Xanthohumol Regulates Th1/Th2 Balance And Promotes Anti-tumor Immunity In Mouse

T helper cells(Th cells) play an important role in the immune system, which regulates T lymphocyte cells or help other lymphocyte cells to complete their function by cytokines secretion. Th1 and Th2 are two major Th cells subgroups. Under normal body circumstances, Th1/Th2 in a balance situation, and maintain the body’s normal immune function. When the body in abnormality, it always associate with Th1/Th2 imbalance, which called "Th1/Th2 balance drift". With the development of tumor immunotherapy, the research of Th1/Th2 balance shift and malignant tumor become one of hot topics in this field and medicinal natural products have been under the favor of people because of lower toxic side-effect. Xanthohumol(XN) is a pentene flavonoids which isolated and purified from hops. Studies have been showed that Xanthohumol can inhibited proliferation of a variety of cancer cells, such as breast cancer, prostate cancer, pancreatic cancer, liver cancer, and so on. However, most of these studies are based on the cellular level, the anti-tumor activity of Xanthohumol in vivo has been reported rarely.In this study, we investigated the mechanism of Xanthohumol antitumor immunity in BALB/c-4T1 mice which Th1/Th2 balance in a core position. Firstly, we constructed the BABL/c-4T1 tumor-bearing mice model. We explored the anti-tumor activity of Xanthohumol in vivo, examined the changes of tumor volume and weight in model mice, the results showed that Xanthohumol significantly inhibited tumor growth in tumor-bearing mice. H&E staining used to examine the tissue damage of liver, kidney and tumor of experimental mice, and results showed that Xanthohumol have no significant effect on the liver and kidney, while promoting tumor tissue damage. ELISA and immunohistochemistry assay used to examine breast cancer-specific marker CA153 tumor proliferation Ki-67 protein expression in mice, the results showed that Xanthohumol down-regulated the expression of CA153 and Ki-67. Then, we explored the effects of Xanthohumol on Th1/Th2 balance. Flow cytometry used to examine the proportion of CD8+/CD25+ and perforin and granzyme B, the results show that the ratio of CD8+/CD25+, the expression of perforin and granzyme B are increased, indicating that anti-tumor CTL responses activity are promoted. ELISA and flow cytometry used to examine the Th1/Th2 cytokines and the ratio changes of Th1/Th2, the results showed that Xanthohumol promoted Th1 cytokines expression, and inhibited Th2 cytokines expression, while promote Th1/Th2 balance towards Th1. At last, we explored the mechanism of Xanthohumol regulates Th1/Th2 balance. Western blot and immunohistochemistry used to examine the key transcription factors of Th1/Th2 development, and the results showed that Xanthohumol increased expression of Th1-specific transcription factor T-bet, and down-regulated expression of Th2-specific transcription factor GATA-3. Simultaneously, we found Xanthohumol promoted activation of STAT4, while have no significant effect on the activity of p-STAT6, suggesting that Xanthohumol may regulate Th1/Th2 balance towards Th1 polarization by regulating STAT4 activity.In summary, this study proved that Xanthohumol have significantly activity of inhibition tumor growth of in vivo, and promoted anti-tumor immunity. We also found that Xanthohumol regulated Th1/Th2 balance towards Th1 polarization maybe via activating STAT4 pathway. The study explored the anti-tumor immune activity of Xanthohumol in vivo, and provide a new direction and theoretical basis tumor immune of Xanthohumol.