OBJECTIVE To explore the protective roles of Astragalus polysaccharide (APS) on acute renal injury (AKI) induced by sepsis. METHODS Firstly, an animal model of sepsis-induced AKI was established by injecting lipopolysaccharide (LPS) into mice. The mice were pretreated with an intraperitoneal injection of 1, 3, and 5 mg/(kg·d) APS for 3 consecutive days. The severity of kidney injury was then scored by histopathological analysis, and the concentrations of serum urea nitrogen (BUN) and serum creatinine (SCr) and the levels of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) were determined as well. In in vitro experiments, lipopolysaccharide (LPS) was used to induce HK-2 cell injury to establish a sepsis-induced AKI cell model, and the cell counting kit-8 (CCK-8) method was performed to determine the cytotoxicity and appropriate experimental concentration of APS. Then, cells were divided into the control, LPS, and APS+LPS groups. Cell apoptosis and inflammation-related TNF-α, IL-1β, IL-6, and IL-8 were determined by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. The microscope was used to observe the morphological changes of cells, and the cell migration ability was measured by wound healing assay. RT-qPCR and Western blot assay were used to determine the mRNA and protein levels of apoptosis-related factors including caspase-3, caspase-9, Bax, and Bcl-2; endoplasmic reticulum stress- (ERS-) related biomarkers including C/EBP homologous protein (CHOP) and glucose-regulated protein78 (GRP78); and epithelial-mesenchymal transition- (EMT-) related biomarkers including E-cadherin, Snail, α-smooth muscle actin (α-SMΑ), and Vimentin. RESULTS In vivo experiments in mice showed that APS can reverse LPS-induced kidney damage in a concentration-dependent manner (P < 0.05); the concentrations of BUN and Scr were increased (all P < 0.05); similarly, the levels of TNF-α and IL-1β were increased as well (all P < 0.05). In in vitro experiments, the results showed that LPS can significantly cause HK-2 cell damage and induce apoptosis, inflammation, ERS, and EMT. When APS concentration was in the range of 0-200 μg/mL, it had no cytotoxicity in HK-2 cells, and 100 μg/mL APS pretreatment could significantly mitigate the decrease of cell activity induced by LPS (P < 0.05). Compared with the LPS group, APS pretreatment could inhibit the expression of inflammatory factors including TNF-α, IL-1 β, IL-6, and IL-8 (all P < 0.05), reducing the number of apoptotic cells (P < 0.05), suppressing the expression of caspase-3, caspase-9, and Bax, but upregulating the expression levels of Bcl-2. In ERS, APS pretreatment inhibited LPS-induced upregulation of CHOP and GRP78. Moreover, in EMT, APS pretreatment could inhibit the morphological changes of cells, downregulate the migration, decrease the expression of EMT biomarkers, and inhibit the process of EMT. CONCLUSION APS could alleviate sepsis-induced AKI by regulating inflammation, apoptosis, ERS, and EMT.

中文翻译：

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黄芪多糖对脓毒症急性肾损伤的保护作用。

目的探讨黄芪多糖（APS）对脓毒症急性肾损伤（AKI）的保护作用。方法首先通过给小鼠注射脂多糖（LPS）建立脓毒症致AKI动物模型。小鼠腹腔注射 1、3 和 5 mg/(kg·d) APS 连续 3 天进行预处理。然后通过组织病理学分析对肾损伤的严重程度进行评分，血清尿素氮（BUN）和血清肌酐（SCr）的浓度以及肿瘤坏死因子α（TNF-α）和白细胞介素-1β（IL-1β）的水平也被确定了。在体外实验中，利用脂多糖（LPS）诱导HK-2细胞损伤建立脓毒症诱导的AKI细胞模型，并采用细胞计数试剂盒8（CCK-8）法测定APS的细胞毒性和适宜的实验浓度。然后，将细胞分为对照、LPS和APS+LPS组。细胞凋亡和炎症相关的 TNF-α、IL-1β、IL-6 和 IL-8 分别通过流式细胞术和酶联免疫吸附试验 (ELISA) 测定。用显微镜观察细胞形态变化，用创面愈合试验测定细胞迁移能力。采用RT-qPCR和Western blot法检测细胞凋亡相关因子caspase-3、caspase-9、Bax、Bcl-2的mRNA和蛋白水平；内质网应激 (ERS-) 相关生物标志物，包括 C/EBP 同源蛋白 (CHOP) 和葡萄糖调节蛋白78 (GRP78)；和上皮间质转化 (EMT-) 相关的生物标志物，包括 E-钙粘蛋白、Snail、α-平滑肌肌动蛋白 (α-SMΑ) 和波形蛋白。结果小鼠体内实验表明，APS能够以浓度依赖性方式逆转LPS引起的肾损伤（P < 0.05）；BUN、Scr浓度升高（均P<0.05）；同样，TNF-α和IL-1β的水平也增加（均P <0.05）。在体外实验中，结果表明LPS可显着引起HK-2细胞损伤并诱导细胞凋亡、炎症、ERS和EMT。当APS浓度在0～200 μg/mL范围内时，对HK-2细胞无细胞毒性，100 μg/mL APS预处理可显着减轻LPS诱导的细胞活性下降（P < 0.05）。与 LPS 组相比，APS预处理可抑制TNF-α、IL-1β、IL-6、IL-8等炎性因子的表达（均P < 0.05），减少凋亡细胞的数量（P < 0.05），抑制炎症因子的表达。 caspase-3、caspase-9 和 Bax，但上调 Bcl-2 的表达水平。在 ERS ​​中，APS 预处理抑制 LPS 诱导的 CHOP 和 GRP78 上调。此外，在 EMT 中，APS 预处理可以抑制细胞的形态变化，下调迁移，降低 EMT 生物标志物的表达，抑制 EMT 的进程。结论 APS可通过调节炎症、细胞凋亡、ERS和EMT来缓解脓毒症引起的AKI。和 Bax，但上调 Bcl-2 的表达水平。在 ERS ​​中，APS 预处理抑制 LPS 诱导的 CHOP 和 GRP78 上调。此外，在 EMT 中，APS 预处理可以抑制细胞的形态变化，下调迁移，降低 EMT 生物标志物的表达，抑制 EMT 的进程。结论 APS可通过调节炎症、细胞凋亡、ERS和EMT来缓解脓毒症引起的AKI。和 Bax，但上调 Bcl-2 的表达水平。在 ERS ​​中，APS 预处理抑制 LPS 诱导的 CHOP 和 GRP78 上调。此外，在 EMT 中，APS 预处理可以抑制细胞的形态变化，下调迁移，降低 EMT 生物标志物的表达，抑制 EMT 的进程。结论 APS可通过调节炎症、细胞凋亡、ERS和EMT来缓解脓毒症引起的AKI。