Mechanism of Shashen-Maidong herb pair in treating hepatocellular carcinoma using network pharmacology and experimental validation

Abstract

Hepatocellular carcinoma (HCC) is among the most prevalent malignant tumors globally and represents a significant public health issue worldwide. Immune cell dysfunction is the crucial factor for the formation of immunosuppression microenvironment of HCC. Glehnia littoralis (A.Gray) F.Schmidt ex Miq. (Shashen) and Ophiopogon japonicus (Thunb.) Ker Gawl. (Maidong) are classic herb pair in traditional Chinese medicine (TCM) of nourishing Yin, and is widely applied in the treatment of HCC and possesses multiple immunomodulatory functions. However, the role of the Shashen-Maidong herb pair (SS-MD) for the management of HCC and the potential mechanisms has not been explicated. The purpose of the research is to investigate the potential mechanism of the SS-MD herb pair for the management of HCC. The known components of the SS-MD herb pair were preliminarily identified using UPLC-Q-Orbitrap-MS/MS. The active ingredients of SS-MD herb pair in treating HCC were screened by constructing herb-component-target network, and the key therapeutic targets were explored by constructing a protein-protein interaction (PPI) network. The binding affinity of the key targets and components were validated through molecular docking and molecular dynamics simulations. GO biological function and KEGG pathway analyses were operated to elucidate the potential mechanisms of the SS-MD herb pair for the management of HCC. And the mechanism was verified in the tumor bearing mice model and cell co-culture experiments. Network pharmacology prediction revealed 39 active components and 138 targets of the SS-MD herb pair for the treatment of HCC. KEGG analysis mainly focused on Notch signaling pathway and Apoptosis signaling pathway. The targets were enriched in biological functions of lymphocyte effector function and lymphocyte apoptosis. In vivo and in vitro experiments proved that the SS-MD herb pair can improve the proportion of CD8 + T cells in the HCC immune microenvironment, regulate its subgroup distribution. SS-MD herb pair promoted CD8 + T cells to secrete IL-2, TNF-α, IFN-γ, Granzyme B and Perforin, and inhibited apoptosis by regulating Notch signaling pathway. This study identified the key components, targets, and signaling pathways of the SS-MD herb pair, confirm that SS-MD herb pair play an immunomodulatory role in treating HCC, provides theoretical support for the collaborative treatment of HCC with TCM. • Network pharmacology prediction revealed 39 active ingredients and 138 targets of the SS-MD herb pair for the treatment of HCC. • SS-MD herb pair can increase the proportion of CD8 + T cells in the tumor microenvironment of H22-bearing mice. • SS-MD herb pair increase the proportion of effector subsets of CD8 + T cells and reduce the proportion of terminally differentiated subsets in HCC. • SS-MD herb pair improve the effector function, and inhibit apoptosis of CD8 + T cells through regulating Notch signaling pathway.

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