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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黄芪多糖对败血症诱导的急性肾脏损伤的保护作用。

目的探讨黄芪多糖（APS）对脓毒症所致急性肾损伤（AKI）的保护作用。方法：首先，通过向小鼠体内注射脂多糖（LPS），建立败血症诱导的AKI动物模型。小鼠连续3天经腹膜内注射1、3和5 mg /（kg·d）APS进行预处理。然后通过组织病理学分析对肾脏损伤的严重程度，血清尿素氮（BUN）和肌酐（SCr）的浓度以及肿瘤坏死因子α（TNF-α）和白介素1β（IL-1β）的水平进行评分。也被确定。在体外实验中，脂多糖（LPS）用来诱导HK-2细胞损伤，从而建立败血症诱导的AKI细胞模型，并采用细胞计数试剂盒8（CCK-8）方法测定APS的细胞毒性和实验浓度。然后，将细胞分为对照组，LPS和APS + LPS组。通过流式细胞术和酶联免疫吸附试验（ELISA）分别测定细胞凋亡和炎症相关的TNF-α，IL-1β，IL-6和IL-8。用显微镜观察细胞的形态变化，并通过伤口愈合法测定细胞的迁移能力。用RT-qPCR和Western blot法测定凋亡相关因子的mRNA和蛋白水平，包括caspase-3，caspase-9，Bax和Bcl-2。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）；以及上皮-间质转化（EMT-）相关的生物标记物，包括E-钙粘着蛋白，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可以以浓度依赖的方式逆转LPS引起的肾脏损害（P <0.05）。BUN和Scr的浓度均升高（均P <0.05）；同样，TNF-α和IL-1β的水平也增加了（所有P <0.05）。在体外实验中，结果显示LPS可以显着引起HK-2细胞损伤并诱导凋亡，炎症，ERS和EMT。当APS浓度在0-200μg/ mL范围内时，它对HK-2细胞没有细胞毒性，而100μg/ mL APS预处理可以显着减轻LPS诱导的细胞活性降低（P <0.05）。与LPS组相比，APS预处理可抑制TNF-α，IL-1β，IL-6和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），从而抑制APS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，并抑制EMT进程。结论APS可通过调节炎症，凋亡，ERS和EMT减轻败血症诱导的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，并抑制EMT进程。结论APS可通过调节炎症，凋亡，ERS和EMT减轻败血症诱导的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，并抑制EMT进程。结论APS可通过调节炎症，凋亡，ERS和EMT减轻败血症诱导的AKI。