Abstract
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.
Highlights
Recent studies have linked inflammation to the development of a number of important diseases [1,2,3,4]
We evaluated the expression of IL-6 to verify whether Astragalus membranaceus (AM) injection could play an anti-inflammatory role in LPS-stimulated macrophages and investigated the effects and mechanisms exerted by LPS on autophagy and whether autophagy was involved in regulating the production of the IL-6 induced by LPS
The level of IL-6 in the cell culture supernatant was measured by ELISA, while the protein level of IL-6 in cells was detected by Western blotting
Summary
Recent studies have linked inflammation to the development of a number of important diseases [1,2,3,4]. Inflammation can be induced by various stimuli, such as damage, pathogens, and their products, and is often accompanied by immune disorders [5,6,7]. It has been shown that antiinflammatory treatment alone might not be usually effective [8]. It is necessary to develop novel anti-inflammatory drugs with high security, which could protect the body from the pathological damage induced by inflammation. Some original and review papers have reported the anti-inflammatory effects of medicinal plants and isolated natural products and proposed their
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