Optimization of protein removal process of polysaccharide and its immunomodulatory mechanism in cyclophosphamide-induced mice by metabolomics and network pharmacology.

In this study, TCA-n-butanol was chosen as the best deproteinization method for polysaccharide (LJP) by comparing the polysaccharide retention rate and the protein clearance rate of five different methods. The response surface methodology (RSM) based on the Box-Behnken design (BBD) was used to optimize the deproteinization conditions as follows: TCA: n-butanol = 1: 5.1, polysaccharide solution: (TCA-n-butanol) = 1: 2.8, and shook for 33 min. LJP could promote the thymus and spleen indexes of cyclophosphamide (CTX)-induced immune-deficient mice. Besides, the contents of cytokine interleukin-2 (IL-2) and hemolysin in mice serum were augmented after treatment with LJP. Based on serum metabolomics analysis, a total of 14 metabolites (VIP >1.0, FC >2 or FC <0.5, and value < .05) were selected as the potential biological biomarkers related to the LJP for treating CTX-induced mice. After the pathway enrichment analysis, these metabolites were mainly involved in the relevant pathways of arginine biosynthesis, Citrate cycle, and other metabolic pathways. Network pharmacology further showed that there were 57 key targeting proteins in the intersection of the potential biological biomarkers and immunodeficiency-related targeting proteins according to protein-protein interactions analysis (PPI). The biological function analysis indicated that the potential biological processes were mainly associated with tricarboxylic acid (TCA) cycle, phospholipid metabolic process, metabolic process, and so on. In conclusion, serum metabolomics combined with network pharmacology could be helpful to clarify the immunomodulatory mechanism of LJP and provide a literature basis for further clinical research on the therapeutic mechanism of LJP.