Screening of immunopeptides from Zhiciweipi (Corium Erinacei Praeparaum) and their immunity to RAW264.7 macrophages

Abstract

Zhiciweipi (Corium Erinacei Praeparaum) is one of the traditional Chinese animal medicines used to treat hematochezia and hemorrhoids. At present, there is a lack of the workflow for ingredient identification of Zhiciweipi (Corium Erinacei Praeparaum) and the studies regarding immunocompetent ingredient screening and evaluation. This study aims to screen immunopeptides in Zhiciweipi (Corium Erinacei Praeparaum) and investigate their immunity to RAW264.7 macrophages. In this study, the workflow for protein polypeptide identification and analysis of Zhiciweipi (Corium Erinacei Praeparaum) was established based on LC-MS/MS. According to Gene ontology_Biological process (GO_BP) functional and signal pathway enrichment analysis results, the identified proteins are related with vascular development, wound healing, IL2 STAT5 signal, and so on. A total of 20 immune regulatory genes were identified from the Venn diagram of protein and immune regulatory genes in Zhiciweipi (Corium Erinacei Praeparaum). The PPI network analysis demonstrated that ALB protein ranks the top in term of significance. According to analysis on physical properties and structural and safety prediction of 61 polypeptide fragments related with serum albumin in Zhiciweipi (Corium Erinacei Praeparaum) which are corresponding to ALB protein, RHPYFYAPELL and RHPYFYAPELLYF (RHP-LYF) are the most optimal. The molecular docking showed that RHPYFYAPELL and RHP-LYF can make effective bonding with IL-2 and STAT5. The molecular dynamic experiment reflected that RHP-LYF has the strongest affinity with ALB. In the present study, CCK8 method was applied to test RHP-LYF treatment of RAW264.7 cells in the mice macrophage system at different concentrations. Results showed that such treatment may not inhibit cell growth when the RHP-LYF concentration is lower than 200 ng/mL. The polypeptide fluorescence marking experiment proved that polypeptides can enter into cells and can be detected in the whole cell. Effects of RHP-LYF on IL-2, TNFα, IFNγ and IL10 levels in RAW264.7 cells and mice serum were detected by ELISA kit. Results showed that polypeptide not only can inhibit secretion of inflammatory factors in vivo and in vitro, but also promote secretion of anti-inflammatory factors, and further test effects of RAW264.7 cells on expressions of p-STAT5 and STAT5 through Western Blot. Results showed that polypeptide treatment may inhibit level of p-STAT5 and has dosage-dependent effect. Polypeptides from Zhiciweipi (Corium Erinacei Praeparaum) are safe and have immunization to RAW264.7 macrophage. All of these results can provide a meaningful method to drug quality assessment, immune bioactive peptide screening and evaluation of Zhiciweipi (Corium Erinacei Praeparaum) and other animals rich of peptides.