Reduction of FoxP3+ Tregs by an immunosuppressive protocol of rapamycin plus Thymalfasin and Huaier extract predicts positive survival benefits in a rat model of hepatocellular carcinoma.

Abstract

BackgroundInvestigate immunoregulation and anti-tumor immunity of FoxP3+Tregs after treatment with rapamycin (RAPA/SRL) plus thymalfasin (Zadaxin) and Huaier extract (PS-T) in a hepatocellular carcinoma (HCC) rat model simulating HCC relapse after liver transplant (LT).MethodsWe successfully established a rat model simulating HCC relapse after LT using an optimized chemical induction method with TACROLIMUS, methylprednisolone, and diethylnitrosamine as identified by visible liver nodules and hematoxylin-eosin staining. The model rats were then treated with RAPA, Zadaxin, and PS-T. Immune status changes were analyzed by flow cytometry, and protein expression of Akt and mTOR was determined by western blotting. Cytokines were measured by ELISAs.ResultsCombined therapy by RAPA plus Zadaxin and PS-T obviously alleviated hepatic pathological changes and significantly decreased the levels of FoxP3+Tregs in peripheral blood, the spleen, and the liver (P<0.05) and expression of mTOR protein (P<0.01) in the liver, obviously improved survival time (P=0.02). Moreover, the levels of CD8+T cells were increased significantly to almost normal levels (P<0.05) in comparison with no SRL monotherapy protocols. Inhibitory cytokines were also decreased in accordance with FoxP3+Tregs. Significant decreases of IL-10 and TGF-β were observed after SRL-based therapy (P<0.01) in comparison with the other groups. Serum alpha fetoprotein (AFP) and vascular endothelial growth factor (VEGF) levels were also decreased significantly (P<0.05). FoxP3+Tregs showed a negative correlation with CD8+ and CD4+/CD8+T cells and a positive correlation with AFP, and VEGF (P<0.05).ConclusionsSRL-based therapy reduces FoxP3+Tregs to decrease secreted inhibitory cytokines which may enhancement the viability and number of CD8+T cells to exert anti-tumor effects that are mainly mediated through the AKT-mTOR signaling pathway.