Morus alba L. (Sangzhi) alkaloids (SZ-A) exert anti-inflammatory effects via regulation of MAPK signaling in macrophages

Ethnopharmacological relevance: Morus alba L. (Sangzhi) alkaloids (SZ-A) tablets have been approved by the China National Medical Products Administration for T2DM treatment. Our previous study (Liu et al., 2021) revealed that SZ-A protected against diabetes and inflammation in KKAy mice. However, the mechanism and components in SZ-A exerting anti-inflammatory effects are unclear. Aim of the study: Investigate the effects and molecular mechanisms of SZ-A on inflammation, and identify antiinflammatory active components in SZ-A. Materials and methods: The major ingredients in SZ-A were analyzed by HPLC and sulfuric acid - anthrone spectrophotometry. The inhibitory activities of SZ-A on lipopolysaccharide (LPS)-stimulated inflammation were determined in bone marrow-derived macrophage (BMDM) and RAW264.7 cells. The cytokine levels of IL-6 and TNF-alpha in cell culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Gene expression levels of IL-6 and TNF-alpha were detected by qRT-PCR. The levels of protein phosphorylation of p38 MAPK, ERK, and JNK were analyzed by Western blot. Results: The main components in SZ-A were found to be 1-deoxynojirimycin (DNJ), 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), fagomine (FAG), polysaccharide (APS), and arginine (ARG). SZ-A reduced the levels of IL-6 and TNF-alpha secreted by LPS-induced RAW264.7 and BMDM cells. Simultaneously, the mRNA expression levels of IL-6 and TNF-alpha were all significantly suppressed by SZ-A in a concentration-dependent manner. Furthermore, SZ-A inhibited the phosphorylation of p38 MAPK, ERK, and JNK in BMDM and the activation of ERK and JNK signaling in RAW264.7 cells. We also observed that DNJ, DAB, FAG, and ARG markedly downregulated IL-6 and TNF-alpha cytokine levels, while APS did not have an obvious effect. Conclusions: SZ-A attenuates inflammation at least partly by blocking the activation of p38 MAPK, ERK, and JNK signaling pathways. DNJ, FAG, DAB, and ARG are the main constituents in SZ-A that exert anti-inflammatory effects.