Water extract of medicinal ink (WEMI) attenuates lipopolysaccharide-induced NO production of Raw264.7 cells via downregulating JAK2/STAT3-mediated iNOS expression

Medicinal ink is used as a traditional topical medicine for treating inflammatory diseases via detoxification, relieving pain, hemostasis, and reducing swelling. However, the effect of medicinal ink on the inhibition of inflammatory responses and the underlying molecular mechanism remain unclear.The present study aimed to investigate the anti-inflammatory function of water extract of medical ink (WEMI) and elucidate its active mechanisms.Cell viability was assessed using crystal violet staining assay. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected by ELISA. Nitric oxide (NO) production was measured by Griess assay. The activation of inflammatory signaling molecules stimulated by lipopolysaccharide (LPS) was evaluated by assessing levels of inducible nitric oxide synthase (iNOS), phosphorylated Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) using Western blot assay.Water extract of medical ink (WEMI) did not present cytotoxic effect on murine macrophage Raw264.7 cells. High dosage of WEMI slightly rescued LPS-suppressed cell viability of Raw264.7 cells. WEMI did not induce NO production or IL-6 secretion, though WEMI significantly induced secretion of TNF-α on Raw264.7 cells not stimulated with LPS. On the other hand, LPS effectively stimulated inflammation on Raw264.7 cells; however, WEMI dramatically reduced LPS-induced NO production. WEMI alleviated LPS-stimulated IL-6 secretion but did not affect the content of TNF-α. In addition, WEMI effectively reduced expression of iNOS by abolishing LPS-mediated phosphorylation of JAK2 and STAT3 but not TLR4-mediated NF-κB and MAPK molecules.Our findings suggest that WEMI targets of the JAK2/STAT3-mediated iNOS expression play a key role in alleviating LPS-induced inflammatory responses in RAW264.7 macrophages. Therefore, medicinal ink may be a potential topical agent for treating fasciitis or synovitis via regulating the immune system.