Water-separated part of Chloranthus serratus alleviates lipopolysaccharide- induced RAW264.7 cell injury mainly by regulating the MAPK and Nrf2/HO-1 inflammatory pathways

Chloranthus serratus (Chloranthaceae) has been used to treat bruises, rheumatoid and bone pain. However, the anti-inflammatory mechanisms of C. serratus in vitro have not been fully elucidated. The present study aimed to explore the anti-inflammatory activity and potential mechanisms of C. serratus's separated part of water (CSSPW) in lipopolysaccharide (LPS)-induced RAW264.7 cells.

CSSPW exerts its anti-inflammatory effect via downregulating the production of pro-inflammatory mediators, inhibiting the activation of NF-κB and MAPK pathways, as well as activating Nrf2/HO-1 pathway in LPS-induced RAW264.7 cells.

The concentrations of CSSPW were optimized by CCK-8 method. Nitric oxide (NO) content was detected by one-step method. The levels of inflammatory cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Gene expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was detected by real-time quantitative PCR (qPCR). Immunofluorescence and DCFH-DA fluorescent probes were used to detect p65 nuclear translocation and reactive oxygen species (ROS) content, respectively. Western blotting was used to assay the protein expression of mitogen-activated protein kinases (MAPK), nuclear factor-kappa B (NF-κB) and nuclear transcription factor E2 related factor 2/haem oxygenase-1 (Nrf2/HO-1) pathways.

The final concentrations of 15 ng/mL, 1.5 μg/mL and 150 μg/mL were selected as low, medium and high doses of CSSPW, respectively. CSSPW treatment significantly reduced the generation of NO, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), prostaglandinE2 (PGE2), iNOS mRNA and COX-2 mRNA in response to LPS stimulation. Furthermore, the protein expression of the MAPK and NF-κB pathways was suppressed by CSSPW treatment, as well as p65 nuclear translocation and ROS production. In contrast, the protein expression of the Nrf2/HO-1 pathway was markedly upregulated. Conclusions: CSSPW exerts its anti-inflammatory effect via downregulating the production of pro-inflammatory mediators, inhibiting the activation of NF-κB and MAPK pathways, as well as activating Nrf2/HO-1 pathway in LPS-induced RAW264.7 cells.