Epimedium brevicornu Maxim. extract activates Natural Killer Cells against Hepatocellular Carcinoma via the cGAS-STING Pathway

Lu Liu^1*Xiaoyan Zhan²Xianling Wang²Jincai Wen²Xiaoyan Chen²Yuanyuan Guo²Xueting Wang³Liu Li⁴Haibo Cheng⁴Zhaofang Bai²Xiaohe Xiao¹

• ¹Chengdu University of Traditional Chinese Medicine, Chengdu, China
• ²5th Medical Center of Chinese PLA General Hospital, Beijing, China
• ³Nankai University, Tianjin, China
• ⁴Nanjing University of Chinese Medicine, Nanjing, China

Epimedium brevicornu Maxim., a traditional Chinese botanical drug, has shown significant therapeutic effects against hepatocellular carcinoma (HCC), with its flavonoid compounds exhibiting anti-HCC activity through various mechanisms. However, it remains unclear whether the anti-HCC effects of Epimedium are mediated through the activation of natural killer (NK) cells. The impact of EPE on NK cell activity was assessed via flow cytometry. A co-culture model of NK-92 cells with K562 target cells was constructed to evaluate EPE-enhanced NK cytotoxicity using Calcein-AM release assay. A murine HCC subcutaneous xenograft model was employed to demonstrate EPE-targeted NK cell activation against HCC in vivo. EPE significantly promoted the synthesis and release of IFN-γ, Granzyme B, and Perforin in NK-92 cells and increased the expression of the activating receptor NKG2D on the NK-92 cell and human primary NK cells surface. Treatment of human primary NK cells with EPE increased the proportion of the CD56brightCD16dim and CD56dimCD16bright NK cell subsets. Calcein release assays demonstrated that EPE enhanced the cytotoxic activity of NK cells against K562 cells. Studies utilizing a murine model of subcutaneous HCC xenografts confirmed that EPE effectively inhibited tumor growth, promoted tumor cell death, and significantly elevated the levels of the NK cell activation markers IFN-γ and CD107a in the spleen and tumor tissues of HCC-bearing mice. NK1.1 cell depletion experiment proved that depletion of NK1.1 cells significantly attenuated the anti-HCC effect of EPE, further demonstrating that the EPE exerts its anti-tumor action by specifically targeting and activating NK cells in vivo. Mechanistic studies revealed that EPE promoted IFN-β release in NK cells, significantly increased the phosphorylation levels of STING and IRF3, and markedly promoted the expression and release of IFN-γ by NK cells. Concurrently, immunofluorescence results indicated that EPE significantly upregulated the expression level of p-STING protein in tumor-infiltrating NK cells within the tumor tissues of HCC mice. There is a strong binding ability between Epimedin C and STING. Moreover, Epimedin C decrease the thermostability of STING but increase the resistance to protein-digesting enzymes. EPE exerts anti- HCC effects by activating NK cells through the activation of the cGAS-STING signaling pathway.