Schisandrin B Induces HepG2 Cells Pyroptosis By Activating NK Cells Mediated Anti-tumor Immunity

Objective: Hepatocellular carcinoma(HCC)has become a tremendous threat to the health of the world’s population with its high incidence and mortality rate.Pyroptosis,an inflammatory programmed cell death,is closely related to the pathogenesis and development of HCC.In addition,studies have shown that human natural killer cells are able to regulate the process of pyroptosis to promote tumor cell death.Schisandrin B(Sch B)is a lignan isolated from the Chinese herb Schisandra chinensis,which has various anti-cancer effects and pharmacological activities.In this study,we propose focusing on Sch B’s role in regulating the pyroptosis of Hep G2 cells by NK cells and its potential mechanisms.The research has provided a theoretical and experimental basis for the development of Sch B as a promising candidate against liver cancer.Methods: Human hepatoma cell line Hep G2 and human natural killer cell line NK-92 were used for the in vitro experiments.(1)A CCK-8 assay was performed to determine the impact of different concentrations of Sch B on the viability of LO2,Hep G2 and NK-92 cells.At the same time,it was possible to determine the optimum effective target cell ratio for the co-culture of Hep G2 and NK-92 for 4 hours.(2)Fluorescent inverted microscopy was used to directly visualize the pyroptosis and apoptotic morphologies of Hep G2 cells after treatment with Sch B alone and co-culture with NK-92.(3)After treatment of Hep G2 cells with Sch B(20 μM)alone or in culture with NK-92 at an effective target cell ratio of 1:1,the mode of cell death was determined by using Annexin V-FITC/(Propidium iodide)PI apoptosis kits and Annexin m Cherry/SYTOX apoptosis kits.(4)Western blot assays were applied to detect the expression and activation levels of the pyroptosis-related proteins gasdermin E(GSDME),N-GSDME and caspase 3.Meanwhile,the extent of lactate dehydrogenase(LDH)release was measured using the lactate dehydrogenase cytotoxicity assay kit.(5)The use of specific inhibitors of caspase 3(Ac-DEVD-CHO),small interfering RNA of caspase 3 and granzyme B(Gzm B)specific inhibitor(Z-AAD-CMK)to further elucidate the mechanism of Sch B regulation of cell pyroptosis.Results: Experiments such as CCK-8,western blot and immunofluorescence assays showed that Sch B could inhibit cell viability and induce apoptosis of Hep G2 cells.However,microscopic observations and the expression level of pyroptosis protein of GSDME,N-GSDME,caspase 3 indicated that NK-92 could transform the apoptotic process into pyroptosis by treatment of Sch B in Hep G2 cells.The studies revealed that this effect of NK cells was related to the activation of caspase 3-GSDME in Sch B-treated Hep G2 cells by experiments such as caspase 3 inhibition or silencing.Moreover,after applying specific inhibitors of Gzm B we found that NK cells might activate caspase 3 through the perforin-granzyme B pathway to promote the pyroptosis of Hep G2 cells.Conclusion: This study explored the correlation between Sch B and NK cells response on pyroptosis and identified perforin-granzyme B-caspase 3-Gasdermin E pathway is involved in the process of pyroptosis.Our study indicated that an immunomodulatory mechanism of Sch B on Hep G2 cells pyroptosis and Sch B as a promising immunotherapy combination partner for the treatment of HCC.