Relationship between AKT and PKCθ and morphine-induced inhibition of differentiation of T helper cells

Objective To evaluate the relationship between protein kinase B (AKT) and protein kinase (PKCθ) and morphine-induced inhibition of differentiation of T helper (Th) cells.Methods Peripheral venous blood samples were taken from 20 healthy volunteers..Peripheral blood mononuclear cells (PBMCs) were extracted by density gradient centrifugation method.CD4+ T lymphocytes extracted were purified by magnetic bead separation.CD4+ T lymphocytes were randomly assigned into 5 groups (n =3 each) using a random number table.CD4 + T lymphocytes were incubated routinely in group C.CD4 + T lymphocytes were incubated in the presence of PMA 25 ng/ml + ionomycin 1 μg/ml (group PI),PMA 25 ng/ml + ionomycin 1 μμg/ml + morphine 50 μμg/ml (group M),or PMA 25 ng/ml + ionomycin 1 μμg/ml + morphine 50 μg/ml + naloxone 50 μμg/ml (group N).The cells were incubated for 4 h in the incubator containing 5％ CO2 at 37 ℃.The expression of interferon (IFN)-γ and interleukin-4 (IL-4) was detected by flow cytometry.The expression of IFN-γ and IL-4 was used to reflect the percentage of Th1 and Th2 cells,respectively.The ratio of Th1/Th2 was calculated.The expression of AKT,phosphorylated AKT (p-AKT),PKCθ and phosphorylated PKCθ (p-PKCθ) was detected by Western blot,and the ratio of p-AKT/AKT and p-PKCθ/PKCθ was calculated to reflect the activities of AKT and PKCθ,respectively.Results Compared with group C,the percentage of Th1 and Th2 cells and ratio of Th1/Th2 were significantly increased in PI,M and N groups,the activities of AKT and PKCθ were increased in PI and N groups,and the activity of PKCθ was increased in group M (P ＜ 0.05).Compared with group PI,the percentage of Th1 cells,ratio of Th1/Th2 and activity of AKT were significantly decreased in group M,the ratio of Th1/Th2 was decreased in group N (P ＜0.05),and no significant change in the activity of PKCθ was found in M and N groups (P ＞ 0.05).Compared with group M,the percentage of Th1 cells,ratio of Th1/Th2 and activity of AKT were significantly increased (P ＜ 0.05),while no significant change in the activity of PKCθ was observed in group N (P ＞ 0.05).Conclusion The mechanism by which morphine inhibits the differentiation of Th cells through activating opioid receptors is related to inhibition of AKT activation,but not related to PKCθ. Key words: Morphine;  T-lymphocytes, helper-inducer;  Protein-serine-threonine kinases;  Protein kinase C