dependently down-regulated the protein expressions of TPL2 and its downstream effector CTGF (P < 0.001), and significantly inhibited the expression levels of renal interstitial fibrosis related proteins (P < 0.05, 0.01, 0.001). In vitro experiments results showed that AS-Ⅳ markedly inhibited the TGF-β1-induced overexpressions of TPL2, CTGF, α-SMA and TNF-α (P < 0.01, 0.001). After transfection with si-TPL2 and continued treatment with AS-Ⅳ, there was no significant difference in the expression levels of TPL2, CTGF, α-SMA and TNF-α compared to AS-Ⅳ treated group alone.Conclusion AS-Ⅳ attenuates post-IRI renal inflammation and fibrosis by inhibiting the TPL2-CTGF pathway, thereby preventing AKI-to-CKD progression, which provide a potential therapeutic strategy for the clinical management of ischemic kidney injury.

R285.5

国家自然科学基金青年项目（82304809）