Supplementary Material for: Iridoids with Genipin Stem Nucleus Inhibit Lipopolysaccharide-Induced Inflammation and Oxidative Stress by Blocking the NF-κB Pathway in Polycystic Ovary Syndrome

<b><i>Background/Aims:</i></b> Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women, and it is usually characterized by chronic inflammation, oxidative stress, and altered microRNA expression. The aim of this study is to investigate how the effects of iridoids with genipin stem nucleus inhibit PCOS complications. The interactions between iridoids were investigated, as well. <b><i>Methods:</i></b> The chronic inflammation cell model was induced using lipopolysaccharide (LPS) in the RAW 264.7 and KGN cell lines. Levels of mRNA and protein expression were quantified using real time-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. The target of the iridoids was identified using the drug affinity responsive target stability (DARTS) method. The ability to scavenge free radicals was evaluated using the DPPH radical scavenging method and the ultra oxygen anion (O<sub>2</sub><sup>-</sup>) radical scavenging method. <b><i>Results:</i></b> The cells recovered from the inflammatory conditions and showed significantly decreased levels of interleukins after treatment with iridoids. The iridoids were demonstrated to target NF-κB, inhibit the phosphorylation and degradation of IκB, inhibit the nuclear entry of NF-κB, and inhibit the expression of inflammatory factors. Though only genipin showed an efficient ability to scavenge O<sub>2</sub><sup>-</sup>, the iridoids, IκB inhibitor (BAY 11-7085), and NF-κB inhibitor (PDTC) could inhibit LPS-induced oxidative stress on the cells, indicating that the iridoids exert their anti-oxidant effects via the NF-κB pathway. The expression levels of microRNAs (miRNAs) were also altered by LPS, but the iridoids could scarcely rescue the abnormal condition. <b><i>Conclusion:</i></b> Chronic inflammation may be an important incentive for oxidative stress and abnormal microRNA expression in PCOS, and iridoids can protect patients from inflammatory damage by regulating the NF-κB pathway.