Multi-component immune knockout: A strategy for studying the effective components of traditional Chinese medicine

Periploca forrestii Schltr., a traditional Chinese medicine (TCM), is commonly used to treat autoimmune diseases such as rheumatoid arthritis (RA). However, its mechanism, involving a variety of cardiac glycosides, remains largely unknown. The immune knockout strategy can highly selectively deplete target components by immunoaffinity chromatography (IAC). We aimed to identify the common structural features of cardiac glycosides in P. forrestii and design IAC to specifically recognize these features to achieve the multi-component knockout of potential active substances from the extracts of P. forrestii. A content detection experiment confirmed that the content of a compound with periplogenin structure (CPS) in the extract of P. forrestii was reduced by 45% by IAC of periplogenin. The immunosuppressive ability of the extract on H9 human T lymphocytic cells was weakened after CPS knockout from P. forrestii extract. Molecular biology experiments showed that mRNA expression of interferon-γ (IFN-γ), interleukin-2 (IL-2), and interleukin-6 (IL-6) in H9 cells was up-regulated after CPS knockout, while no significant changes in the expression of interleukin-4 (IL-4) were found. CPS knockout from P. forrestii extract did not cause significant changes in the proliferation of lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells incubated with this extract. These results indicate that CPS exhibited immunosuppressive effects via inhibiting the T helper 1 (Th1) cell immune response and not via the anti-inflammatory components in P. forrestii. This is the first use of IAC to achieve multi-component knockout in TCM extracts for identifying effective compounds. This method is effective and reliable and warrants further exploration.