Prevention of cyclophosphamide-induced immune suppression by polysaccharides from Apocynum Venetum flowers via enhancing immunity, reducing oxidative stress and regulating gut microbial in mice

Introduction: Emerging proof suggests that Apocynum venetum flowers polysaccharides (AVFP) has immunomodulatory effects in vitro. However, the action mechanism of AVFP is still unclear in vivo. The purpose of this study is to probe into the potential mechanism of AVFP on immunosuppressed mice through investigating organ index, cytokine levels, anti-oxidative stress capacity, transcriptomics and gut microbiota. Methods: Immunocompromised mouse model induced by cyclophosphamide (CXT) was divided into 6 groups. Enzyme-labeled method, HE, transcriptomics and high-throughput sequencing were used to detect the regulatory effects of AVFP on immunocompromised mice, as well as the function of AVFP on the concentration of short-chain fatty acids (SCFAs) by high-performance liquid chromatographic analysis (HPLC). Pearson was used to analyze the correlation between intestinal microbiota and biochemical indexes. Results: The experimental results illustrated that AVFP has protective effects against CXT-induced immunosuppression in mice via prominently increasing organ index, levels of anti-inflammatory factors in serum as well as enhancing antioxidant capacity of the liver. Meanwhile, it could also signally decrease the level of pro-inflammatory cytokines in serum, the activity of transaminase in serum and the content of free radicals in liver. Transcriptomics results discovered that AVFP could play a role in immune regulation via participating in NF-?B signaling pathway, regulating the immune-related genes Bcl3, Hp, Lbp, Cebpd, Gstp2 and Lcn2. Intestinal microbiota results illustrated that AVFP could increase abundance of beneficial bacteria, reduce abundance of harmful bacteria, while dramatically improved the content of SCFAs, modulated immune responses and improved host metabolism. Spearman analysis further assessed the correlation between gut microbiota and biochemical indicators. Conclusion: These findings demonstrated that AVFP could enhance the immune effects of the immunosuppressed mice and improve the body's ability to resist oxidative stress. Overall design: Transcriptomic analysis was performed on 9 spleen samples from Control, Mod and AVFP-M groups. Firstly, took 60 mg of tissue and pulverized it into powder in liquid nitrogen, centrifuged after homogenizing, added 0.3 mL of isoamyl alcohol to the supernatant, and centrifuged. After that, 1 mL75% ethanol was added to wash twice, and then centrifuged. In the end, DEPC-treated water was added to dissolve RNA, followed by identification and quantification of total RNA using Nano Drop and an Agilent 2100 Bioanalyzer.