Natural killer (NK) cells are members of the innate immune system which are known to mediate major histocompatibility complex (MHC) unrestricted cytotoxicity against virally infected and neoplastic cells. Besides their ability to kill target cells directly, NK cells are also known to be potent immune modulators with the ability to produce abundant cytokines capable of modulating immune responses. The NK92-MI cell line displays functional characteristics of activated NK cells, so it is a useful model for the study of NK-cell biology.

In recent studies, we found that Sea buckthorn(SBT; Hippophae rhamnoides L.) oil obtained from the the berries could protect against chronic stress-induced inhibitory function of NK cells.

Flavonoids belong to a group of low-molecular-weight phenylbenzopyrones which have various pharmacological properties including antioxidant activity, anticancer, and immunomodulatory effects. Sea buckthorn fruit is rich in flavonoids. The total flavonoids of Hippophae rhamnoides L. are the main active components extracted from berries of sea buckthorn.

NK cell–mediated lysis of target cells is determined by the balance of signaling between activating and inhibitory receptors on NK cells. Activating receptors, including NCRs (NKp46, NKp44, and NKp30) and NKG2D, bind ligands induced by cellular stress, infection, or tumor transformation. Activating signals are transmitted through immunoreceptor tyrosine–based activating motifs (ITAMs) located in the cytoplasmic tail of the receptor or through ITAMs in adaptor molecules, which associate with activating receptors at the cell surface.

When a target cell lacks or underexpresses HLAI and/or overexpresses activating ligands, NK cells eliminate that target by releasing preformed cytotoxic granzymes and perforin stored as granules or activate apoptosis pathways in the target cell. In our study, we detected expressions of activating receptors (NKp30, NKp44, NKp46, and NKG2D) in NK92-MI cells by flow cytometry analysis.

The results showed that sea buckthorn total flavonoids significantly enhanced expressions of NKp44 and NKp46 in NK92-MI cell. These findings suggest that sea buckthorn total flavonoids may activate NK92-MI cells by up-regulating the expression levels of activating receptors NKp44 and NKp46.

Perforin and granzymes are stored in cytoplasmic granules and function after direct interaction between NK and target cells. Perforin, a membrane pore-forming molecule, permeabilizes cells. Granzymes, a family of serine proteases, disrupt cell-cycle progression, inflict DNA damage, and dissolve the nucleus upon entrance into the cell. Granzyme B is the most abundant serine protease stored in the secretory granules of CTLs and NK cells.

Results showed that sea buckthorn total flavonoids could increase the expressions of both perforin and granzymes B. These findings suggest that sea buckthorn total flavonoids may increase cytotoxicity of NK92-MI cells against K562 cells by up-regulating the expressions of perforin and granzymes B.

NK cells are important sources of various cytokines and chemokines. Results showed that sea buckthorn total flavonoids could up-regulate expressions of IL-1α, IL-2, IL-7, IL-15, CSF-2, CSF-3, MCP-1, MIG, IFN-γ, TNF-α, and TNF-β and down-regulate expressions of MIP-1β, CX3CL-1, IL-16, and MIF. IFN-γ and TNF-α productions by NK cells have been identified as an integral part of NK-cell cytotoxic activity. Activated NK cells are the main source of IFN-γ. IL-2 and IL-15 have been shown to enhance perforin expression in NK cells. IL-15 is an important regulator of NK-cell development, homeostasis, and activation.

Researches have showed that JAK-STAT signal pathway was involved in regulating cytolysis activity of NK cell. STAT1, 4, and 5 were related to the cytotoxicity of NK cells. Results showed that sea buckthorn total flavonoids strongly up-regulated expression of phospho-STAT1 and relatively weakly up-regulated phospho-STAT5. STAT1 is a crucial regulator of IFN-γ production and NK-cell cytotoxicity. Stat1-deficient mice are highly susceptible to bacterial and viral infections. STAT5 transmits signals downstream of IL-2 and IL-15, and its expression is indispensable for the survival of peripheral NK cells. There is evidence that the loss of STAT5 reduces NK-cell numbers and impairs cytolytic responses. Our findings suggest that sea buckthorn total flavonoids may increase cytotoxicity of NK92-MI cells against K562 cells through STAT1 and STAT5 signal pathway.

The above results suggest that sea buckthorn total flavonoids stimulated NK92-MI cells to activate and enhance cytotoxicity of NK92-MI cells. Consequently, sea buckthorn total flavonoids has the potential to be an immuno-potentiating agent, which can potentially be used as functional food and pharmaceutical therapy ingredients.