Jing-Fang n-butanol extract and its isolated JFNE-C inhibit ferroptosis and inflammation in LPS induced RAW264.7 macrophages via STAT3/p53/SLC7A11 signaling pathway

Abstract

Ethnopharmacological relevanceTraditional Chinese medicine has accumulated valuable experience in the treatment of inflammatory diseases caused by Ferroptosis. Jing Jie and Fang Feng are two warm acrid exterior-resolving medicinal herbs that play an important role in the prevention and treatment of inflammatory diseases. The pairing of the two forms a drug pair (Jing-Fang) that shows significant advantages in fighting oxidative stress and inflammation. Whereas, the underlying mechanism needs to be further improved. Aim of the studyIn this study, the anti-inflammatory effect of Jing-Fang n-butanol extract (JFNE) and its isolate C (JFNE-C) on LPS-induced RAW264.7 cells and the regulation effect on ferroptosis were investigated, and also the mechanism of STAT3/p53/SLC7A11 signal pathway-related to ferroptosis. Materials and methodsJing-Fang n-butanol extract (JFNE) and its active isolate (JFNE-C) were extracted and isolated. LPS-induced inflammation model in RAW264.7 cells was established to assess the anti-inflammatory effect and ferroptosis mechanism of JFNE and JFNE-C. The levels of interleukin 6 (IL-6), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were measured. The activity levels of antioxidant substances such as glutathione (GSH), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were measured. Flow cytometry, immunofluorescence and transmission electron microscopy were used to assess ROS level, ferrous iron content and mitochondrial morphological changes. Through administration of Ferrostatin-1 (Fer-1), an ferroptosis inhibitor, to verify the role of JFNE and JFNE-C in regulating ferroptosis in resistance to the inflammatory response. Western blotting was used to determine whether the JFNE and JFNE-C exerted effectiveness by modulating the STAT3/p53/SLC7A11 signaling pathway. In addition, the important role of STAT3/p53/SLC7A11 signaling pathway in drug regulation of ferroptosis and inflammatory response was further validated by administration of S3I-201 (STAT3 inhibitor). Finally, high performance liquid chromatography-mass spectrometry (HPLC-MS) was used to determine the major active components of JFNE and JFNE-C. ResultsThe results showed that treated with JFNE-C significantly reduced the contents of interleukin 6 (IL-6), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in the supernatant of LPS-induced RAW264.7 cells. The pretreatment with JFNE and JFNE-C significantly decreased intracellular oxidative stress levels, including reductions of ROS and MDA levels, and increases of GSH-Px, SOD and GSH levels. In addition, JFNE and JFNE-C obviously reduced intracellular ferrous iron level, and JFNE-C was effective in alleviating mitochondrial damage which includes mitochondrial shrinkage, increase of mitochondrial membrane density and reduction and absence of cristae. Further results indicated that JFNE-C showed a reduction of p53 and p-p53 protein levels in LPS-induced RAW264.7 cells, while significantly increasing the protein expression levels of STAT3, p-STAT3, SLC7A11 and GPX4. Besides, JFNE-C contains key active substances such as 5-O-Methylvisammioside, Hesperidin and Luteolin. Remarkably, this is different from JFNE, which is rich in nutrients such as sucrose, choline and various amino acids. ConclusionThese results suggest that JFNE and JFNE-C may exert anti-inflammatory effect through activating the STAT3/p53/SLC7A11 signaling pathway to inhibit ferroptosis.