Soufeng Sanjie formula alleviates the progression of lupus and joint injury by regulating the ALKBH5-FoxO1-PFKFB3 axis in M-MDSCs

Systemic lupus erythematosus (SLE) cases present with impaired immune function and injured organs, with joint injury being one of the most common complications. Soufeng Sanjie formula (SF) is a traditional Chinese medicine (TCM) that alleviates rheumatoid arthritis and has a significant regulatory effect on T cells. Recently, myeloid-derived suppressor cells (MDSCs) have been considered an essential factor contributing to SLE pathogenesis, as they can mediate the abnormal amplification of Th17 cells. However, it remains unclear whether SF targets MDSCs to alleviate SLE and joint injury. We aim to examine SF for therapeutic effects in lupus mice and the potential molecular mechanisms. We developed an IMQ-induced lupus mouse model for assessing the high and low doses of SF for their effects. The manifestations of joint injury were also examined. Changes in immune cell populations were analyzed by flow cytometry and in vitro co-culture experiments. The key targets and active components of the SF were identified through network pharmacological analysis. Moreover, SF-containing serum was prepared to stimulate TLR7 against R848-induced-MDSCs in vitro. We also developed a pristane-induced lupus model in myeloid FoxO1-deficient mice. ECAR and OCR detection, measurements of glucose and lactic acid levels, luciferase reporter gene assays and ChIP-qPCR were employed to assess the transcriptional regulatory mechanisms of FoxO1. Dot blot analysis in conjunction with RNA immunoprecipitation (RIP) was used to assess post-transcriptional regulation. SF significantly alleviated the symptoms of IMQ-induced lupus in mice, including joint damage. SF decreased the proportion of monocytic MDSCs (M-MDSCs), with no significant effects on granulocytic MDSCs (G-MDSCs), in both blood and spleen. Network pharmacological analysis indicated that FoxO1 was a key target of SF in M-MDSCs. Expectedly, SF-containing serum enhanced the immunosuppressive effect of M-MDSCs on Th17 cells by increasing FoxO1. The therapeutic efficacy of SF was diminished in the pristane-induced lupus model with myeloid FoxO1-deficient mice. Mechanistically, FoxO1 impaired the glycolytic process in M-MDSCs by inhibiting PFKFB3 transcription, thereby enhancing their immunosuppressive effect on Th17 cells. Additionally, delphinidin chloride (DP), an important constituent of SF, increased FoxO1 mRNA stability by downregulating ALKBH5-m6A modification in M-MDSCs. This study confirmed that SF enhanced glycolysis in M-MDSCs by regulating the ALKBH5-FoxO1-PFKFB3 axis, which decreased Th17 cells and alleviated lupus and joint injury. These data firstly indicate SF may represent a potential treatment option for SLE and joint damage, revealing regulatory effects of DP, the key active component of SF, at the post-transcriptional level.