Astragalus membranaceus (AM), used in traditional Chinese medicine, has been shown to enhance immune functions, and recently, its anti-inflammatory effects were identified. However, the mechanisms of action remain unclear. Most studies have shown that autophagy might be involved in the immune response of the body, including inflammation. Here, we developed an inflammatory model by stimulating macrophages with lipopolysaccharides (LPS) to explore the anti-inflammatory effect and mechanisms of AM injection from the perspective of the regulation of autophagy. Immunoblot, immunofluorescence, and ELISA were used to determine the effects of AM injection on the production of interleukin-6 (IL-6) and alterations of autophagy markers. It was found that AM injection reduced the expression of IL-6 in LPS-stimulated macrophages and reversed the LPS-induced inhibition of cellular autophagy. After treatment with inhibitors of signaling pathways, it was shown that LPS downregulated autophagy and upregulated the production of IL-6 in macrophages via the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. AM injection reversed the effects of LPS by activating the AMP-activated protein kinase (AMPK) instead of inhibiting Akt. These results were further confirmed by testing activators and siRNA silencing of AMPK. Hence, these 2 distinct signaling molecules appear to exert opposite effects on mTOR, which integrates information from multiple upstream signaling pathways, negatively regulating autophagy. In addition, we demonstrated that autophagy might play a key role in regulating the production of IL-6 by testing activators and inhibitors of autophagy and siRNA silencing of ATG5. These findings showed that AM injection might enhance autophagy by activating AMPK and might further play a repressive effect on the LPS-stimulated expression of IL-6. This study explored the relationship between autophagy, signaling pathways, and the production of inflammatory factors in a model of endotoxin infection and treatment with AM injection.

中文翻译：

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黄芪注射液通过脂多糖刺激的巨噬细胞中通过AMPK-mTOR途径激活自噬而抑制白细胞介素6的产生。

黄芪已经证明，在传统中药中使用的（AM）可以增强免疫功能，最近，它的抗炎作用得到了确认。但是，作用机理仍不清楚。大多数研究表明自噬可能与机体的免疫反应有关，包括炎症。在这里，我们通过用脂多糖（LPS）刺激巨噬细胞建立了炎症模型，以从自噬调节的角度探讨AM注射剂的抗炎作用和机制。免疫印迹，免疫荧光和ELISA用于确定AM注射对白介素6（IL-6）产生和自噬标记物改变的影响。发现AM注射降低了LPS刺激的巨噬细胞中IL-6的表达，并逆转了LPS诱导的对细胞自噬的抑制。用信号传导途径的抑制剂治疗后，研究表明，LPS通过蛋白激酶B（Akt）/哺乳动物雷帕霉素靶标（mTOR）途径下调了巨噬细胞的自噬，并上调了IL-6的产生。AM注射通过激活AMP激活的蛋白激酶（AMPK）而不是抑制Akt来逆转LPS的作用。通过测试激活剂和AMPK的siRNA沉默进一步证实了这些结果。因此，这两个不同的信号分子似乎对mTOR发挥相反的作用，mTOR整合了来自多个上游信号通路的信息，从而负调控自噬。此外，ATG5。这些发现表明，AM注射可能通过激活AMPK增强自噬，并可能进一步抑制LPS刺激的IL-6表达。这项研究探讨了内毒素感染和AM注射治疗模型中自噬，信号通路和炎症因子产生之间的关系。