Phochinenin I, a Bioactive Constituent from <i>Pholidota chinensis</i>, Attenuates Inflammatory Response and Oxidative Stress In Vitro and In Vivo

A bioactive constituent of <i>P. chinensis</i> Phochinenin I (PI), a dihydrophenanthrene-type monomer, has been isolated from the pseudobulb for more than 15 years, but its activity has not been reported. This study discovered its protective effects on the inflammatory response and oxidative stress for the very first time. The LPS/IFN-γ-induced RAW264.7 model and H₂O₂-induced zebrafish model were used to evaluate the regulatory effects of PI on oxidative stress, and RNA sequencing analysis was performed to explore its possible molecular mechanism. First, PI alleviated the pro-inflammatory response by decreasing tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels and reduced nitric oxide (NO) secretion by inhibiting inducible nitric oxide synthase (iNOS) expression. Next, PI might reduce reactive oxygen species (ROS) production by enhancing nuclear factor erythroid 2-related factor 2 (Nrf2) expression. Western blotting results showed that PI alleviated the cellular inflammatory response mainly by suppressing phosphorylation of signal transducer and activator of transcription 3 (STAT3) and downregulating c-Myc expression, accompanied by reducing autophagy markers LC3B and SQSTM1/p62 accumulation. c-Myc is a target connected with many anti-inflammatory and antioxidant pathways. RNA sequencing data highlighted phagosome pathway regulation and SQSTM1/p62-associated gene networks as key mediators of PI's immunomodulatory effects. In vivo, using H₂O₂-challenged zebrafish embryos, PI conferred significant protection against oxidative injury, manifested by ROS scavenging along with antioxidant enhancement, which included elevated superoxide dismutase (SOD) activity and upregulated expression of Mn-SOD, Cu/Zn-SOD, and NAD-(P)-H:quinone oxidoreductase 1 (NQO1) genes. PI can be used as a pharmaceutical chemistry derivative of anti-inflammatory lead compounds and as a new candidate for future adjuvant therapy.