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

Background: Investigate 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).Methods: We 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.Results: Combined 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-beta 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).Conclusions: SRL-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.