OBJECTIVETo investigate the effect of kirenol on bovine type II collagen (CII)-specific lymphocytes in vivo and in vitro, and explore the mechanism of kirenol-induced immunosuppression in antigen-specific lymphocytes.

METHODSTwenty-four Wistar rats were randomized into control group, collagen-induced arthritis (CIA) model group, kirenol group (2 mg/kg), and prednisolone group (2 mg/kg). After CII injection, the rats in the latter two groups received intragastric administration of kirenol and prednisolone for 30 days, and the spleens and draining lymph nodes of the rats were harvested to prepare single cell suspensions for measurement of the cytokine levels using ELISA. In the in vitro experiment, the lymphocytes from the control rats, with or without 20 µg/ml CII treatment in the presence of 0-80 µg/ml kirenol, were evaluated for cell proliferation and apoptosis using [(3)H]-thymidine incorporation and flow cytometry, respectively.

RESULTSCompared with those in CIA group, IFN-γ and TNF-α production was significantly reduced in splenocyte culture supernatant of kirenol group (P<0.05 and P<0.01, respectively), and the level of IL-10 and IL-4 was up-regulated (P<0.05 and P<0.01, respectively); IFN-γ and TNF-α secretion by the cultured lymph node cells (LNCs) significantly decreased (P<0.05 and P<0.001, respectively) and IL-10 and IL-4 production increased (P<0.05, P<0.001) in kirenol group. In the in vitro experiment, kirenol treatment caused obvious suppression of CII-induced LNC proliferation and dose-dependently induced antigen-specific apoptosis of the splenocytes and LNCs.

CONCLUSIONKirenol treatment reduces pro-inflammatory cytokine secretion, increases anti-inflammatory cytokine production, inhibits cell proliferation and induces apoptosis of CII-specific lymphocytes in vitro, suggesting the potential of kirenol as an immunosuppressant.

Animals ; Anti-Inflammatory Agents ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Arthritis, Rheumatoid ; chemically induced ; drug therapy ; immunology ; Cattle ; Cell Proliferation ; Cells, Cultured ; Collagen Type II ; immunology ; Cytokines ; immunology ; Diterpenes ; pharmacology ; therapeutic use ; Female ; Immunosuppressive Agents ; pharmacology ; therapeutic use ; Lymphocytes ; cytology ; drug effects ; immunology ; Rats ; Rats, Wistar

Objective　To determine the optimal time window to treat spinal cord injury (SCI) by epidermal growth factor receptor (EGFR) inhibitor osimertinib.Methods　60 rats were randomly divided into 6 groups (10 in each group):injury control group and 5 osimertinib-treated groups (0 d,1 d,3 d，5 d and 7 d group).After rats spinal cord injury model was established,the rats in osimertinib-treated group were applied with at different time (0 day,1 day,3 days and 7 days) after injury，while the injury group animals were only given vehicle solution (normal saline) lacking osimertinib.Neurological deficits was evaluated by using the BBB locomotor scale at first day and weekly post-injury.Residual urine volume was recorded everyday in each group.14 days after SCI, 5 rats were randomly selected and sacrificed.Spinal cord tissues were collected,and the expression of GAP-43 was detected by Western blot.Results　(1)The BBB score of rats in 0 d group,1 d group,3 d group was significantly higher than 5 d group,7 d group and injury groups(P<0.05),and there was no significant difference among 0 d group,1 d group and 3 d group(P>0.05).(2)The residual urine volume of rats in 0 d group,1 d group,3 d group was significantly less than that other groups (P<0.05),and there was no significant difference among 0 d group,1 d group and 3 d group(P>0.05).(3)The expression of GAP-43 in 0 d group,1 d group and 3 d group was significantly higher than other groups(P<0.05,or P<0.01),and there was no significant difference among 0 d group,1 d group and 3 d group (P>0.05).Conclusion　The optimal time of EGFR inhibitor osimertinib to treat spinal cord injury in rats was 0 h to 72 h after spinal cord injury.