Effect Of Morphine On T - Assisted Lymphocyte Differentiation And Its Signal Pathway

Morphine is a widely useful opioid analgesic, and regulates the patients’ immune system.Our earlier researches find that the secretion of intracellular cytokines IL-1,IL-2,TNF-α and IFN-γ will decrease and lead to the imbalance of Thl/Th2after morphine treatment, but its mechanism is unclear. PI3K/AKT signal pathway participates in the adaptive immune response, including Th cells proliferation and cytokine secretion. PKCθ signal pathway regulates the expression of transcription factor AP-1, NF-κB, NFAT, involved in Th cells differentiation and proliferation. Thus, our study assumed that morphine could inhibit Th cell differentiation to Thl and Th2cell directions. The mechanism was related to PI3K/AKT or PKCθ signal pathway.Part1Collection of Th cellsObjective To collect Th cells with purity>95%and apoptosis ratio<5%.Methods Twenty healthy volunteers’ peripheral vein blood about20ml were collected. PBMCs were extracted by Density Gradient Centrifugation Methods, and Th cells were purified by MACS method. The purity and apoptosis ratio of Th cells were investigated by Flow Cytometry.Results The purity of Th cells was more than95%, and the apoptosis ratio was less than5%.Conclusion The collected Th cells meet the cell standards in vitro study. Part2Morphine treatment on the differentiation of Th cells via the PI3K/AKT or PKC6signal pathway mechanismObjective To investigate the effect of morphine on Th cells differentiation and the PI3K/AKT or PKCθ signal pathway mechanism.Methods The prepared Th cells were randomly received the following treatment: Control(C group); PMA, Ionomycin(PI group); PMA, Ionomycin and morphine25ng/ml(M1group); PMA, Ionomycin and morphine50ng/ml(M2group); PMA, Ionomycin and morphine100ng/ml(M3group); PMA, Ionomycin and morphine200ng/ml(M4group); morphine200ng/ml(MO group). Culture supernatant were added with10%FBS RMPI1640medium and cells were incubated at37℃,5%CO2for4hours. Intracellular cytokines IFN-y and IL-4were detected by Flow Cytometry, and protein AKT and PKCθ were detected by Western Blotting analysis. All analyses were using ANOVA test between groups, P<0.05was considered significance for all comparisons.Results Compared with group C, IFN-y, IL-4, the ratio of IFN-y/IL-4increased in group M1, M2, M3or M4(P<0.05), and no significant difference in group MO (P>0.05)Compared with group PI, IFN-y and the ratio of IFN-y/IL-4decreased in group M2, M3, M4or MO(P<0.05),and no significant difference in group M1(P>0.05). There was no significant difference about IL-4in group Ml, M2, M3or M4(P>0.05). But IL-4in group C and MO decreased significantly (P<0.05).Compared with group C, p-AKT increased in group PI or M2(P<0.05) There was no significant difference in group M1,M3,M4or MO(P>0.05). p-PKCθ increased in group PI,M1,M2,M3or M4(P<0.05). No significant difference in group MO.Compared with group PI, p-AKT decreased in group M1, M2, M3or M4(P<0.05),and no significance in group MO. p-PKCθ was not changed in group M1, M2, M3or M4respectively (P>0.05),and decreased in group MO(P<0.05).Conclusion Morphine can inhibit the differentiation of Th cells. The mechanism is related to PI3K/AKT signal pathway but not PKCθ signal pathway.Part3Naloxone improved the inhibition of morphine-induced immune-suppression on Th cellsObjective To study whether naloxone can change the inhibition of morphine-induced Th cell differentiation.Methods The prepared Th cells were randomly received the following treatment: control(C group); PMA, ionomycin(PI group); PMA, ionomycin and morphine50ng/ml(M group); PMA, ionomycin, morphine50ng/ml and naloxone25ng/ml(Nl group); PMA, ionomycin, morphine50ng/ml and naloxone50ng/ml(N2group); naloxone50ng/ml(NO group). Cell supernatant were added with10%FBS RMPI1640medium and cells were incubated at37℃,5%CO2for4hours. Intracellular cytokines IFN-y and IL-4were detected by Flow Cytometry, and protein AKT were detected by Western Blotting analysis. All analyses were using ANOVA test between groups,P<0.05was considered significance for all comparisons.Results Compared with group C, IFN-y, IL-4, the ratio of IFN-y/IL-4increased in group PI, M, N1or N2(P<0.05),no significant difference in group NO(P>0.05).Compared with group M, IFN-y and the ratio of IFN-y/IL-4increased in group Nl or N2(P<0.05), and decreased in group NO(P<0.05).IL-4did not change significantly in group N1or N2(P>0.05), but decreased in group NO(P<0.05).Compared with group C, p-AKT increased in group PI, M, N1and N2(P<0.05),no significant difference in group NO(P>0.05).Compared with group M, p-AKT was not changed in group N1(P>0.05), while increased in group N2(P<0.01) and decreased in group NO(P<0.05).Conclusion Morphine can suppress Th cells differentiation and naloxone can antagonize this effect via μ receptor.